HK40013541A - Synthetic immune receptors and methods of use thereof - Google Patents

Description

Synthetic immunoreceptors and methods of use thereof

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application serial No. 62/429,619 filed 2016, 12, month 2 and U.S. provisional patent application No. 62/429,597 filed 2016, 12, month 2, the disclosures of which are incorporated herein by reference in their entireties pursuant to 35 u.s.c. § 119.

Technical Field

The present invention relates to the use of Synthetic Immune Receptor (SIR) polypeptides, polynucleotides, expression constructs, and immune effector cells (e.g., T cells, NKT cells) and stem cells engineered to express Synthetic Immune Receptors (SIRs). The disclosure also provides methods of using such polypeptides, polynucleotides, expression constructs, and recombinant cells to treat diseases and disorders, including but not limited to cancer, infectious diseases, allergic diseases, autoimmune diseases, degenerative diseases, or combinations thereof.

Incorporation by reference of the sequence listing

This document comes with a Sequence Listing, entitled "Sequence _ ST25.txt," which was created in 2017 at 12, month 2 and has 77,085,242 bytes of data, machine formatted on an IBM-PC, MS-Windows operating system. This sequence listing is incorporated by reference herein in its entirety for all purposes.

Background

The strategy of activating immune cells to selectively recognize and destroy tumors, i.e., cancer immunotherapy, provides a powerful approach to cancer therapy. Adoptive transfer of T cells (CAR-T cells) using tumor specific T cells and Chimeric Antigen Receptor (CAR) modified immune therapy mediates durable and complete disease regression in some patients with metastatic cancer.

Despite the success of using CAR-T cells, there are several limitations to this approach. In most patients responding to engineered CAR T cells, excessive release of pro-inflammatory cytokines causes symptoms including fever, hypotension, hypoxemia, cardiac dysfunction, renal failure, and electrolyte abnormalities, collectively referred to as "cytokine release syndrome" (CRS). In some cases, CAR therapy can lead to neurological symptoms, including tremors, epilepsy, and can be fatal. Strategies to counteract CRS include treatment with immunosuppressive agents and antibodies to cytokines to block cytokine release.

In addition, one of the most important challenges for successful cancer immunotherapy is to genetically modify T cells to persist for more than several months after metastasis. This has proven to be a greater challenge for T cells modified with CAR genes.

The latest molecular engineering of CAR constructs to include co-stimulatory domains CD28 or 41BB resulted in improved persistence. However, the inclusion of the co-stimulatory domain in the CAR construct results in non-physiological signaling through the receptor. Some CARs exhibit tonic antigen-independent signaling that results in unrestricted cell activation, ultimately leading to apoptosis, excessive cytokine release independent of the cognate antigen, and immunological depletion. Expression of some CARs containing CD28 and CD3z tandem signaling domains results in the constitutive activation and proliferation of transduced primary human T cells that is associated with low-grade in vivo efficacy (frigualt et al, 2015). One mechanism found to lead to the phenotype of CARs with continuous T cell proliferation is high density of CARs at the cell surface (frigualt et al, 2015).

The T Cell Receptor (TCR) is expressed on the surface of T cells. In humans, these receptors recognize complexes formed between Human Leukocyte Antigen (HLA) molecules and antigenic peptides. Recognition of these peptides results in activation of T cell immune function.

In most T cells, the TCR is a heterodimer of alpha (α) and beta (β) chains. The beta chain has two subtypes: c β 2 (in 80% human T cells) and C β 1 (in 20% human T cells). Each chain of the TCR comprises an N-terminal immunoglobulin (Ig) -like variable (V) domain and an Ig-like constant (C) domain, which in turn comprises a transmembrane region and a short cytoplasmic tail at the C-terminus.

Disclosure of Invention

The present disclosure provides at least one recombinant polynucleotide encoding at least one Synthetic Immune Receptor (SIR) comprising (a) a T Cell Receptor (TCR) constant chain having an amino acid sequence selected from the group consisting of: (i) an amino acid sequence at least 98% identical to SEQ ID NO 3010 and having one or more mutations at positions 48, 61, 91, 92, 93 and/or 94 and which may comprise an optional auxiliary module; (ii) an amino acid sequence which is at least 98% identical to SEQ ID NO:3024 and has one or more mutations at positions 18, 22, 57, 79, 133, 136 and/or 139 and may comprise an optional auxiliary moiety; (iii) an amino acid sequence which is at least 98% identical to SEQ ID NO:3025 and has one or more mutations at positions 18, 22, 57, 79, 133, 136 and/or 139 and may comprise an optional auxiliary moiety; (iv) an amino acid sequence at least 98% identical to SEQ ID NO 3046, 3047 or 3048 and which may comprise an optional auxiliary module; (v) an amino acid sequence at least 98% identical to SEQ id No. 3049 and which may comprise an optional auxiliary module; (vi) an amino acid sequence which is at least 98% identical to SEQ ID NO 3051 or 3052 and which may comprise optional auxiliary modules; and (vii) a dimeric combination of two TCR constant chains selected from (i) and (ii), (i) and (iii), (iv) and (ii), (iv) and (iii), and (v) and (vi); (b) optionally a linker; and (c) one or more non-native TCR antigen binding domains selected from the group consisting of: (1) an antibody; (2) antibody fragments (e.g., Fv, Fab, (Fab') 2); (3) a heavy chain variable region (vH domain) of an antibody or a fragment thereof; (4) a light chain variable region (vL domain) of an antibody or a fragment thereof; (5) a single chain variable fragment (scFv) or a fragment thereof; (6) a Single Domain Antibody (SDAB) or fragment thereof; (7) a camelidae VHH domain or a fragment thereof; (8) a monomeric variable region of an antibody; (9) non-immunoglobulin antigen binding scaffolds such as DARPIN, affibody (affibody), affilin, adnectin, affitin, obodies, repebody, fynomer, alphabody, avimer, atrimer, centryrin, pronectin, anticalin, kunitz-type domain, Armadillo repeat protein, or a fragment thereof; (10) a receptor or fragment thereof; (11) a ligand or fragment thereof; (12) bispecific-antibodies, -antibody fragments, -scFV, -vHH, -SDAB, -non-immunoglobulin antigen-binding scaffolds, -receptors, or-ligands; and (13) an autoantigen or fragment thereof, wherein the mutation of (a) (i) - (a) (iii) and the dimer of (a) (vii) provide different binding affinities for a target antigen of the antigen-binding domain and are at least 5% greater than a tcr with the same binding domain, and when the synthetic immune receptor is expressed in lymphocytes, both the antigen-binding domain and the T cell receptor constant chain are expressed in one or more continuous chains on the surface of lymphocytes, such that lymphocytes are triggered to activate, proliferate, secrete cytokines and/or modulate (induce or inhibit) killing of target cells, and have MHC-restricted and MHC-unrestricted antibody type specificity when the expressed antigen-binding domain binds to its antigen. In one embodiment, comprising the TCR constant chain of (a) (vii), the non-native TCR-binding domain is selected from the group consisting of: (iii) the variable regions of the heavy and light chains of an antibody or fragment thereof specific for a predefined target antigen, such that when expressed, one of said heavy and light chains of the antibody or fragment thereof is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said heavy and light chains of the antibody or fragment thereof is attached to the other of said two chains of said T cell constant region; two single-chain variable fragments (scfvs) specific for one or more predefined target antigens, such that when expressed, one of the scfvs is attached to one of the two chains of (a) (vii) of the T cell constant region and the other of the scfvs is attached to the other of the two chains of the T cell constant region; two antibody fragments specific for one or more predefined target antigens, such that when expressed, one of said antibody fragments is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said antibody fragments is attached to the other of said two chains of said T cell constant region; two Single Domain Antibody (SDAB) fragments specific for one or more predefined target antigens, such that when expressed, one of said SDAB fragments is attached to one of said two chains of (a) (vii) of said T cell constant region, and the other of said SDAB fragments is attached to the other of said two chains of said T cell constant region; two camelidae vHH domains specific for one or more predefined target antigens such that, when expressed, one of said vHH domains is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said vHH domains is attached to the other of said two chains of said T cell constant region; two non-immunoglobulin antigen binding scaffolds that are specific for one or more predefined target antigens, such that when expressed, one of the non-immunoglobulin antigen binding scaffolds is attached to one of the two chains (a) (vii) of the T cell constant region and the other of the non-immunoglobulin antigen binding scaffolds is attached to the other of the two chains of the T cell constant region; two receptors or fragments thereof specific for one or more predefined target antigens such that, when expressed, one of said receptors or fragments thereof is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said receptors or fragments thereof is attached to the other of said two chains of said T cell constant region; two ligands or fragments thereof specific for one or more predefined target antigens such that, when expressed, one of said ligands or fragments thereof is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said ligands or fragments thereof is attached to the other of said two chains of said T cell constant region; two structurally distinct antigen-binding fragments specific for one or more predefined target antigens, such that when expressed, one of the antigen-binding fragments is attached to one of the two chains (a) (vii) of the T cell constant region and the other of the antigen-binding fragments is attached to the other of the two chains of the T cell constant region; two binding fragments, one or both of which is bispecific or multispecific such that, when expressed, one of the antigen-binding fragments is attached to one of the two chains of (a) (vii) of the T cell constant region and the other of the antigen-binding fragments is attached to the other of the two chains of the T cell constant region; two autoantigens or fragments thereof, such that when expressed, one of said autoantigens or fragments thereof is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said autoantigens or fragments thereof is attached to the other of said two chains of said T cell constant region; and two vL or fragments thereof, such that when expressed, one of said vL or fragments thereof is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said vL or fragments thereof is attached to the other of said two chains of said T cell constant region; and two vH or fragments thereof, such that when expressed, one of said vH or fragments thereof is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said vH or fragments thereof is attached to the other of said two chains of said T cell constant region. In another embodiment of any of the foregoing, (a) (iv) the TCR constant chain has a non-native TCR binding domain selected from the group consisting of: a variable region of a heavy chain (vH) of an antibody or fragment thereof specific for a predefined target antigen; a variable region of a light chain (vL) of an antibody or fragment thereof specific for a predefined target antigen; a single-chain variable fragment (scFv) or a fragment thereof specific for a predefined target antigen; antibody fragments specific for predefined target antigens (e.g., Fv, Fab, (Fab') 2); a Single Domain Antibody (SDAB) fragment specific for a predefined target antigen; a camelidae vHH domain specific for a predefined target antigen; a non-immunoglobulin antigen binding scaffold specific for a predefined target antigen; a receptor or fragment thereof specific for a predefined target antigen; a ligand or fragment thereof specific for a predefined target antigen; bispecific-antibodies specific for one or more predefined target antigens, -antibody fragments, -scFV, -vHH, -SDAB, -non-immunoglobulin antigen-binding scaffolds, -receptors, or-ligands; and autoantigens or fragments thereof. In yet another embodiment, a polynucleotide encoding (i), (ii), (iii), (iv), (v), or (vi), the non-native TCR binding domain is selected from the group consisting of: a variable region of a heavy chain (vH) of an antibody specific for a predefined target antigen; a variable region of a light chain (vL) of an antibody specific for a predefined target antigen; a single-chain variable fragment (scFv) specific for a predefined target antigen; antibody fragments specific for predefined target antigens (e.g., Fv, Fab, (Fab') 2); a Single Domain Antibody (SDAB) fragment specific for a predefined target antigen; a camelidae vHH domain specific for a predefined target antigen; a non-immunoglobulin antigen binding scaffold specific for a predefined target antigen; a receptor specific for a predefined target antigen or fragment thereof; a ligand specific for a predefined target antigen or fragment thereof; bispecific-antibodies specific for one or more predefined target antigens, -antibody fragments, -scFV, -vHH, -SDAB, -non-immunoglobulin antigen-binding scaffolds, -receptors, or-ligands; and autoantigens or fragments thereof. In another embodiment of any of the foregoing, the polynucleotide encoding the TCR constant chain is a codon-optimized sequence. In another embodiment of any of the foregoing, the polynucleotide encoding the TCR constant chain of (a) encodes a TCR constant chain comprising a mutation that enhances TCR constant chain expression and/or pairing and reduces its pairing with endogenous T cell receptor chains. In another embodiment of any of the foregoing, the polynucleotide encoding the TCR constant chain of (a) comprises 1-40 modified nucleic acid sequences of nucleic acid sequences SEQ ID NOs 730 to 743 or sequences having at least 70% identity to nucleic acid sequences SEQ ID NOs 730 to 743, and is capable of dimerizing with a TCR β 1 or TCR β 2 chain. In another embodiment of any of the foregoing, the polynucleotide encoding the TCR constant chain of (b) or (c) comprises the nucleic acid sequence SEQ ID NO:744 to 765 or 1-40 modified nucleic acid sequences having at least 70% identity to the nucleic acid sequence SEQ ID NO:744 to 765 and is capable of dimerizing with a TCR alpha chain. In another embodiment of any of the foregoing, the polynucleotide encoding the TCR constant chain of (v) comprises 1-40 modified nucleic acid sequences of nucleic acid sequence SEQ ID NO:769 to 770 or a sequence having at least 70% identity to nucleic acid sequence SEQ ID NO:769 to 770, and is capable of pairing with a TCR delta chain. In another embodiment of any of the foregoing, the polynucleotide encoding the TCR constant chain of (v) comprises 1-40 modified nucleic acid sequences of nucleic acid sequence SEQ ID NOs 771 to 772 or sequences having at least 70% identity to nucleic acid sequence SEQ ID NOs 771 to 772 and is capable of dimerizing with a TCR γ chain. In another embodiment of any of the foregoing, the polynucleotide encoding the TCR constant chain of (iv) comprises 1-40 modified nucleic acid sequences of nucleic acid sequences SEQ ID NOs 766-768 or a sequence having at least 70% identity to nucleic acid sequences SEQ ID NOs 766-768 and is capable of dimerizing with a TCR β 1 or TCR β 2 chain. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains bind to one or more disease-associated antigens selected from the group consisting of: CD 19; CD 123; CD 22; CD 30; CD 171; CS-1 (also known as CD2 subunit 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD 33; epidermal growth factor receptor variant iii (egfrviii); ganglioside G2(GD 2); ganglioside GD3(aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (l-l) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. alpha. -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1(ROR 1); fms-like tyrosine kinase 3(FLT 3); tumor associated glycoprotein 72(TAG 72); CD 38; CD44v 6; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3(CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213a 2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate Stem Cell Antigen (PSCA); protease serine 21 (testis protein or PRSS 21); vascular endothelial growth factor receptor 2(VEGFR 2); lewis (Y) antigen; CD 24; platelet derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD 20; a folate receptor alpha; receptor tyrosine protein kinase ERBB2(Her 2/neu); cell surface associated mucin 1(MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); prostasin; prostatic Acid Phosphatase (PAP); mutant elongation factor 2(ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase ix (caix); proteasome (precursor, megalin) subunit, beta form, 9(LMP 2); glycoprotein 100(gpl 00); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and the Abelson (Abelson) murine leukemia virus oncogene homolog 1 (Abl); a tyrosinase enzyme; ephrin type a receptor 2(EphA 2); fucosyl GM 1; sialylated lewis adhesion molecules (sLe); ganglioside GM3(aNeu5Ac (2-3) bDClalp (l-4) bDGlcp (l-1) Cer); transglutaminase 5(TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); folate receptor beta; tumor endothelial marker 1(TEM1/CD 248); tumor endothelial marker 7 associated (TEM 7R); sealin (claudin)6(CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); class 5 group member D of G protein-coupled receptors C (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); a globoH sugar ceramide hexasaccharide moiety (globoH); mammary differentiation antigen (NY-BR-1); urinary plaque protein (uroplakin)2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenoceptor β 3(ADRB 3); ubiquitin 3(PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex, locus K9 (LY 6K); olfactory receptor 51E2(OR51E 2); TCR γ alternate reading frame protein (TARP); wilm's tumor protein (WT 1); cancer/testis antigen 1(NY-ES 0-1); cancer/testis antigen 2(LAGE-1 a); melanoma-associated antigen 1(MAGE-a 1); ETS translocation variant gene 6 located on chromosome 12p (ETV 6-AML); sperm protein 17(SPA 17); x antigen family member la (xagel); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; tumor protein p53(p 53); a p53 mutant; a prostate-specific protein; survivin (surviving); a telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Rat sarcoma, Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; an inhibitor of melanoma apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyl-transferase V (NA 17); the paired box protein Pax-3(PAX 3); an androgen receptor; a cyclin Bl; a v-myc avian myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); tyrosinase-related protein 2 (TRP-2); cytochrome P450lB 1(CYPlB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site regulator siblings), squamous cell carcinoma antigen recognized by T cells 3(SART 3); the paired box protein Pax-5(PAX 5); the preproepisin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RUl); renal ubiquitin 2(RU 2); legumain; human papilloma virus E6(HPV E6); human papilloma virus E7(HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2(mut hsp 70-2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2(BST 2); EGF-like receptor-module mucin-like hormone-containing sample 2(EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); and immunoglobulin lambda-like polypeptide 1(IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1TROP2, GFR α 54, CDH17, CDH6, NYB 1, CDH19, CD200R, Slea (CA 19.9; sialyl Lewis antigen) fucosyl-GM 1, PTK7, gpNMB, CDH1-CD324, DLL 1, CD276/B7H 1, IL11 1, IL13Ra 1, CD179 1-IGLl 1, ALK TCR γ - δ, NKG 21, CD1 (FCGR 21), Tn Ag, CSPG 1-HMW-MAA, Tim1-/HVCR1, CSF2 CSF- α, TCR β R1, TGF 72/VEGFR, LePG 1-LHS β -PGS, TNF-CSF-TCR β -receptor, TNF-TCR β -receptor, TCR β -receptor, TNF-receptor β -receptor, TNF-receptor β -receptor, TNF- β -receptor, TNF-receptor, and like, HTLV1-Tax, CMV pp65, EBV-EBNA3c, influenza A lectin (HA), GAD, PDL1, Guanylate Cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, desmoglein autoantibody 3(Dsg3), desmoglein autoantibody 1(Dsg1), HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1 TF (1), AFP, GPRC5D, sealer 18.2(CLD18A2 or N18A.2)), P-STEP, glycoprotein AP1, LIV1, BSfibronectin-4, CRIPTO, GPA33, GPT 3936/1, TNT ion channel antigen recognized by low conductivity antibodies. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, Single Domain Antibody (SDAB), vH or vL domain, camelid vHH domain, non-immunoglobulin antigen-binding scaffold, e.g., DARPIN, affibody, affilin, adnectin, affitin, obodies, repebody, fynomer, alphabody, avimer, atrimer, centrin, pronectin, anticalin, kunitz-type domain, Armadillo repeat protein, receptor, or ligand. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: (i) a heavy chain variable region (vH) encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 226 to 400 or 10203 to 10321 or a sequence at least 98% identical thereto and encoding a polypeptide that binds to an antigen thereof; (ii) a light chain variable region (vL) encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 16 to 191 or 10085 to 10202 or a sequence at least 98% identical thereto and encoding a polypeptide that binds to an antigen thereof; (iii) a single chain variable fragment (scFv) encoded by a polynucleotide having the sequence of any one of seq id NOs 488 to 657, 10346 to 10400, or 18098 to 18160, or a sequence at least 98% identical thereto and encoding a polypeptide that binds to an antigen thereof; (iv) a camelid VHH domain encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 421 to 445 or 10322 to 10337 or a sequence at least 98% identical thereto and encoding a polypeptide that binds to an antigen thereof; (v) a non-immunoglobulin scaffold encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 439 to 443 or a sequence at least 98% identical thereto and encoding a polypeptide that binds to an antigen thereof; (vi) a receptor encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 456 to 468 or a sequence at least 98% identical thereto and encoding a polypeptide that binds to a homologue thereof; and (vii) a ligand encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 476 to 486 or 10402 to 10404, or a sequence at least 98% identical thereto and encoding a polypeptide that binds to a homolog thereof. In another embodiment of any of the foregoing, the one or more non-native TCR antigen binding domains comprise one or more of the light chain complementarity determining regions of the selected target antigen represented by any one of SEQ ID Nos 13999 through 14879 or 14880 and/or one or more of the heavy chain complementarity determining regions of the selected target antigen represented by any one of SEQ ID Nos 14881 through 15761 or 15762. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise a variable light chain (vL) domain comprising the sequence of any of SEQ ID Nos. 2307 to 2482 or 12042 to 12159 with up to 10 conservative amino acid substitutions and/or a variable heavy chain (vH) domain comprising the sequence of any of SEQ ID Nos. 2506 to 2680 or 12160 to 12278 with up to 10 conservative amino acid substitutions. In another embodiment of any one of the preceding, the one or more non-native TCR antigen binding domains comprise one or more of the Camelidae vHH complementarity determining regions of the selected antigen as set forth in any one of SEQ ID Nos 2701 to 2725 or 12279 to 12294 and having up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise a non-immunoglobulin antigen-binding domain having the sequence set forth in any one of SEQ ID Nos 2728-2732 or 12296-12301 and having up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise an scFv domain comprising one or more light chain complementarity determining regions of a variable light chain (vL) domain comprising the sequence of any one of SEQ ID Nos 2307 through 2482 or 12042 through 12159 and one or more heavy chain complementarity determining regions of a variable heavy chain (vH) domain comprising the sequence of any one of SEQ ID Nos 2506 through 2680 or 12160 through 12278. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise scFv fragments having a sequence selected from the group consisting of SEQ ID nos 2770 to 2939, 12303 to 12357, or 18162 to 18224, each having up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise one or more receptors comprising the amino acid sequence of any one of SEQ ID nos 2736-2748 and having up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise one or more ligands comprising the sequence of any of SEQ ID NO 2758-2768 or 12359 through 12361 with up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise the extracellular domain of CD16A, NKG2D, CD4, PD1, desmoglein 3(Dsg3), or CD 4-DC-SIGN. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise an extracellular domain of one or more of hTPO, mTPO, CGH α chain, CGH β chain, FH β chain, LH β chain, TSH β chain, APRIL, or a combination thereof. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise any single-chain variable fragment (scFv) comprising the sequence of any one of SEQ ID Nos 2770 to 2939, 12303 to 12357, or 18162 to 18224 and having up to 10 conservative amino acid substitutions, and a) any camelid vHH as set forth in any one of SEQ ID Nos 2701 to 2725 or 12279 to 12294 and having up to 10 conservative amino acid substitutions, or b) any non-immunoglobulin antigen-binding domain having the sequence of any one of SEQ ID Nos 2728-2732 or 12296 to 12301 and having up to 10 conservative amino acid substitutions; or c) any extracellular domain of a receptor comprising the amino acid sequence of any one of SEQ ID Nos 2736 to 2748 and having up to 10 conservative amino acid substitutions; or d) any extracellular domain of a ligand comprising the sequence of any one of SEQ ID NO 2758-2768 or 12359 to 12361 and having up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise Camelidae vHH comprising a sequence set forth in any of SEQ ID Nos 2701 to 2725 or 12279 to 12294 and having up to 10 conservative amino acid substitutions, and a) any single chain variable fragment (scFv) comprising a sequence set forth in any of SEQ ID Nos 2770 to 2939, 12303 to 12357, or 18162 to 18224 and having up to 10 conservative amino acid substitutions, or b) any non-immunoglobulin antigen-binding domain having a sequence set forth in any of SEQ ID Nos 2728-2732 or 12296 to 12301 and having up to 10 conservative amino acid substitutions; or c) any extracellular domain of a receptor comprising the amino acid sequence of any one of SEQ ID Nos 2736 to 2748 and having up to 10 conservative amino acid substitutions; or d) any extracellular domain of a ligand comprising the sequence of any one of SEQ ID NO 2758-2768 or 12359 to 12361 and having up to 10 conservative amino acid substitutions. In another embodiment of any one of the preceding, the one or more non-native TCR antigen-binding domains are optionally linked to each of the TCR constant region chains by a linker region, wherein the linker region nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NOs 2981 to 2992 and any combinations thereof or sequences at least 98% identical thereto; or the linker is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 701 to 714 or a sequence having at least 98% identity thereto. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains have at least 5-fold less binding affinity for its target antigen than the antibody from which it was derived. In another embodiment of any of the foregoing, the polynucleotide encoding the SIR further comprises a leader sequence or signal peptide present at the N-terminus of each strand and comprising a sequence selected from the group consisting of SEQ ID NOs 1-9 and 10. In another embodiment of any of the foregoing, the at least one polynucleotide encodes two SIRs. In another embodiment of any of the foregoing, the polynucleotide encodes two SIRs linked by a nucleotide sequence encoding a cleavable linker. In another embodiment of any of the foregoing, the cleavable linker is a self-cleaving cleavable linker. In another embodiment of any of the foregoing, the cleavable linker is any one or more of a2A linker, a 2A-like linker, or a functional equivalent thereof. In another embodiment of any of the foregoing, the cleavable linker is any one or more of a T2A linker, a P2A, a F2A, an E2A linker, or functional equivalents thereof. In another embodiment of any of the foregoing, the cleavable linker comprises the sequence of any one or more of SEQ ID nos 780 to 785. In another embodiment of any of the foregoing, the polynucleotide sequence encoding a cleavable linker optionally follows a nucleotide sequence encoding a furin cleavage site or a furin-like cleavage site or a functional equivalent thereof. In another embodiment of any of the foregoing, the furin-like cleavage site preceding the cleavable linker comprises the sequence of any one or more of SEQ ID nos 788 to 790. In another embodiment of any of the foregoing, the polynucleotide sequence encoding the cleavable linker follows the nucleotide sequence encoding the flexible linker. In another embodiment of any of the foregoing, the flexible linker preceding the cleavable linker encodes one or more of a Ser-Gly linker, a Ser-Gly-Ser-Gly linker, or a functional equivalent thereof. In another embodiment of any of the foregoing, the flexible linker before the cleavable linker comprises the sequence SEQ ID No:786 or 787. In another embodiment of any of the foregoing, the polynucleotide sequence encoding a furin cleavage site is followed by a polynucleotide encoding a flexible linker, and the polynucleotide encoding a flexible linker is followed by a polynucleotide encoding a cleavable linker, such that the order is furin cleavage site-flexible linker-cleavable linker. In another embodiment of any of the foregoing, the polynucleotide encoding the cleavable linker is present before the sequence encoding the leader sequence (signal peptide), which sequence encodes the second SIR. In another embodiment of any of the foregoing, the SIR can be designed to have different binding affinities for the selected antigen. In another embodiment of any of the preceding, the SIR includes an auxiliary module. In another embodiment of any of the foregoing, the accessory module comprises a CD3z domain. In another embodiment of any of the foregoing, the TCR invariant chain is selected from the group consisting of: (viii) an amino acid sequence at least 98% identical to SEQ ID NO 12401 or 12402 or 12403 or 12408 or 12409; (ix) an amino acid sequence at least 98% identical to SEQ ID NO 12421 or 12422 or 12423 or 12427 or 12428; and (x) a dimeric combination of the two TCR constant chains of (viii) and (ix). In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains bind to CD 19. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2318-2324, 12060-12068, 12108, 12127, or 12156 or any Complementarity Determining Region (CDR) contained in any of the foregoing polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2517-2523, 12178-12186, 1227, 12246 or 12275 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; a polypeptide comprising a sequence at least 98% identical to SEQ ID NO 12288; and polypeptides comprising a sequence at least 98% identical to any of SEQ ID NOs 2770-2774, 12325, 12308, 18162-18170 or 12354; in another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3135-3235, 3250-3346, 3396, 3401-3403, 3406, 3429-3432, 3435-3439, 3540, 3855-3859, 12431-12489, 12491-12493, 12495-12530, 12534, 13195-13203, 13250, 13267, 13289, 13429-13437, 13483, 13501 and 13523. In another embodiment, the one or more non-native TCR antigen-binding domains bind to CD 20. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2325-2326, 12069-12077 or 12078 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2524-2525, 12187-12195 or 12196 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; a polypeptide comprising a sequence at least 98% identical to SEQ ID NO 12289 or 12290; and polypeptides comprising a sequence at least 98% identical to any of SEQ ID NOs 2787-2788, 18177-18186 or 18187. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3263, 3348, 3456-3457, 3876-3877, 12464-12465, 12477-12482, 12492, 12534, 13204-13213, 13438-13446 and 13447. In another embodiment, the one or more non-native TCR antigen-binding domains bind to CD 22. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2327-2329, 12122-12126 or 12132 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID Nos 2526-2528, 12241-12245 or 12251 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; and polypeptides comprising a sequence at least 98% identical to any of SEQ ID NOs 2789-2791, 12320-12330 or 18188. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3332, 3433, 3458-3460, 3878-3880, 12483, 12485, 12488-12490, 13241-13245, 13268, 13475-13479 and 13502. In another embodiment, the one or more non-native TCR antigen-binding domains bind to BCMA. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2310-2313, 12046-12048, 12118-12119, 12139-12145 or 12146 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2509-2512, 12164-12166, 12237-12238, 12258-12264 or 12265 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 12279-12281, 12283-12285, 12287, 12291-12292, 12293 or 12294; and polypeptides comprising a sequence at least 98% identical to any of SEQ ID NOs 2780-2783, 12237-12344, 18174-18175 or 18176. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3445-3449, 3866-3869, 12463, 12533, 12535-12536, 13181-13183, 13261-13262, 13277-13284, 13415-13417, 13495-13496, 13511-13517 and 13518. In another embodiment, the one or more non-native TCR antigen binding domains bind to MPL. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2414-2421, 12120, 12128 or 12129 or any Complementarity Determining Region (CDR) contained in any of the foregoing polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2611-2618, 12239, 12247 or 12248 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; and polypeptides comprising a sequence at least 98% identical to any of SEQ ID NOs 2871-2878, 12326-12327 and 12318. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3347, 3373, 3427-3428, 3495, 3556-3562, 3979-3985, 4025, 12454, 12456, 12458, 12462, 12532, 13259, 13265-13266, 13493, 13499 and 13500. In another embodiment, the one or more non-native TCR antigen-binding domains bind to CS 1. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2355-2358, 12090-12094 or 12095 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2553-2555, 12209-12213 or 12214 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; and a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2817-2819, 18211-18215 or 18216. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3376, 3487, 3489, 3907, 3909, 12455, 12457, 12459, 12461, 12476, 13226, 13231, 13460, 13464 and 13465. In another embodiment, the one or more non-native TCR antigen-binding domains bind to CD 33. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2336-, 12079-12084 or 12085 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2535, 2536, 12197, 12202 or 12203 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; and polypeptides comprising a sequence at least 98% identical to any of SEQ ID NOs 2795-2796, 18189-18193 or 18194. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3464-3465, 3884-3885, 12460, 12473, 12479, 13214-13220, 13448-13453 and 13454. In another embodiment, the one or more non-native TCR antigen-binding domains bind to CD 123. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2315, 2472, 12049-12058 or 12059 or any Complementarity Determining Region (CDR) contained in any of the foregoing polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2514, 2670, 12167-12176 or 12177 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; a polypeptide comprising a sequence at least 98% identical to SEQ ID NO 2716 or 2717; and a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2801, 2929, 18196-18205 or 18206. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3266-3267, 3366-3368, 3375, 3378, 3405, 3409, 3434, 3470, 3492-3497, 3617, 3890, 3912-3913, 4041, 12480, 13184-13194, 13418-13427 and 13428. In another embodiment, the one or more non-native TCR antigen-binding domains bind to folate receptor 1. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to 2373 or any Complementarity Determining Region (CDR) contained therein; a polypeptide comprising a sequence at least 98% identical to any one of SEQ ID NOs 2570 or any Complementarity Determining Regions (CDRs) contained therein; and a polypeptide comprising a sequence at least 98% identical to any one of SEQ ID NO: 2833. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos. 3511 and 3928. In another embodiment, the one or more non-native TCR antigen-binding domains bind to mesothelin. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2413, 12154 or 12155 or any Complementarity Determining Region (CDR) contained by any of the foregoing polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2609-2610, 12273 or 12274 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; a polypeptide comprising a sequence at least 98% identical to SEQ ID NO 2713-2714 or 2725; and a polypeptide comprising a sequence at least 98% identical to any one of SEQ ID NOs 2870, 2899, 12352, or 12353. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3414, 3419, 3554, 3585, 3976, 4008, 13287, 13288, 13521 and 13522. In another embodiment, the one or more non-native TCR antigen-binding domains bind to IL13Ra 2. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: -a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2399 or 2400 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2595 or 2596 or any Complementarity Determining Region (CDR) contained in any of the foregoing polypeptides; and a polypeptide comprising a sequence at least 98% identical to any one of SEQ ID NO:2858 or 2859. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3541-3542, 3963 and 3964. In another embodiment, the one or more non-native TCR antigen-binding domains bind to CD 138. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to SEQ id No. 2316 or any Complementarity Determining Region (CDR) contained therein; a polypeptide comprising a sequence at least 98% identical to SEQ ID NO. 2515 or any Complementarity Determining Region (CDR) contained therein; and a polypeptide comprising a sequence at least 98% identical to SEQ ID NO: 2802. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos. 3268, 3374, 3404, 3471 and 3891. In another embodiment, the one or more non-native TCR antigen-binding domains bind to TCRgd. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to SEQ ID NO 2449 or any Complementarity Determining Region (CDR) contained therein; a polypeptide comprising a sequence at least 98% identical to SEQ ID NO:2646 or any Complementarity Determining Region (CDR) contained therein; and a polypeptide comprising a sequence at least 98% identical to SEQ ID NO: 2907. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos. 3594 and 4017. In yet another embodiment, the one or more non-native TCR antigen-binding domains bind to TCRB 1. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2445 or 2446 or any Complementarity Determining Region (CDR) contained by any of the foregoing polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2642 or 2643 or any Complementarity Determining Region (CDR) contained in any of the foregoing polypeptides; and a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOS 2903 or 2904. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3590-3591, 4013 and 4014. In another embodiment, the one or more non-native TCR antigen-binding domains bind to TCRB 2. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2447 or 2448 or any Complementarity Determining Region (CDR) contained by any of the foregoing polypeptides; a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2644 or 2645 or any Complementarity Determining Region (CDR) contained by any of the foregoing polypeptides; and a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOS 2905 or 2906. In another embodiment of any of the foregoing, the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3353-3364, 3592-3593, 4015 and 4016.

The present disclosure also provides a recombinant expression system comprising any of the recombinant polynucleotides described above co-expressed with a therapeutic control agent, wherein the therapeutic control agent is selected from the group consisting of: truncated epidermal growth factor receptor (tEGFR), truncated epidermal growth factor receptor viii (tEGFRviii), truncated CD30(tCD30), truncated BCMA (tBCMA), truncated CD19(tCD19), CD34, thymidine kinase, cytosine deaminase, nitroreductase, xanthine guanine phosphoribosyltransferase, human caspase 8, human caspase9, inducible caspase9 (icapase 9), purine nucleoside phosphorylase, linalysidase/linalyrin/glucose oxidase, deoxynucleoside kinase, horseradish peroxidase (HRP)/indole-3-acetic acid (IAA), gamma-glutamylcysteine synthetase, CD 20/alpha CD20, CD 34/thymidine kinase chimera, dox-dependent caspase-2, thymidine mutant kinase (HSV-TKSR39), AP1903/Fas system, Chimeric Cytokine Receptors (CCR), selectable markers, and combinations thereof. In one embodiment, the tgegfr and the tgfrviii bind any one or more of an EGFR-specific siRNA, a small molecule, an anti-EGFR antibody or fragment thereof, or a combination thereof. In another embodiment, the tCD30 binds to any one or more of a CD30 specific siRNA, a small molecule, an anti-CD 30 antibody or fragment thereof, or a combination thereof. In another embodiment, the tCD19 binds to any one or more of a CD19 specific siRNA, a small molecule, an anti-CD 19 antibody or fragment thereof, or a combination thereof. In another embodiment, CD34 binds to any one or more of a CD34 specific siRNA, a small molecule, an anti-CD 34 antibody or fragment thereof, or a combination thereof. In another embodiment, the selectable marker comprises any one or more of dihydroxyfolate receptor (DHFR), mutant DHFR, methylated DNA-protein-cysteine methyltransferase, hypoxanthine monophosphate dehydrogenase II (IMDHP2), Puromycin Acetyltransferase (PAC), blasticidin resistance gene, mutant calcineurin a/b (Can/b), CNa12, CNb30, or a combination thereof. In another embodiment, the CCR comprises any one or more of (i) an IL-7 cytokine linker-IL 7Ra, (ii) an IL-7 cytokine linker extracellular domain of the IL-7 Ra-transmembrane domain of the IL-7 Ra-cytoplasmic domain of IL2R β, (iii) an IL-7 cytokine linker-IL 2R β, and (iv) combinations thereof. In another embodiment of any of the foregoing, the recombinant expression system comprises a recombinant polynucleotide of the present disclosure co-expressed with a helper module, wherein the helper module is selected from the group consisting of: 41BBL, CD40L, K13, MC159, cFLIP-L/MRIT alpha, cFLIP-p22, HTLV1Tax, HTLV2Tax, HTLV2Tax-RS mutant, FKBPx2-K13, FKBPx2-HTLV2-Tax, FKBPx2-HTLV2-Tax-RS, IL6R-304-vHH-Alb8-vHH, IL12f, PD1-4H1scFV, PD1-5C4scFV, PD1-4H1-Alb8-vHH, PD1-5C4-Alb8-vHH, HVCTLA 4-Imumab-scFv, CTLA 4-Imumab 4-vHH, IL 4-scFV, IL 72-3619A-3619-scFV-19-Alb 19-hTTL-hTTH, HV3672-hTFHZ-4-CD 4-hTRTP 4-LshT 4-CD 4-LshT, hTTH-4-CD 4-LshT-4-HBT, hTTH-4-HBT-3-HBT, CD-HBT-3, CD-HB, A Chimeric Antigen Receptor (CAR), hTERT, heparinase, CAR, an inhibitory CAR, and any combination thereof. In another embodiment of any of the foregoing, the recombinant polynucleotide encoding a SIR and the one or more therapeutic control agents and/or the one or more adjunct modules are linked by a nucleotide sequence encoding a cleavable linker. In another embodiment, the cleavable linker is a self-cleaving cleavable linker. In another embodiment of any of the foregoing, the polynucleotide sequence encoding a cleavable linker follows a nucleotide sequence encoding a furin cleavage site or a furin-like cleavage site or a functional equivalent thereof. In another embodiment of any of the foregoing, the polynucleotide sequence encoding the cleavable linker optionally follows the nucleotide sequence encoding the flexible linker.

The present disclosure also provides at least one vector comprising a recombinant polynucleotide as described herein above, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, a baculovirus vector, a sleeping beauty transposon vector and a piggybac transposon vector. In one embodiment, the vector backbone has a sequence selected from the group consisting of SEQ ID NOs 870 to 875 and 876. In some embodiments, the vector comprises a promoter selected from the group consisting of an EF-1 promoter, a CMV IE gene promoter, an EF-1 α promoter, a ubiquitin C promoter, a MSCV LTR promoter, or a phosphoglycerate kinase (PGK) promoter. In another embodiment, the EF-1 promoter comprises the sequence of SEQ ID NO 877 or a sequence 80-99% identical thereto. In another embodiment of any of the foregoing, the vector is an in vitro transfected vector, or the vector further comprises a poly (a) tail or 3' UTR.

The present disclosure provides at least one polypeptide encoded by at least one recombinant polynucleotide of the present disclosure.

The present disclosure also provides recombinant cells expressing at least one recombinant polynucleotide as described herein above.

The present disclosure also provides an isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer comprising: (a) a T Cell Receptor (TCR) constant chain having an amino acid sequence selected from the group consisting of: (i) an amino acid sequence at least 98% identical to SEQ ID NO 3010 and having one or more mutations at positions 48, 61, 91, 92, 93 and/or 94 and which may comprise an optional auxiliary module; (ii) an amino acid sequence which is at least 98% identical to SEQ ID NO:3024 and has one or more mutations at positions 18, 22, 57, 79, 133, 136 and/or 139 and may comprise an optional auxiliary moiety; (iii) an amino acid sequence which is at least 98% identical to SEQ ID NO:3025 and has one or more mutations at positions 18, 22, 57, 79, 133, 136 and/or 139 and may comprise an optional auxiliary moiety; (iv) an amino acid sequence at least 98% identical to SEQ ID NO 3046, 3047 or 3048 and which may comprise an optional auxiliary module; (v) an amino acid sequence which is at least 98% identical to SEQ ID NO 3049 and which may comprise an optional auxiliary module; (vi) an amino acid sequence which is at least 98% identical to SEQ ID NO 3051 or 3052 and which may comprise optional auxiliary modules; and (vii) a dimeric combination of two TCR constant chains selected from (i) and (ii), (i) and (iii), (iv) and (ii), (iv) and (iii), or (v) and (vi); (b) optionally a linker; and (c) one or more non-native TCR antigen binding domains selected from the group consisting of: (1) an antibody; (2) antibody fragments (e.g., Fv, Fab, (Fab') 2); (3) a heavy chain variable region (vH domain) of an antibody or a fragment thereof; (4) a light chain variable region (vL domain) of an antibody or a fragment thereof; (5) a single chain variable fragment (scFv) or a fragment thereof; (6) a Single Domain Antibody (SDAB) or fragment thereof; (7) a camelidae VHH domain or a fragment thereof; (8) a monomeric variable region of an antibody; (9) non-immunoglobulin antigen binding scaffolds such as DARPIN, affibody, affilin, adnectin, affitin, obodies, repebody, fynomer, alphabody, avimer, atrimer, centryrin, pronectin, anticalin, kunitz-type domain, Armadillo repeat protein, or a fragment thereof; (10) a receptor or fragment thereof; (11) a ligand or fragment thereof; (12) bispecific-antibodies, -antibody fragments, -scFV, -vHH, -SDAB, -non-immunoglobulin antigen-binding scaffolds, -receptors, or-ligands; and (13) a self-antigen or fragment thereof, wherein the mutations of (a) (i) - (a) (iii) provide different binding affinities for a target antigen of the antigen-binding domain, and the synthetic immune receptor, when expressed in lymphocytes, expresses both the antigen-binding domain and the T cell receptor constant chain in one or more continuous chains on the surface of the lymphocytes, such that the lymphocytes are triggered to activate, proliferate, secrete cytokines and/or modulate (induce or inhibit) killing of the target cells and have MHC-restricted or MHC-unrestricted antibody type specificity when the expressed antigen-binding domain binds to its antigen. In another embodiment of any of the foregoing, (a) (vii) the TCR constant chain has a non-native TCR binding domain selected from the group consisting of: (iii) the variable regions of the heavy and light chains of an antibody or fragment thereof specific for a predefined target antigen, such that when expressed, one of said heavy and light chains of the antibody or fragment thereof is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said heavy and light chains of the antibody or fragment thereof is attached to the other of said two chains of said T cell constant region; two single-chain variable fragments (scfvs) specific for one or more predefined target antigens, such that when expressed, one of the scfvs is attached to one of the two chains of (a) (vii) of the T cell constant region and the other of the scfvs is attached to the other of the two chains of the T cell constant region; two antibody fragments specific for one or more predefined target antigens, such that when expressed, one of said antibody fragments is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said antibody fragments is attached to the other of said two chains of said T cell constant region; two Single Domain Antibody (SDAB) fragments specific for one or more predefined target antigens, such that when expressed, one of said SDAB fragments is attached to one of said two chains of (a) (vii) of said T cell constant region, and the other of said SDAB fragments is attached to the other of said two chains of said T cell constant region; two camelidae vHH domains specific for one or more predefined target antigens such that, when expressed, one of said vHH domains is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said vHH domains is attached to the other of said two chains of said T cell constant region; two non-immunoglobulin antigen binding scaffolds that are specific for one or more predefined target antigens, such that when expressed, one of the non-immunoglobulin antigen binding scaffolds is attached to one of the two chains (a) (vii) of the T cell constant region and the other of the non-immunoglobulin antigen binding scaffolds is attached to the other of the two chains of the T cell constant region; two receptors or fragments thereof specific for one or more predefined target antigens such that, when expressed, one of said receptors or fragments thereof is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said receptors or fragments thereof is attached to the other of said two chains of said T cell constant region; two ligands or fragments thereof specific for one or more predefined target antigens such that, when expressed, one of said ligands or fragments thereof is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said ligands or fragments thereof is attached to the other of said two chains of said T cell constant region; two structurally distinct antigen-binding fragments specific for one or more predefined target antigens, such that when expressed, one of the antigen-binding fragments is attached to one of the two chains (a) (vii) of the T cell constant region and the other of the antigen-binding fragments is attached to the other of the two chains of the T cell constant region; two binding fragments, one or both of which is bispecific or multispecific such that, when expressed, one of the antigen-binding fragments is attached to one of the two chains of (a) (vii) of the T cell constant region and the other of the antigen-binding fragments is attached to the other of the two chains of the T cell constant region; two autoantigens or fragments thereof, such that when expressed, one of said autoantigens or fragments thereof is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said autoantigens or fragments thereof is attached to the other of said two chains of said T cell constant region; and two vL or fragments thereof, such that when expressed, one of said vL or fragments thereof is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said vL or fragments thereof is attached to the other of said two chains of said T cell constant region; two vH or fragments thereof, such that when expressed, one of said vH or fragments thereof is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said vH or fragments thereof is attached to the other of said two chains of said T cell constant region. In another embodiment of any of the foregoing, (a) (iv) the TCR constant chain has a non-native TCR binding domain selected from the group consisting of: a variable region of a heavy chain (vH) of an antibody or fragment thereof specific for a predefined target antigen; a variable region of a light chain (vL) of an antibody or fragment thereof specific for a predefined target antigen; a single-chain variable fragment (scFv) or a fragment thereof specific for a predefined target antigen; antibody fragments specific for predefined target antigens (e.g., Fv, Fab, (Fab') 2); a Single Domain Antibody (SDAB) fragment specific for a predefined target antigen; a camelidae vHH domain specific for a predefined target antigen; a non-immunoglobulin antigen binding scaffold specific for a predefined target antigen; a receptor or fragment thereof specific for a predefined target antigen; a ligand or fragment thereof specific for a predefined target antigen; bispecific-antibodies specific for one or more predefined target antigens, -antibody fragments, -scFV, -vHH, -SDAB, -non-immunoglobulin antigen-binding scaffolds, -receptors, or-ligands; and autoantigens or fragments thereof. In another embodiment of any of the foregoing, the TCR constant domain of (i), (ii), (iii), (iv), (v), or (vi) comprises a non-native TCR binding domain selected from the group consisting of: a variable region of a heavy chain (vH) of an antibody specific for a predefined target antigen; a variable region of a light chain (vL) of an antibody specific for a predefined target antigen; a single-chain variable fragment (scFv) specific for a predefined target antigen; antibody fragments specific for predefined target antigens (e.g., Fv, Fab, (Fab') 2); a Single Domain Antibody (SDAB) fragment specific for a predefined target antigen; a camelidae vHH domain specific for a predefined target antigen; a non-immunoglobulin antigen binding scaffold specific for a predefined target antigen; a receptor specific for a predefined target antigen or fragment thereof; a ligand specific for a predefined target antigen or fragment thereof; bispecific-antibodies specific for one or more predefined target antigens, -antibody fragments, -scFV, -vHH, -SDAB, -non-immunoglobulin antigen-binding scaffolds, -receptors, or-ligands; and autoantigens or fragments thereof. In another embodiment of any of the foregoing, the TCR constant chain comprises a mutation that enhances TCR constant chain expression and/or pairing and reduces its pairing with endogenous T cell receptor chains. In another embodiment of any of the foregoing, the constant region of the TCR is a TCR receptor alpha chain (ca) comprising an amino acid sequence having 1-40 amino acid substitutions or mutations selected from the group consisting of sequences selected from the group consisting of SEQ ID NOs 3010 to 3023 or a sequence at least 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 3010 to 3023. In another embodiment of any of the foregoing, the constant region of the TCR is a TCR receptor beta chain (C β) comprising an amino acid sequence having 1-40 amino acid substitutions or mutations selected from the group consisting of a sequence selected from the group consisting of SEQ ID NOs: 3024 to 3044 or a sequence at least 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3024 to 3044. In another embodiment of any of the foregoing, the constant region of the TCR is a TCR receptor gamma chain (cy) comprising an amino acid sequence having 1-40 amino acid substitutions or mutations selected from the group consisting of the sequence selected from the group consisting of SEQ ID NOs 3049 to 3050 or a sequence at least 98% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs 3049 to 3050. In another embodiment of any of the foregoing, the constant region of the TCR is a TCR receptor delta chain (C δ) comprising an amino acid sequence having 1-40 amino acid substitutions or mutations selected from the group consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 3051-3052, or a sequence at least 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 3051-3052. In another embodiment of any of the foregoing, the constant region of the TCR is a precursor TCR receptor alpha chain (precursor ca) comprising an amino acid sequence having 1-40 amino acid substitutions or mutations selected from the group consisting of the amino acid sequences selected from the group consisting of SEQ ID NOs 3046 to 3048, or a sequence at least 98% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs 3046 to 3048. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains bind to one or more disease-associated antigens selected from the group consisting of: CD 19; CD 123; CD 22; CD 30; CD 171; CS-1 (also known as CD2 subunit 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD 33; epidermal growth factor receptor variant iii (egfrviii); ganglioside G2(GD 2); ganglioside GD3(aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (l-l) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. alpha. -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1(ROR 1); fms-like tyrosine kinase 3(FLT 3); tumor-associated glycoprotein 72(TAG 72); CD 38; CD44v 6; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epidermal cell adhesion molecule (EPCAM); B7H3(CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213a 2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate Stem Cell Antigen (PSCA); protease serine 21 (testis protein or PRSS 21); vascular endothelial growth factor receptor 2(VEGFR 2); a lewis (Y) antigen; CD 24; platelet derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD 20; a folate receptor alpha; receptor tyrosine protein kinase ERBB2(Her 2/neu); cell surface associated mucin 1(MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); prostasin; prostatic Acid Phosphatase (PAP); mutant elongation factor 2(ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase ix (caix); proteasome (precursor, megalin) subunit, beta form, 9(LMP 2); glycoprotein 100(gpl 00); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and the Abelson (Abelson) murine leukemia virus oncogene homolog 1 (Abl); a tyrosinase enzyme; ephrin type a receptor 2(EphA 2); fucosyl GM 1; sialylated lewis adhesion molecules (sLe); ganglioside GM3(aNeu5Ac (2-3) bDClalp (l-4) bDGlcp (l-1) Cer); transglutaminase 5(TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); folate receptor beta; tumor endothelial marker 1(TEM1/CD 248); tumor endothelial marker 7 associated (TEM 7R); sealin 6(CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); class 5 group member D of G protein-coupled receptors C (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); a globoH sugar ceramide hexasaccharide moiety (globoH); mammary differentiation antigen (NY-BR-1); urinary plaque 2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenoceptor β 3(ADRB 3); ubiquitin 3(PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex, locus K9 (LY 6K); olfactory receptor 51E2(OR51E 2); TCR γ alternate reading frame protein (TARP); wilm's tumor protein (WT 1); cancer/testis antigen 1(NY-ES 0-1); cancer/testis antigen 2(LAGE-1 a); melanoma-associated antigen 1(MAGE-a 1); ETS translocation variant gene 6 located on chromosome 12p (ETV 6-AML); sperm protein 17(SPA 17); x antigen family member la (xagel); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; tumor protein p53(p 53); a p53 mutant; a prostate-specific protein; survivin; a telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; an inhibitor of melanoma apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyl-transferase V (NA 17); the paired box protein Pax-3(PAX 3); an androgen receptor; a cyclin Bl; a v-myc avian myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); tyrosinase-related protein 2 (TRP-2); cytochrome P450lB 1(CYPlB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site regulator siblings), squamous cell carcinoma antigen recognized by T cells 3(SART 3); the paired box protein Pax-5(PAX 5); the preproepisin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RUl); renal ubiquitin 2(RU 2); legumain; human papilloma virus E6(HPV E6); human papilloma virus E7(HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2(mut hsp 70-2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2(BST 2); EGF-like receptor-module mucin-like hormone-containing sample 2(EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); and immunoglobulin lambda-like polypeptide 1(IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1TROP2, GFR alpha 54, CDH17, CDH6, NYB 1, CDH19, CD200R, Slea (CA 19.9; sialyl Lewis antigen) fucosyl-GM R, PTK R, gpNMB, CDH R-CD 324, DLL R, CD276/B7H R, IL11R, IL13Ra R, CD 179R-IGLl R, ALK TCR gamma-delta, NKG 2R, CD R (FCGR 2R), Tn Ag, CSPG R-HMW-MAA, Tim R-/HVCR R, CSF2 CSF-alpha, TCR beta R R, TGF 72/VEGFR, Lehr beta-HCS 1-LwS-HA, TNF-TCR beta-receptor, TNF-receptor, receptor and receptor for producing receptor for TNF-beta-receptor, CMV pp65, EBV-EBNA3c, influenza A lectin (HA), GAD, PDL1, Guanylate Cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, desmoglein autoantibody 3(Dsg3), desmoglein autoantibody 1(Dsg1), HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1 (1), AFP, GPRC5D, sealer 18.2(CLD18A2 or N18A.2)), P-glycoprotein, STEAP1, LIV1, laminin-4, CRIPTO, GPA33, EBT 1/CD157, chloride channel antigen recognized by TNT antibodies of low conductivity. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, Single Domain Antibody (SDAB), vH or vL domain, camelid vHH domain, non-immunoglobulin antigen-binding scaffold, e.g., DARPIN, affibody, affilin, adnectin, affitin, obodies, repebody, fynomer, alphabody, avimer, atrimer, centrin, pronectin, anticalin, kunitz-type domain, Armadillo repeat protein, receptor, or ligand. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains are selected from the group consisting of: (i) a heavy chain variable region (vH) comprising the sequence set forth in any one of SEQ ID NOs 2506 to 2680 or 12160 to 12278 or a sequence having at least 98% identity thereto and encoding a polypeptide that binds to an antigen thereof; (ii) a light chain variable region (vL) comprising the sequence set forth in any one of SEQ ID NOs 2307 to 2482 or 12042 to 12159 or a sequence having at least 98% identity thereto and encoding a polypeptide that binds to an antigen thereof; (iii) a single chain variable fragment (scFv) comprising the sequence set forth in any one of SEQ ID NOs 2770 to 2939, 12303 to 12357, or 18162 to 18224 or a sequence having at least 98% identity thereto and encoding a polypeptide that binds to an antigen thereof; (iv) a camelid VHH domain comprising a sequence set forth in any one of SEQ id nos 2701 to 2725 or 12279 to 12294 or a sequence having at least 98% identity thereto and encoding a polypeptide that binds to an antigen thereof; (v) a non-immunoglobulin scaffold encoded by any one of SEQ ID NOs 439 to 443 or a polynucleotide having a sequence at least 98% identical thereto and encoding a polypeptide that binds to an antigen thereof; (vi) a receptor comprising a sequence set forth in any one of SEQ ID NOs 2736 to 2748 or a sequence having at least 98% identity thereto and encoding a polypeptide that binds to a homologue thereof; and (vii) a ligand comprising the sequence set forth in any one of SEQ ID NOs 2758 to 2768 or 12359 to 12361 or a sequence at least 98% identical thereto and encoding a polypeptide that binds to a homologue thereof. In another embodiment of any of the foregoing, the one or more non-native TCR antigen binding domains comprise one or more of the light chain complementarity determining regions of the selected target antigen represented by any one of SEQ ID Nos 13999 through 14879 or 14880 and/or one or more of the heavy chain complementarity determining regions of the selected target antigen represented by any one of SEQ ID Nos 14881 through 15761 or 15762. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise a variable light chain (vL) domain comprising the sequence of any one of SEQ ID nos 2307 to 2482 or 12042 to 12159 with up to 10 conservative amino acid substitutions and/or a variable heavy chain (vH) domain comprising the sequence of any one of SEQ ID nos 2506 to 2680 or 12160 to 12278 with up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise one or more of the camelidae vHH complementarity determining regions of the selected antigen shown in any one of SEQ ID nos 2701 to 2725 or 12279 to 12294 and having up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise a non-immunoglobulin antigen-binding domain having the sequence set forth in any one of SEQ ID Nos 2728-2732 or 12296-12301 and having up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise an scFv domain comprising one or more light chain complementarity determining regions of a variable light chain (vL) domain comprising the sequence of any one of SEQ ID Nos 2307-2482 or 12042-12159 and one or more heavy chain complementarity determining regions of a variable heavy chain (vH) domain comprising the sequence of any one of SEQ ID Nos 2506-2680 or 12160-12278. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise scFv fragments having a sequence selected from the group consisting of SEQ ID nos 2770-2939, 12303-12357, or 18162-18224, each having up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise one or more receptors comprising the amino acid sequence of any one of SEQ ID nos 2736-2748 and having up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise one or more ligands comprising the sequence of any of SEQ ID NO 2758-2768 or 12359 through 12361 with up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise the extracellular domain of CD16A, NKG2D, CD4, PD1, desmoglein 3(Dsg3), or CD 4-DC-SIGN. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise an extracellular domain of one or more of hTPO, mTPO, CGH α chain, CGH β chain, FH β chain, LH β chain, TSH β chain, APRIL, or a combination thereof. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise any single-chain variable fragment (scFv) comprising the sequence of any one of SEQ ID Nos 2770 to 2939, 12303 to 12357, or 18162 to 18224 and having up to 10 conservative amino acid substitutions, and a) any camelid vHH as set forth in any one of SEQ ID Nos 2701 to 2725 or 12279 to 12294 and having up to 10 conservative amino acid substitutions, or b) any non-immunoglobulin antigen-binding domain having the sequence of any one of SEQ ID Nos 2728-2732 or 12296 to 12301 and having up to 10 conservative amino acid substitutions; or c) any extracellular domain of a receptor comprising the amino acid sequence of any one of SEQ ID Nos 2736 to 2748 and having up to 10 conservative amino acid substitutions; or d) any extracellular domain of a ligand comprising the sequence of any one of SEQ ID NO 2758-2768 or 12359 to 12361 and having up to 10 conservative amino acid substitutions. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains comprise Camelidae vHH comprising a sequence set forth in any of SEQ ID Nos 2701 to 2725 or 12279 to 12294 and having up to 10 conservative amino acid substitutions, and a) any single chain variable fragment (scFv) comprising a sequence set forth in any of SEQ ID Nos 2770 to 2939, 12303 to 12357, or 18162 to 18224 and having up to 10 conservative amino acid substitutions, or b) any non-immunoglobulin antigen-binding domain having a sequence set forth in any of SEQ ID Nos 2728-2732 or 12296 to 12301 and having up to 10 conservative amino acid substitutions; or c) any extracellular domain of a receptor comprising the amino acid sequence of any one of SEQ ID Nos 2736 to 2748 and having up to 10 conservative amino acid substitutions; or d) any extracellular domain of a ligand comprising the sequence of any one of SEQ ID NO 2758-2768 or 12359 to 12361 and having up to 10 conservative amino acid substitutions. In another embodiment of any one of the preceding, the one or more non-native TCR antigen-binding domains are optionally linked to each TCR constant region chain by a linker region, wherein the linker region nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ id nos 2981 to 2992 and any combinations thereof or sequences at least 98% identical thereto; or the linker is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 701 to 714 or a sequence having at least 98% identity thereto. In another embodiment of any of the foregoing, the one or more non-native TCR antigen-binding domains have at least 5-fold less binding affinity for its target antigen than the antibody from which it was derived. In another embodiment of any of the foregoing, the polynucleotide encoding the SIR further comprises a leader sequence or signal peptide present at the N-terminus of each strand and comprising a sequence selected from the group consisting of SEQ ID NOs 1-9 and 10. In another embodiment of any of the preceding, the SIR comprises SIR heterodimers. In another embodiment of any of the foregoing, the polypeptide comprises two SIRs linked by a cleavable linker. In another embodiment, the cleavable linker is a self-cleaving cleavable linker. In another embodiment, the cleavable linker is any one or more of a2A linker, a 2A-like linker or a functional equivalent thereof. In yet another embodiment, the cleavable linker is any one or more of a T2A linker, a P2A, F2A, an E2A linker, or functional equivalents thereof. In yet another embodiment, the cleavable linker comprises the sequence of any one or more of SEQ ID Nos 780 to 785. In another embodiment, the cleavable linker is optionally followed by a furin cleavage site or a furin-like cleavage site or a functional equivalent thereof. In yet another embodiment, the furin-like cleavage site preceding the cleavable linker comprises the sequence of any one or more of SEQ ID Nos. 788 to 790. In any one of the preceding embodiments, the cleavable linker is behind the flexible linker. In another embodiment, the flexible linker preceding the cleavable linker encodes one or more of a Ser-Gly linker, a Ser-Gly-Ser-Gly linker, or a functional equivalent thereof. In another embodiment, the flexible linker preceding the cleavable linker comprises the sequence SEQ ID Nos. 786 or 787. In yet another embodiment, the furin cleavage site is followed by a flexible linker, which is followed by a cleavable linker, such that the order is furin cleavage site-flexible linker-cleavable linker. In another embodiment of any of the foregoing, the SIR is designed to have a desired binding affinity for the selected antigen.

The present disclosure also provides an immune effector cell or stem cell comprising at least one polypeptide or heterodimer as described herein above.

The present disclosure also provides an immune effector cell or stem cell comprising at least one recombinant polynucleotide as described herein above.

The present disclosure also provides an immune effector cell or stem cell comprising at least one vector of the present disclosure as described herein above.

In another embodiment of any of the foregoing, any immune cell or stem cell comprises a plurality of SIR polypeptides. In another embodiment of any of the foregoing, at least one SIR polypeptide in the plurality of SIR polypeptides targets an antigen that is different from at least one other SIR polypeptide. In another embodiment of any of the foregoing, at least one SIR polypeptide in the plurality of SIR polypeptides targets the same antigen. In another embodiment of any of the foregoing, at least one SIR polypeptide in the plurality of SIR polypeptides comprises a different binding affinity for the antigen than at least one other SIR polypeptide. In another embodiment of any of the foregoing, the immune cell further comprises at least one Chimeric Antigen Receptor (CAR) polypeptide. In another embodiment of any of the foregoing, the antigen binding domain of the SIR polypeptide targets an antigen that is different from the antigen binding domain of the CAR polypeptide. In another embodiment of any of the foregoing, the CAR polypeptide comprises an intracellular signaling domain comprising a costimulatory signaling domain, but not the primary signaling domain, or an intracellular signaling domain comprising a primary signaling domain, but not the costimulatory signaling domain. In another embodiment of any of the foregoing, the CAR polypeptide comprises a costimulatory signaling domain comprising a functional signaling domain of a protein selected from the group consisting of 4-lBB, CD28, CD27, or OX-40, or the CAR molecule comprises a primary signaling domain comprising a functional signaling domain of CD3 ζ. In another embodiment of any of the foregoing, the CAR polypeptide is an inhibitory CAR polypeptide, wherein the inhibitory CAR polypeptide comprises the antigen binding domain, transmembrane domain, and intracellular domain of an inhibitory molecule, wherein the inhibitory molecule is selected from the group consisting of PDl, PD-Ll, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIRl, CD160, 2B4, TGFR β, CEACAM-1, CEACAM-3, and CEACAM-5. In another embodiment of any of the foregoing, the CAR polypeptide further comprises an intracellular signaling domain comprising a primary signaling domain and/or an intracellular signaling domain, wherein the intracellular signaling domain comprises a primary signaling domain comprising a functional domain of CD3 ζ and a costimulatory signaling domain comprising a functional domain of 4-lBB or CD28, or both. In another embodiment of any of the foregoing, the CAR polypeptide comprises the amino acid sequence SEQ ID NO:3077 to SEQ ID NO: 3083. In another embodiment of any one of the preceding, the immune effector cell is a human T cell, a human NK cell, or a stem cell that can produce an immune effector cell, optionally wherein the T cell is diglycerol (diaglycrol) kinase (DGK) and/or Ikaros (Ikaros) deficient and/or Brd4 deficient.

The present disclosure provides a method of making an immune effector cell that expresses an SIR, the method comprising introducing at least one vector of the present disclosure or at least one recombinant polynucleotide of the present disclosure into an immune effector cell or a hematopoietic stem cell or progenitor cell from which an immune effector cell can be produced under conditions such that the SIR polypeptide is expressed. In another embodiment of any of the foregoing, the method further comprises a) providing a population of immune effector cells; and b) removing T regulatory cells from the population, thereby providing a T regulatory depleted cell population; wherein steps a) and b) are performed prior to introducing the vector or recombinant polynucleotide encoding the SIR into the population. In another embodiment of any of the foregoing, the T regulatory cells are removed from the cell population using an anti-CD 25 antibody or an anti-GITR antibody. In another embodiment of any of the preceding claims, the method further comprises: a) providing a population of immune effector cells; and b) isolating the P-glycoprotein (P-gp or Pgp; MDR1, ABCB1, CD243) positive cells, thereby providing a cell enriched in P-glycoprotein (P-gp or Pgp; MDR1, ABCB1, CD 243); wherein steps a) and b) are performed before or after introduction of the vector or recombinant polynucleotide encoding the SIR. In another embodiment of any of the foregoing, the P-glycoprotein-positive cells are enriched using any one or more cells selected from the group consisting of: i) immunoselection using an antibody specific for P-glycoprotein or mixtures thereof, ii) staining with one or more fluorescent dyes (tetramethylrhodamine methyl ester (TMRM), doxorubicin and actinomycin-D) as substrate for P-glycoprotein under conditions where P-glycoprotein acts as a pump and enriches cells stained less with the dye, iii) selecting cells that are resistant to phototoxic compounds as substrate for P-glycoprotein, such as TH9402, 2- (4, 5-dibromo-6-amino-3-imino-3H-xanthen-9-yl) -benzoic acid methyl ester hydrochloride, 2- (4, 5-dibromo-6-amino-3-imino-3H-xanthen-9-yl) -benzoic acid ethyl ester hydrochloride, ethyl acetate, any one or more of 2- (4, 5-dibromo-6-amino-3-imino-3H-xanthen-9-yl) -octyl benzoate hydrochloride, 2- (4, 5-dibromo-6-amino-3-imino-3H-xanthen-9-yl) -n-butyl benzoate hydrochloride, 2- (6-ethylamino-3-ethylimino-3H-xanthen-9-yl) -n-butyl benzoate hydrochloride or a derivative thereof, or a combination thereof, and iv) selecting cells that are resistant to cytotoxic compounds that are substrates for P-glycoprotein, such as vincristine, vinblastine, taxol, B-carotene, C-E-carotene, C-N-acetyl-benzoate, C-butyl benzoate, paclitaxel, mitoxantrone, etoposide, doxorubicin, rubicin, and actinomycin-D.

The present disclosure also provides a method of generating an RNA-engineered cell population, the method comprising introducing into a cell or cell population one or more RNAs transcribed in vitro or synthesized, wherein the one or more RNAs comprise one or more recombinant polynucleotides described herein above.

The present disclosure provides a method of providing immunity to a disease in a subject, the method comprising administering to the subject an effective amount of an immune effector cell or an immune effector cell-producing stem cell of the present disclosure, wherein the cell is an autologous T cell or an allogeneic T cell, or an autologous NK cell or an allogeneic NK cell, or an autologous or allogeneic hematopoietic stem cell that can produce an immune effector cell. In one embodiment, the allogeneic T cells or allogeneic NK cells lack expression of or have low expression of a functional TCR or functional HLA.

The present disclosure also provides a composition comprising an immune effector cell or a stem cell that can generate an immune effector cell, the cell comprising one or more Synthetic Immune Receptor (SIR) molecules, for use in combination with an agent that increases the efficacy of an immune effector cell for use in treating a subject having a disease associated with expression of a disease-associated antigen or preventing a disease in a subject at increased risk of a disease associated with expression of a disease-associated antigen, wherein: (i) the SIR molecule comprises one or more of a T cell receptor constant chain linked via an optional linker to one or more antigen binding domains that bind to the disease-associated antigen associated with the disease, and said disease-associated antigen is selected from the group consisting of: CD5, CD 19; CD 123; CD 22; CD 30; CD 171; CS-1 (also known as CD2 subunit 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD 33; epidermal growth factor receptor variant iii (egfrviii); ganglioside G2(GD 2); ganglioside GD3(aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (l-l) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. alpha. -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1(ROR 1); fms-like tyrosine kinase 3(FLT 3); tumor-associated glycoprotein 72(TAG 72); CD 38; CD44v 6; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epidermal cell adhesion molecule (EPCAM); B7H3(CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213a 2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate Stem Cell Antigen (PSCA); protease serine 21 (testis protein or PRSS 21); vascular endothelial growth factor receptor 2(VEGFR 2); a lewis (Y) antigen; CD 24; platelet derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD 20; a folate receptor alpha; receptor tyrosine protein kinase ERBB2(Her 2/neu); cell surface associated mucin 1(MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); prostasin; prostatic Acid Phosphatase (PAP); mutant elongation factor 2(ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase ix (caix); proteasome (precursor, megalin) subunit, beta form, 9(LMP 2); glycoprotein 100(gpl 00); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and the Abelson (Abelson) murine leukemia virus oncogene homolog 1 (Abl); a tyrosinase enzyme; ephrin type a receptor 2(EphA 2); fucosyl GM 1; sialylated lewis adhesion molecules (sLe); ganglioside GM3(aNeu5Ac (2-3) bDClalp (l-4) bDGlcp (l-1) Cer); transglutaminase 5(TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); tumor endothelial marker 1(TEM1/CD 248); tumor endothelial marker 7 associated (TEM 7R); sealin 6(CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); class 5 group member D of G protein-coupled receptors C (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); a globoH sugar ceramide hexasaccharide moiety (globoH); mammary differentiation antigen (NY-BR-1); urinary plaque 2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenoceptor β 3(ADRB 3); ubiquitin 3(PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex, locus K9 (LY 6K); olfactory receptor 51E2(OR51E 2); TCR γ alternate reading frame protein (TARP); wilm's tumor protein (WT 1); cancer/testis antigen 1(NY-ES 0-1); cancer/testis antigen 2(LAGE-1 a); melanoma-associated antigen 1(MAGE-a 1); ETS translocation variant gene 6 located on chromosome 12p (ETV 6-AML); sperm protein 17(SPA 17); x antigen family member la (xagel); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; tumor protein p53(p 53); a p53 mutant; a prostate-specific protein; survivin; a telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; an inhibitor of melanoma apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyl-transferase V (NA 17); the paired box protein Pax-3(PAX 3); an androgen receptor; a cyclin Bl; a v-myc avian myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); tyrosinase-related protein 2 (TRP-2); cytochrome P450lB 1(CYPlB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site regulator siblings), squamous cell carcinoma antigen recognized by T cells 3(SART 3); the paired box protein Pax-5(PAX 5); the preproepisin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RUl); renal ubiquitin 2(RU 2); legumain; human papilloma virus E6(HPV E6); human papilloma virus E7(HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2(mut hsp 70-2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2(BST 2); EGF-like receptor-module mucin-like hormone-containing sample 2(EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); and immunoglobulin lambda-like polypeptide 1(IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1TROP2, GFR alpha 54, CDH17, CDH6, NYB 1, CDH19, CD200R, Slea (CA 19.9; sialyl Lewis antigen), fucosyl-GM R, PTK R, gpNMB, CDH R-CD 324, DLL R, CD276/B7H R, IL11R, IL13Ra R, CD 179R-IGLl R, ALK TCR gamma-delta, NKG 2R, CD R (FCGR 2R), CSPG R-HMW-MAA, Tim R-/HVCR R, CSF2R (GM-CSFR-alpha), TGF beta R R, TGF beta TCR 72/VEGFR, VEGFR 1-Ag 2-beta-loop peptide receptor, HIV TCR beta-loop receptor, TCR 72, TCR beta-loop receptor, TCR beta-loop receptor, and hormone, CMV pp65, EBV-EBNA3c, influenza A lectin (HA), GAD, PDL1, Guanylate Cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, desmoglein autoantibody 3(Dsg3), desmoglein autoantibody 1(Dsg1), HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1(TF1), AFP, GPRC5D, sealing protein 18.2(CLD18a2 or cldn18a.2)), P-glycoprotein, STEAP1, LIV1, fibronectin-4, CRIPTO, GPA33, BST1/CD157, low conductivity chloride channels, and antigens recognized by TNT antibodies, (ii) an agent that increases immune cell efficacy is selected from one or more of: inhibitors of protein phosphatases; kinase inhibitors (e.g., PI3K/AKT inhibitors or mTOR inhibitors); a cytokine; an immunosuppressive molecule inhibitor; an agent that decreases TREG cell levels or activity; an agent that increases proliferation and/or persistence of SIR-modified cells; a chemokine; an agent that increases SIR expression; agents that allow for modulation of SIR expression or activity; agents that allow control of the survival and/or persistence of SIR-modified cells; agents that control side effects of SIR-modified cells; brd4 inhibitors; agents that deliver therapeutic (e.g., shvum) or prophylactic agents to the site of disease; an agent that increases the expression of a target antigen to which the SIR is directed; and adenosine A2a receptor antagonists.

The present disclosure provides a method of treating or preventing a disease associated with expression of a disease-associated antigen in a subject, the method comprising administering to the subject an effective amount of an immune effector cell comprising a Synthetic Immune Receptor (SIR) molecule, in combination with an agent that increases the efficacy of the immune cell, wherein: (i) the SIR molecule comprises one or more of a T cell receptor constant chain linked via an optional linker to one or more antigen binding domains that bind to the disease-associated antigen associated with the disease, and said disease-associated antigen is selected from the group consisting of: CD5, CD 19; CD 123; CD 22; CD 30; CD 171; CS-1 (also known as CD2 subunit 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD 33; epidermal growth factor receptor variant iii (egfrviii); ganglioside G2(GD 2); ganglioside GD3(aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (l-l) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. alpha. -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1(ROR 1); fms-like tyrosine kinase 3(FLT 3); tumor-associated glycoprotein 72(TAG 72); CD 38; CD44v 6; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epidermal cell adhesion molecule (EPCAM); B7H3(CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213a 2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate Stem Cell Antigen (PSCA); protease serine 21 (testis protein or PRSS 21); vascular endothelial growth factor receptor 2(VEGFR 2); a lewis (Y) antigen; CD 24; platelet derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD 20; a folate receptor alpha; receptor tyrosine protein kinase ERBB2(Her 2/neu); cell surface associated mucin 1(MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); prostasin; prostatic Acid Phosphatase (PAP); mutant elongation factor 2(ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase ix (caix); proteasome (precursor, megalin) subunit, beta form, 9(LMP 2); glycoprotein 100(gpl 00); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and the Abelson (Abelson) murine leukemia virus oncogene homolog 1 (Abl); a tyrosinase enzyme; ephrin type a receptor 2(EphA 2); fucosyl GM 1; sialylated lewis adhesion molecules (sLe); ganglioside GM3(aNeu5Ac (2-3) bDClalp (l-4) bDGlcp (l-1) Cer); transglutaminase 5(TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); folate receptor beta; tumor endothelial marker 1(TEM1/CD 248); tumor endothelial marker 7 associated (TEM 7R); sealin 6(CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); class 5 group member D of G protein-coupled receptors C (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); a globoH sugar ceramide hexasaccharide moiety (globoH); mammary differentiation antigen (NY-BR-1); urinary plaque 2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenoceptor β 3(ADRB 3); ubiquitin 3(PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex, locus K9 (LY 6K); olfactory receptor 51E2(OR51E 2); TCR γ alternate reading frame protein (TARP); wilm's tumor protein (WT 1); cancer/testis antigen 1(NY-ES 0-1); cancer/testis antigen 2(LAGE-1 a); melanoma-associated antigen 1(MAGE-a 1); ETS translocation variant gene 6 located on chromosome 12p (ETV 6-AML); sperm protein 17(SPA 17); x antigen family member la (xagel); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; tumor protein p53(p 53); a p53 mutant; a prostate-specific protein; survivin; a telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; an inhibitor of melanoma apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyl-transferase V (NA 17); the paired box protein Pax-3(PAX 3); an androgen receptor; a cyclin Bl; a v-myc avian myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); tyrosinase-related protein 2 (TRP-2); cytochrome P450lB 1(CYPlB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site regulator siblings), squamous cell carcinoma antigen recognized by T cells 3(SART 3); the paired box protein Pax-5(PAX 5); the preproepisin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RUl); renal ubiquitin 2(RU 2); legumain; human papilloma virus E6(HPV E6); human papilloma virus E7(HPVE 7); intestinal carboxylesterase; mutated heat shock protein 70-2(mut hsp 70-2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2(BST 2); EGF-like receptor-module mucin-like hormone-containing sample 2(EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); and immunoglobulin lambda-like polypeptide 1(IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1TROP2, GFR alpha 54, CDH17, CDH6, NYB 1, CDH19, CD200R, Slea (CA 19.9; sialyl Lewis antigen), fucosyl-GM R, PTK R, gpNMB, CDH R-CD 324, DLL R, CD276/B7H R, IL11R, IL13Ra R, CD 179R-IGLl R, ALK TCR gamma-delta, NKG 2R, CD R (FCGR 2R), CSPG R-HMW-MAA, Tim R-/HVCR R, CSF2R (GM-CSFR-alpha), TGF beta R R, TGF beta TCR 72/VEGFR, VEGFR 1-Ag 2-beta-loop peptide receptor, HIV TCR beta-loop receptor, TCR 72, TCR beta-loop receptor, TCR beta-loop receptor, and hormone, CMV pp65, EBV-EBNA3c, influenza A lectin (HA), GAD, PDL1, Guanylate Cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, desmoglein autoantibody 3(Dsg3), desmoglein autoantibody 1(Dsg1), HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1(TF1), AFP, GPRC5D, sealing protein 18.2(CLD18a2 or cldn18a.2)), P-glycoprotein, STEAP1, LIV1, fibronectin-4, CRIPTO, GPA33, BST1/CD157, low conductivity chloride channels, and antigens recognized by TNT antibodies, (ii) an agent that increases immune cell efficacy is selected from one or more of: inhibitors of protein phosphatases; kinase inhibitors (e.g., PI3K/AKT inhibitors or mTOR inhibitors); a cytokine; an immunosuppressive molecule inhibitor; an agent that decreases TREG cell levels or activity; an agent that increases proliferation and/or persistence of SIR-modified cells; a chemokine; an agent that increases SIR expression; agents that allow for modulation of SIR expression or activity; agents that allow control of the survival and/or persistence of SIR-modified cells; agents that control side effects of SIR-modified cells; brd4 inhibitors; agents that deliver therapeutic (e.g., shvum) or prophylactic agents to the site of disease; an agent that increases the expression of a target antigen to which the SIR is directed; and an adenosine A2a receptor antagonist, thereby treating or preventing a disease in a subject.

The present disclosure provides a method of treating or preventing a disease associated with expression of a disease-associated antigen in a subject, the method comprising administering to the subject an effective amount of an immune effector cell comprising a Synthetic Immune Receptor (SIR) molecule, wherein: (i) the SIR molecule comprises one or more of a T cell receptor constant chain linked via an optional linker to one or more antigen binding domains that bind to the disease-associated antigen associated with the disease, and said disease-associated antigen is selected from the group consisting of: CD5, CD 19; CD 123; CD 22; CD 30; CD 171; CS-1 (also known as CD2 subunit 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD 33; epidermal growth factor receptor variant iii (egfrviii); ganglioside G2(GD 2); ganglioside GD3(aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (l-l) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. alpha. -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1(ROR 1); fms-like tyrosine kinase 3(FLT 3); tumor-associated glycoprotein 72(TAG 72); CD 38; CD44v 6; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epidermal cell adhesion molecule (EPCAM); B7H3(CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213a 2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate Stem Cell Antigen (PSCA); protease serine 21 (testis protein or PRSS 21); vascular endothelial growth factor receptor 2(VEGFR 2); a lewis (Y) antigen; CD 24; platelet derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD 20; folate receptor alpha (FRa or FR 1); folate receptor beta (FRb); receptor tyrosine protein kinase ERBB2(Her 2/neu); cell surface associated mucin 1(MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); prostasin; prostatic Acid Phosphatase (PAP); mutant elongation factor 2(ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase ix (caix); proteasome (precursor, megalin) subunit, beta form, 9(LMP 2); glycoprotein 100(gpl 00); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and the Abelson (Abelson) murine leukemia virus oncogene homolog 1 (Abl); a tyrosinase enzyme; ephrin type a receptor 2(EphA 2); fucosyl GM 1; sialylated lewis adhesion molecules (sLe); ganglioside GM3(aNeu5Ac (2-3) bDClalp (l-4) bDGlcp (l-1) Cer); transglutaminase 5(TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); tumor endothelial marker 1(TEM1/CD 248); tumor endothelial marker 7 associated (TEM 7R); sealin 6(CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); class 5 group member D of G protein-coupled receptors C (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); a globoH sugar ceramide hexasaccharide moiety (globoH); mammary differentiation antigen (NY-BR-1); urinary plaque 2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenoceptor β 3(ADRB 3); ubiquitin 3(PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex, locus K9 (LY 6K); olfactory receptor 51E2(OR51E 2); TCR γ alternate reading frame protein (TARP); wilm's tumor protein (WT 1); cancer/testis antigen 1(NY-ES 0-1); cancer/testis antigen 2(LAGE-1 a); melanoma-associated antigen 1(MAGE-a 1); ETS translocation variant gene 6 located on chromosome 12p (ETV 6-AML); sperm protein 17(SPA 17); x antigen family member la (xagel); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; tumor protein p53(p 53); a p53 mutant; a prostate-specific protein; survivin; a telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; an inhibitor of melanoma apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyl-transferase V (NA 17); the paired box protein Pax-3(PAX 3); an androgen receptor; a cyclin Bl; a v-myc avian myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); tyrosinase-related protein 2 (TRP-2); cytochrome P450lB 1(CYPlB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site regulator siblings), squamous cell carcinoma antigen recognized by T cells 3(SART 3); the paired box protein Pax-5(PAX 5); the preproepisin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RUl); renal ubiquitin 2(RU 2); legumain; human papilloma virus E6(HPV E6); human papilloma virus E7(HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2(mut hsp 70-2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2(BST 2); EGF-like receptor-module mucin-like hormone-containing sample 2(EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); and immunoglobulin lambda-like polypeptide 1(IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1TROP2, GFR alpha 54, CDH17, CDH6, NYB 1, CDH19, CD200R, Slea (CA 19.9; sialyl Lewis antigen), fucosyl-GM R, PTK R, gpNMB, CDH R-CD 324, DLL R, CD276/B7H R, IL11R, IL13Ra R, CD 179R-IGLl R, ALK TCR gamma-delta, NKG 2R, CD R (FCGR 2R), CSPG R-HMW-MAA, Tim R-/HVCR R, CSF2R (GM-CSFR-alpha), TGF beta R R, TGF beta TCR 72/VEGFR, VEGFR 1-Ag 2-beta-loop peptide receptor, HIV TCR beta-loop receptor, TCR 72, TCR beta-loop receptor, TCR beta-loop receptor, and hormone, CMV pp65, EBV-EBNA3c, influenza A lectin (HA), GAD, PDL1, Guanylate Cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, desmoglin autoantibody 3(Dsg3), desmoglin autoantibody 1(Dsg1), HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1 (1), AFP, GPRC5D, sealer 18.2(CLD18A2 or N18A.2)), P-glycoprotein, STEAP1, LIV1, laminin-4, CRIPTO, GPA33, EBT 1/CD157, chloride channel antigen recognized by TNT antibody of low conductivity; and (ii) the binding domain of the SIR molecule has at least 5-fold less binding affinity than the antibody from which the antigen binding domain is derived.

In another embodiment of any of the foregoing methods or uses, the disease associated with expression of a disease-associated antigen is selected from the group consisting of a proliferative disease, a precancerous condition, a cancer, and a non-cancer related indication associated with expression of the disease-associated antigen. In another embodiment of any of the foregoing methods or uses, the cancer is selected from Chronic Lymphocytic Leukemia (CLL), acute leukemia, Acute Lymphocytic Leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), Chronic Myelogenous Leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, burkitt lymphoma, diffuse large B-cell lymphoma, primary effusion lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-hodgkin lymphoma, lymphomas, Hodgkin's lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor, waldenstrom's macroglobulinemia, or hematological cancers of one or more of the pre-stages of leukemia. In another embodiment of any of the foregoing methods or uses, the cancer is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, hodgkin's disease, non-hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors of childhood, bladder cancer, renal or ureter cancer, renal pelvis cancer, tumors of the Central Nervous System (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, and cervical cancer, Merkel cell carcinoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of said cancers and metastatic lesions of said cancers. In another embodiment of any of the foregoing methods or uses, the disease is associated with infection by a virus including, but not limited to, HIV1, HIV2, HTLV1, Epstein Barr Virus (EBV), Cytomegalovirus (CMV), adenovirus, adeno-associated virus, BK virus (EBV), human herpesvirus 6, human herpesvirus 8 influenza virus, parainfluenza virus, avian influenza virus, MERS and SARS coronavirus, crimean congo hemorrhagic fever virus, rhinovirus, enterovirus, dengue virus, west nile virus, ebola virus, marburg virus, lassa fever virus, seca virus, RSV, measles virus, mumps virus, rhinovirus, varicella virus, herpes simplex virus 1 and 2, varicella-zoster virus, HIV-1, HTLV1, hepatitis virus, enterovirus, hepatitis b virus, Hepatitis C virus, Nipah virus and rift valley fever virus, Japanese encephalitis virus, Merkel cell polyoma virus, or with Mycobacterium tuberculosis, atypical mycobacterial species, Yersinia pneumocystis, toxoplasmosis, Rickettsia, Nocardia, Aspergillus, Mucor or Candida infections. In another embodiment of any of the foregoing methods or uses, the disease is an immune or degenerative disease, including but not limited to diabetes, multiple sclerosis, rheumatoid arthritis, pemphigus vulgaris, ankylosing spondylitis, hypothyroidism (Hoshimoto's thyroiditis), SLE, sarcoidosis, scleroderma, mixed connective tissue disease, graft-versus-host disease, or alzheimer's disease. In another embodiment of any of the foregoing methods or uses, (i) the protein phosphatase inhibitor is a SHP-1 inhibitor and/or a SHP-2 inhibitor; (ii) the kinase inhibitor is selected from one or more of a CDK4 inhibitor, a CDK4/6 inhibitor, an mTOR inhibitor, a MNK inhibitor or a dual P13K/mTOR inhibitor; (iii) the agent that inhibits the immunosuppressive molecule comprises an antibody or antibody fragment, an inhibitory nucleic acid, an aggregated regularly interspaced short palindromic repeats (CRISPR), a transcription activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN) that inhibits expression of an inhibitory molecule; (iv) the agent that reduces the level or activity of the T REG cells is selected from cyclophosphamide, anti-GITR antibodies, CD25 depletion, or a combination thereof; and/or (v) the Brd4 inhibitor is selected from JQ1, MS417, OTXO15, LY 303511 and Brd4 inhibitors or derivatives thereof as described in US 20140256706a 1. In another embodiment of any of the foregoing methods or uses, the immunosuppressive molecule is selected from the group consisting of PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGF β, CEACAM-1, CEACAM-3, and CEACAM-5. In another embodiment of any of the foregoing methods or uses, the agent that inhibits an inhibitory molecule comprises a first polypeptide comprising an inhibitory molecule or fragment thereof and a second polypeptide that provides a positive signal to a cell, and wherein the first and second polypeptides are expressed on an immune cell comprising a CAR, wherein (i) the first polypeptide comprises PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGF β, CEACAM-1, CEACAM-3, and CEACAM-5 or fragments thereof; and/or (ii) the second polypeptide comprises an intracellular signaling domain comprising a primary signaling domain and/or a costimulatory signaling domain. In another embodiment of any one of the foregoing methods or uses, the primary signaling domain comprises a functional domain of CD3 ζ and/or the costimulatory signaling domain comprises a functional domain of a protein selected from 41BB, CD27, and CD 28. In another embodiment of any of the foregoing methods or uses, the cytokine is selected from IL-15 or IL-21 or both. In another embodiment of any of the foregoing methods or uses, the immune effector cell comprising one or more SIR molecules and the agent that increases the efficacy of the immune effector cell are administered substantially simultaneously or sequentially. In another embodiment of any of the foregoing methods or uses, the immune cell comprising the SIR molecule is administered in combination with a molecule that targets GITR and/or modulates GITR function. In another embodiment of any of the foregoing methods or uses, the molecule that targets GITR and/or modulates GITR function is administered prior to the cell or population of cells expressing SIR or prior to apheresis. In another embodiment of any of the foregoing methods or uses, the subject is a human.

The present disclosure also provides a composition comprising at least one polynucleotide of the present disclosure, SIR polypeptide molecule of the present disclosure, vector of the present disclosure, or cell of the present disclosure and a pharmaceutically acceptable excipient.

The present disclosure also provides a kit comprising at least one polynucleotide of the present disclosure, SIR polypeptide molecule of the present disclosure, vector of the present disclosure, or cell of the present disclosure and/or composition of the present disclosure.

The present disclosure also provides a polynucleotide encoding a synthetic immunoreceptor comprising a sequence selected from the group consisting of SEQ ID NOS 900 to 2264, SEQ ID NOS 4531 to 6013, SEQ ID NOS 7519 to 8160, SEQ ID NOS 8803 to 9230, SEQ ID NOS 9659 to 9856, SEQ ID NOS 10474 to 12041, SEQ ID NOS 15786 to 16011, SEQ ID NOS 16240 to 16465, SEQ ID NOS 16694 to 16926, SEQ ID NOS 17162 to 17394, SEQ ID NOS 17864 to 17979, SEQ ID NOS 18321 to 18322, SEQ ID NOS 18242 to 18259, SEQ ID NOS 18280 to 18588, SEQ ID NOS 18899, SEQ ID NOS 18915 to 18916, or SEQ ID NOS 900 to 2264, SEQ ID NOS 4531 to 453, SEQ ID NOS 8119 to 8160, SEQ ID NOS 758930 to 1209864, SEQ ID NOS 12048, A sequence having at least 75% identity to a nucleotide sequence encoding a synthetic immunoreceptor as set forth in any one of SEQ ID NOS 15786 to 16011, SEQ ID NOS 16240 to 16465, SEQ ID NOS 16694 to 169926, SEQ ID NOS 17162 to 17394, SEQ ID NOS 17864 to 17979, SEQ ID NOS 18321 to 18322, SEQ ID NOS 18242 to 18259, SEQ ID NOS 18280 to 18588, SEQ ID NO 18899, and SEQ ID NOS 18915 to 18916.

The present disclosure also provides an amino acid sequence encoding a synthetic immunoreceptor polypeptide selected from the group consisting of SEQ ID NOs 3135 to 4498, SEQ ID NOs 6044 to 7518, SEQ ID NOs 8161 to 8802, SEQ ID NOs 9231 to 9658, SEQ ID NOs 9873 to 10070, SEQ ID NOs 12431 to 13998, SEQ ID NOs 16013 to 16238, SEQ ID NOs 16467 to 16692, SEQ ID NOs 16928 to 17160, SEQ ID NOs 17396 to 17628, SEQ ID NOs 17981 to 18096, SEQ ID NOs 18239 to 18240, SEQ ID NOs 18261 to 18278, SEQ ID NOs 18590 to 18898, SEQ ID NOs 18900 and SEQ ID NOs 18919 to 18928920, or from SEQ ID NOs 3135 to 4498, SEQ ID NOs 7544 to 7518, SEQ ID NOs 8161 to 8161, SEQ ID NOs 9658 to 924631, SEQ ID NOs 100594631 to 1395998, SEQ ID NOs 3135 to 139594698, The sequence having at least 75% identity to the amino acid sequence of the synthetic immunoreceptor polypeptide shown in any one of SEQ ID NOS 16013 to 16238, SEQ ID NOS 16467 to 16692, SEQ ID NOS 16928 to 17160, SEQ ID NOS 17396 to 17628, SEQ ID NOS 17981 to 18096, SEQ ID NOS 18239 to 18240, SEQ ID NOS 18261 to 18278, SEQ ID NOS 18590 to 18898, SEQ ID NO 18900, and SEQ ID NOS 18919 to 18920.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Drawings

Figure 1 shows a schematic depiction of an antibody, a TCR, a two-chain chimeric receptor, and different generations of a CAR.

Figure 2 illustrates an exemplary vector construct of the present disclosure.

Fig. 3A-Q show depictions of various formats that the SIR of the present disclosure may have when expressed. In these depictions, each TCR of a TCR pair is linked to a vH or vL binding domain. In other embodiments, each TCR may be bound to the vHH domain, rather than vH or vL.

Fig. 4A-Q show depictions of various formats that the SIR of the present disclosure may have when expressed. In these depictions, one TCR of a TCR pair is connected to vH or vL and vH or vL is then connected to the opposite vH or vL. Although vL is shown connected to the TCRb chain in (a), it will be appreciated that the orientation in all (a) - (Q) depictions may be switched, such that vL is connected to TCRa in (a), and so on.

Fig. 5A-Q show depictions of various formats that the SIR of the present disclosure may have when expressed. In these constructs, the SIR is based on a single domain antibody (vHH) that binds to only one of the two TCR constant chains and the other chain remains empty. An exemplary such construct is CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-Her3-17B05So-vHH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC [ SEQ ID NO:1715 ]. Similar constructs based on other non-immunoglobulin binding domains other than the vHH domain can be made. For example, the non-immunoglobulin binding domain may be based on an affibody, DARPIN, an autoantigen (e.g., Dsg3), a ligand (e.g., MPL or TRAIL), or a receptor (e.g., CD 16).

Fig. 6A-Q show depictions of various formats that the SIR of the present disclosure may have when expressed. In these constructs, the SIR contains two different types of structurally distinct antigen binding domains. In one example, one antigen binding domain comprises a single domain antibody (vHH), while the other antigen binding domain comprises a scFV fragment in the vL-linker-vH orientation or in the vH-linker-vL orientation. In another example, one antigen binding domain comprises a single domain antibody (vHH) and the other antigen binding domain comprises a receptor (e.g., CD 16). In another example, one antigen binding domain comprises a single domain antibody (vHH) and the other antigen binding domain comprises an affibody. An advantage of using two different types of antigen binding domains is that they are less likely to interfere with each other. The first antigen-binding domain (e.g., vHH) may be directed against one antigen and the second antigen-binding domain (e.g., scFV fragment) may be targeted against another antigen. Alternatively, they may be directed against the same antigen to increase avidity.

Fig. 7A-Q show depictions of various formats that the SIR of the present disclosure may have when expressed. In these constructs, the SIR was based on two scFV fragments. Two scFv fragments can be directed against two different antigens (e.g., CD8SP-CD19Bu12-scFv-V5- [ hTCRb-KACIAH ] -F-P2A-SP-CD20-2F2-scFv-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (040716-B04) [ SEQ ID NO:1028 ]). Alternatively, both may be directed against the same antigen to increase avidity (e.g., CD8SP-CD19Bu12-scFv-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-scFv-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (020216-B07) [ SEQ ID NO:1026 ]). The format of the scFV may be vH-linker-vL or vL-linker-vH.

Fig. 8A-T show depictions of various formats that the SIR of the present disclosure may have when expressed. In some SIR constructs, scFv fragments consisting of a signal peptide fused in frame with a vL region, a Gly-Ser linker (GGGGSx3) and a vH region (e.g., derived from human CD8 signal peptide) are fused to the C α, C β, Pre-C α, C δ or C γ chain and have no complementary chain.

Fig. 9A-B show NLuc assays measuring CAR expression in 293FT cells. Untransfected 293FT cells and those transfected with CD19 (FMC63-BBZ-PAC) and 161-BBZ-PAC CAR were incubated with CD19-GGSG-NLuc-AcV5 and MPL-GGSG-NLuc-AcV5 supernatants, followed by washing with PBS and measurement of NLuc activity by Coeleoentrazine (CTZ; Nanolight) diluted in PBS. Fluorescence was quantified using a BioTek plate reader. Data represent mean +/-Standard Deviation (SD) of triplicate wells.

Figure 10 shows that strong binding of T cells expressing 161(vL + vH) -Myc-BBz-PAC R07, 175(vL + vH) -Myc-28Z-PAC Q04, VB22(vL + vH) -Myc-28Z-PAC B06CAR to MPL-GGSG-NLuc AcV5 supernatant and moderate binding of T cells expressing 161(vL + vH) -Myc-28Z-PAC Z07, AB317(vL + vH) -Myc-28Z-PAC T04, and 12E10(vL + vH) -Myc-28Z-PAC B06 to this supernatant were not observed on uninfected T cells or those expressing 4C3(vL + vH) -Myc-28Z-PAC control CAR. Similarly, no specific binding to CD19-GGSG-NLuc-AcV5 supernatant was observed on any MPL CAR-T cells, demonstrating the specificity of the assay.

Fig. 11 shows a graph demonstrating that TCR α and TCR β constant regions encoded by wild-type nucleotide sequences containing SIRs are not efficiently expressed in human primary T cells. In contrast, the SIR of the codon optimized human TCRa/b chain containing additional cysteine residues that promote interchain disulfide bonds is efficiently expressed. The murine formation of the human TCR α/β constant chain as seen in (081415-D06) [ SEQ ID NO:992] SIR leads to a further increase in SIR expression. In addition, as can be seen in the (082815-G07) [ SEQ ID NO:1620] and (082815-E05) [ SEQ ID NO:1622] constructs, the scFv fragment can be expressed fused to the TCRa constant region when co-expressed with the TCRb constant chain, even if the TCRb does not carry any antigen binding moiety.

Fig. 12 illustrates a method of generating a set of SIRs with desired or different binding affinities.

FIGS. 13A-B show representative FACS analyses. (A) Control Jurkat-NFAT-GFP cells or those expressing SIRs targeting CD19 (clone ID 051716-I08), MPL (clone ID:040716-A07) and BCMA (clone ID:011116-A07) were incubated with RAJI (top), HEL (middle) or U266 (bottom) cells, respectively. The induction of GFP expression was evident when Jurkat-NFAT-GFP cells expressing SIR were co-cultured with their corresponding target cells. (B) Jurkat cells expressing SIRs targeting CDH6 (clone ID:051716-J05), CD276 (clone ID:050516-Q06) and Her2/neu (clone ID:050516-I03) were incubated with SKOV3 (top) and MC7 (middle and bottom) cells, respectively. The induction of GFP expression was evident when Jurkat-NFAT-GFP cells expressing SIR were co-cultured with their corresponding target cells.

Fig. 14 shows exemplary results for retroviral vectors expressing SIRs of the present disclosure.

FIG. 15 shows the results using the sleeping beauty transposon vector, Jurkat-NFAT-GFP cells transfected with construct pSBbi-puro-FMC63vL-V5- [ TCRb-KACIAH ] -F-P2A-FMC63vH-MYC- [ TCRa-CSDVP ] -F-F2A [010616-B01] (SEQ ID NO:875) show GFP induction when co-cultured with the corresponding RAJI target cell line.

FIG. 16 shows lower cell surface expression of Bu12 SIR (CD8SP-CD19Bu12-vL-V5- [ TCRb-S57C-opt1] -F-P2A-SP-CD19Bu12-vH-Myc- [ TCRa-T48C-opt1] -F-F2A-P AC (070215-M03) [ SEQ ID NO:1021], as determined by staining with APC-MYC-APC and biotin-protein-L plus APC-streptavidin, respectively, compared to Bu12CAR CD8SP-CD19Bu12- (vL-vH) -Myc-BBz-T2A-PAC (082815-P08) [ SEQ ID NO: 4503] on T cells.

FIG. 17 shows RAJI cells injected and received expression of SIR CD 8-FMC 1-vL-V1- [ TCRb-S57 1-opt 1] -F-P2 1-SP-72-SP-F2 1-PAC (1-U1) [ SEQ ID 1112] against CD1 in comparison to control SIR CD8SP-MPL-161-vL-V5- [ hTCCRb-S57C-opt 1] -F-P2A-MPL-161-vH-Myc- [ hTCRa-T-48-opt 1] -F-2 1-PAC (1-L1) [ SEQ ID NO:900] and CD8 1-CD 19-Bu-T-48-opt 1-OPT-OCT-1-F-F2 1-PAC (1-L1) [ SEQ ID NO:900] against CD 1-OC-V- [ hTCRb-T-1-OCP-V-OCR-T-1-OCP-F-1-OCR-S-1-OCR Survival of mice with T cells from opt1] -F-F2A-PAC (070215-M03) [ SEQ ID NO:1021 ].

FIG. 18 shows GFP induction by co-culturing Jurkat-NFAT-GFP cells expressing constructs of the disclosure with protein L beads.

FIGS. 19A-D show that co-culture with 293-protein L-II cells resulted in strong induction of GFP expression in Jurkat-NFAT-GFP cells expressing: (B) CD8SP-FMC63(vL-vH) -Myc-BBz-T2A-PAC (112014-A13) [ SEQID NO:4501] CAR, (C) CD8SP-HuLuc64-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-HuLuc64-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (092916-E07) [ SEQ ID NO:1253] and (D) CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-CD19Bu 12-vL-Gly-Ser-linker-CD 19Bu 12-CRH-Myc- [ hTCa-CSP ] -F-F2 DVP 5-PAC 082815-E05 [ SIR ID NO:1622] constructs.

Detailed Description

As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "a polynucleotide" includes reference to one or more polynucleotides and the like.

Also, the use of "or" means "and/or" unless otherwise indicated. Similarly, "comprise/comprising" and "include/include" are interchangeable and not intended to be limiting.

It is also to be understood that where the term "comprising" is used to describe various embodiments, one skilled in the art will understand that in some specific instances an embodiment may alternatively be described using the language "consisting essentially of or" consisting of.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al, Remington: The Science and practice of Pharmacy [ Remington: science and practice of medicine]22 nd edition, Pharmaceutical Press](9 months and 15 days 2012); hornyak et al, Introduction to Nano science and Nanotechnology]CRC Press](2008) (ii) a Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology Dictionary]Revision 3, revision, j.wiley&Sons [ J. Willi father-son Co., Ltd](new york, NY 2006); smith, March's Advanced Organic Chemistry Reactions, Mechanisms and structures [ March Advanced Organic Chemistry Reactions, Mechanisms and structures]7 th edition, j.wiley&Sons [ J. Willi father-son Co., Ltd](new york, NY 2013); singleton, Dictionary of DNA and Genome Technology]3 rd edition, Wiley-Blackwell, Inc](11 months and 28 days 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual [ Molecular Cloning: laboratory manual]4 th edition, Cold Spring Harbor Laboratory Press](cold spring harbor, NY 2012) provides one skilled in the art with a general guidance for many of the terms used in this application. For references on how to prepare Antibodies, see Greenfield, Antibodies A Laboratory Manual]2 nd edition, Cold Spring Harbor Press](cold spring harbor NY, 2013);and Milstein, degradation of specific antibody-producing tissue culture and tumor lines by cell fusion [ obtaining specific antibodies by cell fusion]Eur.J.Immunol. [ journal of European immunology ]]Month 7 1976, 6(7): 511-9; queen and Selick, Humanized immunoglobulins]U.S. Pat. No. 5,585,089(1996 month 12); and Riechmann et al, rehaping human antibodies for therapy [ reshape human antibodies for therapy]Nature (Nature)]323-7All, provided herein for ease of reading only and should not be construed as limiting the invention. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and specific examples are illustrative only and are not intended to be limiting.

The Synthetic Immune Receptors (SIRs) of the present disclosure comprise an antigen binding domain (e.g., an antibody or antibody fragment) that can bind to an antigen, e.g., in an MHC-dependent or MHC-independent manner. Typically, peptides derived from endogenous proteins fill the pocket of Major Histocompatibility Complex (MHC) class I molecules and are recognized by T Cell Receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and/or tumor-specific peptide/MHC complexes represent a unique class of cell surface targets for immunotherapy. Peptides targeting TCR-like antibodies derived from viral or tumor antigens in the context of Human Leukemia Antigen (HLA) -a1 or HLA-a2 have been described (see, e.g., Sastry et al, jvirial [ journal ]201185(5): 1935-. For example, TCR-like antibodies can be identified from a screening library (e.g., a human scFv phage display library).

The present disclosure generally provides Synthetic Immune Receptors (SIRs) comprising an antigen binding site operably linked to a T cell receptor domain. The present disclosure further provides one or more recombinant nucleic acid constructs comprising a sequence encoding a SIR, wherein the SIR comprises one or more antigen binding domains (e.g., antibodies, antibody fragments, non-immunoglobulin antigen binding domains, autoantigens, ligands, or receptors) that bind to an antigen or target molecule (as described further herein below) and are linked to one or more T cell receptor constant chains (including mutants or variants thereof). One or more antigen binding domains of the SIR specifically bind to one or more disease-associated antigens or homologues as described herein, wherein the coding sequence for each antigen binding domain is operably linked to each T cell receptor constant chain to which it is encoded such that the antigen binding domain is operably expressed with the T cell constant chain. In some embodiments, the SIR may comprise a single antigen binding domain linked to a single T cell receptor constant chain. In some embodiments, the SIR comprises two antigen binding domains each linked to a separate T cell receptor constant chain. For example, antigen binding domain 1 is linked to a constant chain of T cell receptor α (TCR α) to construct "functional unit 1" and antigen binding domain 2 is linked to a constant chain of T cell receptor β (TCR β) to construct "functional unit 2". Two functional units of such SIRs are co-expressed in the same cell to become functionally active (e.g., heterodimerization). In some embodiments, the SIR comprises an antigen binding domain that is in frame with one T cell receptor constant chain (functional unit 1) but is co-expressed with a second T cell receptor constant chain. The purpose of the second T cell receptor constant chain in such SIRs is to facilitate cell surface expression of functional unit 1 (e.g., antigen binding domain 1 linked to the T cell receptor constant chain). Thus, the second T cell receptor constant chain may be expressed by itself or as a fusion protein carrying an epitope tag (e.g. MYC, V5, AcV5, G4Sx2, StrepTagII, etc.) or as a fusion protein carrying any unrelated protein fragment (e.g. vL or vH fragment) that does not interfere with the assembly and function of functional unit 1. As an example, the SIR may comprise an antigen binding domain 1 operably linked in frame to a constant chain of T cell receptor α (TCR α) and a blank (i.e., lacking the antigen binding domain) constant chain of T cell receptor β (TCR β). Two functional units of such SIRs are co-expressed in the same cell to become functionally active. In some embodiments, two functional units of the SIR are co-expressed by transfecting a single polynucleotide encoding both functional units, while in other embodiments, two functional units are co-expressed by transfecting two different polynucleotides each encoding one functional unit. In some embodiments, two functional units of the SIR are inserted at a single genomic locus, while in other embodiments, two functional units are inserted at two genomic loci. For example, in some embodiments, two functional units may be inserted at the TCR alpha constant chain (TRAC) locus and expressed as a single polynucleotide. In other embodiments, functional unit 1 may be inserted at the TCR α constant chain (TRAC) locus, while functional unit 2 may be inserted at the TCR constant chain β 1(TRBC1) locus. In some embodiments, two functional units of the SIR are co-expressed by transfecting a single polynucleotide encoding both functional units, while in other embodiments, two functional units are co-expressed by transfecting two different polynucleotides each encoding one functional unit. In some embodiments, the SIR comprises an antigen binding domain that is in frame with one T cell receptor constant chain (functional unit 1) but is co-expressed with a second T cell receptor constant chain. The purpose of the second T cell receptor constant chain in such SIRs is to facilitate cell surface expression of functional unit 1 (e.g., antigen binding domain 1 linked to the T cell receptor constant chain). Thus, the second T cell receptor constant chain may be expressed by itself or as a fusion protein carrying an epitope tag (e.g. MYC, V5, AcV5, G4Sx2, StrepTagII, etc.) or as a fusion protein carrying any unrelated protein fragment (e.g. vL or vH fragment) that does not interfere with the assembly and function of functional unit 1. As an example, the SIR may comprise an antigen binding domain 1 operably linked in frame to a constant chain of T cell receptor α (TCR α) and a blank (i.e., lacking the antigen binding domain) constant chain of T cell receptor β (TCR β). Two functional units of such SIRs are co-expressed in the same cell to become functionally active. In some embodiments, the two functional units of the SIR are co-expressed using a single vector, while in other embodiments, the two functional units are co-expressed in the same cell using different vectors. In some embodiments, two functional units of the SIR are co-expressed by transfecting a single polynucleotide encoding both functional units, while in other embodiments, two functional units are co-expressed by transfecting two different polynucleotides each encoding one functional unit. Different configurations of the SIR of the present disclosure are provided in fig. 3-8.

The present disclosure provides a class of chimeric T cell receptors (synthetic immune receptors (SIRs)) that can be used in adoptive cell therapy to treat cancer, infections, autoimmune and degenerative diseases. In contrast to Chimeric Antigen Receptors (CARs), the SIRs of the present disclosure engage the full power of physiological T cell receptor signaling pathways and are therefore less likely to cause complications associated with CARs, such as cytokine release syndrome, neurotoxicity, and lack of persistence in vivo. In contrast to CAR, the SIRs of the present disclosure have a smaller tendency for the antigen binding domain to self-aggregate, a smaller chance of complement signaling, and a smaller chance of early T cell depletion. SIRs of the present disclosure contain one or more antigen binding domains fused to a constant chain of TCR α (ca), TCR β (cp), TCR δ (C δ), TCR γ (C γ), or preTCR α (ca), including variants and mutants of any of the foregoing. The antigen binding domain may comprise an antibody or antibody fragment, a vL or/and vH fragment of an antibody, a scFv fragment derived from an antibody, a single domain antibody, an affibody, a DARPIN, any antigen binding ligand or receptor, an autoantigen, or any other non-immunoglobulin antigen binding fragment. The antigen binding domain may target a single antigen or multiple antigens (bispecific or multispecific SIR). The TCR constant domains of SIRs may be expressed individually, but are typically expressed in pairs (e.g., ca vs. C β, or precca vs. C β, or C δ vs. C γ, etc.) for optimal cell surface expression. TCR constant chain fragments are typically codon optimized to allow optimal cell surface expression. The TCR constant fragments may carry additional mutations or substitutions to facilitate their optimal expression and pairing with complementary chains and/or to reduce the interaction with the pairing of endogenous TCR chains and/or to stabilize the antigen binding domain. SIR may also express one or more additional domains (e.g., Myc, streptag, V5, FLAG, Ritx tag, etc.) as fusion proteins. The SIRs of the present disclosure can be introduced into cells using a number of techniques including, but not limited to, the use of lentiviral vectors, retroviral vectors, adeno-associated viral vectors, baculovirus vectors, sleeping beauty transposons, piggybac transposons, or by mRNA transfection, or using a combination of the above methods. An optimized carrier for delivering SIR is also disclosed. SIRs of the disclosure can be expressed such that they are under the control of an endogenous promoter (e.g., a TCR α or TCR β promoter). In some embodiments, the SIRs of the present disclosure are expressed using a foreign promoter (e.g., CMV promoter). SIRs of the disclosure may co-express additional modules, such as promoting SIR expression or function (e.g., CD3z, CD3 epsilon, CD3 delta, CD3z-41BB fusion protein, etc.), promoting T cell proliferation, persistence, expansion and activation (e.g., 41BBL, CD40L, IL12f, K13, Tax2, MC159L, cflp, scFV-targeted PD1, shRNA-targeted BRD4, etc.), cDNA-encoding molecules that reduce toxicity (e.g., vHH or scFV-targeted IL6R, IL6, TNF α, etc.), selection markers (e.g., tfegfr, tgfrviii, cmtba, tCD19, etc.), and/or suicide genes (e.g., spicatase 9, HSV-thymidine kinase). The SIRs of the present disclosure may be expressed in immune effector cells (e.g., T cells) or in stem cells (including induced pluripotent stem cells) that can give rise to immune effector cells. The disclosure also provides subsets of immune effector cells for expressing SIR and methods for activating and expanding immune effector cells that express SIR. The disclosure also describes agents that can be used to enhance the activity and persistence or reduce the toxicity of immune effector cells expressing SIR. The present disclosure describes in vitro and in vivo assays that can be used to identify SIRs suitable for different applications.

The term "about," when referring to a measurable value such as an amount, time interval, or the like, is intended to encompass variations from the specified value of ± 20%, or in some cases ± 10%, or in some cases ± 5%, or in some cases ± 1%, or in some cases ± 0.1%, as such variations are suitable for performing the disclosed methods or describing the compositions herein. Thus, for example, "one to five mutations" specifically includes 1, 2, 3, 4, and/or 5 mutations.

The term "secondary module" refers to any one or more of the following that may be co-expressed with the SIR: 41BBL, CD40L, K13, MC159, cFLIP-L/MRIT alpha, cFLIP-p22, HTLV1Tax, HTLV2Tax-RS mutant, FKBPx2-K13, FKBPx2-HTLV2-Tax, FKBPx2-HTLV2-Tax-RS, IL6R-304-vHH-Alb8-vHH, IL12f, PD1-4H1scFV, PD1-5C4scFV, PD1-4H1-Alb8-vHH, PD1-5C4-Alb8-vHH, CTLA 4-ipilimumab-scFv, CTLA 4-ipilimumab-Alb 8-vHH, IL6-19A-scFV, IL6-19A-scFV-Alb8-vHH, sHVEM-Alb8-vHH, hTERT, Fx06, CD3z, CD3z-GGGS-41BB, CD3-BBz, CD3-CD28z, CD3-CD28-Lck fusion protein, targeting Brd shRNA 4, and combinations thereof. The auxiliary module may be co-expressed with the SIR using a single vector or using two or more different vectors. In one embodiment, the auxiliary module comprises the amino acid sequence of SEQ ID NOS 3087 to 3117(DNA coding sequence SEQ ID NO 812-842) or a sequence having 80-99% identity thereto. In other embodiments, the nucleic acid sequence encoding the helper module comprises the sequence of SEQ ID NO:800 or SEQ ID NO:842 or a sequence having 80-99% identity thereto.

"autoantibodies" refers to antibodies produced by B cells specific for self-antigens.

As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be monoclonal or polyclonal, multi-or single-chain, or whole immunoglobulins and may be derived from natural sources or from recombinant sources. The antibody may be a tetramer of immunoglobulin molecules. Antibodies can be 'humanized', 'chimeric', or non-human.

The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an antigenic epitope (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab ', F (ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdfv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments (such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), and isolated CDRs, or other epitope-binding fragments of an antibody. Antigen-binding fragments may also be incorporated into single domain antibodies, macroantibodies (maxibodes), minibodies (minibodies), nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-. Antigen-binding fragments may also be grafted to polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. patent No. 6,703,199, which describes fibronectin polypeptide miniantibodies).

The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains in an antibody molecule that exist in its naturally occurring conformation, and generally determines the class to which an antibody belongs.

The term "antibody light chain" refers to the smaller of the two types of polypeptide chains in an antibody molecule that exists in its naturally occurring conformation. Kappa (. Kappa.) and lambda (. lamda.) light chains refer to the two major antibody light chain isotypes.

The term "anti-cancer effect" refers to a biological effect that can be manifested in a variety of ways, including, but not limited to, reduction in tumor volume, reduction in the number of cancer cells, reduction in the number of metastases, increased life expectancy, reduction in cancer cell proliferation, reduction in cancer cell survival, or improvement in various physiological symptoms associated with a cancerous condition. An "anti-cancer effect" can also be manifested by the ability of SIR to first prevent the development of cancer.

"anti-cancer agent" refers to an agent that inhibits abnormal cell division and growth, inhibits tumor cell migration, inhibits invasion, or prevents cancer growth and metastasis. The term includes chemotherapeutic agents, biological agents (e.g., siRNA, viral vectors such as engineered MLV, adenovirus, herpes virus that delivers cytotoxic genes), antibodies, and the like.

The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immunocompetent cells or both. One skilled in the art will appreciate that virtually any macromolecule, including all proteins or peptides, can serve as an antigen. Furthermore, the antigen may be derived from recombinant or genomic DNA. The skilled person will understand that any DNA comprising a nucleotide sequence or part of a nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" (as that term is used herein). Furthermore, one skilled in the art will appreciate that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. It will be apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Furthermore, one skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It will be apparent that the antigen may be synthetically produced or may be derived from a biological sample, or may be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or fluids with other biological components.

Non-limiting examples of target antigens include: CD5, CD 19; CD 123; CD 22; CD 30; CD 171; CS-1 (also known as CD2 subunit 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD 33; epidermal growth factor receptor variant iii (egfrviii); ganglioside G2(GD 2); ganglioside GD3(aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (l-l) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. alpha. -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1(ROR 1); fms-like tyrosine kinase 3(FLT 3); tumor-associated glycoprotein 72(TAG 72); CD 38; CD44v 6; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3(CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213a 2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate Stem Cell Antigen (PSCA); protease serine 21 (testis protein or PRSS 21); vascular endothelial growth factor receptor 2(VEGFR 2); lewis (Y) antigen; CD 24; platelet derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD 20; folate receptor alpha (FRa or FR 1); folate receptor beta (FRb); receptor tyrosine protein kinase ERBB2(Her 2/neu); cell surface associated mucin 1(MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); prostasin; prostatic Acid Phosphatase (PAP); mutant elongation factor 2(ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase ix (caix); proteasome (precursor, megalin) subunit, beta form, 9(LMP 2); glycoprotein 100(gpl 00); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and the Abelson (Abelson) murine leukemia virus oncogene homolog 1 (Abl); a tyrosinase enzyme; ephrin type a receptor 2(EphA 2); sialylated lewis adhesion molecules (sLe); ganglioside GM3(aNeu5Ac (2-3) bDClalp (l-4) bDGlcp (l-1) Cer); transglutaminase 5(TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); tumor endothelial marker 1(TEM1/CD 248); tumor endothelial marker 7 associated (TEM 7R); sealin (claudin)6(CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); class 5 group member D of G protein-coupled receptors C (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); a globoH sugar ceramide hexasaccharide moiety (globoH); mammary differentiation antigen (NY-BR-1); urinary plaque protein (uroplakin)2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenoceptor β 3(ADRB 3); ubiquitin 3(PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex, locus K9 (LY 6K); olfactory receptor 51E2(OR51E 2); TCR γ alternate reading frame protein (TARP); wilm's tumor protein (WT 1); cancer/testis antigen 1(NY-ES 0-1); cancer/testis antigen 2(LAGE-1 a); melanoma-associated antigen 1(MAGE-a 1); ETS translocation variant gene 6 located on chromosome 12p (ETV 6-AML); sperm protein 17(SPA 17); x antigen family member la (xagel); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; tumor protein p53(p 53); a p53 mutant; a prostate-specific protein; survivin (surviving); a telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Rat sarcoma, Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; an inhibitor of melanoma apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyl-transferase V (NA 17); the paired box protein Pax-3(PAX 3); an androgen receptor; a cyclin Bl; a v-myc avian myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); tyrosinase-related protein 2 (TRP-2); cytochrome P450lB 1(CYPlB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site regulator siblings), squamous cell carcinoma antigen recognized by T cells 3(SART 3); the paired box protein Pax-5(PAX 5); the preproepisin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RUl); renal ubiquitin 2(RU 2); legumain; human papilloma virus E6(HPV E6); human papilloma virus E7(HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2(mut hsp 70-2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2(BST 2); EGF-like receptor-module mucin-like hormone-containing sample 2(EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); and immunoglobulin lambda-like polypeptide 1(IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1TROP2, GFR alpha 54, CDH17, CDH6, NYB 1, CDH19, CD200R, Slea (CA 19.9; sialyl Lewis antigen) fucosyl-GM R, PTK R, gpNMB, CDH R-CD 324, DLL R, CD276/B7H R, IL11R, IL13Ra R, CD 179R-IGLl R, TCR gamma-delta, NKG 2R, CD R (FCGR 2R), Tn Ag, Tim R-/HVCR R, GR 2R (GM-CSFR-alpha), TGF beta R R, Lews Ag, TCR-beta 1 chain TCR-beta 2, TCR-gamma-CMV-chain, genital-CMV-HCH chain, genital-HCH chain-HCH 72, FSH chain receptor (HALF-SLF-HCH 72), FSH 72, FSH-R, FSH-PGF-R, FSH 72, FSH receptor (SLF-R), FSH 72, FSH-R, FSH receptor 5, FSH 72, FSH-PGF-R, FSH-receptor, FSH 72, FSH-R, FSH-, KSHV-gH, influenza A lectin (HA), GAD, PDL1, Guanylate Cyclase C (GCC), desmoglein autoantibody 3(Dsg3), desmoglein autoantibody 1(Dsg1), HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IgE, CD99, Ras G12V, tissue factor 1(TF1), AFP, GPRC5D, sealing protein 18.2(CLD18A2 or N18A.2)), P-glycoprotein, STEAP1, Liv1, laminin-4, Cripto, gpA33, BST1/CD157, low chloride conductivity ion channels, and antigens recognized by TNT antibodies.

The term "antigen presenting cell" or "APC" refers to an immune system cell, such as a helper cell (e.g., B cell, dendritic cell, etc.), that displays on its surface a foreign antigen complexed with a Major Histocompatibility Complex (MHC). T cells can recognize these complexes using their T Cell Receptor (TCR). The APC processes and presents antigens to T cells.

The term "anti-infective effect" refers to a biological effect that can be manifested by various means including, but not limited to, for example, a reduction in the potency of an infectious agent, a reduction in the colony count of an infectious agent, an improvement in various physiological symptoms associated with an infectious condition. The "anti-infective effect" may also be manifested by the ability of peptides, polynucleotides, cells and antibodies to first prevent the development of cancer.

The term "anti-tumor effect" or "anti-cancer effect" refers to a biological effect that can be manifested in a variety of ways, including but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

As used herein, "affinity" means a measure that describes the strength of binding. In some cases, affinity depends on the stereochemical fit proximity between the binding agent and its target (e.g., between the antibody and the antigen (including the epitope specific to the binding domain)), the size of the contact area between them, and the distribution of charged and hydrophobic groups. Affinity generally refers to the "ability" of a binding agent to bind its target. There are many ways in the art for measuring "affinity". For example, methods for calculating the affinity of an antibody for an antigen are known in the art, including the use of affinity-calculating binding experiments. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interference, bipolar interference, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry. An exemplary method for determining binding affinity employs surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows for the analysis of real-time biospecific interactions by detecting changes in protein concentration within the Biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, n.j.), Uppsala, Sweden and Piscataway, Sweden, usa).

An "antigen binding domain" or "antigen binding module" or "antigen binding segment" refers to a polypeptide or peptide that binds to an antigen with a high degree of specificity due to its primary, secondary or tertiary sequence and or post-translational modification and/or charge. The antigen binding domain may be derived from different sources, such as antibodies, non-immunoglobulin binding proteins, ligands, or receptors. The present disclosure provides SIRs comprising an antigen binding domain that binds to one or more target antigens. The disclosure also provides SIRs comprising antigen binding domains not derived from antibodies.

"avidity" refers to the strength of interaction between a binder and a target (e.g., the strength of interaction between an antibody and its antigen target, a receptor, homolog thereof, etc.). The affinity may be weak or strong. Methods for calculating the affinity of an antibody for an antigen are known in the art, including the use of affinity-calculating binding experiments. Antibody activity in functional assays (e.g., flow cytometry assays) also reflects antibody affinity. Antibodies and affinities can be characterized phenotypically and compared using functional assays (e.g., flow cytometry assays).

The term "association constant (Ka)" is defined as the equilibrium constant for the association of a receptor with a ligand.

The term "autoantigen" refers to an endogenous antigen that stimulates the production of an autoimmune response, such as the production of autoantibodies. Autoantigens also include self-antigens or antigens from normal tissues that are targets of cell-mediated or antibody-mediated immune responses that can lead to the development of autoimmune diseases. Examples of autoantigens include, but are not limited to, desmoglein 1, desmoglein 3, and fragments thereof.

As used herein, a "beneficial outcome" may include, but is in no way limited to, lessening or lessening the severity of a disease condition, preventing the worsening of a disease condition, curing a disease condition, preventing the progression of a disease condition, reducing the chances of a patient developing a disease condition, and prolonging the life or life expectancy of a patient.

As used herein, the term "binding domain" or "antibody molecule" refers to a protein, such as an immunoglobulin chain or fragment thereof, that comprises at least one domain, such as an immunoglobulin variable domain sequence that can bind to a target with higher affinity than a non-specific domain. The term encompasses antibodies and antibody fragments. In another embodiment, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence in the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence in the plurality has binding specificity for a second epitope. In another embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies are specific for both antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope.

"binding to the same epitope as" means the ability of an antibody, scFv or other antigen binding domain to bind to a target antigen and which has the same epitope as the enumerated antibody, scFv or other antigen binding domain. As an example, the epitopes of the listed antibodies, scfvs or other binding agents and other antibodies can be determined using standard epitope mapping techniques. Epitope Mapping techniques well known in the art include Epitope Mapping Protocols in Methods in Molecular Biology [ Epitope Mapping protocol for Molecular Biology Methods ], volume 66 (Glenn E. Morris editor, 1996) Humana Press, Totowa, New Jersey [ Lemama Press, Totowawa, N.J. ]. For example, linear epitopes can be determined by, for example, simultaneously synthesizing a large number of peptides on a solid support, peptides corresponding to portions of a protein molecule, and reacting these peptides with an antibody while the peptides are still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. nos. 4,708,871; geysen et al, (1984) Proc. Natl. Acad. Sci. USA [ Proc of national academy of sciences USA ]8: 3998-4002; geysen et al, (1985) Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA ]82: 78-182; geysen et al, (1986) mol. lmmunol. [ molecular immunology ]23: 709-. Epitopes bound by the antigen binding domain of the SIR can also be determined by epitope binning assays. Epitope binning is a competitive immunoassay for characterizing and then sorting monoclonal antibody libraries against a target protein. Antibodies against similar targets were tested in a pairwise fashion against all other antibodies in the library to see if the antibodies block the binding of another antibody to an epitope. After each antibody had a characteristic curve created for all other antibodies in the library, a competitive blocking characteristic curve was created for each antibody in the library relative to the other antibodies. Closely related binning profiles indicate that antibodies have the same or closely related epitopes and are "binned" together. Similarly, conformational epitopes are readily identified by determining the spatial conformation of amino acids, such as by, for example, hydrogen/deuterium exchange, x-ray crystallography, and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenic and hydrophilic profiles, such as those calculated using the Omiga version 1.0 software program, available, for example, from Oxford Molecular Group (Oxford Molecular Group). The computer program was developed using the method of Hopp/Woods, Hopp et al, (1981) Proc. Natl. Acad. Sci USA [ Proc. Natl. Acad. Sci. USA ]78: 3824-; for determining the antigenic profile, and using the Kyte-Doolittle technique, Kyte et al, (1982) J.mol.Bioi. [ J.M. 157: 105-; for hydrophilicity mapping. To determine whether a selected monoclonal antibody directed against a target (e.g., CD19) binds to a unique epitope, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, il.). Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using CD19 extracellular domain coated ELISA plates. Biotinylated mAb binding can be detected with a streptavidin-alkaline phosphatase probe.

As used herein, the term "CDR" or "complementarity determining region" is intended to mean the discrete antigen combining sites found within the variable regions of both heavy and light chain polypeptides. These specific regions have been identified by Kabat et al, J.Bioi.Chern. [ J.Biol.Chem ]252: 6609-; kabat et al, U.S. dept.of Health and Human Services [ U.S. department of Health and public service ], "Sequences of proteins of immunological interest [ protein Sequences of immunological interest ]" (1991); chothia et al, J.mol.Bioi. [ J.M. J.biol. ]196:901-917 (1987); and MacCallum et al, J.mol.Bioi. [ J.Mol.Biol. ] 25262: 732-745(1996), wherein a repetitive part or subset is defined which includes amino acid residues when compared to each other. However, any definition is used to refer to the CDRs of an antibody or grafted antibody or variant thereof, and is intended to be within the scope of the terms as defined and used herein. As used herein, different CDRs of an antibody may also be defined by a combination of different definitions. For example, vHCDR1 may be defined based on Kabat and vHCDR2 may be defined based on Chothia. The amino acid residues encompassing the CDRs as defined in each of the above-cited references are as follows:

CDR definition

(residue numbers correspond to identifying references).

The SEQ IDs of the CDRs of the different vL and vH segments targeting the antigen binding domains of different antigens that make up the SIRs of the present disclosure are provided in table 5.

In some embodiments, reference to an antigen binding moiety that specifically binds to a target antigen (such as a Fab-like or Fv-like antigen binding moiety) means that the antigen binding moiety binds to the target antigen with (a) an affinity that is at least 10 (e.g., about 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or more) times its binding affinity to other molecules; or (b) K_dIs K in combination with other molecules_dNo more than 1/10 (e.g., 1/10, 1/20, 1/30, 1/40, 1/50, 1175, 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000 or less) times. Binding affinity can be determined by methods known in the art, such as ELISA, Fluorescence Activated Cell Sorting (FACS) sorting, or radioimmunoprecipitation assay (RIA). K_dCan be determined by methods known in the art, such as Surface Plasmon Resonance (SPR) using, for example, a Biacore instrument or kinetic exclusion assay (KinExA) using, for example, a Sapidyne instrument.

The terms "cancer" and "cancerous" refer to or describe the physiological condition of a mammal that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, B-cell lymphoma (hodgkin's lymphoma and/or non-hodgkin's lymphoma), T-cell lymphoma, myeloma, myelodysplastic syndrome, skin cancer, brain tumor, breast cancer, colon cancer, rectal cancer, esophageal cancer, anal cancer, cancer of unknown primary focus, endocrine cancer, testicular cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, cancer of the genitalia, thyroid cancer, renal cancer, tumors, melanoma, head and neck cancer, brain cancer (e.g., glioblastoma multiforme), prostate cancer (including, but not limited to, androgen-dependent prostate cancer and androgen-independent prostate cancer), and leukemia. Other cancers and cell proliferative disorders will be readily recognized in the art. The terms "tumor" and "cancer" are used interchangeably herein, for example, the terms include solid and liquid, such as a diffuse or circulating tumor. As used herein, the term "cancer" or "tumor" includes pre-malignant as well as malignant cancers and tumors.

"chemotherapeutic agents" are compounds known for use in cancer chemotherapy. Non-limiting examples of chemotherapeutic agents may include alkylating agents such as thiotepa and thiotepaCyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzotepa (benzodopa), carboquone (carboquone), meturedpa (meturedpa), and uredepa (uredpa); ethyleneimine and methylmelamine, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; annonaceous acetogenins (especially bullatacin and bullatacin); camptothecin (including the synthetic analog topotecan); bryostatin; kelitin (callystatin); CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin (dolastatin); duocarmycins (duocarmycins) (including the synthetic analogs KW-2189 and CB1-TM 1); shogaol (eleutherobin); pancratistatin; sarcandra glabra alcohol (sarcodictyin); spongistatin (spongistatin); nitrogen mustards, such as chlorambucil, chlorambucil (chlorethazine), cholorphamide (cholephosphamide),Estramustine, ifosfamide (ifosfamide), dichloromethyl diethylamine (mechlororethamine), dichloromethyl diethylamine oxide hydrochloride, melphalan (melphalan), novembichin (novembichin), benzene mustard cholesterol (phenesterine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil mustard; nitrosoureas such as carmustine (carmustine), chlorozotocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranimustine); antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin γ 1I and calicheamicin ω I1) (see, e.g., Agnew, chem. intl.ed. Engl. [ english edition international chemical reaction)]33:183-186 (1994)); daptomycin (dynemicin), including daptomycin a; diphosphonates (bisphosphates), such as clodronate; esperamicin (esperamicin); and neocarzinostatin (neocarzinostatin) chromophores and related chromendine antibiotic chromophores, aclacinomycin (acarinomycin), actinomycin (actinomycin), amphenicol (aurramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), karabixin (carabicin), carminomycin (carminomycin), carcinomycin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), ditorexin (detorbicin), 6-diazo-5-oxo-L-norleucine,Doxorubicin (doxorubicin) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolo-doxorubicin, and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcellomycin), mitomycin (such as mitomycin C), mycophenolic acid (mycophenolic acid), nogomycin (nogalamycin), olivomycin (oleamycin), pelomycin (peplomycin), pofiomycin (potfiromycin), puromycin (puromycin), apramycin (queamycin), rodobicin (rodorubicin), streptonigrosins (stremogrin), streptozomycins (strezocins), tubercidines (tretinomycins), and tubercidins (tretinomycins)ubercidin), ubenimex (ubenimex), zinostatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thiamine, thioguanine; pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trostane (trilostane); folic acid supplements such as folinic acid (frilic acid); acegulonone (aceglaone); an aldophosphamide glycoside (aldophosphamideglycoside); (ii) aminolevulinic acid; eniluracil (eniluracil); amsacrine (amsacrine); doubly-branched betuzucil; bisantrene; edatrexate (edatraxate); deflazafamine (defofamine); dimecorsine (demecolcine); diazaquinone (diaziqutone); edenisol (elfornitine); ammonium etitanium acetate; epothilone (epothilone); etoglut (etoglucid); gallium nitrate; a hydroxyurea; lentinan (lentinan); lonidamine (lonidamine); maytansinoids such as maytansine and ansamitocin (ansamitocin)); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllic acid); 2-ethyl hydrazide; procarbazine (procarbazine);polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane (rizoxane); rhizomycin(rhizoxin); sizofuran (sizofiran); germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; trichothecene toxins (trichothecenes), especially T-2 toxin, veracurin a, bacillocin a and serpentine (anguidine); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; arabinoside ("Ara-C"); cyclophosphamide; taxanes (taxoids), e.g.Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.),Unhydrogenated castor oil albumin engineered nanoparticle paclitaxel formulations (American Pharmaceutical Partners, Schaumberg, Ill.) anddocetaxel (doxetaxel) (Rhone-Poulenc Rorer, Antony, France); chlorambucil (chlorenbucil);gemcitabine (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin (oxaplatin), and carboplatin; vinblastine (vinblastine); platinum; etoposide (VP-16); ifosfamide; mitoxantrone (mitoxantrone); vincristine (vincristine); catharanthine (NAVELBINE); vinorelbine (vinorelbine); norfloxacin (novantrone); teniposide (teniposide); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin; (xiloda); ibandronate (ibandronate); irinotecan (CPT-11) (therapeutic regimens that include irinotecan with 5-FU and folinic acid); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); a retinoid and a vitamin E in an amount sufficient to inhibit the formation of a retinoid,such as retinoic acid; capecitabine (capecitabine); combretastatin (combretastatin); leucovorin (LV); oxaliplatin, including oxaliplatin treatment regimen (FOLFOX); lapatinib (typerb); PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib) to reduce cell proliferation) And an inhibitor of VEGF-a, and a pharmaceutically acceptable salt, acid, or derivative of any of the foregoing, or a combination thereof.

"chimeric antigen receptors" (CARs) are artificial T cell receptors that are expected to be used as cancer therapies using a technique known as adoptive cell transfer. The essential antigen binding, signaling, and stimulatory functions of the complex have been reduced to a single polypeptide chain by genetic recombination methods, which are commonly referred to as Chimeric Antigen Receptors (CARs). See, e.g., Eshhar, U.S. patent No. 7,741,465; eshhar, U.S. patent application publication No. 2012/0093842. The CAR is specifically constructed to stimulate T cell activation and proliferation in response to the specific antigen to which the CAR binds. The term "chimeric antigen receptor" or alternatively "CAR" refers to a group of polypeptides, typically two polypeptides in the simplest embodiment, which when expressed in an immune effector cell, provide the cell with specificity for a target cell (typically for a cancer cell) and provide intracellular signal generation. In some embodiments, the CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule and/or a co-stimulatory molecule. In some aspects, the set of polypeptides are contiguous to each other. In one aspect, the stimulatory molecule is a zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one aspect, the co-stimulatory molecule is selected from the co-stimulatory molecules described herein, such as 4-1BB (i.e., CD137), CD27, and/or CD 28. In one aspect, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane. Typically "CAR-T cells" are used, which have been essentially engineered to contain T cells of a chimeric antigen receptor. Thus, T lymphocytes carrying such CARs are commonly referred to as CAR-T lymphocytes.

"codon optimization" or "control of species codon bias" refers to the preferred codon usage of a particular host cell. As will be appreciated by those of ordinary skill in the art, it is advantageous to modify the coding sequence to enhance its expression in a particular host. There are 64 possible codons for gene codon redundancy, but most organisms generally use a subset of these codons. Codons that are most frequently used in a species are called optimal codons, and those that are less frequently used are classified as rare or low-use codons.

Optimized coding sequences containing codons preferred by particular prokaryotic or eukaryotic cells can be prepared (see also Murray et al (1989) nucleic acids Res. [ nucleic acid research ]17:477-508), for example to increase the rate of translation or to produce recombinant RNA transcripts with desired properties, such as longer half-lives, compared to transcripts produced from non-optimal sequences. Translation stop codons can also be modified to reflect host preferences. One skilled in the art will recognize that due to the degenerate nature of the genetic code, a number of DNA compounds differing in nucleotide sequence may be used to encode a given polypeptide of the disclosure.

As used herein, "co-expression" refers to the expression of two or more genes. A gene can be, for example, a nucleic acid encoding a single protein or a chimeric protein as a single polypeptide chain. The SIRs described herein can be encoded by a single polynucleotide chain and synthesized as a single polypeptide chain that is subsequently cleaved into different polypeptides, each representing a different functional unit. In some embodiments, where the SIR consists of two or more functional polypeptide units, the functional units cannot be co-expressed using one or more polynucleotide strands. In another embodiment, the different polynucleotide strands are linked by a nucleic acid sequence encoding a cleavable linker (e.g., T2A, F2A, P2A, E2A, etc.). In another embodiment, a Ser-Gly-Ser-Gly (SGSG) motif (SEQ ID NO:3065) is also added upstream of the cleavable linker sequence to enhance the efficiency of cleavage. A potential disadvantage of a cleavable linker is the possibility that a small 2A tag located at the terminus of the N-terminal protein may affect protein function or cause protein antigenicity. To overcome this drawback, in some embodiments, a furin cleavage site (RAKR) (SEQ ID NO:3066) is added upstream of the SGSG motif to facilitate cleavage of the residual 2A peptide after translation. Polynucleotides encoding different units of SIR may be linked by IRES (internal ribosome entry site) sequences. Alternatively, different functional units of the SIR are encoded by two different polynucleotides which are not linked via a linker but are encoded by, for example, two different vectors. The nucleic acid sequences of the cleavable linker and furin cleavage site are provided in SEQ ID NO 780 to SEQ ID NO 790.

"conservative substitutions" or "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics or function of the encoded protein. For example, "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics or function of a TCR constant chain, antibody fragment, or non-immunoglobulin binding domain. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the TCR constant chains, antibodies or antibody fragments, non-immunoglobulin binding domains or other proteins or polypeptides of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PPCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the SIRs of the present disclosure can be replaced with other amino acid residues from the same side chain family, and the altered SIRs can be tested using the binding and/or functional assays described herein.

The term "constant region of a T cell receptor alpha" or "constant chain of a T cell receptor alpha" or "TCR alpha" or "C alpha" is defined as a protein from a non-human species, e.g., mouse, rodent, monkey, ape, etc., provided as SEQ ID NO:3010 or equivalent residues (i.e., homologs). The disclosure also provides certain mutations of TCR alpha polypeptides. For example, mutation sites in ca demonstrating increased expression of the Synthetic Immune Receptors (SIRs) of the present disclosure and reduced mismatches are located at positions 91, 92, 93 and 94 of SEQ ID NO 3010. SIR with a Thr 48Cys (T48C) mutation in C α and a Ser-57-Cys (S57C) mutation in either the C β 1 or C β 2 chains (described more fully elsewhere herein) forms an additional disulfide bond between the two TCR constant chains (α and β). This in turn leads to reduced mismatch and enhanced functionality with endogenous TCR chains in immune cells. Similarly, SIRs with Ser 61Arg (S61R) mutations in C α and Arg 79Gly (R79G) mutations in C β 1 or C β 2 chains (described more fully elsewhere herein) resulted in reduced mismatch and enhanced functionality with endogenous TCR chains due to paired "knob and hole" design. The present disclosure provides a C α polypeptide having one or more or all mutations according to table 1 below.

The human genome encodes two highly homologous TCR β constant chains; TCR β 1(TCR β 1 or TCRb1 or c β 1) and TCR β 2(TCR β 2 or TCRb2 or c β 2). The SIR of the present disclosure may comprise either of these two chains. Similarly, TCR β 1 or TCR β 2 chains of other mammalian species may be used in the methods of making SIRs of the present disclosure.

The term "constant chain of T cell receptor β 1" or "constant region of T cell receptor β 1" (TCR-. beta.1 or TCR. beta.1 or TCRb1 or hTCR. beta. -1 or C.beta.1) is defined as a protein from a non-human species, such as mouse, rodent, monkey, ape, etc., provided as SEQ ID NO:3024 or equivalent residues (i.e., homologues).

The term "constant chain of T cell receptor β 2" or "constant region of T cell receptor β 2" (TCR- β 2 or TCR β 2 or TCRb2 or C β 2) is defined as a protein from a non-human species, e.g. mouse, rodent, monkey, ape etc. provided as SEQ ID NO:3025 or equivalent residues (i.e. homologues).

The term "constant chain of T cell receptor beta" or "constant region of T cell receptor beta" (TCR-. beta.or TCR. beta. or TCRb or C.beta.) is defined as a protein from a non-human species, e.g., mouse, rodent, monkey, ape, etc., provided as SEQ ID NO:3024 or SEQ ID NO:3025 or equivalent residues (i.e., homologues).

The protein sequences of both C.beta.2 (SEQ ID NO:3025) and C.beta.1 (SEQ ID NO:3024) are known. Differences between the sequences of C β 2 and β 1 are readily identified by aligning the sequences using techniques typical and common in the art. The disclosure also provides certain mutations of TCR β. For example, provided herein are mutation sites in C β that demonstrate increased expression of Synthetic Immune Receptors (SIRs) and decreased mismatch with endogenous TCR α chains. These mutation sites in C β 1 and C β 2 are located at positions 18, 22, 57, 79133, 136 and 139 of SEQ ID NOs 3025 and 3024 and are summarized in tables 2 and 3 below. The mutation sites in C β 1 and C β 2 are identical in their positions. The only difference between the two sequences is the mutation at position 136. At this position, glutamic acid (E) is present in C β 2, while valine is present in C β 1.

The term "constant chain of preTCRa" (preTCR-a or preTCR a or preTCRa or preC a) or "constant region of preTCRa" is defined as a protein from a non-human species, e.g.mouse, rodent, monkey, ape etc. provided as SEQ ID NO:3046 or SEQ ID NO:3047 or equivalent residues (i.e.homologues).

The term "constant chain of preTCRa-Del 48" (preTCR- α -Del48 or preTCR α -Del48 or preTCRa-Del48 or preC α -Del48) or "constant region of preTCRa-Del 48" is defined as a protein from a non-human species, e.g., mouse, rodent, monkey, ape, etc., provided as SEQ ID NO:3048 or an equivalent residue (i.e., homolog).

The term "constant chain of TCR- γ" or "constant region of TCR- γ" (TCR- γ or TCR γ or TCRg or TCR- γ 1 or TCR γ 1 or TCRg1 or Cγ) is defined as a protein from a non-human species, such as mouse, rodent, monkey, ape etc. provided as SEQ ID NO:3049 or equivalent residues (i.e. homologues).

The term "constant chain of TCR-delta" or "constant region of TCR-delta" (TCR-delta or TCR delta or TCRd or C delta) is defined as a protein from a non-human species, e.g., mouse, rodent, monkey, ape, etc., provided as SEQ ID NO:3052 or equivalent residues (i.e., homologues).

It will be appreciated that the proteins may have identity or homology to each other and retain similar or identical functions. The present disclosure includes TCR constant regions that are 85%, 90%, 95%, 97%, 98%, 98.5%, 99%, or 99.9% identical to any of the sequences described herein while retaining biological activity.

Accordingly, the present disclosure provides a T cell receptor constant chain having a sequence selected from the group consisting of: (a) an amino acid sequence at least 98% identical to SEQ ID NO 3010 and which may have one or more mutations at positions 61, 91, 92, 93 and/or 94; (b) an amino acid sequence that is at least 98% identical to SEQ ID NO:3024 and that may have one or more mutations at positions 18, 22, 57, 79, 133, 136 and/or 139; (c) an amino acid sequence which is at least 98% identical to SEQ ID NO:3025 and which may have one or more mutations at positions 18, 22, 57, 79, 133, 136 and/or 139; (d) an amino acid sequence at least 98% identical to SEQ ID NO 3046 or 3047; (e) an amino acid sequence at least 98% identical to SEQ ID NO 3048; (f) an amino acid sequence at least 98% identical to SEQ ID NO 3049; and (g) an amino acid sequence at least 98% identical to SEQ ID NO 3051 or 3052. (a) The T cell receptor constant chain of any one of (a) - (g) retains at least one biological activity of the wild type T cell receptor constant chain with which it has identity or homology.

The term "co-stimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response (such as, but not limited to, proliferation) of the T cell. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which contribute to a highly efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CD8, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), and 4-1BB (CD 137). Other examples of such co-stimulatory molecules include CD8, ICAM-1, GITR, BAFFR, HVEM (LIGHT TR), SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 α, CD8 β, IL2R β, IL2R γ, IL7R α, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11B, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IP0-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83, and a ligand that specifically binds to CD 83. The costimulatory intracellular signaling domain may be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activated NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CS8, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind to CD83, and the like. The intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

The term "TCR" refers to a wild-type TCR nucleic acid encoding sequence and the corresponding wild-type TCR protein linked to an antigen-binding domain. In some embodiments, a tcr is used and is subject to reference. For example, a TCR with a CD19 binding domain and a CD19-SIR (comprising a mutant TCR chain and a CD19 binding domain) will have different expression and/or different binding affinity to the target antigen.

The term "degenerative condition" refers to a disease that is based on the result of a continuous process of degenerative cellular changes affecting tissues or organs, which will gradually worsen over time, whether due to normal physical wear or due to lifestyle choices such as exercise or eating habits. Exemplary degenerative diseases include alzheimer's disease, peroneal muscular atrophy (Charcot-Marie-Tooth disease), Creutzfeldt-Jakob disease, friedrich's ataxia, diabetes (type II), and atherosclerosis.

"derived from" (as that term is used herein) refers to the relationship between a first molecule and a second molecule. It generally refers to the structural similarity between a first molecule and a second molecule and does not imply or include limitations on the process or source of the first molecule from the second molecule. For example, in the case of an antigen binding domain derived from the CD3 ζ molecule, the antigen binding domain retains sufficient antibody structure such that it has the desired function, i.e., ability to bind to an antigen. It does not imply or include limitations on the specific process of producing an antibody, for example, it does not mean that in order to provide an antigen binding domain, it is necessary to start with an antibody sequence and delete unwanted sequences, or impose mutations to reach the antigen binding domain.

The phrase "a disease associated with expression of a target antigen" or "a disease-associated antigen as described herein" includes, but is not limited to, a disease associated with expression of a target antigen as described herein or a condition associated with a cell expressing a target antigen as described herein, including, for example, a proliferative disease (such as a cancer or malignancy) or a precancerous condition (such as myelodysplasia, myelodysplastic syndrome, or pre-leukemia); or a non-cancer related indication associated with a cell expressing a target antigen as described herein. In one aspect, the cancer associated with expression of a tumor antigen as described herein is a hematologic cancer. In one aspect, the cancer associated with expression of a tumor antigen as described herein is a solid cancer. Other diseases associated with expression of a tumor antigen described herein include, but are not limited to, atypical and/or non-classical cancers, malignancies, pre-cancerous conditions, or proliferative diseases associated with expression of a tumor antigen as described herein. Non-cancer related indications associated with expression of target antigens as described herein include, but are not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory disorders (allergy and asthma), and transplantation. In some embodiments, the cells expressing the target antigen express or at any time express mRNA encoding the target antigen. In another embodiment, the cell expressing the target antigen produces a target antigen protein (e.g., wild-type or mutant), and the target antigen protein may be present at normal levels or at reduced levels. In another embodiment, a cell expressing a target antigen produces detectable levels of the target antigen protein at one time point and subsequently produces substantially no detectable target antigen protein.

As used herein, "disease targeted by genetically modified cells" includes targeting any cell involved in any disease in any way by a genetically modified cell of an abrasive disease or a target tissue or cell type, whether the genetically modified cell is targeted to diseased cells or to healthy cells to achieve a therapeutically beneficial result.

The term "dissociation constant (Kd)" is defined as the equilibrium constant for dissociation of receptor-ligand interactions.

As used herein, a "different set of SIRs" or a "different set of synthetic immune receptors"and" refers to a plurality of SIRs having the same binding domain linked to a collection of different T cell receptor constant chains, wherein each construct comprising a binding domain and a different T cell constant chain provides a different range of binding to a target antigen and/or altered expression levels. For example, the binding affinity of a binding domain to its target is altered according to the mutational composition of the constant domains (e.g., mutant TCRa + TCRb). In some embodiments, SIRs (single chain or heterodimers) of the present disclosure comprise a binding affinity that is greater than the binding affinity of a wild-type TCR (e.g., a TCR) to the same binding domain. In one embodiment, the expression level of SIR is at least 1.25-fold to about 10000-fold higher than the TCR (and any value in between), where the SIR and the TCR differ only by a mutation in the TCR domain. In another embodiment, the SIR has a binding affinity for the target that is at least 1.5-fold to about 10000-fold higher than the binding affinity of a tcr with a binding domain to the same antigen. In another embodiment, the SIR has a binding affinity that is higher than the binding affinity of the tcr for the same antigen, but less than a Chimeric Antigen Receptor (CAR) with the same binding domain. In some embodiments, effector cells expressing SIR bind to the target antigen at least 1.25-fold greater than the binding of corresponding effector cells expressing tcr, but 100000-fold less than the corresponding tcr. In some embodiments, the antigen binding domain has about 10^-4M to 10^-8Dissociation constant (K) between M_DReflecting its binding affinity). In some embodiments, the antigen binding domain binds to one or more antigens described above. In some embodiments, the antigen binding domain has a K for the target antigen_DIs at about 10^-4M to 10^-8M, e.g. between about 10^-5M to 10^-7M, e.g. between about 10^-5M to 10^-6M is greater than or equal to the total weight of the composition. In one embodiment, the antigen binding domain has at least five, 10, 20, 30, 50, 100 or 1000 times less binding affinity than a reference antibody. In one embodiment, the encoded antigen binding domain has a binding affinity that is at least 5-fold less than a reference antibody. In some embodiments, the reference antibody is an antigenAn antibody from which the binding domain is derived.

As used herein, an "epitope" is defined as an antigenic portion capable of eliciting an immune response or binding to an antibody or antibody fragment. An epitope may be a protein sequence or a subsequence.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

The term "Functional Polypeptide Unit (FPU)" of the SIR refers to a polypeptide comprising an amino-terminal signal sequence functionally linked to the TCR constant chain. In some embodiments, the FPU contains an antigen binding domain located between the signal sequence and the TCR constant chains. In other embodiments, the FPU lacks an antigen binding domain located between the signal sequence and the TCR constant chains. The FPU may contain additional sequences, such as linkers. As an example, the FPU may contain a MYC2-tag (eqkliseedlgsg) linker between the antigen binding domain and the TCR constant chain. FPUs can also contain a cleavable linker (e.g., P2A, F2A), Ser-Gly (SGSG) linker, and a furin cleavage site (RAKR).

The term "functional part" when used with reference to SIR refers to any part or fragment of the SIR that retains the biological activity of the SIR (parent SIR) of which it is a part. Functional proteins include, for example, those portions of the SIR that retain the ability to recognize a target cell or detect, treat or prevent a disease to a similar extent, to the same extent, or to a greater extent, as the parent SIR. The functional portion may comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent SIR, with reference to the parent SIR.

As used herein, "genetically modified cell," "redirected cell," "genetically engineered cell," or "modified cell" refers to a cell that has been modified to express a Synthetic Immune Receptor (SIR) and may optionally comprise a chimeric antigen receptor. For example, a genetically modified T lymphocyte that expresses SIR is a genetically modified cell.

The term "immune disorder" refers to a disease characterized by a disturbance of the immune system. Autoimmune diseases are pathologies caused by an abnormal immune response to a normal body part. There are at least 80 types of autoimmune disease.

An "immune effector cell" (as that term is used herein) refers to a cell that participates in an immune response (e.g., promotes an immune effector response). Examples of immune effector cells include T cells, such as α/β T cells and γ/δ T cells, B cells, Natural Killer (NK) cells, natural killer T (nkt) cells, mast cells, and bone marrow-derived phagocytes.

The term "immune effector function or immune effector response" (as that term is used herein) refers to a function or response (e.g., of an immune effector cell) that enhances or facilitates immune attack on a target cell. For example, an immune effector function or response refers to the property of a T cell or NK cell to promote killing or growth inhibition or proliferation of a target cell. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

The term "intracellular signaling domain" (as that term is used herein) refers to the intracellular signaling portion of a molecule. The intracellular signaling domain generates a signal that promotes the immune effector function of the TCR-containing cell. Examples of immune effector functions include cytolytic activity and helper activity (including secretion of cytokines). The TCR α/β/γ/δ chains do not have their own intracellular signaling domains, but transmit signals by associating with other chains of the TCR signaling complex (e.g., CD3z, CD3e, CD3d, and CD3g) that have signaling domains.

In another embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent simulation. In another embodiment, the intracellular signaling domain may comprise a co-stimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signaling or antigen-independent stimulation. For example, the primary intracellular signaling domain may comprise the cytoplasmic sequence of CD3z, and the costimulatory intracellular signaling domain may comprise the cytoplasmic sequence from a co-receptor or a co-stimulatory molecule such as CD28 or 41 BB.

The primary intracellular signaling domain may comprise a signaling motif referred to as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3 ζ, common FcR γ (FCER1G), fcyriia, FcR β (fcepsilonr 1b), CD3 γ, CD3 δ, CD3 ε, CD79a, CD79b, DAP10, and DAP 12.

As used herein, the term "linker" (also a "linker domain" or "linker region") refers to an oligopeptide or polypeptide that joins two or more domains or regions of the SIRs disclosed herein together. The linker may be anywhere from 1 to 500 amino acids in length. In some embodiments, a "linker" is cleavable or non-cleavable. The term "linker" as used herein means a non-cleavable linker unless otherwise specified. Exemplary non-cleavable linkers that can be used to generate SIRs are provided in table 6D. The non-cleavable linker may be composed of flexible residues that allow free movement of adjacent protein domains relative to each other. Non-limiting examples of such residues include glycine and serine. In some embodiments, the linker comprises a non-flexible residue. Exemplary embodiments of linkers with inflexible linkers are EAAAK (SEQ ID NO:18933), E-coil (SEQ ID NO:18931), K-coil (SEQ ID NO:18932) or PG4SP (18929). The SIR targeting CD19 and containing the antigen binding domain derived from FMC63 antibody showed about 1.5 fold higher binding affinity for the target antigen when constructed with a non-flexible linker (e.g., EAAAK (SEQ ID NO:18933), E-coil (SEQ ID NO:18931), K-coil (SEQ ID NO:18932) or PG4SP (18929)) between the antigen binding domain and the TCR constant chain compared to the SIR without the linker. Thus, in some embodiments, a non-flexible linker (e.g., EAAAK (SEQ ID NO:18933), E-coil (SEQ ID NO:18931), K-coil (SEQ ID NO:18932), or PG4SP (18929) represents a preferred linker for construction of a SIR.in other embodiments, the antigen binding domain of a double-stranded SIR shares a similar length with two linkers that are TCR constant links together.in other embodiments, the lengths of the two linkers that are holding the antigen binding domain of a double-stranded SIR and the TCR constant links together differ by NO more than 20 amino acids, typically NO more than 10 amino acids, preferably NO more than 5 amino acids, more preferably NO more than 2 amino acids v2(SEQ ID NO: 18923). In some embodiments, the two linkers that hold the antigen binding domain of the double-stranded SIR and the TCR constant chain together are PG4SP (DNA SEQ ID NO: 18922; PRT SEQ ID NO:18929) and PG4SP-v2(DNA SEQ ID NO: 18923; PRT SEQ ID NO:18929 or 18930). In some embodiments, the two linkers that hold the antigen binding domain of the double-stranded SIR and the TCR constant chain together are EAAAK (SEQ ID NO: 18926; PRT SEQ ID NO:18933 and 18934) and EAAAK-v 2(DNA SEQ ID NO: 18927). In some embodiments, the two linkers that hold the antigen binding domain of the double-stranded SIR and the TCR constant chain together are E-coil (DNA SEQ ID NO:18924) and K-coil (DNA SEQ ID NO: 18925). In some embodiments, the linker may comprise an epitope tag. In some embodiments, the epitope tag is selected from the group consisting of a MYC tag, a V5 tag, an AcV5 tag, StreptagII, a FLAG tag, or HA. In some embodiments, the non-cleavable linker is of a length sufficient to ensure that two adjacent domains do not sterically interfere with each other. In one embodiment of the disclosure, three amino acid residues (Gly-Ser-Gly) are added to the carboxy terminus of a linker (e.g., Myc tag or V5 tag) that is located between the antigen binding domain of the SIR and the TCR constant chain. In certain embodiments, the linker may carry additional sequences, such as restriction enzyme sites. The nucleic acid sequences of several exemplary linkers are provided in SEQ ID NO 701 to SEQ ID NO 725 and the amino acid sequences of several exemplary linkers are provided in SEQ ID NO 2981 to SEQ ID NO 3003.

The term "flexible polypeptide linker" as used herein refers to a peptide linker composed of amino acid (such as glycine and/or serine) residues used alone or in combination to link polypeptide chains together (e.g., to link a variable heavy chain region and a variable hydrogen chain region together). In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)_nWherein n is a positive integer equal to or greater than 1. For example, n is 1, n is 2, n is 3, n is 4, n is 5, and n is 6, n is 7, n is 8, n is 9, and n is 10. In one embodiment, the flexible polypeptide linker includes, but is not limited to (Gly)₄Ser)₄Or (Gly)₄Ser)₃(SEQ ID NO: 2500). In another embodiment, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NOS: 2501 and 2502). The linkers described in WO2012/138475, which is incorporated herein by reference, are also included within the scope of the present disclosure.

Non-limiting examples of cleavable linkers include a 2A linker (e.g., T2A), a picornavirus 2A-like linker, a CHYSEL sequence of porcine teschovirus (P2A), a Thosea asigna virus (T2A), a 2A-like linker, or functional equivalents thereof, and combinations thereof. In some embodiments, the linker sequence may comprise a motif that causes cleavage between 2A glycine and 2B proline (see, e.g., the T2A sequence, SEQ ID NO:3061, C-terminal Gly-Pro). The nucleic acid sequences of several exemplary cleavable linkers are provided in SEQ ID NO:780 to SEQ ID NO:785 and the amino acid sequences of several exemplary linkers are provided in SEQ ID NO:3060 to SEQ ID NO: 3064. Other cleavable linkers that may be used herein will be readily apparent to those skilled in the art.

The term "lentivirus" refers to a genus of the family retroviridae. Lentivirus is unique in the family of retroviruses in that it is capable of infecting non-dividing cells; they can deliver significant amounts of genetic information into the DNA of host cells, and are therefore one of the most efficient methods of gene delivery vehicles. HIV, SIV and FIV are all examples of lentiviruses.

The term "lentiviral vector" refers to a vector derived from at least a portion of the lentiviral genome, and specifically includes, for example, Milone et al, mol]17(8) 1453-. Other examples of lentiviral vectors that may be used in the clinic include, but are not limited to, for example, those from Oxford biomedical corporation (Oxford biomedical)Gene delivery technology, LENTIMAX from Lentigen^TMVector systems, and the like. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art. Other examples of lentiviral vectors are pLENTI-EF1 alpha (SEQ ID NO:870) and pLENTI-EF1 alpha-DWPRE (SEQ ID NO: 871).

As used herein, "mammal" refers to any member of the mammalian family, including, but not limited to, humans and non-human primates, such as orangutans and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs, and the like. The term does not indicate a particular age or gender. Thus, adult and infant subjects, as well as male or female fetuses, are intended to be included within the scope of this term.

As used herein, "non-naturally occurring TCR antigen binding domain" refers to a binding domain that is operably linked to a TCR constant region that is chimeric and non-naturally occurring relative to a TCR that occurs in nature. In other words, a non-naturally occurring TCR antigen-binding domain is "engineered" using recombinant molecular biology techniques to be operably linked to a TCR and, in addition, the antigen-binding domain is derived or derived from a molecule other than a TCR found in nature. Antigen binding domains that differ from TCRs found in nature include antibody vH and vL fragments, humanized antibody fragments, chimeric antibody fragments, receptor ligands, and the like.

The term "operably linked" refers to a functional connection or association between a first component and a second component such that each component may be functional. For example, operably linked includes an association between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. In the context of two polypeptides that are operably linked, the first polypeptide functions in such a way that it will be independent of any linkage and the second polypeptide functions in the absence of a linkage between the two.

In the context of two or more nucleic acid or polypeptide sequences, "percent identity" refers to two or more identical sequences. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are identical (e.g., 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, over a specified region, or, when unspecified, over the entire sequence) when compared and aligned for maximum correspondence over a comparison window (or specified regions measured using one of the following sequence comparison algorithms or by manual calibration and visual inspection). Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm will then calculate the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. Methods of sequence alignment for comparison are well known in the art. For example, the homology alignment algorithm of Needleman and Wunsch, (1970) adv.appl.Math. [ applied mathematical progression ]2:482c, the homology alignment algorithm of Needleman and Wunsch, (1970) J.mol.biol. [ journal of Molecular Biology ]48:443, the similarity method of Pearson and Lipman, (1988) Proc.nat' l.Acad.Sci.USA [ Proc. Acad. Sci.USA ]85:2444, the computerized alignment of GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package of Madison, Mass., Inc. No. 575 genetic Computer Group,575Science Dr., Madison, Wis, etc., or the alignment of sequences of the best Molecular sin, Biological for Molecular comparison by manual alignment and visual inspection (see, Brent et al., (see: ceramics, et al.).

Two examples of algorithms that can be used to determine percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al, (1977) Nuc. acids Res. [ nucleic acid research ]25: 3389-; and Altschul et al, (1990) J.. mol.. Bioi. [ J. Mobiol. ]215: 403-. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

The percentage of identity between two amino acid sequences can also be determined using the algorithm of e.meyers and w.miller, (1988) comput.appl.biosci. [ computer application in bioscience ]4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a table of PAM120 weight residues, a gap length penalty of 12 and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970) J.mol.biol. [ J.M. J.biol. ]48:444-453 algorithm in the GAP program already incorporated into the GCG software package (available at www.gcg.com), using either the bloom 62 matrix or the P AM250 matrix, as well as the GAP weights of 16, 14, 12, 10, 8, 6, or 4 and the length weights of 1,2,3,4, 5, or 6.

The terms "polynucleotide", "nucleic acid" or "recombinant nucleic acid" refer to a polymer of nucleotides, such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA).

The two terms "protein" or "polypeptide" are used interchangeably herein and comprise one or more chains of chemical building blocks called amino acids, which are linked together by chemical bonds called peptide bonds.

The term "retroviral vector" refers to a vector derived from at least a portion of a retroviral genome. Examples of retroviral vectors include MSCVneo, MSCV-pac (or MSCV-puro), MSCV-hygro, available from Addgene or Clontech. Other examples of retroviral vectors are MSCV-Bgl2-AvrII-Bam-EcoR1-Xho-BstB1-Mlu-Sal-ClaI.I03(SEQ ID NO: 872).

The term "sleeping beauty transposon" or "sleeping beauty transposon vector" refers to a vector derived from at least a portion of the sleeping beauty transposon genome. An example of a sleeping beauty transposon vector is pSBbi-Pur (SEQ ID NO: 874). Other examples of sleeping beauty transposon vectors encoding SIR are provided in SEQ ID NO 875 and SEQ ID NO 876.

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are contiguously linked, e.g., via a synthetic linker (e.g., a short flexible polypeptide linker), and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it was derived. Unless otherwise specified, as used herein, a scFv can have VL and VH variable regions in any order, e.g., relative to the N-terminus and C-terminus of a polypeptide, the scFv can comprise a VL-linker-VH or can comprise a VH-linker-VL. In the present invention, scFv is also described as vL-Gly-Ser-linker-vH. For example, FMC 63-vL-Gly-Ser-linker-FMC 63-vH refers to an scFv containing fragments of vL and vH of the FMC63 monoclonal antibody linked via a linker consisting of Gly and Ser. The amino acid sequence of an exemplary Gly-Ser linker is provided in SEQ ID NO: 2500. Alternatively, the scFv is described as (vL + vH). For example, FMC6- (vL + vH) refers to an scFv comprising a vL and vH fragment of FMC63 antibody linked via a linker, wherein the vL fragment is located at the N-terminus.

The term "signaling domain" refers to a functional part of a protein that transmits information within a cell to modulate cellular activity via a defined signaling pathway, either by generating second messengers or by acting as an effector in response to such messengers.

The term "synthetic immune receptor" or alternatively "SIR" refers to a group of polypeptides, typically two polypeptides in some embodiments, which when expressed in an effector cell, provides the cell with specificity for a target cell (typically for a cancer cell) and provides intracellular signal generation. In a typical embodiment, the SIR comprises one or more antigen binding domains (e.g., antibodies or antibody fragments, ligands or receptors) that bind to an antigen as described herein and are linked to one or more T cell receptor constant chains via an optional linker. In some embodiments, the set of polypeptides are contiguous with each other. In some embodiments, the SIR comprises two or more sets of two or more polypeptides. The polypeptides of each group SIR are contiguous with each other (functional polypeptide unit 1) but not with the other group (functional polypeptide unit 2). In some aspects, the T cell receptor constant chain (or region) of the SIR is selected from the group consisting of: human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or hTCR-alpha or hTCRa or hTCRb or calpha), human T cell receptor beta 1 (TCR-beta 1 or TCR beta 1 or TCRb1 or hTCR-beta 1 or hTCRbeta 1 or hTCRb1 or cbeta 1), human T cell receptor beta 2 (TCR-beta 2 or TCR beta 2 or TCRb2 or hTCCR-beta 2 or hTCRbeta 2 or hTCRb2 or cbeta 2, also known as TCR-beta, TCR beta or TCRb or cbeta), human Pre-T cell receptor alpha ((preTCR-alpha or preTCRa or preCalpha), human T cell receptor gamma (TCR-gamma or TCR gamma or TCRg or hTCRg or hTCCR gamma 1 or hTCgamma), or hTCRd or some embodiments, the TCR constant chain of the SIR is encoded by its wild-type nucleotide sequence, while in other aspects the TCR constant chain of the SIR is encoded by a non-wild-type nucleotide sequence. In some embodiments, the TCR constant chain of the SIR is encoded by its codon-optimized sequence. In some embodiments, the TCR constant chain of the SIR encodes a wild-type polypeptide sequence, while in other embodiments, the TCR constant chain of the SIR encodes a polypeptide carrying one or more mutations. In some embodiments, the TCR constant chain of the SIR is encoded by a codon-optimized sequence that carries one or more mutations. SIRs (such as those described herein) comprising an antigen binding domain (e.g., scFv or TCR) targeted to a particular tumor marker X are also referred to as X-SIRs or XSIR. For example, a SIR comprising an antigen binding domain targeted to CD19 is referred to as CD19-SIR or CD19 SIR. The TCR constant chains/domains of the SIRs can be derived from the same species that will ultimately use the SIRs. For example, for use in humans, it may be advantageous for the TCR invariant chain of the SIR to be derived from or comprise a human TCR invariant chain. However, in some cases it may be advantageous for the TCR constant chains to be derived from the same species that will eventually SIR, but modified to carry amino acid substitutions that enhance TCR constant chain expression. For example, for use in humans, it may be advantageous for the TCR invariant chain of the SIR to be derived from or comprise a human TCR invariant chain, but in which certain amino acids are replaced by corresponding amino acids from a murine TCR invariant chain. Such murine TCR constant chains provide increased SIR expression. The amino acid sequences of exemplary murine TCR constant chains are provided in SEQ ID NO:3017, SEQ ID NO:3033 to 3039 (see also tables 1-3). The SIR or functional portion thereof may comprise additional amino acids at the amino terminus or the carboxy terminus or both, which are not found in the amino acid sequence of the TCR or antigen binding domain that makes up the SIR. It is desirable that the additional amino acids do not interfere with the biological function of the SIR or functional moiety, such as identifying target cells, detecting cancer, treating or preventing cancer, and the like. More desirably, the additional amino acids enhance the biological activity as compared to the biological activity of the parent SIR.

The term "stimulation" refers to a primary response induced by the binding of a stimulating molecule (e.g., TCR/CD3 complex or CAR) to its cognate ligand (or target antigen in the case of a SIR) to mediate a signaling event, such as, but not limited to, signaling via TCR/CD 3. Stimulation may mediate altered expression of certain molecules.

The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides one or more cytoplasmic signaling sequences that modulate immune cell activation in a stimulatory manner with respect to at least some aspect of the immune cell signaling pathway. In one aspect, the signal is a primary signal initiated by, for example, binding of the TCR/CD3 complex to a peptide-loaded MHC molecule and resulting in the mediation of a T cell response including, but not limited to, proliferation, activation, differentiation, and the like. The primary cytoplasmic signaling sequence (also referred to as the "primary signaling domain") that functions in a stimulatory manner may contain signaling motifs referred to as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs-containing cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 ζ, common FcR γ (FCER1G), fcyriia, FcR β (fcepsilonr 1b), CD3 γ, CD3 δ, CD3 ε, CD79a, CD79b, DAP10, and DAP 12.

The term "subject" is intended to include living organisms (e.g., any domesticated mammal or human) in which an immune response can be elicited.

The terms "T cell" and "T lymphocyte" are used interchangeably and synonymously herein. Examples include, but are not limited to, naive T cells ("lymphocyte progenitors"), central memory T cells, effector memory T cells, memory T stem cells (T cells)_scm) iPSC-derived T cells, synthetic T cells, or combinations thereof.

The term "therapeutic effect" refers to a biological effect that can be exhibited by various means, including, but not limited to, e.g., reduction in tumor volume, reduction in the number of cancer cells, reduction in the number of metastases, increase in life expectancy, reduction in cancer cell proliferation, reduction in cancer cell survival, reduction in infectious agent titer, reduction in infectious agent colony count, improvement in various physiological symptoms associated with a disease condition. "therapeutic effect" can also be demonstrated by the ability of peptides, polynucleotides, cells and antibodies to first prevent disease development or prevent disease recurrence.

The term "transfer vector" refers to a composition of matter that comprises an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes an autonomously replicating plasmid or virus. The term should also be construed to also include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

As used herein, "Treatment" refers to therapeutic Treatment or preventative or prophylactic measures, wherein the object is to prevent or slow down (lessen) the targeted pathological condition, prevent the pathological condition, obtain or obtain a beneficial result, or reduce the chance of an individual developing the condition, even if Treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in which the condition is to be prevented.

As used herein, "tumor" refers to all neoplastic cell growth and proliferation (whether malignant or benign) as well as all precancerous and cancerous cells and tissues.

The term "zeta" or alternatively "zeta chain", "CD 3-zeta" or "TCR-zeta" is defined as being the protein provided under GenBan accession number BAG36664.1 or the equivalent residues from a non-human species such as mouse, rodent, monkey, ape etc., and the "zeta stimulating domain" or alternatively "CD 3-zeta stimulating domain" or "TCR-zeta stimulating domain" is defined as being the amino acid residues or functional derivatives thereof from the cytoplasmic domain of the zeta chain which are sufficient to functionally convey the initial signal necessary for T cell activation. In one aspect, the cytoplasmic domain of ζ comprises residues 52 to 54 of GenBank accession No. BAG36664.1 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) that are functional orthologs thereof. In one aspect, the "zeta stimulating domain" or "CD 3-zeta stimulating domain" is the sequence provided as SEQ ID NO: 18. In one aspect, the "zeta stimulating domain" or "CD 3-zeta stimulating domain" is the sequence provided as SEQ ID NO: 20.

As described above and elsewhere herein, each chain of the SIR of the present disclosure has the general structure: signal peptide- (binding domain) - (optional linker) - (T cell receptor constant region) - (optional accessory molecule). The T cell receptor constant region of the SIR comprises a fusion between the T cell receptor constant chain and the CD3 signaling chain with an optional co-stimulatory domain. Exemplary TCR β constant chain and CD3z fusion proteins are provided in SEQ ID NO 12401-12407. Exemplary TCR β constant chains and CD3z fusion proteins with additional costimulatory domains are provided in SEQ ID NO 12408-12409. Exemplary TCR α constant chain and CD3z fusion proteins are provided in SEQ ID NO 12422-12426. Exemplary TCR α constant chains and CD3z fusion proteins with additional costimulatory structures are provided in SEQ ID NO 12427-12428. Each "domain" or "segment" of the SIRs of the present disclosure is described below. One skilled in the art will recognize that various TCRs can be shuffled and combined with different binding domains and the like.

The present disclosure provides polynucleotide sequences encoding SIRs of the disclosure, SIR polypeptides, expression constructs, recombinantly engineered cells comprising SIRs or constructs of the disclosure, and methods of making and using such polypeptides, polynucleotides, and cells.

The present disclosure provides isolated nucleic acid molecules encoding a Synthetic Immune Receptor (SIR), wherein the SIR comprises one or more antigen binding domains (e.g., antibodies or antibody fragments, autoantigens, ligands, or receptors) that bind to an antigen as described herein and are linked to one or more T cell receptor constant chains.

In some embodiments, the SIR may comprise or consist of a single antigen binding domain linked to a single T cell receptor constant chain. In some embodiments, the SIR may comprise or consist of more than one antigen binding fragment (e.g., a vL and vH fragment or two vHH fragments) linked via a linker and in turn linked to a single T cell receptor constant chain (e.g., SEQ ID NO:1169, SEQ ID NO:1182, SEQ ID NO: 10497-10508; 10524-10538). In another embodiment, the SIR comprises or consists of two antigen binding domains each linked in-frame to a separate T cell receptor constant chain (e.g., SEQ ID NO: 1200). For example, antigen binding domain 1 is linked to the constant chain of TCR α (ca) to construct functional unit 1 and antigen binding domain 2 is linked to the constant chain of TCR β (cp) to construct functional unit 2. Two functional units of such SIRs are co-expressed in the same cell to become functionally active. In some embodiments, the two functional units of the SIR are co-expressed using a single vector, while in other embodiments, the two functional units are co-expressed in the same cell using different vectors. In some embodiments, two functional units of the SIR are co-expressed by transfecting a single mRNA sequence encoding both functional units, while in other embodiments, two functional units are co-expressed by transfecting two different mRNA sequences each encoding one functional unit.

In another embodiment, the SIR comprises or consists of an antigen binding domain linked to one T cell receptor constant chain (functional Unit 1) but co-expressed with a second T cell receptor constant chain (e.g., SEQ ID NO: 1620). The purpose of the second T cell receptor constant chain in such SIRs is to facilitate cell surface expression of functional unit 1 (e.g., antigen binding domain 1 linked to the T cell receptor constant chain). Thus, the second T cell receptor constant chain may be expressed by itself or as a fusion protein carrying an epitope tag (e.g. MYC, V5, AcV5, G4Sx2, StrepTagII, etc.) or as a fusion protein carrying any unrelated protein fragment (e.g. vL or vH fragment) that does not interfere with the assembly and function of functional unit 1. As an example, the SIR may comprise antigen binding domain 1 linked to C α or blank (lacking the antigen binding domain) C β (e.g., SEQ ID NO: 1620). Two functional units of such SIRs are co-expressed in the same cell to become functionally active. In some embodiments, the two functional units of the SIR are co-expressed using a single vector, while in other embodiments, the two functional units are co-expressed in the same cell using different vectors. In some embodiments, two functional units of the SIR are co-expressed by transfecting a single mRNA sequence encoding both functional units, while in other embodiments, two functional units are co-expressed by transfecting two different mRNA sequences each encoding one functional unit.

The SIRs described herein can be encoded by a single polynucleotide chain and translated into a single polypeptide chain that is subsequently cleaved into different proteins. Nucleic acid molecules encoding SIRs may comprise one or more leader sequences (also referred to as signal peptides). In one embodiment, each functional unit of the SIR (e.g., the antigen binding domain linked to the T cell receptor constant chain plus a furin-SGSG-cleavable linker or the T cell receptor constant chain plus a furin-SGSG-cleavable linker) can be preceded by a leader sequence that directs the SIR to the cell surface as a type I transmembrane protein. In one embodiment, the antigen binding domain of the SIR is cell-outward facing. In some embodiments, the leader sequence comprises the nucleic acid sequence of any one of SEQ ID NOs 1 to 9 and the amino acid sequence of SEQ ID NOs 2300 to 2302. In some embodiments, short nucleic acid sequences (3-9 nucleic acids) comprising restriction enzyme sites are located between different subunits of the SIR, e.g., between the signal sequence and the antigen binding domain of the SIR or between the antigen binding chain and the TCR chain.

Synthetic Immune Receptors (SIRs) with different TCR constant chains can be generated. The TCR constant chain may be encoded by its wild-type sequence, non-wild-type sequence or codon-optimized sequence. In addition, the TCR constant chains may carry specific mutations (e.g., TCR β constant chains having one or more of the mutations shown in table 2 or 3 and TCR α constant chains having one or more of the mutations in table 1) to enhance their cell surface expression and/or pairing with each other and reduce pairing with endogenous TCRs. Mutations in the TCR domain of the SIR alter the binding affinity and/or expression of the SIR to the target or cell, respectively. For example, the present disclosure encompasses different populations of SIRs for specific antigen targets that can be designed and screened based on nucleic acid sequence codon optimization and/or mutations in TCR chains to facilitate pairing or expression between binding domains and TCR domains and/or use of linkers. In some embodiments, an immune effector cell expressing a SIR from a collection exhibits greater than a 2-fold, greater than a 5-fold, greater than a 10-fold, and even greater than a 100-fold difference in one or more characteristics selected from the group consisting of: antigen binding affinity, cell surface expression, cell signaling, NFAT reporter activity, cytotoxicity, cytokine secretion, proliferation, in vivo persistence, expression of depletion markers, and in vivo activity. The present disclosure encompasses libraries of X-SIR molecules, where X is an antigen binding domain target, such that the library or "repertoire" provides SIRs with altered binding affinities, expression levels, and functional characteristics (e.g., cytotoxicity, cytokine production, and long-term persistence). In some embodiments, the SIRs in the collection have a greater than 2-fold, preferably greater than 5-fold, even more preferably greater than 10-fold, and even more preferably greater than 100-fold difference in one or more features selected from the group consisting of: antigen binding affinity, cell surface expression, cytotoxicity, cytokine secretion, T cell proliferation, T cell persistence, T cell depletion, and in vivo activity. Different SIRs in the pool can be labeled with different DNA barcodes to allow their identification by next generation sequencing or other techniques known in the art. Exemplary barcodes are presented by SEQ ID NOs 864 to 869. Barcodes may be inserted at convenient locations on the SIR-encoded carrier so that they do not interfere with the expression of the SIR. In one exemplary embodiment, the barcode may be inserted immediately downstream of the stop codon of the SIR. One skilled in the art can screen such a collection using one or more of the assays described herein to identify X-SIRs with a desired binding affinity, expression level, or functional characteristic. Different SIRs or different sets of SIRs may be appropriate for different diseases and disease conditions and may be combined to generate different and polyclonal immune responses. Thus, T cells expressing SIRs with high affinity for the target may be more effective in short-term killing of tumor cells, but are rapidly depleted and/or have short-term in vivo persistence. Such T cells expressing high affinity SIRs can be combined with T cells expressing low affinity SIRs that are not effective in short-term killing of tumor cells, but do not contain rapid depletion and/or persist in vivo for longer periods of time. The SIRs of the present disclosure (including different SIR sets) can also be combined with other genetically engineered T cells, such as CAR-T cells, to generate different immune responses. Accordingly, the present disclosure provides a library of X-SIRs.

As described above and herein, the SIR comprises one or more antigen binding domains operably linked to one or more T Cell Receptor (TCR) constant chain regions. The SIRs of the present disclosure may comprise a human β 1 chain constant region (C β 1) or a human β 2 chain constant region (C β 2). In one embodiment, the human constant β region (1 or 2) of the SIR comprises a basic amino acid at position 18. The basic amino acid is selected from the group consisting of arginine (R) and lysine (K). The term "position 18" refers to the 18 th amino acid residue in the sequence SEQ ID NO:3025 (for C β 2) or SEQ ID NO:3024 (for C β 1). In addition to this mutation at position 18, C β 1 or C β 2 of the SIR may comprise other mutations as long as the biological function of the SIR remains intact. If the biological function is performed similarly (e.g., better or worse), albeit differently, then the biological activity is complete. The term "function of an SIR" means the ability of an SIR to specifically bind to a given antigen, e.g., with a particular affinity, and/or respond to that antigen by activating cell signaling that results in activation of T cell functions such as activation, proliferation, cytokine secretion, and/or cytotoxicity.

In one embodiment, the SIR comprises at least one additional mutation in the C β 1 or C β 2 chain in addition to the basic amino acid at position 18. The mutation is selected from the group consisting of alanine (A) at position 22, isoleucine (I) at position 133, alanine (A) at position 136 and histidine (H) at position 139, wherein the positions mentioned are those in the sequence SEQ ID NO:3025 (for C β 2) or SEQ ID NO:3024 (for C β 1). In another embodiment, the SIR comprises a basic amino acid at position 18 of the C β 1 or C β 2 chain and two or more additional mutations selected from the group consisting of alanine (a) at position 22, isoleucine (I) at position 133, alanine (a) at position 136 and histidine (H) at position 139, wherein the mentioned positions are those in the sequence SEQ ID NO:3025 (for C β 2) or SEQ ID NO:3024 (for C β 1).

The present disclosure demonstrates that SIRs containing additional cysteines in the constant regions of the alpha and beta chains can promote preferential pairing with each other, increase the overall surface expression of the introduced SIRs, improve binding affinity to the target antigen and improve functionality. For example, SIRs with a Thr 48Cys (T48C) in C α and Ser-57-Cys (S57C) mutation in the C β 1 or C β 2 chains have an additional disulfide bond between the two chains that reduces mismatches with endogenous TCR chains and enhances functionality. Other disulfide bond positions (or combinations thereof) are as follows: c alpha-T48C and C beta 1 or C beta 2-S57C, C alpha-S15C and C beta 1 or C beta 2-E15C, C alpha-T45C and C beta 1 or C beta 2-D59C, C alpha-T45C and C beta 1 or C beta 2-S77C, C alpha-Y10C and C beta 1 or C beta 2-S17C.

Another approach to overcome the problem of unwanted pairing of the introduced TCR α -chains and TCR β -chains with endogenous TCR chains involves the use of a "knob-in-hole" or "knob-in-knob" configuration and an electrostatic environment. The present disclosure demonstrates that SIRs with Ser 61Arg (S61R) mutations in C α and Arg79Gly (R79G) mutations in either C β 1 or C β 2 mutations also result in reduced mismatch and enhanced functionality with endogenous TCR chains. Other methods of enhancing the expression of the introduced TCR chains (such as removal of N-glycosylation sites) can also be used in the methods of the disclosure to generate SIRs with increased surface expression and functionality.

Another approach to overcome the problem of unwanted pairing of the introduced TCR α -and TCR β -chains with endogenous TCR chains of SIR involves the use of genetic targeting to knock out the expression of endogenous TCR α or/and TCR β chains. Knockout of endogenous TCR α or/and TCR β chains can be achieved using a number of techniques known in the art, such as using CRISP/Cas9 and Zn finger nucleases. Sequences of gRNAs targeting the TCR α and TCR β loci are provided by SEQ ID Nos 897 and 898. These grnas can be introduced into T cells or ipscs or stem cells along with Cas9mRNA to knock down the expression of endogenous TCR α and TCR β chains. Such TCR α/β knockout cells can be used to introduce SIRs of the present disclosure. In an alternative embodiment, the same method can be used to enhance expression and chain pairing of a tcr, including the tcr of the present disclosure shown in table 7A. When expressed in T cells with reduced or eliminated expression of endogenous TCR chains, the TCR requires some functional properties of the SIR. In an alternative embodiment of the present disclosure, the SIR or TCR may be first introduced in T cells or ipscs or stem cells, followed by the knockout of TCR α and TCR β chains. If expression of SIR or TCR in TCR γ δ cells is desired, a substantially similar approach can be used to reduce or eliminate expression of endogenous TCR γ or/and TCR δ chains.

Non-limiting examples of suitable substitutions for the C.alpha.C.beta.1 and C.beta.C.beta.2 polypeptide chains are provided in tables 1-3. Other substitutions are also possible. For example, equivalent amino acids (i.e., conservative substitutions) may be used in the same "mutated" position.

Additional mutations in the constant regions of TCR α, TCR β 1, and TCR β 2 that result in increased expression of the introduced SIR chains and reduced mismatch with endogenous TCR chains may be incorporated in the design of SIRs of the present disclosure. In addition, it is known in the literature that murine TCRs are better expressed than human TCRs. The present disclosure demonstrates that SIRs comprising murine human TCR α and β chains (i.e., wherein certain amino acid residues of the human TCR α and β constant regions are replaced by corresponding amino acids of the mouse TCR α and β chains) are better expressed than SIRs comprising wild-type amino acid sequences of the human TCR α and β constant chains. Additional mutations of the human TCR α and TCR β chains can be similarly generated based on the sequences of the mouse TCR α and TCR β chains. SIRs containing such murine TCR α and TCR β chains can be readily tested in the assays described herein (e.g., binding to target antigens using NLuc binding assays, cytokine secretion, cell killing, Jurkat NFAT-GFP assay, etc.) to identify variants that result in increased expression and/or functional activity of SIRs.

Nucleic acids encoding SIRs of the disclosure encode one or more T cell receptor constant chains or regions. Nucleic acid sequences of exemplary T cell receptor constant chains or regions that can be used to prepare SIRs are provided in SEQ ID NOs 730-775, 10427-10452 and 10464-10471. Corresponding amino acid sequences are provided in SEQ ID NOs 3010 to 3055, 12384-12409 and 12421-12428 (Table 4). In some embodiments, the nucleic acid sequence encoding the T cell receptor constant chain of the encoded SIR molecule comprises a wild-type sequence of a constant chain of human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or Calpha; SEQ ID NOS: 730 and 731), human T cell receptor beta 1 (TCR-beta 1 or TCR beta 1 or TCRb1 or Cbeta 1; SEQ ID NO:744), human T cell receptor beta 2 (TCR-beta 2 or TCR beta 2 or TCRb2 or Cbeta 2, also known as TCR-beta, TCR beta or TCRb or Cbeta; SEQ ID NOS: 745 and 746), human Pre-T cell receptor alpha (preTCR-alpha or preTCR alpha or preTCRa or preCalpha), human T cell receptor gamma (TCR-gamma or TCR gamma or TCRg or Cgamma; SEQ ID NO:769), or human T cell receptor delta (TCR-delta or TCRd or TCR delta or C delta).

Table 4:

in some embodiments, the nucleic acid sequence encoding the T cell receptor constant chain of the encoded SIR molecule comprises human T cell receptor- α (TCR- α or TCR α or TCRa or Cα), human T cell receptor- β 1(TCR- β 1 or TCR β 1 or TCRb1 or Cβ 1), human T cell receptor- β 2(TCR- β 2 or TCR β 2 or TCRb2 or Cβ 2, also known as TCR- β, TCR β or TCRb or Cβ), for example, a non-wild-type nucleic acid sequence for human Pre-T cell receptor alpha ((preTCR-alpha or preTCR alpha or preTCRa or preC alpha), human T cell receptor-gamma (TCR-gamma or TCR gamma or TCRg or C gamma), or human T cell receptor-delta (TCR-delta or TCRd or TCR delta or C delta) can be a codon-optimized sequence and/or a sequence comprising one or more mutations that result in a mutation in the encoded polypeptide.

In some embodiments, the nucleic acid sequence encoding the T cell receptor invariant chain of the encoded SIR molecule comprises a codon optimized sequence of human T cell receptor- α (TCR- α or TCR α or TCRa or ca), human T cell receptor- β 1(TCR- β 1 or TCR β 1 or TCRb1 or ca), human T cell receptor- β 2(TCR- β 2 or TCR β 2 or TCRb2 or ca, also known as TCR- β, TCR β or TCRb or ca), human Pre-T cell receptor α (preTCR- α or preTCR α or preTCRa or preC α), human T cell receptor- γ (TCR- γ or TCR γ or TCRg or ca), or human T cell receptor- δ (TCR- δ or TCRd or TCR δ or ca). An exemplary codon optimized human TCR β 1 constant region nucleic acid sequence is provided in SEQ ID NO 752. Exemplary codon-optimized human TCR β 2 constant region nucleic acid sequences are provided in SEQ ID NOS 749 and 750.

In some embodiments, the nucleic acid sequence encoding the T cell receptor constant chain of the encoded SIR molecule comprises a constant chain of human T cell receptor- α (TCR- α or TCR α or TCRa or ca), human T cell receptor- β 1(TCR- β 1 or TCR β 1 or TCRb1 or ca), human T cell receptor- β 2(TCR- β 2 or TCR β 2 or TCRb2 or C β 2, also known as TCR- β, TCR β or TCRb or C β), human Pre-T cell receptor α (preTCR- α or preTCR α or preTCRa or preC α), human T cell receptor- γ (TCR- γ or TCR γ or TCRb or C γ), or human T cell receptor- δ (TCR- δ or TCRd or TCR δ or C δ), these constant chains carry specific mutations (point mutations or deletions or both) that enhance expression and pairing of SIR-enhancing chains and reduce their pairing with endogenous T cell receptor chains.

In some embodiments, the nucleic acid sequence encoding the T cell receptor constant chains of the encoded SIR molecules comprises constant chains of human T cell receptor- α (TCR- α or TCR α or TCRa or ca), human T cell receptor- β 1(TCR- β 1 or TCR β 1 or TCRb1 or ca), human T cell receptor- β 2(TCR- β 2 or TCR β 2 or TCRb2 or C β 2, also known as TCR- β, TCR β or TCRb or C β), human Pre-T cell receptor α (preTCR- α or preTCR α or preTCRa or preC α), human T cell receptor- γ (TCR- γ or TCR or C γ), or human T cell receptor- δ (TCR- δ or TCRd or TCR δ or C δ) codon optimized and carrying expression of chains that enhance SIR and reduce their pairing with endogenous T cell receptor chains (point mutations or deletions or specific pairing with endogenous T cell receptor chains Both).

In some embodiments, the nucleic acid sequence encoding the T cell receptor constant chain of the encoded SIR molecule comprises a wild-type, non-wild-type, or codon-optimized constant chain of canine T cell receptor- α (TCR- α or TCR α or TCRa or ca), canine T cell receptor- β (TCR- β or TCR β or TCRb or ca), canine Pre-T cell receptor α ((preTCR- α or preTCR α or preTCRa or preC α), canine T cell receptor- γ (TCR- γ or TCR γ or TCRg or cy), or canine T cell receptor- δ (TCR- δ or TCRd or TCR δ or C δ).

In some embodiments, the nucleic acid sequence encoding the T cell receptor constant chains of the encoded SIR molecules comprises canine T cell receptor- α (TCR- α or TCR α or TCRa or ca), canine T cell receptor- β (TCR- β or TCR β or TCRb or ca), canine Pre-T cell receptor α (preTCR- α or preTCR α or preTCRa or preC α), canine T cell receptor- γ (TCR- γ or TCR γ or TCRg or ca), or canine T cell receptor- δ (TCR- δ or TCRd or TCR δ or C δ), which are codon optimized and carry specific mutations (point mutations or deletions or both) that enhance expression and pairing of the chains of SIR and reduce their pairing with endogenous T cell receptor chains.

In some embodiments, the nucleic acid sequence encoding the T cell receptor constant chain of the encoded SIR molecule comprises a wild-type, non-wild-type or codon optimized constant chain of murine T cell receptor- α (TCR- α or TCR α or TCRa or ca), murine T cell receptor- β (TCR- β or TCR β or TCRb or ca), murine Pre-T cell receptor α (preTCR- α or preTCR α or preTCRa or preca), murine T cell receptor- γ (TCR- γ or TCR γ or TCRg or C γ), or murine T cell receptor- δ (TCR- δ or TCRd or TCR δ or C δ) that may or may not carry specific mutations (point mutations or deletions or both) that enhance expression and pairing of the SIR-enhanced chain and reduce its pairing with endogenous T cell receptor chains.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO:730, SEQ ID NO:731, or SEQ ID NO:733 of the human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or hTCRa or C alpha) constant chain. In certain embodiments, the nucleic acid sequence of the SIR molecule encodes an amino acid sequence of the constant chain of human T cell receptor alpha having at least one, five or nine modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3010 or SEQ ID No. 3011 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3010 or SEQ ID No. 3011. In certain embodiments, the nucleic acid sequence of the SIR molecule encodes the constant strand of human TCRa comprising the sequence SEQ ID NO 3010 or SEQ ID NO 3011.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises a nucleic acid sequence encoding the amino acid sequence SEQ ID NO:3010 but which carries one or more mutations in the human T cell receptor alpha constant region (chain) including: serine (S) at position 91, (D) at position 92, valine (V) at position 93, proline (P) at position 94, cysteine (C) at position 48, and arginine (R) at position 61 (e.g., SEQ ID NOs: 732, 735, 736, 737, 738, 739, or 740).

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises a nucleic acid sequence encoding the amino acid sequence SEQ ID NO:3010 but wherein one or more amino acids are replaced with the corresponding amino acids of a mouse TCR α constant chain (SEQ ID NO:3022) human T cell receptor α constant region (chain).

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO:732 of the constant chain of human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or hTCRa). In certain embodiments, the nucleic acid sequence of the SIR molecule encodes a constant chain of human TCRa that carries amino acid substitutions (CSDVP) that enhance expression of the encoded polypeptide and its chain pairing with the complementary TCRb constant chain of the SIR and reduce chain pairing with the endogenous TCR β chain. In certain embodiments, the nucleic acid sequence of the SIR comprises a sequence encoding the amino acid sequence of the constant chain of human T cell receptor alpha having at least one, five or ten modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3012 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3012. In certain embodiments, the constant chain of human TCRa encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO: 3012.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence of human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or hTCRa or C alpha) constant chain as set forth in SEQ ID NO: 737. In certain embodiments, the nucleic acid sequence of the SIR molecule encodes a constant chain of human TCRa that carries amino acid Substitutions (SDVP) that enhance expression of the encoded polypeptide. In certain embodiments, the nucleotide sequence of the SIR comprises a sequence encoding an amino acid sequence of a constant chain of human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or ca) having at least one, five or ten modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3017 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3017. In certain embodiments, the constant chain of human TCRa encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO: 3017.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO:740 of the constant chain of human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or hTCRa). In certain embodiments, the nucleic acid sequence of the SIR molecule encodes a constant chain of human TCRa that carries amino acid Substitutions (SD) that enhance expression of the encoded polypeptide. In certain embodiments, the nucleic acid sequence of the SIR comprises a sequence encoding the amino acid sequence of the constant chain of human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or ca) having at least one, five or ten modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID NO:3020 or a sequence having 80-99% identity to the amino acid sequence SEQ ID NO: 3020. In certain embodiments, the constant chain of human TCRa encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO: 3020.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO:736 of the constant chain of human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or hTCRa or calpha). In certain embodiments, the nucleic acid sequence of the SIR molecule encodes a constant chain of human TCR α with a Thr 48Cys (T48C) substitution that promotes additional interchain disulfide bond formation and reduces chain pairing with endogenous TCR β chains when co-expressed with an introduced human mutant TCR β constant chain carrying a S57C (Ser57Cys) substitution. In certain embodiments, the nucleic acid sequence of the SIR comprises a sequence encoding an amino acid sequence of a constant chain of human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or ca) having at least one, five or nine modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3016 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3016. In certain embodiments, the constant chain of human TCRa encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO: 3016.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO:738 of the constant chain of human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or hTCRa). In certain embodiments, the nucleic acid sequence of the SIR molecule encodes a constant chain of human TCR α having a Ser 61Arg (S61R) substitution that promotes chain pairing and reduces chain pairing with endogenous TCR β chains when co-expressed with an introduced human mutant TCR β constant chain carrying a R79G (Arg79Gly) substitution. In certain embodiments, the nucleic acid sequence of the SIR comprises a sequence encoding an amino acid sequence of a constant chain of human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or ca) having at least one, five or nine modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3018 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3018. In certain embodiments, the constant chain of human TCRa encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO: 3018.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO:739 of the constant chain of human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or hTCRa). In certain embodiments, the nucleic acid sequence of the SIR molecule encodes a constant chain of human TCRa that carries amino acid Substitutions (SDVPR) that enhance expression of the encoded polypeptide and its pairing with a complementary TCR β constant chain and reduce chain pairing with endogenous TCR β chains. In certain embodiments, the nucleotide sequence of the SIR comprises a sequence encoding an amino acid sequence of a constant chain of human T cell receptor alpha (TCR-alpha or TCR alpha or TCRa or ca) having at least one, five or ten modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID NO:3019 or a sequence having 80-99% identity to the amino acid sequence SEQ ID NO: 3019. In certain embodiments, the constant chain of human TCRa encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO: 3019.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence of the constant chain of human T cell receptor beta (TCR-beta or TCRb or TCR beta or hTCR-beta or hTCRb or hTCR beta or C beta) as shown in SEQ ID NO:744 or SEQ ID NO: 745. In certain embodiments, the nucleotide sequence of the SIR encodes an amino acid sequence of the constant chain of human T cell receptor β having at least one, five or nine modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3024 or SEQ ID No. 3025 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3024 or SEQ ID No. 3025. In certain embodiments, the constant chain of human TCRb encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO:3024 or SEQ ID NO: 3025.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises an amino acid sequence encoding the amino acid sequence SEQ ID NO:3024 or SEQ ID NO:3025 but carrying a human T cell receptor β (TCR- β or TCRb or TCR β or hTCR- β or hTCRb or hTCR β or C β) constant chain comprising one or more mutations of: a basic amino acid at position 18 (Arg or Lys), alanine at position 22 (a), isoleucine at position 22 (I), alanine at position 136 (a), histidine at position 139 (H), cysteine at position 57 (C) and/or glycine at position 79 (G).

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises a nucleic acid sequence encoding the constant domain (chain) of human T cell receptor beta (TCR-. beta.or TCRb or TCR. beta.or hTCR-. beta.or hTCRb or hTCrB or hTCRbeta. or C.beta.) in which one or more amino acids have been replaced with the corresponding amino acid of the constant chain of mouse TCR. beta. (SEQ ID NO:3047) of human TCR. beta.1 (SEQ ID NO:3024) or TCR. beta.2 (SEQ ID NO: 3025).

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence of the constant chain of human T cell receptor beta (TCR-. beta.or TCRb or TCR. beta. or hTCR-. beta. or hTCRb or hTCR. beta. or C.beta.) as shown in SEQ ID NO: 748. In certain embodiments, the nucleic acid sequence of the SIR molecule encodes a constant chain of human TCRb that carries an amino acid substitution (KACIAH) as set forth in SEQ ID NO:3028 that enhances expression of the encoded polypeptide and its chain pairing with the complementary TCRa constant chain of the SIR and reduces chain pairing with the endogenous TCR β chain. In certain embodiments, the nucleic acid sequence of the SIR comprises a sequence encoding the amino acid sequence of the constant chain of human TCRb having at least one, five, or ten modifications, but NO more than 20 modifications, of the amino acid sequence of SEQ ID NO:3028 or a sequence having 80-99% identity to the amino acid sequence of SEQ ID NO: 3028. In certain embodiments, the constant chain of human TCRb encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO: 3028.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO. 747 of the constant chain of the human T cell receptor beta (TCR-. beta.or TCRb or TCR. beta. or hTCR-. beta.or hTCRb or hTCrB or hTCrbeta. or C.beta.). In certain embodiments, the nucleic acid sequence of the SIR molecule encodes a constant chain as shown in SEQ ID NO:3027 of human TCRb carrying a Ser57Cys (S57C) substitution, which when co-expressed with a human mutant TCR α constant chain carrying a T48C (Thr57Cys) substitution promotes additional interchain disulfide formation and reduces chain pairing with endogenous TCR α chains. In certain embodiments, the nucleic acid sequence of the SIR comprises a sequence encoding the amino acid sequence of the constant chain of human TCRb having at least one, five, or ten modifications, but NO more than 20 modifications, of the amino acid sequence of SEQ ID NO:3027 or a sequence having 80-99% identity to the amino acid sequence of SEQ ID NO: 3027. In certain embodiments, the constant chain of human TCRb2 encoded by the SIR molecule comprises the amino acid sequence SEQ ID NO: 3027.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO 762 of the constant chain of the human T cell receptor β (TCR-. beta.or TCRb or TCR. beta. or hTCR-. beta.or hTCRb or hTCRbeta. or cbeta.). In certain embodiments, the nucleic acid sequence of the SIR molecule encodes a constant chain as shown in SEQ ID NO:3042 of human TCRb carrying an Arg79Gly (R79G) substitution that promotes chain pairing with the introduced human mutant TCR α constant chain carrying a Ser 61Arg (S61R) substitution and reduces chain pairing with the endogenous TCR α chain. In certain embodiments, the nucleic acid sequence of the SIR comprises a sequence encoding the amino acid sequence of the constant chain of human T cell receptor β having at least one, five or ten modifications, but NO more than 20 modifications, of the amino acid sequence of SEQ ID No. 3042 or a sequence having 80-99% identity to the amino acid sequence of SEQ ID No. 3042. In certain embodiments, the constant chain of human TCRb encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO 3042.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NOs 753 through 759 of the constant chain of the human T cell receptor β (TCR-. beta.or TCRb or TCR. beta. or hTCR-. beta. or hTCRb or hTCrB or hTCrbeta. or C.beta.). In certain embodiments, the nucleic acid sequence of the SIR molecule encodes the invariant chain of human TCRb, as shown in SEQ ID NOS 3034, 3035, 3036, 3037, 3038, or 3039, carrying amino acid substitutions that enhance its expression. In certain embodiments, the nucleic acid sequence of the SIR comprises a sequence encoding the amino acid sequence of the constant chain of human T cell receptor β having at least one, five or ten modifications of the amino acid sequence SEQ ID No. 3034 to 3038 or 3039 or a sequence having 80-99% identity with the amino acid sequence SEQ ID No. 3034, 3035, 3036, 3037, 3038 or 3039, but NO more than 20 modifications. In certain embodiments, the constant chain of human TCRb encoded by the SIR molecule comprises the amino acid sequence SEQ ID NO 3034, 3035, 3036, 3037, 3038, or 3039.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence of the constant chain of human T cell receptor beta (TCR-. beta.or TCRb or TCR. beta. or hTCR-. beta. or hTCRb or hTCR. beta. or cbeta.) as set forth in SEQ ID NO: 753. In certain embodiments, the nucleic acid sequence of the SIR molecule encodes the constant chain of human TCRb as shown in SEQ ID NO:3033 carrying amino acid substitutions (KAIAH) that enhance its expression. In certain embodiments, the nucleic acid sequence of the SIR comprises a sequence encoding the amino acid sequence of the constant chain of human T cell receptor β having at least one, five or ten modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3033 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3033. In certain embodiments, the constant chain of human TCRb encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO 3033.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO:761 of the constant chain of human T cell receptor β (TCR-. beta.or TCRb or TCR. beta. or hTCR-. beta.or hTCRb or hTCRbeta. or cbeta.). In certain embodiments, the nucleic acid sequence of the SIR molecule encodes the constant chain of human TCRb as shown in SEQ ID NO:3041 carrying amino Acid Substitutions (KAGs) that enhance its expression and pairing with the introduced TCRa constant chain. In certain embodiments, the nucleotide sequence of the SIR comprises a sequence encoding the amino acid sequence of the constant chain of human T cell receptor β having at least one, five or ten modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3041 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3041. In certain embodiments, the constant chain of human TCRb encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO 3041.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO 760 of the constant chain of the human T cell receptor β (TCR-. beta.or TCRb or TCR. beta. or hTCR-. beta.or hTCRb or hTCR. beta. or C.beta.). In certain embodiments, the nucleic acid sequence of the SIR molecule encodes the invariant chain shown in SEQ ID NO:3040 of human TCRb carrying amino acid substitutions (KAIHAG) that enhance its expression and pairing with the introduced TCRa invariant chain. In certain embodiments, the nucleotide sequence of the SIR comprises a sequence encoding the amino acid sequence of the constant chain of human T cell receptor β having at least one, five or ten modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3040 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3040. In certain embodiments, the constant chain of human TCRb encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO 3040.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the sequence SEQ ID NOs 755, 756, 757, 758 or 759 encoding the invariant chain of human TCRb2 carrying paired amino acid substitutions (K18I133 or K18a136 or K18H139 or R18a22 or R18) that enhance its expression as well as the introduced TCRa invariant chain. In certain embodiments, the nucleotide sequence of the SIR comprises a sequence encoding an amino acid sequence of a invariant chain of human T cell receptor β 2(TCR- β 2 or TCR β 2 or TCRb2 or C β 2; also known as TCR- β, TCR β or TCRb or C β) having at least one, five or ten modifications of the amino acid sequence SEQ ID NO:3035, 3036, 3037, 3038 or 3039 or a sequence having 80-99% identity with the amino acid sequence SEQ ID NO:3035, 3036, 3037, 3038 or 3039, but NO more than 20 modifications. In certain embodiments, the constant chain of human TCRb2 encoded by the SIR molecule comprises the amino acid sequence SEQ ID NOs 3035, 3036, 3037, 3038, or 3039.

In certain embodiments, the nucleic acid sequence of the SIR encodes an amino acid sequence of the constant chain of human pre T cell receptor alpha (pre-TCR-alpha-Del 48 or pre-TCR alpha-Del 48 or pre-TCRa-Del48 or pre C alpha-Del 48) missing the C-terminal 48 amino acids and has the nucleic acid sequence shown as SEQ ID NO: 768. In certain embodiments, the nucleic acid sequence of the SIR encodes an amino acid sequence of the preC α -Del48 constant chain having at least one, five or nine modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3048 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3048. In certain embodiments, the constant chain of human pre-TCRa-Del48 encoded by the SIR molecule comprises the amino acid sequence SEQ ID NO 3048.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence of human pre T cell receptor alpha (pre-TCR-alpha or pre-TCR alpha or pre-TCRa or pre C alpha) constant chain as set forth in SEQ ID NO 766 or 767. In certain embodiments, the nucleic acid sequence of the SIR encodes an amino acid sequence of the constant chain of human pre T cell receptor alpha as set forth in SEQ ID No. 3046 or 3047 and has at least one, five or nine modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3046 or 3047 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3046 or 3047. In certain embodiments, the constant chain of human pre-TCRa encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO 3046 or 3047.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO:769 or 770 of the human T cell receptor gamma (TCR-gamma or TCR gamma or TCRg or hTCR-gamma, or hTCRgamma or hTCRg or cgamma) constant chain. In certain embodiments, the nucleic acid sequence of the SIR encodes the sequence of the amino acid sequence of the constant chain of human T cell receptor gamma, and the amino acid sequence has at least one, five or nine modifications of the amino acid sequence SEQ ID No. 3049 or 3050 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3049 or 3050, but NO more than 20 modifications. In certain embodiments, the constant chain of human TCRg encoded by the SIR molecule comprises the amino acid sequence of SEQ ID NO 3049 or 3050.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO:771 or 772 of the constant chain of human T cell receptor delta (TCR-delta or TCR delta or TCRd or hTCR-delta or hTCR delta or hTCRD or C delta). In certain embodiments, the nucleic acid sequence of the SIR encodes an amino acid sequence of the constant chain of human T cell receptor delta, as set forth in SEQ ID NO:3052 and having at least one, five or nine modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID NO:3052 or a sequence having 80-99% identity to the amino acid sequence SEQ ID NO: 3052. In certain embodiments, the constant chain of human TCR-delta encoded by the SIR molecule comprises the amino acid sequence SEQ ID NO: 3052.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence set forth in SEQ ID NO:743 of the canine T cell receptor alpha (canine TCR-alpha or canine TCR alpha or canine TCRa or canine or TCR alpha, or TCR alpha or cTCR alpha or cC alpha) constant chain. In certain embodiments, the nucleic acid sequence of the SIR encodes an amino acid sequence of the constant chain of canine T cell receptor alpha, as set forth in SEQ ID NO:3023 and has at least one, five or nine modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID NO:3023 or a sequence having 80-99% identity to the amino acid sequence SEQ ID NO: 3023. In certain embodiments, the constant chain of the canine TCR- α encoded by the SIR molecule comprises the amino acid sequence SEQ ID NO: 3023.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence shown as SEQ id No. 764 of the canine T cell receptor β (canine TCR- β or canine TCR β or canine TCRb or canine-C β or TCR β, or TCR β or ctrb or cC β) constant chain. In certain embodiments, the nucleic acid sequence of the SIR encodes an amino acid sequence of the constant chain of canine T cell receptor β, which amino acid sequence is set forth in SEQ ID No. 3044 and has at least one, five or nine modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3044 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3044. In certain embodiments, the constant chain of the canine TCR- β encoded by the SIR molecule comprises the amino acid sequence SEQ ID NO 3044.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence shown as SEQ ID NO:742 of the murine T cell receptor alpha (murine TCR-alpha or murine TCR alpha or murine TCRa or murine-C alpha or mTCR alpha, or mTCR alpha or mTCRa or mC alpha) constant chain. In certain embodiments, the nucleic acid sequence of the SIR encodes an amino acid sequence of the constant chain of murine T cell receptor alpha as set forth in SEQ ID NO:3022 and has at least one, five or nine modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID NO:3022 or a sequence having 80-99% identity to the amino acid sequence SEQ ID NO: 3022. In certain embodiments, the constant chain of murine TCR- α encoded by the SIR molecule comprises the amino acid sequence SEQ ID NO: 3022.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence shown as SEQ ID NO:763 of the murine T cell receptor β (murine TCR- β or murine TCR β or murine-C β or mTCR β, or mTCR β or mTCRb or mC β) invariant chain. In certain embodiments, the nucleic acid sequence of the SIR encodes an amino acid sequence of the constant chain of murine T cell receptor β, as set forth in SEQ ID No. 3043 and has at least one, five or nine modifications, but NO more than 20 modifications, of the amino acid sequence SEQ ID No. 3043 or a sequence having 80-99% identity to the amino acid sequence SEQ ID No. 3043. In certain embodiments, the constant chain of the murine TCR-. beta.encoded by the SIR molecule comprises the amino acid sequence SEQ ID NO 3043.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises a nucleic acid sequence in which the extracellular domain of the human TCRa constant chain is fused to the extracellular domain, transmembrane domain, and cytoplasmic domain of the human CD3 zeta (CD3 zeta) chain as set forth in SEQ ID NO: 741. In certain embodiments, the nucleic acid sequence of the SIR encodes an amino acid sequence as set forth in SEQ ID NO:3021 and having at least one, five or nine modifications of the amino acid sequence SEQ ID NO:3021 or a sequence having 80-99% identity to the amino acid sequence SEQ ID NO:3021, but NO more than 20 modifications. In certain embodiments, the constant chain in which the extracellular domain of the human TCRa constant chain is fused to the extracellular domain, transmembrane domain, and cytoplasmic domain of the human CD3 ζ (CD3 ζ) chain encoded by the SIR molecule comprises the amino acid sequence SEQ ID NO: 3021.

In certain embodiments, the nucleic acid sequence of the SIR molecule comprises the nucleic acid sequence of a human TCRb constant chain fused to the extracellular domain, transmembrane domain, and cytoplasmic domain of the human CD3 zeta (CD3 zeta) chain as shown in SEQ ID NO: 765. In certain embodiments, the nucleic acid sequence of the SIR encodes an amino acid sequence as set forth in SEQ ID No. 3045 and having at least one, five or nine modifications of the amino acid sequence SEQ ID No. 3045 or a sequence having 80-99% identity with the amino acid sequence SEQ ID No. 3045, but NO more than 20 modifications. In certain embodiments, the constant chain of human TCRa constant chain extracellular domain fused to the extracellular domain, transmembrane domain, and cytoplasmic domain of the human CD3 ζ (CD3 ζ) chain encoded by the SIR molecule comprises the amino acid sequence SEQ ID NO 3045.

In certain embodiments, a nucleic acid encoding a SIR of the disclosure encodes a single T cell receptor constant chain comprising or derived from a constant chain of TCRa, TCRb, pre-TCRa, TCR- γ, or TCR- δ chains of human, mouse, or canine origin. An exemplary SIR with a single TCR constant chain is represented by clone ID:051216-F04, whose nucleic and amino acid sequences are given as SEQ ID Nos. 1023 and 3258, respectively.

In certain embodiments, a nucleic acid encoding a SIR of the disclosure encodes two T cell receptor constant chains comprising or derived from TCRa, TCRb, pre-TCRa, TCR- γ, or TCR- δ chains of human, mouse, or canine origin. An exemplary SIR with two TCR constant chains is represented by clone ID:102615-C08, whose nucleic and amino acid sequences are given in SEQ ID NO:1200 and 3435, respectively.

In certain embodiments, the two T cell receptor constant chains of the SIR may be of the same type (e.g., TCRa/TCRa; TCRb/TCRb; preTCRa/preTCRa; TCR γ/TCR γ; and TCR- δ/TCR- δ). Exemplary SIRs having two TCR constant chains of the same type are clone ID:021116-E08(SEQ ID NO:905), clone ID:012216-P08(SEQ ID NO:906), clone ID NO:012216-Q05(SEQ ID NO:907), clone ID NO:012216-R04(SEQ ID NO:908) and clone ID NO:012216-S02(SEQ ID NO: 909). In another embodiment, the two T cell receptor constant chains of the SIR are of different types (e.g., TCRa/TCRb; preTCRa/TCRb; TCR γ/TCR- δ, etc.). Exemplary SIRs with different types of two TCR constant chains are represented by clone ID:102615-C08, whose nucleic and amino acid sequences are given in SEQ ID NO:1200 and 3435, respectively.

As described above, SIRs of the present disclosure comprise a TCR domain linked to an antigen-binding domain. Thus, the SIRs of the present disclosure may comprise one or more antigen binding domains (e.g., antibodies or antibody fragments, ligands, or receptors) and one or more T cell receptor constant chains (as described herein above), wherein the one or more antigen binding domains bind to a target antigen. Non-limiting exemplary target antigens include: CD 19; CD 123; CD 22; CD23, CD 30; CD 171; CS-1 (also known as CD2 subunit 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD 33; epidermal growth factor receptor variant iii (egfrviii); ganglioside G2 (GD 2); ganglioside GD3(aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (l-l) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. alpha. -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1(ROR 1); fms-like tyrosine kinase 3(FLT 3); tumor-associated glycoprotein 72(TAG 72); CD 38; CD44v 6; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epidermal cell adhesion molecule (EPCAM); B7H3(CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213a 2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate Stem Cell Antigen (PSCA); protease serine 21 (testis protein or PRSS 21); vascular endothelial growth factor receptor 2(VEGFR 2); a lewis (Y) antigen; CD 24; platelet derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD 20; folate receptor alpha (FRa or FR 1); folate receptor beta (FRb); receptor tyrosine protein kinase ERBB2(Her 2/neu); cell surface associated mucin 1(MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); prostasin; prostatic Acid Phosphatase (PAP); mutant elongation factor 2(ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase ix (caix); proteasome (precursor, megalin) subunit, beta form, 9(LMP 2); glycoprotein 100(gpl 00); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and the Abelson (Abelson) murine leukemia virus oncogene homolog 1 (Abl); a tyrosinase enzyme; ephrin type a receptor 2(EphA 2); sialylated lewis adhesion molecules (sLe); ganglioside GM3(aNeu5Ac (2-3) bDClalp (l-4) bDGlcp (l-1) Cer); transglutaminase 5(TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); \ tumor endothelial marker 1(TEM1/CD 248); tumor endothelial marker 7 associated (TEM 7R); sealin 6(CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); class 5 group member D of G protein-coupled receptors C (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); a globoH sugar ceramide hexasaccharide moiety (globoH); mammary differentiation antigen (NY-BR-1); urinary plaque 2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenoceptor β 3(ADRB 3); ubiquitin 3 (PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex, locus K9 (LY 6K); olfactory receptor 51E2 (OR51E 2); TCR γ alternate reading frame protein (TARP); wilm's tumor protein (WT 1); cancer/testis antigen 1(NY-ES 0-1); cancer/testis antigen 2(LAGE-1 a); melanoma-associated antigen 1(MAGE-a 1); ETS translocation variant gene 6 located on chromosome 12p (ETV 6-AML); sperm protein 17(SPA 17); x antigen family member la (xagel); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; tumor protein p53(p 53); a p53 mutant; a prostate-specific protein; survivin; a telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; an inhibitor of melanoma apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyl-transferase V (NA 17); the paired box protein Pax-3(PAX 3); an androgen receptor; a cyclin Bl; a v-myc avian myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); tyrosinase-related protein 2 (TRP-2); cytochrome P450lB1(CYPlB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site regulator siblings), squamous cell carcinoma antigen recognized by T cells 3(SART 3); the paired box protein Pax-5(PAX 5); the preproepisin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RUl); renal ubiquitin 2(RU 2); legumain; human papilloma virus E6(HPV E6); human papilloma virus E7(HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2(mut hsp 70-2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2 (BST 2); EGF-like receptor-module mucin-like hormone-containing sample 2(EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); and immunoglobulin lambda-like polypeptide 1(IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1TROP2, GFRalpha 4, CDH17, CDH6, NYB 1, CDH19, CD200R, Slea (CA 19.9; sialyl Lewis antigen) fucosyl-GM R, PTK R, gpNMB, CDH R-CD 324, DLL R, CD276/B7H R, IL11R, IL13Ra R, CD 179R-IGLl R, TCR gamma-delta, NKG 2R, CD R (FCGR 2R), CSF Tn, Tim R-/HVCR R, GM-CSFR-alpha, TGF-R, Lews Ag, TCR-beta 1 chain TCR-beta 2, TCR-beta 2 chain, TCR-gamma-CMV-gamma chain, gonadotropin-CMV-HCH chain, TNF-HCH-HCR R, TNF-HCH chain receptor (SLF-HCH 72), FSH 72, FSH-R, FSH-PGF-R, FSH 72, FSH-R, FSH receptor, FSH 72, FSH-R, FSH-PGF-receptor, TNF-PGF-5, and PGF-PGS, KSHV-gH, influenza A lectin (HA), GAD, PDL1, Guanylate Cyclase C (GCC), desmoglein autoantibody 3(Dsg3), desmoglein autoantibody 1(Dsg1), HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RASG12V, tissue factor 1(TF1), AFP, GPRC5D, sealing protein 18.2(CLD18A2 or N18A.2)), P-glycoprotein, STEAP1, LIV1, laminin-4, CRIPTO, GPA33, BST1/CD157, low conductivity chloride channels and antigens recognized by TNT antibodies.

In some embodiments, the antigen binding domain of the SIR polypeptide molecule binds to a tumor antigen. Non-limiting examples of tumor antigens that can be targeted by SIR polypeptides include TSHR, CD171, CS-1, CLL-1, GD, TnAg, FLT, CD44v, B7H, KIT, IL-13Ra, IL-11Ra, PSCA, PRSS, VEGFR, LewisY, CD, PDGFR-beta, SSEA-4, MUC, EGFR, NCAM, CAlX, LMP, EphA, fucosylGM, sLe, GM, TGS, HMWMAA, o-acetyl-GD, folate receptor beta, TEM/CD 248, TEM, CLDN, GPRC5, CXORF, CD179, ALK, polysialic acid, PLAC, GlobH, NY-BR-1, UPK, HAVCR, ADRB, PANX, GPR, LY6, OR51E, TARP, WT, ETV-AML, sperm protein 17, XAGE, Tie 2, MAD-CT-1, MOLD-CT-related site-mutant, sarcoma, hTERT-associated site-2, sarcoma, hTRP-associated mutants, hTERT-2, hTRP-associated with antigen, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLLl.

In some embodiments, the antigen binding domain of the SIR polypeptide molecule binds to an antigen associated with HLA-a 2. Non-limiting examples of antigens recognized in association with HLA-A2 include TARP, WT1, hTERT, gp100, tyrosinase, MART1, NY-ESO1, CMV pp65, EBV EBNA3c, HIV1gag, HTLV1-Tax, PR1, CMV pp65, EBV-EBNA3c, Ras G12V mutant, and GAD.

In some embodiments, the antigen binding domain of the SIR polypeptide molecule comprises an autoantigen or fragment thereof that binds to an autoantibody. Non-limiting examples of autoantigens include Dsg1 and Dsg 3.

In some embodiments, the antigen binding domain of the SIR polypeptide molecule is derived from or comprises a wild-type or non-immunoglobulin wild-type sequence of an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, Single Domain Antibody (SDAB), vH or vL domain, camelidae vHH domain, or a non-immunoglobulin antigen binding scaffold, e.g., DARPIN, affibody, affilin, adnectin, affitin, obodies, repebody, fynomer, alphabody, avimer, atrimer, centyrin, pronectin, anticalin, kunitz-type domain, Armadillo repeat protein, autoantigen, receptor, or ligand. In some embodiments, the encoded SIR polypeptide contains more than one antigen binding domain. In embodiments, the antigen binding domain is operably linked to the NH2 terminus of the TCR domain (i.e., the constant chain of TCR- α, TCR- β 1, TCR- β 2, preTCR- α, pre-TCR- α -Del48, TCR- γ, or TCR- δ), either directly or via an optional linker. The nucleic acid and amino acid sequences of several exemplary linkers are provided in SEQ ID NOs 701-18927, 18922-18927 and 2981-3003, 18929-18934. Constructs encoding exemplary such SIRs are provided in clone ID NO: 082815-G07. The amino acid sequence of the encoded SIR polypeptide corresponds to SEQ ID NO 3855.

In some embodiments, the antigen binding domains of the SIR polypeptide molecule are derived from or constitute the vL and vH domains of an antibody, which domains are individually linked to the NH2 ends of the two constant chains of the T cell receptor (i.e., the constant chains of TCR- α, TCR- β 1, TCR- β 2, preTCR- α, pre-TCR- α -Del48, TCR- γ, or TCR- δ, or mutants or variants thereof, as described herein) to collectively constitute a single antigen binding domain. Exemplary such SIRs targeting CD19 are provided in clone ID NO: 102615-C08. The amino acid sequence of the SIR corresponds to SEQ ID NO: 3435. In this SIR, the vL fragment derived from the CD19 monoclonal antibody FMC63 was linked via a linker to the constant region of the mutant (KACIAH) human TCRb chain, while the vH fragment derived from the FMC63 monoclonal antibody was linked via a linker to the constant region of the mutant (CSDVP) human TCR α chain.

In some embodiments, the SIR polypeptide has two or more antigen binding domains derived from or constituting antibodies expressed as single chain variable fragments (scfvs) and individually linked to the NH2 termini of two constant chains of the T cell receptor (i.e., constant chains of TCR- α, TCR- β 1, TCR- β 2, preTCR- α, pre-TCR- α -Del48, TCR- γ, or TCR- δ, or variants or mutants thereof). In some embodiments, the two (or more) antigen-binding domains of the encoded SIR molecule are encoded by a nucleotide sequence encoding two single-chain variable fragments (scfvs) fused in frame to two constant chains derived from the T cell receptor (i.e., TCR- α, TCR- β 1, TCR- β 2, pre-TCR- α -Del48, TCR- γ, or TCR- δ constant chains). The two scFv fragments encoded may target the same antigen (i.e. non-specific SIR) or different antigens (i.e. bispecific or multispecific SIR). In the case of a non-specific SIR, the two scfvs may encode polypeptides having the same amino acid sequence or different amino acid sequences. In addition, in the case of a non-specific SIR, where two scfvs are encoded by polypeptides having the same amino acid sequence, the nucleotide sequences encoding the two same scFv polypeptides may be the same or different. An exemplary nonspecific SIR with two scFv is represented by SEQ ID NO: 1026. The two antigen-binding domains of the SIR constitute scfvs derived from two different monoclonal antibodies, CD19Bu12 and FMC63, which target the human CD19 antigen. An exemplary multispecific SIR with two scFv is represented by SEQ ID NO: 1028. The two antigen-binding domains of the SIR constitute scfvs derived from two different monoclonal antibodies, CD19Bu12 and CD20-2F2, which target the human CD19 and CD20 antigens, respectively. An example of such a SIR is represented by 040716-B04 (CD8SP-CD19Bu12-scFv-V5- [ hTCRB-KACIAH ] -F-P2A-SP-CD20-2F2-scFv-Myc- [ hTCRA-CSDVP ] -F-F2A-PAC), which targets CD19 and CD20, and the corresponding amino acid sequence is represented by SEQ ID NO: 1028.

An exemplary SIR with two binding domains is represented by 040716-B04 (CD8SP-CD19Bu12-scFv-V5- [ hTCRb-KACIAH ] -F-P2A-SP-CD20-2F2-scFv-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC) which targets CD19 and CD20, the corresponding amino acid sequence being represented by SEQ ID NO: 1028. The two scFv polypeptide fragments can target the same antigen (i.e., a non-specific SIR) or different antigens (i.e., a bispecific or multispecific SIR). In the case of a non-specific SIR, the two scfvs may have the same amino acid sequence or different amino acid sequences. Exemplary SIRs targeting two different antigens are represented by SEQ ID NOS: 1028 and 1163.

In certain embodiments, the antigen binding domains of the two SIR polypeptides are structurally similar (e.g., the two antigen binding domains are scFv or camelid VHH domains or affibodies or vL or vH). For example, the antigen binding domain of the first SIR polypeptide comprises a camelid VHH domain targeted to Her2 and the antigen binding domain of the second SIR polypeptide comprises a VHH domain targeted to Her 3. The SIR consisting of both antigen binding domains by the vL chain is CD8SP-FMC63-11-vL-V5- [ TCRb-KACIAH ] -F-P2A-FMC63vL-Myc- [ TCRa-CSDVP ] -F-F2A-PAC and is represented by SEQ ID NO: 10474. In one embodiment, the antigen binding domains of the two SIR polypeptides are structurally dissimilar (e.g., the first antigen binding domain is an scFv and the second antigen binding domain is a camelidae VHH). An exemplary such SIR is CD8SP-IL6R-304-vHH-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC 63-vL-Gly-Ser-Gly-linker-vH-MYC- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1166). In certain embodiments, the antigen binding domain of the first SIR polypeptide (functional polypeptide unit 1) comprises a camelid VHH domain targeted to CD123 and the antigen binding domain of the second SIR polypeptide (functional polypeptide unit 2) comprises an scFv targeted to MPL.

In some embodiments, the antigen binding domain of the encoded SIR polypeptide is encoded by a codon optimized nucleotide sequence corresponding to a wild-type sequence or non-wild-type sequence antibody, a Single Domain Antibody (SDAB), a VH domain, a VL domain, a camelid VHH domain, or a non-immunoglobulin scaffold, such as DARPIN, an affibody, an affilin, an adnectin, an affitin, an obodies, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centryrin, a pronectin, an anticalin, a kunitz-type domain, an ardillo repeat protein, an autoantigen, a receptor, or a ligand.

In some embodiments, the one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the light chain variable domain (vL or vL) amino acid sequences of SEQ ID NOs 2307 to 2482 and 12042 to 12159 (in which NO more than 9 amino acid residues, but NO more than 10 amino acids, are replaced by any other amino acid residues), or sequences having 80-100% identity to the amino acid sequences of SEQ ID NOs 2307 to 2482 and 12042 to 12159, or sequences having 98-100% identity to the Complementarity Determining Regions (CDRs) of SEQ ID NOs 2307 to 2482 and 12042 to 12159. Table 5 shows the target antigen, name, SEQ ID NO (DNA), SEQ ID NO (PRT) of CDR1-3 of the exemplary vL domains used in the present disclosure.

In some embodiments, the one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the heavy chain variable domain (vH or vH) amino acid sequences of SEQ ID NOs 2506 to 2680 and 12160 to 12278 (in which up to 9 amino acid residues but NO more than 10 amino acids are replaced by any other amino acid residues), or sequences having 80-100% identity to the amino acid sequences of SEQ ID NOs 2506 to 2680 and 12160 to 12278, or sequences having 98-100% identity to the Complementarity Determining Regions (CDRs) of SEQ ID NOs 2506 to 2680 and 12160 to 12278. Table 5 shows target antigens for CDR1-3 of exemplary vH domains used in the present disclosure, showing target antigen, name, SEQ ID NO (DNA), SEQ ID NO (PRT). The name of the vH segment can be used to identify the corresponding vL segment based on the name of the latter. For example, the vH fragment Alk-48-vH (SEQ ID NO:226) is derived from the same antibody or scFv as the vL fragment Alk-48-vL (SEQ ID NO:16), and the two components can be used together to make an scFv or SIR that targets ALK. In some cases, Table 5 lists two or more vL or vH fragments having the same name followed by a number, such as FMC63(SEQ ID NO:30), FMC63- [2] -vL (SEQ ID NO:31), and FMC63- [3] -vL (SEQ ID NO: 32). In such cases, any of the above FMC63vL chains may be linked to any of the FMC63-vH chains (SEQ ID NOs: 241 and 242) to generate a corresponding SIR based on the FMC 63-based binding domain.

In some embodiments, the one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the camelidae single domain antibody (vHH or VHH) amino acid sequences of SEQ ID NOs 2701 to 2725 and 12279-12294 (in which NO more than 9 amino acid residues but NO more than 10 amino acids are substituted by any other amino acid residue), or sequences having 80-100% identity to the amino acid sequences of SEQ ID NOs 2701 to 2725 and 12279-12294, or sequences having 98-100% identity in the three Complementarity Determining Regions (CDRs) of SEQ ID NOs 2701 to 2725 and 12279-12294. Table 5 shows the target antigen, name, SEQ ID NO (DNA) and SEQ ID NO (PRT) of the exemplary vHH domain used in the present disclosure.

In some embodiments, the one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the non-immunoglobulin antigen binding scaffold amino acid sequences of SEQ ID NOs 2728 to 2732 and 12296-12301 (in which NO more than 9 amino acid residues but NO more than 10 amino acids are substituted by any other amino acid residues), or sequences having 80-100% identity to the amino acid sequences of SEQ ID NOs 2728 to 2732 and 12296-12301. Table 6A shows target antigens, names, SEQ ID NOs (DNA), SEQ ID NOs (PRT), names of exemplary non-immunoglobulin antigen binding scaffolds used in the present disclosure.

In some embodiments, the one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the receptor amino acid sequences of SEQ ID NOs 2736 to 2747 (in which up to 19 amino acid residues but NO more than 20 amino acids are substituted by any other amino acid residues), or sequences having 80-100% identity to the amino acid sequences of SEQ ID NOs 2736 to 2747. Table 6A shows the target antigen, SEQ ID NO (DNA), SEQ ID NO (PRT) and name.

In some embodiments, the one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the autoantigen amino acid sequence of SEQ ID NO 2748 (in which up to 19 amino acid residues but NO more than 20 amino acids are substituted by any other amino acid residues), or a sequence having 80-100% identity to the amino acid sequence of SEQ ID NO 2748. Table 6A shows the target antigen, SEQ ID NO (DNA), SEQ ID NO (PRT) and name.

In some embodiments, the one or more encoded antigen binding domains of the SIR molecule comprise any one or more of the ligand amino acid sequences of SEQ ID NOs 2758 to 2768 and 12359-12361 and 18918 (in which up to 19 amino acid residues but NO more than 20 amino acids are substituted by any other amino acid residues), or sequences having 80-100% identity with the amino acid sequences SEQ ID NOs 2758 to 2768 and 12359-12361 and 18918. Table 6A shows target antigens, SEQ ID NOs (DNA), SEQ ID NOs (PRT) and names.

In some embodiments, the one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the scFv amino acid sequences of SEQ ID NOs 2770 to 2939, 12303-12357 and 18162-18224 (in which NO more than 18 amino acid residues but NO more than 20 amino acids are replaced by any other amino acid residues), or sequences having 80-100% identity with the amino acid sequences SEQ ID NOs 2770 to 2939, 12303-12357 and 18162-18224, or sequences having 98-100% identity among the six Complementarity Determining Regions (CDRs) of SEQ ID NOs 2770 to 2939, 12303-12357 and 18162-182244. Table 5 shows target antigens, SEQ ID NOs (DNA), SEQ ID NOs (PRT), names and amino acid sequences of exemplary scFV used in the present disclosure.

In some embodiments, one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the antigen binding portions (e.g., CDRs) that target the vL and vH fragments of the antigen. The SEQ ID NOs of CDR1-3 of the vL and vH fragments targeting different antigens are listed in table 5.

In some embodiments, the one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the vL and antigen binding portions (e.g., CDRs) of the vH fragment of the scFv comprising the SIR polypeptide. The SEQ ID NOs comprising vL targeting scFv fragments of different antigens and CDR1-3 of vH fragments are listed in table 5. The SEQ ID NOs (DNA) and SEQ ID NOs (PRT) of scFv fragments targeting different antigens and the sequences of their corresponding CDRs 1-3 are listed in table 6A can be determined by methods known in the art or from their SEQ ID NOs of CDR1-3 of the component vL and vH fragments listed in table 5.

In some embodiments, one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the antigen binding portions (e.g., CDRs) of the vHH fragment that target the antigen. The SEQ ID NOs of vHH fragments targeting different antigens and the sequences of their corresponding CDRs 1-3 are listed in table 5 and can be determined by methods known in the art.

In one embodiment, the antigen binding domain of the SIR is an antigen binding portion of a receptor known to bind this target antigen.

In some embodiments, the one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the antigen binding portions of a receptor comprising the SIR polypeptide.

In some embodiments, the one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the antigen binding portions of a ligand comprising the SIR polypeptide.

In some embodiments, the one or more encoded antigen binding domains of the SIR polypeptide comprise any one or more of the antigen binding portions of a non-immunoglobulin scaffold comprising the SIR polypeptide.

In another embodiment, the disclosure provides SIRs that bind to the same epitope on the different targets described in tables 7A-7H as any one of the SIRs of the disclosure (i.e., SIRs that have the ability to cross-compete with any one of the SIRs of the disclosure for binding to a different target). In some embodiments, the antigen-specific domains of these SIRs may be determined by vL fragments, vH fragments, and/or scFv fragments of antibodies used as components of SIRs. In some embodiments, the reference antibody used in the cross-competition study to determine the target epitope recognized by the SIRs of the present disclosure described in tables 7A-7H is an scFv having the sequence shown as SEQ ID NOs 2770 to 2939, 12303-12357 and 18162-18224 (Table 6B). In an exemplary embodiment, the reference scFv FMC63, represented by SEQ ID NO:2770, can be used in cross-competition studies to determine the target epitope recognized by the FMC 63-based SIRs of the present disclosure described in tables 7A-7H. In some embodiments, the reference vHH fragment used in the cross-competition study to determine the target epitope recognized by the SIRs of the present disclosure described in tables 7A-7H is the vHH fragment having the sequence shown as SEQ ID NOs 2701 to 2725 and 12279-12294 (Table 5). In some embodiments, the reference non-immunoglobulin antigen binding scaffold used in the cross-competition study for determining the target epitope recognized by the SIR of the present disclosure described in tables 7A-7H is a non-immunoglobulin antigen binding scaffold having a sequence as set forth in SEQ ID NOS 2728 to 2732 and 12296-12301 (Table 6A). In some embodiments, the reference ligands used in the cross-competition studies to determine the target epitopes recognized by the SIRs of the present disclosure described in tables 7A-7H are ligands having the sequences shown as SEQ ID NOs: 2758 to 2768 and 12359-12361 (Table 6A). In some embodiments, the reference SIR for cross-competition studies against SIRs targeting different targets is the SIR with the sequence shown in SEQ ID NO: 3435-.

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the MPL-targeting SIRs of the present disclosure (e.g., SEQ ID NOS: 3566-3562, 13259, and 13265-13266) are the corresponding scFv shown in Table 6B (e.g., SEQ ID NOS: 2871-2878, 12318, 12326-12327). In one embodiment, the reference scFv for use in the cross-competition study to determine the target epitope recognized by the SIR targeting MPL of the present disclosure is represented by SEQ ID NO: 2871-2874.

In another embodiment, the reference ligand used in the cross-competition study to determine the target epitope recognized by the SIR targeting MPL of the present disclosure is the corresponding ligand shown in Table 6A (e.g., SEQ ID NO: 2758-2759).

In another embodiment, the reference SIR used in the cross-competition study to determine the target epitope recognized by the SIR targeting MPL of the present disclosure is MPL-SIR as shown in tables 7D and 7E (e.g., SEQ ID NO:3566-3562, 13259, and 13265-13266).

In another embodiment, the SIR of MPL-targeting of the present disclosure binds to an epitope of MPL that corresponds to or overlaps with the peptide sequence PWQDGPK- (SEQ ID NO: 15784).

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR targeting CD19 of the present disclosure (e.g., SEQ ID NOS: 3645. about. 3649, 13195. about. 13203, 13249, and 13267) is the corresponding scFv shown in Table 6B (e.g., SEQ ID NOS: 2770. about. 2774, 12308, 12325, 18162. about. 18170). In one embodiment, the reference scfvs used to determine the target epitope recognized by the SIRs targeting CD19 of the present disclosure in a cross-competition study are represented by SEQ ID NOs 2771, 2772, 12308, and 18169.

In another embodiment, the reference SIR used in the cross-competition studies to determine the target epitope recognized by the SIR targeting CD19 of the present disclosure is the SIR targeting CD19 shown in tables 7A-7H (e.g., SEQ ID NOs: 3645-3649, 13195-13203, 13249 and 13267).

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR targeting CD20 of the present disclosure (e.g., SEQ ID NO:3456-3457, 13204-13213) is the corresponding scFv shown in Table 6B (e.g., SEQ ID NO:2787-2788, 18177-18187). In one embodiment, the reference scfvs used to determine the target epitope recognized by the SIRs targeting CD20 of the present disclosure in a cross-competition study are represented by SEQ ID NOs 18182, 18185, and 2787.

In another embodiment, the reference SIR used in the cross-competition study to determine the target epitope recognized by the SIR of the targeted CD20 of the present disclosure is the CD20-SIR shown in tables 7A-7H (e.g., SEQ ID NO:3456-3457, 13204-13213).

In preferred embodiments, SIRs of the present disclosure that target CD20 bind to an epitope corresponding to one or more of the following sequences: : -PAGOYAPI- (SEQ ID NO:18902), -FLKMESLNFIRAHTP- (SEQ ID NO:18903), -HFLKMESLNFIRAHTPY- (SEQ ID NO:18904), -YNAEPANPSEKNSPSTQY- (SEQ ID NO:18905), -YNAEPANPSEKNSPST- (SEQ ID NO:18906) and-YNCEPANPSEKNSP- (SEQ ID NO: 18907).

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the BCMA-targeting SIRs of the present disclosure (e.g., SEQ ID NOS: 3446. about. 3449, 3632. about. 3634, 13277. about. 13284) are the corresponding scFv shown in Table 6B (e.g., SEQ ID NOS: 2780. about. 2783, 12337. about. 12344, and 18174. about. 18176). In one embodiment, the reference scFv for use in the cross-competition study to determine the target epitope recognized by the BCMA-targeting SIR of the present disclosure is represented by SEQ ID NO:2780-2781, 18175-18176.

In another embodiment, the reference SIR used in the cross-competition study to determine the target epitope recognized by the BCMA-targeting SIRs of the present disclosure is the BCMA-SIR shown in tables 7A-7H (e.g., SEQ ID NOS: 3446-3449, 3632-3634, 13277-13284).

In preferred embodiments, BCMA-targeted SIRs of the present disclosure bind to epitopes corresponding to one or more of the sequences set forth in SEQ ID NO 18908-18912.

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR targeting CD22 of the present disclosure (e.g., SEQ ID NOs: 3458-3460, 13241-13245, 13268) is the corresponding scFv set forth in Table 6B (e.g., SEQ ID NOs: 2789-2791, 12320-12324, 12330, 18188). In one embodiment, the reference scfvs used to determine the target epitope recognized by the SIRs targeting CD22 of the present disclosure in a cross-competition study are represented by seq id NOs 18188, 12330, and 12320.

In another embodiment, the reference SIR used in the cross-competition study to determine the target epitope recognized by the SIR of the targeted CD22 of the present disclosure is the CD22-SIR shown in tables 7A-7H (e.g., SEQ ID NOS: 3458-3460, 13241-13245, 13268).

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR targeting CD123 of the present disclosure (e.g., SEQ ID NOS: 2929, 3470, 13184-. In one embodiment, the reference scFv used in the cross-competition studies to determine the target epitope recognized by the SIRs targeting CD123 of the present disclosure is represented by SEQ ID NOs 2929, 18196, 18197, 18200, 18202, and 18205.

In another embodiment, the reference SIR used in the cross-competition study to determine the target epitope recognized by the SIR targeting CD123 of the present disclosure is the CD123-SIR shown in tables 7A-7H (e.g., SEQ ID NOS: 3470, 13184-13194).

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR targeting CD33 of the present disclosure (e.g., SEQ ID NO: 3464. sup. 3465, 13214. sup. 13220) is the corresponding scFv shown in Table 6B (e.g., SEQ ID NO: 2795. sup. 2796, 18189. sup. 18194). In one embodiment, the reference scfvs used to determine the target epitope recognized by the SIRs targeting CD33 of the present disclosure in a cross-competition study are represented by SEQ ID NOs 2795, 2796, and 18127.

In another embodiment, the reference SIR used in the cross-competition study to determine the target epitope recognized by the SIR of the targeted CD33 of the present disclosure is the CD33-SIR shown in tables 7A-7H (e.g., SEQ ID NO:3464-3465, 13214-13220).

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR targeting CS1 of the present disclosure (e.g., SEQ ID NO: 3487. sub.3489, 13226. sub.1323) is the corresponding scFv shown in Table 6B (e.g., SEQ ID NO: 2817. sub.2819, 18211. sub.18216). In one embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR of the targeting CS1 of the present disclosure is represented by SEQ ID NOs 2818, 18212, 18213, 18215, and 18216.

In another embodiment, the reference SIR used in the cross-competition studies to determine the target epitope recognized by the SIR of the targeted CS1 of the present disclosure is the CS1-SIR shown in tables 7A-7H (e.g., SEQ ID NO: 3487. sub. 3489, 13226. sub. 1323).

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR targeting CLL1 of the present disclosure (e.g., SEQ ID NOS: 3484. about. 3485, 13222. about. 13225) is the corresponding scFv shown in Table 6B (e.g., SEQ ID NOS: 2814. about. 2815, 18207. about. 18210, 12345. about. 12346).

In another embodiment, the reference SIR used in the cross-competition study to determine the target epitope recognized by the SIR targeting CLL1 of the present disclosure is the CLL1-SIR shown in tables 7A-7H (e.g., SEQ ID NO:3484-3485, 13222-13225).

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the mesothelin-targeting SIR of the present disclosure (e.g., SEQ ID NO:3554, 13287-.

In another embodiment, the reference SIR used in the cross-competition studies to determine the target epitope recognized by the SIR of the targeted mesothelins of the present disclosure is the mesothelin-SIR shown in tables 7A-7H (e.g., SEQ ID NO:3554, 13287- -.

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIRs targeting BST1/CD157 of the present disclosure is the corresponding ligand shown in table 6B.

In another embodiment, the reference SIR used in the cross-competition studies to determine the target epitope recognized by the SIRs targeting BST1/CD157 of the present disclosure is the BST1/CD157-SIR shown in tables 7A-7H.

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR of the targeted DLL3 of the present disclosure is the corresponding scFv shown in table 6B.

In another embodiment, the reference SIR used in the cross-competition studies to determine the target epitope recognized by the SIRs of the targeted DLL3 of the present disclosure is DLL3-SIR shown in tables 7A-7H.

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR of the targeting PTK7 of the present disclosure is the corresponding scFv shown in table 6B.

In another embodiment, the reference SIR used in the cross-competition studies to determine the target epitope recognized by the SIRs of the targeted PTK7 of the present disclosure is the PTK7-SIR shown in tables 7A-7H.

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIRs targeting IL13Ra2 of the present disclosure is the corresponding scFv shown in table 6B.

In another embodiment, the reference SIR used in the cross-competition studies to determine the target epitope recognized by the SIRs of the targeting IL13Ra2 of the present disclosure is IL13Ra2-SIR shown in tables 7A-7H.

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR targeting ROR1 of the present disclosure is the corresponding scFv shown in table 6B.

In another embodiment, the reference SIR used in the cross-competition studies to determine the target epitope recognized by the SIRs of the targeted ROR1 of the present disclosure is ROR1-SIR shown in tables 7A-7H.

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the TCRgd-targeting SIR of the present disclosure is the corresponding scFv shown in table 6B.

In another embodiment, the reference SIR used in the cross-competition studies to determine the target epitope recognized by the TCRgd-targeting SIRs of the present disclosure is the TCRgd-SIR shown in tables 7A-7H.

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR targeting TCRB1 of the present disclosure is the corresponding scFv shown in table 6B.

In another embodiment, the reference SIR used in the cross-competition studies to determine the target epitope recognized by the SIRs of the targeted TCRB1 of the present disclosure is the TCRB1-SIR shown in tables 7A-7H.

In another embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR targeting TCRB2 of the present disclosure is the corresponding scFv shown in table 6B.

In another embodiment, the reference SIR used in the cross-competition studies to determine the target epitope recognized by the SIRs of the targeted TCRB2 of the present disclosure is the TCRB2-SIR shown in tables 7A-7H.

In some embodiments, the SIRs targeting gp100, MART, tyrosinase, hTERT, MUC1, CMV-pp65, HTLV1-Tax, HIV1-gag, NY-ESO, WT1, AFP, HPV-16-E7, PR1, and Ras G12V bind to targeting peptides complexed to MHC class I (e.g., HLA-A201) as shown in Table 7I.

In one embodiment, the reference scfvs used to determine the target epitope recognized by the AFP/MHC I-targeted SIRs of the present disclosure in a cross-competition study are represented by SEQ ID NOs 18171 and 18173.

In one embodiment, the reference scFv for use in a cross-competition study to determine the target epitope recognized by the SIR targeting WT1/MHC I of the present disclosure is represented by SEQ ID NO: 2926-2928.

In one embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR targeting ALK of the present disclosure is represented by SEQ ID NO: 2777.

In one embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR of the targeting B7H4 of the present disclosure is represented by SEQ ID NO: 2934-2935.

In one embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR of the targeted CD30 of the present disclosure is represented by SEQ ID NO: 2792-2793.

In one embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIRs targeting CD138 of the present disclosure is represented by SEQ ID NO: 2802.

In one embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIRs targeting EGFRviii of the present disclosure is represented by SEQ ID NO: 2826.

In one embodiment, the reference scFv used in the cross-competition study to determine the target epitope recognized by the SIR targeting FR1 (folate receptor 1) of the present disclosure is represented by SEQ ID NO: 2833.

In another embodiment, the epitope for use in a cross-competitive study to determine the epitope to be recognized by a targeting trpp 2, LAMP1, CDH19, CDH17, CD70, CD79 2, CDH6, TSHR, ALK, WT1/MHC 1, NY-ESO-1/MHC I, HIV1env gp, NYBR1, Lym1, Lym2, TSLRP, folate receptor alpha, B7H4, CD200R, Igk-light chain, CD 179R, Cripto, STEAP R, hLiv R, ILRAP, laminin-4, gppa R, PSCA, PSMA, Muc R/MHC I, GFRa R, EGFRviii, EGFR, Her R, CSF2R, CLEC 5R, GPRC 5R, prc 5R, PSCA-Muc R, FLT R/MHC 695 72, MHC 72/MHC I, EGFR R, MHC R/MHC I, scFv R, and scFv 366 in a scFv R of the scFv R are references in the scFv R.

In another embodiment, the epitope for use in a cross-competitive study to determine targeting of trpp 2, LAMP1, CDH19, CDH17, CD70, CD79B, CDH6, TSHR, ALK, WT1/MHC 1, NY-ESO-1/MHC I, HIV1 coated glycoprotein, NYBR1, Lym1, TSLRP, folate receptor alpha, B7H 1, CD200 1, Igk-light chain, CD179 1, Cripto, STEAP1, hLiv1, ILRAP, laminin-4, gppa 1, PSCA, PSMA, Muc1/MHC I, GFRa 1, EGFRviii, EGFR, Her 1, CSF2 CSF 1, CLEC 51, GPRC 51, prc 51, Muc-Muc 1, FLT 1, SIR 1/SIR 1, SIR-l 1, SIR-MHC I, and SIR-7A of the present disclosure is a reference epitope for recognition in a reference table.

Table 6A:

table 6B:

table 6C: MHC I (HLA-A2) restricted peptides for generating SIR

Protein	Segment names	Peptide SEQ	SEQ ID
				gp100	G9-209	IMDQVPFSV	15764
gp100	G9-280	YLEPGPVTV	15765
				gp100	G9-154	KTWGQYWQV	15766
MUC1-A7(130-138)	A7	NLTISDVSV	15767
				MUC1-D6(13-21)	D6	LLLTVLTVV	15768
TAX(11-19)		LLFGYPVYV	15769
				hTERT(540-548)	T540	ILAKFLHWL	15770
hTERT(865-873)	T865	RLVDDFLLV	15771
				HIV1gag(77-85)	SL9	SLYNTVATL	15772
CMV-pp65(495-503)		NLVPMVATV	15773
				MART(26-35)		EAAGIGILTV	15774
EBNA-3A(596-604)		SVRDRLARL	15775
				EBNA-3c		LLDFVRFMGV	15776
WT1		RMFPNAPYL	15777
				PR1		VLQELNVTV	15778
Ras	Ras9-G12V	LVWGAVGV	15779
				HPV	HPV16-E7	YMLDLQPET	15780
NY-ESO-1	NY-ESO-1-(155-163)	QLSLLMWIT	15781
				NY-ESO-1	NY-ESO-1-(157-165)	SLLMWITQC	15782
NY-ESO-1	NY-ESO-1-(157-167)	SLLMWITQCFL	15783

Table 6D: exemplary Joint for generating SIR

In one embodiment of the disclosure, the SIR construct comprises scFv domains, wherein the scFv may be preceded by an optional leader sequence such as the sequence provided by any of SEQ ID NOS: 2300, 2301 or 2302, and followed by an optional linker sequence such as the sequence provided by any of SEQ ID NOS: 2981 and 2986 and a T cell receptor constant chain such as the constant chains provided by any of SEQ ID NOS: 3010 to 3020, SEQ ID NOS: 3022 to 3044, SEQ ID NOS: 3045 to 3052 (including mutants and variants as described herein), wherein these domains are contiguous and in the same reading frame to form a single fusion protein. The linker sequence may or may not be present in the SIR construct. In one embodiment, the SIR contains two functional polypeptide units, in which case the two units are separated by a cleavable linker such as the linkers provided in SEQ ID NOs 3060 to 3064. The cleavable linker may be preceded by a short flexible linker (e.g., SGSG) such as SEQ ID NO:3065 and a furin cleavage site (e.g., RAKR) such as SEQ ID NO: 3066. The two functional polypeptide units of the SIR may also be encoded by two different polynucleotides separated by an IRES sequence. Alternatively, two different polynucleotides encoding two functional polypeptide units of the SIR may be encoded by two different vectors.

In one embodiment, an exemplary SIR construct comprises a leader sequence (e.g., a leader sequence described herein), an extracellular antigen-binding domain (e.g., an antigen-binding domain described herein), an optional linker (e.g., a linker region described herein), and a T cell receptor constant chain (e.g., a T cell receptor constant chain described herein, including mutants and variants).

For example, the SIR of the present disclosure comprises an exemplary leader sequence selected from SEQ ID NOs 2300, 2301 and 2302 linked to an antigen binding domain such as any of the antigen binding domains identified in Table 5 and tables 6A-B; an optional linker sequence selected from SEQ ID NOs 2981 to 2985 and 2986 (see also table 6D) and linked to a T cell receptor constant chain domain comprising a sequence selected from the group consisting of SEQ ID NOs 3010 to 3020, 3022 to 3044, 3045 to 3051 and 3052 (and mutants and variants thereof as described herein). The SIR may further comprise a fusion of the extracellular domain of the T cell receptor constant chain and the extracellular transmembrane cytosolic domain of CD3z (such as provided in SEQ ID NO:3021 and SEQ ID NO: 3045), as well as mutants and variants.

In any of the embodiments described herein, effector cells expressing the SIR exhibit higher binding to the target antigen than do corresponding effector cells expressing the tcr (such as effector cells presenting on their surface a tcr comprising an antigen binding domain of the SIR, such as a tcr comprising an scFv, vL, and/or vH fragment comprising the antigen binding domain of the SIR) when compared under similar conditions. An exemplary SIR targeting CD19 is represented by CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1200). The cTCR corresponding to the target CD19 is represented by CD8SP-FMC63-vL- [ hTCRb-WT ] -F-P2A-SP-FMC63-vH- [ hTCRa-WT ] -F-F2A-PAC (SEQ ID NO: 18280). The nucleotides and amino acid SEQ ID nos of several exemplary tcr of the present disclosure are provided in tables 7A-7B. For example, in some embodiments, SIR-expressing effector cells targeted to CD19 have higher binding to CD19-ECD-GGSG-NLuc-AcV5 fusion protein after incubation for 60 minutes at 4 ℃ than corresponding tcr-expressing effector cells under similar conditions. In some embodiments, effector cells expressing SIR have higher specific binding to the target antigen than corresponding effector cells expressing tcr under similar conditions. Specific binding of the target antigen to SIR-expressing effector cells is measured by subtracting the value obtained using control effector cells from the binding value obtained using SIR-expressing effector cells. In some embodiments, the control effector cell is a parent effector cell that does not express any SIR, e.g., an untransduced T cell. In an alternative embodiment, the control effector cell is a control SIR that expresses an antigen that targets an antigen other than the antigen targeted by the test SIR. An exemplary control SIR to compare the binding activity of CD19-SIR is CD8SP-MPL-161-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-161-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1322). In some embodiments, the binding of effector cells expressing SIR to the target antigen after incubation for 60 minutes at 4 ℃ is at least 5%, 10%, 20%, 30%, 40%, 50% or 100% greater than the binding of effector cells corresponding to tcr expression. In some embodiments, effector cells expressing SIR bind to the target antigen at least 1.25-fold (e.g., 1.5-fold, 2-fold, 5-fold, or 10-fold) greater than the binding of corresponding effector cells expressing tcr. In some embodiments, effector cells expressing SIR bind to the target antigen no more than 100000-fold (e.g., 5000-fold, 10000-fold, or 50000-fold) higher than the corresponding effector cells expressing tcr. In some embodiments, the binding of an effector cell expressing an SIR to a target antigen is about 1.25-fold to about 100000-fold (e.g., about 5-fold to about 50000-fold, about 10-fold to about 10000-fold, or about 100-fold to about 1000-fold, and any value between any of the foregoing ranges) greater than the binding of a corresponding effector cell expressing a tcr. In some embodiments, under similar conditions, the binding of an effector cell expressing SIR to a target antigen after incubation for 60 minutes at 4 ℃ is at least 1.25-fold to less than about 100000-fold greater than the binding of a corresponding effector cell expressing tcr. In some embodiments, the effector cell expressing SIR is an SIR T cell. In some embodiments, the effector cell expressing SIR is a Jurkat T cell expressing SIR.

In other embodiments described herein, effector cells expressing a SIR exhibit lower binding to a target antigen than corresponding effector cells expressing a CAR (such as a CAR presenting an antigen binding domain comprising a SIR on its surface, such as effector cells of a CAR comprising an scFv comprising an antigen binding domain of a SIR) when compared under similar conditions. An exemplary SIR targeting CD19 is represented by CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1200). The corresponding CD 19-targeted CAR was represented by CD8SP-FMC63(vL-vH) -Myc-BBz-T2A-PAC (SEQ ID NO: 4501). Provided herein are the nucleotides and amino acid SEQ ID nos of several exemplary CARs. For example, in some embodiments, SIR-expressing effector cells that target CD19 have lower binding to CD19-ECD-GGSG-NLuc-AcV5 than corresponding CAR-expressing effector cells under similar conditions. In some embodiments, under similar conditions, binding of an effector cell expressing an SIR to a target antigen after incubation for 60 minutes at 4 ℃ is at least 5% (e.g., 10%, 20%, 30%, 40%, or 50%, or any value between any of the foregoing integers) less than the binding of a corresponding effector cell expressing a CAR. In some embodiments, binding of the SIR-expressing effector cell to the target antigen is at least about 1.5-fold less than the binding of the corresponding CAR-expressing effector cell (e.g., 2-fold, 5-fold, 10-fold, 20-100-fold, 500-fold, 1000-fold, 10000-fold, 50000-fold, or 50000-fold, 100000-fold, or any integer therebetween). In some embodiments, under similar conditions, the binding of an effector cell expressing an SIR to a target antigen after incubation for 60 minutes at 4 ℃ is at least 1.25-fold to 100000-fold (or any value therebetween) less than the binding of a corresponding effector cell expressing a CAR. In some embodiments, the effector cell expressing SIR is an SIR T cell. In some embodiments, the effector cell expressing SIR is a Jurkat T cell expressing SIR.

In other embodiments described herein, effector cells expressing SIR exhibit higher binding to the target antigen than do corresponding effector cells expressing tcr, but exhibit lower binding to the target antigen than do corresponding effector cells expressing CAR, when compared under similar conditions. For example, in some embodiments, SIR-expressing effector cells that target CD19 have higher binding to CD19-ECD-GGSG-NLuc-AcV5 than the corresponding tcr-expressing effector cells, but lower binding than the corresponding CAR-expressing effector cells, under similar conditions. In some embodiments, under similar conditions, binding of effector cells expressing SIR to target antigen after incubation for 60 minutes at 4 ℃ is at least 5% greater (e.g., 10%, 20%, 30%, 40%, 50%, or 100%, or any value therebetween) than binding of effector cells corresponding to tcr, but at least 5% less (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any value therebetween) than binding of effector cells corresponding to CAR. In some embodiments, the binding of effector cells expressing SIR to target antigen after incubation for 60 minutes at 4 ℃ is at least 1.25-fold (e.g., 1.5-fold, 2-fold, 5-fold, or 10-fold, or any value therebetween) greater than the binding of corresponding effector cells expressing tcr under similar conditions, but at least 1.5-fold (e.g., 2-fold, 5-fold, or 10-fold, or any value therebetween) less than the binding of corresponding effector cells expressing CAR. In some embodiments, the effector cell expressing SIR is an SIR T cell. In some embodiments, the effector cell expressing SIR is a Jurkat T cell expressing SIR.

In other embodiments described herein, the SIR exhibits higher cell surface expression compared to the corresponding tcr when expressed in effector cells and compared under similar conditions. An exemplary SIR targeting CD19 is represented by CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1200). The cTCR corresponding to the target CD19 is represented by CD8SP-FMC63-vL- [ hTCRb-WT ] -F-P2A-SP-FMC63-vH- [ hTCRa-WT ] -F-F2A-PAC (SEQ ID NO: 18280). For example, in some embodiments, the cell surface expression of SIR (as measured by binding to APL-conjugated protein L) is higher than the cell surface expression of the corresponding tcr when examined under similar conditions. In some embodiments, effector cells expressing SIR have greater than about 5% (up to greater than about any of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, including any range between these values) binding to APC-conjugated protein L after 60 minutes of incubation compared to corresponding effector cells expressing tcr. In some embodiments, the effector cell expressing SIR is an SIR T cell. SIR and the expression of the corresponding tcr on the surface of effector cells can be measured by alternative methods, including binding to the CD8 SP-protein L-GGSG-NLuc-4xFLAG-x2STREP-8xHis fusion protein or staining with an epitope tag (e.g., MYC tag) inserted within the extracellular domain of SIR and tcr.

In other embodiments described herein, the SIR exhibits lower cell surface expression compared to the corresponding CAR when expressed in effector cells and compared under similar conditions. An exemplary SIR targeting CD19 is represented by CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1200). The corresponding CD 19-targeted CAR was represented by CD8SP-FMC63(vL-vH) -Myc-BBz-T2A-PAC (SEQ ID NO: 4501). For example, in some embodiments, the cell surface expression of SIR (as measured by binding to APL-conjugated protein L) is lower than the cell surface expression of the corresponding CAR when examined under similar conditions. In some embodiments, under similar conditions, binding of SIR-expressing effector cells to APC-protein L is at least 5% (e.g., 10%, 20%, 30%, 40%, or 50%, or any value in between) less than the binding of corresponding CAR-expressing effector cells. In some embodiments, binding of SIR-expressing effector cells to APC-protein L is at least 1.5-fold to about 1000-fold (or any value in between) less than the binding of corresponding CAR-expressing effector cells. In one embodiment, binding of SIR-expressing effector cells to APC-protein L is at least 2-fold to about 100-fold (or any value in between) less than the binding of corresponding CAR-expressing effector cells. In some embodiments, the effector cell expressing SIR is an SIR T cell. In some embodiments, the effector cell expressing SIR is a Jurkat T cell expressing SIR. SIR and the expression of the corresponding CAR on the surface of effector cells can be measured by alternative methods, including binding to the CD8 SP-protein L-GGSG-NLuc-4xFLAG-x2STREP-8xHis fusion protein or staining with an epitope tag (e.g., MYC tag) inserted in a comparable location (e.g., N-terminal region) within the extracellular domain of SIR and CAR.

In any of some of such embodiments described herein, the SIR, when expressed in effector cells and compared under similar conditions, exhibits higher cell surface expression compared to the corresponding tcr, but lower expression compared to the corresponding CAR. For example, in some embodiments, the cell surface expression of SIR (as measured by binding of protein L conjugated to APL) is higher than the cell surface expression of the corresponding tcr, but lower than the cell surface expression of the corresponding CAR, when examined under similar conditions. In some embodiments, an effector cell that expresses SIR has greater than about 5% (such as greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45%, including any range between these values) binding to APC-conjugated protein L after 60 minutes of incubation as compared to a corresponding effector cell that expresses CAR, but has less than about 50% (such as less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, including any range between these values) binding to APC-conjugated protein L after 60 minutes of incubation as compared to a corresponding effector cell that expresses CAR. In some embodiments, the effector cell expressing SIR is an SIR T cell.

In other embodiments described herein, effector cells expressing a SIR exhibit higher cytotoxicity to cells expressing a target antigen than corresponding effector cells expressing a tcr (such as effector cells presenting on their surface a tcr comprising an antigen binding domain of a SIR, such as a tcr comprising an scFv, vL, and/or vH fragment comprising an antigen binding domain of a SIR) when compared under similar conditions. An exemplary SIR targeting CD19 is represented by CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1200). The cTCR corresponding to the target CD19 is represented by CD8SP-FMC63-vL- [ hTCRb-WT ] -F-P2A-SP-FMC63-vH- [ hTCRa-WT ] -F-F2A-PAC (SEQ ID NO: 18280). The nucleotides and amino acid seq id nos of several exemplary SIRs and ctcrs of the present disclosure are provided in tables 7A and 7D. For example, in some embodiments, SIR-expressing effector cells that target CD19 exhibit higher cytotoxicity against RAJI-GLuc cells after incubation at 37 ℃ for 4 to 96 hours compared to corresponding tcr-expressing effector cells under similar conditions. In some embodiments, effector cells expressing SIR have higher specific cytotoxicity against cells expressing their target antigen than corresponding effector cells expressing tcr under similar conditions. Specific cytotoxicity of effector cells expressing SIR was measured by subtracting the value obtained using control effector cells from the cytotoxicity value obtained using effector cells expressing SIR. In some embodiments, the control effector cell is a parent effector cell that does not express any SIR, e.g., an untransduced T cell. In an alternative embodiment, the control effector cell is a control SIR that expresses an antigen targeted to an antigen other than the test SIR. For example, an exemplary control SIR to compare the cytotoxic activity of CD19SIR is CD8SP-MPL-161-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-161-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1322). In some embodiments, the cytotoxicity of an effector cell expressing an SIR is at least 5%, 10%, 20%, 30%, 40%, 50%, or 100% greater than the cytotoxicity of a corresponding effector cell expressing a tcr after co-culturing for 4 to 96 hours at 37 ℃. In some embodiments, the cytotoxicity of the effector cell expressing the SIR on the target cell is at least 1.25-fold, 1.5-fold, 2-fold, 5-fold, or 10-fold greater than the cytotoxicity of the corresponding effector cell expressing the tcr. In some embodiments, the effector cell expressing SIR is an SIR T cell.

In another or other embodiment of any of the preceding embodiments described herein, when compared under similar conditions, effector cells expressing a SIR exhibit greater in vivo activity against cells expressing a target antigen than corresponding effector cells expressing a tcr (such as effector cells presenting on their surface a tcr comprising an antigen binding domain of a SIR, such as a tcr comprising an scFv, vL, and/or vH fragment of an antigen binding domain comprising a SIR). An exemplary SIR targeting CD19 is represented by CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1200). The cTCR corresponding to the target CD19 is represented by CD8SP-FMC63-vL- [ hTCRb-WT ] -F-P2A-SP-FMC63-vH- [ hTCRa-WT ] -F-F2A-PAC (SEQ ID NO: 18280). The nucleotides and amino acid SEQ ID nos of several exemplary SIRs and ctcrs of the present disclosure are provided in tables 7A and 7D. For example, in some embodiments, SIR-expressing effector cells that target CD19 exhibit greater in vivo activity against RAJI-FLuc cells in an NSG mouse xenograft model than corresponding tcr-expressing effector cells under similar conditions. In some embodiments, effector cells expressing SIR have higher in vivo activity against cells expressing their target antigen than corresponding effector cells expressing tcr under similar conditions. The specific in vivo activity of effector cells expressing SIR is measured by subtracting the value obtained using control effector cells (e.g. the reduction in tumor or the reduction in bioluminescence value obtained from a tumor expressing FLuc) from the in vivo activity obtained using effector cells expressing SIR. In some embodiments, the control effector cell is a parent effector cell that does not express any SIR, e.g., an untransduced T cell. In an alternative embodiment, the control effector cell is a control SIR that expresses an antigen that targets an antigen other than the antigen targeted by the test SIR. For example, an exemplary control SIR for comparing the in vivo activity of CD19SIR is CD8SP-MPL-161-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-161-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1322). In some embodiments, the in vivo activity of an effector cell expressing an SIR against a cell expressing a target antigen (i.e., a target cell) is at least 5%, 10%, 20%, 30%, 40%, 50%, or 100% greater in an NSG mouse xenograft model than the in vivo activity of a corresponding effector cell expressing a tcr. In some embodiments, the in vivo activity of an effector cell expressing an SIR on a target cell is at least 1.25-fold, 1.5-fold, 2-fold, 5-fold, or 10-fold greater than the in vivo activity of a corresponding effector cell expressing a tcr. In some embodiments, the effector cell expressing SIR is an SIR T cell. In some embodiments, the in vivo activity of effector cells expressing SIR is measured by other methods, such as improvement in survival or reduction in tumor volume using caliper measurements.

In another or other embodiment of any of the preceding embodiments described herein, when compared under similar conditions, effector cells expressing an SIR exhibit higher TNF α production when co-cultured with their target cells (e.g., cells expressing their target antigen) compared to corresponding effector cells expressing a tcr (such as effector cells presenting on their surface a tcr comprising an antigen binding domain of an SIR, such as a tcr comprising an scFv, vL, and/or vH fragment comprising an antigen binding domain of an SIR). An exemplary SIR targeting CD19 is represented by CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1200). The cTCR corresponding to the target CD19 is represented by CD8SP-FMC63-vL- [ hTCRb-WT ] -F-P2A-SP-FMC63-vH- [ hTCRa-WT ] -F-F2A-PAC (SEQ ID NO: 18280). The nucleotides and amino acid SEQ ID nos of several exemplary SIRs and ctcrs targeting different antigens are provided in tables 7A and 7D. For example, in some embodiments, SIR-expressing effector cells that target CD19 have higher TNF α production when co-cultured with Nalm6 target cells at 37 ℃ for 4 to 96 hours, as measured by ELISA, than corresponding tcr-expressing effector cells under similar conditions. In some embodiments, effector cells expressing SIR have higher fold-induced TNF α production under similar conditions as compared to corresponding effector cells expressing tcr. The fold-induced TNF α production by SIR expressing effector cells is measured by dividing the TNF α value obtained when SIR expressing effector cells are cultured alone by the TNF α level obtained when SIR expressing cells are co-cultured with their target cells. In some embodiments, effector cells expressing SIR have higher specific TNF α production when co-cultured with their target cells compared to corresponding effector cells expressing tcr under similar conditions. Specific TNF α production by SIR-expressing effector cells upon exposure to target cells is measured by subtracting the value obtained using control effector cells from the TNF α value obtained using SIR-expressing effector cells when co-cultured with target cells at 37 ℃ for 4 hours to 96 hours under similar conditions. In some embodiments, the control effector cell is a parent effector cell that does not express any SIR, e.g., an untransduced T cell. In an alternative embodiment, the control effector cell is a control SIR that expresses an antigen that targets an antigen other than the antigen targeted by the test SIR. An exemplary control SIR to compare the binding activity of CD19SIR is CD8SP-MPL-161-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-161-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1322). In some embodiments, the specific TNF α production by the effector cells expressing SIR to the target antigen is at least 5%, 10%, 20%, 30%, 40%, 50% or 100% greater than the specific TNF α production by the corresponding effector cells expressing tcr after 24 hours incubation at 37 ℃. In some embodiments, the specific TNF α production of the effector cell expressing the SIR is at least 1.25-fold, 1.5-fold, 2-fold, 5-fold, or 10-fold greater than the specific TNF α production of the corresponding effector cell expressing the tcr. In some embodiments, the specific TNF α production by the effector cell expressing the SIR is less than 100000 times greater than the specific TNF α production by the corresponding effector cell expressing the tcr. In some embodiments, the specific TNF α production by the effector cell expressing the SIR is at least 1.25-fold, 1.5-fold, 2-fold, or 5-fold or 10-fold greater than the specific TNF α production by the effector cell corresponding to the tcr expressing under similar conditions, but is greater than 100000-fold (e.g., less than 50000-fold, 10000-fold, or 1000-fold) greater than the specific TNF α production by the effector cell corresponding to the tcr expressing under similar conditions. In some embodiments, the effector cell expressing SIR is an SIR T cell.

In another or other embodiment of any of the preceding embodiments described herein, when compared under similar conditions, effector cells expressing a SIR exhibit higher IL2 production when co-cultured with their target cells (e.g., cells expressing their target antigen) than do corresponding effector cells expressing a tcr (such as effector cells presenting on their surface a tcr comprising an antigen-binding domain of a SIR, such as a tcr comprising an scFv, vL, and/or vH fragment comprising an antigen-binding domain of a SIR). An exemplary SIR targeting CD19 is represented by CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1200). The cTCR corresponding to the target CD19 is represented by CD8SP-FMC63-vL- [ hTCRb-WT ] -F-P2A-SP-FMC63-vH- [ hTCRa-WT ] -F-F2A-PAC (SEQ ID NO: 18280). The nucleotides and amino acid SEQ ID nos of several exemplary SIRs and ctcrs targeting different antigens are provided in tables 7A and 7D. For example, in some embodiments, SIR-expressing effector cells that target CD19 have higher IL2 production when co-cultured with Nalm6 target cells at 37 ℃ for 4 to 96 hours, as measured by ELISA, compared to corresponding tcr-expressing effector cells under similar conditions. In some embodiments, effector cells expressing SIR have higher fold-induced IL2 production under similar conditions compared to corresponding effector cells expressing tcr. The fold-induced IL2 production by SIR expressing effector cells was measured by dividing the IL2 value obtained when SIR expressing effector cells were cultured alone by the IL2 level obtained when SIR expressing cells were co-cultured with their target cells. In some embodiments, effector cells expressing SIR have higher specific IL2 production when co-cultured with their target cells compared to corresponding effector cells expressing tcr under similar conditions. Specific IL2 production by SIR expressing effector cells upon exposure to target cells was measured by subtracting the value obtained using control effector cells from the IL2 value obtained using SIR expressing effector cells when co-cultured with target cells at 37 ℃ for 4 hours to 96 hours under similar conditions. In some embodiments, the control effector cell is a parent effector cell that does not express any SIR, e.g., an untransduced T cell. In an alternative embodiment, the control effector cell is a control SIR that expresses an antigen that targets an antigen other than the antigen targeted by the test SIR. An exemplary control SIR for comparison of binding activity of CD19SIR is CD8SP-MPL-161-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-161-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1322). In some embodiments, specific IL2 production by effector cells expressing SIR against a target antigen is at least 5%, 10%, 20%, 30%, 40%, 50% or 100% greater than specific IL2 production by corresponding effector cells expressing tcr after 24 hours of incubation at 37 ℃. In some embodiments, specific IL2 production by effector cells expressing SIR is at least 1.25-fold, 1.5-fold, 2-fold, 5-fold, or 10-fold greater than specific IL2 production by corresponding effector cells expressing tcr. In some embodiments, the specific IL2 production by effector cells expressing SIR is less than 100000 times greater than the specific IL2 production by corresponding effector cells expressing tcr. In some embodiments, specific IL2 of an effector cell expressing SIR produces at least 1.25-fold, 1.5-fold, 2-fold, or 5-fold, or 10-fold greater than specific IL2 of a corresponding effector cell expressing tcr under similar conditions, but produces a size of 100000-fold (e.g., less than 50000-fold, 10000-fold, or 1000-fold) greater than specific IL2 of a corresponding effector cell expressing tcr under similar conditions. In some embodiments, the effector cell expressing SIR is an SIR T cell. In some embodiments, the effector cell expressing SIR is a Jurkat T cell expressing SIR.

In another or other embodiment of any of the preceding embodiments described herein, when compared under similar conditions, effector cells expressing a SIR exhibit lower TNF α and/or IL2 production when compared to corresponding effector cells expressing a CAR (such as effector cells presenting on their surface a CAR comprising an antigen binding domain of a SIR, such as a CAR comprising an scFv, vL, and/or vH fragment comprising an antigen binding domain of a SIR) when co-cultured with their target cells (e.g., cells expressing their target antigens). An exemplary SIR targeting CD19 is represented by CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (SEQ ID NO: 1200). For example, in some embodiments, SIR-expressing effector cells that target CD19 have higher TNF α and/or IL2 production when co-cultured with Nalm6 target cells at 37 ℃ for 4 to 96 hours, as measured by ELISA, under similar conditions than corresponding tcr-expressing effector cells, but lower higher TNF α and/or IL2 production under similar conditions than corresponding CAR-expressing effector cells. In some embodiments, under similar conditions, effector cells expressing SIR have higher fold-induced TNF α and/or IL2 production than corresponding effector cells expressing tcr, but lower fold-induced TNF α and/or IL2 production than corresponding effector cells expressing CAR. In some embodiments, under similar conditions, effector cells expressing SIR have higher specific TNF α and/or IL2 production when co-cultured with their target cells compared to corresponding tcr-expressing effector cells, but lower specific TNF α and/or IL2 production compared to CAR-expressing effector cells. In some embodiments, the specific TNF α and/or IL2 production by the SIR-expressing effector cells for the target antigen is at least 5%, 10%, 20%, 30%, 40%, 50%, or 100% greater than the specific TNF α and/or IL2 production by the corresponding tcr-expressing effector cells after incubation for 24 hours at 37 ℃, but at least 5%, 10%, 20%, 30%, 40%, 50%, or 100% less than the specific TNF α and/or IL2 production by the corresponding CAR-expressing effector cells. In some embodiments, under similar conditions, specific TNF α and/or IL2 production by effector cells expressing SIR is at least 1.25-fold, 1.5-fold, 2-fold, 5-fold, or 10-fold greater than specific TNF α and/or IL2 production by effector cells corresponding to tcr expression, but at least 1.25-fold, 1.5-fold, 2-fold, 5-fold, or 10-fold less than specific TNF α and/or IL2 production by effector cells corresponding to CAR expression. In some embodiments, the effector cell expressing SIR is an SIR T cell. In some embodiments, the effector cell expressing SIR is a Jurkat T cell expressing SIR.

In any of the embodiments described herein, effector cells expressing one type of SIR exhibit different characteristics when compared under similar conditions as compared to effector cells expressing a different type of SIR (such as effector cells presenting on their surface an SIR comprising the antigen binding domain of the first SIR but having a different TCR chain, e.g., an SIR comprising an scFv, vL, and/or vH fragment comprising the antigen binding domain of the first SIR but having a different TCR chain). Tables 7A-7C provide SEQ IDs for various types of exemplary SIRs. Where the SIR is modular in design, one skilled in the art can generate additional SIR types by replacing one module with another. Exemplary characteristics that different types of SIRs may exhibit diversity when expressed in immune effector cells include, but are not limited to, binding affinity, cell surface expression, cytotoxicity, cytokine production, cell proliferation, terminal differentiation, depletion, and in vivo biological activity. In exemplary embodiments, effector cells expressing SIR1(SEQ ID NO:1200) containing a CD19 targeting domain based on FMC63 have higher binding to CD19-ECD-GGSG-NLuc-AcV5 fusion protein after incubation at 4 ℃ for 60 minutes than corresponding effector cells expressing SIR2(SEQ ID NO:1410) or SIR3(SEQ ID NO:4531) targeting CD19 when examined under similar conditions and when both SIR types are targeted to the TRAC (TCR alpha constant chain) genomic locus to rule out any expression changes due to random integration sites of the different SIR constructs. In some embodiments, when examined under similar conditions and when both SIR types are targeted to the TRAC (TCR α constant chain) genomic locus, target antigen binding of effector cells expressing one type of SIR (e.g., SIR1) is at least 5%, 10%, 20%, 30%, 40%, 50% or 100% greater than target antigen binding of effector cells expressing another type of SIR (e.g., SIR2) containing the same binding domain after incubation for 60 minutes at 4 ℃. In some embodiments, when examined under similar conditions and when both SIR types are targeted to the TRAC (TCR α constant chain) genomic locus, target antigen binding of effector cells expressing different types of SIRs containing the same binding domain (e.g., SIR1, SIR2, SIR3, etc.) changes by more than 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold after incubation for 60 minutes at 4 ℃. Techniques for targeting genomic inserts to specific genomic loci are known in the art. In some embodiments, when examined under similar conditions and when both SIR types are targeted to the TRAC (TCR α constant chain) genomic locus, target antigen binding of effector cells expressing different types of SIRs containing the same binding domain (e.g., SIR1, SIR2, SIR3, etc.) changes by more than 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold after incubation for 60 minutes at 4 ℃. In some embodiments, the standard deviation of target antigen binding of effector cells expressing different types of SIRs (e.g., SIR1, SIR2, SIR3, etc.) containing the same binding domain is more than 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold after incubation at 4 ℃ for 60 minutes compared to the standard deviation of target antigen binding of an independently isolated population of effector cells expressing the corresponding tcr, when examined under similar conditions and when the different SIR types and tcr are targeted to the TRAC locus. In other embodiments, when each SIR type and tcr is inserted at the TRAC locus, the standard deviation of cytotoxicity of effector cells expressing different types of SIRs containing the same binding domain (e.g., SIR1, SIR2, SIR3, etc.) is more than 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold greater than the standard deviation of cytotoxicity of an independently isolated population of effector cells expressing the corresponding tcr after incubation with the target cells for 4 hours at 37 ℃. The standard deviation is the square root of the variance and can be measured by methods known in the art. In some embodiments, the effector cell expressing SIR is an SIR T cell. In some embodiments, the effector cell expressing SIR is a Jurkat T cell expressing SIR.

In any or some of these embodiments described herein, the SIR comprises the wild type and variant TCRa (e.g., SEQ ID NO:732-740) and TCRb (e.g., SEQ ID NO:747-762) constant chains encoded by the human codon optimized polynucleotide, while the corresponding cTCR comprises the wild type TCRa (SEQ ID NO:730-731) and TCRb constant chain (SEQ ID NO:744-746) encoded by the wild type polynucleotide sequence. In some embodiments, the SIR further comprises an optional linker connecting the one or more antigen binding domains to the TCRa and TCRb constant chains. In some embodiments, the SIR comprises the wild type and variant pre-TCRa (e.g., SEQ ID NO:767-747-762) and the corresponding cTCR comprises the wild-type TCRa and TCRb constant chains encoded by the wild-type polynucleotide sequence. In some embodiments, the SIR further comprises an optional linker connecting one or more antigen binding domains to the pre-TCRa and TCRb chains. In any or some of such embodiments described herein, the SIR comprises the wild-type and variant TCRg (e.g., SEQ ID NO:770) and TCRd (e.g., SEQ ID NO:772) constant chains encoded by the human codon-optimized polynucleotide, while the corresponding tcr comprises the wild-type TCRg (SEQ ID NO:769) and TCRd constant chains (SEQ ID NO:771) encoded by the wild-type polynucleotide sequence. In some embodiments, the SIR further comprises an optional linker connecting the one or more antigen binding domains to the TCRa and TCRb constant chains. In some embodiments, the SIR comprises wild type and variant hCRbECD-Bam-CD 3 zECDPCP (SEQ ID NO:10444-10452) and hCRaECD-Kpn-CD 3 zECDPCP-opt 2(SEQ ID NO:10464-10471) constant chains, while the corresponding cTCR comprises the wild type TCRa and TCRb constant chains encoded by the wild type polynucleotide sequence. In some embodiments, the SIR further comprises an optional linker attaching one or more antigen binding domains to the constant chains of hCRbECD-Bam-CD 3 zECCDMP (SEQ ID NO:10444-10452) and hCRaECD-Kpn-CD 3 zECDPCP-opt 2(SEQ ID NO: 10464-10471). CD19-ECD-GGSG-NLuc-AcV5 in some embodiments, the antigen binding domain has about 10^-4M to 10^-8Dissociation constant (K) between M_DReflecting its binding affinity). In some embodiments, the antigen binding domain binds to one or more antigens described above. In some embodiments, the antigen binding domain has a K for the target antigen_DIs at about 10^-4M to 10^-8M, e.g. between about 10^-5M to 10^-7M, e.g. between about 10^-5M to 10^-6M is greater than or equal to the total weight of the composition. In one embodiment, the antigen binding domain has at least five, 10, 20, 30, 50, 100 or 1000 times less binding affinity than a reference antibody. In one embodiment, the encoded antigen binding domain has a binding affinity that is at least 5-fold less than a reference antibody. In some embodiments, the reference antibody is an antibody from which the antigen binding domain is derived.

In some embodiments, the binding of the antigen binding domain of said first chain of a double-stranded SIR to its cognate antigen is not substantially reduced by the presence of said second chain of the SIR or the presence of the CAR when present on the surface of the cell. In some embodiments, the binding of the antigen binding domain of the first chain of a SIR to its cognate antigen in the presence of the second chain of a SIR (or CAR) is 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the binding of the antigen binding domain of the first chain of a SIR to its cognate antigen in the absence of the second chain of a SIR (or CAR). For example, if a cell expresses a double-stranded SIR (wherein the first chain comprises a scFV targeting CD19 linked to TCR α and the second chain comprises a camelidae vHH fragment targeting CD123 linked to TCR β 2) then the binding of the antigen binding domain of said first chain of the SIR to its cognate antigen (i.e. CD19) in the presence of said second chain of the SIR is 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the binding of the antigen binding domain of said first chain of the SIR to its cognate antigen (i.e. CD19) in the absence of said second chain of the SIR to its cognate antigen (i.e. CD 123). For example, if a cell expresses a double-stranded SIR (wherein the first chain comprises a vL fragment of a FMC63 antibody linked to TCR α targeting CD19 and the second chain comprises a vH fragment of a FMC63 antibody linked to TCR β 2 targeting CD19) together with a CAR comprising a vHH fragment targeting CD123, then the binding of the antigen binding domains of said first and said second chains of the double-stranded SIR in the presence of said CAR to their cognate antigen (i.e., CD19) is 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the binding of the antigen binding domains of said first and said second chains of the SIR duplex to their cognate antigen (i.e., CD19) in the absence of said CAR to their cognate antigen (i.e., CD 123).

In some embodiments, the antigen binding domains of the first and second strands of a two-stranded SIR, when present on the surface of a cell, associate with each other less than if both were scFv antigen binding domains. In some embodiments, the antigen binding domains of the first and second chains of a double-stranded SIR associate with each other by less than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% than if both were scFv antigen binding domains.

The SIR function polypeptide units described herein can be encoded by a single polynucleotide chain and synthesized as a single polypeptide chain that is subsequently cleaved into different polypeptides. SIR polypeptides may be initially synthesized that include one or more leader sequences (also referred to as signal peptides) that are subsequently removed from the mature polypeptide. In a preferred embodiment, each functional polypeptide unit of the SIR polypeptide (i.e., the antigen binding domain linked in-frame to the T cell receptor constant strand plus the furin-SGSG-cleavable linker or the T cell receptor constant strand plus the furin-SGSG-cleavable linker) is preceded by a leader sequence that directs the functional polypeptide unit of the SIR to the cell surface as a type I transmembrane protein. In a preferred embodiment, the antigen binding domain of the SIR is cell-outward facing. In some embodiments, the leader sequence of the SIR polypeptide comprises the sequence of SEQ ID NO 2300 to SEQ ID NO 2302.

In certain embodiments, the two T cell receptor constant chains of the SIR may be of the same type (i.e., TCRa and TCRa; TCRb and TCRb; preTCRa and preTCRa; TCR γ and TCR γ; and TCR- δ). Some exemplary SIRs having two TCR constant chains of the same type are clone ID:021116-E08(SEQ ID NO:905), clone ID:012216-P08(SEQ ID NO:906), clone ID NO:012216-Q05(SEQ ID NO:907), clone ID NO:012216-R04(SEQ ID NO:908) and clone ID NO:012216-S02(SEQ ID NO: 909). In preferred embodiments, the two T cell receptor invariant chains of the SIR are of different types (e.g., TCRa and TCRb; preTCRa and TCRb; TCR γ and TCR- δ, etc.). An exemplary SIR with two TCR constant chains of different types is represented by clone ID:102615-C08, whose nucleic and amino acid sequences are given in SEQ ID NO:1200 and SEQ ID NO:3435, respectively.

In certain embodiments, the SIR polypeptide comprises a single T cell receptor constant chain comprising or derived from TCRa, TCRb, pre-TCRa, TCR-gamma or TCR-delta chains of human, mouse or canine origin. An exemplary SIR with a single TCR constant chain is represented by clone ID:051216-F04, the amino acid sequence of which is SEQ ID NO: 3258.

In certain embodiments, the SIR polypeptides of the present disclosure encode two T cell receptor constant chains comprising or derived from TCRa, TCRb, pre-TCRa, TCR- γ, or TCR- δ chains of human, mouse, or canine origin. An exemplary SIR with two TCR constant chains is represented by clone ID:102615-C08, whose nucleic and amino acid sequences are given in SEQ ID NO:1200 and SEQ ID NO:3435, respectively.

In certain embodiments, the two T cell receptor constant chains of the SIR polypeptide are of the same type (i.e., TCRa and TCRa; TCRb and TCRb; preTCRa and preTCRa; TCR γ and TCR γ; and TCR- δ). An exemplary SIR for two TCR constant chains of the same type is clone ID 021116-E08, the amino acid sequence of which is given in SEQ ID NO 3140. In preferred embodiments, the two T cell receptor constant chains of the SIR polypeptide are of different types (e.g., TCRa and TCRb; preTCRa and TCRb; TCR γ and TCR- δ, etc.). An exemplary SIR with two TCR constant chains of different types is represented by clone ID:102615-C08, whose nucleic and amino acid sequences are given in SEQ ID NO:1200 and SEQ ID NO:3435, respectively.

In some embodiments, neither T cell receptor constant chain of the SIR polypeptide is wild-type TCRa or wild-type TCRb or wild-type TCRg or wild-type TCRd or wild-type preTCRa.

The following table summarizes the target antigens, clone IDs, SEQ ID (DNA), SEQ ID (PRT), and names of several exemplary SIRs described in the present disclosure. These constructs are typically made by combining the antigen-binding fragments described in tables 5-6 with exemplary variants of the TCR constant chains described herein, including the variants provided in tables 1-3. The SIR is classified into different types (e.g., SIR1-SIR18) based on its backbone, i.e., the type of TCR constant chain present therein. However, it should be understood that the SIR is modular in design and the scope of the present disclosure is not limited to the SIR described in the table below and different SIRs may be generated by converting different modules. Thus, the antigen binding domain can be combined with other variants of the TCR constant chain, but they are not included in the SIRs described in table 7 below. SIR can be designed using antigen binding domains not listed in tables 5-6. Different therapeutic and auxiliary modules may also be added to the SIR or replaced or removed. Thus, while the table below contains several SIRs with antibiotic resistance genes (e.g., PAC), this module can be removed. In addition to the SIRs of the present disclosure, table 7 also describes several Chimeric Antigen Receptors (CARs) containing a CD3z primary signaling domain and a 41BB co-stimulatory domain. It is understood that similar CARs may be generated using other primary and costimulatory domains (e.g., from CD 28). These CARs may be expressed in combination with the SIRs described herein.

In tables 7A-C, SIR types refer to the construct backbones listed under "exemplary SIR", each sequence having the same "backbone" but different antigen binding domains. Tables 7A-C can be used to determine the DNA and PRT SEQ ID NO of constructs containing a specific binding domain and belonging to a specific SIR type tcr or CAR. Thus, SEQ ID NO 1200 has the FMC63 binding domain, while SEQ ID NO 1201 has the same SIR backbone but has the huFMC63 antigen binding domain. The target antigen, DNA and PRT SEQ ID NO and name (including binding domain) of several exemplary SIR1 type constructs are listed in table 7D. The order of the DNA and PRT SEQ ID NO referencing different binding domains of SIR1 type DNA and PRT SEQ ID NO on the backbone SIR2-9 and tcr is presented in tables 7A-B. Thus, by using tables 7A and 7D, the DNA and PRT SEQ ID NO of any antigen binding domain on the SIR1-SIR 6-type backbone and tcr can be determined. Similarly, by using tables 7B and 7D, the DNA and PRT SEQ ID NO of any antigen binding domain on the SIR7-SIR9 type backbone can be determined. The target antigen, DNA and PRT SEQ ID NO and name (including binding domain) of several exemplary SIR10 type constructs are listed in table 7E. The order of the DNA and PRT SEQ ID NO referencing different binding domains of SIR10 type DNA and PRT SEQ ID NO on backbone SIR10-18 and CAR is presented in table 7C. Thus, by using tables 7E and 7C, the DNA and PRT SEQ ID NO of the SIR10-SIR18 type backbone and any antigen binding domain on the CAR can be determined. Alternatively, the sequence of the SIR containing a particular antigen binding domain of the present disclosure can be determined by homology search of the SEQ listing files accompanying the present disclosure. Finally, since SIRs are modular in design, DNA and amino acid sequences containing the SIRs of a particular module can be generated by simply replacing the modules present in SIR1 and SIR10 types with new modules.

TABLE 7A- -SIR1- -guidance for sequence identification of SIR6 and cTCR reference SIR1

Table 7B-guidance for sequence identification of SIR1 and SIR 7-9 types, where SIR1 is used as a reference.

Table 7C-SIR 10-guidance for sequence identification of SIR18 type and CAR, where SIR10 is used as a reference.

TABLE 7D- -SIR targeting different antigens on SIR 1-type scaffold

Table 7E: targeting SIR of different antigens on SIR 10-type scaffold

Table 7F: exemplary SIR constructs targeting CD19 with FMC 63-based binding domain

Table 7G: exemplary SIR targeting Bu12 binding domain-based CD19

Table 7H: different types of SIR targeting different antigens

TABLE 7I- -antigen LUC fusion constructs

In another aspect, the present disclosure provides an isolated SIR polypeptide molecule comprising one or more antigen binding domains (e.g., antibodies or antibody fragments, ligands, or receptors) that bind to an antigen as described herein and are linked to one or more T cell receptor constant chains.

In some embodiments, the SIR may comprise or consist of a single polypeptide comprising a single antigen binding domain linked to the NH2 terminus of a single T cell receptor constant chain (class I). Constructs encoding exemplary class 1 SIRs are provided in clone ID NO: 051216-F04. The nucleic acid sequence encoding the SIR is presented in SEQ ID NO 1023 and the amino acid sequence encoding the SIR corresponds to SEQ ID NO 3258.

In some embodiments, the SIR comprises or consists of two polypeptides assembled to form a functional SIR (class 2). Each polypeptide of this double-chain class 2 SIR contains a T cell receptor constant chain and either contains (as in class 2A) or does not contain (as in class 2B) an antigen binding domain. In class 2A SIRs, each antigen binding domain is linked to the N-terminus of a separate T cell receptor constant chain. For example, antigen binding domain 1 (e.g., a vL fragment of an antibody) is linked to a constant chain of T cell receptor β (TCR β) to construct functional polypeptide unit 1 and antigen binding domain 2 (a vH fragment of an antibody) is linked to a constant chain of T cell receptor α (TCR α) to construct functional polypeptide unit 2. Two functional polypeptide units of such SIRs are co-expressed in the same cell and pair with each other to become functionally active. It should be noted that each antigen binding domain may in turn be bi-specific or multi-specific, such that the class 2 SIRs target more than 2 antigens. An exemplary class 2A SIR targeting CD19 is provided in CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (102615-C08) [ SEQ ID NO:1200 ]. The nucleic acid sequence of this SIR is presented in SEQ ID No. 1200 and its amino acid sequence corresponds to SEQ ID No. 3435. In this SIR, the vL fragment derived from the CD19 monoclonal antibody FMC63 was linked via a linker to the constant region of a mutant (KACIAH) form of human TCRb chain, while the vH fragment derived from the FMC63 monoclonal antibody was linked via a linker to the constant region of a mutant (CSDVP) human TCR alpha chain.

In some embodiments, the polypeptide double-chain SIR comprises or consists of an antigen binding domain linked to the NH2 terminus of only one T cell receptor constant chain (functional polypeptide unit 1) but co-expressed with a second T cell receptor constant chain. Such SIR is designated as class 2B. The purpose of the second T cell receptor constant chain in such 2B SIRs is to promote cell surface expression of functional polypeptide unit 1 (i.e., antigen binding domain 1 linked to the T cell receptor constant chain). Thus, the second T cell receptor constant chain in a 2B-class SIR may be expressed by itself or as a fusion protein carrying an epitope tag (e.g. MYC, V5, AcV5, G4Sx2, StrepTagII, etc.) or as a fusion protein carrying any irrelevant protein fragment (e.g. vL or vH fragment) as long as the irrelevant protein does not interfere with the assembly and function of functional unit 1. As an example, a class 2B SIR may comprise or consist of an empty (i.e. lacking the antigen binding domain) constant chain linked to the constant chain of the T cell receptor alpha (TCR alpha) to constitute the antigen binding domain 1 of functional polypeptide unit 1 and the T cell receptor beta (TCR beta) to constitute functional polypeptide unit 2. Two functional polypeptide units of such SIRs are co-expressed in the same cell. Constructs encoding exemplary class 2B SIRs are provided in CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-FMC 63-vL-Gly-Ser-linker-FMC 63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (082815-G07) [ SEQ ID NO:1620 ]. The nucleic acid and amino acid sequences encoding SIR correspond to SEQ ID NO 1620 and SEQ ID NO 3855, respectively.

In some embodiments, two functional polypeptide units of class 2 SIRs are co-expressed in a cell using different vectors. In some embodiments, two functional polypeptide units of class 2 SIRs are co-expressed in a cell using a single vector that encodes two polynucleotides encoding the two functional polypeptide units of class 2 SIRs using two separate regulatory elements (e.g., promoters). In some embodiments, two functional polypeptide units of class 2 SIR are co-expressed in a cell using a single vector that uses a single promoter to express a polynucleotide containing an IRES sequence that separately encodes nucleotide fragments of the two polypeptides of the SIR. In some embodiments, two functional polypeptide units of class 2 SIRs are co-expressed in a cell using a single vector that uses a single promoter to express a polynucleotide encoding a single polypeptide containing a cleavable linker (e.g., F2A, T2A, E2A, P2A, furin-SGSG-F2A, furin-SGSG-T2A, furin-SGSG-E2A, furin-SGSG-P2A, etc.). The resulting mRNA encodes a single polypeptide that subsequently produces two functional polypeptide units of SIR. In some embodiments, two functional polypeptide units of class 2 SIR are co-expressed by transfecting a single mRNA sequence encoding the two functional polypeptide units, while in other embodiments, two functional polypeptide units are co-expressed by transfecting two different mRNA sequences each encoding one functional polypeptide unit. In some embodiments, the vector or mRNA encoding the SIR may encode additional genes/proteins (therapeutic control, inhibitory molecules, accessory modules, etc.) that may be separated from the SIR coding sequence by an IRES or cleavable linker or a combination thereof. In another embodiment, the therapeutic control or adjunct module or both can be expressed in cells expressing SIR using a separate vector or mRNA. Exemplary therapeutic controls are provided in Table 8(SEQ ID NOs:3070 to 3076). It will be appreciated that the therapeutic control or auxiliary module is not necessary for the function of the SIR and any SIR of this embodiment may be used without the therapeutic control or auxiliary module. For example, an antibiotic resistance cassette such as PAC (puromycin resistance gene) can be removed from the SIR encoding vectors of the present disclosure without compromising SIR function.

Also provided are functional variants of the SIRs described herein, which variants have substantial or significant sequence identity or similarity to the parent SIRs, which functional variants retain the biological activity of the SIRs to which they are mutated. Functional variants include those variants such as the SIRs described herein (parent SIRs) that retain the ability to recognize a target cell to a similar degree, to the same degree, or to a greater degree, as the parent SIRs. The amino acid sequence of a functional variant may, for example, be at least about 30%, or 50%, or 75%, or 80%, or 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%), or 97%, or 98%, or 99% or more identical to the parent SIR, with reference to the parent SIR.

A functional variant may, for example, comprise an amino acid sequence of a parent SIR with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant may comprise an amino acid sequence of a parent SIR with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. A non-conservative amino acid substitution can enhance the biological activity of a functional variant, such that the biological activity of the functional variant is increased as compared to the parent CAR.

The SIR (including functional portions and functional variants) may be of any length, i.e. may comprise any number of amino acids, provided that the SIR (or a functional portion or functional variant thereof) retains its biological activity, e.g. the ability to specifically bind an antigen, detect a diseased cell in a subject, or treat or prevent a disease in a mammal, etc. For example, the SIR may be about 300 to about 5000 amino acids in length, such as 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

SIRs (including functional portions and functional variants of the disclosure) may comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, a-aminon-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β -phenylserine, β -hydroxyphenylalanine, phenylglycine, α -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid monoamide, N '-benzyl-N' -methyl-lysine, N-phenylglycine, n ', N' -dibenzyl-lysine, 6-hydroxylysine, ornithine, alpha aminocyclopentanecarboxylic acid, alpha aminocyclohexanecarboxylic acid, oc-aminocycloheptane carboxylic acid, - (2-amino-2-norbornane) -carboxylic acid, gamma-diaminobutyric acid, alpha, beta-diaminopropionic acid, homophenylalanine and alpha-tert-butylglycine.

SIRs (including functional moieties and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, a disulfide bridge, or converted to an acid addition salt and/or optionally dimerized or polymerized or conjugated.

The present disclosure provides a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a SIR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding one or more antigen binding domains, wherein the nucleotide sequence encoding each antigen binding domain is contiguous with and in the same reading frame as the nucleic acid sequence encoding the T cell receptor constant chain. Exemplary T cell receptor constant chains that can be used to construct SIRs include, but are not limited to, constant chains of TCR α, TCR β 1, TCR β 2, TCR γ, TCR δ, preTCR α and variants and mutants thereof (see, e.g., tables 1-3). In some cases, the SIR may comprise a constant chain of TCR α, TCR β 1, TCR β 2, TCR γ, TCR δ, preTCR α, etc. The present disclosure provides a SIR comprising a TCR constant chain pair selected from: TCR α and TCR β 1, TCR α and TCR β 2, pretra and TCR β 1, pretra and TCR β 2, and TCR γ and TCR δ. The present disclosure provides fusions of TCR invariant chains with CD3z chains that can replace TCR invariant chains in the construction of SIRs. In addition, the human preTCR α constant chain in the SIR lacks the carboxy-terminal 48 amino acids of the wild-type human preTCR α constant chain. The amino acid sequence of preTCR α lacking the carboxy-terminal 48 amino acids is provided in SEQ ID NO 3048.

The disclosure also provides one or more vectors comprising a nucleic acid sequence or a sequence encoding a SIR as described herein. In one embodiment, the SIR is encoded by a single carrier. In another embodiment, the SIR is encoded by more than one carrier. In another embodiment, the two functional polypeptide units of the SIR are each encoded by a separate vector or by separate nucleic acids. In one embodiment, each of the two functional polypeptide units of the SIR is encoded by a single vector or a single nucleic acid. In one embodiment, the one or more vectors are selected from one or more DNA vectors, one or more RNA vectors, one or more plasmids, one or more lentiviral vectors, one or more adenoviral vectors, one or more retroviral vectors, one or more baculovirus vectors, one or more sleeping beauty transposon vectors, or one or more piggy back (piggyback) transposons. In one embodiment, the vector is a lentiviral vector or a retroviral vector. In another embodiment, the vector is a sleeping beauty transposon vector. The nucleic acid sequences of several exemplary vectors are provided in SEQ ID NOS: 870 to 876. The vectors pLenti-EF1 alpha (SEQ ID NO:870) and pLenti-EF1 alpha-DWPRE (SEQ ID NO:871) are empty lentiviral vectors which differ from the fact that pLenti-EF1 alpha-DWPRE lacks the WPRE region. The SIR coding sequences of the present disclosure may be cloned between Nhe I and SalI sites in these vectors. The vector MSCV-Bgl2-AvrII-Bam-EcoR1-Xho-BstB1-Mlu-Sal-ClaI.I03(SEQ ID NO:872) is a retroviral vector and the SIR coding sequence of the present disclosure can be cloned between the multiple cloning sites of this vector. The vector MSCV-FMC63vL-V5- [ TCRb-KACIAH ] -F-P2A-2-Spe-FMC63vH-MYC- [ TCR α -CSDVP ] -F-F2A-Pac.N01(SEQ ID NO:873) is also a retroviral vector in which the SIR coding sequence is already present. The SIR coding sequence of the present disclosure may be cloned in this vector by removing the existing SIR and inserting a nucleic acid encoding the new SIR. The vector pSBbi-Pur (SEQ ID NO:874) is a sleeping beauty transposon vector. Vectors pSBbi-pur-EF1-FMC63vL-V5- [ TCRb-KACIAH ] -F-P2A-FMC63vH-MYC- [ TCRa-CSDVP ] -F-F2A-Xba.B01(SEQ ID NO:875) and pSBbi-pur-EF1-Nhe-FMC63vL-Xho-V5- [ TCRb-S57C-opt ] -F-P2A-Spe-FMC63vH-Mlu-MYC- [ TCRa-T48C-opt ] -F2A-MCS-I01 (SEQ ID NO:876) are sleeping beauty transposon vectors containing SIR nucleic acids that can be used to subclone the SIRs of the present disclosure after removal of existing SIRs using standard recombinant DNA techniques known in the art.

The present disclosure also includes RNA constructs that can be transfected directly into cells. Methods for generating mRNA for transfection involve In Vitro Transcription (IVT) of a template with specially designed primers, followed by addition of polya to produce constructs containing 3 'and 5' untranslated sequences ("UTRs") (e.g., 3 'and/or 5' UTRs described herein), 5 'caps (e.g., 5' caps described herein), and/or Internal Ribosome Entry Sites (IRES) (e.g., IRESs described herein), the nucleic acid to be expressed, and a polya tail, typically 50-2000 bases in length (SEQ ID NOs: 860 and 861). The RNA thus produced can efficiently transfect cells of various kinds. In one embodiment, the template contains a sequence for the SIR. In one embodiment, the RNA SIR vector is transduced into a cell (e.g., a T cell or NK cell) by electroporation. In another embodiment, the RNA SIR vector is transduced into a cell (e.g., a T cell or NK cell) by causing transient perturbations in the cell membrane using a microfluidic device. Different chains of SIRs (or functional polypeptide units) may also be introduced into a cell using one or more than one vector, different vectors or combinations of techniques. As an example, the vectors CLONE ID NO:050216-T02 and CLONE ID NO:050216-S08 encode SEQ ID NO:913 (CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-PAC) which comprises functional polypeptide unit 1 of SIR, wherein FMC63-vL chain is linked to hTCRb-KACIAH chain via a V5 linker. These vectors also express the puromycin resistance gene (PAC). The vectors CLONE ID NO:041916-A02 and 041916-B03 encode SEQ ID NO:997(CD8SP-FMC63-vH-MYC- [ TCR α -CSDVP ] -F-F2A-BlastR), which comprises functional polypeptide unit 2 of SIR, wherein FMC63-vH chain is linked to hTCRa-CSDVP chain via a MYC linker. These vectors also express the blasticidin resistance gene (BlastR). The cells can be infected simultaneously or sequentially with lentiviruses encoding SEQ ID NO:913 and SEQ ID NO:997 and then optionally selected for resistance to both puromycin and blasticidin to enrich for doubly infected cells. Cells infected with both viruses express functional polypeptide units, which will then assemble to express a functional SIR on the cell surface. In another embodiment, one strand or functional polypeptide unit of the SIR may be introduced using a retroviral vector, while another functional polypeptide unit is introduced using a lentiviral vector. In another aspect, one functional polypeptide unit of the SIR can be introduced using a lentiviral vector, while another functional polypeptide unit is introduced using a sleeping beauty transposon. In another aspect, one functional polypeptide unit of the SIR can be introduced using a lentiviral vector, while another functional polypeptide unit is introduced using RNA transfection. In another aspect, one functional polypeptide unit is produced in a cell by genetic recombination at an endogenous TCR chain locus using gene targeting techniques known in the art, while another functional polypeptide unit is introduced using a lentiviral or retroviral vector.

RNA can be introduced into the target cell using any of a number of different methods, such as commercially available methods, including but not limited to: electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany, Conn.), ECM 830(BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg, Germany); cationic liposome-mediated transfection (using lipofection); polymer encapsulation; peptide-mediated transfection; or biolistic particle delivery systems such as "gene guns" (see, for example, nimhikawa et al Hum Gene Ther [ human Gene therapy ],12(8):861-70(2001)) or by using a microfluidic device in the cell membrane to cause transient perturbations (see, e.g., patent applications WO 2013/059343a1 and PCT/US 2012/060646).

In some embodiments, the non-viral method comprises the use of a transposon (also known as a transposable element). In some embodiments, a transposon is a DNA fragment that can insert itself into a location in the genome, e.g., a DNA fragment that is capable of self-replication and inserting a copy thereof into the genome, or a DNA fragment that can be spliced from a longer nucleic acid and inserted into another location in the genome. For example, transposons contain a DNA sequence consisting of inverted repeats flanking a gene for transposition.

Exemplary methods of nucleic acid delivery using transposons include the Sleeping Beauty Transposon System (SBTS) and piggybac (pb) transposon system. See, e.g., Aronovich et al, hum.mol.genet. [ human molecular genetics ]20.R1(2011): R14-20; singh et al Cancer Res [ Cancer research ]15(2008) 2961-; huang et al mol. [ molecular therapy ]16(2008) 580-; mol. ther. molecular therapy [ molecular therapy ]18(2010) 1200-1209; kebriaiei et al Blood 122.21(2013): 166; molecular Therapy [ molecular Therapy ]16.9(2008): 1515-16; bell et al Nat. Protoc. [ Nature laboratory Manual ]2.12(2007): 3153-65; and Ding et al Cell 122.3(2005):473-83, all of which are incorporated herein by reference.

The SBTS comprises two components: 1) a transposon containing the transgene and 2) a source of transposase. Transposases can transfer transposons from a vector plasmid (or other donor DNA) to a target DNA, such as a host cell chromosome/genome. For example, a transposase is combined with a vector plasmid/donor DNA, a transposon (including one or more transgenes) is excised from the plasmid, and inserted into the genome of a host cell. See, e.g., Aronovich et al, supra.

Exemplary transposons include pT 2-based transposons. See, e.g., Grabundzija et al Nucleic Acids Res [ Nucleic Acids research ] 41.3(2013): 1829-47; and Singh et al Cancer Res. [ Cancer research ]68.8(2008): 2961-. The nucleic acid sequences of exemplary transposons are provided in SEQ ID NO:874 and SEQ ID NO: 875. Exemplary transposases include Tc 1/mariner-type transposases, such as SB 10 transposase or SB 11 transposase (an overactive transposase that can be expressed, for example, from a cytomegalovirus promoter). See, e.g., Aronovich et al; kebriaiei et al; and Grabundzija et al, all of which are incorporated herein by reference.

The use of SBTS allows for efficient integration and expression of transgenes (e.g., nucleic acids encoding SIRs as described herein). Provided herein are methods of generating cells (e.g., T cells or NKT cells or stem cells or ipscs or synthetic T cells) that stably express SIRs described herein, e.g., using a transposon system such as SBTS.

According to the methods described herein, in some embodiments, one or more nucleic acids (e.g., plasmids) containing the SBTS component are delivered to a cell (e.g., a T or NKT cell or stem cell or iPSC or synthetic T cell). For example, one or more nucleic acids are delivered by standard methods of nucleic acid (e.g., plasmid DNA) delivery, such as the methods described herein, e.g., electroporation, transfection, or lipofection. In some embodiments, the nucleic acid comprises a transposon comprising a transgene (e.g., a nucleic acid encoding a SIR as described herein). In some embodiments, the nucleic acid comprises a transposon comprising a transgene (e.g., a nucleic acid encoding a SIR as described herein) and a nucleic acid sequence encoding a transposase. In some embodiments, a system having two nucleic acids is provided, such as a two plasmid system, e.g., where a first plasmid contains a transposon that comprises a transgene and a second plasmid contains a nucleic acid sequence encoding a transposase. For example, the first nucleic acid and the second nucleic acid are co-delivered into the host cell.

In some embodiments, cells expressing SIRs described herein, e.g., T or NKT or stem cells or ipscs or synthetic T cells, are generated by using a combination of gene insertion (using SBTS) and gene editing (using nucleases (e.g., Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas systems, or engineered meganucleases re-engineered homing endonucleases)).

In some embodiments, the use of non-viral delivery methods allows for reprogramming of cells, such as T cells or NKT cells or stem cells or ipscs or synthetic T cells, and direct infusion of these cells into a subject. Advantages of non-viral vectors include, but are not limited to, the ease and relatively low cost of producing sufficient quantities, stability during storage, and lack of immunogenicity needed to meet a patient population.

In some embodiments, a vector comprising a nucleic acid sequence encoding a SIR may further comprise a nucleic acid sequence encoding one or more inhibitory molecules. Non-limiting examples of inhibitory molecules included herein include, for example, inhKIR cytoplasmic domains; a transmembrane domain, such as a KIR transmembrane domain; and an inhibitor cytoplasmic domain, e.g., an ITIM domain, e.g., an inhKIR ITIM domain. In one embodiment, the inhibitory molecule is a wild-type inhKIR, or a sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% homology or differs by no more than 1,2,3,4, 5, 6, 7, 8, 9, 10, 15, or 20 residues with wild-type inhKIR; a SLAM family cytoplasmic domain; transmembrane domains, such as SLAM family transmembrane domains; and an inhibitor cytoplasmic domain, e.g., a SLAM family ITIM domain. In another embodiment, the inhibitory molecule is a wild-type SLAM family member, or a sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% homology or differs by no more than 1,2,3,4, 5, 6, 7, 8, 9, 10, 15 or 20 residues with a wild-type SLAM family member.

In some embodiments, the vectors of the present disclosure may further comprise a promoter. Non-limiting examples of promoters include, for example, the EF-1 promoter, the CMV IE gene promoter, the EF-1 α promoter, the ubiquitin C promoter, the core promoter, or the phosphoglycerate kinase (PGK) promoter. In some embodiments, the promoter is the EF-1 promoter. In another embodiment, the EF-1 promoter comprises SEQ ID NO 877. In some embodiments, the vector is an RNA nucleic acid. In some embodiments, the carrier comprises a poly (a) tail. For example, poly (A) tails comprising about 150 adenosine bases (SEQ ID NO:860 through SEQ ID NO:864) are contemplated herein. In some embodiments, the vector comprises a 3' UTR.

In another aspect, the disclosure provides a method of making a cell (e.g., an immune effector cell or population thereof), the method comprising introducing (e.g., transducing) a vector comprising a nucleic acid encoding a SIR (e.g., a SIR described herein) or a nucleic acid encoding a SIR molecule (e.g., a SIR described herein) into a cell (e.g., a T cell, NKT cell or stem cell or iPSC or synthetic T cell described herein).

The cell may be an immune effector cell (e.g., a T cell or NKT cell or a combination thereof) or a stem/progenitor cell that can produce an immune effector cell or a synthetic T cell. In some embodiments, the cells in these methods are diacylglycerol kinase (DGK) and/or Ikaros deficient. In some embodiments, the cells in these methods lack constant chains of endogenous T cell receptors α, β 1, β 2, pre-TCR α, γ, or δ, or combinations thereof. In some embodiments, the cells in these methods are devoid of HLA antigens. In some embodiments, the cells in these methods lack β 2 microglobulin. In some embodiments, the cells in these methods lack expression of a target antigen for SIR. For example, a T cell expressing a SIR lacks endogenous CD5 if the SIR is against CD5, or lacks TCR- β 1 constant chains if the SIR is against TCR- β 1 constant chains, or lacks TCR- β 2 constant chains if the SIR is against TCR- β 2, or lacks CS1 if the SIR is against CS 1.

In some embodiments, introducing a nucleic acid molecule encoding a SIR comprises transducing a vector comprising the nucleic acid molecule encoding the SIR, or transfecting the nucleic acid molecule encoding the SIR, wherein the nucleic acid molecule is an in vitro transcribed RNA. In some embodiments, the nucleic acid molecule encodes two or more components of the SIR, introduced by transducing the cell with more than one vector or transfecting with two or more nucleic acid molecules encoding different subunits of the SIR. For example, a cell may be transduced with two separate vectors each encoding one of the two functional polypeptide units of the SIR. An exemplary SIR construct encoded by two separate vectors is provided by SIR lentiviral construct 050216-S08, which contains a SIR sequence corresponding to SEQ ID NO:913 and encoding SIR fragment CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-PAC, wherein the vL fragment derived from CD19 monoclonal antibody FMC63 is linked via a V5 linker to the constant chain of hTCRb with a KACIAH mutation. This SIR FPU is linked to the PAC (puromycin resistance) gene via a F-P2A cleavable linker. The vector for the 050216-S08 construct was pLenti-EF1 α (SEQ ID NO: 870). SIR lentiviral construct 041916-a02 contained a SIR sequence corresponding to SEQ ID NO:997 and encoding the SIR fragment CD8SP-FMC63-vH-MYC- [ TCR α -CSDVP ] -F2A-BlastR, wherein the vH fragment derived from CD19 monoclonal antibody FMC63 was linked via a MYC linker to the constant chain of hTCRa with CSDVP mutation. This SIR FPU is linked to the blasticidin resistance gene via a F-F2A cleavable linker. The vector for the 041916-A02 construct was pLenti-EF1 α -DWPRE (SEQ ID NO: 871). Exemplary selection markers are presented in SEQ ID NO 795 through SEQ ID NO 801. Similarly, cells may be transduced with two separate in vitro transcribed RNAs each encoding one of the two functional polypeptide units of the SIR. In addition to the functional polypeptide units of the SIR, each RNA may carry a different selection marker or reporter gene (e.g. tfegfr or CD34 or CNB30 or mutant DHFR), which can be used to select cells transduced with two RNAs and thus expressing two functional polypeptide units of the SIR.

In some embodiments, the method further comprises: a) providing a population of immune effector cells (e.g., T cells or NK cells); and b) removing the T regulatory cells from the population, thereby providing a population of T regulatory-depleted cells; wherein steps a) and b) are performed prior to introducing into the population a nucleic acid encoding a SIR. In embodiments of these methods, the T regulatory cell comprises CD25⁺T cells, and are removed from the cell population using an anti-CD 25 antibody or fragment thereof. The anti-CD 25 antibody or fragment thereof can be attached to a substrate (e.g., a bead). In other embodiments, the population of immune effector cells depleted of T regulatory cells provided from step (b) contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells. In other embodiments, the method further comprises: cells expressing a disease-associated antigen that does not comprise CD25 are removed from the population to provide a population of cells depleted of T regulatory and tumor antigens, after which nucleic acids encoding SIRs are introduced into the population. The disease-associated antigen may be selected from CD19, CD30, CD123, CD20, CD22, CD33, CD138, BCMA, Lym1, Lym2, CD79b, CD170, CD179b, CD14, or CD11b, or a combination thereof.

In other embodiments, the method further comprises depleting cells expressing the checkpoint inhibitor from the population to provide a population of cells depleted of T regulatory and inhibitory molecules, prior to introducing the nucleic acid encoding the SIR into the population. Checkpoint inhibitors may be selected from CTLA-4, PD-1, LAG-3, TIM3, B7-H1, CD160, P1H, 2B4, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), TIGIT, BTLA and LAIRl.

The present disclosure also provides recombinant cells, e.g., immune effector cells (e.g., a population of cells, e.g., a population of immune effector cells) and/or stem cells (e.g., hematopoietic stem cells, peripheral blood stem cells, bone marrow derived stem cells, immune stem cells, induced pluripotent stem cells, or ipscs) comprising a nucleic acid molecule, SIR polypeptide molecule, or vector as described herein.

In some embodiments, the cell is an immune cell. Non-limiting examples of immune cells include T cells and NK cells. Additionally, non-limiting examples of T cells include tregs, CD8+ T cells, and CD4+ T cells. In one embodiment, the cell is a human T cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a dog cell.

In one embodiment, the human T cells are T cells expressing P-glycoprotein ((P-gp or Pgp; MDR1, ABCB1, CD 243). In one embodiment, the human T cells are T cells that are tarnished with a dye that is a substrate for P-glycoprotein-mediated efflux.

In some embodiments, the cell is a diacylglycerol kinase (DGK) and/or Ikaros deficient T cell.

In one embodiment, the cell is a T cell and the T cell lacks one or more endogenous T cell receptor chains. T cells stably lacking expression of a functional TCR according to the present disclosure can be generated using a variety of methods, such as using Zn Finger Nuclease (ZFN), CRISP/Cas9, and shRNA targeting endogenous T cell receptor chains. Non-limiting exemplary methods related to shRNA are described in US 2012/0321667a1, which is incorporated herein by reference. Another non-limiting exemplary method associated with elimination of endogenous TCR expression using ZFNs targeting the constant regions of the α and β chains of the TCR is described in Torikai H et al (Blood [ Blood ],119(24), 6/14/2012). It should be noted that in some embodiments, SIRs of the present disclosure comprise constant chains that are codon optimized or designed to a TCR with a nucleotide sequence different from that of the endogenous TCR constant chains and thus escape targeting by CRISP/Cas9, ZFNs, and/or shrnas that target the endogenous TCR constant chains.

A T cell lacking a functional endogenous TCR may, for example, be engineered such that it does not express any functional endogenous TCR on its surface, engineered such that it does not express a constant chain comprising one or more subunits of a functional endogenous TCR (e.g., endogenous TCR α, TCR β 1, TCR β 2, TCR γ, TCR δ, or pre-TCR α), or engineered such that it produces very little functional endogenous TCR on its surface. Alternatively, T cells may express a severely impaired endogenous TCR, for example by expressing a mutated or truncated form of one or more subunits of the TCR. The term "severely impaired TCR" means that the TCR does not elicit an adverse immune response in the host. Unmodified TCRs are often poorly expressed on primary human T cells when ectopically expressed (e.g., using retroviral or lentiviral vectors), suggesting that they inefficiently compete with endogenous TCR chains for cell surface expression. However, it has been shown that optimization of TCR chains for efficient translation in human cells allows better expression of the introduced TCR. More importantly, ectopic expression of such dominant TCRs prevents surface expression of a large proportion of the endogenous TCR repertoire from occurring in human T cells.

In one embodiment, the cell is a stem cell and the stem cell lacks one or more endogenous T cell receptor chains. In another embodiment, the cell is a stem cell in which one or more target antigens of the SIR (e.g., MPL, CD33, CD123, CD19, etc.) have been deleted or mutated to a form that is no longer recognized by the SIR. As an example, SIRs targeting CD19 are expressed in stem cells that have been made CD19 deficient using CRISP/Cas9 or Zn finger nucleases such that B cells produced by such stem cells fail to be eliminated by T cells expressing SIRs targeting CD 19. Alternatively, SIRs targeting CD19 are expressed in stem cells in which endogenous CD19 has been mutated to a form that is not SIR-triggerable using CRISP/Cas9 or Zn-finger nucleases, such that B cells produced by such stem cells fail to be eliminated by T cells expressing SIRs targeting CD 19. In another embodiment, the SIR is expressed in immune effector cells and stem cells from an autologous or allogeneic donor are genetically engineered to lack expression of the SIR target antigen or to express a mutant form that is not recognized by the SIR of the SIR target antigen. For example, SIRs targeting CD19 are expressed in T cells infused into patients along with autologous or allogeneic hematopoietic stem cells that have been made to lack CD19 using CRISP/Cas9 or Zn-finger nucleases, such that B cells produced by such stem cells fail to be eliminated by T cells expressing SIRs targeting CD 19. Alternatively, SIRs targeting CD19 are expressed in T cells infused into patients along with autologous or allogeneic hematopoietic stem cells in which endogenous CD19 has been mutated to a form that is not SIR-triggerable using CRISP/Cas9 or Zn-finger nucleases, such that B cells produced by such stem cells fail to be eliminated by T cells expressing SIRs targeting CD 19. Similar approaches can be used using shRNA, CRISP/Cas9, or Zn to nuclease mutations or elimination of other endogenous antigens (e.g., MPL, CD33, CD123, etc.) in stem cells in subjects receiving SIR-T cells targeting these antigens for the treatment of specific diseases where these antigens are expressed on disease-associated cells or on disease-causing cells.

T cells or Natural Killer (NK) cells or stem cells may be obtained from a subject. The term "subject" is intended to include living organisms (e.g., mammals) in which an immune response can be elicited. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. The T cells may be tissue resident γ - δ T cells, which may be cultured and expanded in vitro, before expressing the SIR.

Immune effector cells expressing SIR and/or CAR and/or Ab-TCR and/or TRUC and/or TCR can be expanded by stimulation with protein L. In one aspect, protein L is immobilized on a bead or another surface such as a plate. In one aspect, protein L is immobilized on the same bead as the CD3 antibody. In one aspect, protein L is immobilized on the same bead as the CD28 antibody. In one aspect, protein L is immobilized on beads to which CD3 antibody and CD28 antibody are immobilized. In one aspect, protein L is expressed on the surface of an artificial antigen presenting cell. In one aspect, protein L is expressed on the surface of an artificial antigen presenting cell in association with one or more costimulatory molecules. In one aspect, the co-stimulatory molecule comprises one or more of CD28, 41BB, or OX 40. In one aspect, the cell expressing protein L on its surface is a mammalian cell. In one aspect, the cell is a human cell. In one aspect, the cells are 293FT cells. In one aspect, the cell is a K562 cell. In one aspect, protein L is stably expressed in a cell. In another aspect, protein L is transiently expressed in a cell. Protein L may be expressed in cells by any method known in the art. In one aspect, immune effector cells expressing SIR or CAR or Ab-TCR or TRUC or TCR are expanded by co-culturing with protein L-coated beads or APCs for a period of 10min to several days or weeks (or any period in between).

In certain aspects of the disclosure, immune effector cells, e.g., T cells, can be obtained from a blood unit collected from a subject using any number of techniques known to those skilled in the art (such as ficoll (tm) isolation). In a preferred aspect, the cells from the circulating blood of the individual are obtained by apheresis. Apheresis products typically contain lymphocytes (including T cells), monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one aspect, cells collected by apheresis may be washed to remove the plasma fraction and optionally placed in an appropriate buffer or culture medium for subsequent processing steps. In one embodiment of the invention, the cells are washed using Phosphate Buffered Saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium, or may lack many, if not all, divalent cations.

An initial activation step in the absence of calcium may result in amplified activation. As will be readily understood by one of ordinary skill in the art, the washing step may be accomplished by methods known to those of skill in the art, such as by using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 Cell processor, pocter (Baxter) CytoMate, or hanidick (Haemonetics) Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers (e.g., Ca-free, Mg-free PBS, PlasmaLyte a, or other salt solutions with or without buffers). Alternatively, the sample from apheresis may be freed of unwanted components and the cells resuspended directly in culture medium.

It will be appreciated that the methods of the present application may utilize media conditions comprising 5% or less (e.g., 2%) human AB serum, and employ known media conditions and compositions, such as Smith et al, "Ex vivo expansion of human T cells for adoptive immunization using the novel Xeno-free CTS Immune cell serum Replacement [ Ex vivo expansion of human T cells for adoptive immunotherapy using a novel Xeno-free CTS Immune cell serum Replacement ]" Clinical & Translational immunological Immunology [ 2015)4, e 31; 10.1038/ct.2014.31.

In one aspect, the monocytes are depleted by lysing the erythrocytes and depleting the monocytes (e.g., by counter-current centrifugal elutriation or by PERCOLL)^TMGradient centrifugation) to separate T cells from peripheral blood lymphocytes.

In another embodiment, an immune effector cell expressing an SIR described herein may further express an agent that enhances the activity of the cell expressing the SIR. In some embodiments, the agent is an agent that inhibits an inhibitory molecule. Non-limiting examples of inhibitory molecules include PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGF β. Non-limiting examples of agents that inhibit these inhibitory molecules are provided in SEQ ID NOS 3102 through 3107 (coding sequence SEQ ID NO:827-832) (see Table 8). In one embodiment, an agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an scFv or VHH or a receptor or ligand fragment, that binds the inhibitory molecule in association with a second polypeptide that provides a positive signal to a cell, e.g., an intracellular signaling domain, such as 41BB, CD27, OX40, CD28, Dap10, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-1, TNFR-II, Fas, CD30, CD40, or a combination thereof) and/or a primary signaling domain (e.g., CD3 signaling zeta domain). Exemplary SIRs for expression of such polypeptides are presented in SEQ ID NOS: 3217 through 3219 and SEQ ID NOS: 3221 and 3222. In one embodiment, an agent that inhibits an inhibitory molecule comprises a first polypeptide that binds to an inhibitory molecule, such as a scFv or VHH fragment or a receptor or ligand fragment, that associates with a T cell receptor invariant chain described herein (e.g., a invariant chain of TCRa, TCRb1, TCRb2, pre-TCRa-Del48, TCR- γ, or TCR- δ). Exemplary SIRs for binding inhibitory molecules are presented in SEQ ID NOs 3572, 3573, 3574 and 3575.

In another embodiment, a cell expressing a SIR as described herein may further express an accessory module, such as an agent that enhances the activity of the cell expressing the SIR. Several examples of accessory modules comprising agents that enhance the activity of SIR-expressing cells are provided in SEQ ID NOs 3087 to 3116 (table 8). For example, in one embodiment, the agent may be an agent that increases expression and/or activity of a SIR chain (e.g., CD3 ζ, CD3 δ, CD3 ε, CD3 γ, or a combination thereof). In another embodiment, the agent may be one that provides a co-stimulatory signal to a cell expressing SIR (e.g., vflp K13, vflp c159, cflp-L, cFLIP-p22, HTLV1Tax, HTLV2Tax, 41BB, or CD 28). In another embodiment, the agent can be an agent that provides a costimulatory signal to a cell expressing SIR in an inducible manner (e.g., FKBPx2-K13, FKBPx2-MC159, FKBPx2-cFLIP, FKBPx2-cFLIP-L, FKBPx2-cFLIP-p22, FKBPx2-HTLV1Tax, FKBPx2-HTLV2Tax, FKBPx2-41BB, or FKBPx2-CD28, Myr-MYD88-CD40-Fv' -Fv, etc.). In another embodiment, the agent may be a cytokine or chemokine that promotes the proliferation or persistence of SIR-expressing cells (e.g., CD40L, IL2, IL-7, IL-15, IL12f, or IL-21). In another embodiment, the agent may be a soluble receptor (e.g., sHVEM or sHVEM-Alb8-vHH) that promotes the activity of and/or synergizes with SIR-expressing cells. In one embodiment, the agent may be an agent that inhibits an inhibitory molecule. In some embodiments, an inhibitory molecule (e.g., PD1) can reduce the ability of a cell expressing SIR to produce an immune effector response. In another embodiment, the agent may be a scFV targeted to PD1 or CTLA 4. Exemplary scFV targeting PD1 and CTLA4 are provided in SEQ id nos 3102 to 3107. In one embodiment, the agent comprises a first polypeptide, e.g., an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGF β, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide that is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein), in one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., an extracellular domain of PD1), and at least a portion of an intracellular signaling domain (e.g., an extracellular domain of a signaling domain described herein) A CD28 signaling domain as described herein and/or a CD3 zeta signaling domain as described herein).

Table 8:

in one embodiment, the agent comprises a first polypeptide of PD-1 or a fragment thereof and a second polypeptide of an intracellular signaling domain described herein (e.g., CD28, CD27, OX40, or 4-IBB signaling domain and/or CD3 zeta signaling domain). In another embodiment, the agent comprises a first polypeptide of PD-1 or a fragment thereof and a second polypeptide which is a constant chain of a T cell receptor as described herein (e.g., a constant chain of TCRa, TCRb1, TCRb2, pre-TCRa-Del48, TCR- γ, or TCR- δ).

In one embodiment, an effector cell expressing a SIR described herein can further comprise a second SIR that can induce a different antigen binding domain to the same or a different target. In some embodiments, the second SIR may target the same or a different cell class as the first SIR.

In one embodiment, the SIR-expressing effector cells described herein may further comprise a CAR having the same or different antigen binding domains, optionally to the same or different targets. In some embodiments, the CAR may target the same or different cell class as the first SIR. The nucleic acid and amino acid sequences of several exemplary CARs are presented in SEQ ID NOs 9659 to 9854 and 9873 to 10068, respectively. In one embodiment, the CAR comprises an antigen binding domain against a target expressed on the same disease cell type as the disease-associated antigen (e.g., cancer). In one embodiment, the SIR-expressing cell comprises a SIR that targets a first antigen and a CAR that targets a second, different antigen and comprises an intracellular signaling domain that does not have a primary signaling domain but has a costimulatory signaling domain. While not wishing to be bound by theory, placement of a costimulatory signaling domain, such as 4-1BB, CD28, CD27, or OX-40, on a CAR may modulate the activity of the SIR on cells expressing both targets. In one embodiment, a cell expressing SIR comprises i) a first disease-associated antigen SIR comprising one or more antigen binding domains that bind a target antigen described herein, and one or two TCR constant chains; and ii) a CAR that targets a different target antigen (e.g., an antigen expressed on the same disease-associated (e.g., cancer) cell type as the first target antigen) and comprises an antigen binding domain, a transmembrane domain, and a primary signaling domain, and a costimulatory domain. The nucleic acid and amino acid sequences of the exemplary constructs having this configuration are presented in SEQ ID NO:983 and SEQ ID NO:3218, respectively. The antigen binding domain of the SIR in this construct comprises vL and vH fragments derived from a FMC63 monoclonal antibody targeting CD19, while the antigen binding domain of the CAR comprises the extracellular domain of PD1, the primary signaling domain of the CAR in this construct comprises the CD3z cytosolic domain, and the co-stimulatory domain comprises the 4-1BB cytosolic domain. In another embodiment, a cell expressing a SIR comprises i) a SIR comprising an antigen binding domain that binds a target antigen described herein and one or two TCR constant chains; and ii) a CAR that targets an antigen other than the first target antigen (e.g., an antigen expressed on the same cancer cell type as the first target antigen) and comprises an antigen binding domain, a transmembrane domain, and a costimulatory signaling domain for the antigen. The nucleic acid and amino acid sequences of the exemplary constructs having this configuration are presented in SEQ ID NO:982 and SEQ ID NO:3217, respectively. This construct is similar to that shown in SEQ ID NO:983 except that the CAR lacks the CD3z domain. In another embodiment, a cell expressing SIR comprises i) a first disease-associated antigen SIR comprising one or more antigen binding domains that bind a target antigen described herein, and one or two TCR constant chains; and ii) a CAR that targets a different target antigen (e.g., an antigen expressed on the same disease-associated (e.g., cancer) cell type as the first target antigen) and comprises an antigen binding domain, a transmembrane domain, and a primary signaling domain, but does not have a costimulatory domain.

In one embodiment, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain (such as, but not limited to, one or more intracellular signaling domains from 41BB, CD27, OX40, CD28, Dap10, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-1, TNFR-II, Fas, CD30, CD40, or a combination thereof) and/or a primary signaling domain (such as, but not limited to, CD3 zeta signaling domain). Exemplary SIRs co-expressed with CAR are presented in SEQ ID NOS: 3217 through 3219 and SEQ ID NOS: 3221 and 3222.

In one embodiment, an effector cell expressing a SIR comprises a SIR and an inhibitory CAR described herein. In one embodiment, the inhibitory CAR comprises an antigen binding domain that binds to an antigen found on normal cells but not cancer cells. In one embodiment, the inhibitory CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. For example, the intracellular domain of the inhibitory CAR may be an intracellular domain of any one of PD1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGF β. An exemplary SIR polypeptide co-expressed with an inhibitory CAR is presented in SEQ ID NO: 3220. In this polypeptide the inhibitory CAR expresses vHH targeting CXCR4 fused to the transmembrane and cytosolic domains of LAIR 1.

In certain embodiments, the antigen binding domain of the SIR molecule comprises an scFv and the antigen binding domain of the CAR molecule does not comprise an scFv. For example, the antigen binding domain of the SIR molecule comprises a scFv and the antigen binding domain of the CAR molecule comprises a camelidae VHH domain.

In one embodiment, the present disclosure provides an immune effector cell (e.g., T cell, NK cell) that expresses a SIR comprising an antigen binding domain that binds to a tumor antigen as described herein; and a CAR comprising a PD1 extracellular domain or a fragment thereof. In some embodiments, the cell further comprises an inhibitory molecule comprising an inhKIR cytoplasmic domain; a transmembrane domain, such as a KIR transmembrane domain; and an inhibitor cytoplasmic domain, e.g., an ITIM domain, e.g., an inhKIR ITIM domain. In one embodiment, the inhibitory molecule is a naturally occurring inhKIR, or a sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% homology or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues with a naturally occurring inhKIR; or a SLAM family cytoplasmic domain; transmembrane domains, such as SLAM family transmembrane domains; and an inhibitor cytoplasmic domain, e.g., a SLAM family ITIM domain. In another embodiment, the inhibitory molecule is a naturally occurring SLAM family member, or a sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% homology or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 residues with a naturally occurring SLAM family member.

The disclosure also provides a method comprising administering to a subject an SIR molecule, a cell that expresses an SIR molecule, or a cell comprising a nucleic acid that encodes an SIR molecule. In one embodiment, the subject has a disorder described herein, e.g., the subject has a cancer, an infectious disease, an allergic disease, a degenerative disease, or an autoimmune disease, which disorder expresses a target antigen described herein. In one embodiment, the subject has an increased risk of a disorder described herein, e.g., the subject has an increased risk of cancer, an infectious disease, an allergic disease, a degenerative disease, or an autoimmune disease, which disorder expresses a target antigen described herein. In one embodiment, the subject is a human. In another embodiment, the subject is an adult. In another embodiment, the subject is a companion animal such as a dog.

The present disclosure provides methods for treating or preventing a disease associated with expression of a disease-associated antigen described herein.

In one embodiment, the present disclosure provides methods of treating or preventing a disease by providing to a subject in need thereof an immune effector cell (e.g., T cell) or a stem cell that can produce an immune effector cell engineered to express a targeted X-SIR, wherein X represents a disease-associated antigen as described herein, and wherein the disease-causing cell or disease-associated cell expresses the X antigen. Table 9 provides a list of different antigens and exemplary diseases that can be prevented, inhibited or treated using immune effector cells expressing SIRs targeting these antigens.

Table 9:

in another embodiment, the present disclosure provides methods of treating or preventing cancer by providing to a subject in need thereof immune effector cells (e.g., T cells) engineered to express an XSIR (or X-SIR) as described herein, wherein the cancer cells express an antigen target "X". In one embodiment, X is expressed on both normal and cancer cells, but at a lower level on normal cells. In one embodiment, the method further comprises selecting a SIR that binds with an affinity for X that allows the XSIR to bind to and kill X-expressing cancer cells, but less than 30%, 25%, 20%, 15%, 10%, 5% or less of X-expressing normal cells are killed, e.g., as determined by an assay described herein. For example, the Gluc release cytotoxicity assay described herein can be used to identify XSIR that targets, for example, cancer cells. In one embodiment, the selected SIR has an antigen binding domain with a binding affinity for the target antigen KD of about 10^-4M to 10^-8M, more typically about 10^-5M to 10^-7M, and typically about 10^-6M or 10^-7And M. In one embodiment, the binding affinity of the selected antigen binding domain is at least 2-fold, at least 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or 1000-fold lower than a reference antibody (e.g., an antibody described herein) from which the binding domain of the SIR is derived.

In another embodiment, the present disclosure provides methods of treating or preventing a disease by providing an immune effector cell (e.g., a T cell) or a stem cell that can produce an immune effector cell engineered to express TCRB1-SIR to a subject in need thereof, wherein the disease-causing cell or disease-associated cell expresses TCRB1(T cell receptor β 1 chain). In one embodiment, the disease to be treated or prevented is cancer or an immune disease. In one embodiment, the cancer to be treated or prevented is a T cell leukemia or a T cell lymphoma. In one embodiment, the immune disorder to be treated or prevented is multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, diabetes, graft-versus-host disease, or autoimmune thyroiditis.

In another embodiment, the disclosure provides methods of treating or preventing a disease by providing an immune effector cell (e.g., a T cell) or a stem cell that can produce an immune effector cell engineered to express TCRB2-SIR to a subject in need thereof, wherein the disease-causing cell or disease-associated cell expresses TCRB2(T cell receptor β 2 SIR). In one embodiment, the disease to be treated or prevented is cancer or an immune disorder. In one embodiment, the cancer to be treated or prevented is a T cell leukemia or a T cell lymphoma. In one embodiment, the immune disorder to be treated or prevented is multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, diabetes, graft-versus-host disease, or autoimmune thyroiditis.

In another embodiment, the present disclosure provides methods of treating or preventing a disease by providing to a subject in need thereof an immune effector cell (e.g., T cell) or a stem cell that can produce an immune effector cell engineered to express a T cell receptor gamma-delta-SIR, wherein the disease-causing cell or disease-associated cell expresses the T cell receptor gamma-delta. In one embodiment, the disease to be treated or prevented is cancer or an immune disorder. In one embodiment, the cancer to be treated or prevented is a T cell leukemia or a T cell lymphoma. In one embodiment, the immune disorder to be treated or prevented is multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, diabetes, graft-versus-host disease, or autoimmune thyroiditis.

In another embodiment, the present disclosure provides methods of treating or preventing a disease by providing to a subject in need thereof immune effector cells (e.g., T cells) or stem cells that can produce immune effector cells engineered to express the SIR encoding CD 4-DC-SIGN. In one embodiment, the disease to be treated or prevented is HIV 1/AIDS.

In another embodiment, the present disclosure provides methods of treating or preventing an autoimmune disease by providing to a subject in need thereof an immune effector cell (e.g., a T cell) or a stem cell that can produce an immune effector cell engineered to express an SIR encoding a self-antigen or fragment thereof. In one embodiment, the autoimmune disease is diabetes, rheumatoid arthritis, multiple sclerosis, pemphigus vulgaris, pemphigus paraneoplastic, glomerulonephritis, ankylosing spondylitis, ulcerative colitis, or Crohn's disease. In one aspect, the disease is pemphigus vulgaris and the antigen binding domain of the SIR comprises the extracellular domain of desmoglein 3(Dsg 3).

In another embodiment, the present disclosure provides methods of treating or preventing cancer, infection, autoimmune or allergic disease by providing to a subject in need thereof immune effector cells (e.g., T cells) or stem cells that can produce immune effector cells engineered to express a universal SIR encoding CD16 or a deletion or point mutant fragment thereof and an antibody or antibody fragment that binds to the CD16 domain of the SIR and an antigen expressed on disease-associated cells. In one aspect, the disease-associated cell is a cancer cell, an infected cell, or a plasma cell or a B cell or a T cell.

In another embodiment, the present disclosure provides methods of treating or preventing cancer, infection, autoimmune or allergic disease by providing to a subject in need thereof immune effector cells (e.g., T cells) or stem cells producing immune effector cells engineered to express a universal SIR encoding an immunoglobulin-binding receptor or a deletion or point mutant fragment thereof. The patient is administered immune effector cells that express the SIR, as well as one or more antibodies or antibody fragments that bind to the immunoglobulin binding domain of the SIR receptor and one or more antigens expressed on disease-associated cells. In one aspect, the disease-associated cell is a cancer cell, an infected cell, or a plasma cell or a B cell or a T cell.

In another embodiment, the disclosure provides methods of treating or preventing cancer, infection, autoimmune or allergic disease by providing to a subject in need thereof immune effector cells (e.g., T cells) or stem cells that can produce immune effector cells engineered to express a universal SIR encoding an immunoglobulin binding receptor or a deletion or point mutant fragment thereof linked to a T cell receptor constant chain (e.g., the constant chain of TCR α) and an antigen binding domain (e.g., scFv, vHH, vL, vH or non-immunoglobulin antigen binding domain) linked to a T cell receptor constant chain (e.g., the constant chain of TCR β). Administering to the patient immune effector cells expressing the SIR and one or more antibodies or antibody fragments that bind to the immunoglobulin binding domain of the first SIR receptor and one or more antigens expressed on disease-associated cells. In one aspect, the disease-associated cell is a cancer cell, an infected cell, or a plasma cell or a B cell or a T cell.

In another embodiment, the present disclosure provides methods of treating or preventing cancer, infection, autoimmune or allergic diseases by providing to a subject in need thereof immune effector cells (e.g., T cells) or stem cells that can produce immune effector cells engineered to express a universal SIR that encodes an immunoglobulin receptor or a deletion or point mutant fragment thereof along with antibodies or antibody fragments that bind to antigens expressed on the above receptors and disease-associated cells.

In another embodiment, the disclosure provides methods of treating or preventing cancer, infection, autoimmune or allergic disease by providing to a subject in need thereof immune effector cells (e.g., T cells) or immune effector cell-producing stem cells engineered to express a universal SIR encoding CD16 or a deletion or point mutant (e.g., V158 mutant) fragment thereof linked to a T cell receptor constant chain and a SIR encoding an antigen binding domain (e.g., scFv, vHH, vL, vH or a non-immunoglobulin antigen binding domain) linked to a T cell receptor constant chain. The patient is administered immune effector cells expressing the SIR and one or more antibodies or antibody fragments that bind to the CD16 domain of the SIR and one or more antigens expressed on disease-associated cells. In one aspect, the disease-associated cell is a cancer cell, an infected cell, or a plasma cell or a B cell or a T cell.

In another embodiment, the present disclosure provides methods of treating or preventing cancer, infection, autoimmune or allergic disease by providing to a subject in need thereof immune effector cells (e.g., T cells) or stem cells that can produce immune effector cells engineered to express a universal SIR encoding CD16 or a deletion or point mutant fragment thereof (e.g., a V158 mutant) and one or more antibodies or antibody fragments that bind to the CD16 domain of the SIR and one or more antigens expressed on disease-associated cells. In one aspect, the disease-associated cell is a cancer cell, an infected cell, or a plasma cell or a B cell or a T cell.

In another embodiment, the present disclosure provides methods of treating or preventing cancer, infection, autoimmune or allergic disease by providing to a subject in need thereof immune effector cells (e.g., T cells) engineered to express an SIR encoding NKG2D or a deletion or point mutant fragment thereof. In one aspect, the disease-associated cell is a cancer cell, an infected cell, or a plasma cell or a B cell or a T cell.

In another embodiment, the present disclosure provides methods of treating or preventing a disease by providing to a subject in need thereof immune effector cells (e.g., T cells) or stem cells that can produce immune effector cells engineered to express CD19 SIR. In one aspect, the disease is an immunological or allergic disease.

In another embodiment, the present disclosure provides methods of treating or preventing a disease by providing to a subject in need thereof immune effector cells (e.g., T cells) engineered to express CD20 SIR. In one aspect, the disease is an immunological or allergic disease.

In another embodiment, the present disclosure provides methods of treating or preventing a disease by providing to a subject in need thereof immune effector cells (e.g., T cells) engineered to express CD22 SIR. In one aspect, the disease is an immunological or allergic disease.

In another embodiment, the present disclosure provides methods of treating or preventing cancer, infection, autoimmune or allergic disease by providing to a subject in need thereof immune effector cells (e.g., T cells) or stem cells that can produce immune effector cells engineered to express FITC-SIR and a FITC-labeled antibody or antibody fragment or receptor or ligand or non-immunoglobulin scaffold that binds to an antigen expressed on disease-associated cells. In one aspect, the disease-associated cell is a cancer cell, an infected cell, or a plasma cell or a B cell or a T cell.

In another embodiment, the present disclosure provides methods of treating or preventing cancer, infection, autoimmune or allergic disease by providing to a subject in need thereof immune effector cells (e.g., T cells) or stem cells that can produce immune effector cells engineered to express avidin-SIR and a biotin-labeled antibody or antibody fragment or receptor or ligand or non-immunoglobulin scaffold that binds to an antigen expressed on a disease-associated cell. In one aspect, the disease-associated cell is a cancer cell, an infected cell, or a plasma cell.

In another embodiment, the present disclosure provides methods of treating or preventing cancer, infection, autoimmune or allergic disease by providing to a subject in need thereof immune effector cells (e.g., T cells) or stem cells that can produce immune effector cells engineered to express Streptag-SIR and Streptag-containing antibodies or antibody fragments or receptors or ligands or non-immunoglobulin scaffolds that bind to antigens expressed on disease-associated cells. In one aspect, the disease-associated cell is a cancer cell, an infected cell, or a plasma cell.

In another embodiment, the present disclosure provides methods of treating or preventing a disease by providing to a subject in need thereof an immune effector cell (e.g., a T cell) engineered to express an IgE-SIR, the antigen binding domain of which comprises an antibody or antibody fragment that binds to IgE. In one aspect, the disease is an immunological or allergic disease.

In another embodiment, the disclosure relates to treating a subject with PD1SIR (i.e., SIR containing the extracellular domain of PD1 as its antigen binding domain) in vivo such that the growth of a cancerous tumor is inhibited. The nucleic acid sequence of an exemplary PD1-SIR is provided in SEQ ID NO: 1337. PD1SIR alone can be used to inhibit the growth of cancerous tumors. Alternatively, the PD1SIR may be used in combination with other SIRs, CARs, immunogenic agents, standard cancer therapy, or other antibodies. In one embodiment, the subject is treated with PD1SIR and XSIR as described herein. In another embodiment, the PD1SIR is used in combination with another SIR or CAR (e.g., a SIR or CAR described herein) and a kinase inhibitor (e.g., a kinase inhibitor described herein).

In another embodiment, the disclosure relates to treating a subject with XSIR and PD1-CAR or CTL4-CAR in vivo such that growth of a cancerous tumor is inhibited. In one embodiment, a subject is treated with PD1-CAR or CTLA4-CAR and XSIR as described herein. The nucleic acid sequences of exemplary constructs encoding PD1-CAR and XSIR (e.g., CD19-SIR) are provided in SEQ ID NO: 982-984. Nucleic acid sequences encoding exemplary constructs of CTLA4-CAR and XSIR (e.g., CD19-SIR) are provided in SEQ ID NO:986 and 987. In another embodiment, the PD1-CAR is used in combination with another SIR or CAR (e.g., a SIR or CAR described herein) and a kinase inhibitor (e.g., a kinase inhibitor described herein). In one embodiment, XSIR is used in combination with PD1-CAR or CTL 4-CAR.

In another aspect, a method of treating a subject, e.g., reducing or ameliorating a hyperproliferative disorder or condition (e.g., cancer), e.g., a solid tumor, a soft tissue tumor, a hematologic cancer, or a metastatic lesion, in a subject is provided. The term "cancer" as used herein is intended to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasion. Examples of solid tumors include malignancies, e.g., adenocarcinomas, sarcomas, and carcinomas, of various organ systems such as those affecting the breast, liver, lung, brain, lymph, gastrointestinal (e.g., colon), genitourinary tract (e.g., kidney, urothelial cells), prostate, and pharynx. Adenocarcinoma includes cancers such as most colon, rectal, renal cell, liver, non-small cell lung, small bowel and esophageal cancers. In one embodiment, the cancer is melanoma, e.g., advanced melanoma. The methods and compositions of the present disclosure may also be used to treat or prevent metastatic lesions of the aforementioned cancers. Examples of other cancers that may be treated or prevented include pancreatic cancer, bone cancer, skin cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, head and neck cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, non-hodgkin's lymphoma, carcinoma of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias (including acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, solid tumors of children, lymphocytic lymphoma, bladder cancer, renal or ureter cancer, renal pelvis cancer, Central Nervous System (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, cancer angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), and combinations of said cancers. Treatment of metastatic cancer, such as metastatic cancer expressing PD-L1, can be achieved using the antibody molecules described herein (Iwai et al (2005) int. Immunol. [ International immunology ]17: 133-.

Exemplary cancers in which growth may be inhibited include cancers that typically respond to immunotherapy. Non-limiting examples of cancers to be treated include renal cancer (e.g., clear cell carcinoma), melanoma (e.g., metastatic malignant melanoma), breast cancer, prostate cancer (e.g., hormone refractory prostate adenocarcinoma), colon cancer, and lung cancer (e.g., non-small cell lung cancer). In addition, the molecules described herein can be used to treat recurrent or refractory malignancies.

In one embodiment, the disclosure relates to a vector comprising an SIR operably linked to a promoter for expression in a mammalian immune effector cell (e.g., T cell) or a stem cell that can produce an immune effector cell. In one aspect, the invention provides recombinant immune effector cells expressing a SIR of the invention for use in the treatment or prevention of a cancer expressing a cancer-associated antigen as described herein. In one aspect, SIR-expressing cells of the present disclosure are capable of contacting tumor cells with at least one cancer-associated antigen expressed on their surface such that the SIR-expressing cells target cancer cells and inhibit the growth of cancer. In one aspect, the disclosure provides recombinant immune effector cells expressing the SIRs of the invention for use in treating or preventing a disease expressing a disease-associated antigen as described herein. In one aspect, the SIR-expressing cells of the present disclosure are capable of contacting a disease-causing cell or a disease-associated cell with at least one disease-associated antigen expressed on its surface such that the SIR-expressing cell targets the disease-causing cell or the disease-associated cell and inhibits growth of the disease.

In one embodiment, the disclosure relates to a method of inhibiting the growth of a disease (e.g., cancer, an autoimmune disease, an infectious disease, or an allergic disease or a degenerative disease) comprising contacting a disease-causing or disease-associated cell with a SIR-expressing cell of the invention such that SIRT is activated and targeted to the disease-causing or disease-associated cell in response to the antigen, wherein the growth of the disease-causing or disease-associated cell is inhibited. In one aspect, the disclosure relates to a method of preventing a disease, the method comprising administering to a patient at risk of a disease SIR-expressing cell or a cell capable of generating the SIR-expressing cell of the invention, such that the SIRT activates in response to the antigen and targets a disease-causing cell or a disease-associated cell, wherein growth of the disease-causing cell or the disease-associated cell is prevented. In one aspect, the disease is cancer, an infectious disease, an immune disease, an allergic disease, or a degenerative disease.

In another embodiment, the present disclosure relates to a method of treating cancer in a subject. The method comprises administering to a subject a SIR-expressing cell of the invention, such that the cancer in the subject is treated. In one aspect, the cancer associated with expression of a cancer-associated antigen as described herein is a hematologic or hematological cancer. In one aspect, the hematologic cancer is leukemia or lymphoma. In one aspect, cancers associated with the expression of cancer-associated antigens as described herein include cancers and malignancies, including but not limited to, for example, one or more acute leukemias, including but not limited to, for example, B-cell acute lymphocytic leukemia ("BALL"), pre-B-cell acute lymphocytic leukemia, T-cell acute lymphocytic leukemia ("TALL"), Acute Lymphocytic Leukemia (ALL); one or more chronic leukemias, including but not limited to, for example, Chronic Myelogenous Leukemia (CML), Chronic Lymphocytic Leukemia (CLL). Additional cancers or hematological conditions associated with expression of a cancer-associated antigen as described herein include, but are not limited to, for example, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-hodgkin's lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor, waldenstrom's macroglobulinemia, and "preleukemia" which is a collection of various hematological conditions linked together by inefficient production (or dysplasia) of myeloid blood cells, among others. Other diseases associated with expression of a cancer-associated antigen as described herein include, but are not limited to, for example, atypical and/or non-classical cancers, malignancies, pre-cancerous conditions, or proliferative diseases associated with expression of a cancer-associated antigen as described herein.

In another aspect, the present disclosure relates to a method of treating a disease in a subject. The method comprises administering to the subject a SIR-expressing cell of the invention, such that the disease in the subject is treated. In one aspect, the disease associated with expression of a disease-associated antigen as described herein is an infectious disease. In one aspect, the infectious disease is a disease associated with infection by: HIV1, HIV2, HTLV1, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), adenovirus, adeno-associated virus, BK virus (EBV), human herpesvirus 6, human herpesvirus 8, influenza A virus, influenza B virus, parainfluenza virus, avian influenza virus, MERS and SARS coronavirus, Crimean Congo hemorrhagic fever virus, rhinovirus, enterovirus, dengue virus, West Nile virus, Ebola virus, Marburg virus, Lassa virus, Seca virus, RSV, measles virus, mumps virus, rhinovirus, varicella virus, herpes simplex virus 1 and 2, varicella-zoster virus, HIV-1, HTLV1, hepatitis virus, enterovirus, hepatitis B virus, hepatitis C virus, Nipah virus and rift valley fever virus, Japanese encephalitis virus, Mycobacterium tuberculosis, atypical mycobacterial species, Mycobacterium species, HIV-1, HTLV1, hepatitis B virus, hepatitis C virus, Nipah virus and rift valley fever virus, Pneumocystis yeri, toxoplasmosis, rickettsia, Nocardia, Aspergillus, Mucor or Candida.

In another aspect, the present disclosure relates to a method of treating a disease in a subject. The method comprises administering to the subject a SIR-expressing cell of the invention, such that the disease in the subject is treated. In one aspect, the disease associated with expression of a disease-associated antigen as described herein is an immune or allergic or reproductive disease. In one aspect, the immune or degenerative disease is diabetes, multiple sclerosis, rheumatoid arthritis, pemphigus vulgaris, ankylosing spondylitis, hypothyroidism (Hoshimoto's thyroiditis), SLE, sarcoidosis, scleroderma, mixed connective tissue disease, graft-versus-host disease, peanut allergy, chronic idiopathic urticaria, food allergy, hay fever, seasonal allergy, pollen allergy, HLH (lymphocytosis), amyloidosis, or alzheimer's disease.

In some embodiments, the cancer that can be treated or prevented with the SIR-expressing cells of the invention is multiple myeloma. Multiple myeloma is a cancer characterized by the accumulation of plasma cell clones in the bone marrow. Current therapies for multiple myeloma include, but are not limited to, treatment with the thalidomide analog lenalidomide. Lenalidomide has activities including anti-tumor activity, angiogenesis inhibition and immunomodulation. Typically, myeloma cells are considered negative for the cancer-associated antigen CD19 as described herein by flow cytometry. Thus, in some embodiments, CD19 SIRs, e.g., as described herein, can be used to target myeloma cells. In some embodiments, the SIR therapies of the invention may be used in combination with one or more additional therapies (e.g., lenalidomide treatment). Other SIRs described herein (e.g., BCMA-SIR, CD138-SIR, CSI-SIR, GPRC5D-SIR, etc.) may also be used to treat or prevent multiple myeloma.

The present disclosure includes a type of cell therapy in which immune effector cells (e.g., T cells or T cell-provokable stem cells) are genetically modified to express synthetic antigen receptors (SIRs), and the SIRs expressing T cells or stem cells are infused to a recipient in need thereof. The infused cells are capable of killing disease-associated cells (e.g., tumor cells or virus-infected cells) in the recipient. Unlike antibody therapies, SIR-modified immune effector cells (e.g., T cells, stem cells) are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control. In various aspects, following administration of T cells or stem cells to a patient, immune effector cells (e.g., T cells or stem cells that can cause T cells) or progeny thereof administered to the patient persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years.

The disclosure also includes a type of cell therapy in which immune effector cells (e.g., T cells) are modified, e.g., by in vitro transcribed RNA, to transiently express a synthetic antigen receptor (SIR), and the SIRT cells are infused to a receptor in need thereof. The infused cells are capable of killing disease-associated cells (e.g., tumor cells or virus-infected cells) in the recipient. Thus, in various aspects, following administration of T cells to a patient, immune effector cells (e.g., T cells) administered to the patient are present for less than one month, e.g., three weeks, two weeks, one week.

The disclosure also includes a type of cell therapy in which stem cells (e.g., hematopoietic stem cells or lymphoid stem cells or embryonic stem cells or induced pluripotent stem cells) capable of producing immune effector cells (e.g., T cells) are modified to express synthetic antigen receptors (SIRs) and these stem cells are administered to a receptor in need thereof. The administered stem cells, upon implantation into a recipient, generate immune effector cells (e.g., T cells), which (i.e., immune effector cells) are capable of killing disease-related cells in the recipient. Thus, in various aspects, immune effector cells (e.g., T cells) produced in a patient following administration of stem cells expressing an SIR are present in the patient for at least one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, five years, ten years, or twenty years following administration of the T cells or stem cells to the patient. The disclosure also includes a type of cell therapy in which stem cells capable of producing immune effector cells (e.g., T cells) are modified to express synthetic antigen receptors (SIRs) and differentiated in vitro to generate immune effector cells infused to a receptor in need thereof. The infused immune effector cells (e.g., T cells) are capable of killing disease-associated cells in the recipient after infusion into the recipient. Thus, in various aspects, the immune effector cells (e.g., T cells) generated in administration to a patient persist in the patient for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, five years, ten years, or twenty years.

The disclosure also includes a type of cell therapy in which immune effector cells (e.g., T cells) are modified to express SIRs encoding autoantigens (e.g., Dsg3 or Dsg 1). Such SIRs expressing autoantigens of the present disclosure can be used to eliminate B cells and plasma cells that express autoantibodies against autoantigens. Such autoantigens-SIRs may be used in the treatment and prevention of autoimmune disorders, such as pemphigus vulgaris.

The disclosure also includes a type of cell therapy in which a regulatory immune effector cell (e.g., T)_REGOr CD25+ T cells) are modified to express SIRs that target specific antigens. Administering such SIR-T to a patient_REGTo suppress the immune response against specific antigens. SIR-T_REGCan be used for preventing and treating autoimmune diseases and enhancing immune tolerance. Without wishing to be bound by any particular theory, the anti-tumor immune response elicited by the SIR-modified immune effector cells (e.g., T cells) may be an active or passive immune response, or alternatively may be due to a direct versus indirect immune response. In one aspect, SIR-transduced immune effector cells (e.g., T cells) exhibit specific pro-inflammatory cytokine secretion and high potency cytolytic activity in response to human diseased cells (e.g., cancer or diseased cells) expressing a disease-associated antigen as described herein, are resistant to a soluble disease-associated antigen as described herein, mediate bystander (bystander) killing and mediate regression of established human tumors, including cancers. For example, antigen-poor tumor cells within a heterogeneous region of a tumor expressing a cancer-associated antigen as described herein may be susceptible to immune effector cells redirected by a cancer-associated antigen as described herein that have previously reacted against adjacent antigen-positive cancer cells(e.g., T cells).

In one aspect, a fully human SIR-modified immune effector cell (e.g., T cell) of the present disclosure may be a type of vaccine for ex vivo immunization and/or in vivo therapy of a mammal. In one aspect, the mammal is a human. In one aspect, the mammal is a dog.

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells to a mammal: i) amplifying the cells, ii) introducing into the cells a nucleic acid encoding a SIR or iii) cryopreserving the cells.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector that expresses a SIR disclosed herein. SIR-modified cells can be administered to mammalian recipients to provide therapeutic benefits. The mammalian recipient may be a human, and the SIR-modified cell may be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic, or allogeneic with respect to the recipient.

In another embodiment, SIR-modified cells are used ex vivo to deplete disease-associated cells (e.g., cancer cells) of bone marrow or peripheral blood hematopoietic stem cells. As an example, CD19-SIR expressing T cells are co-cultured with bone marrow or peripheral blood stem cell samples taken from patients with acute lymphoblastic leukemia or non-hodgkin's lymphoma in order to kill the leukemia or lymphoma present in the bone marrow or peripheral blood stem cell preparation. After in vitro (ex vivo) culture for a suitable duration, which may range from 6 hours to several days, the depleted bone marrow and peripheral blood samples are used for autologous transplantation in the patient.

Ex vivo expansion procedures for hematopoietic stem and progenitor cells are described in U.S. Pat. No. 5,199,942, incorporated herein by reference, which can be applied to the cells of the present invention. Other suitable methods are known in the art, and thus the present invention is not limited to any particular method of expanding cells ex vivo. Briefly, ex vivo culture and expansion of immune effector cells (e.g., T cells) includes: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammalian peripheral blood harvest or bone marrow explant; and (2) ex vivo expansion of such cells. In addition to the cell growth factors described in U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligands can also be used to culture and expand cells.

In addition to using cell-based vaccines in ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

In general, cells activated and expanded as described herein can be used to treat and prevent diseases that occur in immunocompromised individuals. In particular, the SIR-modified immune effector cells (e.g., T cells) of the present disclosure are useful for treating diseases, disorders, and conditions associated with expression of a disease-associated antigen (e.g., a cancer antigen or a viral antigen) as described herein. In certain aspects, the cells of the present disclosure are used to treat patients at risk of developing diseases, disorders, and conditions associated with expression of a disease-associated antigen as described herein. Accordingly, the present disclosure provides methods for treating or preventing diseases, disorders, and conditions associated with expression of a disease-associated antigen as described herein, the methods comprising administering to a subject in need thereof a therapeutically effective amount of a SIR-modified immune effector cell (e.g., a T cell) of the present disclosure or a stem cell capable of generating an immune effector cell.

In one aspect, SIR-expressing cells of the disclosure are useful for treating a proliferative disease, such as a cancer or malignancy, or a precancerous condition (such as myelodysplasia, myelodysplastic syndrome, or pre-leukemia). Other diseases associated with expression of a cancer-associated antigen as described herein include, but are not limited to, for example, atypical and/or non-classical cancers, malignancies, pre-cancerous conditions, or proliferative diseases expressing a cancer-associated antigen as described herein. Non-cancer related indications associated with expression of a disease-associated antigen as described herein include, but are not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory disorders (allergy and asthma), infectious conditions (e.g., HIV1, CMV, EBV, influenza), and transplantation.

The SIR-modified immune effector cells (e.g., T cells) of the present disclosure can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components, such as IL-2 or other cytokines or cell populations.

Hematologic cancers or hematologic cancer conditions are types of cancer such as leukemia, lymphoma, and malignant lymphoproliferative conditions that affect the blood, bone marrow, and lymphatic systems.

Leukemias can be classified as acute leukemia and chronic leukemia. Acute leukemias can be further classified as Acute Myelogenous Leukemia (AML) and Acute Lymphoid Leukemia (ALL). Chronic leukemias include Chronic Myelogenous Leukemia (CML) and Chronic Lymphocytic Leukemia (CLL). Other related disorders include myelodysplastic syndrome (MDS, formerly known as "preleukemia"), which is a diverse collection of hematological disorders combined by ineffective production (or dysplasia) of myeloid lineage blood cells, and has a risk of conversion to AML.

Lymphomas are a group of blood cell tumors that develop from lymphocytes. Exemplary lymphomas include non-hodgkin lymphoma and hodgkin lymphoma.

The present invention provides compositions and methods for treating and preventing cancer. In one aspect, the cancer is a hematologic cancer or hematologic cancer, including but not limited to a hematologic cancer, which is a leukemia or lymphoma. In one aspect, SIR-expressing cells of the present disclosure are useful for treating cancers and malignancies, such as, but not limited to, for example, acute leukemias, including, but not limited to, for example, B-cell acute lymphocytic leukemia ("BALL"), T-cell acute lymphocytic leukemia ("TALL"), Acute Lymphocytic Leukemia (ALL); one or more chronic leukemias, including but not limited to, for example, Chronic Myelogenous Leukemia (CML), Chronic Lymphocytic Leukemia (CLL); additional hematologic cancers or hematologic conditions, including but not limited to, e.g., B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumors, burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-hodgkin lymphoma, plasmablatic lymphoma, plasmacytoid dendritic cell tumors, waldenstrom's macroglobulinemia, and "preleukemia" which is a collection of various blood conditions linked together by inefficient production (or dysplasia) of myeloid blood cells, and the like. Other diseases associated with expression of a cancer-associated antigen as described herein include, but are not limited to, for example, atypical and/or non-classical cancers, malignancies, pre-cancerous conditions, or proliferative diseases expressing a cancer-associated antigen as described herein.

The present disclosure also provides methods for inhibiting proliferation of or reducing a population of cells expressing a disease-associated antigen as described herein, the methods comprising contacting a population of cells comprising cells expressing a disease-associated antigen as described herein with SIR-expressing T cells of the present disclosure that bind to cells expressing a disease-associated antigen as described herein. In a particular aspect, the invention provides methods for inhibiting the proliferation of or reducing a population of diseased cells expressing a disease-associated antigen as described herein, the methods comprising contacting a population of cancer cells expressing a disease-associated antigen as described herein with SIR-expressing T cells of the present disclosure that bind to cells expressing a disease-associated antigen as described herein. In one aspect, the invention provides methods for inhibiting the proliferation of or reducing a diseased cell population expressing a disease-associated antigen as described herein, the methods comprising contacting the diseased cell population expressing a disease-associated antigen as described herein with SIR-expressing T cells of the present disclosure that bind to cells expressing a disease-associated antigen as described herein. In certain aspects, a T cell expressing SIR of the present disclosure reduces the number (quality), number (number), amount (amount), or percentage of cells and/or diseased cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% relative to a negative control in a subject having myeloid leukemia or another disease associated with cells expressing a disease-associated antigen as described herein, or an animal model of myeloid leukemia or another disease associated with cells expressing a disease-associated antigen as described herein. In one aspect, the subject is a human. In one aspect, the disease is cancer, an infectious disease, an immune disease, an allergy, or a degenerative disease.

The invention also provides methods for preventing, treating and/or managing a disease associated with cells expressing a disease-associated antigen as described herein (e.g., a hematologic cancer or atypical cancer or infectious disease or immune disease or allergic disease or degenerative disease expressing a disease-associated antigen as described herein), comprising administering to a subject in need thereof SIR T cells of the present disclosure that bind to cells expressing a disease-associated antigen as described herein. In one aspect, the subject is a human. Non-limiting examples of disorders associated with cells expressing a disease-associated antigen as described herein include autoimmune disorders (such as lupus), inflammatory disorders (such as allergy and asthma), infections (such as HIV1, HTLV1, influenza virus, CMV, adenovirus, EBV and HHV8), and cancers (such as hematologic cancers or atypical cancers that express a cancer-associated antigen as described herein).

The present disclosure also provides methods for preventing, treating, and/or managing a disease associated with a cell expressing a disease-associated antigen as described herein, the methods comprising administering to a subject in need thereof a SIRT cell of the present disclosure that binds to a cell expressing a disease-associated antigen as described herein. In one aspect, the subject is a human.

The present disclosure provides methods for preventing relapse of cancer associated with cells expressing a disease-associated antigen as described herein, the methods comprising administering to a subject in need thereof SIR T cells of the present disclosure that bind to cells expressing a disease-associated antigen as described herein. In one aspect, the methods comprise administering to a subject in need thereof an effective amount of a T cell expressing an SIR described herein that binds to a cell expressing a disease-associated antigen as described herein in combination with an effective amount of another therapy.

The present disclosure also provides a method of treating or preventing a disease in a subject having, or at increased risk for, a disease associated with expression of a target antigen, the method comprising administering to the subject an effective amount of a cell comprising an SIR molecule.

The present disclosure also provides a method of treating or preventing a disease in a subject having, or at increased risk for, a disease associated with expression of a target antigen, the method comprising administering to the subject an effective amount of a cell, e.g., an immune effector cell (e.g., a population of immune effector cells), comprising an SIR molecule, wherein the SIR molecule comprises one or more antigen binding structures and one or more T cell receptor constant chains, wherein the antigen binding domain binds to a target antigen associated with the disease. Non-limiting examples of target antigens are disclosed herein above.

The present disclosure provides a method of administering to a subject cells, e.g., immune effector cells or populations thereof (each cell comprising an SIR molecule), optionally in combination with increasing the efficacy and/or safety of the immune cells. In various embodiments, the agent that increases the efficacy and/or safety of an immune cell is selected from the group consisting of: (i) protein phosphatase inhibitors; (ii) a kinase inhibitor; (iii) a cytokine; (iv) inhibitors of immunosuppressive molecules; (v) reduction of T_REGAn agent of the level or activity of a cell; (vi) an agent that increases proliferation and/or persistence of SIR-modified cells; (vii) a chemokine; (viii) an agent that increases expression of SIR; (ix) an agent that allows for modulation of expression or activity of a SIR; (x) Agents that allow control of the survival and/or persistence of SIR-modified cells; (xi) Agents that control side effects of SIR-modified cells; (xii) Brd4 inhibitors; (xiii) Agents that deliver therapeutic (e.g., shvum) or prophylactic agents to the site of disease; (xiv) An agent that increases the expression of a target antigen to which the SIR is directed; (xv) Adenosine A2a receptor antagonists; and (xvi) (i) - (xv) in any combination.

In some embodiments, the disease to be treated or prevented is a hematologic cancer. In other embodiments, the hematologic cancer is leukemia. Non-limiting examples of acute leukemias include, but are not limited to, B-cell acute lymphocytic leukemia ("BALL"), T-cell acute lymphocytic leukemia ("TALL"), Acute Lymphocytic Leukemia (ALL); one or more chronic leukemias, including but not limited to Chronic Myelogenous Leukemia (CML), Chronic Lymphocytic Leukemia (CLL); additional hematologic cancers or hematologic conditions, including but not limited to B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumors, burkitt lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-hodgkin lymphoma, plasmacytoma, plasmacytoid dendritic cell tumors, waldenstrom's macroglobulinemia, and "leukemia" which is a collection of various blood conditions linked together by inefficient production (or dysplasia) of myeloid blood cells, and diseases associated with expression of a tumor antigen described herein include, but are not limited to, atypia and/or expression of a tumor antigen as described herein Non-classical cancer, malignancy, precancerous condition, or proliferative disease; and any combination thereof. In another embodiment, the disease associated with a tumor antigen described herein is a solid tumor.

In some embodiments, the tumor antigen associated with the disease is selected from the group consisting of CD, CD123, CD171, CS-1, CLL-1 (CLECL), CD, EGFRviii, GD, BCMA, TnAg, PSMA, ROR, FLT, TAG, CD44v, CEA, EPCAM, B7H, KIT, IL-13Ra, mesothelin, IL-11Ra, PSCA, PRSS, VEGFR, LewisY, CD, PDGFR-beta, SSEA-4, CD, folate receptor alpha, ERBB (Her/neu), MUC, EGFR, NCAM, protease, PAP, ELF2, ephrin B, FAP, IGF-I receptor, CAlX, LMP, gpl, bcr-abl, tyrosinase, EphA, Fucosyl, sLe, GM, TGS, HMAA, WMO-acetyl-HR, WMO-TSHR, TEM-7, TLCP, GMH 179, GMH, TSH, GMH, BCH, BCAR, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ES0-1, LAGE-1a, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, protein (prostate specific protein), survivin and telomerase, PCTA-1/galectin 8, MelanA/82MART 56, Ras mutant, hT, sarcoma translocation point, ML-IAP, ERG (TMPRSS 2S fusion gene), NA17, PAX3, androgen receptor, cyclin B1, CN, RhoC, TRP-2, CYP 874-7, BORT 7, SART-72, human RAKE-5, RAKE-5, RAKE-5, and CALT, E7, intestinal carboxylipase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL 75, IGLLI, MPL, FITC, biotin, c-MYC epitope tag, CD 75, LAMP 75, TROP 75, GFR α 4, CDH 75, NYBR 75, CDH 75, CD200 75, IL13Ra 75, CD179 75-LIGTCR, ALK 75, gpNMB, CDH 75-CD 324, LH3672, CD/B7H 75, IL11 75, IL13Ra 75, TGF 75-LIGTCR, ALK, NKG 2-delta-CSF, DLL 72, TGFR-75, TGFHG-75, TGFHGF-75, TGFcGRC-75, TGFcR-75, TGCsCR 72, TGFcR-75, TGCsCR-75, TGFcR-TCR-75, TGFcR-75, TGCsCR-TCR-75, TGFcR-TCR, EBV-EBNA3c, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1(TF1), AFP, GPRC5D, oncoprotein 18.2(CLD18A2 or CLDN18A.2)), P-glycoprotein, STEAP1, LIV1, fibronectin-4, CRIPTO, GPA33, BST1/CD157, low conductivity chloride channel, antigen recognized by TNT antibody, HR, CD171, CS-1, CLL-1, GD 84, TnAg, FLT3, CD38, CD44v6, epitope expressed on acute leukemia or lymphoma but not expressed on hematopoietic progenitor CD43, epitope expressed on glycosylated CD43, KIL 3723, 585737-Ra 5, epitope expressed on hematopoietic progenitor cells, IL 24, Ra-24-Ra-24, and Ra-13 epitope expressed on hematopoietic progenitor cells, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAlX, LMP2, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179 97, ALK, polysialic, PLAC 97, Globoh, NY-BR-1, UPK 97, HAVCR 97, AD3672, PANX 97, GPR 97, LY 697, OR51E 97, TAWT, 97, ETV 72-AML, sperm protein 17, XA 97, Tie 2, MAD-1, LR-GPR 97, SALR 6-97, SARCT 97, SALCS 97, SALCERCT-97, SALCP 97, SARCS 97, SALCR 97, SALCS 97, SALCX 97, SARCS 97, SALCS 97, SARCS 97, SALCS 97, SARCS, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, TSHR, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, and OR51E 2.

In some embodiments, the disease to be treated is an infectious disease, including but not limited to infections of: HIV1, HIV2, HTLV1, Epstein Barr Virus (EBV), Cytomegalovirus (CMV), adenovirus, adeno-associated virus, BK virus (EBV), human herpesvirus 6, human herpesvirus 8, influenza virus, parainfluenza virus, avian influenza virus, MERS and SARS coronavirus, Crimean Congo hemorrhagic fever virus, rhinovirus, enterovirus, dengue virus, West Nile virus, Ebola virus, Marburg virus, Lassa virus, Seca virus, RSV, measles virus, mumps virus, rhinovirus, varicella virus, herpes simplex virus 1 and 2, varicella zoster virus, HIV-1, HTLV1, hepatitis virus, enterovirus, hepatitis B virus, hepatitis C virus, Nipalv virus and rift fever virus, Japanese encephalitis virus, Mycobacterium tuberculosis, atypical mycobacteria species, Yersinia pneumocystis, sporozoon, Toxoplasmosis, rickettsia, Nocardia, Aspergillus, Mucor or Candida. In such diseases, the target antigen associated with the disease is selected from HIV1 envelope glycoprotein, HIV1-gag, HTLV1-Tax, CMVpp65, EBV-EBNA3c, influenza A Hemagglutinin (HA) and GAD.

The disease to be treated or prevented by the methods and compositions of the present disclosure may be an immune or degenerative disease, such as diabetes, multiple sclerosis, rheumatoid arthritis, pemphigus vulgaris, ankylosing spondylitis, hypothyroid thyroiditis, SLE, sarcoidosis, scleroderma, mixed connective tissue disease, graft-versus-host disease, or alzheimer's disease. In such embodiments, the target antigen associated with the disease is an autoantibody. Exemplary autoantibodies that are suitable targets for SIRs are autoantibodies directed against Dsg3 or Dsg 1.

Other non-limiting examples of diseases associated with expression of a target antigen include any of the following cancers or related conditions: colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, non-hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumor of the child, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, tumor of the Central Nervous System (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, Combinations of said cancers, and metastatic lesions of said cancers.

In certain embodiments of the methods or uses described herein, the SIR molecule is administered in combination with an agent that increases the efficacy of immune effector cells, such as one or more of: inhibitors of protein phosphatases, kinase inhibitors, cytokines, chemokines, scFV fragments, bispecific antibodies, immunosuppressive molecules; cell signaling proteins, viral signaling, or reduction of T_REGA level or activity of a cell. Non-limiting examples of protein phosphatase inhibitors include SHP-1 inhibitors and/or SHP-2 inhibitors. LaserNon-limiting examples of enzyme inhibitors include: a CDK4 inhibitor, a CDK4/6 inhibitor (e.g., palbociclib), a BTK inhibitor (e.g., ibrutinib or RN-486), an mTOR inhibitor (e.g., rapamycin or everolimus (RAD001)), a MNK inhibitor, or a dual P13K/mTOR inhibitor. In one embodiment, the BTK inhibitor does not decrease or inhibit the kinase activity of interleukin 2 inducible kinase (ITK). Non-limiting examples of A2a receptor antagonists include Vipadenant. In some embodiments, the agent that inhibits an immunosuppressive molecule can be one or more of: an antibody or antibody fragment, an inhibitory nucleic acid, an aggregated regularly interspaced short palindromic repeats (CRISPR), a transcription activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN) that inhibits expression of an inhibitory molecule. In other embodiments of the methods or uses described herein, the agent that reduces the level or activity of TREG cells is selected from cyclophosphamide, an anti-GITR antibody, CD25 depletion, or a combination thereof. In certain embodiments of the methods or uses described herein, the immunosuppressive molecule is selected from the group consisting of: PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGF β, CEACAM-1, CEACAM-3, and CEACAM-5. In other embodiments, the cytokine is selected from IL2, IL-7, IL-15 or IL-21, or both. In other embodiments, the immune effector cell comprising the SIR molecule and the second combination therapy (e.g., an agent that increases the efficacy of the immune effector cell), e.g., any of the combination therapies disclosed herein, are administered substantially simultaneously or sequentially.

In other embodiments, an agent that inhibits an inhibitory molecule comprises a first polypeptide comprising an inhibitory molecule or fragment thereof and a second polypeptide that provides a positive signal to a cell, and wherein the first polypeptide and second polypeptide are expressed on immune cells comprising a SIR, wherein (i) the first polypeptide comprises PD1, PD-L1, CTLA-4, TIM-3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGF β, CEACAM-1, CEACAM-3, and CEACAM-5 or fragment thereof; and/or (ii) the second polypeptide comprises an intracellular signalling domain comprising a primary signalling domain and/or a co-stimulatory signalling domain. In one embodiment, the primary signaling domain comprises a functional domain of CD3 ζ and/or the co-stimulatory signaling domain comprises a functional domain of a protein selected from the group consisting of 41BB, CD27, and CD 28.

In one embodiment, lymphocyte infusion (e.g., allogeneic lymphocyte infusion) is used to treat cancer, infection, or an immune disease, wherein the lymphocyte infusion comprises at least one SIR-expressing cell of the present disclosure. In one embodiment, autologous lymphocyte infusion is used to treat cancer, infection, or immune disease, wherein autologous lymphocyte infusion comprises at least one SIR-expressing cell described herein.

In one embodiment, the method comprises administering a cell expressing an SIR molecule as described herein in combination with an agent that enhances the activity of the cell expressing an SIR, wherein the agent is a cytokine, e.g., IL-2, IL-7, IL-15, IL-21, or a combination thereof. The cytokine may be delivered in combination with, e.g., simultaneously with or shortly after administration of the SIR expressing cell. Alternatively, the cytokine may be delivered after an extended period of time following administration of SIR-expressing cells, e.g., after assessing the subject's response to SIR-expressing cells. In one embodiment, the cytokine is administered to the subject concurrently with (e.g., on the same day as) or shortly after (e.g., 1, 2, 3, 4, 5, 6, or 7 days after) administration of the cell or population of cells according to any one of claims 143 to 161. In other embodiments, the cytokine is administered to the subject after the cell or population of cells or after an extended period of time (e.g., at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or more) after assessing the subject's response to the cells.

In other embodiments, the cell expressing the SIR molecule is administered in combination with an agent that ameliorates one or more side effects associated with the administration of the cell expressing the SIR molecule. The side effects associated with SIR expressing cells may be selected from Cytokine Release Syndrome (CRS), Hemophagocytic Lymphohistiocytosis (HLH) or neurological complications. Examples of such agents include steroids (e.g., prednisone, dexamethasone), IL6R antagonists (e.g., toclizumab), src kinase inhibitors (e.g., dasatinib), kinase inhibitors (e.g., ibrutinib), calcineurin inhibitors (e.g., tacrolimus or cyclosporine a), or chemotherapeutic drugs (e.g., cyclophosphamide, methotrexate, or vinblastine).

In an embodiment of any of the foregoing methods or uses, the cell expressing the SIR molecule is administered in combination with an agent (e.g., any of the second or third therapies disclosed herein) that treats a disease associated with expression of a tumor antigen. Further exemplary combinations include one or more of the following.

In another embodiment, a cell expressing a SIR molecule (e.g., as described herein) can be administered in combination with another agent that increases expression of a target antigen against which the SIR is directed. For example, classical hodgkin lymphoma is characterized by the actual absence of genes expressed in B cells. B cell phenotypes that are lost in the disease are reported to be reduced by promoter hypermethylation and histone deacetylation and B cell committed transcription factor expression. Du, J et al identified compounds (compounds 27, 40, 49) that promoted the re-expression of the B cell phenotype in classical hodgkin lymphoma cells (Blood; online republishing at 10, 12 days 2016). It has also been reported that the anti-leukemia drugs arsenic trioxide and ATRA, when used alone or in combination with the identified compounds 27, 40 and 49, promote the re-expression of the B cell phenotype in classical hodgkin lymphomas. In one embodiment, cells expressing SIRs targeting B cell markers such as CD19, CD20, CD22, and the like, can be administered in combination with one or more of compounds 27, 40, 49, arsenic trioxide, and ATRA.

In another embodiment, SIR-expressing immune effector cells of the present disclosure (e.g., T cells, NK cells, or hematopoietic stem cells) are administered to a subject along with an agent that disrupts the immunosuppressive pathway in certain cancer tumor environments. In one embodiment, the agent that disrupts an immunosuppressive pathway in the cancer tumor microenvironment is an adenosine A2a receptor antagonist. An exemplary adenosine A2a receptor antagonist that may be administered in conjunction with SIR-expressing immune effector cells of the present disclosure is vipadentan.

In one embodiment, SIR-expressing immune effector cells of the present disclosure (e.g., T cells, NK cells, or hematopoietic stem cells) are administered to a subject who has received a prior stem cell transplant (e.g., an autologous stem cell transplant or an allogeneic stem cell transplant).

In one embodiment, SIR-expressing immune effector cells of the present disclosure (e.g., T cells, NK cells, or hematopoietic stem cells) are administered to a subject who has received a prior dose of chemotherapy, such as melphalan, fludarabine, or cyclophosphamide.

In one embodiment, immune effector cells expressing SIRs of the present disclosure (e.g., T cells, NK cells, or hematopoietic stem cells) are administered to a subject who has received a previous dose of an agent that enhances expression of a SIR target antigen, such as compounds 27, 40, and 49(Du, J et al, Blood, republished online at 10/12 days 2016), arsenic trioxide, or ATRA.

In one embodiment, a cell expressing an SIR molecule (e.g., an SIR molecule described herein) is administered in combination with an agent that increases the efficacy of the cell expressing the SIR molecule (e.g., an agent described herein).

In one embodiment, cells expressing an SIR molecule (e.g., an SIR molecule described herein) are administered in combination with a low immunopotentiating dose of an mTOR inhibitor. While not wishing to be bound by theory, it is believed that treatment with low immunopotentiating doses (e.g., doses that do not completely suppress the immune system but are sufficient to improve immune function) is accompanied by a decrease in PD-1 positive T cells or an increase in PD-1 negative cells. PD-1 positive T cells, but not PD-1 negative T cells, can be depleted by engaging with cells expressing a PD-1 ligand (e.g., PD-L1 or PD-L2).

Animal models can also be used to measure SIR activity. For example, a xenograft model can be used that uses the human cancer associated antigen specific SIR described herein⁺T cells are used to treat primary human pre-B-ALL in immunodeficient mice. See, e.g., Milone et al, Molecular Therapy]17(S): 1453-. Briefly, after establishment of ALL, mice were randomly assigned to treatsAnd (4) treating the groups. Co-injection of varying numbers of cancer-associated antigen-specific SIR (e.g., CD19-SIR) engineered T cells at a 1:1 ratio into NOD-SCID- γ carrying B-ALL^-/-In mice. The number of copies of the cancer-associated antigen-specific SIR vector in spleen DNA from mice was evaluated at various times after T cell injection. Animals were evaluated for leukemia at weekly intervals. Peripheral blood count was measured in mice injected with B-ALL-SIR + T cells or mock transduced T cells. The survival curves of the groups were compared using a time series test. In addition, the expression can be analyzed in NOD-SCID-gamma^-！-Absolute peripheral blood CD4+ and CD8+ T cell counts 4 weeks after T cell injection in mice. Mice were injected with leukemia cells and after 3 weeks with T cells engineered to express SIR through a bicistronic lentiviral vector encoding SIR linked to eGFP. T cells were normalized to 45% -50% infused GFP + T cells by mixing with mock-transduced cells prior to injection and confirmed by flow cytometry. Animals were evaluated for leukemia at 1 week intervals. Survival curves of the SIR + T cell group were compared using a time series test.

Dose-dependent SIR treatment response can be evaluated. See, e.g., Milone et al, Molecular Therapy [ Molecular Therapy ]17 (S):1453-1464 (2009). For example, peripheral blood is obtained 35-70 days after establishment of leukemia in mice with SIR T cells, the same number of mock-transduced T cells, or no T cell injection on day 21. Each group of mice was randomly bled to determine peripheral blood B + ALL blast counts and then sacrificed on days 35 and 49. The remaining animals were evaluated on day 57 and day 70.

Evaluation of cell proliferation and cytokine production has been previously described, for example in Milone et al, Molecular Therapy [ Molecular Therapy ]]17(S):1453 and 1464 (2009). Briefly, the assessment of SIR-mediated proliferation was performed in microtiter plates by mixing washed T cells with K562 cells expressing the disease-associated antigen described herein (K19) or CD32 and CD137(KT32-BBL) (final T cell: K562 ratio of 2: 1). K562 cells were irradiated with gamma radiation prior to use. anti-CD 3 (clone OKT3) and anti-CD 28 (clone 9.3) monoclonal antibodies were added to cultures with KT32-BBL cells to serve as a vaccineIn positive controls that stimulate T cell proliferation, as these signals support long-term CD8+ T cell ex vivo expansion. CountBright was used^TMFluorescent beads (Invitrogen, Carlsbad, CA) and flow cytometry (as described by the manufacturer) counted T cells in culture. SIR + T cells were identified by GFP expression using T cells engineered with eGFP-2A linked lentiviral vector expressing SIR. For SIR + T cells that do not express GFP, SIR + T cells were detected using biotinylated recombinant cancer-associated antigens and secondary avidin-PE conjugates as described herein. Specific monoclonal antibodies (BD biosciences) were also used to simultaneously detect CD4+ and CD8+ expression on T cells. Cytokine measurements were performed on supernatants collected 24 hours after restimulation using a human TH1/TH2 cytokine cytometry bead array kit (BD Biosciences, San Diego, CA), according to the manufacturer's instructions. Fluorescence was assessed using a FACScalibur flow cytometer and the data was analyzed according to the manufacturer's instructions.

Can pass standard⁵¹Cr-release assay to assess cytotoxicity. See, e.g., Milone et al, Molecular Therapy]17(8):1453-1464(2009). Briefly, target cells (K562 line and primary pro-B-ALL cells) were loaded at 37 deg.C⁵¹Cr (e.g. NaCrO4, New England Nuclear, Boston, MA) was washed twice in complete RPMI with frequent stirring for 2 hours and plated into microtiter plates. Effector T cells were mixed with target cells in wells of complete RPMI in varying ratios of effector to target cells (E: T). Additional wells containing either media only (spontaneous release, SR) or 1% triton-X100 detergent solution (total release, TR) were also prepared. After 4 hours incubation at 37 ℃, the supernatant from each well was harvested. The released was then measured using a gamma particle counter (Packard Instrument co., Waltham, MA)⁵¹And Cr. At least triplicate for each condition was performed and percent solubilization was calculated using the formula: percent cell lysis ═ ER-SR)/(TR-SR), where ER denotes release under each experimental conditionAverage of (2)⁵¹And Cr. This assay or similar assays can be used to detect the presence of SIR cells in any population. This assay can also be used to measure the expansion and persistence of SIR cells in any population.

Imaging techniques can be used to assess specific trafficking and proliferation of SIRs in tumor-bearing animal models. For example Barrett et al, Human Gene Therapy]Such assays have been described in 22:1575-1586 (2011). Briefly, NOD/SCID/γ^-！-(NSG) mice were IV injected with Nalm-6 cells, 7 days later with T cells 4 hours after electroporation with SIR encoding mRNA. T cells were stably transfected with lentiviral constructs to express firefly luciferase, and mice were imaged for bioluminescence. Alternatively, the therapeutic effect and specificity of a single injection of SIR + T cells in a Nalm-6 xenograft model can be measured as follows: NSG mice are injected with Nalm-6 transduced to stably express firefly luciferase, followed by a single tail vein injection 7 days later with T cells electroporated with a SIR of the present disclosure (e.g., CD 19-SIR; SEQ ID NO: 1200). Animals were imaged at different time points after injection. For example, photon density heatmaps of firefly luciferase-positive leukemia in representative mice at day 5 (2 days before treatment) and day 8 (24 hours after SIR + PBL) can be generated. Similar methods can be used to evaluate SIR targeting other cancers or other diseases.

Other assays, including those described in the examples section herein and those known in the art, can also be used to evaluate the SIR described herein.

The pharmaceutical compositions of the present disclosure may comprise SIR-expressing cells (e.g., a plurality of SIR-expressing cells as described herein) in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, sorbitol; a protein; polypeptides or amino acids, such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. In one aspect, the compositions of the present disclosure are formulated for intravenous administration. The composition may further comprise a second active agent (e.g., an anti-cancer agent, an anti-viral agent, or an antibiotic agent).

The pharmaceutical compositions of the present disclosure may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administrations will be determined by factors such as the condition of the patient and the type and severity of the patient's disease. When an "immunologically effective amount", "anti-tumor effective amount", "tumor inhibiting effective amount" or "therapeutic amount" or "anti-infective" is indicated, the physician can determine the amount of the disclosed composition to be administered in view of the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). In general, it can be said that a pharmaceutical composition comprising an immune effector cell (e.g., T cell, NK cell) described herein can be administered at 10⁴To 10⁹Individual cells/kg body weight, in some cases 10⁵To 10⁶Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. T cell compositions may also be administered multiple times at these doses. Cells can be administered by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med. [ New England journal of medicine ]]319:1676,1988)。

In certain aspects, it may be desirable to administer activated immune effector cells (e.g., T cells, NK cells) to a subject, and then subsequently redraw the blood (or perform apheresis), activate immune effector cells (e.g., T cells, NK cells) therefrom according to the present disclosure, and use these activated and expanded immune effector cells (e.g., T cells, NK cells) for transfusion back to the patient. This process may be performed multiple times every few weeks. In certain aspects, immune effector cells (e.g., T cells, NK cells) from 10cc to 400cc blood draws can be activated. In certain aspects, immune effector cells (e.g., T cells, NK cells) from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc blood draws are activated.

In some embodiments, the subject may undergo a leukapheresis procedure in which leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate cells of interest (e.g., T cells). These T cell isolates may be expanded and treated and/or transformed by methods known in the art such that one or more SIR constructs of the invention may be introduced to produce SIR T cells of the invention. The subject in need thereof may then receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain aspects, after or concurrently with transplantation, the subject receives an infusion of the expanded SIR T cells of the invention. In another aspect, the expanded cells are administered before or after surgery.

Kits for practicing the disclosure are also provided. For example, a kit for treating cancer in a subject or preparing SIR T cells that express one or more SIRs disclosed herein. The kit may include a nucleic acid molecule or polypeptide molecule encoding a SIR or a vector encoding a SIR and a method of introducing the nucleic acid into an immune effector cell. The kit may include a virus comprising a nucleic acid encoding a SIR and a chemical such as polybrene to enhance viral transduction. The kit may contain components for isolating T cells expressing SIR. Alternatively, the kit may contain immune effector cells (e.g., T cells or NK cells) or stem cells that express the SIR. More than one disclosed SIR is included in the kit. The kit may comprise a container and a label or package insert on or associated with the container.

Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed of various materials, such as glass or plastic. The container typically contains a composition comprising one or more of a nucleic acid molecule, a virus, a vector, a T cell expressing an SIR. In several embodiments, the container may have a sterile access port (e.g., the container may be an intravenous bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is for treating a particular condition in an individual. The label or package insert will typically also include instructions for use of the disclosed nucleic acid molecules, SIRs, or T cells expressing SIRs in methods of treating or preventing a tumor or preparing SIR T cells. Package insert refers to an insert typically included in commercial packaging for therapeutic products that contains information regarding indications, usage, dosage, administration, contraindications, and/or warnings for use of such therapeutic products. The illustrative material may be written, in electronic form (such as a computer diskette or compact diskette), or may be visual (such as a video file). The kit may also include additional components to facilitate the design of the particular application for which the kit is intended. Thus, for example, the kit may additionally contain means for measuring SIR expression on T cells or means for measuring the number or percentage of T cells expressing SIR or means for determining SIRT cell functionality. The kit may additionally include buffers and other reagents conventionally used to carry out particular methods. Such kits and appropriate contents are well known to those skilled in the art.

The disclosure is further described by reference to the following experimental examples. These implementations are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the disclosure should in no way be construed as limited to the following examples, but rather should be construed to cover any and all variations which become evident as a result of the teachings provided herein.

Examples

SIR activity can be tested by several in vitro and in vivo assays, described herein and below. The following provides a general scheme for generating, selecting and using a suitable SIR.

Identification of targets for SIR generation based on a search of a library or gene expression database to select suitable targets for which SIRs are designed. In general, suitable targets for SIRs show higher expression on disease-causing or disease-associated cells compared to normal healthy cells.

Once candidate target antigens for SIR are identified, the antigen binding domain of SIR is designed based on the information available in the library. In general, the antigen binding domain of the SIR is typically based on an antibody, antibody fragment, scFV, or camelidae vHH domain. The sequences of the variable chains of the heavy (vH) and light (vL) chains of antibodies, camelidae vHH domains and various receptors and ligands can be obtained by sequencing or by publicly available databases and can be used to synthesize SIRs using the methods described herein, as shown in the different examples. The sequence of the antigen binding domain containing the SIR was codon optimized and synthesized artificially using publicly available software (e.g., ThermoFisher or IDT) and commercial suppliers (e.g., IDT). The resulting fragments were PCR amplified and cloned in different vectors containing different SIR backbones using standard molecular biology techniques. Generally, SIR constructs are typically cloned in lentiviral vectors. The sequence of the SIR construct was confirmed using automated sequencing.

An exemplary SIR construct is pLenti-EF1a-CD8SP-MYC3-WT1-Ab13-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-WT1-Ab13-vH-Myc4- [ hTCRa-CSDVP ] -F-F2A-PAC-DWPRE [ CLONE ID:071516-J04] (SEQ ID NO: 1380). This construct has a number of convenient restriction sites so that antigen binding domain fragments (e.g., vL and vH domains) can be cleaved and replaced with antigen binding domains that target other antigens. For example, the vL domain can be cut from the vector using EcoR I and Xho I restriction enzyme sites and replaced with a new DNA fragment containing EcoRI and Xho I sites and encoding a vL domain targeted to another antigen. The vector carries a Nhe I site upstream of the CD8 signal peptide (CD8SP), which can also be used together with the Xho I site for cloning in a new vL fragment carrying the 5' signal peptide. The BstB I and Mlu I sites can be used to replace the vH fragment. The Xho I and Spe I sites may be used to replace the module encoding V5- [ hTCRb-KACIAH ] -F-P2A. Similarly, MluI and Xba sites can be used to replace the module containing Myc4- [ hTCRa-CSDVP ] -F-F2A. The ancillary modules of the encoded PAC can be replaced with Xba I (or Nde I) and SalI restriction sites. Thus, one of ordinary skill in the art can use the sequences of this vector and antigen binding domains (e.g., the vL and vH domains of an antibody) to generate SIRs that target any other novel antigen.

Another exemplary construct that can be used to treat SIR where the antigen binding domain is linked to the TCRb constant chain and the TCRa constant chain remains empty is pLenti-EF1a-CD8SP-MYC- [ hTCRa-T48C-opt1] -F-F2A-FMC63-vH-V5- [ hTCRb-S57C-opt1] -F-P2A-Pac-DWPRE [042616-B03] (SEQ ID NO 896). The antigen binding domain can be cloned in this vector between the BstB I and Mlu I restriction enzyme sites. Alternatively, an antigen-binding domain with its own signal sequence may be cloned in this vector between the Spe I and Mlu I restriction enzyme sites.

Another exemplary construct that can be used to treat SIR in which the antigen binding domain is linked to the TCRa constant chain and the TCRb constant chain remains empty is pLenti-EF1a-CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-FMC63-vH-MYC- [ hTCRa-CSDVP ] -F-F2A-Pac-DWPRE [081415-F04] (SEQ ID NO 895). The antigen binding domain can be cloned in this vector between the BstB I and Mlu I restriction enzyme sites. Alternatively, an antigen-binding domain with its own signal sequence may be cloned in this vector between the Spe I and Mlu I restriction enzyme sites.

Secreted antigens-production of NLuc fusion proteins (optional step) to measure expression and binding activity of SIRs on immune effector cells, the extracellular domain of the antigen target of the SIR is fused via a short Gly-Ser linker to a luciferase, such as NLuc or GLuc or MLuc7 or TurboLuc16 or PaLuc. For example, the extracellular domain of CD19 is fused in-frame to NLuc via a Gly-Ser-linker. The fusion protein carries an N-terminal signal sequence. The resulting constructs were transiently transfected in 293FT cells and supernatants containing secreted fusion proteins were collected after 48-72 hours.

To identify suitable cell lines for testing SIR activity, the antigen binding domain of SIR is fused to a luciferase, such as NLuc or GLuc or MLuc7 or TurboLuc16 or PaLuc, via a short Gly-Ser linker. For example, in the case where the SIR is based on the FMC63 monoclonal antibody targeting CD19, the scFV fragment of FMC63 (vL-Gly-Ser-linker-vH) is fused in-frame to the NLuc via a Gly-Ser-linker. The fusion protein carries an N-terminal signal sequence. The resulting constructs were transiently transfected in 293FT cells and supernatants containing secreted fusion proteins were collected after 48-72 hours. A panel of cell lines were tested for binding to ABD-NLuc fusion proteins to identify cell lines that express high levels of SIR targets and therefore can be used to test SIR activity. Table a provides an exemplary list of cell lines expressing different antigenic targets that can be used to determine the activity of the SIRs of the present disclosure. Cell lines expressing targets of SIR can also be identified using alternative methods such as library searches, immunostaining using commercially available antibodies, or by searching publicly available gene expression databases.

Immune effector cells expressing SIR were tested in the following assay to identify functional SIR.

(A) NLuc binding assay: Jurkat-NFAT-GFP or T cells expressing control vectors and SIRs were stained with target antigen-Nluc fusion protein (as described above) and their ability to bind to the target antigen was determined by measuring Nluc activity. For example, Jurkat-NFAT-GFP cells expressing an SIR targeting CD19 based on FMC63 show increased binding to CD19-NLuc fusion protein compared to Jurkat-NFAT-GFP cells expressing a control vector or parental Jurkat-NFAT-GFP cells.

(B) Jurkat-NFAT-GFP cells expressing the control vector and SIR were co-cultured for 4-24 hours with a cell line expressing the target antigen (as described above) and their ability to bind to the target antigen was determined by measuring induction of GFP expression using flow cytometry. Cell supernatants were collected and assayed for induction of cytokines (e.g., IL 2).

(C) Measurement of cytokine production: Jurkat-NFAT-GFP or T cells expressing the control vector and SIR were co-cultured with the target cell line for 4-96 hours and the supernatants were examined for induction of cytokine (e.g., IL2, IFN γ, TNF α, etc.) expression using ELISA.

(D) Determination of cytotoxic activity in vitro and in vivo: uninfected T cells or those expressing control vectors or SIRs were co-cultured with luciferase expressing a non-secreted form (such as GLuc, NLuc, Turboluc16, etc.) for 4-96 hours and the induction of cell lysis was examined by measuring luciferase activity as described in PCT/US 17/52344. Alternative methods for measuring cytotoxic activity may also be used (e.g.⁵¹Cr release assay or LDH release assay). The activity of SIR expressing T cells can also be measured in vivo using an appropriate xenograft model of immunodeficient mice.

Based on the above methods, one of ordinary skill in the art can readily design, construct, test, and select an appropriate functionalized SIR or SIR library for any antigen. The SIR or pool of SIRs may be used in human clinical trials and clinical uses for the prevention and treatment of various disease conditions. Table 9 provides an exemplary list of human disease conditions that can be treated using the SIRs of the present disclosure.

It is likely that different SIRs or subsets of SIRs are best suited for different disease conditions depending on various factors including, but not limited to, prevalence and levels of expression of target antigens on disease-causing and disease-associated cells, disease burden, and rate of disease progression. Depending on its efficacy and toxicity profile and the condition of the patient, different SIRs may even be best suited for a single disease condition in different patients. The present disclosure provides a solution to the significant technical and logical hurdles to generating diverse adoptive immune responses.

Normal TCR diversity is generated by gene rearrangement. The strict positive and negative selection process in the thymus ensures that only T cells expressing α β TCR restricted to recognition of self peptides/MHC in the low affinity range can be populated in the periphery. Thus, the thymic environment allows the generation of self-restricted, but not self-reactive α β T cell banks.

The generation of different SIR libraries from different antigen binding domains is limited by the technical and financial barriers to generating and testing multiple antigen binding domains. More importantly, since each antigen binding domain (e.g., vL and vH fragments of an antibody) has the potential to bind to other antigens and cause off-target toxicity, different SIR libraries based solely on multiple antigen binding domains may have an increased risk of toxicity. Thus, the potential diversity of this library would have to be limited to reducing off-target toxicity. The present disclosure overcomes this problem by generating different SIR libraries from a single or several antigen binding domains by attaching the single or several antigen binding domains to different variants of TCR chains. The diversity of the SIR library is further increased by using different linkers. The diversity of T cells expressing this pool can be further increased by using different helper modules and therapeutic controls as described in the present disclosure.

This diverse pool of SIRs can be used to provide diverse immune responses against disease-causing or disease-associated cells expressing the antigen. Alternatively, different SIR libraries may optionally be DNA barcoded using techniques known in the art and subsequently used to select a single SIR or SIR subset with the best biological and clinical characteristics. These characteristics may include, but are not limited to, performance of in vitro bioassays (e.g., cytotoxicity, cytokine secretion, binding affinity, cell surface expression, off-target effects, T cell proliferation, depletion marker expression, and terminal differentiation, etc.), performance of in vivo assays (e.g., survival, tumor reduction, T cell persistence, T cell expansion, etc.), and clinical experience (e.g., disease regression, recurrence rate, toxicity, etc.). The SIRs of the present disclosure can be used alone or in combination with other SIRs, CARs, ctcrs, and other natural and synthetic immune receptors known in the art to generate a diverse pool of immune effector cells to prevent and treat various disease conditions caused by or associated with cells expressing their target antigens.

The SIRs of the present disclosure are modified forms of chimeric T cell receptors that have been optimized not only for diversity, but also for binding affinity, cell surface expression, chain pairing, signaling, and cell delivery. To generate a double-stranded SIR suitable for clinical applications, several technical and conceptual hurdles are addressed. The major limitation of the double-chain tcr platform is the technical difficulty of delivering two different plasmid constructs. Although it is possible to transduce T cells with two strands of double-stranded SIR in two separate vectors or a single vector with two separate promoters, these two approaches risk unbalanced introduction of expression of the SIR chains, thereby increasing the likelihood that an over-expressed chain will be mismatched to the endogenous TCR chain. An additional limitation of the two vector approach is the lower infection efficiency of the target cells compared to the single vector approach. The use of two separate promoters in a single vector has the disadvantage of increasing the insert size and thus increasing the vector size beyond the optimal packaging limits of most retroviral and lentiviral vectors, resulting in reduced viral titer. In addition, internal promoters can be silenced in retroviral vectors by promoter interference. Although Internal Ribosome Entry Site (IRES) sequences of encephalomyocarditis virus have been widely used to construct bicistronic viral vectors, IRES-mediated translation is relatively inefficient.In addition, the size of IRES is relatively large and the use of IRES elements can lead to competition for translation factors and/or homologous recombination. In this regard, the ` self-cleaving ` 2A peptide sequence derived from picornavirus or porcine teschovirus is selected to bind between the two SIR chains to achieve equimolar expression of the two introduced SIR chains. These sequences induce translation of two separate peptides from a single mRNA transcript by the ribosome skipping mechanism, thereby achieving approximately stoichiometric production of each peptide. Examples of cleavable "2A" peptides are provided in SEQ ID Nos: 3060-3062 and 3064. In some embodiments, (SG)₂Also added upstream of the 2A sequence to enhance cleavage efficiency. In addition, to avoid any remaining 2A peptide sequence attached to the SIR, a furin cleavage site was added at (SG)₂Upstream was continued to facilitate cleavage of residual 2A peptide after translation.

Lentiviral and retroviral vectors were initially selected to develop a double stranded SIR system suitable for clinical use. Subsequently, experiments used sleeping beauty transposon and mRNA transfection to successfully express SIR in T cells.

The pLENTI-Blast vector was derived from the pLenti6v5gw _ LacZ vector (Invitrogen; Sammer Feishel Scientific) by removing the LacZ gene. pLenti-MP2 was gifted by Pattelis Toulfas (Edison plasmid number 36097; Enomoto et al. Exp. neuron. [ Experimental neurology ]248: 170. sub.82, 2013) and was used to generate pLENTI-EF 1. alpha. lentiviral vectors (SEQ ID NO:870) using standard molecular biology techniques by replacing the CMV promoter with the human EF 1. alpha. promoter. psPAX2 was awarded by Didier Trono (Edy Gene plasmid number 12260). pLP/VSVG envelope plasmids and 293FT cells were obtained from the Acceptor (Seimer Feishell science). Retroviral transfer vectors MSCVneo, MSCVhygro and MSCVpac and the packaging vector pKAT were obtained from the laboratory of Robert, Illaria. The phRGTK renilla luciferase plasmid was from Promega (Promega).

Gene segments encoding different signal peptides, antibody binding domains, linkers, TCR constant chains, cleavable linkers and selection markers (e.g., PAC, EGFP, CNB30, etc.) were synthesized in single or multiple fragments using commercial suppliers (IDT) and used as templates in PCR reactions using primers containing appropriate restriction enzymes. The amplified fragment was digested with the appropriate restriction enzymes and then cloned using standard molecular biology techniques in pLENTI-EF1 alpha (SEQ ID NO:870), pLENTI-EF1 alpha-DWPRE (SEQ ID NO:871) or MSCV-Bgl2-AvrII-Bam-EcoR1-Xho-BstB1-Mlu-Sal-ClaI.I03(SEQ ID NO:872) vectors. The pLENTI-EF1 α -DWPRE vector differs from pLENTI-EF1 α in the absence of the WPRE region. Alternatively, a gene segment encoding the entire SIR cassette (e.g., CD8 signal peptide-161-vL-TCRb-F-2A-IgH-signal peptide-161-vH-TCRa-F-2A-PAC) may be synthesized artificially. The resulting fragment can then be used as a template in a PCR reaction using primers containing appropriate restriction enzymes. The amplified fragments can be digested with appropriate restriction enzymes and then cloned in appropriate vectors using molecular biology techniques.

A number of different lentiviral constructs were made containing vL and vH fragments derived from FMC63 monoclonal antibody fused in-frame with the constant regions of human TCR- β (TCRb) chain and human TCR- α (TCRa) chain, respectively. Most constructs also carry different selection markers (e.g. puromycin N-acetyl-transferase (PAC), Enhanced Green Fluorescent Protein (EGFP) and secretory NLuc) fused to the SIR expression cassette via a linker encoding furin-SGSG-2A.

An exemplary construct is CD8SP-FMC63-vL-V5- [ hTCRb-WT ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-WT ] -F-F2A-PAC (080815-F02) cloned in the pLenti-EF1a vector (SEQ ID NO:870) [ SEQ ID NO:922 ]. The SIR expression cassette in this vector is driven by the human EF1 α (elongation 1 α) promoter. The SIR expression cassette comprises variant forms selected from the group consisting of a V5 linker containing Gly-Ser-Gly amino acids at the C-terminus, the constant region (C region) of the human TCR- β 2(TCRb) chain, a furin cleavage site (RAKR), an SGSG linker, a P2A ribosome skipping sequence, a human IgH signal peptide, a codon optimized FMC63vH fragment, a Myc linker derived from a commonly used Myc epitope tag and containing Gly-Ser-Gly amino acids at the C-terminus, the constant region (C region) of the human TCR- α (TCRa) chain, a furin cleavage site (RAKR), an SGSG linker, the 5' -terminal nucleotide of the F2A ribosome skipping sequence, and a puromycin resistance gene (PAC). There is an Xho I restriction enzyme site between the vL fragment and the V5 epitope tag, a Spe I restriction site before the human IgH signal peptide, a Mlu I site before the Myc tag, and a short linker containing Xba I and Nde I sites before the PAC gene. The entire expression cassette was cloned between the Nhe I and Sal I sites in the pLenti-EF1a vector (SEQ ID NO: 870). This vector can be used to clone antigen binding domains targeting other antigens by removing FMC63-vL and-vH fragments by digestion with Nhe I and XhoI and Spe I and Mlu I enzymes, respectively, and replacing them with DNA fragments targeting antigen binding domains of other antigens (e.g., vL, vH, vHH, scFv, receptors or ligands) using standard molecular biology techniques.

Several variants of the above construct were also created:

pLenti-EF1-FMC63vL-V5- [ mTCRb-WT ] -F-P2A-FMC63-vH-Myc- [ mTCRa-WT ] -F-F2A-Pac-B06: this construct is similar to the pLenti-EF1-FMC63vL-V5- [ hTCRb-WT ] -F-P2A-FMC63-vH-Myc- [ hTCRa-WT ] -F-F2A-Pac-F02 construct, except that it has a mouse TCR- β (TCRb) and a constant region of the mouse TCR α chain instead of the corresponding human chain (C region).

2. pLenti-EF1-FMC63-vL-V5- [ TCRb-S57C-opt1] -F-P2A-FMC63-vH-Myc- [ TCR-ca-T48C-opt1] -F-F2A-PAC-L05 (050515-L05): this construct differs from pLenti-EF1-FMC63vL-V5- [ hTCRB-WT ] -F-P2A-FMC63-vH-Myc- [ hTCRa-WT ] -F-F2A-Pac-F02 in the following ways:

a. the nucleotide sequences encoding the constant regions of the TCRb and TCRa chains were codon optimized to increase SIR expression.

b. To reduce the mismatch of the introduced TCR chains with endogenous TCR chains, additional cysteine residues are added to each chain to facilitate the formation of additional interchain disulfide bonds. Thus, the TCRb sequence carries a Ser 57 to cysteine mutation and the TCRa sequence carries a threonine 48 to cysteine mutation.

pLenti-EF1-FMC63-vL-V5- [ TCRb-KACIAH ] -F-P2A-FMC63-vH-Myc- [ TCRa-CSDVP ] -F-F2A-PAC-D06 (081415-D06): this construct differs from pLenti-EF1-FMC63vL-V5- [ hTCRB-WT ] -F-P2A-FMC63-vH-Myc- [ hTCRa-WT ] -F-F2A-Pac-F02 in the following ways:

a. the nucleotide sequences encoding the constant regions of the TCRb and TCRa chains were codon optimized to increase SIR expression.

b. To reduce the mismatch of the introduced TCR chains with endogenous TCR chains, additional cysteine residues are added to each chain to facilitate the formation of additional interchain disulfide bonds. Thus, the TCRb sequence carries a Ser 57 to cysteine mutation and the TCRa sequence carries a threonine 48 to cysteine mutation.

c. Murine TCRs have been shown to express better than their human counterparts and the murine version of human TCRs has been shown to improve their expression. In this construct, five amino acids of the human TCR β (TCRb) constant region were replaced with corresponding murine amino acids. These murine amino acids are K-18, A-22, I-133, A-136 and H-139. In addition, in the human TCR alpha (TCRa) constant chain (SEQ ID NO:3010), the region of the murine TCR alpha (TCRa) constant chain containing the four amino acids serine (S-91), aspartic acid (D-92), valine (V-93) and proline (P-94) was substituted.

In addition to lentiviral vectors, SIR expression cassettes were cloned into retroviral vectors. For this purpose, the pMSCV-puro retroviral vector was modified by digesting with Bgl II and Cla I enzymes and ligating Bgl II and ClaI-cleaved double-stranded oligonucleotides containing restriction enzyme sites for AvrII, BamHI, EcoRI, XhoI, BstBI, MluI and SalI. The sequence of the resulting vector is designated MSCV-Bgl2-AvrII-Bam-EcoR1-Xho-BstBI-Mlu-Sal-ClaI.I03 in SEQ ID NO: 872. The expression cassettes for the different SIR constructs were digested from the pLenti-EF1 vector using NheI and SalI enzymes and cloned in the above vectors which had been digested with AvrII and SalI. The sequence of the exemplary SIR construct MSCV-FMC63vL-V5- [ TCRb-KACIAH ] -F-P2A-2-Spe-FMC63vH-MYC- [ TCRa-CSDVP ] -F-F2A-Pac.N01[ CLONE ID: 032216-N01] is presented in SEQ ID NO: 873. Other SIR constructs in MSCV-based retroviral vectors can be readily constructed by cloning the NheI to SalI fragments from their corresponding pLenti-EF 1.

The SIR expression cassette was also cloned into sleeping beauty vectors pSBbi-pur (SEQ ID NO: 874; Edison; plasmid No. 60523) and pSBbi-GP (Edison; plasmid No. 60511). For this purpose, the expression cassettes for the different SIR constructs were digested from the pLenti-EF1 vector using AgeI and XbaI enzymes and cloned in pSBbi-pur and pSBbi-GP vectors which had been digested with AgeI and XbaI. The sequence of an exemplary vector encoding the SIR targeting CD19 is provided in SEQ ID No. 875. The sequence of another exemplary sleeping beauty vector with an extended multiple cloning site is provided in SEQ ID NO 876.

Cell lines engineered to express luciferases (e.g. GLuc or NLuc) for measuring cytotoxicity of different constructs targeting different cell surface and intracellular antigens are provided in table a. The cell lines used in this experiment, the target antigens on these cell lines and their growth media are shown in table a below. Cells were cultured at 37 ℃ in a 5% CO2 humidified incubator. Cell lines were obtained from ATCC, NIH AIDS reagent procedure or available in laboratory.

Table a:

the Jurkat cell line (clone E6-1) engineered with the NFAT-dependent GFP reporter was gifted by Dr. Arthur Weiss, UCSF. Jurkat cells were maintained in RPMI-1640 medium supplemented with 10% FBS, penicillin and streptomycin.

Lentiviruses and retroviruses were generated by calcium phosphate-based transfection in 293FT cells essentially as described previously (Matta, Hozayev, Tomar, Chugh, & Chaudhary, 2003). Growth of 293FT cells in DMEM with 10% FCS 4mM L-glutamate, 0.1mM MEM non-essential amino acids and 1mM MEM sodium pyruvate (herein referred to as DMEM-10) to generate lentiviruses, 293FT cells were seeded in tissue culture plates in 10ml of antibiotic-free DMEM-10 medium so that they would be approximately 80 confluent on the day of transfection. The next day, cells were transfected by the calcium phosphate transfection method using 10. mu.g of the lentiviral expression plasmid encoding the different genes, 7.5. mu.g of the PSPAX2 plasmid, and 2. mu.g of the PLP/VSVG plasmid. Approximately 15-16 hours after transfection, 9ml of medium was removed and replaced with 5ml of fresh medium. Approximately 48 hours after transfection, 5ml of supernatant (first collection) was collected and replaced with fresh 5ml of medium. Approximately 72 hours after transfection, the entire medium was collected (second collection, typically about 6 ml). The collected supernatants were combined and centrifuged at 1000rpm for 1 minute to remove any cell debris and non-adherent cells. The cell-free supernatant was filtered through a 0.45 μm syringe filter. In some cases, the supernatant was further concentrated by ultracentrifugation at 18500rpm for 2 hours at 4 ℃. The viral pellet was resuspended in 1/10 initial volume of XVIVO medium. Target cells were then either freshly infected with virus or cryopreserved in aliquots at-80 ℃.

Buffy coat cells were obtained from healthy unidentified adult donors in the blood bank of the los angeles children hospital and used to isolate Peripheral Blood Mononuclear Cells (PBMCs) by serodextran (Ficoll-Hypaque) gradient centrifugation. CD3 magnetic microbeads (Miltenyi Biotech) were used as such or for the isolation of T cells, following the manufacturer's instructions. PBMC or isolated T cells were resuspended in XVIVO medium (Longsha, Lonza) supplemented with 10ng/ml CD3 antibody, 10ng/ml CD28 antibody and 100IU recombinant human-IL 2. Cells were cultured at 37 ℃ in a 5% CO2 humidified incubator. Cells were activated in the above medium for 1 day before infection with lentiviral vectors. In general, use of a suspension that has been resuspended in the presence of 8. mu.g/ml of(Sigma, Cat H9268) infected primary cells (e.g., T cells) with 300. mu.l of concentrated virus in XVIVO medium. The medium was changed in the evening and the infection was repeated for more than two days for a total of 3 infections. After infection 3, cells were pelleted and resuspended in fresh XVIVO medium containing 10ng/ml CD3 antibody, 10ng/ml CD28 antibody, and 100IU of recombinant human-IL 2 supplemented with the corresponding antibiotics (as indicated) and placed in cell culture flasks for selection unless otherwise indicated. The cells were cultured in the above medium,for 10-15 days without drug selection and for 20-30 days with drug selection. If cells are infected with an EGFP-expressing lentivirus, the cells are expanded without drug selection or flow sorted to enrich for EGFP-expressing cells. For infecting cancer cell lines, in(Sigma, Cat. H9268) approximately 500000 cells were infected with 2ml of unconcentrated virus supernatant in a total volume of 3 ml. The next morning, cells were then pelleted and resuspended in media with the corresponding antibiotic and placed in cell culture flasks for selection.

Procedures substantially similar to those described above for lentiviral vector production were used to generate retroviruses, except that 293FT cells were generally transfected with 10. mu.g of the retroviral construct, 4. mu.g of pKAT and 2. mu.g of VSVG plasmid in 10ml of DMEM-10 medium in 10cm tissue culture plates. Viral collection and target cell infection are performed essentially as described above for lentiviral vectors.

Jurkat cells were electroporated using sleeping beauty vehicle for electroporation 5X 10⁶The Jurkat cells were centrifuged at 90g for 10 minutes, resuspended in 100. mu.L buffer and mixed with 20. mu.g of the plasmid encoding sleeping beauty SIR and 5. mu.g of SB100X transposase plasmid. AmaxaCell Line Nuclear optoelector kit V (VCA-1003) from Dragon Sand is provided to the electroporation buffer and cuvette. The resuspended cells were transferred to a cuvette and electroporated using procedure X-001. After electroporation, cells were incubated in the cuvette for 10 minutes at room temperature and then 1ml of pre-warmed RPMI medium supplemented with 20% FBS was added to the cells in the cuvette. Cells were transferred to 6-well plates containing 1ml of pre-warmed medium per well and incubated overnight at 37 ℃. The next day, cells were centrifuged and the medium was replaced by RPMI supplemented with 10% FBS and 250ng/ml puromycin to select Jurkat cells expressing sleeping beauty-SIR.

Digitonin was purchased from sigma (catalog No. D141) and made as a100 mg/ml stock solution in DMSO. Diluted 1mg/ml stock solutions were prepared in PBS. The final concentration of digitonin used for cell lysis was 30 μ g/ml unless otherwise indicated.

IL2ELISA. human IL2 was measured in cell culture supernatants of SIR expressing Jurkat-NFAT-GFP effector cells or T cells that had been co-cultured with specific target cell lines for 24 to 96 hours using IL2-ELISA kit from R & D systems, Minneapolis, MN, following the manufacturer's recommendations.

Mouse anti-human c-Myc APC-conjugated monoclonal antibody (catalog No. IC3696A) was from R & D systems, inc (minneapolis, mn). Biotinylated protein L was purchased from GeneScript (Piscataway, NJ), reconstituted at 1mg/ml with Phosphate Buffered Saline (PBS) and stored at 4 ℃. streptavidin-APC (SA1005) was purchased from Saimer Feishell scientific Co.

To detect CAR and SIR using Myc staining, 1 × 10 was harvested⁶Cells were washed three times with 3ml ice-cold 1 × PBS containing 4% Bovine Serum Albumin (BSA) wash buffer. After washing, the cells were resuspended in 0.1ml ice-cold wash buffer containing 10 μ Ι apc-conjugated Myc antibody and incubated in the dark for 1 hour, followed by two washes with ice-cold wash buffer.

To detect CAR and SIR using protein L staining, 1 × 10 was harvested⁶Cells were washed three times with 3ml ice-cold 1 × PBS containing 4% Bovine Serum Albumin (BSA) wash buffer. After washing, the cells were resuspended in 0.1ml ice-cold wash buffer containing 1. mu.g protein L for 1 hour at 4 ℃. Cells were washed three times with ice-cold wash buffer and then incubated (in the dark) with 10 μ Ι APC-conjugated streptavidin in 0.1ml wash buffer for 30 minutes, followed by two washes with ice-cold wash buffer. FACS was analyzed using a FACSVerse analyzer from BD Biosciences (BD Biosciences).

Measurement of cell death Ex-situ cells based on Gluc or NLuc were usedNovel assays for cytosolic expression, e.g. PCT/US17/52344 "A Non-Radioactive cytotoxic Assay]"is said. The method involves expressing the reporter gene in the target cell in a manner such that it is preferentially retained within healthy cells, but released from or its activity can be preferentially measured in dead and dying cells. Preferred reporter genes for this assay are 1) non-secreted forms of luciferases from radial-legged animals such as Gaussiaprinceps, Metridia abdominalis (Pleuromamma abdominalis), Pacific flea pacificus (Metridia acificana), Metridia capitata (Metridia curculida), asymmetric Metridia abdominalis (Metridia asymmetrica), Metridia okhotenis, Metridia elongata (Metridia longa), Lucicutia ovaliformis, Heterorhabdus tanneri and Pedalis peltata (Pleuromamma sciulus), 2) engineered reporter luciferase genes from deep shrimps such as NanoLuc. The sequences of several such reporter vectors are provided in SEQ ID NO 881 through SEQ ID NO 887. The above vectors are used to generate retroviruses and lentiviruses, which in turn are used to generate polyclonal populations of several target cell lines stably expressing GLuc, NLuc, TurboLuc or MLuc7 after selection with appropriate antibiotics. Unless otherwise indicated, target cells stably expressing different luciferases (GLuc, Nluc, MLuc7, or TurboLuc16) were seeded in 384-well plates in three replicates in medium for growing the target cells. Target cells grown in suspension are typically grown at 2-3x10 per well⁴And the target cells grown as adherent monolayers were seeded at a concentration of 1-2x10 per well⁴The concentration of (4) is inoculated. Unless otherwise indicated, target cells were co-cultured with genetically modified T cells (i.e., those expressing SIR or CAR) at effector: target (E: T) ratios varying between 1:1 to 10:1 for 4 hours to 96 hours. In the case of target cells grown as adherent cells (e.g., HeLa cells), they were attached to the bottom of the wells overnight, after which T cells were added. T cell mediated induction of target cell lysis was determined by increasing luciferase activity by direct injection of 0.5xCTZ assay buffer containing native coelenterazine (nanaoght), as measured by a BioTek synergy plate reader.

CTZ assay A100X stock solution of native coelenterazine (CTZ; Nanolight, Cat. 303) was prepared by dissolving 1mg of lyophilized CT powder in 1.1ml of 100% methanol supplemented with 30. mu.l of 6N HCl to avoid oxidation of the CTZ over time. To prepare the CTZ assay buffer, 100X CTZ stock solution was diluted to 0.5X concentration in PBS. Unless otherwise indicated, a total volume of 15 μ Ι of CTZ assay buffer (prepared as above) was added to each well of a 384-well whiteboard (greenner, catalog No. 384-well whiteboard 781075), which contained cells expressing luciferase in non-secreted form in a volume of about 50-60 μ Ι of medium, and the fluorescence of the plates was read in end-point mode using a BioTek synergy h4 plate reader. For 96-well plates, cells were seeded in 200 μ l medium and approximately 50 μ l of 0.5X CTZ assay buffer was added. Unless otherwise indicated, 0.5X CTZ assay buffer was used to determine the activity of GLuc, TurboLuc16 and MLuc 7. CTZ assay buffer (diluted to 0.125X concentration) was also used in some experiments to measure NLuc activity (see below). Generally, unless otherwise indicated, the volume of 0.5X CTZ assay buffer added is about 1/4 of the liquid volume in the well containing the cells, although the assay is useful when the 0.5X CTZ assay is added to the medium containing the cells at a 1:1 volume. When indicated, Gluc activity in wells containing medium alone (Med) and wells in which target cells were incubated with T cells not infected with any SIR construct (T-UI) was used as a control.

The fluorescence of the plates was read in endpoint mode using a BioTek synergyH4 plate reader without prior cell lysis. In some experiments, NLuc activity was measured using a CTZ assay buffer, but here the buffer was diluted to a final concentration of 0.125X. When CTZ assay buffer is used to measure NLuc activity, a total volume of about 15 μ Ι (unless otherwise indicated) of 0.125X CTZ assay buffer is added by syringe to each well of a 384-well whiteboard (greenner, 384-well whiteboard catalog No. 781075) containing a volume of cells in culture medium of about 50-60 μ Ι and the fluorescence of the plate is read in endpoint mode using a bioteksyergy h4 plate reader. For 96-well plates, cells are typically seeded in 200 μ l of medium and approximately 50 μ l of 0.125X CTZ assay buffer is added.

Assays to detect expression of CD19 And MPL (thrombopoietin receptor) antigens to detect expression of SIRs And their target antigens, Luciferase-Based Reporter assays are utilized, such as PCT/US2017/025602 "a high hlysensitive And Specific Luciferase-Based Reporter Assay For antigen detection ]". Both CD19 and MPL (also known as thrombopoietin receptor or TPO-R) are expressed on hematopoietic cells, but show different expression in cells of different lines. FMC63 is a well-characterized mouse monoclonal antibody that specifically recognizes human CD 19. Similarly, 161 is a monoclonal antibody that recognizes human MPL. Based on the known sequence of FMC63vL and the vH fragment, an FMC63 single chain fv (scfv) fragment was generated. The cDNA encoding the FMC63scFv fragment consisted of a nucleotide sequence encoding, from 5 'to 3', a signal peptide derived from human CD8 molecule, an FMC63vL fragment, a (Gly4Ser) x3 linker and an FMC63-vH fragment. The cDNA encoding the FMC63scFv fragment was then fused in-frame at its 3' end by a Gly-Gly-Ser-Gly linker to the cDNA encoding AcV 5-labeled NLuc to generate FMC63-GGSG-NLuc, which was then cloned downstream of the human EF1a promoter of the lentiviral vector pLenti-EF1 to make the construct Plenti-EF1a-FMC63(vL-vH) -GGSG-NLuc-AcV5-U09(SEQ ID NO: 880). The insert fragment sequence is provided in SEQ ID NO: 4516. Constructs encoding 161-GGSG-NLuc were similarly generated using vL and vH fragments of 161 monoclonal antibody against MPL. The nucleic acid sequence of the insert fragment is provided in SEQ ID NO: 4517. The pLenti-EF1-FMC63-GGSG-NLuc-AcV5 and pLenti-EF1-161-GGSG-NLuc-AcV5 plasmids were transfected into 293FT cells by the calcium phosphate co-precipitation method. Approximately 20h after transfection, the cell culture medium was replaced with XVIVO medium. After 48-72h, the conditioned medium containing secreted FMC63-GGSG-NLuc-AcV5 and 161-GGSG-NLuc-AcV5 proteins was collected.

Supernatants containing FMC63-GGSG-NLuc-AcV5 and 161-GGSG-NLuc-AcV5 proteins were used to detect expression of CD19 and MPL on the surface of Jurkat, K562, RAJI, RS-4-11(RS411) and HEL.92.1.7(HEL) cells that had been engineered to express c-MPL cDNA by transducing these cells with lentiviral vectors or empty vectors expressing human c-MPL cDNA. These cells also expressed humanized Gluc cDNA lacking its signal peptide. Jurkat-Gluc, K562-Gluc, HEL.92.1.7-Gluc, RAJI-Gluc and RS411-Gluc cells of the expression vector and MPL were incubated with FMC63-GGSG-NLuc-AcV5 and 161-GGSG-NLuc-AcV5 supernatants for 1h at 4 ℃ followed by extensive washing with cold PBS supplemented with 0.1% BSA. Cells were resuspended in cold PBS and seeded with 30 μ l of cell supernatant per well in flat bottom 384 well plates (greenner, 384 well whiteboard catalogue number 781075) in triplicate. NLuc assay buffer containing native Coelenterazine (CTZ) as NLuc substrate (30 μ Ι/well of native coelenterazine diluted in PBS) was added to each well in well mode by an automatic dispenser using a BioTek synergyH4 plate reader and the light emission measured as a measure of NLuc activity. The strong binding observed with 161-GGSG-NLuc-AcV5 was observed on hel.92.1.7-Gluc-vector cells, indicating an endogenous significant expression of MPL. Ectopic expression of MPL in HEL.92.1.7-Gluc-MPL cells caused a slight increase in 161-GGSG-NLuc-AcV5 binding. In contrast, very weak binding to 161-GGSG-NLuc-AcV5 was observed on Jurkat, RAJI and RS411 cells of the expression vector, and only moderately increased when MPL was ectopically expressed. Binding of 161-GGSG-NLuc-AcV5 was observed on K562-carrier cells and was significantly increased on K562-MPL cells. Compared to 161-GGSG-NLuc-AcV5, FMC63-GGSG-NLuc-AcV5 supernatant showed the strongest binding on RAJI cells expressing vector and MPL, moderately strong binding on RS411 cells, and very weak to negligible binding on other cells.

A common problem in the field of adoptive cell therapy is the lack of sensitive and specific assays that can detect cells expressing chimeric antigen receptors and SIRs. Although staining with protein-L can be used to detect cell surface expression of scFv containing CAR and SIR, it fails to measure the interaction of CAR and SIR with their target antigens. To detect the binding affinity of CARs targeting CD19 and MPL, a high sensitivity luciferase reporter based antigen detection assay was used, as described in PCT/US2017/025602, which is incorporated by reference in its entirety. The extracellular domains of human CD19 and human MPL (including their signal peptides) are fused in-frame to nucleotide sequences encoding Gly-Ser-Gly linkers, NLuc (no secretion signal) and AcV5 epitope tags. In the case of the CD19 construct, a FLAG tag was inserted between the signal peptide and the beginning of the extracellular domain. The entire cassette was cloned downstream of the human EF1 alpha promoter of the lentiviral vector pLenti-EF1 to make constructs pLenti-EF1-CD19-GGSG-NLuc-AcV5 and pLenti-EF1-MPL-GGSG-NLuc-AcV5, respectively. The nucleic acid sequences of the insert fragments are provided in SEQ ID NO:4518 and 4519, respectively. Constructs were transfected into 293FT cells by calcium phosphate co-precipitation method. Approximately 20h after transfection, the cell culture medium was replaced with fresh medium. After 48-72h, the conditioned medium containing secreted Flag-CD19-GGSG-NLuc-AcV5 and MPL-GGSG-NLuc-AcV5 proteins was collected.

293 FT-cells (500 ul volume in 24-well plates) were transiently transfected or untransfected with lentiviral constructs expressing chimeric antigen receptors targeting CD19(FMC 63-BBZ-PAC; SEQ ID NO:4501), MPL (161-BBZ-PAC-R07; SEQ ID NO:4502 or 161-28Z-PAC-Z07) using a calcium phosphate co-transfection method. The next morning, approximately 18 hours after transfection, cells were harvested by pipetting up and down in 1.5ml tubes. The tube was spun at 1500RPM for 5 minutes. The cells were then washed once with wash buffer (1% FBS in PBS) and subsequently incubated with 100 μ l of the indicated secreted form of GGS NLuc supernatant. Cells were incubated at 4 ℃ for 1 hour.

After incubation, cells were washed 5 times with wash buffer (1 ml per wash). Finally, the pellet was resuspended in 200. mu.l of wash buffer. The resuspended cells were placed in 384-well plates in triplicate (25. mu.l each). Luciferase activity was measured using a BioTek synergy H4 plate reader after adding NLuc assay buffer (plomega corporation) containing native coelenterazine (25 μ l per well) directly to each well (one well at a time).

As shown in fig. 9A-B, 293FT cells expressing CD19(FMC63-BBZ-PAC) -CAR demonstrated strong binding to Flag-CD19-GGSG-NLuc-AcV5 as measured by the NLuc assay, while very little binding was seen on uninfected T cells (UI) or those expressing the control 161-BBZ-PAC CAR. Similarly, 293FT cells expressing 161-CD28Z-PAC CAR showed strong binding to MPL-GGSG-NLuc-AcV5 supernatant compared to 293FT cells that were not transfected or those transfected with FMC 63-BBZ-PACCAR. The results demonstrate the ability of the GGSG-NLuc assay (or NLuc binding assay) to measure the binding of cell surface expressed chimeric receptors to their antigen targets in a sensitive and quantitative manner. A number of other NLuc fusion proteins containing CARs and extracellular domains of different potential targets of SIRs were constructed and validated using 293T or T cells expressing their corresponding CARs. The names, DNA and amino acid SEQ ID NOs of these constructs are provided in table 7I. Similar constructs can be generated against other antigen targets of SIR by fusing the extracellular domain that is the CAR/SIR target to NLuc via a short flexible linker. CD20 is a type III membrane protein with two extracellular loops. A CD20-ECx2-ECD-GGSG-TurboLuc16-4xFlag-2xStreptag-8xHis-T2A-Pac (060816-I04) fusion construct was successfully generated and validated against cells expressing CD20 CAR. The amino acid sequence of this construct is represented by SEQ ID NO: 12374. Thus, NLuc (or TurboLuc16) fusion proteins can be generated using any protein antigen target of the CAR or SIR that can be used to detect the expression and binding affinity of cells expressing the CAR/SIR.

Protein L is known to bind to the kappa light chain. To examine the expression of CAR and SIR containing kappa light chain, two NLuc fusion constructs containing the N-terminal protein L coding region downstream of the CD8 signal peptide were constructed. Both constructs were identical except that construct CD8SP-Protein-L-2-GGSG-NLuc-4xFLAG-x2STREP-8xHis-T2A-PAC (101916-P03) [ SEQ ID NO:12382] lacked a single amino acid in the Protein L coding region, which amino acid was present in construct CD8 SP-Protein-L-GGSG-NLuc-4 xFLAG-x2STREP-8xHis-T2A-PAC (112316-Q02) [ SEQ ID NO:12381 ]. The conditioned supernatant containing the fusion protein was generated by transfection of the construct in 293FT cells and was shown to bind to a CAR construct containing a kappa light chain, wherein the kappa light chain binds to protein L. antigen-Nluc fusion proteins such as CD19-ECD-GGSG-Nluc-AcV5 bind to the antigen binding domain of a CAR or SIR and can therefore be used to measure the binding affinity of immune effector cells expressing the CAR or SIR. In contrast, the protein L-GGSG-NLuc fusion protein binds to the CAR or the kappa light chain component of the SIR. Thus, the primary utility of these agents is for detecting the expression of a CAR or SIR and they cannot be used to measure the binding affinity of a CAR or SIR to its target antigen.

T cells expressing MPL CAR are able to bind MPL-GGSG-NLuc fusion proteins. Jurkat T cells expressing different MPL CAR constructs or control CARs (4C3) were incubated with MPL-GGSG-NLuc-AcV5 and CD19-GGSG-NLuc-AcV5 supernatants and NLuc activity was determined after extensive washing, essentially as described in the previous examples. The results are presented in figure 10 and demonstrate that Jurkat cells expressing different MPL CAR constructs show different levels of binding to MPL-GGSG-NLuc-AcV5 fusion protein. This difference may reflect a difference in the expression level of the different constructs or a difference in the binding affinity of their corresponding scFv fragments to MPL or both.

Expression of SIR on its primary human T cells and detection by NLuc assay human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing indicated SIR constructs targeted to CD 19. Lentiviral vectors encoding SIR targeting MPL (161-SIR-U01) were used as negative controls. Following infection, cells were expanded in XVIVO medium containing 10ng/ml soluble anti-CD 3, 10ng/ml soluble anti-CD 28, and 100IU recombinant human-IL 2 and selected with puromycin, unless otherwise indicated. T cells expressing different SIR constructs were incubated with CD19-GGSG-NLuc-AcV5 supernatant and NLuc activity was determined after extensive washing, essentially as described in the previous examples. The data show binding of CD19-GGSG-NLuc-AcV5 to T cells expressing different SIRs. T cells expressing CD8SP-FMC63-vL-V5- [ hTCRb-WT ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-WT ] -F2A-PAC (080815-F02) [ SEQ ID NO:922] did not show any significant binding to CD19-GGSG-NLuc-AcV5 (average NLuc value 1190) compared to T cells expressing the negative control SIR 161-SIR-U01 (average NLuc value 1580) containing TCR α and TCR β constant regions encoded by wild-type human TCR α and human TCR β 2. In contrast, T cells expressing CD8SP-FMC63-vL-V5- [ mTCRb-opt ] -F-P2A-SP-FMC63-vH-Myc- [ mTCRa-opt ] -F2A-PAC (080815-B06) [ SEQ ID NO:953] showed significant binding to CD19-GGSG-NLuc-AcV5 (mean NLuc value 5359), the antibody containing TCR α and TCR β constant regions encoded by codon-optimized murine TCR α and murine TCR β nucleotide sequences. T cells expressing CD8SP-FMC63-vL-V5- [ TCRb-S57C-opt1] -F-P2A-SP-FMC63-vH-Myc- [ TCRa-T48C-opt1] -F2A-PAC (050515-L05) [ SEQ ID NO:900] showed strong binding to CD19-GGSG-NLuc-AcV5 (mean NLuc value 19178). This construct contains TCR α and TCR β constant regions encoded by codon optimized human TCR α and human TCR β 2 nucleotide sequences and carries the S57C mutation in the TCR β constant chain and the T48C mutation in the TCR α constant chain. CD8SP-FMC63-vL-V5- [ TCRb-S57C-opt1] -F-P2A-SP-FMC63-vH-Myc- [ TCRa-T48C-opt1] -F-F2A-PAC (050515-L05) [ SEQ ID NO:900] SIR construct also carries the V5 epitope tag between FMC63-vL region and TCR β chain and the Myc tag between FMC63-vH region and TCR α chain. T cells expressing CD8SP-CD19Bu12-vL-V5- [ TCRb-S57C-opt1] -F-P2A-SP-CD19Bu12-vH-Myc- [ TCRa-T48C-opt1] -F2A-PAC (070215-M03) [ SEQ ID NO:1021] SIR show stronger binding to CD19-GGSG-NLuc-AcV5 (mean NLuc value 39575). This construct was similar to the FMC 63-based 050515-L05 construct, except that it had vL and vH fragments derived from the hCD19-Bu12 antibody, which is a humanized monoclonal antibody directed against human CD 19. T cells expressing CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F2A-PAC (081415-D06) [ SEQ ID NO:992] showed the strongest binding to CD19-GGSG-NLuc-AcV5 (mean NLuc value 107077). This construct contains TCR α (TCRa) and TCR β (TCRb) constant regions encoded by codon-optimized human TCR α and human TCR β 2 nucleotide sequences and carries the S57C mutation in the TCR β constant chain and the T48C mutation in the TCR α constant chain. The FMC63-SIR-D06 construct also carried the V5 epitope tag between the FMC63-vL region and the TCR β chain and the Myc tag between the FMC63-vH region and the TCR α chain. Finally, in this construct, TCR α and TCR β constant chains were murine. Thus, five amino acids of the human TCR-b constant region were replaced with corresponding murine amino acids. These murine amino acids are K-18, A-22, I-133, A-136 and H-139. In addition, in the human TCRa constant chain, the region of the murine TCRa constant chain containing the four amino acids serine (S-90), aspartic acid (D-91), valine (V-92) and proline (P-93) was substituted.

T cells expressing CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-FMC 63-vL-Gly-Ser-linker-FMC 63-vH-Myc- [ hTCRa-CSDVP ] -F2A-PAC (082815-G07) [ SEQ ID NO:1620] and CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-CD19Bu 12-vL-Gly-Ser-linker-CD 19Bu12-vH-Myc- [ hTCRa-CSDVP ] -F2A-PAC (082815-E05) [ SEQ ID NO:1622] SIR constructs also showed significant binding to CD 19-gg-NLuc-AcV 5 (mean NLuc values ═ 29262 and 4671.5, respectively). In the 082815-G07 construct, the FMC63-scFv fragment [ i.e., FMC63(vL + vH) ] was fused to the TCRa-CSDVP fragment, while the TCRb-KACIAH constant region fragment was expressed without any antigen binding moiety. The 082815-E05 construct was similar to 082815-G07 except that the CD19-Bu12scFv fragment replaced the FMC63-scFv present in the 082815-G07 construct.

Taken together, these results indicate that SIRs containing TCR α and TCR β constant regions encoded by wild-type nucleotide sequences failed to show significant binding to CD19, probably due to poor expression of this construct in human primary T cells. In contrast, SIR of codon optimized human TCRa/b chain containing additional cysteine residues that promote interchain disulfide bonds showed efficient CD19 binding and functional expression. The murine activation of the human TCR α/β constant chain as seen in (081415-D06) [ SEQ ID NO:992] SIR resulted in a further increase in CD19 binding. In addition, as can be seen in the (082815-G07) [ SEQ ID NO:1620] and (082815-E05) [ SEQ ID NO:1622] constructs, the scFv fragment can be functionally expressed as a fusion to the TCRa constant region when co-expressed with the TCRb constant chain, even though the TCRb does not carry any antigen binding moiety (see FIG. 11). Alternatively, the TCRb chain in such constructs may express an unrelated vL or vH moiety, so long as it does not interfere with the functional assembly of the vL and vH chains present in the scFv fragment.

Jurkat-NFAT-Luc cells were transduced with different constructs (SEQ ID NO:922, 953, 900, 992, 1110 and 1021) and selected among puromycin. Cells were incubated with CD19-GGSG-NLuc-AcV5 supernatant and NLuc activity was determined after extensive washing, essentially as described previously. The NLuc values for the parent Jurkat and those cells expressing the different constructs were 996, 3606, 12128, 37216, 503043, 101958 and 128996, respectively. Experiments have shown that the Jurkat expressing the construct CD8SP-FMC63-vL-V5- [ hTCRb-WT ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-WT ] -F2A-PAC (080815-F02) [ SEQ ID NO:922] containing a TCRa/b constant chain with a wild type nucleotide sequence does not bind significantly to the CD19-GGSG-NLuc AcV5 supernatant, whereas different levels of binding to other constructs containing TCRa/b constant chains with codon optimized nucleotide sequence and/or carrying specific amino acid substitutions enhance chain pairing and/or expression. In particular, constructs with SEQ ID NO:900 and 992 showed more than 10-fold and 15-fold increases in CD19-GGSG-NLuc binding, respectively.

Jurkat-NFAT-Luc cells were stably transduced with different SIR constructs and selected among puromycin. Cells were incubated with CD19-GGSG-NLuc-AcV5 supernatant and NLuc activity was determined after extensive washing, essentially as described previously. Different SIR constructs showed different levels of binding to CD19-GGSG-NLuc-AcV5 fusion protein. Specifically, the construct CD8SP-FMC63-vL-V5- [ hTCRG-opt ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRd-opt ] -F-F2A-PAC (091015-A06) [ SEQ ID NO:949] containing TCR constant chains derived from TCR- γ and TCR- δ also showed binding to the CD19-GGSG-NLuc-AcV5 fusion protein. The NLuc values of the parent Jurkat and those cells expressing the constructs with SEQ ID NOs: 1620, 1623, 1622, 926, 949, 1112 are 1515, 27594, 6357, 10254, 693, 2176 and 179, respectively. Thus, the construct with SEQ ID:926 again showed the lowest binding to soluble CD 19.

Jurkat-NFAT-Luc cells were stably transduced with the indicated constructs and selected for puromycin. Cells were incubated with CD19-GGSG-NLuc-AcV5 supernatant and NLuc activity was determined after extensive washing, essentially as described previously. Different SIR constructs showed different levels of binding to CD19-GGSG-NLuc-AcV5 fusion protein.

T cells expressing CD19SIR induced cytotoxicity in RAJI lymphoma cells expressing CD 19. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing indicated SIR constructs targeted to CD 19. Cells were selected with puromycin and expanded. RAJI cells stably expressing hGLuc in the cells were co-cultured with SIR-expressing T cells at an effector to target (E: T) ratio of 10:1 for 4 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity (as measured by a BioTek synergy plate reader) by direct injection of 0.5xCTZ assay buffer containing native coelenterazine (nanlight). The data indicate that CD 1-specific SIR CD 8-FMC 1-vL-V1- [ TCRb-S57 1-opt 1] -F-P2 1-MPL-161-vH-Myc- [ hTCCra-T48-opt 1] -F-F2 1-PAC (1-U1) [ SEQ ID NO:1112] compared to MPL-targeted control SIR CD8SP-MPL-161-vL-V5- [ hTCCRb-S57-C-opt 1] -F-P2 1-PAC [ SEQ ID NO: 72 ] -CD 1-FMC 1-vH-Myc- [ TCRa-T48-opt 1] -F-F2 1-PAC (1-L1) [ SEQ ID NO:900] and CD8 1-CD19 BuvL-V1-opt 1-OPS-P-19-OCR-T1-OPT 1-OC-OCR-F2 1-OCR-1-OCR-P-1-OCR-S-1-OCR- Increased GLuc activity after co-culture of T cells of-F-F2A-PAC (070215-M03) [ SEQ ID NO:1021], indicating target cell lysis. Treatment with digitonin was used to show maximal cell death. The average Gluc values of T cells exposed to the expression construct (040315-U02) [ SEQ ID NO:1112], (050515-L05) [ SEQ ID NO:900], (070215-M03) [ SEQ ID NO:1021], and RAJI cells after digitonin treatment were 119, 3042, 2547, and 3869, respectively.

T cells expressing CD19SIR induced cytotoxicity in RAJI lymphomas expressing CD 19. Human peripheral blood T cells isolated with CD3 magnetic beads were infected or left uninfected with lentiviruses expressing indicated SIR constructs targeted to CD19 (T-UI). Cells were selected with puromycin and expanded. RAJI cells stably expressing hGLuc were co-cultured with T cells expressing SIR at an effector to target (E: T) ratio of 10:1 for 4 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity (as measured by a BioTek synergy plate reader) by direct injection of 0.5xCTZ assay buffer containing native coelenterazine (nanlight). When indicated, Gluc activity in wells containing medium alone (Med) and wells in which target cells were incubated with T cells not infected with any SIR construct (T-UI) was used as a control in this and subsequent experiments. The data show that CD19 specific SIR CD8SP-FMC63-vL-V5- [ TCRb-S57C-opt1] -F-P2 1-SP-FMC 1-vH-Myc- [ TCRa-T48-opt 1] -F-F2 1-SP-F72-SP-1-PSD-1-PAC (1-L-PSC-MRT-V-MRT-X-T-CRF-72-OC-T-TCRa-T-48-opt 1] -F-2 1-PAC (1-PSC-1-L1) compared to MPL targeted control SIR CD8SP-MPL-161-vL-V5- [ hTCCRb-S57C-opt 1] -F-P2C-OPT-T-48-OPT-T-5-OPT-OCT-No 6-OC-100-F-PSI-OC-1-OCR Increased GLuc activity after co-culture of T cells of-Del 48] -F-F2A-PAC (091015-Y08) [ SEQ ID NO:926] and CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (081415-D06) [ SEQ ID NO:992], indicating target cell lysis. Treatment with digitonin was used to show maximal cell death. The results using the (091015-Y08) [ SEQ ID NO:926] construct show that a fragment of the preTCR α -del48 constant chain can be substituted for the TCR α constant chain region to make a functional double-stranded SIR when co-expressed with the TCR β constant chain. Note that med indicates medium alone, while T-UI represents T cells not infected with any SIR construct.

T cells expressing the CD19 single chain SIR, in which the antigen binding domain is linked to one of the TCR constant chains and lacks the complementary TCR chain, failed to induce significant cytotoxicity in RAJI lymphomas expressing CD 19. SIR constructs were generated in which scFv fragments derived from CD19Bu12 and FMC63 monoclonal antibodies against human CD19 were fused to the TCRb constant chain and did not express the complementary TCRa chain. In the construct CD8SP-CD19Bu 12-vL-Gly-Ser-linker-CD 19Bu12-vH-V5- [ hTCRb-WT ] -F-P2A-PAC (051216-D08) [ SEQ ID NO:1022], the scFV fragment of CD19Bu12 (denoted CD19Bu 12-vL-Gly-Ser-linker-CD 19Bu12-vH) was linked via a V5 linker to a TCRb constant chain containing a wild-type nucleotide sequence. In the construct CD8SP-FMC 63-vL-Gly-Ser-linker-FMC 63-vH-V5- [ hTCRb-S57C-opt ] -F-P2A-PAC (051216-G01) [ SEQ ID NO:912], the scFV fragment of FMC63 (denoted as FMC 63-vL-Gly-Ser-linker-FMC 63-vH) was linked via a V5 linker to a TCRb constant chain containing its codon optimized nucleotide sequence and carrying the S57C mutation. In the construct CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-FMC 63-vL-Gly-Ser-linker-FMC 63-vH-Myc- [ hTCRa-CSDVP ] -F2A-PAC (082815-G07) [ SEQ ID NO:1620], the scFV fragment of FMC63 was linked via a Myc linker to a TCRa constant chain containing its codon optimized nucleotide sequence and carrying a CSDVP mutation, which constant chain was co-expressed via a V5 linker with a TCRb constant chain containing its codon optimized nucleotide sequence and carrying a KACIAH mutation. The construct CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (081415-D06) [ SEQ ID NO:992] and CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-K13-FLAG-F-T2A-P AC (051216-K04) [ SEQ ID NO: 35918 ] is a double-stranded fragment in which a vL and a CRH fragment derived from FMC63 are attached to the hTC-KACIAH and hTCRa-CSDVP chains, respectively.

Different constructs were expressed in T cells and still tested for their ability to lyse RAJI-Gluc cells. The data show that T cells expressing (051216-D08) [ SEQ ID NO:1022] and (051216-G01) [ SEQ ID NO:912] SIR lacking the complementary TCRa constant strand fail to induce RAJI cell lysis compared to uninfected T cells (T-UI) or wells containing medium alone. RAJI-GLuc cells exposed to T cells of the expression constructs (082815-G07) [ SEQ ID NO:1620], (051216-K04) [ SEQ ID NO:918], and (081415-D06)) [ SEQ ID NO:992] showed an increase in GLuc activity of more than 2-fold, and more than 4-fold compared to those exposed to T-UI cells.

T cells expressing CD19 single chain sir (sc sir) in which the antigen binding domain is linked to one of the TCR constant chains and lacks the complementary TCR chain failed to induce cytotoxicity in RAJI lymphomas expressing CD 19. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing indicated SIR constructs targeted to CD 19. Cells were selected with puromycin and expanded. RAJI cells stably expressing hGLuc were co-cultured with T cells expressing SIR at an effector to target (E: T) ratio of 10:1 for 4 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity (as measured by a BioTek synergy plate reader) by direct injection of 0.5xCTZ assay buffer containing native coelenterazine (nanlight). The data show that T cells expressing SIR constructs (051216-D08) [ SEQ ID NO:1022], (051216-F03) [ SEQ ID NO:1023], and (051216-G01) [ SEQ ID NO:912] SIR in which the antigen binding domain is linked to the TCRb constant strand and lacks the complementary TCRa constant strand fail to induce RAJI cell lysis compared to uninfected T cells (T-UI) or wells containing medium alone.

T cells expressing CD19SIR induced cytotoxicity in RAJI lymphomas expressing CD 19. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing different SIR constructs targeting CD 19. Constructs targeting the TCR β 2 chain (CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-TCRB2-CP 01-E05-vL-Gly-Ser-linker-TCRB 2-CP01-E05-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (030816-C05) [ SEQ ID NO:1781]) were used as negative controls. Cells were either unselected (for constructs 022216-A04 and 031416-A18) or selected with puromycin (for other constructs) and amplified. The cells were tested for their ability to lyse RAJI-Gluc cells. The results show efficient target cell lysis by T cells expressing all constructs compared to uninfected T cells (T-UI) or wells with medium alone (Med). In particular, efficient target cell lysis was observed by constructs (031616-B05) [ SEQ ID NO:1019], (031616-C05) [ SEQ ID NO:1020], (021816-N02) [ SEQ ID NO:1016] in which the CD19Bu12scFv fragment was attached to the TCRb constant chain and co-expressed with the empty hTCRa constant chain. Efficient target cell lysis was also observed with the construct CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-2-CD19 MM-vL-Gly-Ser-linker-CD 19MM-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (031616-A050[ SEQ ID NO:1623], in which construct CD19MM scFv is attached to the hTCRa-CSDVP constant chain via a Myc linker and is co-expressed with the empty hTCRb-KACIAH constant chain carrying a V5 linker, and with the construct CD8SP-CD19Bu12-scFv-V5- [ hTCb-KACIAH ] -F-P2A-SP-FMC 63-Myc- [ hTCRa-CSP ] -F-F2 DVP-F2-2A-PAC 020216-B07) [ PAC ID: 391026 ], thus, it was demonstrated that a functional SIR containing two different scFv fragments could be constructed in which CD19Bu12scFv was attached to the hTCRb-KACIAH chain and FMC63-scFv was attached to the hTCRa-CSDVP chain. Finally, efficient target cell lysis was also observed by the constructs CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-K13-FLAG-F-T2A-C NB30(022216-A04) [ SEQ ID NO:920] and CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63vH-MYC- [ hTCRa-CSDVP ] -F-P2A-CD3z-41BB-T2A-CNB 30(031416-A18) [ SEQ ID NO:998], in which FMC 63-derived hTCLvL and hTCVH fragments are attached to the CICICICICAH-KAAH-P chain and constant, respectively. However, these constructs co-expressed the viral FLICE inhibitory protein (vFLIP) K13 and the fusion protein CD3z-41BB, respectively, encoded by Kaposi's sarcoma-associated herpesvirus. The K13 protein is known to selectively activate the NF-. kappa.B pathway by binding to the cellular protein NEMO, whereas the CD3z-41BB fusion protein contains the cytosolic signaling domain of the costimulatory molecule 41BB linked to the CD3z chain of the TCR complex. By providing a co-stimulatory signal, K13 and CD3z-41BB proteins will enhance activation and proliferation of SIR expressing cells, resulting in better functionality and long-term persistence. The 022216-A04 and 031416-A18 constructs also expressed CNB30 mutants of the calcineurin B chain, which confers resistance to calcineurin inhibitors such as FK506 (tacrolimus).

Expression of double-stranded (DC) and one-half-stranded (OAH) SIRs targeting CD19 induced cytotoxicity in RAJI lymphomas expressing CD 19. T cells were infected with lentiviruses expressing different SIR constructs targeting CD19 and tested for cytotoxicity against RAJI-Gluc cells. The data show that the expression of MPL in combination with the MPL-targeting negative control construct CD8SP-MPL-161-vL-V5- [ hTCRB-S57C-opt1] -F-P2A-MPL-161-vH-Myc- [ hTCRa-T48C-opt1] -F-F2A-PAC (040315-U02) [ SEQ ID NO:1112] CD8SP-FMC63-vL-V5- [ hTCRB-WT ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-WT ] -F-F2A-PAC (080815-F02) containing TCR β and TCR α constant chains encoded by their wild type nucleotide sequences (i.e. SEQ ID NO:746 and SEQ ID NO:731, respectively) [ SEQ ID NO:922] the SIR construct failed to cause significant target cell lysis. In contrast, all other SIR constructs (e.g., (050515-L05) [ SEQ ID NO:900], (070215-M03) [ SEQ ID NO:1021], (081415-D06) [ SEQ ID NO:992], (080815-B06) [ SEQ ID NO:953], (082815-G07) [ SEQ ID NO:1620], and (082815-E05) [ SEQ ID NO:1622]) showed increased GLuc activity, indicating target cell lysis. The (082815-G07) [ SEQ ID NO:1620] and (082815-E05) [ SEQ ID NO:1622] constructs express the empty hTCRb-KACIAH (SEQ ID NO:748) constant strand fused to FMC63 and CD19Bu12scFV fragments, respectively, along with the hTCRa-CSDVP (SEQ ID NO:732) constant strand fragment. Thus, SIRs in which the antigen binding domain is fused to the TCRa constant chain can induce efficient target cell lysis when co-expressed with the empty complementary TCRb constant chain. Empty TCRb constant chains in such SIR constructs may contribute to cell surface expression of the antigen binding domain carrying TCRa constant chains.

T cells expressing CD19Bu12SIR and having TCRa and TCRb wild-type nucleotide sequences failed to induce cytotoxicity in RAJI lymphomas expressing CD 19. RAJI-Gluc cells were co-cultured with T cells expressing different SIRs targeting CD19 at an effector to target (E: T) ratio of 10:1 for 4 hours. SIR-T cell mediated induction of target cell lysis was determined as before. The data show that T cells expressing CD19Bu 12-based CD8SP-CD19Bu12-vL-V5- [ hTCRb-WT ] -F-P2A-CD19Bu12-vH-Myc- [ hTCRa-WT ] -F2A-PAC (021916-Q03) [ SEQ ID NO:1038] SIR constructs and containing TCRb and TCRa constant chains encoded by their wild-type nucleotide sequences failed to cause significant target cell lysis compared to negative control construct 111815-O05 or uninfected T cells, which may be due to poor expression of the 021916-Q03SIR construct in human primary T cells. Thus, similar to the CD8SP-FMC63-vL-V5- [ hTCRb-WT ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-WT ] -F-F2A-PAC (080815-F02) [ SEQ ID NO:922] construct, another SIR based on the wild type nucleotide sequences of TCRa and TCRb failed to induce significant toxicity to the target cell line.

T cells expressing one half-chain (OAH SIR) targeting CD19 induced cytotoxicity in RAJI lymphomas expressing CD 19. Human peripheral blood T cells were infected with lentiviruses encoding different SIRs targeting CD19 and tested for cytotoxicity using RAJI-Gluc cells with an effector to target (E: T) ratio of 1:1 for 96 hours. The data show that the CD8SP-FMC63-vL-V5- [ hTCRb-WT ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-WT ] -F2A-PAC (080815-F02) [ SEQ ID NO:922] SIR construct containing TCRb and TCRa constant chains encoded by the wild type nucleotide sequence failed to cause significant target cell lysis compared to the negative control construct 040315-U02, probably due to the poor expression of this (i.e., 080815-F02) construct in human primary T cells. In contrast, all other SIR constructs (e.g., 050515-L05, 070215-M03, 081415-D06, 080815-B06, 082815-G07, and 082815-E05) showed increased GLuc activity, indicating target cell lysis. The 082815-G07 and 082815-E05 constructs expressed the TCRb constant chain (KACIAH version) along with a fragment of tcra (csdvp) fused to fragments of FMC63 and CD19Bu12scFV, respectively. Thus, SIRs based on antigen binding domains fused to TCRa invariant chains can induce efficient cell lysis when co-expressed with empty TCRb invariant chains. This SIR is specified as a half of the OAH SIR. Thus, OAH SIR is more efficient than single chain SIR (SC SIR). This is probably because TCRa and TCRb require the complementary strand to be efficiently expressed on T cells.

T cells expressing double strand (DC SIR) targeting CD19 induced cytotoxicity in RAJI lymphomas expressing CD 19. RAJI-Gluc cells were co-cultured with T cells expressing different SIRs targeting CD19 at an effector to target (E: T) ratio of 10:1 for 4 hours, followed by measurement of Gluc activity. The data show that T cells expressing the CD8SP-FMC63-vL-Myc- [ hTCRa-T48C-opt1] -F2A-FMC63-vH-V5- [ hTCRb-C57C-opt1] -F-P2A-PAC (100515-E03) [ SEQ ID NO:902] construct, in which the FMC63-vL chain is linked to the hTCRa-T48C-opt1 constant chain and the FMC63-vH is linked to the hTCRb-C57C-opt1 constant chain, can induce efficient target cell lysis compared to T cells that are not infected with T cells or T cells expressing negative control CARs targeting CD4 or KSHV proteins. Similarly, CD8SP-FMC63-vL-V5- [ TCRb-S57C-opt1] -F-P2A-SP-FMC63-vH-Myc- [ TCRa-T48C-opt1] -F-F2A-PAC (050515-L05) [ SEQ ID NO:900] and CD8SP-FMC63-vL-V5- [ hTCRB-S57C-opt ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-T48C-opt ] -F-F2A-PAC (100815-B04) [ SEQ ID NO:951] the T cells of the construct induced efficient target cell lysis, in these constructs the FMC63-vL chain was linked to either the TCRb-S57C-opt1 or hTCRb-S57C-opt invariant chain and FMC63-vH was linked to either the TCRa-T48C-opt1 or hTCRa-T48C-opt invariant chain. Finally, T cells expressing the IgHSP-FMC63-vH- [ hTCRB-C57C-opt ] -F-P2A-CD8SP-FMC63-vL-MYC- [ hTCRa-T48C-opt ] -F-F2A-Pac (101415-M05) [ SEQ ID NO:901] construct, in which FMC63-vH chain is linked to hTCRB-C57C-opt constant chain and FMC63-vL is linked to hTCRA-T48C-opt constant chain, can also induce efficient target cell lysis. Thus, in a double-stranded SIR construct, the vL fragment of the antibody may be linked to the TCRa or TCRb constant chain and the vH fragment to either of the complementary TCRb or TCRa constant chains. In addition, in a double-stranded SIR, the TCRb or TCRa chain containing the two Functional Polypeptide Units (FPUs) may be the first (or 5') FPU when the FPUs are expressed from the same vector.

T cells expressing CD20SIR induced cytotoxicity in RAJI cells expressing CD 20. RAJI-Gluc cells were co-cultured with T cells expressing different SIRs targeting CD19 and CD20 at an effector to target (E: T) ratio of 10:1 for 4 hours, followed by measurement of Gluc activity. The data show that T cells expressing CD8SP-CD20-2F2-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-CD20-2F2-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (100615-D05) [ SEQ ID NO:1221] SIR efficiently induce target cell lysis compared to uninfected T cells or wells containing medium alone (Med). Moderate cytotoxicity was also observed with SIR constructs targeting CD19 (CD8SP-FMC63-vL-V5- [ TCRb-S57C-opt1] -F-P2A-SP-FMC63-vH-Myc- [ TCRa-T48C-opt1] -F-F2A-PAC (050515-L05) [ SEQ ID NO:900] and CD8SP-FMC63-vL-V5- [ hTCRbb-S57C-opt ] -F-P2A-SP-FMC63-vH-Myc- [ hTCRa-T48C-opt ] -F-F2A-PAC (100815-B04) [ SEQ ID NO:951 ]).

Expression of T cells containing canine TCRa and canine TCRb constant chains induced cytotoxicity of RAJI cells expressing CD 20. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing a CD 20-targeted SIR construct in which monoclonal antibodies against the vL and vH fragments of human CD20 (2F2) were linked to codon-optimized canine TCRa and TCRb constant chains. Cells were tested for cytotoxicity against RAJI-Gluc cells after co-culture for 4 hours at an E: T ratio of 10: 1. Data show that T cells expressing CD8SP-CD20-2F2-vL- [ canine-TCRb-opt ] -F-P2A-CD20-2F2-vH- [ canine-TCRa-opt ] -F2A-PAC (051716-E02) [ SEQ ID NO:1113] based on canine TCRb and TCRa constant chains efficiently induce target cell lysis compared to uninfected T cells.

Lym1 SIR-expressing T cells induced cytotoxicity in Lym 1-expressing Kasumi-1 cells. Human T cells were infected with lentiviruses encoding CD8SP-Lym1-vL- [ hTCRB-opt2] -F-P2A-SP-Lym1-vH- [ hTCRa-opt2-Del ] -F-F2A-PAC (012716-B01) [ SEQ ID NO:1185] SIR and tested for cytotoxicity against Kasumi-1-GLuc cells after selection with puromycin at an effector to target (E: T) ratio of 10:1 for 4 hours or uninfected T cells (T-UI) as a control. Cytotoxicity was determined by increasing GLuc activity. The data show that T cells expressing (012716-B01) [ SEQ ID NO:1185] SIR induced efficient target cell lysis and increased Gluc activity by nearly 9-fold compared to uninfected T cells or wells containing medium alone.

T cells expressing Lym1 and Lym2SIR induced cytotoxicity in RAJI cells expressing Lym1 and Lym 2. T cells expressing SIRs targeting Lym1 and Lym2 were tested for cytotoxicity against RAJI-Gluc cells after co-culture for 4 hours at an E: T ratio of 10: 1. Data from the Gluc-cytotoxicity assay showed that CD8SP-Lym1-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-Lym1-vH-Myc- [ hTCRa-CSDVP ] -F2A-PAC (021216-H02) [ SEQ ID NO:1314] and CD8SP-Lym2-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-Lym2-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (100615-B07) [ SEQ ID NO:1315] the SIR construct induced lysis of target cells efficiently and Gluc values were nearly more than 20-fold and 10-fold higher, respectively. 082815-P08 is a conventional CAR containing CD19Bu12scFv and targeting the CD19 antigen. Again, T cells expressing the CD19Bu 12-based (021916-Q03) [ SEQ ID NO:1038] SIR construct containing TCRb and TCRa constant chains encoded by its wild-type nucleotide sequence failed to cause significant target cell lysis.

T cells expressing SIR against CS1(SLAMF7 or CD319) induced cytotoxicity in CS 1-expressing L363 and U266 cells. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing SIR constructs targeting CS1(SLAMF 7). Cells were selected using puromycin and tested for cytotoxicity against L363-Gluc and U266-Gluc cells after 4 hours at an E: T ratio of 10: 1. The Gluc-cytotoxicity assay showed that T cells expressing CD8SP-CS1-huLuc90-vL-V5- [ hTCRB-KACIAH ] -F-P2A-SP-huLuc90-vH-Myc- [ hTCRA-CSDVP ] -F-F2A-PAC (012716-A02) [ SEQ ID NO:1254] SIR induced L363-Gluc and U266-Gluc lysis efficiently as demonstrated by near 15-fold and 10-fold increases in Gluc values compared to uninfected T cells or wells containing medium alone.

T cells expressing SIRs against BCMA (B cell maturation antigen or TNFRSF17 or CD269) and CSI induced cytotoxicity in BCMA expressing L363 and U266 cells. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing SIR constructs targeting CS1(SLAMF7) and BCMA. Cells were selected using puromycin and tested for cytotoxicity against L363-Gluc and U266-Gluc cells after 4 hours at an E: T ratio of 10: 1. The Gluc cytotoxicity assay showed that CD8SP-BCMA-huC12A3-L3H3-vL-V5- [ hTCrb-KACIAH ] -F-P2A-SP-12A 3-L3H3-vH-Myc- [ hTCRa-CSP ] -F-F2A-PAC (011116-A07) [ SEQ ID NO:1212] and CD 8-KSHV-4C 3-vL-363672-LucHUH-363672-LucHU-1-LucHU-T-48-OPt ] -F-F2A-PAC (111815-O05) [ SEQ ID NO:4639] expressed in comparison with uninfected T cells, T cells expressing control SIR CD 8-KSHV-4C 3-vL-V5- [ hTCRA-T-48-opt ] -F-A-PAC (05-O-PAC (05) [ SEQ ID NO:4639 ]) T cells of-vH-Myc- [ hTCra-CSDVP ] -F-F2A-PAC (012716-A02) [ SEQ ID NO:1254] SIR efficiently induce target cell lysis. Mild to moderate target cell lysis was also observed by T cells expressing SIR CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-LAMP1-Mb 4-vL-Gly-Ser-linker-LAMP 1-Mb4-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (050216-F05) [ SEQ ID NO:1732] targeting LAMP1 and bispecific CAR 041316-F06 targeting cMet and Her 3.

T cells expressing SIRs against CD138, CS1, GPRC5D, and WT1 induced cytotoxicity in U266 and L363 cells. T cells expressing SIR constructs targeting CD138, CS1, GPRC5D and WT1 were selected using puromycin and tested against U266 and L363 cells stably expressing GLuc after 72 hours at 2: 1E: T. The Gluc-cytotoxicity assay showed that T cells expressing CD8SP-CD138-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-CD138-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (100815-A05) [ SEQ ID NO:1236] SIR induced target cell lysis compared to uninfected T cells or wells containing medium alone (Med). T cells expressing SIR CD8SP-CD138-vL-V5- [ hTCRB-WT ] -F-P2A-SP-CD138-vH-Myc- [ hTCRa-WT ] -F-F2A-PAC (021916-R04) [ SEQ ID NO:1139] containing the wild type TCRa and TCRb constant chains were only least effective in U266 cells and ineffective in L363 cells. The single chain SIR CD8SP-CD 138-vL-Gly-Ser-linker-CD 138-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (030316-G03) [ SEQ ID NO:1169] was only effective in U266 cells, where CD138scFv was attached to the hTCRa-CSDVP constant chain. The bispecific construct LAMP1-humab1-2-vL-V5- [ TCRb-KACIAH ] -F-P2A-GPRC5D-ET150-18-vH-MYC- [ TCRa-CSDVP ] -F-F2A-Pac-E05 (092916-E05-VN) (SEQ ID NO:1163), which carries an antibody vL fragment against LAMP1 and an antibody vH fragment against GPRC5D, also efficiently induced cytotoxicity in both cell types. Finally, mild to moderate cytotoxicity was observed with constructs targeting CS1 and WT1, especially in U266 cells. The limited cytotoxicity of the constructs against CS1(SEQ ID NO:1674, 1253) and WT1(SEQ ID NO:1804 and 1805) may be due to the use of lower E: T ratios in this experiment.

T cells expressing SIR against CLL1 induced cytotoxicity in HL60 cells expressing CLL 1. T cells expressing SIRCD 8SP-CLL1-M26-vL- [ hTCRB-opt2] -F-P2A-SP-CLL1-M26-vH- [ hTCRA-opt2] -F-F2A-PAC (012616-A05) [ SEQ ID NO:4790] targeted to CLL1 were selected using puromycin and tested against HL60-Gluc cells after 4 hours at an E: T ratio of 10: 1. The Gluc cytotoxicity assay showed that T cells expressing (012616-A05) [ SEQ ID NO:4790] induced target cell lysis efficiently compared to uninfected T cells or wells containing medium alone.

T cells expressing SIRs against CLEC5A and CLL1 induced cytotoxicity in HL60 cells expressing CLEC5A and CLL 1. The experiment was repeated using T cells expressing SIRs against CLEC5A and CLL 1. The results showed that expression of CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-CLEC5A-8H8F 5-vL-Gly-Ser-linker-CLEC 5A-8H8F5-vH-Myc- [ hTCra-CSDVP ] -F-F2A-PAC (042816-E05) [ SEQ ID NO:1666], CD8SP-CLL 1-M1-vL- [ hTCRb-opt 1] -F-P2 1-SP-CLL 1-M1-vH- [ hTCRa-opt 1] -F-F2 1-PAC (1-A1) [ SEQ ID NO:4790] and CD8 1-CLL 1-vCRV-CLV-CLL-1-vH-CLL-CLF-Gly-Ser-linker-CLEC 5-8H-8-MyC-M-D-F2A-PAC (042816-E05) [ SEQ ID NO:1666], and CD 8-CLL-1-CD 1] -My-CLL T cells of-F-F2A-PAC (021216-I03) [ SEQ ID NO:1250] SIR efficiently induced HL60-Gluc cell lysis. In cells treated with T cells expressing three SIRs, the Gluc values were approximately 9-fold, 4-fold, and 7-fold higher, respectively.

T cells expressing SIRs targeting CSF2RA, LAMP1 and CLL1 induced cytotoxicity in THP1 cells expressing CSF2RA, LAMP1 and CLL 1. T cells expressing different SIRs were cultured with THP-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using a Gluc-cytotoxicity assay. The results showed that CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-CSF2RA-Ab 1-vL-Gly-Ser-linker-CSF 2RA-Ab RA-vH-Myc- [ hTCRa-CSDVP ] -F-F2 RA-PAC (RA-B RA) [ SEQ ID NO:1676], CD8 RA-V RA- [ hTCRb-KACIAH ] -F-P2 RA-CD 8-LAMP RA-Mb RA-vL-Gly-Ser-linker-LAMP RA-Mb RA-vH-Myc- [ hTCRa-CSDVP ] -F-F2 RA-PAC RA-vH-Myc- [ hTCRb-CSDVP ] -F-F2 RA-RA (SEQ ID-F RA-F17372 ] and CLAH-C-RA-CLH-RA-CLC-CLH-CLT-RA-CLC-CSF 72-CSF 72-CD 72-RA-C-RA-Mb-RA-C-F-RA-L-A-a T cells of-SP-CLL 1-M32-vH-Myc- [ hTCra-CSDVP ] -F-F2A-PAC (021216-I03) [ SEQ ID NO:1250] SIR efficiently induce target cell lysis.

T cells expressing SIRs targeting CSF2RA induced cytotoxicity in Molm13 cells expressing CSF2 RA. T cells expressing SIRs targeting CSF2RA were cultured with Molm13-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using a Gluc-cytotoxicity assay. The results show that T cells expressing SIR targeting CSF2RA, CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-CSF2RA-Ab 1-vL-Gly-Ser-linker-CSF 2RA-Ab1-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (050216-B02) [ SEQ ID NO:1676] induced target cell lysis efficiently compared to uninfected T cells or T cells expressing SIR targeting KSHV protein (111815-O05) or wells containing medium alone.

T cells expressing SIRs targeting TSHR (thyroid stimulating hormone receptor) and TnAg induced cytotoxicity in Jurkat and PEER cells expressing TSHR and TnAg. T cells expressing different SIRs targeting TSHR and Tn Ag were cultured with Jurkat-Gluc and PEER1-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using the Gluc-cytotoxicity assay. The results show that SIR targeting TSHR and TnAg, expressed as SIR CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD SP-TSHR-KB 1-vL-Gly-Ser-linker-TSHR-KB 1-vH-Myc- [ hTCRa-CSDVP ] -F2A-PAC (042916-E03) [ SEQ ID NO:1795] and CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8 SP-TnAg-vL-Gly-Ser-linker-TnAg-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (050216-A04) [ SEQ ID NO:1788] effectively induces lysis of the target cells.

T cells expressing SIRs targeting TnAg induced cytotoxicity in Jurkat and PEER cells expressing TnAg. T cells expressing SIRs targeting TnAg were cultured with Jurkat-Gluc and PEER1-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using the Gluc-cytotoxicity assay. The results show that T cells expressing SIR CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8 SP-TnAg-vL-Gly-Ser-linker-TnAg-vH-Myc 4- [ preTCRa-Del48] -F-F2A-PAC (080816-H06) [ SEQ ID NO:2003] targeting TnAg efficiently induce lysis of target cells compared to uninfected T cells or wells containing medium alone.

T cells expressing SIR targeting MPL (TPO receptor) induce cytotoxicity in cells expressing MPL. T cells expressing SIR targeting MPL were cultured with HEL-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using a Gluc-cytotoxicity assay. The results show that T cells expressing SIRCD8SP-MPL-175-vL- [ hTCRb-opt2] -F-P2A-SP-175-vH- [ hTCRa-opt2] -F-F2A-PAC (042116-G01) [ SEQ ID NO:4862] targeted MPL induced target cell lysis efficiently compared to uninfected T cells or wells containing medium alone.

T cells expressing SIRs targeting FLT3 induced cytotoxicity in RS:411 (or RS411) cells expressing FLT 3. T cells expressing SIR targeting FLT3 were cultured with RS:411-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using the Gluc-cytotoxicity assay. The data show that T cells expressing SIR CD8SP-FLT3-NC7-vL-V5- [ hTCRB-KACIAH ] -F-P2A-SP-FLT3-NC7-vH-Myc- [ hTCRA-CSDVP ] -F-F2A-PAC (050316-C01) [ SEQ ID NO:1273] targeted to FLT3 efficiently induce target cell lysis compared to uninfected T cells, T cells expressing negative control SIR (111815-O05) or wells containing medium alone.

T cells expressing the SIR of the NKG2D extracellular domain and SIRs targeting FLT3 and CSF2RA induced cytotoxicity in MV411 target cells. Human peripheral blood T cells expressing the extracellular domain of NKG2D (linked to hTCR-CSDVP constant chain via GGGGS-ggd-Myc linker) and those targeting FLT3 and CSF2RA were tested against MV411-Gluc cells after 4 hours at an effector: target (E: T) ratio of 10: 1. The Gluc cytotoxicity assay showed that CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-NKG2D- (GGS-GGGGD) -Myc- [ hTCHA-CSDVP ] -F-F2A-PAC (9-A6862) [ SEQ ID NO: 6865 ] and CD8SP-FLT 53-NC 27-vL-CSV-V- [ 5-CRH-86A-My H-7-NCH-361757-My-N72-C-NCH-T-48-T48-OPT-S57-OPT-F-4C 3-vH-Myc- [ hTCRA-T-48-OPT-T-48-OPT-T-48-T-05) [ SEQ ID NO:4639] SIR or wells containing medium alone expressed in comparison with uninfected T cells, CD 8-KSHV-4-C-S-O-9-OCD-OCG 2-9-PAC (05) [ SEQ ID NO:4639] SIR ] alone T cells of-CSDVP ] -F-F2A-PAC (050316-C01) [ SEQ ID NO:1273] and CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-CSF2RA-Ab 1-vL-Gly-Ser-linker-CSF 2RA-Ab1-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (050216-B02) [ SEQ ID NO:1676] SIR efficiently induce target cell lysis. The above results demonstrate that SIRs in which the antigen binding domain comprises a receptor (i.e., NKG2D) have functional activity.

T cells expressing CD30 and WT1SIR induced cytotoxicity in target cells expressing U266 and L363. SIR can be generated against intracellular peptides that are recognized in association with specific HLA antigens. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing indicated SIR constructs targeted to CD30 or WT 1. WT1SIR recognized the peptide derived from WT1 (RMFPNAPYL) along with the HLA-A2 molecule. Cells were selected with puromycin and expanded. U266(WT1+/HLA-A2+) and L-363(WT1+/HLA-A2+) cells stably expressing hGLuc were co-cultured with SIR-expressing T cells at an effector to target (E: T) ratio of 10:1 for 4 hours. The Gluc cytotoxicity assay showed that compared to uninfected T cells (T-UI), although expressing SIR CD8SP-WT1-Ab1-vL-V5- [ hTCRb-S57C-opt ] -F-P2A-SP-WT1-Ab1-vH-Myc- [ hTCRa-T48C-opt ] -F2A-PAC (012816-G01) [ SEQ ID NO:4709] the T cells are the least potent, but expressing SIR CD8SP-WT1-Ab5-vL-V5- [ hTCRb-S57C-opt ] -F-P2A-SP-WT1-Ab5-vH-Myc- [ hTCRa-T48C-opt ] -F-F2A-PAC (111815-C04) [ SEQ ID NO:4710] the T cells kill target cells efficiently. In addition, SIR CD8SP-CD30-Ac10-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-CD30-Ac10-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (010716-K01) [ SEQ ID NO:1227] targeted to CD30 effectively kills target cells expressing CD 30.

T cells expressing SIRs targeting CD33 and CD179b induced cytotoxicity in HL60 and Molm13 cells expressing CD33 and CD179 b. T cells expressing SIRs targeting CD33 and CD179b were cultured with HL60-Gluc and Molm13-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using a Gluc-cytotoxicity assay. The data show that T cells expressing SIRCD 8SP-CD33-AF5-vL-V5- [ TCR β -KACIAH ] -F-P2A-SP-CD33-AF5vH-MYC- [ TCR α -CSDVP ] -F-F2A-Pac (052416-K05) [ SEQ ID NO:1229] and CD179b-vL-V5- { TCR β -KACIAH } -F-P2A-SP-179 CD179b-vH-MYC- [ TCR α -CSDVP ] -F-F2A-Pac (063016-Y06) [ SEQ ID NO:1237], targeted to CD33 and CD179b, respectively, induce lysis of the target cells compared to uninfected T cells or wells containing medium alone.

T cells expressing SIR targeting CD33 induced cytotoxicity in HL60 cells expressing CD 33. T cells expressing SIR targeting CD33 were cultured with HL60-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using a Gluc-cytotoxicity assay. Experiments have shown that SIRCD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-CD33-huMyc 9-vL-Gly-Ser-linker-CD 9-huMyc 9-vH-Myc- [ hTCRa-CSDVP ] -F-F2 9-PAC (9-C9) [ SEQ ID NO:1650] and CD8 9-V9- [ hTCRb-KACIAH ] -F-P2 9-CD 9-AF 9-vL-Gly-Ser-linker-CD 9-AF 9-vH-Myc 9- [ preTCRa-Del 9] -F-F2-72-PAC (9-E9) [ SEQ ID NO: 364 ] and CD 8-hTCRa-9-CD 9-MyC 9-MyP-CD 9-Y-Gly-Ser-linker-CD 9] express CD 9-Y-MyC 9-My-CD 9-CD-Y-peptide-CTC 9-CD-Y-peptide- T cells of-Ser-linker-CD 33-huMyc9-vH-Myc4- [ preTCRa-Del48] -F-F2A-PAC (083116-C06) [ SEQ ID NO:1865] efficiently induced lysis of target cells.

T cells expressing SIRs targeting CXCR4 induced cytotoxicity in THP cells expressing CXCR 4. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing the indicated bispecific SIR construct targeting CXCR4, CD8SP-CXCR4-1-vHH-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FMC63-vH-MYC- [ hTCRa-CSDV ] -F-F2A-PAC (101415-V01) [ SEQ ID NO:1171 ]. The SIR construct also expressed the vH fragment of FMC63 antibody against CD 19. Cells were cultured with HL60-GLuc cells at an E: T ratio of 5:1 for 4 hours and cytotoxicity was measured using the GLuc assay. The results show that T cells expressing SIRs targeting CXCR4 effectively induced target cell lysis compared to uninfected T cells or wells containing medium alone.

T cells expressing SIR against IL11Ra induced cytotoxicity in BV173 cells expressing IL11 Ra. T cells expressing SIRs targeting IL11Ra were cultured with THP-1-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using a Gluc-cytotoxicity assay. The results show that T cells expressing CD8SP-IL11Ra-8E2-Ts107-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-IL11Ra-8E2-Ts107-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (050516-R06) [ SEQ ID NO:1304] SIR efficiently induce lysis of Bv173 target cells compared to T-UI cells or medium alone.

Binding of T cells expressing CD16SIR to the CD20 monoclonal antibody rituximab induced cytotoxicity in RAJI lymphomas expressing CD 20. Peripheral blood T cells isolated from CD3 magnetic beads were infected with a lentivirus expressing the SIR construct CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP2-CD16A-V158-V2-Myc- [ hTCRa-T48C-opt ] -F-F2A-PAC (020416-A08) [ SEQ ID NO:1186, which expresses the CD16A variant V158A with high affinity for human IgG. The 4C3SIR against the KSHV protein was used as a negative control. Cells were selected with puromycin and expanded. RAJI cells stably expressing hGLuc were co-cultured with SIR-expressing T cells at an effector to target (E: T) ratio of 10:1 for 4 hours in the presence and absence of rituximab (1. mu.g/ml). SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity (as measured by a BioTek synergy plate reader) by direct injection of 0.5xCTZ assay buffer containing native coelenterazine (nanlight). The results show that the CD16A-v158SIR construct performs efficient target cell lysis in the presence of rituximab only. Thus, the CD16A v158SIR can be used as a universal SIR, which is used with any monoclonal antibody, thus avoiding the need to make individual SIRs for different antigen targets.

T cells expressing CD 123-161 bispecific SIR induced cytotoxicity in Bv173 and HEL cells expressing MPL. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing IgHSP-CD123-2-vHH-V5- [ hTCRB-S57C-opt ] -F-P2A-CD8SP-MPL-161-HL-Myc- [ hTCRa-T48C-opt ] -F-F2A-PAC (022516-M08) [ SEQ ID NO:4591] SIR construct targeting CD123 and MPL (161). Cells were selected with puromycin and expanded. Bv173 and HEL cells stably expressing hGLuc were co-cultured with T cells expressing SIR at an effector to target (E: T) ratio of 10:1 for 4 hours. Gluc cytotoxicity assays show that bispecific SIRs can induce efficient lysis of target cells expressing one or more of their target antigens.

T cells expressing SIR against CD123 induced cytotoxicity in CD123 expressing L428 cells. T cells expressing SIR targeting CD123 were cultured with L428-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using a Gluc-cytotoxicity assay. Experiments have shown that T cells expressing CD8SP-CD123-CSL362-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-CD123-CSL362-vH-Myc- [ preTCRa-Del48] -F-F2A-PAC (041416-K04) [ SEQ ID NO:1445] SIR efficiently induce lysis of L428 target cells compared to T-UI cells or medium alone.

T cells expressing SIR against CD123 induced cytotoxicity in Bv173 cells expressing CD 123. T cells expressing SIR targeting CD123 were cultured with Bv173-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using a Gluc-cytotoxicity assay. The results show that T cells expressing IgHSP-CD123-2-vHH-V5- [ hTCRb-KACIAH ] -F-P2A-SP-CD123-1-vHH-Myc- [ preTCRa-Del48] -F-F2A-PAC (041416-V03) [ SEQ ID NO:1467] efficiently induce lysis of Bv173 target cells compared to T-UI cells or medium alone (Med). This SIR contains two different Camelidae vHH (CD123-2 and CD123-1) attached to hTCrb-KACIAH and preTCRa-Del48 constant chains, respectively. These results indicate that SIRs containing two different antigen binding domains are both functionally active.

T cells expressing SIRs targeting CD79b and CD138 induced cytotoxicity in RAJI and L363 cells expressing CD79b and CD 138. T cells expressing SIRs targeting CD79b and CD138 were cultured with RAJI-Gluc and L363-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using the Gluc-cytotoxicity assay. The results show that T cells expressing CD8SP-CD79b-2F2-vL-V5- [ hTCRB-S57C-opt ] -F-P2A-SP-CD79b-2F2-vH-Myc- [ preTCRa-Del48] -F-F2A-PAC (041216-H05) [ SEQ ID NO:1130] and CD8SP-CD138-vL-V5- [ hTCRB-KACIAH ] -F-P2A-SP-CD138-vH-Myc- [ preTCRa-Del48] -F-F2A-PAC (041416-I03) [ SEQ ID NO:1446] targeted to CD79b and CD138, respectively, induce SIR lysis of the target cells compared to uninfected T cells or wells containing medium alone.

T cells expressing TCR β 1SIR induced cytotoxicity in Jurkat cells expressing TCR β 1. Targeting CD8SP-V5- [ hTCRB-KACIAH ] -F-P2A-CD8SP-TCRB1-CP 01-E01-vL-Gly-Ser-linker-TCRB 01-CP 01-E01-vH-Myc- [ hTCra-CSDVP ] -F-F2 01-PAC (01-D01) [ SEQ ID NO:1778] and CD8 01-V01- [ hTCRb-KACIAH ] -F-P2 01-CD 8 01-TC3672-Jovi 01-vL-Gly-Ser-linker-TCRB 01-Job 01-vH-Myc- [ CRhTA-CSP ] -F-F2 72-PAC (01-B01) [ SEQ ID NO:1779] CD 72-CD 01-PAC (01-B01) and CD 72-KACIV-01-PSc-01-KACIAH-P-CP-01-CD 01-TCRB-01-GCvL-Gly-Ser-linker-CT-CD 01-CD-GCH-OCH-C- [ Lentiviruses of CD30-Ac10-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (063016-K02) [ SEQ ID NO:1227] SIR infected human peripheral blood T cells isolated with CD3 magnetic beads. Cells were selected with puromycin and expanded. Jurkat cells stably expressing hGLuc were co-cultured with SIR-expressing T cells at an effector to target (E: T) ratio of 10:1 for 4 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity. The results showed that CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-TCRB1-Jovi 1-vL-Gly-Ser-linker-TCRB 1-Jovi1-vH-Myc- [ hTCRa-CSDVP ] -F-F2 1-PAC (1-B1) [ SEQ ID NO:1779] and CD8 1-CD 1-Ac 1-vL-V1- [ hTCRb-KACIAH ] -F-P2 1-SP-CD 1-Ac 1-vH-Myc- [ hTCRa-CSDVP ] -F-F2 1-PAC (1-K1) [ SEQ ID NO:1227] induced efficient target cell lysis, whereas CD8 1-V1- [ hTCRB-KACIAH ] -F-P2 1-CD 1-TCRB-TCCP-1-TCRB-1-TCCP-1-TCRB-1-TCRB-1-36 vH-Myc- [ hTCra-CSDVP ] -F-F2A-PAC (030816-D04) [ SEQ ID NO:1778] was less effective.

T cells expressing TCR β 2SIR induced cytotoxicity in Jurkat cells expressing SIR containing TCR β 2 constant chains. In initial experiments, T cells expressing SIR against the TCR β 2 chain were nonfunctional in cytotoxicity assays. The reason is that this may be due to the fact that: the TCR β constant chains in these SIRs, and hence the T cells expressing the SIRs, will have suicide or suicide (i.e. kill off neighbouring T cells expressing the SIRs). To avoid this problem, the TCR β 1-opt4 constant chain based on the TCR β 1 chain (nucleic acid SEQ ID NO:752 and amino acid SEQ ID NO:3032) was used to generate SIRs targeting TCR β 2. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with constructs expressing the indicated SIR targeting the TCRB2 (TCR. beta.2) constant chain (CD8SP-TCRB2-D05-vL- [ hTCRB-opt4] -F-P2A-SP-TCRB2-D05-vH-MYC- [ hTCRa-CSDVP ] -F-F2A-Pac-K06 (072816-K06) [ SEQ ID NO:1129] and CD8SP-TCRB2-CP01-E05-vL- [ hTCRB-opt4] -F-P2A-SP-TCRB2-CP01-E05-vH-Myc- [ hTA-CSDVP ] -F2A-PAC (072816-L06) [ SEQ ID NO:1128 ]). Cells were selected with puromycin and expanded. For the target cell line, Jurkat cells stably expressing SIRs targeting PSMA containing the TCRB2 constant chain were used. Jurkat cells also co-express a signal peptide deficient version of TurboLuc as a reporter gene. Jurkat cells were co-cultured with SIR expressing T cells at an effector to target (E: T) ratio of 10:1 for 4 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing TurboLuc activity by direct injection of 0.5xCTZ assay buffer containing native coelenterazine (nanlight), as measured by a BioTek synergy plate reader. The results show that T cells expressing CD8SP-TCRB2-D05-vL- [ TCRb-opt4] -F-P2A-SP-TCRB2-D05-vH-MYC- [ TCR α -CSDVP ] -F-F2A-Pac-K06 (072816-K06) [ SEQ ID NO:1129] and CD8SP-TCRB2-CP01-E05-vL- [ hTCRB-opt4] -F-P2A-SP-TC 2-CP01-E05-vH-Myc- [ hTCCA-DVCSP ] -F-F2A-PAC (072816-L06) [ SEQ ID NO:1128] SIR induce efficient lysis of target cells when compared to uninfected T cells (T-UI) or medium alone, as shown by the increased TurboLuc activity.

T cells expressing the folate receptor 1(FR1) SIR induced cytotoxicity in SKOV3, PC3 and LNCAP cells expressing FR 1. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing the indicated SIR construct targeting FR1 (CD8SP-FR1-huMov19-vL-V5- [ hTCRB-KACIAH ] -F-P2A-SP-FR1-huMov19-vH-Myc- [ hTCRA-CSDVP ] -F-F2A-PA C (102915-P07) [ SEQ ID NO:1276 ]). Cells were selected with puromycin and expanded. SKOV3, PC3 and LNCAP cells stably expressing hGLuc were co-cultured with SIR expressing T cells at an effector to target (E: T) ratio of 10:1 for 4 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity. The results show that T cells expressing CD8SP-FR1-huMov19-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FR1-huMov19-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (102915-P07) [ SEQ ID NO:1276] SIR efficiently induce SKOV3 and PC3 cell death compared to uninfected T cells (T-UI). T cells expressing SIRs targeting Epcam1 and L1CAM also showed weak induction of cell death in SKOV3 and PC3 cell lines compared to uninfected T cells.

T cells expressing SIRs against the intracellular antigens TERT, MART1, MUC1, gp100, tyrosinase, and NYESO induced cytotoxicity in target cells. Expression of the indicated SIR construct (CD 8-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-TERT-3G 3-T865-vL-Gly-Ser-linker-TERT-3G 3-T865-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (021216-L07) [ SEQ ID NO:1784], CD8SP-TERT-3G3-T865-vL- [ hTCRb-opt2] -F-P2A-SP-TERT-3G3-T865-vH- [ hTCRa-opt2] -F-F2A-PAC (050316-A9) [ SEQ ID NO: 4969502 ], CD 8-V5- [ hTCRb-CIb-CICIH ] -F-P2-P8-CD SP-CAAH-CAV SP-CAvT-8458-Gly-MAR 8658-368672-Gly-MAR-368672-MAR-T-opt 2] -F-P2 -Myc- [ hTCra-CSDVP ] -F-F2A-PAC (021216-N03) [ SEQ ID NO:1739], CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-Muc 1-D1-M3B 1-vL-Gly-Ser-linker-Muc 1-D1-M3B 1-vH-Myc- [ hTCra-CSDVP ] -F-F2 1-PAC (021616-B1) [ SEQ ID NO:1751], CD8 1-MUC 1-D1-M3A 1-vL- [ hTCRb-opt 1] -F-P2 1-SP-MUC 1-D1-M3A 1-vH- [ hTCra-opt 1] -F-F2 8272-PAC-OCP-1-pOCP-F-OCP-1-OCP-1-OCP-36 SP-NYESO-T2-vH- [ hTCRa-opt2] -F-F2A-PAC (040416-D01) [ SEQ ID NO:4877], CD8SP-gp100-vL- [ hTCRb-opt2] -F-P2A-SP-gp100-vH- [ hTCRa-opt2] -F-F2A-PAC (031516-B03) [ SEQ ID NO:4828] and CD8SP-Tyros-B2-vL- [ hTCRb-opt2] -F-P2A-SP-Tyros-B2-vH- [ hTCRa-opt2] -F-F2A-PAC (032816-B03) [ SEQ ID NO:4915] were transfected with the lentiviruses isolated human peripheral blood T cells using CD3 magnetic beads. SIRs targeting TERT, MART1, MUC1, gp100, tyrosinase and NYESO recognize peptides derived from these intracellular proteins along with HLA-a2 molecules as previously described. Cells were selected with puromycin and expanded. Cells stably expressing hGLuc, designated target (HLA-a2), were co-cultured with SIR expressing T cells at an effector to target (E: T) ratio of 10:1 for 4 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity. The results show that SIRs targeting TERT, MART1, MUC1, gp100, tyrosinase, and NYESO show lysis of HLA-a2 positive target cell lines expressing these intracellular antigens. In addition, T cells expressing the SIR CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-GD 3-KM-641-vL-Gly-Ser-linker-GD 3-KM-641-vH-Myc- [ hTCra-CSDVP ] -F-F2A-PAC (042816-A04) [ SEQ ID NO:1697] targeting GD3 showed lysis of the MEL624 target cells expressing GD 3.

T cells expressing SIR against EGFR induce cytotoxicity in HeLa cells expressing EGFR. Human peripheral blood T cells were infected with lentiviruses expressing SIR constructs targeting EGFR. Cells were selected with puromycin and expanded. HeLa cells stably expressing hGLuc were co-cultured with SIR expressing T cells or uninfected T cells (T-UI) at an effector to target (E: T) ratio of 5:1 for 24 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity. The results show that T cells expressing CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8 SP-cetuximab-vL-Gly-Ser-linker-cetuximab-vH-Myc 4- [ preTCRa-Del48] -F-F2A-PAC (062916-G04) [ SEQ ID NO:1880] SIR efficiently induce lysis of HeLa target cells compared to T-UI cells or medium alone.

T cells expressing SIR against CD324 induce cytotoxicity in MDA- -MB- -231 cells expressing CD 324. Isolated human peripheral blood T cells were infected with lentiviruses expressing the indicated SIR constructs targeting CD 324. Cells were selected with puromycin and expanded. Stably hGLuc-expressing MDA-MB-231 cells were co-cultured with SIR-expressing T cells or uninfected T cells (T-UI) at an effector to target (E: T) ratio of 5:1 for 24 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity. The data show that T cells expressing CD8SP-CD324-SC10-6-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-CD324-SC10-6-vH-Myc4- [ hTCRa-CSDVP ] -F-F2A-P AC (071516-L04) [ SEQ ID NO:1239] SIR efficiently induce CD324 target cell lysis compared to T-UI cells or medium alone.

T cells expressing SIRs targeting CD276 and IL13Ra2 induced cytotoxicity in U87 — MG target cells expressing these antigens. T cells were infected with lentiviruses expressing SIR constructs targeting CD276 and IL13Ra 2. Cells were selected with puromycin and expanded. U87-MG-GLuc cells were co-cultured with SIR-expressing T cells or uninfected T cells (T-UI) at an E: T ratio of 10:1 for 4 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity. The data show that T cells expressing CD8SP-V5- [ hTCRB-KACIAH ] -F-P2A-CD8SP-CD 276-17-vL-Gly-Ser-linker-CD 276-17-vH-Myc- [ hTCRA-CSDVP ] -F-F2A-PAC (042816-C03) [ SEQ ID NO:1658] SIR induced target cell lysis efficiently compared to T-UI cells or medium alone.

T cells expressing SIRs targeting GD2 induced cytotoxicity in SKMEL-31 and SKMEL-37 target cells expressing these antigens. T cells were infected with lentiviruses expressing SIR constructs targeting GD 2. Cells were selected with puromycin and expanded. SKMEL-31 and SKMEL-37 cells stably expressing GLuc were co-cultured with SIR-expressing T cells or uninfected T cells (T-UI) at an E: T ratio of 10:1 for 4 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity. The data show that T cells expressing CD8SP-GD2-hu3F8-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-GD2-hu3F8-vH-Myc- [ preTCRa-Del48] -F-F2A-PAC (041816-E01) [ SEQ ID NO:1489] SIR efficiently induce target cell lysis compared to T-UI cells or medium alone.

T cells expressing SIR against L1CAM induced cytotoxicity in SKOV3 cells expressing L1 CAM. T cells expressing SIRs targeting L1CAM were cultured with SKOV3-GLuc cells at an E: T ratio of 10:1 for 24 hours and tested using a GLuc-cytotoxicity assay. The data show that T cells expressing CD8SP-MYC- [ hTCRa-T48C-opt1] -F-F2A-CD8SP-L1CAM-9-3-Hu3-V5- [ hTCRB-T57C-opt1] -F-P2A-PAC (080316-T02) [ SEQ ID NO:1136] SIR efficiently induce lysis of SKOV3 target cells compared to T-UI cells or medium alone.

T cells expressing SIR against CDH6 induced cytotoxicity in SKOV3 cells expressing CDH 6. T cells expressing SIRs targeting CDH6 were cultured with SKOV3-Gluc cells at an E: T ratio of 10:1 for 24 hours and tested using the Gluc-cytotoxicity assay. The data show that T cells expressing CD8SP-CDH6-NOV712-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-CDH6-NOV712-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (062816-U01) [ SEQ ID NO:1242] and CD8SP-CDH6-NOV710-vL-V5- [ hTCRRb-KACIAH ] -F-P2A-SP-CDH6-NOV710-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (063016-T05) [ SEQ ID NO:1241] induced efficient target cell lysis of SKOV3 compared to T-UI cells or medium alone.

T cells expressing SIR against TROP2 induced cytotoxicity in PC3 cells expressing TROP 2. T cells expressing SIRs targeting TROP2 were cultured with PC3-Gluc cells at an E: T ratio of 10:1 for 24 hours and tested using a Gluc-cytotoxicity assay. The data show that T cells expressing CD8SP-TROP2-ARA47-HV3KV3-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-TROP2-ARA47-HV3KV3-vH-Myc- [ hTCRa-CSD VP ] -F-F2A-PAC (062816-S01) [ SEQ ID NO:1367] SIR efficiently induce lysis of PC3 target cells compared to T-UI cells or medium alone.

T cells expressing SIR against GFRA4(GDNF family receptor α 4) induced cytotoxicity in TT cells expressing GFRA 4. T cells expressing SIRs targeting GFRA4 were cultured with TT-Gluc cells at an E: T ratio of 10:1 for 24 hours and tested using the Gluc-cytotoxicity assay. The data show that T cells expressing CD8SP-GFRa4-P4-10-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-GFRa4-P4-10-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (062816-W05) [ SEQ ID NO:1282] SIR efficiently induce TT target cell lysis compared to T-UI cells or medium alone.

T cells expressing SIR targeting MPL (thrombopoietin receptor) induce cytotoxicity in MPL-expressing HEL-92.. 1..7 cells. T cells expressing SIR targeting MPL were cultured with HEL-92.1.7-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using a Gluc-cytotoxicity assay. The results show that T cells expressing CD8SP-MPL-161-vL-V5- [ hTCCRb-S57-C-opt 1] -F-P2A-SP-FMC63-vH-Myc- [ TCRa-T48C-opt1] -F-F2A-PAC (050515-L05) [ SEQ ID NO:900] SIR or medium alone induce SIR HEL-92.1.7 target cell lysis in comparison to T-UI cells or T cells expressing CD8SP-FMC 63-vL-V5-F-P2A-PAC (050515-L05) [ SEQ ID NO:900] SIR.

T cells expressing SIR targeting MPL (thrombopoietin receptor) induce cytotoxicity in MPL-expressing HEL-92.. 1..7 cells. T cells expressing SIR targeting MPL were cultured with HEL-92.1.7-Gluc cells at an E: T ratio of 10:1 for 4 hours and tested using a Gluc-cytotoxicity assay. The results show that T cells expressing CD8SP-MPL-161-vL-V5- [ hTCRB-S57C-opt1] -F-P2A-MPL-161-vH-Myc- [ hTCRA-T48C-opt1] -F-F2A-PAC (040315-U02) [ SEQ ID NO:1112] SIR induce HEL-92.1.7 target cell lysis efficiently compared to T-UI cells or T cells expressing CD8SP-FMC63(vL-vH) -Myc-BBz-T2A-PAC (112014-A13) [ SEQ ID NO:4501] CAR or medium alone. Induction of target cell lysis was also observed when cells expressing single chain SIRCD8 SP-MPL-161-vL-Ser-Gly-linker-MPL-161-vH-Myc- [ hTCRa-T48C-opt1] -F-T2A-PAC (040915-X03) [ SEQ ID NO:1192] and CD8 SP-MPL-161-vL-Ser-Gly-linker-MPL-161-vH-V5- [ hTCRb-S57C-opt1] -T2A-PAC (032415-E07) [ SEQ ID NO:1193] were co-cultured with HEL-92.1.7 target cells. Finally, T cells expressing the single chain SIR CD8 SP-MPL-161-vL-Ser-Gly-linker-MPL-161-vH-Myc- [ hTCRa-T48C-opt1] -F-T2A-PAC (040915-X03) [ SEQ ID NO:1192] induced lysis of Jurkat-MPL cells. Other exemplary single chain SIRs in which the different scfvs of the disclosure were attached to the mutant TCR α chain are represented by DNA SEQ ID NOs 7519 to 7715 and 16694 to 16809 and PRT SEQ ID NOs 8161 to 8357 and 16928 to 17043. These single chain SIRs are preferably expressed in cells in which expression of the endogenous TCR α chain has been reduced or eliminated, without the complementary exogenous TCR β chain. The single chain SIR in which the different scFv of the present disclosure were attached to the mutant TCR β chain is represented by DNA SEQ ID NO:7733 to 7929 and 16811 to 169926 and PRT SEQ ID NO:8375 to 8571 and 17045 to 17160. These single chain SIRs are preferably expressed in cells in which the expression of the endogenous TCR β 1 and TCR β 2 chains has been reduced or eliminated, without the complementary exogenous TCR α chain.

T cells expressing SIR against TSLPR (thymic stromal lymphopoietin receptor) induce cytotoxicity in tsurkat cells expressing TSLPR. T cells expressing SIRs targeting TSLPR were cultured with Jurkat-Gluc cells at an E: T ratio of 10:1 for 24 hours and tested using a Gluc-cytotoxicity assay. The data show that T cells expressing CD8SP-V5- [ hTCRB-KACIAH ] -F-P2A-CD8 SP-TSLPR-vL-Gly-Ser-linker-TSLPR-vH-Myc- [ hTCra-CSDVP ] -F-F2A-PAC (091216-C03) [ SEQ ID NO:1797] SIR induced efficient lysis of Jurkat target cells compared to T-UI cells or medium alone.

T cells expressing SIR against SSEA4 induced cytotoxicity in P19 and F9 embryonal cancer cells expressing SSEA 4. T cells expressing SIR targeting SSEA4 were cultured with HEL-92.1.7-Gluc cells at an E: T ratio of 10:1 for 24 hours and tested using the Gluc-cytotoxicity assay. The results show that T cells expressing CD8SP-V5- [ hTCRB-KACIAH ] -F-P2A-CD8SP-SSEA 4-vL-Gly-Ser-linker-SSEA 4-vH-Myc- [ hTCRA-CSDVP ] -F-F2A-PAC (091516-I06) [ SEQ ID NO:1776] and CD8SP-V5- [ hTCRB-KACIAH ] -F-P2A-CD 8-SSEA 4-vL-Gly-Ser-linker-SSEA 4-vH-Myc4- [ preTCRa-Del48] -F-F2A-PAC (091516-K06) [ SEQ ID NO:1991] induced efficiently the lysis of SSEA4 target cells compared to T-UI cells.

T cells expressing SIR against CDH17 induced cytotoxicity in LoVo cells expressing CDH 17. T cells expressing SIRs targeting CDH7 were cultured with LoVo-Gluc cells at an E: T ratio of 10:1 for 24 hours and tested using the Gluc-cytotoxicity assay. The results show that T cells expressing CD8SP-CDH17-PTA001A4-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-CDH17-PTA001A4-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (816-X02) [ SEQ ID NO:1243] SIR efficiently induce lysis of LoVo target cells compared to T-UI cells or medium alone.

T cells expressing SIR against mesothelin induce cytotoxicity in mesothelin-expressing SKOV3 ovarian cancer cells. T cells expressing SIRs targeting mesothelin were cultured with SKOV3-Gluc cells at an E: T ratio of 10:1 for 24 hours and tested using a Gluc-cytotoxicity assay. The data show that T cells expressing CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8 SP-mesothelin-m 912-vL-Gly-Ser-linker-m 912-vH-Myc4- [ preTCRa-Del48] -F-F2A-PAC (090616-E04) [ SEQ ID NO:1956] SIR efficiently induce lysis of SKOV3 target cells compared to T-UI cells or medium alone.

T cells expressing SIR against FSHR (follicle stimulating hormone receptor) induce cytotoxicity in FSHR-expressing MDAMB-231 human breast cancer cells. T cells were infected with lentiviruses expressing SIR constructs targeting FSHR. The antigen binding domain of SIR comprises FSHb (follicle stimulating hormone β chain) linked to CGHa (chorionic gonadotropin α chain) via a Gly-to-Ser linker. Cells were selected with puromycin and expanded. MDAMB-231 cells stably expressing hGLuc were co-cultured with SIR expressing T cells or uninfected T cells (T-UI) at an effector to target (E: T) ratio of 10:1 for 24 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity. The results show that T cells expressing CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8 SP-FSHb-Gly-Ser-linker-CGHa-Myc 4- [ preTCRa-Del48] -F-F2A-PAC (091516-N06) [ SEQ ID NO:1909] SIR efficiently induce lysis of MDAMB-231 target cells compared to T-UI cells or medium alone. The results demonstrate that SIRs in which the antigen binding domain comprises a heterodimeric cytokine have functional activity.

T cells expressing SIR for LHR (luteinizing hormone receptor) induce cytotoxicity in LHR-expressing MCF7 human breast cancer cells. T cells were infected with lentiviruses expressing SIR constructs targeted to LHR. The antigen binding domain of this SIR comprises LHb (luteinizing hormone β chain) linked to CGHa (chorionic gonadotropin α chain) via a Gly-to-Ser linker. Cells were selected with puromycin and expanded. MCF7 cells stably expressing hGLuc were co-cultured with SIR expressing T cells or uninfected T cells (T-UI) at an effector to target (E: T) ratio of 2:1 for 24 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity. The results show that T cells expressing CD8SP-V5- [ hTCRB-KACIAH ] -F-P2A-SP-LHb-Gly-Ser-linker-CGHa-Myc- [ hTCRA-CSDVP ] -F-F2A-PAC (091616-R03) [ SEQ ID NO:1735] moderately induce lysis of MDAMB-231 target cells compared to T-UI cells or medium alone. The results demonstrate that SIRs in which the antigen binding domain comprises a heterodimeric cytokine have functional activity.

T cells expressing SIR against DLL3(δ -like 3) induced cytotoxicity in DLL 3-expressing SKMEL31 and SKMEL37 melanoma cells. T cells expressing SIRs targeting DLL3 were cultured with SKMEL31-Gluc and SKMEL37-Gluc cells at an E: T ratio of 10:1 for 24 hours and tested using a Gluc-cytotoxicity assay. The data show that T cells expressing CD8SP-DLL3-hSC16-13-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-DLL3-hSC16-13-vH-Myc4- [ hTCRa-CSDVP ] -F-F2A-PAC (071516-N04) [ SEQ ID NO:1263] SIR efficiently induced lysis of SKMEL31 and SKMEL37 target cells compared to T-UI cells or medium alone.

T cells expressing SIR against EGFRvIII mutants induced cytotoxicity in HeLa cells expressing EGFRvIII. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing SIR constructs targeting EGFRvIII. Cells were selected with puromycin and expanded. HeLa cells were engineered to express EGFRvIII and hGLuc by infection with a retroviral vector (MSCV-EGFRvIII) and a retroviral vector expressing hGLuc. Hela-EGFRvIII-hGluc cells stably expressing hGLuc were co-cultured with SIR expressing T cells or uninfected T cells (T-UI) at an effector to target (E: T) ratio of 2:1 for 24 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity. Experiments have shown that T cells expressing CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8 SP-EGFRvIII-2173-vH-Gly-Ser-linker-EGFRvIII-2173-vH-Myc 4- [ preTCRa-Del48] -F-F2A-PAC (090616-D03) [ SEQ ID NO:1902] SIR efficiently induce target cell lysis compared to T-UI cells or medium alone.

T cells expressing SIR against EGFR induce cytotoxicity in HeLa cells expressing EGFR. T cells expressing SIRs targeting EGFR were cultured with Hela-Gluc cells at an E: T ratio of 2:1 for 24 hours and tested using a Gluc-cytotoxicity assay. The data show that T cells expressing CD8 SP-cetuximab-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-cetuximab-vH-Myc 4- [ hTCRa-CSDVP ] -F-F2A-PAC (071516-H04) [ SEQ ID NO:1245] SIR induced lysis of EGFR target cells compared to T-UI cells or medium alone.

T cells expressing SIR against HIV1 envelope glycoprotein induced cytotoxicity in HL2/3 cells expressing HIV1 envelope glycoprotein. Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing indicated SIR constructs targeting the HIV1 envelope glycoprotein. Cells were selected with puromycin and expanded. HL2/3 cells expressing HIV1 envelope glycoprotein and engineered to stably express hGLuc were co-cultured with SIR expressing T cells or uninfected T cells (T-UI) at an effector to target (E: T) ratio of 2:1 for 24 hours. SIR-T cell mediated induction of target cell lysis was determined by increasing GLuc activity. The results show that T cells expressing CD8SP-HIV1-3BNC117-vL-MYC2- [ hTCRb-KACIAH ] -F-P2A-SP-HIV1-3BNC117-vH-Myc4- [ hTCRa-CSDVP ] -F-F2A-PAC (091616-Y01-RP) [ SEQ ID NO:1297] SIR induce lysis of EHIV1 envelope target cells compared to T-UI cells or medium alone.

The SIR targeting BST1/CD157 exerts cytotoxicity against Molm13 leukemia cells. T cells were engineered to express SIRs targeting BST1 with SEQ ID NOs 11333 to 11335 and tested for cytotoxicity against Molm13-Gluc cells after co-culture for 24 hours at an E: T ratio of 10: 1. Cytotoxicity was measured by Gluc assay. SIR-T cells targeting BST1 were shown to cause significant cell death as measured by increased GLuc activity.

The SIR targeting IL1RAP exerts cytotoxicity against Molm13 leukemia cells. T cells were engineered to express the SIR of targeted IL1RAP with SEQ ID NOs 18242, 18248 and 18254 and tested for cytotoxicity against Molm13-Gluc cells after co-culture for 24 hours at an E: T ratio of 10: 1. Cytotoxicity was measured by Gluc assay. SIR-T cells targeting IL1RAP were shown to cause significant cell death as measured by increased GLuc activity.

SIR- -TREG cells blocked the cytotoxic activity of SIR- -T cells. T-Reg cells were isolated using the T-Reg isolation kit (130-091-301) from America whirlpool (Miltenyi) and following the manufacturer's recommendations. T-Reg cells were cultured in T cell culture medium supplemented with rapamycin 100ng/ml T. Total T cells and T-Reg cells were infected with the indicated CAR and SIR lentiviruses. Cells were selected with puromycin 400ng/ml and expanded. T cells and T-Reg cells expressing CAR and SIR targeting CD19 were co-cultured with RAJI-Gluc cells alone or in combination at an E: T ratio of 10:1 for 4 hours and cytotoxicity was measured by direct injection of 0.5xCTZ assay buffer containing native coelenterazine (nanlight). The results show that T-Reg cells expressing SIR targeting CD19 can partially block the cytotoxic activity of T cells expressing SIR.

Jurkat NFAT-GFP cells used to synthesize immune receptors were engineered in such a way that the IL-2 promoter carrying the NFAT binding site was cloned upstream of the GFP gene. These cells have been used to study signaling via TCRs and CARs. Different SIRs were stably expressed in Jurkat NFAT-GFP cells by lentivirus-mediated gene transfer followed by selection with puromycin. Jurkat-NFAT-GFP cells expressing SIR were co-cultured with the target cells at an E: T ratio of about 1:2 for about 4 to 18 hours. GFP expression is induced when the interaction between the SIR and its target antigen causes activation of the NFAT pathway. Thus, Jurkat-NFAT-GFP cells expressing SIR show increased levels of GFP expression when interacting with a target cell line expressing a receptor for SIR.

Studies induction of GFP expression was achieved by co-culturing Jurkat-NFAT-GFP cells expressing different SIR constructs with different target cells, essentially as described previously (Wu, Roybal, Puchner, onsffer, & Lim, 2015). GFP expression was monitored by FACS analysis. Several representative examples of this assay are shown in FIGS. 13A-B. In Panel A, control Jurkat-NFAT-GFP cells or those expressing SIRs targeting CD19 (clone ID 051716-I08), MPL (clone ID:040716-A07) and BCMA (clone ID:011116-A07) were incubated with RAJI (top), HEL (middle) or U266 (bottom) cells, respectively. The induction of GFP expression was evident when Jurkat-NFAT-GFP cells expressing SIR were co-cultured with their corresponding target cells. In panel B, Jurkat cells expressing SIRs targeting CDH6 (clone ID: 051716-J05), CD276 (clone ID:050516-Q06) and Her2/neu (clone ID:050516-I03) were incubated with SKOV3 (top) and MC7 (middle and bottom) cells, respectively. The induction of GFP expression was evident when Jurkat-NFAT-GFP cells expressing SIR were co-cultured with their corresponding target cells. Jurkat-NFAT-GFP cells were engineered to express different SIRs targeting other antigens and the experiment was repeated while co-cultured with target cells expressing their corresponding target antigens. Jurkat-NFAT-GFP (parental) cells were used as controls. The results using different SIRs are summarized in the following summary table 10A. The names of the different SIRs, their SEQID NOs, component antigen binding domains and TCR chains can be determined by reference to tables 7A-7H. SIR was considered positive in the assay in the case that Jurkat-NFAT-GFP cells expressing SIR showed a greater% GFP positive cells when cultured with the target cell line compared to the parental Jurkat-NFAT-GFP cells. Thus, Jurkat-NFAT-GFP cells expressing the SIR represented by SEQ ID NO:1207 showed greater induction of GFP expression when co-cultured with LAN5 cells compared to the parental Jurkat-NFAT-GFP cells, but greater induction of GFP when co-cultured with Karpass 299, SUDHL-1 or H460 cell lines. The symbols +/-, +, 2+, etc. following the cell line name indicate the relative degree of positivity in the Jurkat-NFAT-GFP assay, as measured by% GFP-positive cells after culturing the SIR-expressing Jurkat-NFAT-GFP cells with the cell line. The results demonstrate that there are different SIRs containing binding domains derived from the same antibody (e.g. FMC63), and using this assay, a greater diversity of its ability to activate NFAT signaling is shown depending on the TCR chains and linkers used in its structure when exposed to the same cell line. In addition, greater diversity of responses to the same target cell line was observed using Jurkat cells expressing SIRs containing different antigen binding domains targeting the same antigen (e.g., having SIRs derived from antigen binding domains of different CD19 antibodies), even when the SIRs share the same TCR chain and linker. Finally, Jurkat cells expressing SIRs targeting different antigens (e.g., CD19 versus CD20) showed response diversity when exposed to the same target cell line. Thus, different immune responses against a single target cell are generated by combining SIRs with different TCR chains, linkers, antigen binding domains and target specificity. Table 10A also summarizes the results of GLuc-based T cell cytotoxicity assays observed when exposed to their target cell lines using different SIRs. The symbols +/-, + and 2+ etc. indicate that when the assay is performed under similar conditions, the degree of cytotoxicity is observed using the Gluc cytotoxicity assay after co-culturing the target cell line with T cells expressing SIR for 4-96 hours, compared to control T cells, i.e. T cells expressing no SIR or expressing an irrelevant SIR (SIR targeting an antigen not expressed on the particular target cell line). Moreover, similar to the results obtained using Jurkat-NFAT-GFP cells, T cells expressing different SIRs show a greater diversity in their ability to exert cytotoxicity when exposed to cells expressing their target antigens, depending on their TCR chains, linkers, antigen binding domains, target specificity and target cell line. Similar diversity in the ability to induce cytokine production (e.g., IL2, TNF α, and IFN γ) was observed in T cells expressing SIR when they were exposed to target cell lines under comparable conditions based on their TCR chain/linker/antigen binding domain/target specificity and the target cell line. However, when assays are performed under similar conditions, cytokine production by SIR-T cells when exposed to their target cells is typically lower than that observed with comparable CAR-T cells. Differences in expression of proliferation and depletion markers were also observed for T cells expressing different SIRs containing the same antigen binding domain but containing different TCR chains and linkers when assayed under similar conditions.

Table 10A also summarizes the results obtained using SIRs of fusions of the extracellular domain containing TCR α and TCR β chains with the extracellular, transmembrane and cytosolic domains of CD3z chains with and without the optional costimulatory domain derived from 41BB or CD 28. Such a similar exemplary SIR based on the FMC63 binding domain is CD8SP-FMC63-vL-TCRb-KAC-ECD-Bam-CD3 zECDP TMCP-opt-F-P2A-SP-FMC63-vH-hTCRa-CSDVP-ECDn-CD3zECDT MCP-opt2-F-F2A-PAC (SEQ ID NO: 10554). In this construct, the vL fragment of FMC63 was attached to the chain TCRb-KAC-ECD-Bam-CD3 zECCDTMCP-opt (SEQ ID NO:12402), while the vH fragment of FMC63 was attached to the chain hTCRA-CSDVP-ECDn-CD3 zECCDTMCP-opt 2 (SEQ ID NO: 12422). This SIR was strongly positive in the Jurkat-NFAT-GFP assay, but showed low antigen binding activity as measured by binding to soluble CD19-NLuc fusion protein. Several other constructs with similar design but with different TCRa and TCRb chain variants are represented by SEQ ID NOs 10552 to 10557 and show strong but different levels of activity in the Jurkat-NFAT-GFP assay and T-cell cytotoxicity assay. In the SIR construct CD8SP-FMC63-vL-hTCRAECdn-CD3 zECDP-opt 2-F-F2A-SP-FMC63-vH-TCRbECD-Bam-CD3 zECDP-opt-F-P2A-PAC (SEQ ID NO:10564), fragment FMC63vL was attached to TCRaECDend-CD 3 zECDP-opt 2 chain (SEQ ID NO:12421) and fragment FMC63vH was attached to TCRbECD-Bam-CD3 zTMCP-opt (SEQ ID NO: 12401). This construct also showed strong positives in the Jurkat-NFAT-GFP assay. The construct CD8SP-FMC63-vL-TCRbECD-Bam-CD3 zECDCTMCP-BBz-opt-F-P2A-SP-FMC 63-vH-Myc4-hTCRAECdn-CD3 zECDPCP-BBz-opt 2-F-F2A-PAC (SEQ ID NO:10565) containing the co-stimulatory domain from 41BB inserted in the cytoplasmic domains of CD3z of each of the two chains is also active in the Jurkat-NFAT-GFP assay. The design of the construct (SEQ ID NO:10563) is similar to SEQ ID NO:10565, but only one strand contains the 41BB co-stimulatory domain. This construct was also active in the Jurkat-NFAT-GFP assay. Finally, the construct CD8SP-FMC63-vL-V5-TCRbECD-Bam-CD3 zECDPCP-opt-F-P2A-SP-FMC 63-vH-Myc-hTCrAECdn-CD3 zECDP-28 z-opt2-F-F2A-PAC (SEQ ID NO:10557) containing a CD28 co-stimulatory domain in one strand was also active in the Jurkat-NFAT-GFP assay.

TABLE 10A summary of NFAT-GFP and T cell cytotoxicity assays using different SIR libraries targeting different antigens

To demonstrate the ability to generate different SIR pools containing the same antigen binding domain, Jurkat-NFAT-GFP cells stably expressing SIR containing FMC 63-based antigen binding domains but containing different TCR chains and linkers were compared for the following: 1) binding to CD19-GGSG-NLuc-AcV5 fusion protein (i.e., antigen binding assay); 2) induction of GFP expression after 18 hours of co-culture with RAJI cells (i.e., cell signaling assay); 3) Inducing IL2 production (i.e., cytokine production assay) after 18 hours of co-culture with RAJI cells; 4) staining with APC-conjugated protein-L (i.e. measurement of cell surface expression). The results are summarized in table 10B and demonstrate the great diversity in SIRs with the same antigen binding domain in the above assay. The construct SEQ ID NO 992 containing TCR α and TCR β chains encoded by the wild-type nucleotide sequences showed the lowest levels of CD19-GGSG-NLuc binding, GFP induction and protein L staining. Jurkat-NFAT-GFP cells stably expressing CD8SP-FMC63(vL-vH) -Myc-BBzT2A-PAC (SEQ ID NO:4501) CAR were included for comparison in an assay of IL2 production and protein L staining. Strikingly, CAR showed significantly higher IL2 production (1159pg/ml) and protein-L staining (61%) compared to all SIRs.

Table 10B: diversity in SIR targeting CD19 and containing FMC63 binding domain

Diversity in SIRs targeting the same antigen but containing different antigen binding domains. To generate SIRs for specific targets with even greater diversity, several SIRs targeting CD19 were generated using antigen binding domains derived from different antibodies. These SIRs also differ in their TCR chain, linker and antigen-binding domain formats. Corresponding CAR (i.e. CAR that also generated contained the same antigen binding domain as present in SIR). Different SIRs and CARs were stably transduced into Jurkat-NFAT-GFP cells and the resulting cells were compared for their ability to bind to CD19-GGSG-NLuc-AcV5 and activate NFAT signaling after overnight co-culture with RAJI cells. Table 9C demonstrates the significant diversity in different SIRs and CARs containing different CD19 antigen binding domains for the ability to bind to CD19 and activate NFAT signaling upon exposure to target cells. SIR CD8SP-CD19Bu12-vL-V5- [ hTCRb-WT ] -F-P2A-CD19Bu12-vH-Myc- [ hTCRa-WT ] -F-F2A-PAC (SEQ ID NO:1038) based on the Bu12 antibody containing wild type nucleotide sequences of TCR alpha and TCR beta chains showed very little CD19 binding and minimal NFAT signaling. There is also a significant difference in their ability to bind CD19 and activate NFAT signaling in different SIRs and these two parameters are not directly related when comparing SIRs containing different antigen binding domains. Thus, the SIR represented by SEQ ID NO:11240 shows nearly 10-fold higher binding of CD19-NLuc-AcV5, but lower NFAT-signaling, compared to the SIRs represented by SEQ ID NO:10815 and 11245. The results demonstrate the ability to generate immune effector cells expressing SIR libraries with different properties for generating different adoptive immune responses. Immune effector cells expressing SIR can be combined with immune cells expressing CARs and other chimeric immune receptors for generating even more diverse immune responses.

Table 10C: diversity in SIR and CAR targeting CD19 and containing different antigen binding domains

SIR showed lower antigen binding compared to CAR but comparable NFAT signaling. Jurkat-NFAT-GFP cells expressing SIRCD8SP-FMC63-vL-V5- [ hTCRB-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ hTCra-CSDVP ] -F-F2A-PAC (010616-C01) [ SEQ ID NO:1200] and CAR CD8SP-FMC63(vL-vH) -Myc-BBz-T2A-PAC (112014-A13) [ SEQ ID NO:4501] were tested along with Jurkat-NFAT-GFP cells for induction of NFAT-inducible GFP expression after overnight exposure to RAJI cells. Cells expressing SIR (SEQ ID NO:1200) and CAR (SEQ ID NO:4501) showed induction of equivalent and significantly higher GFP induction compared to parental cells, demonstrating that SIR and CAR efficiently induce NFAT signaling. The antigen binding of the test cells was then determined in triplicate using CD19-GGSG-NLuc binding assay. The average NLuc binding activity to parental Jurkat cells was 93, while Jurkat cells expressing SIR (SEQ ID NO:1200) and CAR (SEQ ID NO:4501) showed average NLuc values of 16422 and 186567, respectively. Thus, Jurkat cells expressing SIR SEQ ID NO:1200 and having nearly equivalent NFAT signaling activity to the corresponding CAR (SEQ ID NNO:4501) when both are exposed to the target antigen show nearly 10-fold lower antigen binding affinity.

The SIR based on Bu12 showed significantly lower cell surface expression compared to the comparable Bu12 CAR. Increased expression of CARs on the surface of T cells is known to lead to self-aggregation, complement signaling, and early cell depletion. Human peripheral T cells were infected with lentiviruses encoding CD 19-targeted SIR-based Bu12 (CD8SP-CD19Bu12- (vL-vH) -Myc-BBz-T2A-PAC (SEQ ID NO:4503) and equivalent CAR (CD8SP-CD19Bu12-vL-V5- [ TCRb-S57C-opt1] -F-P2A-SP-CD19Bu12-vH-Myc- [ TCRa-T48C-opt1] -F-F2A-PAC (070215-M03) [ SEQ ID NO:1021] cells were selected and expanded with puromycin T cells when measured by either method showed significantly lower cell surface expression compared to T cells expressing the corresponding CAR (SEQ ID NO: 4503).

SIR-T cells show lower TNF α production compared to corresponding CAR-T cells. T cells were engineered to express CARCD8SP-FMC 63-vL-Gly-Ser-linker-FMC 63-vH-Myc-CD8TM-BBz (SEQ ID NO:9659) and the corresponding SIR (CD8SP-FMC63-vL- [ hTCRa-CSDVP ] -F-F2A-SP-FMC63-vH- [ hTCRb-KACIAH ] -F-P2A-PAC (SEQ ID NO: 10596). these cells were co-cultured with Nalm6 and BV173 target cell lines for 24 hours at 37 ℃ and the induction of TNF α production was measured by ELISA.

Use of two vectors to express SIR. In the previous experiments, a single vector was used to express two Functional Polypeptide Units (FPUs) of SIR. Second, if two FPUs expressing SIR can be expressed using two different vectors, the vector is tested. SIR lentiviral constructs 050216-T02 and 050216-S08 contained a SIR sequence corresponding to SEQ ID NO:913 and encoding the SIR fragment CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH ] -F-P2A-PAC, wherein the vL fragment derived from CD19 monoclonal antibody FMC63 was linked via a V5 linker to the constant chain of hTCRa with a KACIAH mutation. This SIR FPU is linked to the PAC (puromycin resistance) gene via a F-P2A cleavable linker. The vector for the 050216-T02 construct was pLenti-EF1 α -DWPRE (SEQ ID NO:871), while the vector for 050216-S08 was pLenti-EF1 α (SEQ ID NO: 870). SIR lentiviral constructs 041916-a02 and 041916-B03 contained SIR sequences corresponding to SEQ ID NO:997 and encoding SIR fragment CD8SP-FMC63-vH-MYC- [ TCR α -CSDVP ] -F2A-BlastR, where the vH fragment derived from CD19 monoclonal antibody FMC63 was linked via a MYC linker to the constant chain of hTCRa with CSDVP mutation. This SIR FPU is linked to the blasticidin resistance gene via a F-F2A cleavable linker. The vector for the 041916-A02 construct was pLenti-EF1 α -DWPRE (SEQ ID NO:871), while the vector for 041916-B03 was pLenti-EF1 α (SEQ ID NO: 870). Jurkat-NFAT-GFP cells were infected with 050216-S08 and selected with puromycin. Jurkat-NFAT-GFP cells were also infected with the 041916-A02 and 041916-B03 constructs and selected with blasticidin. Finally, Jurkat-NFAT-GFP cells were infected with 050216-S08 and, when selected with puromycin, infected with either the 041916-A02 or 041916-B03 constructs and selected with blasticidin to select cells expressing both FPUs of SIR. Single-infected or double-infected cells were tested for binding to CD19-GGS-NLuc using the NLuc-binding assay. The results show that Jurkat-NFAT-GFP cells infected with constructs 050216-S08[ SEQ ID NO:913], 041916-A02[ SEQ ID NO:997] or 041916-B03[ SEQ ID NO:997] alone do not show any significant binding to CD 19-GGS-NLuc. In contrast, Jurkat-NFAT-GFP cells infected with construct 050216-S08 plus 041916-A02[ SEQ ID NO:997] and 050216-S08[ SEQ ID NO:913] plus 041916-B03[ SEQ ID NO:997] showed strong binding to CD 19-GGS-NLuc. Jurkat-NFAT-GFP cells infected with constructs 050216-S08[ SEQ ID NO:913] plus 041916-A02[ SEQ ID NO:997] and 050216-S08[ SEQ ID NO:913] plus 041916-B03[ SEQ ID NO:997] also showed strong induction of GFP expression when co-cultured with RAJI target cells, whereas Jurkat-NFAT-GFP cells infected individually with these constructs failed. These results demonstrate that two FPUs of SIR can be expressed using two different vectors and can aggregate to form functional receptors in doubly infected cells. Exemplary SIR constructs expressing different vL fragments of the present disclosure, which are linked to the TCRb chain (hTCRb-S57C-opt) via a V5 linker, are represented by DNA SEQ ID NOs 8803 to 8978 and 17162 to 17277 and PRT SEQ ID NOs 9231 to-9406 and 17396 to 17511. Exemplary corresponding SIR constructs expressing the different vH fragments of the present disclosure, which were linked to the TCRa strand (hTCRa-T48C-opt) via Myc linkers, are represented by DNA SEQ ID NOS: 9017 to 9191 and 17279-17394 and PRT SEQ ID NOS: 9445 to 9619 and 17513 to 17628.

Use of retroviral vectors for expression of SIR A number of SIR inserts were cleaved from the lentiviral vector by enzymatic digestion with Nhe I and Sal I and subcloned into the AvrII and Sal I digested retroviral vector MSCV-Bgl2-AvrII-Bam-EcoR1-Xho-BstB1-Mlu-Sal-ClaI.I03(SEQ ID NO: 872). The clone ID, SEQ ID and name of the insert are shown in table 11 below.

Table 11:

the above constructs were used to generate corresponding retroviruses, which were then used to infect Jurkat-NFAT-GFP cells. These infected cells were selected using puromycin and tested for SIR expression and activity in different assays. Jurkat-NFAT-GFP cells infected with the above construct show GFP induction after co-culture with the corresponding target cell line. These results demonstrate that retroviral vectors can be used to express the SIRs of the present disclosure. The results using the exemplary construct (SEQ ID NO:1212) are shown in FIG. 14.

A number of SIR inserts were excised from the lentiviral vector by enzymatic digestion with Age I and Xba I and subcloned into Age I and Xba I digested sleeping beauty transposon vector pSBbi-Pur (SEQ ID NO: 874). The resulting construct was transfected into Jurkat NFAT-GFP cells along with a vector pCMV/SB10 (Eddy Gene plasmid No. 2451) encoding a transposase. Cells were selected with puromycin as above and expanded. As shown in FIG. 15 below, Jurkat-NFAT-GFP cells transfected with construct pSBbi-puro-FMC63vL-V5- [ TCRb-KACIAH ] -F-P2A-FMC63vH-MYC- [ TCRa-CSDVP ] -F-F2A [010616-B01] (SEQ ID NO:875) showed GFP induction when co-cultured with the corresponding RAJI target cell line. These results demonstrate that a sleeping beauty turret can be used to express the SIR of the present disclosure.

Use of In Vitro Transcribed (IVT) RNA for expressing SIR IVT for generating SIR-encoding RNA is performed, essentially as described (ZHao Y et al, MOLECULAR THERAPY [ MOLECULAR THERAPY]Vol 13, No. 1, 2006). IVT RNA was generated using the mMESSAGE mMACHINE high-yield lidded RNA transcription kit (Invitrogen). IVT RNA was purified using RNeasy mini-kit (Qiagen, Inc., Valencia, Calif., USA) and the purified RNA was eluted at 1-0.5. mu.g/ml in RNase-free water. Electroporation of unstimulated PBMCs with 5 μ g of CD 19-targeted SIR CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH]-F-P2A-SP-FMC63-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC(102615-C08) [SEQ ID NO:1200]The cells (0.1ml) were electroporated with RNA of (1). Cells and cuvettes were pre-cooled by placing them on ice for 5 minutes, followed by electroporation. Subsequently, 0.05 to 0.2ml of cells were mixed with 2. mu.g/110⁶An IVT RNA T cell (e.g.Indicated) were mixed and electroporated in a 2-mm cuvette (Harvard Apparatus BTX, Holliston, MA, USA) using an ECM830 square wave electroporator (Harvard Apparatus BTX). Immediately after electroporation, cells were transferred to fresh CM with 300 IU/ml IL-2 and incubated at 37 ℃. Cells transcribed with IVT RNA encoding SIR were assayed after 48-72 h. SIR transfected cells showed increased binding to CD19-GGS-NLuc fusion protein and increased lysis of RAJI-GLuc target cells.

In vivo efficacy of SIR targeting CD19 with expression of CD8SP-FMC63-vL-V5- [ hTCRb-WT]-F-P2A-SP-FMC63-vH-Myc-[hTCRa-WT]-F-F2A-PAC(080815-F02)[SEQ ID NO:922]、CD8SP-FMC63-vL-V5-[mTCRb-opt]-F-P2A-SP-FMC63-vH-Myc-[mTCRa-opt]-F-F2A-PAC (080815-B06)[SEQ ID NO:953]、 CD8SP-FMC63-vL-V5-[hTCRb-KACIAH]-F-P2A-SP-FMC63-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC(081415-D06) [SEQ ID NO:992]Lentiviruses of the SIR construct and the Gluc-PAC-G07 control construct encoding a non-secreted form of Guassia luciferase infect human peripheral blood T cells isolated with CD3 magnetic beads. Approximately half of the cells were selected with puromycin, while the other half was amplified without puromycin selection. NSG mice were sublethally irradiated at 175cGy doses (Jackson Lab). Approximately 24 hours after irradiation (day 2), mice were injected 2.5x10 via tail vein⁴And (3) RAJI cells. On day 3, mice (n-5 per group) were treated with 5 million T cells (50% puromycin selected + 50% unselected) that had been infected with either lentivirus or Gluc-PAC-G07 constructs encoding the indicated SIRs. Control mice (n-5) received no T cells or uninfected T cells. Human IL2(400IU intraperitoneally) was administered to mice every other day until all mice in the control group died. Table 12 shows the survival of mice in each group. FMC 63-based SIR (080815-F02) in which TCRb and TCRa constant chains are encoded by their wild-type nucleotide sequences failed to confer survivability, while SIR constructs containing codon-optimized mouse TCRb and TCRa constant chain sequences (080815-B06) [ SEQ ID NO:953 ]]Conferring survival advantages. Similarly, SIR constructs containing codon-optimized murine human TCRb and TCRa constant chains (081415-D06) [ SEQ ID NO:992]Conferring survivalHas the advantages of simple process and low cost.

Table 12:

expression of the indicated SIR construct (040315-U02) [ SEQ ID NO:1112]、(050515-L05)[SEQ ID NO:900]、 (082815-G07)[SEQ ID NO:1620]、(082815-E05)[SEQ ID NO:1622]And (091015-Y08) [ SEQ ID NO:926]The lentivirus of (3) infects human peripheral blood T cells isolated with CD3 magnetic beads. Approximately half of the cells were selected with puromycin, while the other half was amplified without puromycin selection. NSG mice were sublethally irradiated at 175cGy doses (Jackson Lab). Approximately 24 hours after irradiation (day 2), mice were injected 2.5x10 via tail vein⁴And (3) RAJI cells. On day 3, mice (n-5 per group) were treated with 5 million T cells (50% puromycin selected + 50% unselected) which had been infected with a lentivirus encoding the indicated SIR. Control mice (n-5) received no T cells or uninfected T cells. Human IL2(400IU intraperitoneally) was administered to mice every other day until all mice in the control group died. Table 13 shows the survival of mice in each group. Double-stranded FMC 63-based SIR (050515-L05) [ SEQ ID NO:900]Conferring a survival advantage in which the TCRb and TCRa constant chains are encoded by codon-optimized nucleotide sequences and carry amino acid substitutions to facilitate chain pairing. Importantly, a double stranded SIR construct (091015-Y08) containing FMC63-vL fused to the constant strand of wild type TCRb and FMC63-vH fused to the chain of preTCRa-Del48 [ SEQ ID NO:926]Giving an even greater survival advantage (median survival of 31 days).

(082815-G07) [ SEQ ID NO:1620] construct in which the TCRb-kaliah constant chain does not have any vL (or vH) fragment fused to it and the FMC 63-derived scFV fragment (vL-linker-vH) is expressed fused to TCRa-CSDVP confers an even greater survival advantage (median survival ═ 34 days). Finally, (082815-E05) [ SEQ id no:1622] construct in which the TCRb-kaliah constant chain does not have any vL (or vH) fragment fused to it and the CD19-Bu12 derived scFV fragment (vL-linker-vH) is expressed fused to TCRa-CSDVP confers the greatest survival advantage (median survival ═ 36 days).

Table 13:

the expression of CD8SP-FMC63-vL-V5- [ TCRb-S57C-opt1]-F-P2A-SP-FMC63-vH-Myc-[TCRa-T48C-opt1]-F-F2A-PAC (050515-L05)[SEQ ID NO:900]Lentiviral infection of SIR constructs NSG mice were sublethally irradiated with 175cGy doses of human peripheral blood T cells isolated from CD3 magnetic beads (Jackson Lab). Approximately 24 hours after irradiation (day 2), mice were injected 2.5x10 via tail vein⁴And (3) RAJI cells. On day 3, mice were treated with 5 million T cells (n ═ 5 per group) that had been treated with a peptide encoding the designated CD8SP-FMC63-vL-V5- [ TCRb-S57C-opt1]-F-P2A-SP-FMC63-vH-Myc-[TCRa-T48C-opt1]-F-F2A-PAC (050515-L05)[SEQ ID NO:900]Lentiviral infection of SIR. Control mice (n-5) were injected with 5 million uninfected T cells or given no T cells. Mice in each group are shown to survive. Median survival of mice given RAJI cells alone and uninfected T cells was 22 days. In contrast, it received (050515-L05) [ SEQ ID NO:900]Median survival of mice with SIR T cells was 27 days, which survival was significantly increased. Thus, expression by infusion (050515-L05) [ SEQ ID NO:900]T cells of SIR lead to a significant improvement in mouse survival in this RAJI xenograft lymphoma model compared to mice given no T cells or mice given non-infected T cells.

The expression of CD8SP-FMC63-vL-V5- [ TCRb-S57C-opt1]-F-P2A-SP-FMC63-vH-Myc-[TCRa-T48C-opt1]-F-F2A-PAC (050515-L05)[SEQ ID NO:900]And CD8SP-CD19Bu12-vL-V5- [ TCRb-S57C-opt1]-F-P2A-SP-CD19Bu12-vH-Myc-[TCRa-T48C-opt1]-F-F2A-PAC (070215-M03)[SEQ ID NO:1021]Lentiviral infection of SIR constructs human peripheral blood T cells isolated with CD3 magnetic beads included expression control SIRCD8SP-MPL-161-vL-V5- [ TCRb-S57C-opt1]-F-P2A-MPL-161-vH-Myc-[TCRa-T48C-opt1]-F-F2A-PAC (040315-U02)[SEQ ID NO:1112]T cells at SIR served as control. NSG mice (n-5) were sublethally irradiated at 175cGy doses. At 24 hours post irradiation (day 2), mice were injected with 2.5x10 via tail vein⁴And (3) RAJI cells. On day 3, mice were treated with 5 million T cells (n-5 per group), which had been infected with a lentivirus encoding the indicated SIR. Human IL2(400IU i.p.) was given to mice every other day until all mice in the control group died. Figure 17 shows the survival of mice in each group. The median survival of mice given RAJI cells and T cells expressing control SIR (SEQ ID NO:1112) was 12 days. In contrast, it received (050515-L05) [ SEQ ID NO:900]-SIR-T and (070215-M03) [ SEQ ID NO:1021]Median survival of mice with SIR-T cells was 28.0 days and 51 days, which was significantly increased (p ═ 0004).

Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing indicated SIR constructs targeting folate receptor 1(FR1), L1CAM and Epcam 1. On day 1, NSG mice were sublethally irradiated at 175cGy doses. At 24 hours post irradiation (day 2), mice were injected intraperitoneally with 1x10⁶SKOV-3 cells. On day 3, mice were treated intraperitoneally with 5 million designated CAR-T cells. Mice were i.p. injected with 400 IU/mouse of human IL-2 every other day after CAR-T cell injection, starting on day 4, until all mice in the control group died.

Human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing indicated SIR constructs targeted to CS 1. On day 1, NSG mice were sublethally irradiated at 175cGy doses. At 24 hours post irradiation (day 2), mice were injected intravenously with 0.5x10⁶And L363 cells. On day 3, mice were treated intraperitoneally with 10 million designated CAR-T cells. Mice were i.p. injected with 400 IU/mouse of human IL-2 every other day after CAR-T cell injection, starting on day 4, until all mice in the control group died.

Infection of human peripheral blood T cells isolated with CD3 magnetic beads with lentiviruses expressing indicated SIR constructs targeting Lym1 and Lym2. On day 1, NSG mice were sublethally irradiated at 175cGy doses. At 24 hours post irradiation (day 2), mice were injected intravenously with 25x10³And Raji cells. On day 3, mice were treated intraperitoneally with 10 million designated CAR-T cells. Mice were i.p. injected with 400 IU/mouse of human IL-2 every other day after CAR-T cell injection, starting on day 4, until all mice in the control group died.

A substantially similar experimental plan as described in the previous examples will be used to test the in vivo efficacy of other SIR constructs described herein using NSG mice and appropriate cell lines expressing the target of the SIR.

A number of human cell-based artificial antigen presenting cells for cancer immunotherapy have been described. CAR for in vitro amplification⁺And SIR⁺The method of T cells involves the use of irradiated artificial antigen presenting cells (aapcs) expressing a targeted Tumor Associated Antigen (TAA). However, this approach requires that aapcs be engineered to express each individual TAA. To overcome this problem, a universal antigen presenting cell was developed. Protein L is an immunoglobulin (Ig) hydrogen chain binding protein expressed by Streptococcus macrodigestns, an anaerobic bacterial species. Protein L binds to the framework region of the vL domain of the kappa hydrogen chain and does not interfere with the antigen binding domain. As an alternative to expressing individual TAAs on the aAPC, experiments were performed to determine whether protein L can activate CAR independent of specificity⁺T and SIR⁺T cells. In an initial experiment, the co-culture of Jurkat-NFAT-GFP cells expressing different CAR and SIR constructs was tested to determine if protein L magnetic beads would cause induction of GFP expression. Pierce protein L magnetic beads (catalog number 88849; concentration 10mg/ml) were purchased from Seimer Feishel corporation (ThermoFisher) and diluted 1:10 in PBS. Approximately 10. mu.l of diluted beads were added to each well of a U-bottom 96-well plate. Jurkat-NFAT-GFP parental cells or expressing CD8SP-FMC63(vL-vH)-Myc-BBz-T2A-PAC(112014-A13)[SEQ ID NO:4501]Jurkat-NFAT-GFP cells of CAR were added to each well and incubated with beads for 18 hours. The induction of GFP expression was examined by flow cytometry. FIG. 18 below shows the expression of CD8SP-FMC63(vL-vH) -Myc-BBz-T2A-PAC (112014-A13) [ SEQ ID NO:4501]Jurkat-NFAT-GFP cells of the CAR were co-cultured with protein L beads for moderate induction of GFP expression.

Generation of human aapcs expressing protein L on the cell surface lentivirus Chimeric Antigen Receptors (CARs) were generated, which express fusions of different regions of protein L with the human CD8, 41BB co-stimulatory domain and the hinge and transmembrane regions of the CD3z chain. The nucleotide sequence of protein L is codon optimized for optimal expression in human cells. It is noted that for the purposes of the present invention, the 41BB co-stimulatory domain and CD3z chain are not essential for the function of protein L. The vector also carries an N-terminal signal peptide derived from human CD8 or IgH to allow the transport of the protein L on the cell surface. The complete nucleic acid and amino acid sequences of the vector are provided in SEQ ID NO 888 and SEQ ID NO 889.

Different protein L constructs were transiently transfected into 293FT cells. The ability of cell surface expressed protein L to bind to different scFv fragments was examined by incubating the cells with scFv-GGSG-NLuc fusion protein supernatant. 293FT cells expressing different protein L regions showed increased binding to scFV-GGSG-NLuc supernatant, thus demonstrating that protein L can be successfully expressed as a cell surface protein in mammalian cells.

Next, 293 cells were infected with a lentiviral vector encoding a protein L-II construct (072716-K01) and a polyclonal population of cells stably expressing protein L was generated after selection with 750ng/ml puromycin. These 293-protein L-II cells were used as antigen presenting cells and tested for their ability to activate Jurkat-NFAT-GFP cells expressing different SIR and CAR constructs. For this purpose, 293-protein L-II cells were seeded in 24-well plates and overlaid with Jurkat-NFAT-GFP cells expressing different SIR and CAR constructs after 12-24h, and after approximately 18 h of co-culture the induction of GFP expression was examined by flow cytometry. As shown in FIGS. 19A-D, co-culture with 293-protein L-II cells resulted in strong induction of GFP expression in Jurkat-NFAT-GFP cells expressing: CD8SP-FMC63(vL-vH) -Myc-BBz-T2A-PAC (112014-A13) [ SEQ ID NO:4501] CAR and CD8SP-HuLuc64-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-HuLuc64-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (092916-E07) [ SEQ ID NO:1253], CD8SP-V5- [ hTCRb-KACIAH ] -F-P2A-CD8SP-CD19 12-vL-Gly-Ser-linker-CD 19Bu12-vH-Myc- [ CRhTCa-CSDVP ] -F2A-PAC (082815-E05) [ SEQ ID NO:1622] SIR construct. These results demonstrate that mammalian cells expressing protein L on their surface can be used as universal antigen presenting cells for stimulating immune effector cells expressing SIR and CAR. However, the use of aapcs expressing protein L is not limited to the above types of cells. These cells can be used as universal aapcs for any immune effector cell that carries an antigen binding domain that binds to protein L derived from an antibody containing the appropriate kappa chain.

Expression of SIR in Pgp positive lymphocytes peripheral blood mononuclear cells (1 million cells) were isolated using diatrizoate. The cells were centrifuged and the cell pellet was blocked with 200. mu.l human AB serum for 1h at 4 ℃. Cells were washed with ice cold PBS containing 1% FCS and stained with three monoclonal antibodies UIC2 (200. mu.g/ml; Santa Cruz Biotechnology; SC-73354), MRK16 and 4E3 to P-glycoprotein (Pgp) for 1h at 4 ℃. Using a concentration of 0.5 μ g/million cells) per antibody increases the sensitivity of the assay. After extensive washing with PBS containing 1% FCS, cells were stained with 5. mu.l (2.5. mu.g) FITC-conjugated goat F (ab)2 anti-mouse IgG (H + L) human-absorbed antibody (Southern Biotechnology; Cat. 1032-02). After 2 washes, cells were labeled with PE-conjugated human CD3 antibody for 1h at 4 ℃. Cells were washed and analyzed by flow cytometry. Sorting CD3 positive T lymphocytes into Pgp⁺And Pgp^-And (4) in the fractions. The gene encoding CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH]-F-P2A-SP-FMC63-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC(081415-D06) [SEQ ID NO:992]SIR or negative control SIR CD8SP-KSHV-4C3-vL-V5- [ hTCRb-S57C-opt]-F-P2A-SP-4C3-vH-Myc-[hTCRa-T48C-opt]-F-F2A-PAC (111815-O05)[SEQ ID NO:4639]The lentiviral vector of (a) infects cells in each fraction. NSG mice were sublethally irradiated at 175cGy doses (jackson laboratories). Approximately 24 hours after irradiation (day 2), mice were injected 2.5x10 via tail vein⁴And (3) RAJI cells. On day 3, mice were treated with 5 million lymphocytes (n ═ 6 per group), which had been infected with lentiviruses encoding the indicated SIRs. Control mice (n-6) received no T cells or uninfected T cells. Mice were administered human IL2 every other day. Administration of CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH]-F-P2A-SP-FMC63-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC(081415-D06) [SEQ ID NO:992]Pgp of SIR infection⁺T cell mice are administered with CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH]-F-P2A-SP-FMC63-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC(081415-D06) [SEQ ID NO:992]Pgp of SIR infection^-T cells or negative control SIR CD8SP-KSHV-4C3-vL-V5- [ hTCRb-S57C-opt]-F-P2A-SP-4C3-vH-Myc-[hTCRa-T48C-opt]-F-F2A-PAC (111815-O05)[SEQ ID NO:4639]Infected Pgp⁺Or Pgp^-T cell mice survive longer. Administration of CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH]-F-P2A-SP-FMC63-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC(081415-D06) [SEQ ID NO:992]Pgp of SIR infection⁺Longer survival of T cell mice correlates with longer in vivo persistence of SIR-T cells. The above experiment was repeated by other methods as described in international application No. PCT/US2017/042248 (the disclosure of which is incorporated herein by reference), including MACS (magnetically activated cell sorting), and enrichment of Pgp-expressing T lymphocytes by exposure to TH9402 followed by exposure to light. Again, CD8SP-FMC63-vL-V5- [ hTCRb-KACIAH]-F-P2A-SP-FMC63-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC (081415-D06)[SEQ ID NO:992]Pgp of SIR infection⁺T cell mice survive longer, which correlates with longer in vivo persistence of SIR-T cells.

Use of dasatinib (dasatinib) to block the activity of SIR-T cells expressing SIR CD8SP-CD19Bu12-vL-V5- [ TCRb-S57C-opt1] -F-P2A-SP-CD19Bu12-vH-Myc- [ TCRa-T48C-opt1] -F2A-PAC (070215-M03) [ SEQ ID NO:1021] and CD8SP-CD20-2F2-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-CD20-2F2-vH-Myc- [ hTCRa-CSDVP ] -F2A-PAC (100615-D05) [ SEQ ID NO:1221] were pre-incubated with dasatinib at 100nM at 37 ℃ for about 30min or left untreated. Drug-treated and untreated T cells were seeded in white 384-well plates. RAJI cells stably expressing GLuc were added to wells containing T cells at a concentration of 30K cells/well to give an E: T ratio of 5: 1. Dasatinib was also added to the wells to maintain a final concentration of 100 nM. After co-cultivation for 4-24h, induction of SIR-T cell mediated target cell lysis was determined by increasing GLuc activity by direct injection of 0.5X CTZ assay buffer containing native coelenterazine. The results show that 100nM dasatinib significantly inhibited SIR-induced cell death. Treatment with dasatinib was found to significantly inhibit IFN γ and TNF α production after co-culturing SIR-T cells with a target cell line. The results demonstrate the ability of dasatinib to block cytotoxicity and cytokine production by SIR T cells. Since dasatinib is an FDA-approved drug and can cross the blood-brain barrier, it can be used to control SIR-T cell-induced cytotoxicity, including neurotoxicity, and to control SIR-T cell activity after administration to patients.

Use of PI3K inhibitors to expand SIR-T cells human peripheral blood T cells isolated with CD3 magnetic beads were infected with lentiviruses expressing indicated SIR constructs targeted to CD 19. Cells were either unselected or selected with puromycin and expanded using standard protocols as previously described using CD3/CD28 beads and IL2 but in the presence or absence of the dual PI3K/mTOR inhibitor PF-04691502(0.10 μ M to 0.5 μ M). NSG mice were sublethally irradiated at 175cGy doses (jackson laboratories). At 24 hours post irradiation (day 2), mice were injected with 2.5x10 via tail vein⁴And (3) RAJI cells. On day 3, mice were treated with 5 million T cells (n ═ 5 per group), which had been infected with lentiviruses encoding the indicated SIRs and expanded in the presence or absence of PF-04691502. Control mice (n-5) were injected with 5 million uninfected T cells. Human IL2(400IU i.p.) was given to mice every other day until all mice in the control group died. Receiving (050515-L05) [ SEQ ID NO:900 ] amplified in the presence of PI3K/AKT inhibitor]Median survival of mice receiving SIR-T cells expanded in the absence of PI3K/AKT inhibitor was higher than in mice receiving SIR-T cells expanded in the absence of PI3K/AKT inhibitor.

Use of CD 16-expressing universal SIR-T cells as antigen binding domains by intraperitoneal injection (i.p.; 0.3X 10) in NSG mice (Jackson laboratories)⁶Individual cells/mouse) luciferase-expressing Daudi cells. Some mice received rituximab (150mg) i.p. Four days after Daudi inoculation, CD8SP-MYC- [ hTCRa-T48C-opt1 was expressed by injection]-F-F2A-SP-CD16A-V158-ECD-v2-V5-[hTCRb-S57C-opt1]Human T cells (1X 10) of-F-P2A-PAC (SEQ ID NO:2069) SIR⁷). Control mice received tissue culture medium instead of rituximab or T cells. Rituximab injections were repeated weekly for 4 weeks without further T lymphocyte injections. All mice received intraperitoneal injections of 1000-. Mice receiving rituximab plus SIR-T cells showed reduced tumor growth.

Use of TRAC gRNAs (SEQ ID NO:896) and techniques known in the art to target a range of SIRs targeting CD19 as shown in tables 7A-7H to the TRAC locus in T cells. The targeting vector also carries a DNA barcode located downstream of the stop codon of the SIR insert. T cells may be derived from peripheral blood. In an alternative embodiment, the T cells are derived from a single clone of ipscs or hematopoietic stem cells using techniques known in the art. T cells expressing the SIR series were co-cultured with RAJI cells in vitro for 1 to 21 days. Aliquots of the SIR-T cell bank were collected before they were cultured with the target cells and continued for different days after co-culture. The samples were subjected to next generation sequencing to determine the relative frequency of different SIRs after exposure to the target cells. Bioinformatic analysis was used to determine the SIR associated with better proliferative responses after co-culture with target cells. Substantially similar methods are used to determine that T cells are endowed with higher proliferative potential in vivo and/or persist long in vivo and/or are present at a higher frequency when normalized to their frequency in the starting T cell population of surviving animals as compared to animals yielded to tumor challenge. In alternative embodiments of the present disclosure, substantially similar methods are used for human clinical samples to identify SIRs associated with different characteristics and/or outcomes including, but not limited to, longer term survival, lower incidence of cytokine release syndrome, lower neurotoxins, and/or higher long term persistence. Such SIRs can then be used singly or in different combinations to develop different SIR sub-libraries containing SIRs targeted to the same or different antigen binding domains, with different properties for treating different disease conditions and different patients. In other embodiments, SIR-T cells are exposed to their target cell line and then sorted into different groups based on the intracellular degree of IFN γ (as determined by flow cytometry). The frequency of different SIRs in the low contrast high IFN γ population was determined by next generation sequencing and normalized to its frequency in SIR-T cells of the control SIR-T cell population, i.e., cell lines that have not been exposed to the target cell line or exposed to a cell line that does not express the SIR target. From this analysis, the SIR associated with different levels of IFN γ production can be determined. Similar methods are used to screen and select for SIRs having any desired property or attribute, or combination thereof, including but not limited to lower TNF α production, lower expression of depletion markers, lower expression of terminal differentiation markers, and/or higher expression of cytotoxic markers.

IL7 use in conjunction with SIR-T cells studies were performed as described in the previous examples except that the mice were given an exogenous recombinant human IL-7 administered to three 200 ng/mouse doses by intraperitoneal injection 1 day after SIR-T cell injection, while control mice received saline. Mice receiving IL-7 showed rejection of their tumors and survived longer than control mice.

Use of an shRNA targeting BRD4 along with SIR-T cells was studied as described in the previous examples, except that T cells expressing a SIR construct represented by SEQ ID NO:893 (pLenti-EF1 α -CD8SP-FMC63-vL-V5- [ hTCrb-KACIAH ] -F-P2A-SP-FMC63-vH-Myc- [ preTCRa-Del48] -F-F2A-PAC-shRNA-BRD 4-DWPRE) were used, which constructs co-express an shRNA driven against the H1 promoter of BRD 4. This construct was compared to a construct expressing the SIR represented by SEQ ID NO:1200 but lacking the shRNA targeting BRD 4. Mice receiving shRNA constructs co-expressing BRD4-shRNA (i.e., SEQ ID NO:893) showed longer SIR-T cell persistence and survived longer.

The experiment was repeated using mice that received T cells expressing the SIR represented by SEQ ID NO:1200, except that half of the mice (n ═ 6) received twice daily intraperitoneal injections of the BRD4 inhibitor JQ1(+) (50mg/kg), while control mice received vehicle control (10% β -cyclodextrin, sigma). Mice receiving JQ1(+) showed longer SIR-T cell persistence and survived for longer periods of time compared to control mice.

The disclosed SIR-T cells may be used in adoptive cell therapy. As an example, patients with relapsed Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), or high risk intermediate grade B cell lymphoma may receive immunotherapy with adoptive transfer of autologous SIR-T cells targeted to CD 19. CliniMACS from Miltenyi Biotech Inc. (Miltenyi Biotec) was usedThe system and according to the manufacturer's recommendations, subjects the leukapheresis product collected from each patient to selection of CD3 positive T lymphocytes. T lymphocytes are enriched for Pgp positive T cells, optionally with flow sorting after staining with Pgp antibody, MACS after staining with Pgp antibody, or photodynamic selection after exposure to TH9402 plus light. The cells are treated with clinical grade CD19-SIR virus (e.g., CD8SP-CD19Bu12-vL-V5- [ hTCrb-KACIAH)]-F-P2A-CD19Bu12-vH-Myc-[hTCRa-CSDVP]-F-F2A-GMCSF-SP-tEGF R()[SEQ ID NO:1087]Transduction and then selection and expansion of SIR-T cells occurs in a closed system. After the resulting cell products are subjected to quality control tests (including sterility and tumor-specific cytotoxicity assays), they are cryopreserved. Meanwhile, after leukopheresis, study participants were treated with lymphocyte depletion (lymphodepletion) chemotherapy (30 mg/m)²Rifludarabine plus 500mg/m²Cyclophosphamide x3 days/day). One day after completion of its lymphocyte depletion protocol, the previously stored SIR-T cell product was shipped, thawed and infused at the patient's bedside. Study participants received intravenous infusion of SIR-transduced lymphocytes followed every 8 hoursTolerance was achieved by receiving a high dose (720000IU/kg) of IL-2 (aldesleukin; Prometheus, San Diego, Calif.). The dosage of SIR-T product was from 1x10 according to the study protocol⁴SIR + CD3 cells/kg to 5x10⁹Changes between SIR + CD3 cells/kg. The SIR-T product may be administered as a single infusion or as a split infusion. Study participants were pre-dosed at least 30 minutes prior to T cell infusion with 15mg/kg acetaminophen P.O (max 650mg) and diphenhydramine 0.5-1mg/kg i.v. (max 50 mg). Study participants may optionally receive daily injections of human IL-2. Clinical and laboratory-related follow-up studies can then be performed at the discretion of the physician, and these studies can comprise quantitative RT-PCR studies for the presence of CD 19-expressing ALL/lymphoma cells and/or adoptively transferred T cells; FDG-PET and/or CT scans; bone marrow examination for disease-specific pathology assessment; and/or long-term follow-up according to the guidelines set forth by the FDA's biological response modifiers Advisory Committee (FDA's biologics Advisory Committee) for gene transfer studies. Substantially similar methods can be used to treat other diseases using autoimmune cells (e.g., T cells) that have been engineered to express the disclosed SIRs, in which the SIRs target one or more antigens expressed on the cells causing the disease or disease-associated cells.

Registration of patients with a number of different diseases including infectious diseases (e.g. HIV1, EBV, CMV, HTLV1 etc.), degenerative diseases (e.g. alzheimer's disease), autoimmune diseases (e.g. pemphigus vulgaris), allergic diseases (e.g. chronic idiopathic urticaria) and various cancers in IRB-approved phase I clinical trials and taking immunotherapy using autologous SIR-T cells targeted to different adoptive transfers causing disease antigens or disease-associated antigens. The SIR for different diseases is selected based on the known expression of the target antigen in the disease-causing or disease-associated cells. Where possible, expression of SIR targets on disease-causing or disease-associated cells was confirmed by binding of ABD-GGS-NLuc fusion proteinsWherein the antigen binding domain of SIR is fused to the non-secreted form of the NLuc protein via a flexible linker. Alternatively, immunohistochemistry or flow cytometry using commercially available antibodies was used to confirm expression of SIR targets on disease-causing or disease-associated cells. T cells were collected from subjects using leukapheresis, transduced with lentiviral vectors encoding appropriate SIRs and expanded ex vivo in a closed system using CD3/CD28 beads. After the resulting cell products are subjected to quality control tests (including sterility and tumor-specific cytotoxicity assays), they are cryopreserved. At the same time, study participants used lymphocyte depletion chemotherapy (30 mg/m)²Rifludarabine plus 500mg/m²Cyclophosphamide x3 days/day). On the day after completion of their lymphocyte depletion protocol, study participants received intravenous infusions of transduced lymphocytes followed by high doses (720000IU/kg) of IL-2 (aldesleukin; Promilx corporation, san Diego, Calif.) every 8 hours to achieve tolerance. Previously stored SIR-T cell products were shipped, thawed and infused at the patient's bedside. The dosage of SIR-T product was from 1x10 according to the study protocol⁴SIR + CD3 cells/kg to 5x10⁹Changes between SIR + CD3 cells/kg. The SIR-T product may be administered as a single infusion or as a split infusion. Study participants were pre-dosed at least 30 minutes prior to T cell infusion with 15mg/kg acetaminophen P.O (max 650mg) and diphenhydramine 0.5-1mg/kg i.v. (max 50 mg). Study participants may optionally receive daily injections of human IL-2. Clinical and laboratory-related follow-up studies can then be performed at the discretion of the physician.

Use of an mTOR inhibitor RAD001 in combination with SIR-T cells studies were performed as described in the previous examples except that the mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, was administered to study participants at a dose that provided a target trough level of 0.1 to 3ng/ml beginning 1 day after infusion of SIR-T cells, where trough level "refers to the drug concentration in the plasma just prior to the next dose or the minimum drug concentration between the two doses.

Study was conducted as described in the previous example except that oral ibrutinib at doses from 140mg/d to 420mg/d was administered to study participants 1 day after infusion of SIR-T cells. It should be noted that study participants who received ibrutinib had a smaller incidence of severe cytokine release syndrome than participants who received SIR-T cells without ibrutinib.

Patients with relapsed acute lymphoblastic leukemia or high risk intermediate grade B-cell lymphoma and undergoing allogeneic bone marrow transplantation may receive immunotherapy with adoptively transferred allogeneic SIR-T cells. CliniMACS from America, whirlpool Biotech IncThe leukopheresis product collected from each donor (the same donor as used for the allograft) was subjected to selection of CD3 positive T lymphocytes systematically and according to the manufacturer's recommendations. T lymphocytes are enriched for Pgp positive T cells, optionally with flow sorting after staining with Pgp antibody, MACS after staining with Pgp antibody, or photodynamic selection after exposure to TH9402 plus light. Cells were activated using artificial antigen presenting cells based on CD3 and CD28 magnetic beads and treated with clinical grade CD19-SIR virus (e.g., CD8SP-CD19Bu12-vL-V5- [ hTCrb-KACIAH)]-F-P2A-CD19Bu12-vH-Myc-[hTCRa-CSDVP]-F-F2A-icasapase9[SE Q ID NO:1080]Transduction. Cells were expanded in a closed system for 9-12 days. After the resulting cell products are subjected to quality control tests (including sterility and tumor-specific cytotoxicity assays), they are cryopreserved. At the same time, study participants used lymphocyte depletion chemotherapy (30 mg/m)²Rifludarabine plus 500mg/m²Cyclophosphamide x3 days/day). On the day after completion of their lymphocyte depletion protocol, study participants received intravenous infusions of transduced lymphocytes followed by high doses (720000IU/kg) of IL-2 (aldesleukin; Promilx corporation, san Diego, Calif.) every 8 hours to achieve tolerance. SIR-T cell products were shipped, thawed and infused at the patient's bedside. According to the studyProtocol, dosage of SIR-T product can be from 1x10⁴SIR + CD3 cells/kg to 5x10⁹Changes between SIR + CD3 cells/kg. The SIR product may be administered as a single infusion or as a split infusion. Study participants were pre-dosed at least 30 minutes prior to SIR-T cell infusion with 15mg/kg acetaminophen P.O. (maximum 650mg) and diphenhydramine 0.5-1mg/kg i.v. (maximum 50 mg). Clinical and laboratory-related follow-up studies can then be performed at the discretion of the physician, and these studies can comprise quantitative RT-PCR studies for the presence of CD 19-expressing ALL/lymphoma cells and/or adoptively transferred T cells; FDG-PET and/or CT scans; bone marrow examination for disease-specific pathology assessment; and/or long-term follow-up according to the guidelines set forth by the FDA's biological Response modifier Advisory Committee (FDA's biological Response Modifiers Advisory Committee) applicable to gene transfer studies. Immunosuppressive drugs are also used at the discretion of the physician. Other diseases can be treated using substantially similar methods using allogeneic immune cells (e.g., T cells) expressing the disclosed SIRs, in which the SIRs target one or more antigens expressed on the cells causing the disease or disease-associated cells.

SIR-T cells hepatic arterial infusion in addition to intravenous infusion, SIR-T cells may be intra-arterially infused to provide a high concentration of SIR-T cells in a localized area or organ involved in disease. In the following examples, this method is used in the context of patients with liver metastatic cancer from gastrointestinal cancer expressing folate receptor alpha (FR 1). A substantially similar approach can be used to intra-arterially infuse SIR-T cells targeted to other tumor antigens.

The mapping angiography is performed via the right common femoral artery method at baseline. In addition to extra-hepatic perfusion from other potential sources, the stomach and duodenum and right gastric artery were embolized with microcoils. The same arterial access procedure was performed for the administration of the gene expressing CD8SP-FR1-huMov19-vL-V5- [ hTCRb-KACIAH]-F-P2A-SP-FR1-huMov19-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC (102915-P07)[SEQ ID NO:1276]T cells of SIR. T cells were collected from the patient on day 0 and infected with a lentivirus encoding SIR and expanded as described in the previous example. On day 14 (10)⁸Individual SIR-T cells), day 28 (10)⁹Individual SIR-T cells) and day 44 (10)¹⁰Individual SIR-T cells) are administered in a dose-escalating manner to the SIR-T cells. Through a 60cc syringe to<SIR-T cells were manually injected at a rate of 2 cc/s. The total volume infused was approximately 100 cc. Angiography with calibrated contrast was performed after the first 50cc infusion and after completion of the SIR-T infusion to confirm retained arterial blood flow. Infusions were delivered to the appropriate hepatic artery when possible. Some patients have abnormal hepatic artery anatomy, where the right or left hepatic artery does not start from the appropriate hepatic artery. In such cases, doses to segment SIR-T cells are calculated based on lobe volume. In such cases, the divided doses are delivered to the right and left hepatic arteries separately to ensure that proportional SIR-T is delivered to both lobes.

Clinical assessments were performed at baseline, on infusion days, and 1, 2, 4, and 7 days post-infusion. Planned imaging assessments using liver MRI and PET examinations were scheduled within one month before the first infusion and then within one month after the last SIR-T infusion. The response was ranked by the research radiologist (BS) according to the modified RECIST (mRECIST) and immune-related response criteria (Wolchok et al, 2009, Clin Cancer Res [ clinical Cancer research ],15: 7412-. Percutaneous biopsies were performed before treatment and three weeks after the final dose. The blind pathologist scored tumor necrosis and fibrosis on the biopsy samples. Safety assessments were made according to the protocol. The severity of adverse events was graded using the National Cancer Institute Common Terminology standard adverse event version 3.0(National Cancer Institute Common Terminology for additive events version 3.0).

SIR- -intraperitoneal administration of T cells SIR-T cells may also be administered intraperitoneally, essentially as described by Koneru M et al (Journal of transformed Medicine 2015; 13: 102). In the following examples, this method was used in the context of patients with ovarian cancer with peritoneal involvement of folate receptor alpha (FR1) expression. Substantially similar methods can be used to intraperitoneally infuse SIR-T cells targeted to other tumor antigens.

Patients with recurrent advanced severe ovarian cancer will be provided with screening informed consent to test for cancers that express FR 1. In case the expression of FR1 was confirmed by immunohistochemistry, then the patient would have leukopheresis product obtained from peripheral blood. Excess platelet and red blood cell contamination is removed from the leukapheresis product and the frozen product. During the treatment period of the study, the leukapheresis product will be thawed and washed. Subsequently, CD3+ T cells will be isolated from the thawed leukapheresis product by magnetic separation using CD3/CD28 beads. Activated T cells will be transduced with CD8SP-FR1-huMov19-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FR1-huMov19-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (102915-P07) [ SEQ ID NO:1276] SIR lentivirus and further amplified using a CD3/CD28 bead amplification protocol.

This is a phase I clinical trial that tested the safety of Intravenous (IV) and Intraperitoneal (IP) infusion (with or without prior cyclophosphamide chemotherapy) of genetically modified autologous T cells in patients with recurrent FR1+ ovarian cancer, fallopian tubes, or primary peritoneal cancer. These autologous T cells will be genetically engineered to express CD8SP-FR1-huMov19-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-FR1-huMov19-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (102915-P07) [ SEQ ID NO:1276] SIR. Patients with recurrent advanced severe ovarian, primary peritoneal or fallopian tube cancer that have been shown to express FR1 antigen confirmed by Immunohistochemical (IHC) analysis of deposited (banked) (paraffin embedded) or freshly biopsied tumors would be eligible for this study. Only a moderate to strong immune response score (3-5) will be considered positive, with score 3 being described as 51% -75% strong staining or 51% -100% weak staining, 4 being described as 76% -99% strong staining, and 5 being described as 100% strong staining. All patients will receive existing chemotherapy for relapsed disorders, with up to five existing classes of chemotherapy being allowed. Patients with other active malignancies, a lifespan of <3 months, or a Karnofsky Performance Status (KPS) score of < 70% at the time of planned treatment would be ineligible.

Phase I dose escalation will be used in this trial. Will be oriented to 3-6 positionsGroups of patients were infused with increasing doses of modified T cells to establish the Maximum Tolerated Dose (MTD). There are four planned dose levels: 3X10⁵、1×10⁶、3×10⁶And 1X10⁷ CD8SP-FR1-huMov19-vL-V5-[hTCRb-KACIAH]-F-P2A-SP-FR1-huMov19-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC (102915-P07)[SEQ ID NO:1276]SIR-T cells/kg. The recombinant plasmid will use 3X 105 CD8SP-FR1-huMov19-vL-V5- [ hTCRb-KACIAH]-F-P2A-SP-FR1-huMov19-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC (102915-P07)[SEQ ID NO:1276]SIR-T cells/kg treatment groups I and II, but group II patients will also receive lymphocyte depletion of cyclophosphamide. Groups II-V will receive increasing doses of modified T cells following pretreatment with cyclophosphamide. Cyclophosphamide depletion of lymphocytes at 750mg/m2 administration will be given 2-4 days prior to the initial T cell infusion. Standard 3+3 dose escalation protocols will be followed. If the first dose level exceeds the MTD, a subsequent group of 3-6 patients will have a 1X10 dose level at-1⁵ CD8SP-FR1-huMov19-vL-V5-[hTCRb-KACIAH]-F-P2A-SP-FR1-huMov19-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC( 102915-P07)[SEQ ID NO:1276]SIR-T cells/kg treatment without addition of lymphocyte-depleted cyclophosphamide (-group I).

An IP catheter will be placed followed by T cell infusion. The catheter will be placed when the modified T cells are ready for administration. The patient will be admitted to an inpatient ward of the hospital, after which the SIR T cells will be infused for the first time, and will remain hospitalized until at least 3 days after the second infusion of SIR T cells. Admitting a first group of patients to be treated and a first patient treated in each subsequent group into an Intensive Care Unit (ICU); subsequent patients may be admitted to medical oncology hospitalization (at the clinical discretion of the treating physician).

Patients will receive a single dose of cyclophosphamide (750mg/m2IV) chemotherapy with lymphocyte depletion 2 to 4 days before starting treatment with SIR-modified T cells. Transduced T cells will be quality tested for quantity, purity, viability and sterility prior to infusion. The following T cell release criteria must be met, including viability > 80%, CD3+ ≧ 95% and flow cytometry analysis based on expanded T cell populations, the infused T cell population must have a transduction fraction > 20%. In addition, the average vector copy number of transduced T cells will be determined by real-time PCR prior to infusion and will need to be in the range of 0.3 to 5 copies/cell, and a lentivirus that will use PCR to ensure replication capacity will not be present in the transduced T cells. All patients will receive a 50% genetically modified T cell dose intravenously. The toxicity of the patient will be closely monitored. After one to 3 days, the remaining doses of T cells will be given as IP infusion. At least 3 patients will be treated at dose level 1 with no more than 2 patients per month having a natural increase within each dose level. The time between treatment of each patient enrolled will be at least one week. All patients treated in the previous group were observed for a minimum of 4 weeks starting on the day of initial T cell infusion, after which the next group was escalated. Based on the significant risk of neutropenia (ANC ≦ 1000/mm3) following cyclophosphamide therapy, patients treated with cyclophosphamide can be treated with growth factor support (either a single subcutaneous injection of pegylated filgrastim or 3 consecutive days of subcutaneous filgrastim) at the discretion of the investigator.

Blood samples will be obtained from all patients before and after assessing toxicity, therapeutic efficacy and survival of the genetically modified T cells. Post-treatment blood samples will be collected approximately 1h, 1 day, and 1, 2, 3, 4,5, 6, 7, 8, and 12 weeks after T cell infusion, then up to 1 year monthly thereafter, then up to 15 years after T cell infusion. The ascites of the patient can also be sampled before cyclophosphamide or T cell therapy (first performed) and during follow-up if technically feasible. Patients will undergo CT scans approximately 6 weeks, 3 months, 6 months, 9 months and 12 months after T cell infusion and thereafter at the time of clinical indication.

Use of SIR-T cells for intratumoral injection, and SIR-T cells can also be administered intratumorally, essentially as in Brown CE et al, Clin Cancer Res. [ clinical Cancer research ] 9/15 days 2015; 4062-. In the following examples, this method is used in the context of patients with recurrent Glioblastoma (GBM) expressing IL13Ra 2. A substantially similar approach can be used for intratumoral infusion of SIR-T cells targeted to other tumor antigens.

Preliminary safety and feasibility studies will be performed to test expression of CD8SP-IL13Ra2-hu107-vL-V5- [ hTCRb-KACIAH in recurrent GBM]-F-P2A-SP-IL13Ra2-hu107vH-Myc-[hTCRa-CSDVP]-F-F2A-P AC(051816-Y03)[SEQ ID NO:1306]T cells of SIR. All participating patients will be required to sign informed consent. The clinical protocol will be approved by the University of Southern california institute Review Board and conducted in accordance with the Investigational new drug Application and registered on clinical trials. Eligible patients will include adults (18-70 years old) who have recurrent or refractory single-lesion supratentorial stage III or IV gliomas and whose tumors do not show communication with the ventricular/CSF pathway and are easy to resect. This assay will be independent of IL13R α 2(or IL13Ra2) tumor antigen status. Patients will be enrolled after initial diagnosis of high-grade glioma (WHO grade III or IV) at which time they will undergo leukapheresis to collect Peripheral Blood Mononuclear Cells (PBMCs). These cells will be used to engineer T cells to express CD8SP-IL13Ra2-hu107-vL-V5- [ hTCRb-KACIAH ] containing puromycin resistance gene (PAC) following infection with the corresponding lentiviral vectors as described in the previous examples]-F-P2A-SP-IL13Ra2-hu107vH-Myc-[hTCRa-CSDVP]-F-F2A-P AC(051816-Y03)[SEQ ID NO:1306]SIR. Alternatively, SIR-T cells can be generated after infection with a retrovirus either using a sleeping beauty transposon or by transfecting IVT mRNA. Subsequently, the cryopreserved released the tested therapeutic SIR-T cells and stored for later use. Upon first recurrence of the tumor, study participants will undergo tumor resection and placement of a Rickham reservoir/catheter. At the same time, therapeutic SIR-T cells were thawed and re-expanded in vitro using a rapid expansion protocol based on CD3/CD28 beads. After recovery from surgery and after baseline MR imaging, CD8SP-IL13Ra2-hu107-vL-V5- [ hTCRb-KACIAH will be delivered via an indwelling catheter]-F-P2A-SP-IL13Ra2-hu107vH-Myc-[hTCRa-CSDVP]-F-F2A-P AC(051816-Y03)[SEQ ID NO:1306]SIR administration directly into the resection cavity, essentially as described (Brown CE et al, Clin Cancer Res. [ clinical Cancer research ]]9 months and 15 days 2015; 21(18):4062-4072)The method is as follows. Cells will be manually injected into the Rickman reservoir using a No. 21 butterfly needle to deliver a 2ml volume in 5-10 minutes followed by a 2ml flush with preservative-free physiological saline in 5 minutes. Protocol treatment plan an in-patient dose escalation schedule is specified, which targets 12 CAR T cell doses administered intracranially over a 5 week period including a weekly treatment cycle. Within cycles 1, 2, 4 and 5, T cell infusions will be made on days 1, 3 and 5 of the cycle week, and week 3 will be the rest period. For safety, during cycle 1 we will utilize an intra-patient escalation strategy with 10 given on days 1, 3 and 5, respectively⁷、5×10⁷And 10⁸SIR T cell dose per cell/infusion, and this will be followed by 9 additional SIR T cell infusions over 4 weeks 10⁸And (4) cells. Imaging will be performed during the 3 week rest period and after the 5 week to assess the response. The guidelines provided in NCT Common Toxicity Criteria version 2.0 (NCI Common Toxicity criterion 2.0, https:// ctep. if. nih. gov/l) will be followed for monitoring Toxicity and adverse event reporting

SIR-use of T cells for ex vivo purging of bone marrow or peripheral blood stem cell preparations prior to transplantation. In the following examples, bone marrow or peripheral blood stem cells were obtained from patients with multiple myeloma using SIR-T cell purging expressing CD8SP-HuLuc64-vL-V5- [ hTCRb-KACIAH ] -F-P2A-SP-HuLuc64-vH-Myc- [ hTCRa-CSDVP ] -F-F2A-PAC (092916-E07) [ SEQ ID NO:1253] prior to autologous stem cell (or bone marrow) transplantation.

The patient will undergo leukapheresis to collect Peripheral Blood Mononuclear Cells (PBMCs). T cells will be purified using CD3 beads. These cells will be used to engineer T cells to express CD8SP-HuLuc64-vL-V5- [ hTCRb-KACIAH ] containing puromycin resistance gene (PAC) following infection with the corresponding lentiviral vectors as described in the previous examples]-F-P2A-SP-HuLuc64-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC (092916-E07)[SEQ ID NO:1253]SIR. This SIR targets the antigen CS1 expressed on myeloma cells. Expressing CD8SP-CS1-huLuc90-vL-V5- [ hTCRb-KACIAH]-F-P2A-SP-huLuc90-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC (012716-A02)[SEQ ID NO:1254]、 CD8SP-CD138-vL-V5-[hTCRb-KACIAH]-F-P2A-SP-CD138-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC(100815-A05) [SEQ ID NO:1236]Or CD8SP-GPRC5D-ET150-5-vL-Myc2- [ hTCRb-KACIAH]-F-P2A-SP-GPRC5D-ET150-5-vH-Myc4-[hTCRa-CSDVP]-F -F2A-PAC(100616-C03)[SEQ ID NO:1285]Will be used as an alternative or in combination with the above SIR-T cells targeted to CS 1. Alternatively, SIR-T cells can be generated after infection with a retrovirus either using a sleeping beauty transposon or by transfecting IVT mRNA. Subsequently, the cryopreserved released the tested therapeutic SIR-T cells and stored for later use or fresh use. Bone marrow cells and peripheral blood progenitor cell products will be collected from patients with multiple myeloma after standard procedures. For mobilization of peripheral blood stem cells, patients will receive 3gm/m2 cyclophosphamide, followed by 10 μ G/kg G-CSF subcutaneously daily starting 24h after cyclophosphamide until the extraction is complete. Once the peripheral blood CD34+ -cell count is 15 cells/μ l, peripheral blood stem cells will be collected. The collection target would be to treat three blood volumes per day until a minimum of 2.0 times 10 is reached after treatment⁶CD34+ cells/kg. CliniMACS from America, whirlpool Biotech IncThe system and according to the manufacturer's recommendations will optionally deplete red blood cells and/or enrich for cells expressing CD34 in bone marrow and peripheral blood stem cell products. These products will be used to purify fresh or cryopreserved products ex vivo. For purging, bone marrow or peripheral blood stem cell products were co-cultured with thawed SIR-T cells in XVIVO medium (loxa) supplemented with 100IU of recombinant human IL2 at effector to target ratios ranging from 5:1 to 30:1 for 4 to 24 hours. Cells will be cultured in a 5% CO2 humidified incubator at 37 ℃. After the co-culture period was over, cell aliquots were taken for sterility and quality testing (including flow cytometry to measure CFU-GM as well as CD34 and CD138 positive cells). Will receive spinal cord inhibitory chemotherapy (e.g., high dose melphalan, two divided doses of 70 mg/m) previously²The total dose is 140mg/m²) The remaining samples were administered intravenously.

Patients with pemphigus vulgaris will be enrolled in clinical trials to test the safety and efficacy of SIRs containing the extracellular domain of Dsg 3. Patients will be enrolled after diagnosis of pemphigus vulgaris that are resistant to treatment, at which time they will undergo leukapheresis to collect Peripheral Blood Mononuclear Cells (PBMCs). These cells will be used to engineer T cells to express SIRCD8SP-MYC- [ hTCRa-T48C-opt1 following infection with the corresponding lentiviral vectors as described in the previous examples]-F-F2A-SP-Dsg3-ECD-V5-[hTCRb-S57C-opt1]F-P2A-PAC (SEQ ID NO: 1144). Alternatively, SIR-T cells will be generated after infection with retrovirus either using a sleeping beauty transposon or by transfection of IVT mRNA. Subsequently, the cryopreserved released the tested therapeutic SIR-T cells and stored for later use. Patients will receive a single dose of cyclophosphamide (750mg/m2IV) chemotherapy with lymphocyte depletion 2 to 4 days before starting treatment with SIR-modified T cells. Transduced T cells will be quality tested for quantity, purity, viability and sterility prior to infusion. The dosage of SIR-T product can be from 1x10 according to the study protocol⁴SIR + CD3 cells/kg to 5x10⁹Changes between SIR + CD3 cells/kg. The SIR product may be administered as a single infusion or as a split infusion. Study participants were pre-dosed at least 30 minutes prior to SIR-T cell infusion with 15mg/kg acetaminophen P.O. (maximum 650mg) and diphenhydramine 0.5-1mg/kg i.v. (maximum 50 mg). Clinical and laboratory-related follow-up studies can then be performed at the discretion of the physician.

Many embodiments have been set forth above to illustrate the disclosure. The following claims will further set forth the summary of the applicant's invention.

Claims (198)

1. At least one recombinant polynucleotide encoding at least one Synthetic Immune Receptor (SIR), the at least one SIR comprising:

(a) a T Cell Receptor (TCR) constant chain having an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence at least 98% identical to SEQ ID NO 3010 and having one or more mutations at positions 48, 61, 91, 92, 93 and/or 94 and which may comprise an optional auxiliary module;

(ii) an amino acid sequence which is at least 98% identical to SEQ ID NO:3024 and has one or more mutations at positions 18, 22, 57, 79, 133, 136 and/or 139 and may comprise an optional auxiliary moiety;

(iii) an amino acid sequence which is at least 98% identical to SEQ ID NO:3025 and has one or more mutations at positions 18, 22, 57, 79, 133, 136 and/or 139 and may comprise an optional auxiliary moiety;

(iv) an amino acid sequence at least 98% identical to SEQ ID NO 3046, 3047 or 3048 and which may comprise an optional auxiliary module;

(v) an amino acid sequence which is at least 98% identical to SEQ ID NO 3049 and which may comprise an optional auxiliary module;

(vi) an amino acid sequence which is at least 98% identical to SEQ ID NO 3051 or 3052 and which may comprise optional auxiliary modules; and

(vii) (iii) a dimeric combination of two TCR constant chains selected from (i) and (ii), (i) and (iii), (iv) and (ii), (iv) and (iii), and (v) and (vi);

(b) optionally a linker; and

(c) one or more non-native TCR antigen binding domains selected from the group consisting of:

(1) an antibody;

(2) antibody fragments (e.g., Fv, Fab, (Fab') 2);

(3) a heavy chain variable region (vH domain) of an antibody or a fragment thereof;

(4) a light chain variable region (vL domain) of an antibody or a fragment thereof;

(5) a single chain variable fragment (scFv) or a fragment thereof;

(6) a Single Domain Antibody (SDAB) or fragment thereof;

(7) a camelidae VHH domain or a fragment thereof;

(8) a monomeric variable region of an antibody;

(9) non-immunoglobulin antigen binding scaffolds such as DARPIN, affibody, affilin, adnectin, affitin, obodies, repebody, fynomer, alphabody, avimer, atrimer, centryrin, pronectin, anticalin, kunitz-type domain, Armadillo repeat protein, or a fragment thereof;

(10) a receptor or fragment thereof;

(11) a ligand or fragment thereof;

(12) bispecific-antibodies, -antibody fragments, -scFV, -vHH, -SDAB, -non-immunoglobulin antigen-binding scaffolds, -receptors, or-ligands; and

(13) (ii) an autoantigen or a fragment thereof,

wherein the mutations of (a) (i) - (a) (iii) and the dimers of (a) (vii) provide different binding affinities for the target antigen of the antigen binding domain and are at least 5% greater than a tcr with the same binding domain, and when the synthetic immune receptor is expressed in a lymphocyte, both the antigen binding domain and the T cell receptor constant chain are expressed in one or more continuous chains on the surface of the lymphocyte such that the lymphocyte is triggered to activate, proliferate, secrete cytokines and/or modulate (induce or inhibit) killing of the target cell, and when the expressed antigen binding domain binds to its antigen has MHC-restricted and MHC-unrestricted antibody type specificity.

2. The recombinant polynucleotide of claim 1, comprising the TCR constant chains of (a) (vii), wherein the non-native TCR-binding domain is selected from the group consisting of:

-the variable regions of the heavy and light chains of an antibody or fragment thereof specific for a predefined target antigen, such that when expressed, one of said heavy and light chains of said antibody or fragment thereof is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said heavy and light chains of said antibody or fragment thereof is attached to the other of said two chains of said T cell constant region;

-two single-chain variable fragments (scfvs) specific for one or more predefined target antigens, such that when expressed, one of the scfvs is attached to one of the two chains of (a) (vii) of the T cell constant region and the other of the scfvs is attached to the other of the two chains of the T cell constant region;

-two antibody fragments specific for one or more predefined target antigens, such that when expressed one of said antibody fragments is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said antibody fragments is attached to the other of said two chains of said T cell constant region;

-two Single Domain Antibody (SDAB) fragments specific for one or more predefined target antigens, such that when expressed, one of said SDAB fragments is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said SDAB fragments is attached to the other of said two chains of said T cell constant region;

-two camelidae vHH domains specific for one or more predefined target antigens such that, when expressed, one of the vHH domains is attached to one of the two chains of (a) (vii) of the T cell constant region and the other of the vHH domains is attached to the other of the two chains of the T cell constant region;

-two non-immunoglobulin antigen binding scaffolds specific for one or more predefined target antigens, such that when expressed, one of the non-immunoglobulin antigen binding scaffolds is attached to one of the two chains (a) (vii) of the T cell constant region and the other of the non-immunoglobulin antigen binding scaffolds is attached to the other of the two chains of the T cell constant region;

-two receptors or fragments thereof specific for one or more predefined target antigens such that, when expressed, one of said receptors or fragments thereof is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said receptors or fragments thereof is attached to the other of said two chains of said T cell constant region;

-two ligands or fragments thereof specific for one or more predefined target antigens such that, when expressed, one of said ligands or fragments thereof is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said ligands or fragments thereof is attached to the other of said two chains of said T cell constant region;

-two structurally distinct antigen-binding fragments specific for one or more predefined target antigens, such that when expressed, one of said antigen-binding fragments is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said antigen-binding fragments is attached to the other of said two chains of said T cell constant region;

-two binding fragments, one or both of which is bispecific or multispecific such that, when expressed, one of the antigen-binding fragments is attached to one of the two chains of (a) (vii) of the T cell constant region and the other of the antigen-binding fragments is attached to the other of the two chains of the T cell constant region;

-two autoantigens or fragments thereof, such that when expressed, one of said autoantigens or fragments thereof is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said autoantigens or fragments thereof is attached to the other of said two chains of said T cell constant region; and

-two vls or fragments thereof, such that when expressed, one of said vls or fragments thereof is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said vls or fragments thereof is attached to the other of said two chains of said T cell constant region; and

-two vH or fragments thereof, such that when expressed, one of said vH or fragments thereof is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said vH or fragments thereof is attached to the other of said two chains of said T cell constant region.

3. The recombinant polynucleotide of claim 1, comprising the TCR constant chains of (a) (iv), wherein the non-native TCR-binding domain is selected from the group consisting of:

-the variable region of the heavy chain (vH) of an antibody or fragment thereof specific for a predefined target antigen;

-a variable region of a light chain (vL) of an antibody or fragment thereof specific for a predefined target antigen;

-a single-chain variable fragment (scFv) or a fragment thereof specific for a predefined target antigen;

antibody fragments specific for predefined target antigens (e.g. Fv, Fab, (Fab') 2);

-a Single Domain Antibody (SDAB) fragment specific for a predefined target antigen;

-camelidae vHH domains specific for a predefined target antigen;

-a non-immunoglobulin antigen binding scaffold specific for a predefined target antigen;

-a receptor or fragment thereof specific for a predefined target antigen;

-a ligand or fragment thereof specific for a predefined target antigen;

-bispecific-antibodies specific for one or more predefined target antigens, -antibody fragments, -scFV, -vHH, -SDAB, -non-immunoglobulin antigen-binding scaffold, -receptors or-ligands; and

-an autoantigen or a fragment thereof.

4. The recombinant polynucleotide of claim 1, comprising:

(iv) a polynucleotide encoding (i), (ii), (iii), (iv), (v), or (vi), wherein the non-native TCR binding domain is selected from the group consisting of:

-the variable region of the heavy chain (vH) of an antibody specific for a predefined target antigen;

-a variable region of a light chain (vL) of an antibody specific for a predefined target antigen;

-a single-chain variable fragment (scFv) specific for a predefined target antigen;

antibody fragments specific for predefined target antigens (e.g. Fv, Fab, (Fab') 2);

-a Single Domain Antibody (SDAB) fragment specific for a predefined target antigen;

-camelidae vHH domains specific for a predefined target antigen;

-a non-immunoglobulin antigen binding scaffold specific for a predefined target antigen;

-a receptor specific for a predefined target antigen or fragment thereof;

-a ligand specific for a predefined target antigen or fragment thereof;

-bispecific-antibodies specific for one or more predefined target antigens, -antibody fragments, -scFV, -vHH, -SDAB, -non-immunoglobulin antigen-binding scaffold, -receptors or-ligands; and

-an autoantigen or a fragment thereof.

5. The recombinant polynucleotide according to claim 1, wherein the polynucleotide encoding the TCR constant chains is a codon-optimized sequence.

6. The recombinant polynucleotide according to claim 1, wherein the polynucleotide encoding the TCR constant chains of (a) encodes mutated TCR constant chains that enhance TCR constant chain expression and/or pairing and reduce pairing thereof with the endogenous T cell receptor chains.

7. The recombinant polynucleotide of claim 1, wherein the polynucleotide encoding the TCR constant chains of (a) comprises 1-40 modified nucleic acid sequences of nucleic acid sequences SEQ ID NOs 730 to 743 or sequences having at least 70% identity to nucleic acid sequences SEQ ID NOs 730 to 743, and is capable of dimerizing with a TCR β 1 or TCR β 2 chain.

8. The recombinant polynucleotide according to claim 1, wherein the polynucleotide encoding the TCR constant chain of (b) or (c) comprises the nucleic acid sequence SEQ ID No.: 744 to 765 or 1-40 modified nucleic acid sequences having at least 70% identity to the nucleic acid sequence SEQ ID No.: 744 to 765 and is capable of dimerizing with a TCR alpha chain.

9. The recombinant polynucleotide according to claim 1, wherein the polynucleotide encoding the TCR constant chain of (v) comprises 1-40 modified nucleic acid sequences of nucleic acid sequence SEQ ID NO:769 to 770 or a sequence having at least 70% identity to nucleic acid sequence SEQ ID NO:769 to 770, and is capable of pairing with a TCR delta chain.

10. The recombinant polynucleotide of claim 1, wherein said polynucleotide encoding said TCR constant chains of (vi) comprises 1-40 modified nucleic acid sequences of nucleic acid sequence SEQ ID NOs 771 to 772 or sequences having at least 70% identity to nucleic acid sequences SEQ ID NOs 771 to 772 and is capable of dimerizing with TCR gamma chains.

11. The recombinant polynucleotide according to claim 1, wherein the polynucleotide encoding the TCR constant chains of (iv) comprises 1-40 modified nucleic acid sequences of nucleic acid sequences SEQ ID NOs 766 to 768 or sequences having at least 70% identity to nucleic acid sequences SEQ ID NOs 766 to 768 and is capable of dimerizing with TCR β 1 or TCR β 2 chains.

12. The recombinant polynucleotide of claim 1, wherein said one or more non-native TCR antigen-binding domains bind to one or more disease-associated antigens selected from the group consisting of: CD 19; CD 123; CD 22; CD 30; CD 171; CS-1 (also known as CD2 subunit 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD 33; epidermal growth factor receptor variant iii (egfrviii); ganglioside G2(GD 2); ganglioside GD3(aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (l-l) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. alpha. -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1(ROR 1); fms-like tyrosine kinase 3(FLT 3); tumor-associated glycoprotein 72(TAG 72); CD 38; CD44v 6; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epidermal cell adhesion molecule (EPCAM); B7H3(CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213a 2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate Stem Cell Antigen (PSCA); protease serine 21 (testis protein or PRSS 21); vascular endothelial growth factor receptor 2(VEGFR 2); a lewis (Y) antigen; CD 24; platelet derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD 20; a folate receptor alpha; receptor tyrosine protein kinase ERBB2(Her 2/neu); cell surface associated mucin 1(MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); prostasin; prostatic Acid Phosphatase (PAP); mutant elongation factor 2(ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase ix (caix); proteasome (precursor, megalin) subunit, beta form, 9(LMP 2); glycoprotein 100(gpl 00); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and the Abelson murine leukemia virus oncogene homolog 1 (Abl); a tyrosinase enzyme; ephrin type a receptor 2(EphA 2); fucosyl GM 1; sialylated lewis adhesion molecules (sLe); ganglioside GM3(aNeu5Ac (2-3) bDClalp (l-4) bDGlcp (l-1) Cer); transglutaminase 5(TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); folate receptor beta; tumor endothelial marker 1(TEM1/CD 248); tumor endothelial marker 7 associated (TEM 7R); sealin 6(CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); class 5 group member D of G protein-coupled receptors C (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); a globoH sugar ceramide hexasaccharide moiety (globoH); mammary differentiation antigen (NY-BR-1); urinary plaque 2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenoceptor β 3(ADRB 3); ubiquitin 3(PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex, locus K9(LY 6K); olfactory receptor 51E2(OR51E 2); TCR γ alternate reading frame protein (TARP); wilm's tumor protein (WT 1); cancer/testis antigen 1(NY-ES 0-1); cancer/testis antigen 2(LAGE-1 a); melanoma-associated antigen 1(MAGE-a 1); ETS translocation variant gene 6 located on chromosome 12p (ETV 6-AML); sperm protein 17(SPA 17); x antigen family member la (xagel); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; tumor protein p53(p 53); a p53 mutant; a prostate-specific protein; survivin; a telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; an inhibitor of melanoma apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyl-transferase V (NA 17); the paired box protein Pax-3(PAX 3); an androgen receptor; a cyclin Bl; a v-myc avian myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); tyrosinase-related protein 2 (TRP-2); cytochrome P450 lB1(CYPlB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site regulator siblings), squamous cell carcinoma antigen recognized by T cells 3(SART 3); the paired box protein Pax-5(PAX 5); the preproepisin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RUl); renal ubiquitin 2(RU 2); legumain; human papilloma virus E6(HPV E6); human papilloma virus E7(HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2(mut hsp 70-2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2(BST 2); EGF-like receptor-module mucin-like hormone-containing sample 2(EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); and immunoglobulin lambda-like polypeptide 1(IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFR α 54, CDH17, NYB 17, CDH17, CD200 17, Slea (CA 19.9; sialyl Lewis antigen) fucosyl-GM 17, PTK 17, gpNMB, CDH 17-CD 324, DLL 17, CD276/B7H 17, IL11 17, IL13Ra 17, CD179 17-IGLl 17, ALK TCR γ - δ, NKG 217, CD17 (FCGR2 17), Tnag, CSPG 17-HMW-MAA, Tim 17-/HVCR 17, CSF2 CSF- α, TCR β R17, TGF 72/VEGFR, Lews-1-HMW-MAA, TCHR β -CSF-glycoprotein, TCR β R17, TCR β -receptor, TNF-CSF- β -receptor, TNF- β -receptor, TNF-receptor- β -TCR- β -72, TNF-receptor, TNF- β -receptor, and like receptor, CMV pp65, EBV-EBNA3c, influenza A lectin (HA), GAD, PDL1, Guanylate Cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, desmoglein autoantibody 3(Dsg3), desmoglein autoantibody 1(Dsg1), HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1 (1), AFP, GPRC5D, sealer 18.2(CLD18A2 or N18A.2)), P-glycoprotein, STEAP1, LIV1, laminin-4, CRIPTO, GPA33, EBT 1/CD157, chloride channel antigen recognized by TNT antibodies of low conductivity.

13. The recombinant polynucleotide of claim 12, wherein said one or more non-native TCR antigen-binding domains comprise an antibody, an antibody fragment, a scFv, a Fv, a Fab, (Fab')2, a Single Domain Antibody (SDAB), a vH or vL domain, a camelidae vHH domain, a non-immunoglobulin antigen-binding scaffold, such as a DARPIN, an affibody, an affilin, an adnectin, an affitin, an obodies, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centryrin, a pronectin, an anticalin, a kunitz-type domain, an ardillo repeat protein, a receptor, or a ligand.

14. The recombinant polynucleotide according to claim 12, wherein said one or more non-native TCR antigen-binding domains are selected from the group consisting of:

(i) a heavy chain variable region (vH) encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 226 to 400 or 10203 to 10321 or a sequence at least 98% identical thereto and encoding a polypeptide that binds to an antigen thereof;

(ii) a light chain variable region (vL) encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 16 to 191 or 10085 to 10202 or a sequence at least 98% identical thereto and encoding a polypeptide that binds to an antigen thereof;

(iii) a single chain variable fragment (scFv) encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 488 to 657, 10346 to 10400, or 18098 to 18160, or a sequence at least 98% identical thereto and encoding a polypeptide that binds to an antigen thereof;

(iv) a camelid VHH domain encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 421 to 445 or 10322 to 10337 or a sequence at least 98% identical thereto and encoding a polypeptide that binds to an antigen thereof;

(v) a non-immunoglobulin scaffold encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 439 to 443 or a sequence at least 98% identical thereto and encoding a polypeptide that binds to an antigen thereof;

(vi) a receptor encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 456 to 468 or a sequence at least 98% identical thereto and encoding a polypeptide that binds to a homologue thereof; and

(vii) a ligand encoded by a polynucleotide having the sequence of any one of SEQ ID NOs 476 to 486 or 10402 to 10404 or a sequence having at least 98% identity thereto and encoding a polypeptide that binds to a homologue thereof.

15. The recombinant polynucleotide of claim 1, wherein said one or more non-native TCR antigen binding domains comprise one or more of the light chain complementarity determining regions of a selected target antigen represented by any one of SEQ ID Nos 13999-14879 or 14880 and/or one or more of the heavy chain complementarity determining regions of a selected target antigen represented by any one of SEQ ID Nos 14881-15761 or 15762.

16. The recombinant polynucleotide of claim 1, wherein said one or more non-native TCR antigen-binding domains comprise a variable light chain (vL) domain comprising the sequence of any one of SEQ ID Nos 2307 to 2482 or 12042 to 12159 with up to 10 conservative amino acid substitutions and/or a variable heavy chain (vH) domain comprising the sequence of any one of SEQ ID Nos 2506 to 2680 or 12160 to 12278 with up to 10 conservative amino acid substitutions.

17. The recombinant polynucleotide according to claim 1, wherein said one or more non-native TCR antigen binding domains comprise one or more of the Camelidae vHH complementarity determining regions of a selected antigen as set forth in any one of SEQ ID Nos 2701 to 2725 or 12279 to 12294 and having up to 10 conservative amino acid substitutions.

18. The recombinant polynucleotide of claim 1, wherein said one or more non-native TCR antigen-binding domains comprise a non-immunoglobulin antigen-binding domain having the sequence set forth in any one of SEQ ID Nos 2728-2732 or 12296-12301 and having up to 10 conservative amino acid substitutions.

19. The recombinant polynucleotide of claim 1, wherein the one or more non-native TCR antigen-binding domains comprise an scFv domain comprising one or more light chain complementarity determining regions of a variable light chain (vL) domain comprising the sequence of any one of SEQ ID Nos 2307-2482 or 12042-12159 and one or more heavy chain complementarity determining regions of a variable heavy chain (vH) domain comprising the sequence of any one of SEQ ID Nos 2506-2680 or 12160-12278.

20. The recombinant polynucleotide according to claim 1, wherein said one or more non-native TCR antigen-binding domains comprise scFv fragments having a sequence selected from the group consisting of SEQ ID Nos 2770 to 2939, 12303 to 12357, or 18162 to 18224, each having up to 10 conservative amino acid substitutions.

21. The recombinant polynucleotide according to claim 1, wherein said one or more non-native TCR antigen-binding domains comprise one or more receptors comprising the amino acid sequence of any one of SEQ ID Nos. 2736 to 2748 with up to 10 conservative amino acid substitutions.

22. The recombinant polynucleotide of claim 1, wherein said one or more non-native TCR antigen-binding domains comprise one or more ligands comprising the sequence of any one of SEQ ID Nos 2758-2768 or 12359-12361 with up to 10 conservative amino acid substitutions.

23. The recombinant polynucleotide of claim 1, wherein said one or more non-native TCR antigen-binding domains comprise the extracellular domain of CD16A, NKG2D, CD4, PD1, desmoglein 3(Dsg3), or CD 4-DC-SIGN.

24. The recombinant polynucleotide of claim 1, wherein the one or more non-native TCR antigen-binding domains comprise an extracellular domain of one or more of hTPO, mTPO, CGH α chain, CGH β chain, FH β chain, LH β chain, TSH β chain, APRIL, or a combination thereof.

25. The recombinant polynucleotide of claim 1, wherein the one or more non-native TCR antigen-binding domains comprise:

any single chain variable fragment (scFv) comprising the sequence of any one of SEQ ID Nos 2770 to 2939, 12303 to 12357, or 18162 to 18224 and having up to 10 conservative amino acid substitutions, and

a) any Camelidae vHH as set forth in any of SEQ ID Nos 2701 to 2725 or 12279 to 12294 and having up to 10 conservative amino acid substitutions, or

b) Any non-immunoglobulin antigen-binding domain having the sequence set forth in any one of SEQ ID NOs 2728-2732 or 12296-12301 and having up to 10 conservative amino acid substitutions; or

c) Any extracellular domain of a receptor comprising the amino acid sequence of any one of SEQ ID nos 2736 to 2748 and having up to 10 conservative amino acid substitutions; or

d) Any extracellular domain of a ligand comprising the sequence of any one of SEQ ID NO 2758-2768 or 12359 to 12361 and having up to 10 conservative amino acid substitutions.

26. The recombinant polynucleotide of claim 1, wherein the one or more non-native TCR antigen-binding domains comprise:

camelidae vHH as set out in any of SEQ ID Nos 2701 to 2725 or 12279 to 12294 and having up to 10 conservative amino acid substitutions, and

a) any single chain variable fragment (scFv) comprising the sequence of any one of SEQ ID Nos 2770 to 2939, 12303 to 12357, or 18162 to 18224 and having up to 10 conservative amino acid substitutions, or

b) Any non-immunoglobulin antigen-binding domain having the sequence set forth in any one of SEQ ID NOs 2728-2732 or 12296-12301 and having up to 10 conservative amino acid substitutions; or

c) Any extracellular domain of a receptor comprising the amino acid sequence of any one of SEQ ID nos 2736 to 2748 and having up to 10 conservative amino acid substitutions; or

d) Any extracellular domain of a ligand comprising the sequence of any one of SEQ ID NO 2758-2768 or 12359 to 12361 and having up to 10 conservative amino acid substitutions.

27. The recombinant polynucleotide according to claim 1, wherein said one or more non-native TCR antigen-binding domains are optionally linked to each of said TCR constant region chains by a linker region, wherein said linker region nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NOs 2981 to 3003 and any combinations thereof or sequences at least 98% identical thereto; or the linker is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 701 to 725 or a sequence having at least 98% identity thereto.

28. The recombinant polynucleotide of claim 1, wherein the one or more non-native TCR antigen-binding domains have at least 5-fold less binding affinity for their target antigen than the antibody from which the binding domains were obtained.

29. The recombinant polynucleotide of claim 1, wherein the polynucleotide encoding the SIR further comprises a leader sequence or signal peptide present at the N-terminus of each strand and comprising a sequence selected from the group consisting of SEQ ID NOs 1-9 and 10.

30. The recombinant polynucleotide of claim 1, wherein said at least one polynucleotide encodes two SIRs.

31. The recombinant polynucleotide according to claim 30, wherein said polynucleotide encodes two SIRs linked by a nucleotide sequence encoding a cleavable linker.

32. The recombinant polynucleotide according to claim 31, wherein said cleavable linker is a self-cleaving cleavable linker.

33. The recombinant polynucleotide according to claim 31, wherein said cleavable linker is any one or more of a2A linker, a 2A-like linker or a functional equivalent thereof.

34. The recombinant polynucleotide of claim 31, wherein said cleavable linker is any one or more of a T2A linker, P2A, F2A, E2A or functional equivalents thereof.

35. The recombinant polynucleotide according to claim 31, wherein said cleavable linker comprises the sequence of any one or more of SEQ ID nos 780 to 785.

36. The recombinant polynucleotide according to claim 31, wherein said polynucleotide sequence encoding said cleavable linker optionally follows a nucleotide sequence encoding a furin cleavage site or a furin-like cleavage site or a functional equivalent thereof.

37. The recombinant polynucleotide according to claim 36, wherein said furin cleavage site preceding said cleavable linker comprises the sequence of any one or more of SEQ ID nos 788 to 790.

38. The recombinant polynucleotide according to any one of claims 31-37, wherein said polynucleotide sequence encoding said cleavable linker follows a nucleotide sequence encoding a flexible linker.

39. The recombinant polynucleotide according to claim 38, wherein said flexible linker preceding said cleavable linker encodes one or more of a Ser-Gly linker, a Ser-Gly-Ser-Gly linker, or a functional equivalent thereof.

40. The recombinant polynucleotide according to claim 39, wherein said flexible linker preceding said cleavable linker comprises the sequence SEQ ID No 786 or 787.

41. The recombinant polynucleotide according to claim 38, wherein the polynucleotide sequence encoding the furin cleavage site is followed by a polynucleotide encoding the flexible linker, and the polynucleotide encoding the flexible linker is followed by a polynucleotide encoding the cleavable linker, such that the order is furin cleavage site-flexible linker-cleavable linker.

42. The recombinant polynucleotide according to claim 31, wherein said polynucleotide encoding said cleavable linker is present before a sequence encoding a leader sequence (signal peptide), said sequence encoding a second SIR.

43. The recombinant polynucleotide of claim 1, wherein said SIR can be designed to have different binding affinities for a selected antigen.

44. The recombinant polynucleotide of claim 1, wherein the SIR comprises an auxiliary module.

45. The recombinant polynucleotide according to claim 44, wherein said helper module comprises a CD3z domain.

46. The recombinant polynucleotide according to claim 45, wherein said TCR constant chain is selected from the group consisting of:

(viii) an amino acid sequence at least 98% identical to SEQ ID NO 12401 or 12402 or 12403 or 12408 or 12409;

(ix) an amino acid sequence at least 98% identical to SEQ ID NO 12421 or 12422 or 12423 or 12427 or 12428; and

(x) (viii) and (ix) a dimeric combination of the two TCR constant chains.

47. The recombinant polynucleotide according to claim 12, wherein said one or more non-native TCR antigen-binding domains bind to CD 19.

48. The recombinant polynucleotide according to claim 47, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2318-2324, 12060-12068, 12108, 12127 or 12156 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides;

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2517-2523, 12178-12186, 1227, 12246 or 12275 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides;

-a polypeptide comprising a sequence at least 98% identical to SEQ ID No. 12288; and

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2770-2774, 1232512308, 18162-18170 or 12354.

49. The recombinant polynucleotide of claim 47, wherein the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3135-3235, 3250-3346, 3396, 3401-3403, 3406, 3429-3432, 3435-3439, 3540, 3855-3859, 12431-12489, 12491-12493, 12495-12530, 12534, 13195-13203, 13250, 13267, 13289, 13429-13437, 13483, 13501 and 13523.

50. The recombinant polynucleotide according to claim 12, wherein said one or more non-native TCR antigen-binding domains bind to CD 20.

51. The recombinant polynucleotide according to claim 50, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2325-2326, 12069-12077 or 12078 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides;

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2524-2525, 12187-12195 or 12196 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides;

-a polypeptide comprising a sequence at least 98% identical to SEQ ID No. 12289 or 12290; and

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2787-2788, 18177-18186 or 18187.

52. The recombinant polynucleotide according to claim 50, wherein the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3263, 3348, 3456-3457, 3876-3877, 12464-12465, 12477-12482, 12492, 12534, 13204-13213, 13438-13446 and 13447.

53. The recombinant polynucleotide according to claim 12, wherein said one or more non-native TCR antigen-binding domains bind to CD 22.

54. The recombinant polynucleotide according to claim 53, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2327-2329, 12122-12126 or 12132 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides;

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2526-2528, 12241-12245 or 12251 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides; and

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2789-2791, 12320-12330 or 18188.

55. The recombinant polynucleotide according to claim 53, wherein the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3332, 3433, 3458-3460, 3878-3880, 12483, 12485, 12488-12490, 13241-13245, 13268, 13475-13479 and 13502.

56. The recombinant polynucleotide according to claim 12, wherein said one or more non-native TCR antigen-binding domains bind to BCMA.

57. The recombinant polynucleotide according to claim 56, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2310-2313, 12046-12048, 12118-12119, 12139-12145 or 12146 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides;

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2509-2512, 12164-12166, 12237-12238, 12258-12264 or 12265 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides;

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 12279-12281, 12283-12285, 12287, 12291-12292, 12293 or 12294; and

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2780-2783, 12237-12344, 18174-18175 or 18176.

58. The recombinant polynucleotide of claim 56, wherein the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3445-3449, 3866-3869, 12463, 12533, 12535-12536, 13181-13183, 13261-13262, 13277-13284, 13415-13417, 13495-13496, 13511-13517 and 13518.

59. The recombinant polynucleotide of claim 12, wherein the one or more non-native TCR antigen binding domains bind to MPL.

60. The recombinant polynucleotide according to claim 59, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2414-2421, 12120, 12128 or 12129 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides;

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2611-2618, 12239, 12247 or 12248 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides; and

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2871-2878, 12326-12327 and 12318.

61. The recombinant polynucleotide according to claim 59, wherein said recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3347, 3373, 3427-3428, 3495, 3556-3562, 3979-3985, 4025, 12454, 12456, 12458, 12462, 12532, 13259, 13265-13266, 13493, 13499 and 13500.

62. The recombinant polynucleotide according to claim 12, wherein said one or more non-native TCR antigen-binding domains bind to CS 1.

63. The recombinant polynucleotide according to claim 62, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2355-2358, 12090-12094 or 12095 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides;

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2553-2555, 12209-12213 or 12214 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides; and

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2817-2819, 18211-18215 or 18216.

64. The recombinant polynucleotide according to claim 62, wherein said recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3376, 3487-3489, 3907-3909, 12455, 12457, 12459, 12461, 12476, 13226-13231, 13460-13464 and 13465.

65. The recombinant polynucleotide according to claim 12, wherein said one or more non-native TCR antigen-binding domains bind to CD 33.

66. The recombinant polynucleotide according to claim 65, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2336-2337, 12079-12084 or 12085 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides;

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2535, 2536, 12197, 12202 or 12203 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides; and

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2795-2796, 18189-18193 or 18194.

67. The recombinant polynucleotide according to claim 65, wherein the recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3464-3465, 3884-3885, 12460, 12473, 12479, 13214-13220, 13448-13453 and 13454.

68. The recombinant polynucleotide according to claim 12, wherein said one or more non-native TCR antigen-binding domains bind to CD 123.

69. The recombinant polynucleotide according to claim 68, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2315, 2472, 12049-12058 or 12059 or any Complementarity Determining Region (CDR) contained in any of the aforementioned polypeptides;

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2514, 2670, 12167-12176 or 12177 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides;

-a polypeptide comprising a sequence at least 98% identical to SEQ ID NO 2716 or 2717; and

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2801, 2929, 18196-18205 or 18206.

70. The recombinant polynucleotide of claim 68, wherein said recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3266-3267, 3366-3368, 3375, 3378, 3405, 3409, 3434, 3470, 3492-3497, 3617, 3890, 3912-3913, 4041, 12480, 13184-13194, 13418-13427 and 13428.

71. The recombinant polynucleotide according to claim 12, wherein said one or more non-native TCR antigen-binding domains bind to folate receptor 1.

72. The recombinant polynucleotide according to claim 71, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

-a polypeptide comprising a sequence at least 98% identical to 2373 or any Complementarity Determining Region (CDR) contained therein;

-a polypeptide comprising a sequence at least 98% identical to any of SEQ ID No. 2570 or any Complementarity Determining Region (CDR) contained therein; and

-a polypeptide comprising a sequence at least 98% identical to any one of SEQ ID NO: 2833.

73. The recombinant polynucleotide of claim 71, wherein said recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos. 3511 and 3928.

74. The recombinant polynucleotide of claim 12, wherein the one or more non-native TCR antigen-binding domains bind to mesothelin.

75. The recombinant polynucleotide according to claim 74, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

-a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2413, 12154 or 12155 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides;

a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2609-2610, 12273 or 12274 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides;

-a polypeptide comprising a sequence at least 98% identical to SEQ ID NO 2713-2714 or 2725; and

-a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2870, 2899, 12352 or 12353.

76. The recombinant polynucleotide according to claim 74, wherein said recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3414, 3419, 3554, 3585, 3976, 4008, 13288, 13521 and 13522.

77. The recombinant polynucleotide of claim 12, wherein said one or more non-native TCR antigen-binding domains bind to IL13Ra 2.

78. The recombinant polynucleotide according to claim 77, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

-a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2399 or 2400 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides;

-a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2595 or 2596 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides; and

-a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2858 or 2859.

79. The recombinant polynucleotide according to claim 77, wherein said recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3541-3542, 3963 and 3964.

80. The recombinant polynucleotide according to claim 12, wherein said one or more non-native TCR antigen-binding domains bind to CD 138.

81. The recombinant polynucleotide according to claim 80, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

-a polypeptide comprising a sequence at least 98% identical to SEQ ID No. 2316 or any Complementarity Determining Region (CDR) contained therein;

-a polypeptide comprising a sequence at least 98% identical to SEQ ID No. 2515 or any Complementarity Determining Region (CDR) contained therein; and

a polypeptide comprising a sequence at least 98% identical to SEQ ID NO 2802.

82. The recombinant polynucleotide of claim 80, wherein said recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos. 3268, 3374, 3404, 3471 and 3891.

83. The recombinant polynucleotide according to claim 12, wherein the one or more non-native TCR antigen-binding domains bind to TCRgd.

84. The recombinant polynucleotide according to claim 83, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

-a polypeptide comprising a sequence at least 98% identical to SEQ ID NO 2449 or any Complementarity Determining Region (CDR) contained therein;

-a polypeptide comprising a sequence at least 98% identical to SEQ ID NO:2646 or any Complementarity Determining Region (CDR) contained therein; and

-a polypeptide comprising a sequence at least 98% identical to SEQ ID NO 2907.

85. The recombinant polynucleotide of claim 83, wherein said recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos. 3594 and 4017.

86. The recombinant polynucleotide according to claim 12, wherein said one or more non-native TCR antigen-binding domains bind to TCRB 1.

87. The recombinant polynucleotide according to claim 86, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

-a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2445 or 2446 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides;

-a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2642 or 2643 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides; and

-a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2903 or 2904.

88. The recombinant polynucleotide according to claim 86, wherein said recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3590-3591, 4013 and 4014.

89. The recombinant polynucleotide according to claim 12, wherein said one or more non-native TCR antigen-binding domains bind to TCRB 2.

90. The recombinant polynucleotide according to claim 89, wherein said one or more non-native TCR antigen binding domains are selected from the group consisting of:

-a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2447 or 2448 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides;

-a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NOs 2644 or 2645 or any Complementarity Determining Region (CDR) comprised by any of the aforementioned polypeptides; and

-a polypeptide comprising a sequence at least 98% identical to any of SEQ ID NO 2905 or 2906.

91. The recombinant polynucleotide of claim 89, wherein said recombinant polynucleotide encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos 3353-3364, 3592-3593, 4015 and 4016.

92. A recombinant expression system comprising the recombinant polynucleotide of claim 1 co-expressed with a therapeutic control agent, wherein the therapeutic control agent is selected from the group consisting of: truncated epidermal growth factor receptor (tEGFR), truncated epidermal growth factor receptor viii (tEGFRviii), truncated CD30(tCD30), truncated BCMA (tBCMA), truncated CD19(tCD19), CD34, thymidine kinase, cytosine deaminase, nitroreductase, xanthine guanine phosphoribosyltransferase, human caspase 8, human caspase9, inducible caspase9 (icapase 9), purine nucleoside phosphorylase, linalysidase/linalyrin/glucose oxidase, deoxynucleoside kinase, horseradish peroxidase (HRP)/indole-3-acetic acid (IAA), gamma-glutamylcysteine synthetase, CD 20/alpha CD20, CD 34/thymidine kinase chimera, dox-dependent caspase-2, thymidine mutant kinase (HSV-TKSR39), AP1903/Fas system, Chimeric Cytokine Receptors (CCR), selectable markers, and combinations thereof.

93. The recombinant expression system of claim 92, wherein said tEGFR and tEGFRviii bind to any one or more of an EGFR-specific siRNA, a small molecule, an anti-EGFR antibody or fragment thereof, or a combination thereof.

94. The recombinant expression system of claim 92, wherein said tCD30 binds to any one or more of a CD30 specific siRNA, a small molecule, an anti-CD 30 antibody or fragment thereof, or a combination thereof.

95. The recombinant expression system of claim 92, wherein said tCD19 binds to any one or more of a CD19 specific siRNA, a small molecule, an anti-CD 19 antibody or fragment thereof, or a combination thereof.

96. The recombinant expression system of claim 92, wherein said CD34 binds to any one or more of a CD34 specific siRNA, a small molecule, an anti-CD 34 antibody or fragment thereof, or a combination thereof.

97. The recombinant expression system of claim 92, wherein said selectable marker comprises any one or more of dihydroxyfolate receptor (DHFR), mutant DHFR, methylated DNA-protein-cysteine methyltransferase, hypoxanthine monophosphate dehydrogenase II (IMDHP2), Puromycin Acetyltransferase (PAC), blasticidin resistance gene, mutant calcineurin a/b (Can/b), CNa12, CNb30, or a combination thereof.

98. The recombinant expression system of claim 92, wherein said CCR comprises any one or more of (i) an IL-7 cytokine linker-IL 7Ra, (ii) an IL-7 cytokine linker extracellular domain of the IL-7 Ra-transmembrane domain of the IL-7 Ra-cytoplasmic domain of IL2R β, (iii) an IL-7 cytokine linker-IL 2R β, and (iv) combinations thereof.

99. A recombinant expression system comprising the recombinant polynucleotide of claim 1 co-expressed with a helper module, wherein the helper module is selected from the group consisting of: 41BBL, CD40L, K13, MC159, cFLIP-L/MRIT alpha, cFLIP-p22, HTLV1 Tax, HTLV2 Tax, HTLV2 Tax-RS mutant, FKBPx2-K13, FKBPx2-HTLV2-Tax, FKBPx2-HTLV2-Tax-RS, IL6R-304-vHH-Alb8-vHH, IL12f, PD1-4H1 scFV, PD1-5C 1 scFV, PD1-4H1-Alb 1-vHH, PD1-5C 1-Alb 1-vHH, CTLA 1-Imumab-scFv, CTLA 1-Iipilimumab-Alb 1-vHH, IL 1-19A-FV-72-Alb 3619-vHH, HVFLX-1-CD 1-hVTHH-CD 1-hTHV-1-HBT, CD 1-CD 1-hTHV-1-HBT-3-HBT-72, CD 1-HBT-3-HBT-3, CD-HBT-3, and CD-, The shRNA targets Brd4, Chimeric Antigen Receptor (CAR), hTERT, heparinase, CAR, inhibitory CAR, and combinations thereof.

100. The recombinant expression system of claim 92 or 99, wherein the recombinant polynucleotide encoding the SIR and one or more therapeutic control agents and/or one or more auxiliary modules are linked by a nucleotide sequence encoding a cleavable linker.

101. The recombinant expression system of claim 100, wherein said cleavable linker is a self-cleaving cleavable linker.

102. The recombinant expression system of claim 101, wherein the polynucleotide sequence encoding the cleavable linker follows a nucleotide sequence encoding a furin cleavage site or a furin-like cleavage site or a functional equivalent thereof.

103. The recombinant expression system of claim 100, wherein the polynucleotide sequence encoding the cleavable linker optionally follows the nucleotide sequence encoding the flexible linker.

104. At least one vector comprising the recombinant polynucleotide according to claim 1, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, a baculovirus vector, a sleeping beauty transposon vector, and a piggybac transposon vector.

105. The vector according to claim 104, wherein the vector backbone has a sequence selected from the group consisting of SEQ ID NOs 870 to 875 and 876.

106. The vector of claim 104, comprising a promoter selected from the group consisting of EF-1 promoter, CMV IE gene promoter, EF-1 a promoter, ubiquitin C promoter, MSCV LTR promoter, or phosphoglycerate kinase (PGK) promoter.

107. The vector of claim 104, wherein the EF-1 promoter comprises the sequence of SEQ ID NO 877 or a sequence 80-99% identical thereto.

108. The vector of claim 104, wherein the vector is an in vitro transfected vector, or the vector further comprises a poly (a) tail or 3' UTR.

109. At least one polypeptide encoded by at least one recombinant polynucleotide according to claim 1.

110. A recombinant cell expressing at least one recombinant polynucleotide according to claim 1.

111. An isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer comprising:

(a) a T Cell Receptor (TCR) constant chain having an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence at least 98% identical to SEQ ID NO 3010 and having one or more mutations at positions 48, 61, 91, 92, 93 and/or 94 and which may comprise an optional auxiliary module;

(ii) an amino acid sequence which is at least 98% identical to SEQ ID NO:3024 and has one or more mutations at positions 18, 22, 57, 79, 133, 136 and/or 139 and may comprise an optional auxiliary moiety;

(iii) an amino acid sequence which is at least 98% identical to SEQ ID NO:3025 and has one or more mutations at positions 18, 22, 57, 79, 133, 136 and/or 139 and may comprise an optional auxiliary moiety;

(iv) an amino acid sequence at least 98% identical to SEQ ID NO 3046, 3047 or 3048 and which may comprise an optional auxiliary module;

(v) an amino acid sequence which is at least 98% identical to SEQ ID NO 3049 and which may comprise an optional auxiliary module;

(vi) an amino acid sequence which is at least 98% identical to SEQ ID NO 3051 or 3052 and which may comprise optional auxiliary modules; and

(vii) (iii) a dimeric combination of two TCR constant chains selected from (i) and (ii), (i) and (iii), (iv) and (ii), (iv) and (iii), or (v) and (vi);

(b) optionally a linker; and

(c) one or more non-native TCR antigen binding domains selected from the group consisting of:

(1) an antibody;

(2) antibody fragments (e.g., Fv, Fab, (Fab') 2);

(3) a heavy chain variable region (vH domain) of an antibody or a fragment thereof;

(4) a light chain variable region (vL domain) of an antibody or a fragment thereof;

(5) a single chain variable fragment (scFv) or a fragment thereof;

(6) a Single Domain Antibody (SDAB) or fragment thereof;

(7) a camelidae VHH domain or a fragment thereof;

(8) a monomeric variable region of an antibody;

(9) non-immunoglobulin antigen binding scaffolds such as DARPIN, affibody, affilin, adnectin, affitin, obodies, repebody, fynomer, alphabody, avimer, atrimer, centryrin, pronectin, anticalin, kunitz-type domain, Armadillo repeat protein, or a fragment thereof;

(10) a receptor or fragment thereof;

(11) a ligand or fragment thereof;

(12) bispecific-antibodies, -antibody fragments, -scFV, -vHH, -SDAB, -non-immunoglobulin antigen-binding scaffolds, -receptors, or-ligands; and

(13) (ii) an autoantigen or a fragment thereof,

wherein the mutations of (a) (i) - (a) (iii) provide different binding affinities for the target antigen of the antigen binding domain, and the synthetic immune receptor, when expressed in lymphocytes, expresses both the antigen binding domain and the T cell receptor constant chain in one or more continuous chains on the surface of the lymphocytes, such that lymphocytes are triggered to activate, proliferate, secrete cytokines and/or modulate (induce or inhibit) killing of the target cells, and have MHC-restricted or MHC-unrestricted antibody type specificity when the expressed antigen binding domain binds to its antigen.

112. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, comprising the TCR constant chains of (a) (vii), wherein the non-native TCR-binding domain is selected from the group consisting of:

-the variable regions of the heavy and light chains of an antibody or fragment thereof specific for a predefined target antigen, such that when expressed, one of said heavy and light chains of said antibody or fragment thereof is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said heavy and light chains of said antibody or fragment thereof is attached to the other of said two chains of said T cell constant region;

-two single-chain variable fragments (scfvs) specific for one or more predefined target antigens, such that when expressed, one of the scfvs is attached to one of the two chains of (a) (vii) of the T cell constant region and the other of the scfvs is attached to the other of the two chains of the T cell constant region;

-two antibody fragments specific for one or more predefined target antigens, such that when expressed one of said antibody fragments is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said antibody fragments is attached to the other of said two chains of said T cell constant region;

-two Single Domain Antibody (SDAB) fragments specific for one or more predefined target antigens, such that when expressed, one of said SDAB fragments is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said SDAB fragments is attached to the other of said two chains of said T cell constant region;

-two camelidae vHH domains specific for one or more predefined target antigens such that, when expressed, one of the vHH domains is attached to one of the two chains of (a) (vii) of the T cell constant region and the other of the vHH domains is attached to the other of the two chains of the T cell constant region;

-two non-immunoglobulin antigen binding scaffolds specific for one or more predefined target antigens, such that when expressed, one of the non-immunoglobulin antigen binding scaffolds is attached to one of the two chains (a) (vii) of the T cell constant region and the other of the non-immunoglobulin antigen binding scaffolds is attached to the other of the two chains of the T cell constant region;

-two receptors or fragments thereof specific for one or more predefined target antigens such that, when expressed, one of said receptors or fragments thereof is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said receptors or fragments thereof is attached to the other of said two chains of said T cell constant region;

-two ligands or fragments thereof specific for one or more predefined target antigens such that, when expressed, one of said ligands or fragments thereof is attached to one of said two chains of (a) (vii) of said T cell constant region and the other of said ligands or fragments thereof is attached to the other of said two chains of said T cell constant region;

-two structurally distinct antigen-binding fragments specific for one or more predefined target antigens, such that when expressed, one of said antigen-binding fragments is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said antigen-binding fragments is attached to the other of said two chains of said T cell constant region;

-two binding fragments, one or both of which is bispecific or multispecific such that, when expressed, one of the antigen-binding fragments is attached to one of the two chains of (a) (vii) of the T cell constant region and the other of the antigen-binding fragments is attached to the other of the two chains of the T cell constant region;

-two autoantigens or fragments thereof, such that when expressed, one of said autoantigens or fragments thereof is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said autoantigens or fragments thereof is attached to the other of said two chains of said T cell constant region; and

-two vls or fragments thereof, such that when expressed, one of said vls or fragments thereof is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said vls or fragments thereof is attached to the other of said two chains of said T cell constant region; and

-two vH or fragments thereof, such that when expressed, one of said vH or fragments thereof is attached to one of said two chains (a) (vii) of said T cell constant region and the other of said vH or fragments thereof is attached to the other of said two chains of said T cell constant region.

113. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, comprising the TCR constant chains of (a) (iv), wherein the non-native TCR binding domain is selected from the group consisting of:

-the variable region of the heavy chain (vH) of an antibody or fragment thereof specific for a predefined target antigen;

-a variable region of a light chain (vL) of an antibody or fragment thereof specific for a predefined target antigen;

-a single-chain variable fragment (scFv) or a fragment thereof specific for a predefined target antigen;

antibody fragments specific for predefined target antigens (e.g. Fv, Fab, (Fab') 2);

-a Single Domain Antibody (SDAB) fragment specific for a predefined target antigen;

-camelidae vHH domains specific for a predefined target antigen;

-a non-immunoglobulin antigen binding scaffold specific for a predefined target antigen;

-a receptor or fragment thereof specific for a predefined target antigen;

-a ligand or fragment thereof specific for a predefined target antigen;

-bispecific-antibodies specific for one or more predefined target antigens, -antibody fragments, -scFV, -vHH, -SDAB, -non-immunoglobulin antigen-binding scaffold, -receptors or-ligands; and

-an autoantigen or a fragment thereof.

114. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, comprising:

(i) (iii), (iv), (v), or (vi), wherein the non-native TCR-binding domain is selected from the group consisting of:

-the variable region of the heavy chain (vH) of an antibody specific for a predefined target antigen;

-a variable region of a light chain (vL) of an antibody specific for a predefined target antigen;

-a single-chain variable fragment (scFv) specific for a predefined target antigen;

antibody fragments specific for predefined target antigens (e.g. Fv, Fab, (Fab') 2);

-a Single Domain Antibody (SDAB) fragment specific for a predefined target antigen;

-camelidae vHH domains specific for a predefined target antigen;

-a non-immunoglobulin antigen binding scaffold specific for a predefined target antigen;

-a receptor specific for a predefined target antigen or fragment thereof;

-a ligand specific for a predefined target antigen or fragment thereof;

-bispecific-antibodies specific for one or more predefined target antigens, -antibody fragments, -scFV, -vHH, -SDAB, -non-immunoglobulin antigen-binding scaffold, -receptors or-ligands; and

-an autoantigen or a fragment thereof.

115. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the TCR constant chain comprises a mutation that enhances TCR constant chain expression and/or pairing and reduces its pairing with the endogenous T cell receptor chain.

116. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the constant region of a TCR is a TCR receptor alpha chain (ca) comprising an amino acid sequence having 1-40 amino acid substitutions or mutations selected from the group consisting of sequences selected from the group consisting of SEQ ID NOs 3010-3023 or a sequence at least 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 3010-3023.

117. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the constant region of the TCR is a TCR receptor beta chain (C β) comprising an amino acid sequence having 1-40 amino acid substitutions or mutations selected from the group consisting of sequences selected from the group consisting of SEQ ID NOs: 3024 to 3044, or a sequence at least 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3024 to 3044.

118. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the constant region of the TCR is a TCR receptor gamma chain (cy) comprising an amino acid sequence having 1-40 amino acid substitutions or mutations selected from the group consisting of sequences selected from the group consisting of SEQ ID NOs 3049 to 3050, or a sequence at least 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 3049 to 3050.

119. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the constant region of the TCR is a TCR receptor delta chain (C δ) comprising an amino acid sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs 3051-3052, or 1-40 amino acid substitutions or mutations that are at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs 3051-3052.

120. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the constant region of the TCR is a preTCR receptor alpha chain (preC) comprising an amino acid sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs 3046 to 3048, or 1-40 amino acid substitutions or mutations that are at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs 3046 to 3048.

121. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen binding domains bind to one or more disease-associated antigens selected from the group consisting of: CD 19; CD 123; CD 22; CD 30; CD 171; CS-1 (also known as CD2 subunit 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD 33; epidermal growth factor receptor variant iii (egfrviii); ganglioside G2(GD 2); ganglioside GD3(aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (l-l) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. alpha. -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1(ROR 1); fms-like tyrosine kinase 3(FLT 3); tumor-associated glycoprotein 72(TAG 72); CD 38; CD44v 6; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epidermal cell adhesion molecule (EPCAM); B7H3(CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213a 2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate Stem Cell Antigen (PSCA); protease serine 21 (testis protein or PRSS 21); vascular endothelial growth factor receptor 2(VEGFR 2); a lewis (Y) antigen; CD 24; platelet derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD 20; a folate receptor alpha; receptor tyrosine protein kinase ERBB2(Her 2/neu); cell surface associated mucin 1(MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); prostasin; prostatic Acid Phosphatase (PAP); mutant elongation factor 2(ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase ix (caix); proteasome (precursor, megalin) subunit, beta form, 9(LMP 2); glycoprotein 100(gpl 00); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and the Abelson (Abelson) murine leukemia virus oncogene homolog 1 (Abl); a tyrosinase enzyme; ephrin type a receptor 2(EphA 2); fucosyl GM 1; sialylated lewis adhesion molecules (sLe); ganglioside GM3(aNeu5Ac (2-3) bDClalp (l-4) bDGlcp (l-1) Cer); transglutaminase 5(TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); folate receptor beta; tumor endothelial marker 1(TEM1/CD 248); tumor endothelial marker 7 associated (TEM 7R); sealin 6(CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); class 5 group member D of G protein-coupled receptors C (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); a globoH sugar ceramide hexasaccharide moiety (globoH); mammary differentiation antigen (NY-BR-1); urinary plaque 2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenoceptor β 3(ADRB 3); ubiquitin 3(PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex, locus K9(LY 6K); olfactory receptor 51E2(OR51E 2); TCR γ alternate reading frame protein (TARP); wilm's tumor protein (WT 1); cancer/testis antigen 1(NY-ES 0-1); cancer/testis antigen 2(LAGE-1 a); melanoma-associated antigen 1(MAGE-a 1); ETS translocation variant gene 6 located on chromosome 12p (ETV 6-AML); sperm protein 17(SPA 17); x antigen family member la (xagel); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; tumor protein p53(p 53); a p53 mutant; a prostate-specific protein; survivin; a telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; an inhibitor of melanoma apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyl-transferase V (NA 17); the paired box protein Pax-3(PAX 3); an androgen receptor; a cyclin Bl; a v-myc avian myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); tyrosinase-related protein 2 (TRP-2); cytochrome P450 lB1(CYPlB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site regulator siblings), squamous cell carcinoma antigen recognized by T cells 3(SART 3); the paired box protein Pax-5(PAX 5); the preproepisin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RUl); renal ubiquitin 2(RU 2); legumain; human papilloma virus E6(HPV E6); human papilloma virus E7(HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2(mut hsp 70-2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2(BST 2); EGF-like receptor-module mucin-like hormone-containing sample 2(EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); and immunoglobulin lambda-like polypeptide 1(IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFR α 54, CDH17, NYB 17, CDH17, CD200 17, Slea (CA 19.9; sialyl Lewis antigen) fucosyl-GM 17, PTK 17, gpNMB, CDH 17-CD 324, DLL 17, CD276/B7H 17, IL11 17, IL13Ra 17, CD179 17-IGLl 17, ALK TCR γ - δ, NKG 217, CD17 (FCGR2 17), Tn Ag, CSPG 17-HMW-MAA, Tim 17-/CR 17, CSF 217 (CSF-CSFR- α), TCR β R17, TGF 72/VEGFR, LeHR β -TCR β -CSF 1-HMW-MAA, HIV SLHR β -receptor, TCHR β -TCR 17, TCHR- β -TCR β -receptor, TCHR-17, TCHR- β -receptor, TCHR- β -receptor, and TNF-receptor, CMV pp65, EBV-EBNA3c, influenza A lectin (HA), GAD, PDL1, Guanylate Cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, desmoglein autoantibody 3(Dsg3), desmoglein autoantibody 1(Dsg1), HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1 (1), AFP, GPRC5D, sealer 18.2(CLD18A2 or N18A.2)), P-glycoprotein, STEAP1, LIV1, laminin-4, CRIPTO, GPA33, EBT 1/CD157, chloride channel antigen recognized by TNT antibodies of low conductivity.

122. The isolated Synthetic Immunoreceptor (SIR) polypeptide or polypeptide heterodimer of claim 121, wherein the one or more non-native TCR antigen-binding domains comprise an antibody, an antibody fragment, an scFv, an Fv, a Fab, (Fab')2, a Single Domain Antibody (SDAB), a vH or vL domain, a camelidae vHH domain, a non-immunoglobulin antigen-binding scaffold, such as DARPIN, an affibody, an affilin, an adnectin, an affitin, an obodies, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centryrin, a pronectin, an anticalin, a kunitz-type domain, an ardio repeat protein, a receptor, or a ligand.

123. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 121, wherein the one or more non-native TCR antigen-binding domains are selected from the group consisting of:

(i) a heavy chain variable region (vH) comprising the sequence set forth in any one of SEQ ID NOs 2506 to 2680 or 12160 to 12278 or a sequence having at least 98% identity thereto and encoding a polypeptide that binds to an antigen thereof;

(ii) a light chain variable region (vL) comprising the sequence set forth in any one of SEQ ID NOs 2307 to 2482 or 12042 to 12159 or a sequence having at least 98% identity thereto and encoding a polypeptide that binds to an antigen thereof;

(iii) a single chain variable fragment (scFv) comprising the sequence set forth in any one of SEQ ID NOs 2770 to 2939, 12303 to 12357, or 18162 to 18224 or a sequence having at least 98% identity thereto and encoding a polypeptide that binds to an antigen thereof;

(iv) a camelid VHH domain comprising a sequence set forth in any one of SEQ ID NOs 2701 to 2725 or 12279 to 12294 or a sequence having at least 98% identity thereto and encoding a polypeptide that binds to an antigen thereof;

(v) a non-immunoglobulin scaffold encoded by any one of SEQ ID NOs 439 to 443 or a polynucleotide having a sequence at least 98% identical thereto and encoding a polypeptide that binds to an antigen thereof;

(vi) a receptor comprising a sequence set forth in any one of SEQ ID NOs 2736 to 2748 or a sequence having at least 98% identity thereto and encoding a polypeptide that binds to a homologue thereof; and

(vii) a ligand comprising the sequence set forth in any one of SEQ ID NOs 2758 to 2768 or 12359 to 12361 or a sequence having at least 98% identity thereto and encoding a polypeptide that binds to a homologue thereof.

124. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen binding domains comprise one or more of the light chain complementarity determining regions of a selected target antigen represented by any one of SEQ ID Nos 13999 through 14879 or 14880 and/or one or more of the heavy chain complementarity determining regions of a selected target antigen represented by any one of SEQ ID Nos 14881 through 15761 or 15762.

125. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen-binding domains comprise a variable light chain (vL) domain comprising the sequence of any one of SEQ ID Nos 2307 to 2482 or 12042 to 12159 with up to 10 conservative amino acid substitutions and/or a variable heavy chain (vH) domain comprising the sequence of any one of SEQ ID Nos 2506 to 2680 or 12160 to 12278 with up to 10 conservative amino acid substitutions.

126. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen binding domains comprise one or more of the camelidae vHH complementarity determining regions of a selected antigen as set forth in any one of SEQ ID nos 2701-2725 or 12279-12294 and having up to 10 conservative amino acid substitutions.

127. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111 wherein the one or more non-native TCR antigen-binding domains comprise a non-immunoglobulin antigen-binding domain having the sequence set forth in any one of SEQ ID Nos 2728-2732 or 12296-12301 with up to 10 conservative amino acid substitutions.

128. The isolated Synthetic Immunoreceptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen-binding domains comprise an scFv domain comprising one or more light chain complementarity determining regions of a variable light chain (vL) domain comprising the sequence of any one of SEQ ID Nos 2307 to 2482 or 12042 to 12159 and one or more heavy chain complementarity determining regions of a variable heavy chain (vH) domain comprising the sequence of any one of SEQ ID Nos 2506 to 2680 or 12160 to 12278.

129. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen-binding domains comprise scFv fragments having a sequence selected from the group consisting of SEQ ID Nos 2770-2939, 12303-12357, or 18162-18224, each having up to 10 conservative amino acid substitutions.

130. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen-binding domains comprise one or more receptors comprising an amino acid sequence of any one of SEQ ID Nos. 2736 to 2748 with up to 10 conservative amino acid substitutions.

131. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111 wherein the one or more non-native TCR antigen-binding domains comprise one or more ligands comprising the sequence of any one of SEQ ID Nos 2758-2768 or 12359 through 12361 with up to 10 conservative amino acid substitutions.

132. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen-binding domains comprise the extracellular domain of CD16A, NKG2D, CD4, PD1, desmoglein 3(Dsg3), or CD 4-DC-SIGN.

133. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen-binding domains comprise the extracellular domain of one or more of hTPO, mTPO, CGH α chain, CGH β chain, FH β chain, LH β chain, TSH β chain, APRIL, or a combination thereof.

134. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen-binding domains comprise:

any single chain variable fragment (scFv) comprising the sequence of any one of SEQ ID Nos 2770 to 2939, 12303 to 12357, or 18162 to 18224 and having up to 10 conservative amino acid substitutions, and

a) any Camelidae vHH as set forth in any of SEQ ID Nos 2701 to 2725 or 12279 to 12294 and having up to 10 conservative amino acid substitutions, or

b) Any non-immunoglobulin antigen-binding domain having the sequence set forth in any one of SEQ ID NOs 2728-2732 or 12296-12301 and having up to 10 conservative amino acid substitutions; or

c) Any extracellular domain of a receptor comprising the amino acid sequence of any one of SEQ ID nos 2736 to 2748 and having up to 10 conservative amino acid substitutions; or

d) Any extracellular domain of a ligand comprising the sequence of any one of SEQ ID NO 2758-2768 or 12359 to 12361 and having up to 10 conservative amino acid substitutions.

135. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen-binding domains comprise:

camelidae vHH as set out in any of SEQ ID Nos 2701 to 2725 or 12279 to 12294 and having up to 10 conservative amino acid substitutions, and

a) any single chain variable fragment (scFv) comprising the sequence of any one of SEQ ID Nos 2770 to 2939, 12303 to 12357, or 18162 to 18224 and having up to 10 conservative amino acid substitutions, or

b) Any non-immunoglobulin antigen-binding domain having the sequence set forth in any one of SEQ ID NOs 2728-2732 or 12296-12301 and having up to 10 conservative amino acid substitutions; or

c) Any extracellular domain of a receptor comprising the amino acid sequence of any one of SEQ ID nos 2736 to 2748 and having up to 10 conservative amino acid substitutions; or

d) Any extracellular domain of a ligand comprising the sequence of any one of SEQ ID NO 2758-2768 or 12359 to 12361 and having up to 10 conservative amino acid substitutions.

136. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen binding domains are optionally linked to each of the TCR constant region chains by a linker region, wherein the linker region nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NOs 2981 to 3003, and any combinations thereof, or sequences at least 98% identical thereto; or the linker is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs 701 to 725 or a sequence having at least 98% identity thereto.

137. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the one or more non-native TCR antigen-binding domains have at least 5-fold less binding affinity for its target antigen than the antibody from which the binding domain was obtained.

138. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the polynucleotide encoding the SIR further comprises a leader sequence or signal peptide present at the N-terminus of each chain and comprising a sequence selected from the group consisting of SEQ ID NOs 1-9 and 10.

139. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the SIR comprises a SIR dimer.

140. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the polypeptide comprises two SIRs connected by a cleavable linker.

141. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 140, wherein the cleavable linker is a self-cleaving cleavable linker.

142. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 140, wherein the cleavable linker is any one or more of a2A linker, a 2A-like linker, or a functional equivalent thereof.

143. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 140, wherein the cleavable linker is any one or more of a T2A linker, a P2A, F2A, an E2A linker, or functional equivalents thereof.

144. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 140, wherein the cleavable linker comprises the sequence of any one or more of SEQ ID nos 780-785.

145. The isolated Synthetic Immunoreceptor (SIR) polypeptide or polypeptide heterodimer of claim 140, wherein the cleavable linker is optionally followed by a furin cleavage site or a furin-like cleavage site, or a functional equivalent thereof.

146. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 145, wherein the furin cleavage site preceding the cleavable linker comprises the sequence of any one or more of SEQ ID nos 788 to 790.

147. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of any one of claims 140-146, wherein the cleavable linker is followed by a flexible linker.

148. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 147, wherein the flexible linker preceding the cleavable linker encodes one or more of a Ser-Gly linker, a Ser-Gly-Ser-Gly linker, or a functional equivalent thereof.

149. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 147, wherein the flexible linker preceding the cleavable linker comprises the sequence of SEQ ID nos 786 to 787.

150. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 147, wherein the furin cleavage site is followed by the flexible linker, and the cleavable linker is followed by the flexible linker, such that the order furin cleavage site-flexible linker-cleavable linker is furin.

151. The isolated Synthetic Immune Receptor (SIR) polypeptide or polypeptide heterodimer of claim 111, wherein the SIR is designed to have a desired binding affinity for a selected antigen.

152. An immune effector cell or stem cell comprising at least one polypeptide or heterodimer of claim 111.

153. An immune effector cell or stem cell comprising at least one recombinant polynucleotide of claim 1.

154. An immune effector cell or stem cell comprising at least one vector of claim 104.

155. The immune cell or stem cell of any one of claims 152-154, wherein the cell comprises a plurality of SIR polypeptides.

156. The immune cell or stem cell of claim 155, wherein at least one SIR polypeptide in the plurality of SIR polypeptides targets an antigen that is different from at least one other SIR polypeptide.

157. The immune cell or stem cell of claim 155, wherein at least one SIR polypeptide in the plurality of SIR polypeptides targets the same antigen.

158. The immune cell or stem cell of claim 157, wherein at least one SIR polypeptide in the plurality of SIR polypeptides comprises a different binding affinity for an antigen than at least one other SIR polypeptide.

159. The immune cell or stem cell of any one of claims 152-154, wherein the immune cell further comprises at least one Chimeric Antigen Receptor (CAR) polypeptide.

160. The immune cell or stem cell of claim 159, wherein the antigen binding domain of the SIR polypeptide targets an antigen that is different from the antigen binding domain of the CAR polypeptide.

161. The immune cell or stem cell of claim 159, wherein the CAR polypeptide comprises an intracellular signaling domain comprising a costimulatory signaling domain, but not the primary signaling domain, or an intracellular signaling domain comprising the primary signaling domain, but not the costimulatory signaling domain.

162. The immune cell or stem cell of claim 161, wherein the CAR polypeptide comprises a costimulatory signaling domain comprising a functional signaling domain of a protein selected from the group consisting of 4-lBB, CD28, CD27, or OX-40, or the CAR molecule comprises a primary signaling domain comprising a functional signaling domain of CD3 ζ.

163. The immune cell or stem cell of claim 159, wherein the CAR polypeptide is an inhibitory CAR polypeptide, wherein the inhibitory CAR polypeptide comprises an antigen binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule, wherein the inhibitory molecule is selected from the group consisting of PDl, PD-Ll, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIRl, CD160, 2B4, TGFR β, CEACAM-1, CEACAM-3, and CEACAM-5.

164. The immune cell or stem cell of claim 159, wherein the CAR polypeptide further comprises an intracellular signaling domain comprising a primary signaling domain and/or an intracellular signaling domain, wherein the intracellular signaling domain comprises a primary signaling domain comprising a functional domain of CD3 ζ and a costimulatory signaling domain comprising a functional domain of 4-lBB or CD28, or both.

165. The immune cell or stem cell of claim 159, wherein the CAR polypeptide comprises the amino acid sequence of SEQ ID NO 3077 to SEQ ID NO 3083.

166. The immune cell or stem cell of any one of claims 152-154, wherein the immune effector cell is a human T cell, a human NKT cell or a synthetic T cell, or a stem cell that produces an immune effector cell, optionally wherein the T cell is diglycerol kinase (DGK) and/or incarville deficient and/or Brd4 deficient.

167. A population of immune or effector cells according to claim 152, wherein the population of cells comprises a plurality of different SIR polypeptides.

168. The immune cell or effector cell population of claim 167, wherein the plurality of different SIR polypeptides comprise different sequences but bind to the same target antigen.

169. The immune cell or effector cell population of claim 167, wherein the cell population comprises SIR polypeptides that target different antigens present in a particular disease type.

170. A method of making an immune effector cell that expresses an SIR, comprising introducing at least one vector of claim 104 or at least one recombinant polynucleotide of claim 1 into an immune effector cell or a hematopoietic stem or progenitor cell from which an immune effector cell can be produced under conditions such that the SIR polypeptide is expressed.

171. The method of claim 170, further comprising:

a) providing a population of immune effector cells; and is

b) Removing T regulatory cells from the population, thereby providing a population of T regulatory-depleted cells;

wherein steps a) and b) are performed prior to introducing the vector or recombinant polynucleotide encoding the SIR into the population.

172. The method of claim 171, wherein the T regulatory cells are removed from the population of cells using an anti-CD 25 antibody or an anti-GITR antibody.

173. The method of claim 170, further comprising:

a) providing a population of immune effector cells; and is

b) Enriching the population for P-glycoprotein (P-gp or Pgp; MDR1, ABCB1, CD243) positive cells, thereby providing P-glycoprotein (P-gp or Pgp; MDR1, ABCB1, CD243) enriched cell population;

wherein steps a) and b) are performed before or after the introduction of said vector or recombinant polynucleotide encoding said SIR.

174. The method of claim 173, wherein the P-glycoprotein-positive cells are enriched using any one or more of the methods selected from the group consisting of:

i) immunoselection using a P-glycoprotein specific antibody or a mixture thereof,

ii) staining with one or more of the fluorescent dyes tetramethylrhodamine methyl ester (TMRM), doxorubicin and actinomycin-D) as substrates for P-glycoprotein under conditions in which the P-glycoprotein acts as a pump and enriching for cells which are less stained with said dyes,

iii) selecting cells that are resistant to phototoxic compounds as substrates for P-glycoprotein, such as TH9402, 2- (4, 5-dibromo-6-amino-3-imino-3H-xanthen-9-yl) -benzoic acid methyl ester hydrochloride, 2- (4, 5-dibromo-6-amino-3-imino-3H-xanthen-9-yl) -benzoic acid ethyl ester hydrochloride, 2- (4, 5-dibromo-6-amino-3-imino-3H-xanthen-9-yl) -benzoic acid octyl ester hydrochloride Any one or more of n-butyl ester hydrochloride, 2- (6-ethylamino-3-ethylimino-3H-xanthen-9-yl) -benzoic acid n-butyl ester hydrochloride or derivative thereof, or combination thereof, and

iv) selecting cells that are resistant to cytotoxic compounds that are substrates for the P-glycoprotein, such as vincristine, vinblastine, taxol, paclitaxel, mitoxantrone, etoposide, doxorubicin, rubicin and actinomycin-D.

175. A method of generating an RNA-engineered cell population comprising introducing into a cell or cell population one or more RNAs transcribed in vitro or synthesized, wherein the one or more RNAs comprise one or more recombinant polynucleotides according to claim 1.

176. A method of providing immunity against disease in a subject, comprising administering to said subject an effective amount of an immune effector cell or immune effector cell-producing stem cell according to any one of claims 152-154, wherein said cell is an autologous T cell or an allogeneic T cell, or an autologous NKT cell or an allogeneic NKT cell, or an autologous or allogeneic hematopoietic stem cell or an autologous or allogeneic iPSC that produces an immune effector cell.

177. The method of claim 176, wherein said allogeneic T cells or allogeneic NKT cells or hematopoietic stem cells or ipscs lack expression of or have low expression of a functional TCR or functional HLA.

178. A composition comprising an immune effector cell or a stem cell that can generate an immune effector cell, the cell comprising one or more Synthetic Immune Receptor (SIR) molecules, for use in combination with an agent that increases the efficacy of the immune effector cell for use in treating a subject having a disease associated with expression of a disease-associated antigen or preventing a disease in a subject at increased risk of a disease associated with expression of a disease-associated antigen, wherein:

(i) the SIR molecule comprises one or more of a T cell receptor constant chain linked via an optional linker to one or more antigen binding domains that bind to the disease-associated antigen associated with the disease, and the disease-associated antigen is selected from the group consisting of: CD5, CD 19; CD 123; CD 22; CD 30; CD 171; CS-1 (also known as CD2 subunit 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD 33; epidermal growth factor receptor variant iii (egfrviii); ganglioside G2(GD 2); ganglioside GD3(aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (l-l) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. alpha. -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1(ROR 1); fms-like tyrosine kinase 3(FLT 3); tumor-associated glycoprotein 72(TAG 72); CD 38; CD44v 6; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epidermal cell adhesion molecule (EPCAM); B7H3(CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213a 2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate Stem Cell Antigen (PSCA); protease serine 21 (testis protein or PRSS 21); vascular endothelial growth factor receptor 2(VEGFR 2); a lewis (Y) antigen; CD 24; platelet derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD 20; a folate receptor alpha; receptor tyrosine protein kinase ERBB2(Her 2/neu); cell surface associated mucin 1(MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); prostasin; prostatic Acid Phosphatase (PAP); mutant elongation factor 2(ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase ix (caix); proteasome (precursor, megalin) subunit, beta form, 9(LMP 2); glycoprotein 100(gpl 00); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and the Abelson (Abelson) murine leukemia virus oncogene homolog 1 (Abl); a tyrosinase enzyme; ephrin type a receptor 2(EphA 2); fucosyl GM 1; sialylated lewis adhesion molecules (sLe); ganglioside GM3(aNeu5Ac (2-3) bDClalp (l-4) bDGlcp (l-1) Cer); transglutaminase 5(TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); tumor endothelial marker 1(TEM1/CD 248); tumor endothelial marker 7 associated (TEM 7R); sealin 6(CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); class 5 group member D of G protein-coupled receptors C (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); a globoH sugar ceramide hexasaccharide moiety (globoH); mammary differentiation antigen (NY-BR-1); urinary plaque 2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenoceptor β 3(ADRB 3); ubiquitin 3(PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex, locus K9(LY 6K); olfactory receptor 51E2(OR51E 2); TCR γ alternate reading frame protein (TARP); wilm's tumor protein (WT 1); cancer/testis antigen 1(NY-ES 0-1); cancer/testis antigen 2(LAGE-1 a); melanoma-associated antigen 1(MAGE-a 1); ETS translocation variant gene 6 located on chromosome 12p (ETV 6-AML); sperm protein 17(SPA 17); x antigen family member la (xagel); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; tumor protein p53(p 53); a p53 mutant; a prostate-specific protein; survivin; a telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; an inhibitor of melanoma apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyl-transferase V (NA 17); the paired box protein Pax-3(PAX 3); an androgen receptor; a cyclin Bl; a v-myc avian myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); tyrosinase-related protein 2 (TRP-2); cytochrome P450 lB1(CYPlB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site regulator siblings), squamous cell carcinoma antigen recognized by T cells 3(SART 3); the paired box protein Pax-5(PAX 5); the preproepisin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RUl); renal ubiquitin 2(RU 2); legumain; human papilloma virus E6(HPVE 6); human papilloma virus E7(HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2(mut hsp 70-2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2(BST 2); EGF-like receptor-module mucin-like hormone-containing sample 2(EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); and immunoglobulin lambda-like polypeptide 1(IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFR alpha 54, CDH17, NYB 17, CDH17, CD200 17, Slea (CA 19.9; sialyl Lewis antigen) fucosyl-GM 17, PTK 17, gpNMB, CDH 17-CD 324, DLL 17, CD276/B7H 17, IL11 17, IL13Ra 17, CD179 17-IGLl 17, ALK TCR gamma-delta, NKG 217, CD17 (FCGR2 17), CSPG 17-HMW-MAA, Tim 17-/HVCR 17, CSF 217 (GM-CSFR-alpha), TGF beta R17, TCR 72/TCR HR, Lews 1-Ag, LHR beta-loop receptor, HIV-SLCSF-17, FSHR receptor gamma receptor, FSHR receptor, TNF-receptor gamma-receptor, and TNF receptor, CMV pp65, EBV-EBNA3c, influenza A lectin (HA), GAD, PDL1, Guanylate Cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, desmoglin autoantibody 3(Dsg3), desmoglin autoantibody 1(Dsg1), HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1 (1), AFP, GPRC5D, sealer 18.2(CLD18A2 or N18A.2)), P-glycoprotein, STEAP1, LIV1, laminin-4, CRIPTO, GPA33, EBT 1/CD157, chloride ion channel recognition antigen by TNT antibody with low conductivity

(ii) The agent that increases the efficacy of the immune cell is selected from one or more of the following:

-a protein phosphatase inhibitor;

kinase inhibitors (e.g. PI3K/AKT inhibitors or mTOR inhibitors or LCK inhibitors or BTK inhibitors);

-a cytokine;

-an inhibitor of an immunosuppressive molecule;

-lowering T_REGAn agent of the level or activity of a cell;

-an agent that increases proliferation and/or persistence of SIR-modified cells;

-a chemokine;

-an agent that increases SIR expression;

-agents allowing the modulation of SIR expression or activity;

-agents that allow to control the survival and/or persistence of SIR-modified cells;

-agents that control side effects of SIR-modified cells;

-inhibitors of Brd 4;

-agents that deliver therapeutic (e.g. shvum) or prophylactic agents to the site of disease;

-an agent that increases the expression of a target antigen against which the SIR is directed; and

-adenosine A2a receptor antagonists.

179. A method of treating or preventing a disease associated with expression of a disease-associated antigen in a subject, comprising administering to the subject an effective amount of an immune effector cell comprising a Synthetic Immune Receptor (SIR) molecule in combination with an agent that increases the efficacy of the immune cell, wherein:

(i) the SIR molecule comprises one or more of a T cell receptor constant chain linked via an optional linker to one or more antigen binding domains that bind to the disease-associated antigen associated with the disease, and the disease-associated antigen is selected from the group consisting of: CD5, CD 19; CD 123; CD 22; CD 30; CD 171; CS-1 (also known as CD2 subunit 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD 33; epidermal growth factor receptor variant iii (egfrviii); ganglioside G2(GD 2); ganglioside GD3(aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (l-l) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. alpha. -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1(ROR 1); fms-like tyrosine kinase 3(FLT 3); tumor-associated glycoprotein 72(TAG 72); CD 38; CD44v 6; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epidermal cell adhesion molecule (EPCAM); B7H3(CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213a 2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate Stem Cell Antigen (PSCA); protease serine 21 (testis protein or PRSS 21); vascular endothelial growth factor receptor 2(VEGFR 2); a lewis (Y) antigen; CD 24; platelet derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD 20; a folate receptor alpha; receptor tyrosine protein kinase ERBB2(Her 2/neu); cell surface associated mucin 1(MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); prostasin; prostatic Acid Phosphatase (PAP); mutant elongation factor 2(ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase ix (caix); proteasome (precursor, megalin) subunit, beta form, 9(LMP 2); glycoprotein 100(gpl 00); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and the Abelson (Abelson) murine leukemia virus oncogene homolog 1 (Abl); a tyrosinase enzyme; ephrin type a receptor 2(EphA 2); fucosyl GM 1; sialylated lewis adhesion molecules (sLe); ganglioside GM3(aNeu5Ac (2-3) bDClalp (l-4) bDGlcp (l-1) Cer); transglutaminase 5(TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); tumor endothelial marker 1(TEM1/CD 248); tumor endothelial marker 7 associated (TEM 7R); sealin 6(CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); class 5 group member D of G protein-coupled receptors C (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); a globoH sugar ceramide hexasaccharide moiety (globoH); mammary differentiation antigen (NY-BR-1); urinary plaque 2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenoceptor β 3(ADRB 3); ubiquitin 3(PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex, locus K9(LY 6K); olfactory receptor 51E2(OR51E 2); TCR γ alternate reading frame protein (TARP); wilm's tumor protein (WT 1); cancer/testis antigen 1(NY-ES 0-1); cancer/testis antigen 2(LAGE-1 a); melanoma-associated antigen 1(MAGE-a 1); ETS translocation variant gene 6 located on chromosome 12p (ETV 6-AML); sperm protein 17(SPA 17); x antigen family member la (xagel); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; tumor protein p53(p 53); a p53 mutant; a prostate-specific protein; survivin; a telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; an inhibitor of melanoma apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyl-transferase V (NA 17); the paired box protein Pax-3(PAX 3); an androgen receptor; a cyclin Bl; a v-myc avian myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); tyrosinase-related protein 2 (TRP-2); cytochrome P450 lB1(CYPlB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site regulator siblings), squamous cell carcinoma antigen recognized by T cells 3(SART 3); the paired box protein Pax-5(PAX 5); the preproepisin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RUl); renal ubiquitin 2(RU 2); legumain; human papilloma virus E6(HPVE 6); human papilloma virus E7(HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2(mut hsp 70-2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2(BST 2); EGF-like receptor-module mucin-like hormone-containing sample 2(EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); and immunoglobulin lambda-like polypeptide 1(IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFR alpha 54, CDH17, NYB 17, CDH17, CD200 17, Slea (CA 19.9; sialyl Lewis antigen) fucosyl-GM 17, PTK 17, gpNMB, CDH 17-CD 324, DLL 17, CD276/B7H 17, IL11 17, IL13Ra 17, CD179 17-IGLl 17, ALK TCR gamma-delta, NKG 217, CD17 (FCGR2 17), CSPG 17-HMW-MAA, Tim 17-/HVCR 17, CSF 217 (GM-CSFR-alpha), TGF beta R17, TCR 72/TCR HR, Lews 1-Ag, LHR beta-loop receptor, HIV-SLCSF-17, FSHR receptor gamma receptor, FSHR receptor, TNF-receptor gamma-receptor, and TNF receptor, CMV pp65, EBV-EBNA3c, influenza A lectin (HA), GAD, PDL1, Guanylate Cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, desmoglin autoantibody 3(Dsg3), desmoglin autoantibody 1(Dsg1), HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1 (1), AFP, GPRC5D, sealer 18.2(CLD18A2 or N18A.2)), P-glycoprotein, STEAP1, LIV1, laminin-4, CRIPTO, GPA33, EBT 1/CD157, chloride ion channel recognition antigen by TNT antibody with low conductivity

(ii) The agent that increases the efficacy of the immune cell is selected from one or more of the following:

-a protein phosphatase inhibitor;

kinase inhibitors (e.g. PI3K/AKT inhibitors or mTOR inhibitors or LCK inhibitors or BTK inhibitors);

-a cytokine;

-an inhibitor of an immunosuppressive molecule;

-lowering T_REGAn agent of the level or activity of a cell;

-an agent that increases proliferation and/or persistence of SIR-modified cells;

-a chemokine;

-an agent that increases SIR expression;

-agents allowing the modulation of SIR expression or activity;

-agents that allow to control the survival and/or persistence of SIR-modified cells;

-agents that control side effects of SIR-modified cells;

-inhibitors of Brd 4;

-agents that deliver therapeutic (e.g. shvum) or prophylactic agents to the site of disease;

-an agent that increases the expression of a target antigen against which the SIR is directed; and

-an adenosine A2a receptor antagonist,

thereby treating the subject or preventing a disease in the subject.

180. A method of treating or preventing a disease associated with expression of a disease-associated antigen in a subject, comprising administering to the subject an effective amount of an immune effector cell comprising a Synthetic Immune Receptor (SIR) molecule, wherein:

(i) the SIR molecule comprises one or more of a T cell receptor constant chain linked via an optional linker to one or more antigen binding domains that bind to the disease-associated antigen associated with the disease, and the disease-associated antigen is selected from the group consisting of: CD5, CD 19; CD 123; CD 22; CD 30; CD 171; CS-1 (also known as CD2 subunit 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD 33; epidermal growth factor receptor variant iii (egfrviii); ganglioside G2(GD 2); ganglioside GD3(aNeu5Ac (2-8) aNeu5Ac (2-3) bDGalp (l-4) bDGlcp (l-l) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAc. alpha. -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1(ROR 1); fms-like tyrosine kinase 3(FLT 3); tumor-associated glycoprotein 72(TAG 72); CD 38; CD44v 6; a glycosylated CD43 epitope expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, carcinoembryonic antigen (CEA); epidermal cell adhesion molecule (EPCAM); B7H3(CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213a 2); mesothelin; interleukin 11 receptor alpha (IL-llRa); prostate Stem Cell Antigen (PSCA); protease serine 21 (testis protein or PRSS 21); vascular endothelial growth factor receptor 2(VEGFR 2); a lewis (Y) antigen; CD 24; platelet derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD 20; folate receptor alpha (FRa or FR 1); folate receptor beta (FRb); receptor tyrosine protein kinase ERBB2(Her 2/neu); cell surface associated mucin 1(MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecule (NCAM); prostasin; prostatic Acid Phosphatase (PAP); mutant elongation factor 2(ELF 2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase ix (caix); proteasome (precursor, megalin) subunit, beta form, 9(LMP 2); glycoprotein 100(gpl 00); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and the Abelson (Abelson) murine leukemia virus oncogene homolog 1 (Abl); a tyrosinase enzyme; ephrin type a receptor 2(EphA 2); fucosyl GM 1; sialylated lewis adhesion molecules (sLe); ganglioside GM3(aNeu5Ac (2-3) bDClalp (l-4) bDGlcp (l-1) Cer); transglutaminase 5(TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); tumor endothelial marker 1(TEM1/CD 248); tumor endothelial marker 7 associated (TEM 7R); sealin 6(CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); class 5 group member D of G protein-coupled receptors C (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); a globoH sugar ceramide hexasaccharide moiety (globoH); mammary differentiation antigen (NY-BR-1); urinary plaque 2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenoceptor β 3(ADRB 3); ubiquitin 3(PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex, locus K9(LY 6K); olfactory receptor 51E2(OR51E 2); TCR γ alternate reading frame protein (TARP); wilm's tumor protein (WT 1); cancer/testis antigen 1(NY-ES 0-1); cancer/testis antigen 2(LAGE-1 a); melanoma-associated antigen 1(MAGE-a 1); ETS translocation variant gene 6 located on chromosome 12p (ETV 6-AML); sperm protein 17(SPA 17); x antigen family member la (xagel); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; tumor protein p53(p 53); a p53 mutant; a prostate-specific protein; survivin; a telomerase; prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; an inhibitor of melanoma apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyl-transferase V (NA 17); the paired box protein Pax-3(PAX 3); an androgen receptor; a cyclin Bl; a v-myc avian myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); tyrosinase-related protein 2 (TRP-2); cytochrome P450 lB1(CYPlB 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site regulator siblings), squamous cell carcinoma antigen recognized by T cells 3(SART 3); the paired box protein Pax-5(PAX 5); the preproepisin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX 2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RUl); renal ubiquitin 2(RU 2); legumain; human papilloma virus E6(HPV E6); human papilloma virus E7(HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2(mut hsp 70-2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2(BST 2); EGF-like receptor-module mucin-like hormone-containing sample 2(EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); and immunoglobulin lambda-like polypeptide 1(IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFR α 54, CDH17, CDH6, NYB 1, CDH19, CD200R, Slea (CA 19.9; sialyl Lewis antigen); fucosyl-GM 1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276/B7H3, IL11Ra, IL13Ra2, CD179B-IGLl1, ALK TCR γ - δ, NKG2D, CD32(FCGR2A), CSPG4-HMW-MAA, Tim 4-/HVCR 4, CSF 24 (GM-CSFR- α), TGF β R4, VEGFR 4/KDR, Lews Ag, TCR β 1 chain, TCR β 2 chain, TCR- γ chain, TCR- δ chain, FITC, Luteinizing Hormone Receptor (LHR), follicle stimulating hormone receptor (LHFSHR), chorionic gonadotropin hormone receptor (CGCCR 4), HR 4, GD 4, SLAMF4, guanosine glycoprotein AMSLF 4, HIV 4 envelope 4, HTLV-72, CMV-72, CMV-MAH 3, influenza HA, HLA-derived protein (SLF 4), HLA-cDNA 4, IgG 4, HLA-cDNA-binding protein (D), HLA-cDNA-binding protein (cDNA) and DNA receptor (cDNA-binding protein), HLA-binding protein (cDNA) of the like), and DNA of the like, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, tissue factor 1(TF1), AFP, GPRC5D, Encapsulated protein 18.2(CLD18A2 or CLDN18A.2)), P-glycoprotein, STEAP1, LIV1, fibronectin-4, CRIPTO, GPA33, BST1/CD157, low conductivity chloride channel, and antigen recognized by TNT antibody; and is

(ii) The binding domain of the SIR molecule has at least 5-fold less binding affinity than the antibody from which the antigen binding domain is derived.

181. The use or method of any one of claims 178-180, wherein the disease associated with expression of the disease-associated antigen is selected from the group consisting of a proliferative disease, a precancerous condition, cancer, and a non-cancer related indication associated with expression of the disease-associated antigen.

182. The use or method of claim 181, wherein the cancer is selected from Chronic Lymphocytic Leukemia (CLL), acute leukemia, Acute Lymphocytic Leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), Chronic Myelogenous Leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, burkitt lymphoma, diffuse large B-cell lymphoma, primary effusion lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-hodgkin lymphoma, lymphomatosis, and myelodysplasia syndrome, Hodgkin's lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor, waldenstrom's macroglobulinemia, or hematological cancers of one or more of the pre-stages of leukemia.

183. The use or method of claim 181, wherein the cancer is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, non-hodgkin's lymphoma, cancer of the endocrine system, carcinoma of the thyroid gland, carcinoma of the parathyroid gland, carcinoma of the adrenal gland, sarcoma of soft tissue, carcinoma of the urethra, carcinoma of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the Central Nervous System (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, merkel cell carcinoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of said cancers and metastatic lesions of said cancers.

184. The use or method of claim 183, wherein the disease is associated with infection by viruses including, but not limited to, HIV1, HIV2, HTLV1, epstein-barr virus (EBV), Cytomegalovirus (CMV), adenovirus, adeno-associated virus, BK virus (EBV), human herpesvirus 6, human herpesvirus 8 influenza virus, parainfluenza virus, avian influenza virus, MERS and SARS coronavirus, crimean congo hemorrhagic fever virus, rhinovirus, enterovirus, dengue virus, west nile virus, ebola virus, marburg virus, lassa fever virus, seca virus, RSV, measles virus, mumps virus, rhinovirus, varicella virus, herpes simplex virus 1 and 2, varicella-zoster virus, HIV-1, HTLV1, hepatitis virus, enterovirus, hepatitis b virus, hepatitis c virus, herpes simplex virus, rhinovirus, herpes simplex virus 1 and 2, varicella-zoster virus, HIV-1, HTLV1, hepatitis virus, enterovirus, entero, Nipah virus and rift valley fever virus, japanese encephalitis virus, merkel cell polyoma virus, or associated with infection by mycobacterium tuberculosis, atypical mycobacterium species, yersinia pneumocystis, toxoplasmosis, rickettsia, nocardia, aspergillus, mucor or candida.

185. The use or method of claim 183, wherein the disease is an immune or degenerative disease including, but not limited to, diabetes, multiple sclerosis, rheumatoid arthritis, pemphigus vulgaris, ankylosing spondylitis, hypothyroid thyroiditis, SLE, sarcoidosis, scleroderma, mixed connective tissue disease, graft-versus-host disease, or alzheimer's disease.

186. The use or method of any one of claims 178-180 or 182-184, wherein:

(i) the protein phosphatase inhibitor is a SHP-1 inhibitor and/or a SHP-2 inhibitor;

(ii) the kinase inhibitor is selected from one or more of a CDK4 inhibitor, a CDK4/6 inhibitor, an mTOR inhibitor, a MNK inhibitor, or a dual P13K/mTOR inhibitor;

(iii) the agent that inhibits the immunosuppressive molecule comprises an antibody or antibody fragment, an inhibitory nucleic acid, an aggregated regularly interspaced short palindromic repeats (CRISPR), a transcription activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN) that inhibits expression of an inhibitory molecule;

(iv) the agent that reduces the level or activity of the T REG cells is selected from cyclophosphamide, anti-GITR antibodies, CD25 depletion, or a combination thereof; and/or

(v) The Brd4 inhibitor is selected from JQ1, MS417, OTXO15, LY 303511 and Brd4 inhibitors or derivatives thereof as described in US 20140256706 a 1.

187. The use or method of any one of claims 178-180 or 182-184, wherein the immunosuppressive molecule is selected from the group consisting of PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGF β, CEACAM-1, CEACAM-3 and CEACAM-5.

188. The use or method of any one of claims 178-180 or 182-184, wherein the agent that inhibits the inhibitory molecule comprises a first polypeptide comprising an inhibitory molecule or fragment thereof and a second polypeptide that provides a positive signal to the cell, and wherein the first and second polypeptides are expressed on immune cells comprising SIRs, wherein (i) the first polypeptide comprises PD1, PD-L1, CTLA-4, TIM-3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGF β, CEACAM-1, CEACAM-3, and CEACAM-5, or fragments thereof; and/or (ii) the second polypeptide comprises an intracellular signalling domain comprising a primary signalling domain and/or a co-stimulatory signalling domain.

189. The use or method of claim 188, wherein the primary signaling domain comprises a functional domain of CD3 ζ and/or the co-stimulatory signaling domain comprises a functional domain of a protein selected from 41BB, CD27, and CD 28.

190. The use or method of any one of claims 178-180 or 182-184, wherein the cytokine is selected from IL-15 or IL-21 or both.

191. The use or method of any one of claims 178-180 or 182-184, wherein the immune effector cell comprising the one or more SIR molecules and the agent that increases the efficacy of the immune effector cell are administered substantially simultaneously or sequentially.

192. The use or method of claim 191, wherein the immune cell comprising the SIR molecule is administered in combination with a molecule that targets GITR and/or modulates GITR function.

193. The use or method of claim 192, wherein the molecule that targets GITR and/or modulates GITR function is administered prior to a cell or population of cells expressing SIR or prior to apheresis.

194. The use or method of any one of claims 178-180 or 182-184, wherein the subject is a human.

195. A composition comprising at least one polynucleotide according to claim 1, an SIR polypeptide molecule according to claim 111, a vector according to claim 104 or a cell according to any one of claims 152-153 and a pharmaceutically acceptable excipient.

196. A kit comprising at least one polynucleotide according to claim 1, an SIR polypeptide molecule according to claim 111, a vector according to claim 104 or a cell according to any one of claims 152-153 and/or a composition according to claim 144.

197. A polynucleotide encoding a synthetic immunoreceptor comprising a sequence selected from the group consisting of SEQ ID NOS 900 to 2264, 4531 to 6013, 7519 to 8160, 8803 to 9230, 9659 to 9856, 10474 to 12041, 15786 to 16011, 16240 to 16465, 16694 to 16926, 17162 to 17394, 17864 to 17979, 18321 to 18322, 18242 to 18259, 18280 to 18588, 18899, 18915 to 18916, and 19248 to 19246, or 900 to 2264, 454531 to 96531, 7560119 to 8160130, 7548 to 8130, 7560130, 759 to 8130, 10474 to 12041, 15786 to 16011, 16240 to 16465, 16694 to 16926, 17162 to 17394, 17864 to 17979, 18321 to 18322, 18242 to 18259, 18280 to 18588, 18899 and 18915 to 18916 and 19248 to 19246, respectively.

198. An amino acid sequence encoding a synthetic immunoreceptor polypeptide selected from the group consisting of SEQ ID NOS 3135 to 4498, 6044 to 7518, 8161 to 8802, 9231 to 9658, 9873 to 10070, 12431 to 13998, 16013 to 16238, 16467 to 16692, 16928 to 17160, 17396 to 17628, 17981 to 18096, 18239 to 18240, 18261 to 18278, 18590 to 18898, 8939 18900 and 8918919 to 18920, and 48 to 19246, or 3135 to 608, 31344 to 6018, 44961 to 4492, 8131 to 8131, 13959 9873, 598808, and 1395988098, The sequence encoding the amino acid sequence of the synthetic immunoreceptor polypeptide shown in any one of SEQ ID NOS 16013 to 16238, SEQ ID NOS 16467 to 16692, SEQ ID NOS 16928 to 17160, SEQ ID NOS 17396 to 17628, SEQ ID NOS 17981 to 18096, SEQ ID NOS 18239 to 18240, SEQ ID NOS 18261 to 18278, SEQ ID NOS 18590 to 18898, SEQ ID NOS 18900 and SEQ ID NOS 18919 to 18920, and SEQ ID NOS 19248 to 19246 has at least 75% identity.