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WO2020018964A1 - Compositions et procédés pour réguler l'expression de récepteurs spécifiques à l'antigène - Google Patents

Compositions et procédés pour réguler l'expression de récepteurs spécifiques à l'antigène Download PDF

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WO2020018964A1
WO2020018964A1 PCT/US2019/042696 US2019042696W WO2020018964A1 WO 2020018964 A1 WO2020018964 A1 WO 2020018964A1 US 2019042696 W US2019042696 W US 2019042696W WO 2020018964 A1 WO2020018964 A1 WO 2020018964A1
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cells
antigen
cell
ror1
amino acid
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Stanley R. Riddell
Shivani Srivastava
Alexander SALTER
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Fred Hutchinson Cancer Center
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Fred Hutchinson Cancer Center
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/28—Expressing multiple CARs, TCRs or antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • Figures 1A-1S relate to experiments in which T cells expressing an anti-RORl CAR with a Rl 1 -derived scFv induced lethal toxicity in mice pre-conditioned with radiation.
  • A Schematic illustrations of retroviral constructs used to generate ROR1 CAR and control T cells.
  • TM transmembrane.
  • hIgG4 human immunoglobulin G4.
  • scFv single chain variable fragment.
  • B Percent change in body weight in BALB/c mice treated as indicated.
  • C Representative flow cytometry plots showing frequency of CD8 + CD45.l + donor T cells of live cells and frequency of CD8 + tCDl9 + transduced cells of CD45.l + donor T cells 7 days post-transfer.
  • Figures 2A-2F show that the observed ROR1 CAR-T cell-mediated toxicity is dependent on the degree of lymphodepletion and dose of ROR1 CAR-T cells.
  • A, B Percent change in body weight over time in BALB/c mice left untreated
  • LD lymphodepleted
  • LD and given lxlO 6 CD8 + control T cells or LD and given lxlO 6 CD8 + ROR1 CAR-T cells.
  • LD given was either 100R radiation, 500R radiation, lOOmg/kg cyclophosphamide (Cy), or 200mg/kg Cy.
  • Cy cyclophosphamide
  • N 4 mice per group.
  • C Frequency of
  • N 4 mice per group.
  • D-F Percent change in body weight (D), frequency of
  • CD8 + CD45 CD8 + CD45.
  • N 4 mice per group.
  • FIGS 3A-3I show that lymphopenia is sufficient to induce minor
  • Figures 4A-4I show that ROR1 CAR-T cell-mediated toxicity following lymphodepletion is dependent on ROR1 expression in non-hematopoietic cells.
  • A Percent change in body weight (two-way ANOVA with Tukey post-test (WT + CAR-T vs. KO + CAR-T: D7,8,9, P ⁇ 0.00001).
  • E RBC count in peripheral blood (one-way ANOVA with Tukey post test).
  • C left
  • MSC mesenchymal stem cell
  • pre-B cell C, right
  • granulocyte/macrophage D, left
  • erythrocyte D, middle
  • megakaryocyte D, right
  • Figures 6A-6G show that trigger EpCAM-inducible ROR1 CAR-T cells of the present disclosure selectively target EpCAM + RORl + cells but not EpCAM + RORL or EpCAM RORU cells in vitro.
  • A Representative flow cytometric analysis of EpCAM expression on 4T1 tumor cells, MSC, osteoblasts (OBL), CD45 Terl l9 PDGFRp + spleen cells, and CD45 Terl l9 VE-cadherin + spleen cells from WT BALB/c mice.
  • Figures 7A-7J show that EpCAM-trigger-inducible ROR1 CAR-T cells rescue toxicity to RORl + normal tissues while maintaining activity against RORl + tumors.
  • (A) Flow cytometric analysis showing EpCAM expression and MFI of sorted 4T1- EpCAM low and 4T1 -EpCAM 111 target cells.
  • (B) Change in frequency of tCDl9 CAR marker expression on CD8 + myc + BFP + synNotch EpCAM-inducible ROR1 CAR-T cells at various time points after co-culture with 4Tl-EpCAM low or 4T 1 -EpCAM 111 target cells.
  • (C) Percent specific lysis of the indicated 51 Cr-labeled tumor cells after 24 hours of co-culture with CD8 + untransduced (black) or EpCAM-inducible ROR1 CAR- T cells (open circles).
  • mice B ALB/c mice treated as indicated.
  • ROR1 CAR P0.0003; UT vs. EpCAM-inducible ROR1 CAR, P .0016; ROR1 CAR vs. EpCAM-trigger-inducible ROR1 CAR, PO.OOOl).
  • I Representative flow cytometric analysis of ROR1 CAR and PD-l expression on CD8 + CD45. l + donor T cells from mice treated with Cy and the indicated T cell populations 10 days post-transfer.
  • J Summary of frequency of ROR1 CAR + , CAR + Ki67 + , and CAR + PD-l + T cells of CD8 + CD45. l + donor T cells from mice treated with 200mg/kg Cy and the indicated T cell groups 10 days post-transfer.
  • N 6-l2 mice per group.
  • Unpaired two-way Student’s t-test (Frequency: spleen, PO.OOOl; BM, PO.OOOl; PD-l : spleen, P0.0004; BM, P0.0003; Ki67: spleen, PO.OOOl; BM, PO.OOOl).
  • Data are summarized from 2 independent experiments. All data are presented as the mean values ⁇ SEM.
  • Figures 8A-8H show that primary human B7-H3-trigger-inducible ROR1 CAR- T cells rescue toxicity to RORl + normal tissues while maintaining activity against human RORl + tumors.
  • A Schematic illustration of an exemplary lentiviral construct encoding a constitutively expressed Rl 1 ROR1 CAR. Expression of the CAR is detected by tEGFR expression.
  • FIG. 1 Schematic illustration of lentiviral constructs encoding CDl9-Gal4VP64 trigger polypeptide (top), UAS inducible Rl 1 ROR1 CAR (middle), and constitutive Rl 1 ROR1 CAR (bottom).
  • Expression of the CD 19 trigger polypeptide is detected by myc expression; transduction of the UAS inducible ROR1 CAR construct is detected by BFP expression; and induction of CAR expression is detected by tEGFR expression.
  • FIG. 1 Schematic illustration of lentiviral constructs encoding CDl9-Gal4VP64 trigger polypeptide (top), UAS inducible Rl 1 ROR1 CAR (middle), and constitutive Rl 1 ROR1 CAR (bottom).
  • Expression of the CD 19 trigger polypeptide is detected by myc expression; transduction of the UAS inducible ROR1 CAR construct is detected by BFP expression; and induction of CAR expression is detected by tEGFR expression.
  • FIG. 1 Schematic illustration of lentivi
  • FIG. 1 Schematic illustration of lentiviral constructs encoding B7-H3-Gal4VP64 trigger polypeptide (top), UAS inducible Rl 1 ROR1 CAR (middle), and constitutive Rl 1 ROR1 CAR (bottom). Expression of the B7-H3 trigger polypeptide is detected by myc expression; transduction of the UAS inducible ROR1 CAR construct is detected by BFP expression; and induction of CAR expression is detected by tEGFR expression.
  • C Intracellular cytokine analysis of tEGFR CAR marker and IFNy expression in the indicated primary human CD8 + T cell populations co-cultured with the indicated tumor cells in the presence of Brefeldin A for the last 6 hours of culture.
  • (F) Percent change in body weight in NSG mice implanted with MDA-MB-231 tumors, irradiated 250R and treated with untransduced (UT) T cells, ROR1 CAR-T cells, or B7-H3-trigger-inducible ROR1 CAR-T cells. N 4 mice per group. Two-way ANOVA with Tukey post-test (ROR1- CAR vs.
  • B7-H3 -trigger-inducible ROR1 CAR D13, D14 D19, PO.OOOl).
  • tumor, n.s. PD-l spleen, P ⁇ 0.000l; BM, P ⁇ 0.000l; tumor, n.s.). *, P ⁇ 0.05; **, P ⁇ 0.005; ***, P ⁇ 0.0005; ****, P ⁇ 0.000l .
  • Data are representative of 2 independent experiments. All data are presented as the mean values ⁇ SEM.
  • Figure 10 shows illustrates that a "split CAR approach" in which a ROR1- specific CAR lacking a costimulatory domain did not rescue CAR T cell-mediated toxicity to RORl + normal tissue.
  • Panel A depicts the percent change in body weight over time in BALB/c mice left untreated (No Tx), or irradiated 500R and receiving lxlO 6 CD8 + control T cells, Rl 1- CD3z first-generation ROR1 CAR-T cells, or Rl l-4lBB-CD3z second-generation ROR1 CAR-T cells.
  • N 3 mice per group.
  • Panel B shows red blood cell (RBC) and platelet (PLT) counts in peripheral blood in BALB/c mice irradiated 500R and receiving lxlO 6 CD8 + control T cells, Rl l-CD3z first-generation ROR1 CAR-T cells, or Rl l-4lBB-CD3z second-generation ROR1 CAR-T cells.
  • RBC red blood cell
  • PHT platelet
  • the present disclosure provides trigger polypeptides that can be expressed by a host cell and can bind to an antigen (e.g ., an antigen expressed on a surface of a neighboring cell; a soluble antigen). Binding by the trigger polypeptide to the antigen can release or produce a translational factor that modulates (e.g., drives, activates, increases, decreases, suppresses, or prevents) expression of a gene product of interest by the host cell.
  • the gene product of interest is a ROR1 -specific binding protein (e.g, a CAR or a TCR).
  • human ROR1 (SEQ ID NO: l) is an oncofetal antigen that is overexpressed in a wide variety of tumors.
  • ROR1 is highly expressed in certain solid cancers, including epithelial cancers, and in B-cell chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • ROR1 has minimal expression in most normal tissues. Accordingly, ROR1 is an attractive antigen to target with therapeutics.
  • T cells expressing a chimeric antigen receptor (CAR) have been designed to target ROR1 -expressing tumors (see Hudecek el al, Blood 116: 4532-41, 2010; Hudecek et al, Clin. Cancer Res. 19(12)3153 (2013); Berger el al, Cancer Immunol. Res. 3(2):206 (2015), and PCT Publication No. WO 2014/031687).
  • the trigger polypeptide binds to a non-RORl antigen, such as, for example, a B7-H3 antigen or an EpCAM antigen.
  • a non-RORl antigen such as, for example, a B7-H3 antigen or an EpCAM antigen.
  • the present disclosure shows that expression of a ROR1 -specific binding protein (such as a CAR) by a modified immune cell can be advantageously controlled or directed using a trigger polypeptide that is specific for a non-RORl antigen that is selectively or primarily expressed on RORl + diseased cells.
  • trigger polypeptides targeting B7-H3 or EpCAM surprisingly improved selective expression of a ROR1- specific binding protein to target diseased cells, but not healthy RORl + cells.
  • controlled expression of a ROR1 -specific binding protein by a host immune cell can prevent, reduce, or minimize off-target cytotoxic effects against RORl + non-hematopoietic cells; e.g. , following cytotoxic therapy to treat a RORl + cancer and/or to allow or improve engraftment of transferred cells.
  • host cells that comprise: (i) a polynucleotide that encodes a trigger polypeptide; and (ii) a polynucleotide that encodes a binding protein that specifically binds to a ROR1 antigen.
  • the host cell is a modified immune cell, such as, for example, a modified T cell, NK cell, or NK-T cell.
  • compositions comprising the host cells and methods of using the same to, for example, treat a disease or condition associated with expression of (i) ROR1 and (ii) the antigen bound by the trigger polypeptide (e.g, a non-RORl antigen such as a B7-H3 antigen or an EpCAM antigen) are also provided.
  • a method comprises administering a host immune cell to a subject, wherein the subject has previously been administered an agent (such as an immune cell or an antibody) that targets ROR1, and/or has been administered lymphodepleting chemotherapy.
  • the subject receiving the agent that targets ROR1 and/or the lymphodepleting chemotherapy prior to receiving the host immune cell, the subject receiving the agent that targets ROR1 and/or the lymphodepleting chemotherapy exhibits one or more adverse effects, such as, for example: (i) weight loss; (ii) accumulation of the agent that targets ROR1 in spleen, bone marrow, a peripheral lymph node, and/or liver; (iii) a reduced number of red blood cells (RBCs) and/or platelets; (iv) necrosis of spleen; (v) reduced hematopoiesis; (vi) a reduced number of erythroid cells and/or
  • RBCs red blood cells
  • hematopoiesis a reduced number of erythroid cells and/or
  • compositions and methods are useful for treating diseases and conditions that are characterized by expression of ROR1 and the antigen bound by a trigger polypeptide, such as, for example, various cancers.
  • the disclosed embodiments can modulate the specificity of adoptive cell therapies intended to target ROR1 expressed by diseased cells, but not, for example, healthy cells.
  • polynucleotides that encode disclosed trigger polypeptides proteins, as well as expression vectors that comprise the polynucleotides, and kits that comprise the polynucleotides proteins or host cells.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness is to be understood to include any integer within the recited range, unless otherwise indicated.
  • the term “about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms “a” and “an” as used herein refer to “one or more" of the enumerated components.
  • a protein domain, region, or module e.g ., a binding domain, hinge region, or linker
  • a protein which may have one or more domains, regions, or modules
  • substantially affect i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein (e.g, the target binding affinity of a binding protein).
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g, hydroxyproline, g-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g ., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
  • mutation refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively.
  • a mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).
  • a “conservative substitution” refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1 : Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3 : Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr
  • amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g, acidic, basic, aliphatic, aromatic, or sulfur-containing).
  • an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile.
  • Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins. W.H. Freeman and Company.
  • protein or “polypeptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid and non-naturally occurring amino acid polymers. Variants of proteins, peptides, and polypeptides of this disclosure are also contemplated.
  • variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein.
  • fusion protein refers to a protein that, in a single chain, has at least two distinct domains, wherein the domains are not naturally found together as a single chain protein.
  • a polynucleotide encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be synthesized.
  • a fusion protein may further contain other components, such as a tag, a linker, or a transduction marker.
  • a fusion protein expressed or produced by a host cell locates to the cell surface, where the fusion protein is anchored to the cell membrane (e.g., via a transmembrane component or domain) and comprises an extracellular component (e.g, capable of associating with a MHC molecule) and an intracellular component (e.g, containing a signaling domain, effector domain, co-stimulatory domain or portions or combinations thereof).
  • a host cell e.g a T cell
  • Nucleic acid molecule refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g ., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring).
  • Purine bases include adenine, guanine, hypoxanthine, and xanthine
  • pyrimidine bases include uracil, thymine, and cytosine.
  • Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double-stranded.
  • the nucleic acid molecule may be the coding strand or non-coding (anti-sense strand).
  • a nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.
  • Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and are preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C. Nucleic acid molecule variants retain the capacity to encode a fusion protein or a binding domain thereof having a functionality described herein, such as specifically binding a target molecule.
  • Percent sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences. Preferred methods to determine sequence identity are designed to give the best match between the sequences being compared. For example, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs.
  • Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX).
  • BLAST program e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX.
  • the mathematical algorithm used in the BLAST programs can be found in Altschul et al ., Nucleic Acids Res. 25: 3389-3402, 1997.
  • sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. "Default values" mean any set of values or parameters which originally load with the software when first initialized.
  • isolated means that the material is removed from its original environment (e.g, the natural environment if it is naturally occurring).
  • a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • Such nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g, a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region ("leader and trailer") as well as intervening sequences (introns) between individual coding segments (exons).
  • a “functional variant” refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs slightly in composition (e.g., one base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide.
  • a functional variant of a polypeptide or encoded polypeptide of this disclosure has "similar binding,” “similar affinity” or “similar activity” when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g ., Biacore® or tetramer staining measuring an association (K a ) or a dissociation (KD) constant).
  • binding affinity e.g ., Biacore® or tetramer staining measuring an association (K a ) or a dissociation (KD) constant.
  • a “functional portion” or “functional fragment” refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function).
  • a biological benefit e.g., effector function
  • a “functional portion” or “functional fragment” of a polypeptide or encoded polypeptide of this disclosure has “similar binding” or “similar activity” when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity), such as an assay for measuring binding affinity or measuring effector function (e.g, cytokine release).
  • heterologous or non-endogenous or exogenous refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered.
  • Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules.
  • heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra-chromosomal genetic material (e.g, as a plasmid or other self-replicating vector).
  • homologous or
  • homolog refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain.
  • a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof.
  • a non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity may be from the same species, a different species, or a combination thereof.
  • endogenous or “native” refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject.
  • expression refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene.
  • the process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post- translational modification, or any combination thereof.
  • An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).
  • operably linked refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
  • Unlinked means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.
  • expression vector refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host.
  • control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation.
  • the vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.
  • "plasmid,” “expression plasmid,” “virus” and “vector” are often used interchangeably.
  • the term "introduced” in the context of inserting a nucleic acid molecule into a cell means “transfection", or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g ., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • a cell e.g ., chromosome, plasmid, plastid, or mitochondrial DNA
  • transiently expressed e.g., transfected mRNA
  • the term "engineered,” “recombinant” or “non-natural” refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention).
  • Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene or operon.
  • more than one heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof.
  • the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule ( e.g ., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof.
  • the number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.
  • construct refers to any polynucleotide that contains a recombinant nucleic acid molecule (or, when the context clearly indicates, a fusion protein of the present disclosure).
  • a (polynucleotide) construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome.
  • a "vector” is a nucleic acid molecule that is capable of transporting another nucleic acid molecule.
  • Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi -synthetic or synthetic nucleic acid molecules.
  • Vectors of the present disclosure also include transposon systems (e.g, Sleeping Beauty, see , e.g, Geurts el al., Mol.
  • Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g, viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).
  • the term "host” refers to a cell (e.g., T cell) or microorganism targeted for genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g, a fusion protein of the present disclosure).
  • a host cell may optionally already possess or be modified to include other genetic modifications that confer desired properties related or unrelated to, e.g, biosynthesis of the heterologous protein (e.g, inclusion of a detectable marker; deleted, altered or truncated endogenous TCR; or increased co-stimulatory factor expression).
  • enriched or “depleted” with respect to amounts of cell types in a mixture refers to an increase in the number of the "enriched” type, a decrease in the number of the “depleted” cells, or both, in a mixture of cells resulting from one or more enriching or depleting processes or steps.
  • a mixture or composition may contain 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more (in number or count) of the "enriched" cells.
  • Cells subjected to a depleting process can result in a mixture or composition containing 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% percent or less (in number or count) of the "depleted" cells.
  • amounts of a certain cell type in a mixture will be enriched and amounts of a different cell type will be depleted, such as enriching for CD4 + cells while depleting CD8 + cells, or enriching for CD62L + cells while depleting CD62L- cells, or combinations thereof.
  • T cell receptor refers to an immunoglobulin superfamily member (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway el al., Immunobiology: The Immune System in Health and Disease, 3 rd Ed., Current Biology Publications, p. 4:33, 1997) capable of specifically binding to an antigen peptide bound to a MHC receptor.
  • a TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and b chains (also known as TCRa and TOIb, respectively), or g and d chains (also known as TCRy and TCR6, respectively).
  • each TCR chain contains two immunoglobulin domains: a variable domain (e.g., a-chain variable domain or Vo, b-chain variable domain or V ; typically amino acids 1 to 116 based on Rabat numbering (Rabat et al., "Sequences of Proteins of Immunological Interest, US Dept.
  • TCR variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. U.S.A.
  • a TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex.
  • the source of a TCR as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit or other mammal.
  • variable region refers to the domain of a TCR a-chain or b-chain (or g-chain and d-chain for gd TCRs), or of an antibody heavy or light chain, that is involved in binding to antigen.
  • the variable domains of the a-chain and b-chain (Va and nb, respectively) of a native TCR generally have similar structures, with each domain comprising four generally conserved framework regions (FRs) and three CDRs.
  • FRs generally conserved framework regions
  • VH antibody heavy
  • VL light chains each also generally comprise four generally conserved framework regions (FRs) and three CDRs.
  • CDR complementarity determining region
  • HVR hypervariable region
  • CDR3 is thought to be the main CDR responsible for recognizing processed antigen.
  • CDR1 and CDR2 mainly interact with the MHC.
  • variable region or domain (e.g, Va, nb, VH, or VL) comprises four FRs and three CDRs arranged as follows: FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4.
  • variable regions of a TCR (or of an antibody) together form an antigen-binding site through their respective CDRs.
  • Variable domain sequences can be aligned to a numbering scheme, which can allow equivalent residue positions to be annotated and for different molecules to be compared using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300).
  • numbering of CDR and framework regions may be according to any known method or scheme, such as the Rabat, Chothia, EU, IMGT, Contact, and AHo numbering schemes (see, e.g. , Rabat el al., "Sequences of Proteins of Immunological Interest, US Dept.
  • CD3 is a multi -protein complex of six chains that is involved in T cell signaling in response to antigen (see, Abbas and Lichtman, 2003; Janeway et al., p. 172 and 178, 1999).
  • the complex generally comprises a CD3y chain, a CD35 chain, two CD3e chains (each of which, in general, associates with a cognate CD3y chain or CD35 chain to form a dimer), and a homodimer of O03z chains.
  • the CD3y, CD35, and CD3e chains are related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain.
  • the transmembrane regions of the CD3y, CD35, and CD3e chains are negatively charged, which is thought to allow these chains to associate with positively charged regions of T cell receptor chains.
  • the intracellular tails of the CD3y, CD35, and CD3e chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each O03z chain has three IT AMS.
  • ITAMs are important for the signaling capacity of a TCR complex.
  • CD3 as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.
  • MHC molecules refer to glycoproteins that deliver peptide antigens to a cell surface.
  • MHC class I molecules are heterodimers consisting of a membrane spanning a chain (with three a domains) and a non-covalently associated b2 microglobulin.
  • MHC class II molecules are composed of two transmembrane glycoproteins, a and b, both of which span the membrane. Each chain has two domains.
  • MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a peptide:MHC complex is recognized by CD8 + T cells.
  • MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4 + T cells.
  • An MHC molecule may be from various animal species, including human (i.e., HLA molecule), mouse, rat, cat, dog, goat, horse, or other mammals.
  • CD8 co-receptor means the cell surface glycoprotein CD8, which is sometimes expressed by T cells as a homodimer comprising two CD8a chains, or as a heterodimer comprising an a chain and a b chain.
  • the CD8 co-receptor is believed to assist in the function of cytotoxic T cells (CD8 + ) and functions through signaling via its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 27:630-636, 2000; Cole and Gao, Cell. Mol.
  • CD8 co-receptor binds to an MHC-I protein complex expressed on the surface of an antigen-expressing cell, and that this binding in the context of
  • TCR:antigen-MHC binding initiates or assists in a T cell signaling pathway that produces an immune response (e.g ., transcription and expression of cytokines, calcium secretion, cytolytic activity, or the like) against the antigen-expressing cell.
  • an immune response e.g ., transcription and expression of cytokines, calcium secretion, cytolytic activity, or the like
  • Ml CD8 beta chain isoforms
  • CD4 refers to an immunoglobulin co-receptor glycoprotein that assists the TCR in communicating with antigen-presenting cells (see, Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002); UniProtKB P01730).
  • CD4 is found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and typically includes four immunoglobulin domains (Dl (comprising an Ig-like V-type domain), D2, D3, and D4 (respectively comprising Ig-like C2-type domains 1,
  • CD4 binds MHCII b2
  • TCR complex binds MHCII a ⁇ /b ⁇
  • CD4-associated kinase molecules to phosphorylate the immunoreceptor tyrosine activation motifs (ITAMs) present on the cytoplasmic domains of CD3. This activity is thought to amplify the signal generated by the activated TCR in order to produce various types of T helper cells.
  • ITAMs immunoreceptor tyrosine activation motifs
  • CAR Chimeric antigen receptor
  • CARs of the present disclosure include an extracellular component comprising an antigen-binding domain (e.g ., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a scFv or scTCR derived from an antibody or TCR specific for a cancer antigen, respectively, or an antigen-binding domain derived or obtained from a killer immunoreceptor from an NK cell) linked to a transmembrane domain and an intracellular component (e.g., containing one or more intracellular signaling domains (optionally containing co-stimulatory domain(s))) (see, e.g, Sadelain et /., Cancer Discov. 3:388, 2013); see also Harris and Kranz, Trends Pharmacol. Sci. 37: 220, 2016; Stone et al, Cancer Immunol. Immunother. 63: 1163, 2014).
  • an antigen-binding domain e.g ., obtained or derived from an immunoglobulin
  • a binding protein comprises a CAR comprising an antigen-specific TCR binding domain (see, e.g., Walseng et al., Scientific Reports 7: 10713, 2017; the TCR CAR constructs and methods of which are hereby incorporated by reference in their entirety), which can be a MHC-I-restricted TCR binding domain, a MHC-II-restricted TCR binding domain, or both.
  • a binding protein comprises a CAR comprising an antigen-specific TCR binding domain (see, e.g., Walseng et al., Scientific Reports 7: 10713, 2017; the TCR CAR constructs and methods of which are hereby incorporated by reference in their entirety), which can be a MHC-I-restricted TCR binding domain, a MHC-II-restricted TCR binding domain, or both.
  • Antigen refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically-competent cells (e.g., T cells), or both.
  • An antigen immunologically-competent cell
  • An antigen immunologically-competent molecule
  • An antigen immunologically-competent molecule
  • An antigen immunologically-competent molecule
  • An antigen immunologically-competent cells
  • glycopeptide polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid or the like. It is readily apparent that an antigen can be synthesized, produced
  • exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof.
  • Antigens can be produced by cells that have been modified or genetically engineered to express an antigen. Antigens can be expressed at a cell surface or presented in complex with a MHC molecule.
  • epitope includes any molecule, structure, amino acid sequence or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain or protein.
  • a cognate binding molecule such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain or protein.
  • Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • an effector domain is an intracellular portion, component, or domain of a fusion protein or receptor that can directly or indirectly promote an immunological response in a cell when receiving an appropriate signal.
  • an effector domain is from a protein or portion thereof or protein complex that receives a signal when bound, or when the protein or portion thereof or protein complex binds directly to a target molecule and triggers a signal from the effector domain.
  • An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an Intracellular Tyrosine-based Activation Motif (ITAM), such as those found in costimulatory molecules.
  • a cellular response comprises an immune response.
  • IT AMs are important for T cell activation following ligand engagement by a T cell receptor or by a fusion protein comprising a T cell effector domain.
  • the intracellular component or functional portion thereof comprises an ITAM.
  • An effector domain may be from a protein of a Wnt signaling pathway (e.g ., LRP, Ryk, or ROR2), NOTCH signaling pathway (e.g., NOTCH1, NOTCH2,
  • RTKs receptor tyrosine kinases
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • IR insulin receptor
  • PDGF platelet-derived growth factor
  • VEGF vascular endothelial growth factor
  • Trk tropomycin receptor kinase
  • Trh ephrin
  • AXL receptor family leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) receptor family, receptor tyrosine kinase-like orphan (ROR) receptor family, discoidin domain (DDR) receptor family, rearranged during transfection (RET) receptor family
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • IR insulin receptor
  • PDGF platelet-derived growth factor
  • VEGF vascular endot
  • Exemplary effector domains include those from, CD3e, CD35, E03z, CD25, CD79A, CD79B, CARD11, DAP 10, FcRa, FcRp, FcRy, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, Wnt, ROR2, Ryk, SLAMF1, Slp76, pTa, TCRa, TCRp, TRIM, Zap70, PTCH2, or any combination thereof.
  • the present disclosure provides trigger polypeptides that can directly or indirectly modulate (e.g ., activate, drive, increase, decrease, suppress, or prevent) expression of a gene product of interest; e.g., of a binding protein that specifically binds to an antigen.
  • trigger polypeptide refers to a polypeptide that can be expressed at the surface of a host cell, comprises an extracellular portion that binds to an antigen, and comprises an intracellular portion including a transcriptional activator, wherein binding of the trigger polypeptide to the antigen results in (or facilitates, or permits, or allows) release of the intracellular portion and/or
  • transcriptional activator from the trigger polypeptide (e.g, by a cleavage event modulated by or involving a protease, such as gamma secretase, a furin-like protease, ADAM10, ADAM17, or the like, as described herein).
  • the transcriptional activator (optionally contained on all or a portion of the intracellular portion) is capable of localizing to the nucleus of the host cell and modulating (e.g ., activating) transcription of a target nucleic acid sequence, such as a nucleic acid sequence encoding a binding protein of interest.
  • binding of a trigger polypeptide to an antigen directly modulates transcription of a target nucleic acid sequence of interest, or modulates transcription of one or mor eintermediary gene product (e.g., a second trigger polypeptide that is specific for a second antigen; a second transcriptional factor) that, in turn, can modulate transcription of the target nucleic acid sequence of interest.
  • a trigger polypeptide to an antigen directly modulates transcription of a target nucleic acid sequence of interest, or modulates transcription of one or mor eintermediary gene product (e.g., a second trigger polypeptide that is specific for a second antigen; a second transcriptional factor) that, in turn, can modulate transcription of the target nucleic acid sequence of interest.
  • trigger polypeptides of the present disclosure comprise: (a) an extracellular component comprising a binding domain that specifically binds to an antigen; (b) an intracellular component comprising a transcriptional factor, and (c) a Notch core domain disposed between the extracellular component and the intracellular component.
  • binding domain refers to a molecule or portion thereof (e.g., peptide, oligopeptide, polypeptide, protein (e.g, a fusion protein)) that possesses the ability to specifically and non-covalently associate, unite, or combine with a target.
  • a binding domain includes any naturally occurring, synthetic, semi -synthetic, or recombinantly produced binding partner for a biological molecule, a molecular complex (i.e., complex comprising two or more biological molecules), or other target of interest.
  • binding domains include single chain immunoglobulin variable regions (e.g, scTCR, scFv, Fab, TCR variable regions), receptor ectodomains, ligands (e.g, cytokines, chemokines), or synthetic polypeptides selected for their specific ability to bind to a biological molecule, a molecular complex or other target of interest.
  • the binding domain is a scFv, scTCR, or ligand.
  • the binding domain is chimeric, human, or humanized.
  • the binding domain of a trigger polypeptide can comprise a scFv, which may be in a VH- linker-VL orientation, or in a VL-linker-VH orientation.
  • the binding domain of a trigger polypeptide can comprise a scTCR, which can, when derived or obtained from an ab TCR, be in a nb-linker-Va orientation, or in a Va-linker-Ub orientation.
  • Sources of binding domains include antibody or TCR variable regions from various species, including human, rodent, avian, leporine, procine, and ovine.
  • binding domains include variable regions of antibodies from other species, such as camelid (from camels, dromedaries, or llamas; Ghahroudi et al, FEBS Letters 414: 521, 1997; Vincke et al, ./. Biol. Chem. 284: 3273, 2009; Hamers- Casterman et al, Nature 363: 446, 1993 and Nguyen et al, ./. Mol Biol 275: 413, 1998), nurse sharks (Roux et al, Proc. Nat'l Acad. Sci.
  • Binding domains of this disclosure can be generated as described herein or by a variety of methods known in the art (see, e.g, U.S. Patent Nos. 6,291,161 and
  • binding domains of this disclosure may be identified by screening a Fab phage library for Fab fragments that specifically bind to a target of interest (see Hoet et al., Nat. Biotechnol. 23:344, 2005). Additionally, traditional strategies for hybridoma development using a target of interest as an immunogen in convenient systems (e.g., mice, HUMAB MOUSE ® , TC MOUSETM, KM-MOUSE ® , llamas, chicken, rats, hamsters, rabbits, etc.) can be used to develop binding domains of this disclosure.
  • convenient systems e.g., mice, HUMAB MOUSE ® , TC MOUSETM, KM-MOUSE ® , llamas, chicken, rats, hamsters, rabbits, etc.
  • a binding domain of a trigger polypeptide of the present disclosure can have at least about 90% amino acid identity (i.e., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to an exemplary binding domain disclosed herein (e.g, an antibody Fab, VH, or VL; a TCR Va or Ub), or can comprise CDRs (e.g, CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3; CDRal, CDRa2, CDRa3; CDRpl, CDRp2, CDRp3) from an exemplary binding domain disclosed herein, one or more of which CDRs may have from one to three or four amino acid substitutions relative to the corresponding CDR of the exemplary binding domain.
  • CDRs e.g, CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3; CDRal, CDRa2, CDRa3; CDRpl
  • binding protein e.g ., a T cell receptor or a chimeric antigen receptor
  • binding domain or fusion protein thereof
  • K a an affinity or K a (i.e., an equilibrium association constant of a particular binding interaction with units of l/M) equal to or greater than 10 5 M 1 (which equals the ratio of the on-rate [K 0n ] to the off rate [K 0ff ] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample.
  • Binding proteins or binding domains may be classified as “high-affinity” binding proteins or binding domains or as “low-affinity” binding proteins or binding domains.
  • “High-affinity” binding proteins or binding domains refer to those binding proteins or binding domains having a K a of at least l0 7 M _1 , at least 10 8 M 1 , at least 10 9 M l , at least 10 10 M 1 , at least 10 11 M 1 , at least l0 12 M _1 , or at least 10 13 M 1 .
  • “Low-affinity” binding proteins or binding domains refer to those binding proteins or binding domains having a K a of up to 10 7 M 1 , up to 10 6 M l , or up to 10 5 M 1 .
  • affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10 5 M to 10 13 M).
  • a receptor or binding domain may have "enhanced affinity," which refers to selected or engineered receptors or binding domains with stronger binding to a target antigen than a wild type (or parent) binding domain.
  • enhanced affinity may be due to a K a (equilibrium association constant) for the target antigen that is higher than the wild type binding domain, due to a Kd
  • fusion proteins or trigger polypeptides may be codon-optimized to enhance expression in a particular host cell, such as T cells (Scholten et al., Clin. Immunol. 119: 135, 2006).
  • codon optimization can be performed using known techniques and tools, e.g ., using the GenScript® OptimumGeneTM tool. Codon-optimized sequences include sequences that are partially codon-optimized (i.e., one or more codon is optimized for expression in the host cell) and those that are fully codon-optimized.
  • binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain or fusion protein affinities, such as Western blot, ELISA, analytical
  • Assays for assessing affinity or apparent affinity or relative affinity are also known.
  • apparent affinity of a fusion protein is measured by assessing binding to various concentrations of tetramers, for example, by flow cytometry using labeled tetramers.
  • apparent KD of a fusion protein is measured using 2-fold dilutions of labeled tetramers at a range of concentrations, followed by determination of binding curves by non-linear regression, apparent KD being determined as the concentration of ligand that yielded half-maximal binding.
  • a binding domain of a trigger polypeptide specifically binds to a B7-H3 (CD276), EpCAM, L1CAM, Her2, and/or EGFR antigen.
  • a binding domain of a trigger polypeptide specifically binds to a B7-H3 (aka CD276) antigen.
  • the amino acid sequence of the human B7-H3 precursor protein is provided in SEQ ID NO:2.
  • Binding domains specific for B7-H3 antigens include, for example, those from: BRCA69D (enoblituzumab, (MGA271)) and other anti-B7-H3 antibodies disclosed in Loo et al. (Clin. Cancer Res. 75:3834, 2012), and including BRCA84D and PRCA157; see also PCT Publication No. WO
  • the extracellular component of a trigger polypeptide includes a binding domain that specifically binds to a B7-H3 antigen and comprises a VH and a VL having at least 90% (i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% ) amino acid sequence identity to the VH and VL of
  • Variable domain and CDR sequences of BRCA69D antibody are set forth in SEQ ID NOs: 104-111.
  • the binding domain comprises a scFv.
  • the scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO:7.
  • Variable domain and CDR sequences of BRCA84D antibody are provided in SEQ ID NOs:3, 5, 7, 9, 11, 13, 15, and 17 of US Pre-Grant Publication No. 2012/0294796A1.
  • Variable domain and CDR sequences of PRCA157 antibody are provided in SEQ ID NOs:35, 37, 39, 41, 43, 45, 47, and 49 of US Pre-Grant Publication No. 2012/0294796A1.
  • a binding domain of a trigger polypeptide specifically binds to an EpCAM (Epithelial Cell Adhesion Molecule, aka CD326)) antigen.
  • EpCAM Epicellular Cell Adhesion Molecule
  • the amino acid sequence of the human EpCAM precursor is provided in SEQ ID NO:3.
  • Binding domains specific for EpCAM antigens include, for example, those from antibodies described in Borquin et al. (./. Nat ⁇ Cancer Inst. 107:364, 2014); in PCT Publication Nos. WO 2010/142990A1, WO 2005/080428A2; WO 2008/122551A9; WO 2011/079283A1; WO 2015/048901 Al; WO 2017/023704A1; WO
  • the extracellular component of a trigger polypeptide includes a binding domain that specifically binds to an EpCAM antigen and comprises a VH and a VL having at least 90% (i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% ) amino acid sequence identity to the VH and VL of G8.8 antibody (available commercially through the Developmental Studies Hybridoma Bank at the University of Iowa; dshb.biology.uiowa.edu/EpCAM), respectively, and/or containing the CDRs of G8.8 antibody.
  • Variable domain sequences of G8.8 antibody are set forth in SEQ ID NOs: 112 and 113.
  • the binding domain comprises a scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO:8.
  • a transcriptional factor refers to a polypeptide capable of activating or increasing, or inhibiting, repressing or reducing, transcription of a target nucleotide sequence (e.g., a gene) or set of target nucleotide sequences.
  • Transcriptional factors include transcription activators and transcription repressors, and, in some embodiments, comprise a DNA-binding domain. In some embodiments, a
  • transcriptional factor binds to an enhancer element present in a nucleotide sequence of interest.
  • transcription activators include GAL4; Gcn4;
  • Tbx2l an inducible activator such as a tetracycline-controlled transcriptional activator (tTA); a homeodomain protein transcription activator; a zinc-finger protein
  • transcription activator a Winged-Helix (Forkhead) protein transcription activator; a Leucine Zipper protein transcription activator; and a Helix-Loop-Helix (HLH) protein transcription activator.
  • Other transcriptional factors include those from ABT1, ACYP2, AEBP1, AEBP2, AES, AFF1, AFF3, AHR, ANK1, ANK2, ANKFY1, ANKIB1, ANKRD 1 , ANKRD 10, ANKRD2, ANKRD32, ANKRD46, ANKRD49, ANKRD56, ANKRD57, ANKS4B, AR, ARHGAP17, ARID 1 A, ARID! B, ARID3A, ARID4A, ARID5B, ARNT, ARNT2, ARNTL, ARNTL2, ARX, ASB 10, ASB11, ASB12,
  • NUDT12 NULL, NUPR1, 1700065013RIK, OLIG1, OLIG2, OLIG2, ONECUT1, ONECUT2, ONECUT3, ORC2L, OSGIN1, OSR1, OSR2, OSTF1, OVOL1, OVOL2, PAPOLA, PAPOLG, PAPPA2, PATZ1, PAWR, PAX2, PAX5, PAX6, PAX7, PAX8, PAX9, PBX1, PBX2, PBX3, PBX4, PCBD1, PCGF6, PDCD11, PDLIM4, PDX1, PEG3, PERI, PFDN1, PGR, PHF1, PHF10, PHF12, PHF13, PHF14, PHF20, PHF21A, PHF5A, PHF7, PHOX2A, PHOX2B, PIAS2, PIR, PITX1, PITX2, PKNOX1,
  • Z SCAN 16 ZSCAN20, ZSCAN21, ZXDC, and/or ZZZ3.
  • a transcriptional factor can comprise a transcription effector domain, such as, for example, a VP 16 activation domain or a VP64 activation domain.
  • a transcriptional factor can be a transcriptional activator or a transcriptional repressor.
  • a trigger polypeptide of the present disclosure comprises or consists of an amino acid sequence having at least 90% (z.e., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequence set forth in SEQ ID NO: 10, optionally not including amino acids 1-10 of SEQ ID NO: 10.
  • a trigger polypeptide of the present disclosure comprises or consists of an amino acid sequence having at least 90% (z.e., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequence set forth in SEQ ID NO: 11, optionally not including the leader sequence consisting of amino acids 1-27 of SEQ ID NO: 11.
  • a “Notch core domain” refers to an amino acid sequence from, or derived from, a Notch receptor polypeptide, that includes one or more antigen-inducible proteolytic cleavage site, wherein the one or more antigen-inducible proteolytic cleavage site is selected from a Sl proteolytic cleavage site, a S2 proteolytic cleavage site, a S3 proteolytic cleavage site, or any combination thereof.
  • Notch receptor polypeptides typically comprise, in N- terminal to C-terminal orientation: an extracellular ligand-binding domain containing 2- 11 EGF Repeats; a core domain containing a Lin 12-Notch Repeat (LNR) segment, two heterodimerization domains (HD-N and HD-C, respectively), and a transmembrane (TM) domain; and an intracellular domain containing a RBP-Jk-associated module (RAM) domain, an ankyrin repeat region, a transcription activation domain, and a PEST domain.
  • LNR Lin 12-Notch Repeat
  • HD-N and HD-C two heterodimerization domains
  • TM transmembrane
  • RAM RBP-Jk-associated module
  • Notch receptor polypeptides typically comprise three proteolytic cleavage sites (Sl, S2, S3) located in the core domain.
  • Sl proteolytic cleavage site is located between the HD-N segment and the HD-C segment; an S2 proteolytic cleavage site is located within the HD-C segment; and an S3 proteolytic cleavage site is located within the TM domain.
  • Cleavage at the Sl site is believed to occur during protein maturation in the Golgi complex and is performed by a furin-like convertase/protease, resulting in a heterodimeric Notch receptor expressed at the cell surface.
  • Binding of the Notch receptor to ligand e.g ., a cognate Delta ligand expressed on the surface of a neighboring cell
  • ADAM10 metalloprotease ADAM10
  • gamma-secretase cleaves remaining Notch receptor fragment at the S3 site, releasing the Notch intracellular domain to localize to the nucleus and activate transcription.
  • a Notch core domain of a trigger polypeptide of the present disclosure comprises an S2 and/or an S3 cleavage site.
  • the S2 proteolytic cleavage site ADAM- 17-type protease cleavage site comprising an Ala-Val dipeptide sequence, wherein cleavage occurs between the Ala and the Val.
  • an amino acid sequence comprising a S2 proteolytic cleavage site can comprise the amino acid sequence KIEAVKSE (SEQ ID NO:9l), where cleavage occurs within the "AV" sequence.
  • an amino acid sequence comprising a S2 proteolytic cleavage site can comprise the amino acid sequence KIEAVQSE (SEQ ID NO:92), where cleavage occurs within the "AV" sequence.
  • the S3 proteolytic cleavage site is a g-secretase cleavage site comprising a Gly-Val dipeptide sequence, wherein cleavage occurs between the Gly and the Val.
  • an amino acid sequence comprising a S3 proteolytic cleavage site can comprise the amino acid sequence VGCGVLLS (SEQ ID NO:93), wherein cleavage occurs within the "GV" sequence.
  • an amino acid sequence comprising a S3 proteolytic cleavage site can comprise the amino acid sequence GCGVLLS (SEQ ID NO:94), wherein cleavage occurs within the "GV" sequence.
  • the Notch core domain further comprises a HD-N segment, an HD-C segment, or both, wherein the S2 cleavage site is optionally located between the HD-N segment and the HD-C segment.
  • Exemplary HD-N and HD-C amino acid sequences and variants thereof are disclosed in PCT Publication No. WO 2016/138034A1, and are incorporated herein by reference.
  • the Notch core domain further comprises a TM domain, wherein the S3 cleavage site is optionally located within the TM domain.
  • TM amino acid sequences and variants thereof are disclosed in PCT Publication No. WO 2016/138034A1, and are incorporated herein by reference.
  • the Notch core domain further comprises a LNR domain.
  • LNR amino acid sequences and variants thereof are disclosed in PCT Publication No. WO 2016/138034A1, and are incorporated herein by reference.
  • the Notch core domain further comprises a Sl cleavage site.
  • the Sl proteolytic cleavage site is a furin-like protease cleavage site comprising the amino acid sequence Arg-X-(Arg/Lys)-Arg, wherein X is any amino acid (SEQ ID NO: 95), and the protease cleaves immediately C-terminal to the canonical sequence.
  • an amino acid sequence comprising an Sl antigen-inducible proteolytic cleavage site comprises the amino acid sequence GRRRRELDPM (SEQ ID NO: 106), wherein cleavage occurs within the "RE" sequence.
  • an amino acid sequence comprising an Sl antigen-inducible proteolytic cleavage site can comprise the amino acid sequence RQRRELDPM (SEQ ID NO: 97), wherein cleavage occurs within the "RE" sequence.
  • the Notch core domain of a trigger polypeptide of the present disclosure lacks any one or two of a Sl, a S2, or a S3 proteolytic cleavage site.
  • the Notch core domain comprises a S2 proteolytic cleavage site, but lacks a Sl and a S3 proteolytic cleavage site.
  • the Notch core domain comprises a S3 proteolytic cleavage site, but lacks an Sl and a S2 proteolytic cleavage site.
  • the Notch core domain comprises a S2 and a S3 proteolytic cleavage site, but lacks a Sl proteolytic cleavage site.
  • a Notch core domain can comprise or consist of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:4, or can be derived from SEQ ID NO:5.
  • binding of a cell-surface expressed trigger polypeptide to an antigen results in or permits or facilitates cleavage within the Notch core domain. This cleavage leads to release of all or a portion of the intracellular domain, wherein the translational factor localizes to the nucleus and modulates transcription of one or more target nucleic acid sequences.
  • a translational factor binds to an enhancer element. In some embodiments, binding of the transcriptional factor to the enhancer element activates expression of the binding protein that specifically binds to the ROR1 antigen.
  • an “enhancer element” refers to a nucleic acid sequence that is bound by at least one transcriptional activator and increases the likelihood that transcription of a sequence of interest (e.g., a gene) will occur. Enhancer elements are generally about 15 to about 1500 base pairs in length, and can be upstream of (5' to), downstream of (3' to), or within the sequence region of interest.
  • An exemplary enhancer element is a GAL4 UAS (Upstream Activation Sequence), which is bound by GAL4 transcriptional activators.
  • Exemplary enhancers are also predicted by the GeneHancer program (see Fishilevich el al., Database 1-17 (2017), Article ID bax028; database of predicted and annotated enhancers accessible at genecards.org); and annotated in the Enhancer Atlas (enhanceratlas.org) and dbSUPER (see Khan and Zheng, Nucl. Acids Res. 44 D164-D171, 2016), and other publicly available resources.
  • an enhancer element is operably linked to a polynucleotide encoding a ROR1 -specific binding protein of this disclosure.
  • Exemplary binding domains specific for ROR1 include those from antibodies disclosed in, for example, Yang et al ., PLoS One ⁇ 5:e2l0l8 doi : 10.1371 , 2011; Paredes-Moscosso et al. , Blood 128:2052, 2016; PCT Publication Nos. WO 2014/031174, WO
  • Variants of these antibodies and binding domains in which one or more mutations has been made to introduce or improve a desired function, or to remove or reduce an undesired function are also within the scope of this disclosure. See , e.g., the humanized anti- ROR1 binding domain sequences disclosed in WO 2018/197675A1, which sequences are incorporated herein by reference.
  • a binding domain that binds to a ROR1 antigen is derived from Rl l antibody, 2A2 antibody, R12 antibody, UC-961 antibody, D10 antibody, Y31 antibody, or H10 antibody.
  • a binding domain that binds to a ROR1 antigen comprises CDR (e.g, HCDR1, HCDR2, HCDR3,
  • LCDR1, LCDR2, and/or LCDR3) VH, VL, and/or scFv amino acid sequences as set forth in SEQ ID NOs: 17-61.
  • a binding protein that specifically binds to a ROR1 antigen comprises: (i) an extracellular component comprising a binding domain that specifically binds to the ROR1 antigen; (ii) an intracellular component comprising a co- stimulatory domain (e.g, from, or derived from: CD27; O ⁇ 3z; CD28; 4-1BB; ICOS; 0X40; CD30; CD40; PD-l; LFA-l; CD2; CD7; LIGHT; NKG2C; B7-H3; GITR; BAFF-R; CD5; HVEM; CD 160; LFA-l; SLAMF7; NKp80; ICAM-l; CD94; DAP 12; a ligand that specifically binds with CD83; or any combination thereof); and (iii) a transmembrane component disposed between the extracellular component and the intracellular component.
  • a co- stimulatory domain e.g, from, or derived from: CD27; O ⁇ 3z
  • transmembrane domain is a portion of a membrane protein that can insert into or span a cell membrane.
  • Transmembrane domains have a three- dimensional structure that is thermodynamically stable in a cell membrane and generally range in length from about 15 amino acids to about 30 amino acids.
  • the structure of a transmembrane domain may comprise an alpha helix, a beta barrel, a beta sheet, a beta helix, or any combination thereof.
  • the structure of a transmembrane domain may comprise an alpha helix, a beta barrel, a beta sheet, a beta helix, or any combination thereof.
  • the structure of a transmembrane domain may comprise an alpha helix, a beta barrel, a beta sheet, a beta helix, or any combination thereof.
  • transmembrane domain comprises or is derived from a known transmembrane protein (e.g ., a CD4 transmembrane domain, a CD8 transmembrane domain, a CD27 transmembrane domain, a CD28 transmembrane domain, a CD3 transmembrane domain, or any combination thereof).
  • a known transmembrane protein e.g ., a CD4 transmembrane domain, a CD8 transmembrane domain, a CD27 transmembrane domain, a CD28 transmembrane domain, a CD3 transmembrane domain, or any combination thereof.
  • the extracellular component of a ROR1 -specific binding protein further comprises a linker disposed between the binding domain and the transmembrane domain.
  • a linker may be an amino acid sequence having from about two amino acids to about 500 amino acids, which can provide flexibility and room for conformational movement between two regions, domains, motifs, fragments, or modules connected by the linker.
  • Linker length in a ROR1 -specific binding protein (or in a trigger polypeptide, which can, in some embodiments, include a linker) of the present disclosure may be varied to maximize antigen or MHC recognition based on the selected target molecule, selected binding epitope, or antigen binding domain size and affinity (see, e.g., Guest el al., J. Immunother. 28: 203-11, 2005; PCT Publication No. WO 2014/031687).
  • Exemplary linkers include those having a glycine-serine amino acid chain having from one to about ten repeats of Gly x Ser y , wherein x and y are each independently an integer from 0 to 10, provided that x and y are not both 0.
  • Exemplary spacers can vary in length, for instance, from about five to about 500 amino acids, from about 10 to about 350 amino acids, from about 15 to about 100 amino acids, from about 20 to about 75 amino acids, or from about 25 to about 35 amino acids. Certain exemplary spacers have a length of 12 amino acids or less, 119 amino acids or less, or 229 amino acids or less.
  • Linkers of the present disclosure also include immunoglobulin constant regions
  • the linker comprises a CH3 domain, a CH2 domain, or both. In certain embodiments, the linker comprises a CH2 domain and a CH3 domain. In further embodiments, the CH2 domain and the CH3 domain are each a same isotype. In particular embodiments, the CH2 domain and the CH3 domain are an IgG4 or IgGl isotype. In other embodiments, the CH2 domain and the CH3 domain are each a different isotype. In specific embodiments, the CH2 comprises a N297Q mutation.
  • the linker comprises a human immunoglobulin constant region or a portion thereof.
  • An exemplary amino acid sequence of a human IgG4 constant region is provided in SEQ ID NO:65. Additional linkers include extracellular domains (or portions thereof) from from CD27, CD28, CD8, CD4, or any combination thereof.
  • a linker may comprise a hinge region or a portion thereof (e.g, comprising all or a portion of a human IgGl, IgG2, or IgG4 hinge region, or modified version thereof).
  • Hinge regions are flexible amino acid polymers of variable length and sequence (typically rich in proline and cysteine amino acids) and connect larger and less-flexible regions of immunoglobulin proteins.
  • hinge regions connect the Fc and Fab regions of antibodies and connect the constant and transmembrane regions of TCRs.
  • the linker comprises an immunoglobulin constant region or a portion thereof and a hinge region or a portion thereof. Exemplary hinge sequences are provided in SEQ ID NOs:62 and 63.
  • the linker comprises a glycine-serine linker of the present disclosure.
  • a hinge is an altered immunoglobulin hinge in which one or more cysteine residues in a wild type immunoglobulin hinge region is substituted with one or more other amino acid residues.
  • exemplary altered immunoglobulin hinges include an immunoglobulin human IgGl, IgG2, or IgG4 hinge region having one, two, or three cysteine residues found in a wild type human IgGl, IgG2, or IgG4 hinge substituted by one, two, or three different amino acid residues (e.g., serine or alanine).
  • a hinge comprises or consists of a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a wild type immunoglobulin hinge region, such as a wild type human IgGl hinge, a wild type human IgG2 hinge, or a wild type human IgG4 hinge.
  • a hinge may include a peptide or polypeptide of about five to about 150 amino acids of the stalk region of type II C-lectins or CD molecules, including peptides or polypeptides of about eight to about 25 amino acids or peptides of about seven to about 18 amino acids, or variants thereof.
  • a "stalk region" of a type II C-lectin or CD molecule refers to the portion of the extracellular domain of the type II C-lectin or CD molecule that is located between the C-type lectin-like domain (CTLD; e.g., similar to CTLD of natural killer cell receptors) and the hydrophobic portion (transmembrane domain).
  • C-type lectin-like domain C-type lectin-like domain
  • hydrophobic portion transmembrane domain
  • AAC50291.1 corresponds to amino acid residues 34-179, but the CTLD corresponds to amino acid residues 61-176, so the stalk region of the human CD94 molecule includes amino acid residues 34-60, which are located between the hydrophobic portion (transmembrane domain) and CTLD (see Boyington et al., Immunity 10: 15, 1999; for descriptions of other stalk regions, see also Beavil et al., Proc. Nat'l. Acad. Sci. USA 89: 153, 1992; and Figdor et al., Nat. Rev. Immunol. 2: 11, 2002).
  • These type II C-lectin or CD molecules may also have junction amino acids between the stalk region and the transmembrane region or the CTLD.
  • the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) ranging from amino acids 71-93 and an extracellular domain ranging from amino acids 94-233.
  • the CTLD includes amino acids 119-231 and the stalk region includes amino acids 99-116, which may be flanked by additional junction amino acids.
  • Other type II C-lectin or CD molecules, as well as their extracellular ligand-binding domains, stalk regions, and CTLDs are known in the art (see, e.g., GenBank Accession Nos. NP 001993.2;
  • a "derivative" of a stalk region hinge, or fragment thereof, of a type II C-lectin or CD molecule includes about an eight to about 150 amino acid sequence in which one, two, or three amino acids of the stalk region of a wild type II C-lectin or CD molecule have a deletion, insertion, substitution, or any combination thereof.
  • a derivative can include one or more amino acid substitutions and/or an amino acid deletion.
  • a derivative of a stalk region is more resistant to proteolytic cleavage as compared to the wild-type stalk region sequence, such as those derived from about eight to about 20 amino acids of NKG2A, NKG2D, CD23, CD64, CD72, or CD94.
  • stalk region hinges may include from about seven to about 18 amino acids and can form an a-helical coiled coil structure. In certain embodiments, stalk region hinges contain 0, 1, 2, 3, or 4 cysteines. Exemplary stalk region hinges include fragments of the stalk regions, such as those portions including from about ten to about 150 amino acids from the stalk regions of CD69, CD72, CD94, NKG2A, and NKG2D.
  • Additional hinges encompassed in this disclosure are from portions of cell surface receptors (interdomain regions) that connect immunoglobulin V-like or immunoglobulin C-like domains.
  • Amino acid sequences found between Ig V-like domains where the cell surface receptor contains multiple Ig V-like domains in tandem and between Ig C-like domains where the cell surface receptor contains multiple tandem Ig C-like regions are also contemplated as hinges useful in this disclosure. Examples of hinges include interdomain regions between the Ig V-like and Ig C-like regions of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD150,
  • CD 166 or CD244.
  • a hinge sequence is stable in plasma and serum, and is resistant to proteolytic cleavage.
  • the first lysine in an IgGl upper hinge region may be mutated or deleted to minimize proteolytic cleavage, and hinges may include junction amino acids.
  • a hinge sequence may contain a naturally occurring or added motif, such as an immunoglobulin hinge core structure CPPCP that confers the capacity to form a disulfide bond or multiple disulfide bonds to stabilize dimer formation.
  • one or more of the extracellular component, the binding domain, the linker, the transmembrane domain, the intracellular component, or the costimulatory domain comprises junction amino acids.
  • Junction amino acids or “junction amino acid residues” refer to one or more ( e.g ., about 2-20) amino acid residues between two adjacent domains, motifs, regions, modules, or fragments of a protein, such as between a binding domain and an adjacent linker, between a transmembrane domain and an adjacent extracellular or intracellular domain, or on one or both ends of a linker that links two domains, motifs, regions, modules, or fragments (e.g., between a linker and an adjacent binding domain or between a linker and an adjacent hinge).
  • Junction amino acids may result from the construct design of a ROR1- specific binding protein (e.g, amino acid residues resulting from the use of a restriction enzyme site or self-cleaving peptide sequences during the construction of a
  • a transmembrane domain of a ROR1 -specific binding protein may have one or more junction amino acids at the amino-terminal end, carboxy -terminal end, or both.
  • a binding protein that specifically binds to the ROR1 antigen comprises a chimeric antigen receptor (CAR), a T cell receptor (TCR), a single-chain T cell receptor (scTCR), or any combination thereof.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • scTCR single-chain T cell receptor
  • the ROR1 -specific CAR constructs including the binding domains, extracellular domains (including spacers), transmembrane domains, intracellular domains, costimulatory domains, transduction markers, and related amino acid and nucleotide sequences) of which are incorporated herein by reference in their entireties.
  • a binding protein that specifically binds to the ROR1 antigen comprises an amino acid sequence having at least 90% identity (i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% ) to the amino acid sequence set forth in SEQ ID NO:79.
  • the binding protein having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:79 comprises CDRs according to Rl 1 antibody (SEQ ID NOs: 17-22, respectively), and optionally comprises a VH and a VL domain having at least 90% identity (i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% ) to the amino acid sequences set forth in SEQ ID NOs:23 and 24, respectively.
  • a binding protein that specifically binds to the ROR1 antigen comprises the amino acid sequence set forth in SEQ ID NO:79.
  • a binding protein that specifically binds to the ROR1 antigen consists of the amino acid sequence set forth in SEQ ID NO:79.
  • ROR1 -specific binding protein or trigger protein of the present disclosure comprises a protein tag.
  • Protein tags are unique peptide sequences that are affixed or genetically fused to, or are a part of, a protein of interest and can be recognized or bound by, for example, a heterologous or non-endogenous cognate binding molecule or a substrate (e.g ., receptor, ligand, antibody, carbohydrate, or metal matrix) or a fusion protein of this disclosure.
  • Protein tags can be useful for detecting, identifying, isolating, tracking, purifying, enriching for, targeting, or biologically or chemically modifying tagged proteins of interest, particularly when a tagged protein is part of a heterogeneous population of cell proteins or cells (e.g., a biological sample like peripheral blood).
  • a tagged protein is part of a heterogeneous population of cell proteins or cells (e.g., a biological sample like peripheral blood).
  • the ability of the tag(s) to be specifically bound by a cognate binding molecule or a protein or polypeptide of this disclosure is distinct from, or is in addition to, the ability of binding domain(s) contained by the cell surface protein (e.g, trigger polypeptide, CAR, TCR) to specifically bind target molecule(s).
  • a protein tag of this disclosure comprises a Myc tag, His tag, Flag tag, Xpress tag, Avi tag, Calmodulin tag, Polyglutamate tag, HA tag, Nus tag, S tag, SBP tag, Softag, V5 tag, CBP, GST, MBP, GFP, Thioredoxin tag, Strep® tag, or any combination thereof.
  • Methods useful for isolating and purifying recombinantly produced soluble binding proteins or fusion proteins may include obtaining supernatants from suitable host cell/vector systems that secrete the soluble protein into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable
  • purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin.
  • One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide.
  • These purification methods may also be employed when isolating an immunogen from its natural environment.
  • Methods for large scale production of one or more of the isolated/recombinant soluble protein described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the soluble protein may be performed according to methods described herein and known in the art and that comport with laws and guidelines of domestic and foreign regulatory agencies.
  • polypeptides and host cells that express the polypeptides.
  • a polynucleotide encoding a desired trigger polypeptide or binding protein of this disclosure can be inserted into an appropriate vector (e.g ., viral vector or non-viral plasmid vector) for introduction into a host cell of interest (e.g., an immune cell, such as a T cell).
  • an appropriate vector e.g ., viral vector or non-viral plasmid vector
  • a host cell of interest e.g., an immune cell, such as a T cell.
  • a trigger polypeptide-encoding polynucleotide and a binding protein encoding polynucleotide can be present on a single nucleic acid molecule, or can be present on separate nucleic acid molecules.
  • a polynucleotide of the present disclosure may be codon-optimized for a host cell containing the polynucleotide (see, e.g, Scholten et al. , Clin. Immunol. 119: 135, 2006).
  • a polynucleotide encoding a binding protein comprises an enhancer element (e.g, an Upstream Activation Sequence (UAS)), wherein a transcriptional activator from a trigger polypeptide can bind to the enhancer element and alter the expression of the binding protein.
  • an enhancer element e.g, an Upstream Activation Sequence (UAS)
  • a polynucleotide of the present disclosure (e.g, encoding a fusion protein, or a trigger polypeptide, or any component, domain, or portion thereof) has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to an exemplary polynucleotide provided herein. It will be understood that variant polynucleotide sequences also include those wherein the variation is due (in whole or in part) to the degeneracy of the genetic code.
  • An exemplary polynucleotide encoding an anti-B7-H3 trigger polypeptide is set forth in SEQ ID NO: 14.
  • Exemplary polynucleotides encoding an anti-EpCAM trigger polypeptide are set forth in SEQ ID NOs: l5, 16, and 199.
  • Exemplary CAR construct- encoding polynucleotides are provided in SEQ ID NOs:80, 82, 100, and 101.
  • An exemplary polynucleotide encoding an anti-CD 19 trigger polypeptide is set forth in SEQ ID NO: 98.
  • expression constructs are provided, wherein the expression constructs comprise a polynucleotide encoding a polypeptide of the present disclosure (e.g, binding protein) operably linked to an expression control sequence (e.g, a promoter).
  • the expression construct is contained in a vector.
  • An exemplary vector may comprise a polynucleotide encoding a polypeptide of the present disclosure, wherein the vector can be introduced or mobilized into a host of interest, or, in certain embodiments, is capable of replication in a host cell or organism.
  • Some examples of vectors include plasmids, viral vectors, cosmids, and others.
  • Some vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g.
  • bacterial vectors having a bacterial origin of replication and episomal mammalian vectors
  • other vectors may be integrated into the genome of a host cell or promote integration of the polynucleotide insert upon introduction into the host cell and thereby replicate along with the host genome (e.g, lentiviral vector, retroviral vector).
  • some vectors are capable of directing the expression of genes to which they are operably linked (these vectors may be referred to as "expression vectors").
  • each agent may reside in separate or the same vectors, and multiple vectors (each containing a different agent or the same agent) may be introduced to a cell or cell population or administered to a subject.
  • polynucleotides of the present disclosure may be operably linked to certain elements of a vector.
  • polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are linked may be operably linked.
  • Expression control sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals;
  • Expression control sequences may be operably linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • the vector comprises a plasmid vector or a viral vector (e.g ., a vector selected from lentiviral vector or a g-retroviral vector).
  • Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).
  • herpesvirus
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLY-BLY group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al ., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
  • “Retroviruses” are viruses having an RNA genome, which is reverse-transcribed into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is then incorporated into the host cell genome.
  • “Gammaretrovirus” refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.
  • Lentiviral vector means HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.
  • the viral vector can be a gammaretrovirus, e.g ., Moloney murine leukemia virus (MLV)-derived vectors.
  • the viral vector can be a more complex retrovirus-derived vector, e.g. , a lentivirus-derived vector. HIV- 1 -derived vectors belong to this category.
  • Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus).
  • Retroviral and lentiviral vector constructs and expression systems are also commercially available.
  • Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., Gene Ther. 5: 1517, 1998).
  • HSVs herpes simplex viruses
  • vectors can be used with the compositions and methods of this disclosure.
  • Such vectors include those derived from baculoviruses and a-viruses. (Jolly, D J. 1999. Emerging Viral Vectors pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as sleeping beauty or other transposon vectors).
  • the viral vector may also comprise additional sequences between the two (or more) transcripts allowing for bicistronic or multi cistronic expression.
  • additional sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, or any combination thereof.
  • a polynucleotide in each recombinant expression construct includes at least one appropriate expression control sequence (also called a regulatory sequence), such as a leader sequence and particularly a promoter operably linked to the nucleotide sequence encoding the immunogen.
  • a regulatory sequence also called a regulatory sequence
  • polynucleotides of the present disclosure are used to transfect/transduce a host cell (e.g ., a T cell) for use in adoptive transfer therapy (e.g, targeting a cancer antigen.
  • a host cell e.g ., a T cell
  • adoptive transfer therapy e.g, targeting a cancer antigen.
  • T cells of desired target-specificity e.g., Schmitt et al., Hum. Gen. 20: 1240, 2009; Dossett et al., Mol. Ther. 77:742, 2009; Till et al, Blood 772:2261, 2008; Wang et al, Hum. Gene Ther. 18:112, 2007; Kuball et al, Blood 109 :233 l, 2007; US 2011/0243972; US 2011/0189141; Leen et al, Ann. Rev. Immunol.
  • desired target-specificity e.g., Schmitt et al., Hum. Gen. 20: 1240, 2009; Dossett et al., Mol. Ther. 77:742, 2009; Till et al, Blood 772:2261, 2008; Wang et al, Hum. Gene Ther. 18:112, 2007; Kuball et al, Blood 109 :233 l, 2007; US 2011/0243972; US 2011/
  • host cells comprise a polynucleotide of the present disclosure and express the encoded trigger polypeptide, binding protein, or both.
  • the host cell is a hematopoietic progenitor cell or a human immune system cell.
  • a "hematopoietic progenitor cell”, as referred to herein, is a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiation into mature cells types ( e.g ., immune system cells).
  • Exemplary hematopoietic progenitor cells include those with a CD24 Lo Lin- CD117 + phenotype or those found in the thymus (referred to as progenitor thymocytes).
  • an “immune system cell” or “immune cell” means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells, natural killer (NK) cells, and NK-T cells).
  • a myeloid progenitor cell which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes
  • lymphoid progenitor cell which give rise to lymphoid cells such as T cells, B cells, natural killer (NK) cells, and NK-T cells.
  • Exemplary immune system cells include a CD4 + T cell, a CD8 + T cell, a CD4 CD8 double negative T cell, a gd T cell, a regulatory T cell, a stem cell memory T cell, a natural killer cell (e.g., a NK cell or a NK-T cell), a B cell, and a dendritic cell.
  • Macrophages and dendritic cells may be referred to as "antigen presenting cells" or "APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.
  • MHC major histocompatibility complex
  • T cell or "T lymphocyte” is an immune system cell that matures in the thymus and produces T cell receptors (TCRs).
  • T cells can be naive (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells (TM) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic).
  • TM can be further divided into subsets of central memory T cells (TCM, increased expression of CD62L, CCR7, CD28, CD 127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naive T cells) and effector memory T cells (TEM, decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naive T cells or TCM).
  • TCM central memory T cells
  • TEM effector memory T cells
  • Effector T cells refers to antigen-experienced CD8 + cytotoxic T
  • helper T cells are CD4 + cells that influence the activity of other immune cells by releasing cytokines.
  • CD4 + T cells can activate and suppress an adaptive immune response, and which of those two functions is induced will depend on presence of other cells and signals.
  • T cells can be collected using known techniques, and the various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as by affinity binding to antibodies, flow cytometry, or immunomagnetic selection.
  • Other exemplary T cells include regulatory T cells, such as CD4 + CD25 + (Foxp3 + ) regulatory T cells and Tregl7 cells, as well as Trl, Th3, CD8 + CD28 , and Qa-l restricted T cells.
  • Cells of T cell lineage refer to cells that show at least one phenotypic characteristic of a T cell, or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages.
  • Such phenotypic characteristics can include expression of one or more proteins specific for T cells (e.g ., CD3 + , CD4 + , CD8 + ), or a physiological, morphological, functional, or immunological feature specific for a T cell.
  • cells of the T cell lineage may be progenitor or precursor cells committed to the T cell lineage; CD25 + immature and inactivated T cells; cells that have undergone CD4 or CD8 linage commitment; thymocyte progenitor cells that are CD4 + CD8 + double positive; single positive CD4 + or CD8 + ; TCRa.p or TCR gd; or mature and functional or activated T cells.
  • the immune system cell is a CD4 + T cell, a CD8 + T cell, a CD4 CD8 double negative T cell, a gd T cell, a natural killer cell (e.g., NK cell or NK-T cell), a dendritic cell, a B cell, or any combination thereof.
  • the immune system cell is a CD4 + T cell.
  • the T cell is a naive T cell, a central memory T cell, an effector memory T cell, a stem cell memory T cell, or any combination thereof.
  • a host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids or express proteins. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Moreover, a cell comprising a "modification" or a "heterologous" polynucleotide or binding protein includes progeny of that cell, regardless of whether the progeny were transduced, transfected, or otherwise manipulated. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells.
  • These cells may be induced to incorporate the vector or other material by use of a viral vector, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See , for example, Sambrook el al ., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989).
  • polynucleotide encoding a first protein or polypeptide can be separated from a polynucleotide encoding a second protein or polypeptide by a polynucleotide that encodes a self-cleaving polypeptide.
  • an encoded self-cleaving polypeptide comprises a 2A peptide from porcine teschovirus-l (P2A), Thoseaasigna virus (T2A), equine rhinitis A virus (E2A), or foot-and-mouth disease virus (F2A)).
  • P2A porcine teschovirus-l
  • T2A Thoseaasigna virus
  • E2A equine rhinitis A virus
  • F2A foot-and-mouth disease virus
  • markers can be used to identify, monitor or isolate a host cell transduced with a heterologous polynucleotide encoding a trigger polypeptide or binding protein as provided herein.
  • Exemplary markers include green fluorescent protein, an extracellular domain of human CD2, a truncated human EGFR (huEGFRt, (see Wang et al. , Blood 118: 1255, 2011), a truncated human CD19 (huCDl9t); a truncated human CD34 (huCD34t); a truncated human NGFR (huNGFRt); a CD 19; an EGFR; or RQR8 (see Philip et al., Blood 124 1277-1287, 2014).
  • an encoded marker comprises EGFRt, CDl9t, CD34t, NGFRt, CD 19, EGFR, or RQR8.
  • a polynucleotide encoding a trigger polypeptide has at least 80% identity to the polynucleotide sequence set forth in SEQ ID NO: 14. In further embodiments, a polynucleotide encoding a trigger polypeptide comprises the polynucleotide sequence set forth in SEQ ID NO: 14. In still further embodiments, a polynucleotide encoding a trigger polypeptide consists of the polynucleotide sequence set forth in SEQ ID NO: 14.
  • a polynucleotide encoding a trigger polypeptide has at least 80% identity to the polynucleotide sequence set forth in SEQ ID NO: 15. In further embodiments, a polynucleotide encoding a trigger polypeptide comprises the polynucleotide sequence set forth in SEQ ID NO: 15. In still further embodiments, a polynucleotide encoding a trigger polypeptide consists of the polynucleotide sequence set forth in SEQ ID NO: 15.
  • a polynucleotide encoding a trigger polypeptide has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 16. In further embodiments, a polynucleotide encoding a trigger polypeptide comprises the polynucleotide sequence set forth in SEQ ID NO: 16. In still further embodiments, a polynucleotide encoding a trigger polypeptide consists of the polynucleotide sequence set forth in SEQ ID NO: 16.
  • a polynucleotide encoding a trigger polypeptide has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO:99.
  • a polynucleotide encoding a trigger polypeptide comprises the polynucleotide sequence set forth in SEQ ID NO:99.
  • a polynucleotide encoding a trigger polypeptide consists of the polynucleotide sequence set forth in SEQ ID NO:99.
  • a polynucleotide encoding a ROR1 -specific binding protein comprises a polynucleotide having at least 80% identity to the polynucleotide sequence set forth in SEQ ID NO:80, 82, 100, or 101.
  • a polynucleotide encoding a ROR1 -specific binding protein comprises the polynucleotide sequence set forth in SEQ ID NO:80, 82, 100, or 101.
  • a polynucleotide encoding a ROR1 -specific binding protein consists of the
  • polynucleotide sequence set forth in SEQ ID NO: 80, 82, 100, or 101 is set forth in SEQ ID NO: 80, 82, 100, or 101.
  • a polynucleotide encodes a fusion protein, wherein the polynucelotide is comprised of a first polynucleotide encoding a ROR1 -specific binding protein, a second polynucleotide encoding a marker polypeptide, and third
  • polynucleotide encoding a cleavage peptide, wherein the cleavage peptide is disposed between and links the ROR1 -specific binding protein to the marker polypeptide, and wherein the fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:78, optionally not including amino acids 1-22 of SEQ ID NO:78.
  • a polynucleotide encodes a fusion protein, wherein the polynucelotide is comprised of a first polynucleotide encoding a ROR1 -specific binding protein, a second polynucleotide encoding a marker polypeptide, and third
  • polynucleotide encoding a cleavage peptide, wherein the cleavage peptide is disposed between and links the ROR1 -specific binding protein to the marker polypeptide, and wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:78, optionally not including amino acids 1-22 of SEQ ID NO:78.
  • polynucleotides that encode a trigger polypeptide, wherein the encoded trigger polypeptide specifically binds to a CD 19 antigen and the polynucleotide has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 98.
  • a polynucleotide encoding a trigger polypeptide comprises the polynucleotide sequence set forth in SEQ ID NO:98.
  • a polynucleotide encoding a trigger polypeptide consists of the polynucleotide sequence set forth in SEQ ID NO: 98.
  • the polynucleotide is comprised in a host cell.
  • a host cell that comprises a heterologous polynucleotide encoding a trigger polypeptide or binding protein is an immune cell that is modified to reduce or eliminate expression of one or more endogenous genes that encode a polypeptide product selected from PD-l, LAG-3, CTLA4, TIM3, TIGIT, an HLA molecule, a TCR molecule, or any component or combination thereof.
  • certain endogenously expressed immune cell proteins may downregulate the immune activity of a modified immune host cell (e.g ., PD-l, LAG-3, CTLA4, TIGIT), or may interfere with the binding activity of a heterologously expressed binding protein of the present disclosure (e.g., CAR, TCR) and interfere with the immune host cell binding to a target cell or antigen, or any combination thereof.
  • a modified immune host cell e.g ., PD-l, LAG-3, CTLA4, TIGIT
  • a heterologously expressed binding protein of the present disclosure e.g., CAR, TCR
  • endogenous proteins e.g., immune host cell proteins, such as an HLA
  • a donor immune cell to be used in a cell transfer therapy may be recognized as foreign by an allogeneic recipient, which may result in elimination or suppression of the donor immune cell by the allogeneic recipient.
  • a modified host immune cell is a donor cell (e.g., allogeneic) or an autologous cell.
  • a modified immune host cell of this disclosure comprises a chromosomal gene knockout of one or more of a gene that encodes PD-l, LAG-3, CTLA4, TIM3, TIGIT, an HLA component (e.g., a gene that encodes an al macroglobulin, an a2 macroglobulin, an a3 macroglobulin, a b ⁇ microglobulin, or a b2 microglobulin), or a TCR component (e.g., a gene that encodes a TCR variable region or a TCR constant region) (see, e.g., Torikai el al., Nature Sci.
  • chromosomal gene knockout refers to a genetic alteration in a host cell that prevents production, by the host cell, of a functionally active endogenous polypeptide product. Alterations resulting in a chromosomal gene knockout can include, for example, introduced nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletion, and strand breaks, as well as the heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.
  • a chromosomal gene knock-out or gene knock-in is made by chromosomal editing of a host cell.
  • Chromosomal editing can be performed using, for example, endonucleases.
  • endonucleases refers to an enzyme capable of catalyzing cleavage of a phosphodiester bond within a polynucleotide chain.
  • an endonuclease is capable of cleaving a targeted gene thereby inactivating or "knocking out" the targeted gene.
  • An endonuclease may be a naturally occurring, recombinant, genetically modified, or fusion endonuclease.
  • the nucleic acid strand breaks caused by the endonuclease are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ).
  • NHEJ non-homologous end joining
  • a donor nucleic acid molecule may be used for a donor gene "knock-in”, for target gene "knock-out”, and optionally to inactivate a target gene through a donor gene knock in or target gene knock out event.
  • NHEJ is an error- prone repair process that often results in changes to the DNA sequence at the site of the cleavage, e.g., a substitution, deletion, or addition of at least one nucleotide.
  • NHEJ may be used to "knock-out" a target gene.
  • Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.
  • a "zinc finger nuclease” refers to a fusion protein comprising a zinc finger DNA-binding domain fused to a non-specific DNA cleavage domain, such as a Fokl endonuclease.
  • ZFN zinc finger nuclease
  • Each zinc finger motif of about 30 amino acids binds to about 3 base pairs of DNA, and amino acids at certain residues can be changed to alter triplet sequence specificity (see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci.
  • ZFNs mediate genome editing by catalyzing the formation of a site-specific DNA double strand break (DSB) in the genome, and targeted integration of a transgene comprising flanking sequences homologous to the genome at the site of DSB is facilitated by homology directed repair.
  • DSB DNA double strand break
  • a DSB generated by a ZFN can result in knock out of target gene via repair by non-homologous end joining (NHEJ), which is an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site.
  • NHEJ non-homologous end joining
  • a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, made using a ZFN molecule.
  • TALEN transcription activator-like effector nuclease
  • a "TALE DNA binding domain” or “TALE” is composed of one or more TALE repeat domains/units, each generally having a highly conserved 33-35 amino acid sequence with divergent l2th and l3th amino acids.
  • the TALE repeat domains are involved in binding of the TALE to a target DNA sequence.
  • the divergent amino acid residues referred to as the Repeat Variable Diresidue (RVD), correlate with specific nucleotide recognition.
  • RVD Repeat Variable Diresidue
  • the natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD (histine-aspartic acid) sequence at positions 12 and 13 of the TALE leads to the TALE binding to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagine-isoleucine) to A, NN (asparagine-asparagine) binds to a G or A nucleotide, and NG (asparagine-glycine) binds to a T nucleotide.
  • Non-canonical (atypical) RVDs are also known (see, e.g., ET.S. Patent Publication No.
  • TALENs can be used to direct site-specific double-strand breaks (DSB) in the genome of T cells.
  • Non- homologous end joining (NHEJ) ligates DNA from both sides of a double-strand break in which there is little or no sequence overlap for annealing, thereby introducing errors that knock out gene expression.
  • homology directed repair can introduce a transgene at the site of DSB providing homologous flanking sequences are present in the transgene.
  • a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a TALEN molecule.
  • CRISPR/Cas nuclease system refers to a system that employs a CRISPR RNA (crRNA)-guided Cas nuclease to recognize target sites within a genome (known as protospacers) via base-pairing complementarity and then to cleave the DNA if a short, conserved protospacer associated motif (PAM) immediately follows 3’ of the complementary target sequence.
  • CRISPR/Cas systems are classified into three types (i.e., type I, type II, and type III) based on the sequence and structure of the Cas nucleases.
  • the crRNA-guided surveillance complexes in types I and III need multiple Cas subunits.
  • Type II system the most studied, comprises at least three components: an RNA-guided Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA).
  • the tracrRNA comprises a duplex forming region.
  • a crRNA and a tracrRNA form a duplex that is capable of interacting with a Cas9 nuclease and guiding the
  • Cas9/crRNA:tracrRNA complex to a specific site on the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA upstream from a PAM.
  • Cas9 nuclease cleaves a double-stranded break within a region defined by the crRNA spacer. Repair by NHEJ results in insertions and/or deletions which disrupt expression of the targeted locus.
  • a transgene with homologous flanking sequences can be introduced at the site of DSB via homology directed repair.
  • the crRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) (see, e.g., Jinek et al., Science 337:816-21, 2012). Further, the region of the guide RNA complementary to the target site can be altered or programed to target a desired sequence (Xie et al, PLOS One 9:el00448, 2014; U.S. Pat. Appl. Pub. No. US 2014/0068797, U.S. Pat. Appl. Pub. No. US 2014/0186843; U.S. Pat. No. 8,697,359, and PCT Publication No. WO 2015/071474; each of which is incorporated by reference).
  • a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a CRISPR/Cas nuclease system.
  • gRNA sequences and methods of using the same to knock out endogenous genes that encode immune cell proteins include those described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), the gRNAs, CAS9 DNAs, vectors, and gene knockout techniques of which are hereby incorporated by reference in their entirety.
  • Meganucleases can be divided into five families based on sequence and structure motifs: LAGLIDADG, GIY- YIG, HNH, His-Cys box and PD-(D/E)XK.
  • Exemplary meganucleases include I-Scel, I-Ceul, PI-PspI, RI-Sce, 1-SceIV, I-Csml, I-Panl, I-Scell, I-Ppol, I-SceIII, I-Crel, I- TevI, I-TevII and I-TevIII, whose recognition sequences are known (see, e.g., U.S. Patent Nos.
  • naturally occurring meganucleases may be used to promote site-specific genome modification of a target selected from PD-l, LAG3,
  • TIM3, CTLA4, TIGIT an HLA-encoding gene, or a TCR component-encoding gene.
  • an engineered meganuclease having a novel binding specificity for a target gene is used for site-specific genome modification (see, e.g., Porteus et al., Nat. Biotechnol. 23:967-73, 2005; Sussman et al., J. Mol. Biol. 342: 31-41, 2004; Epinat et al., Nucleic Acids Res. 37:2952-62, 2003; Chevalier et al., Molec. Cell 70:895-905, 2002; Ashworth et al., Nature 441:656-659, 2006; Paques et al., Curr. Gene Ther. 7:49- 66, 2007; U.S.
  • a chromosomal gene knockout is generated using a homing endonuclease that has been modified with modular DNA binding domains of TALENs to make a fusion protein known as a megaTAL.
  • MegaTALs can be utilized to not only knock-out one or more target genes, but to also introduce (knock in) heterologous or exogenous
  • polynucleotides when used in combination with an exogenous donor template encoding a polypeptide of interest.
  • a chromosomal gene knockout comprises an inhibitory nucleic acid molecule that is introduced into a host cell (e.g, an immune cell) comprising a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor associated antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor and wherein the encoded target-specific inhibitor inhibits endogenous gene expression (i.e., of PD-l, TIM3, LAG3, CTLA4, TIGIT, an HLA component, or a TCR component, or any combination thereof) in the host immune cell.
  • a host cell e.g, an immune cell
  • a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor associated antigen
  • the inhibitory nucleic acid molecule encodes a target-specific inhibitor and wherein the encoded target-specific inhibitor inhibits endogenous gene expression (i.e., of PD-l,
  • a chromosomal gene knockout can be confirmed directly by DNA sequencing of the host immune cell following use of the knockout procedure or agent.
  • Chromosomal gene knockouts can also be inferred from the absence of gene expression (e.g ., the absence of a mRNA or polypeptide product encoded by the gene) following the knockout.
  • the present disclosure also provides methods of treating a disease or condition characterized by expression of ROR1 and a second antigen targeted by ⁇ i.e., bound by, including specifically bound by, a binding domain of) a trigger polypeptide as disclosed herein, wherein the methods comprise administering a host cell (e.g., host immune cell) that expresses the trigger polypeptide and comprises a polynucleotide that encodes a ROR1 -specific binding protein and/or expresses a ROR1 -specific binding protein.
  • a host cell e.g., host immune cell
  • the trigger polypeptide comprises a binding domain as disclosed herein that specifically binds to an antigen that is not a ROR1 antigen and that is specifically expressed (i.e., is only expressed by, or is has an increased or more intense expression as compared to healthy cells) by cells having the disease or condition.
  • the trigger polypeptide binds to a B7-H3 antigen.
  • the trigger polypeptide binds to an EpCAM antigen.
  • the trigger polypeptide binds to a CD 19 antigen.
  • the trigger polypeptide binds to a L1CAM antigen.
  • the trigger polypeptide binds to a HER2 antigen.
  • the trigger polypeptide binds to a EGFR antigen.
  • a modified immune cell comprising: (i) a polynucleotide encoding a trigger polypeptide, wherein the encoded trigger polypeptide comprises (a) an extracellular portion component comprising a binding domain that specifically binds to an antigen that is specifically expressed by cells having the disease or condition, wherein the antigen is not a ROR1 antigen (e.g ., is a B7-H3, EpCAM, L1CAM, HER2, or EGFR antigen), (b) an intracellular component comprising a transcriptional factor, and (c) a Notch core domain disposed between the extracellular component and the intracellular component; and (ii) a polynucleotide comprising: an enhancer element operably linked to a nucleotide sequence encoding a binding protein that specifically binds to a ROR1 antigen, wherein the
  • Treatment refers to medical management of a disease, disorder, or condition of a subject (e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat).
  • a subject e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat.
  • an appropriate dose or treatment regimen comprising a host cell expressing a fusion protein of the present disclosure, and optionally an adjuvant, is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit.
  • Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease; stabilization of disease state; delay of disease progression; remission; survival; prolonged survival; or any
  • a “therapeutically effective amount” or “effective amount” of a fusion protein or host cell expressing a fusion protein of this disclosure refers to an amount of fusion proteins or host cells sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease;
  • a therapeutically effective amount refers to the effects of that ingredient or cell expressing that ingredient alone.
  • a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously.
  • a combination may also be a cell expressing more than one active ingredient.
  • pharmaceutically acceptable excipient or carrier or “physiologically acceptable excipient or carrier” refer to biologically compatible vehicles, e.g ., physiological saline, which are described in greater detail herein, that are suitable for administration to a human or other non-human mammalian subject and generally recognized as safe or not causing a serious adverse event.
  • statically significant refers to a p-value of 0.050 or less when calculated using the Student’s t-test and indicates that it is unlikely that a particular event or result being measured has arisen by chance.
  • adoptive cellular immunotherapy refers to administration of naturally occurring or genetically engineered, disease-antigen-specific immune cells (e.g., T cells).
  • adoptive cellular immunotherapy may be autologous (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species) or syngeneic (immune cells are from a donor genetically identical to the recipient).
  • the host immune cell and/or effector immune cell are administered to treat a hyperproliferative disorder.
  • hyperproliferative disorder refers to excessive growth or proliferation as compared to a normal or undiseased cell.
  • hyperproliferative disorders include tumors, cancers, neoplastic tissue, carcinoma, sarcoma, malignant cells, pre-malignant cells, as well as non-neoplastic or non-malignant hyperproliferative disorders (e.g ., adenoma, fibroma, lipoma, leiomyoma, hemangioma, fibrosis, restenosis, as well as autoimmune diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, or the like). Certain diseases that involve abnormal or excessive growth that occurs more slowly than in the context of a hyperproliferative disease can be referred to as
  • proliferative diseases include certain tumors, cancers, neoplastic tissue, carcinoma, sarcoma, malignant cells, pre-malignant cells, as well as non-neoplastic or non-malignant disorders
  • cancer may refer to any accelerated proliferation of cells, including solid tumors, ascites tumors, blood or lymph or other malignancies;
  • connective tissue malignancies connective tissue malignancies; metastatic disease; minimal residual disease following transplantation of organs or stem cells; multi-drug resistant cancers, primary or secondary malignancies, angiogenesis related to malignancy, or other forms of cancer.
  • Cancers treatable by presently disclosed methods and compositions include carcinomas, sarcomas, gliomas, lymphomas, leukemias, myelomas, cancers of the head or neck, melanoma, pancreatic cancer, cholangiocarcinoma, hepatocellular cancer, breast cancer, gastric cancer, non-small-cell lung cancer, prostate cancer, esophageal cancer, mesothelioma, small-cell lung cancer, colorectal cancer, glioblastoma, Askin's tumor, sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, PNET, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans (DFSP), desmoid tumor, desmoplastic small
  • GIST gastrointestinal stromal tumor
  • hemangiopericytoma hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, undifferentiated pleomorphic sarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, linitis plastic, vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, renal cell carcinoma, Grawitz tumor, ependymoma, astrocytoma, oligodendroglioma, brainstem glioma, optice nerve glioma, a mixed gli
  • adenocarcinoma hypernephroma, Transitional cell carcinoma (renal pelvis), Squamous cell carcinoma, Bellini duct carcinoma, Clear cell adenocarcinoma, Transitional cell carcinoma, Carcinoid tumor of the renal pelvis); an adrenal carcinoma (e.g.,
  • Adrenocortical carcinoma a carcinoma of the testis (e.g., Germ cell carcinoma (Seminoma, Choriocarcinoma, Embryonal carciroma, Teratocarcinoma), Serous carcinoma); Gastric carcinoma (e.g., Adenocarcinoma); an intestinal carcinoma (e.g., Adenocarcinoma of the duodenum); a colorectal carcinoma; or a skin carcinoma (e.g., Basal cell carcinoma, Squamous cell carcinoma); ovarian carcinoma, an ovarian epithelial carcinoma, a cervical adenocarcinoma or small cell carcinoma, a pancreatic carcinoma, a colorectal carcinoma (e.g., an adenocarcinoma or squamous cell carcinoma), a lung carcinoma, a breast ductal carcinoma, or an adenocarcinoma of the prostate.
  • Germ cell carcinoma Seminoma, Choriocarcinoma, Embryonal car
  • the cancer is a solid cancer and/or a hematological malignancy (e.g., triple-negative breast cancer (TBNC), non-small cell lung cancer (NSCLC), mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL)), breast adenocarcimona, ovarian carcinoma, r any combination thereof).
  • TBNC triple-negative breast cancer
  • NSCLC non-small cell lung cancer
  • MCL mantle cell lymphoma
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • breast adenocarcimona ovarian carcinoma, r any combination thereof.
  • the subject can have been previously been administered an agent that targets ROR1 (e.g., antibody or antigen-binding fragment; CAR-T cell; TCR-T cell; small organic molecule) to treat the disease or condition.
  • ROR1 e.g., antibody or antigen
  • the subject has been previously been administered lymphodepleting chemotherapy (e.g, oxaliplatin, cyclophosphamide, fludarabine, or any combination thereof), radiation therapy, or both.
  • the agent that targets ROR1 comprises: (i) an immune cell that expresses at its cell surface a binding protein that binds to a ROR1 antigen; and/or (ii) an antibody, or an antigen-binding fragment thereof, or an antibody-drug conjugate comprising the antibody or antigen-binding fragment.
  • the subject exhibits one or more of: (i) weight loss; (ii) accumulation of the agent that targets ROR1 in spleen, bone marrow, a peripheral lymph node, and/or liver (e.g, as compared to another tissue, such as lung, kidney, or pancreas); (iii) a reduced number of red blood cells (RBCs) and/or platelets (e.g., as compared to a prior number in the same subject, or as compared to a reference subject); (iv) necrosis of spleen; (v) reduced
  • hematopoiesis e.g, as compared to a prior level of hematopoiesis in the same subject, or as compared to a reference subject
  • a reduced number of erythroid cells and/or megakaryocytes e.g, as compared to a prior number in the same subject, or as compared to a reference subject
  • any combination of (i)-(vi) Thereafter, the subject may be administered a modified immune cell of the present disclosure, and may, in certain embodiments, thereafter exhibit a reduced amount, duration, and/or severity of any one or more of (i)-(vi) above.
  • a modified immune cell of this disclosure may minimize or mitigate one or more on-target/off- tumor toxicities in a subject to be treated relative to a subject treated with a host cell or a compostion including a non-logic-gated system.
  • the on-target/off-tumor toxicity may include splenic necrosis, weight loss, anemia, thrombocytopenia, myelofibrosis, hematopoietic toxicity, pulmonary toxicity, hepatic toxicity, renal toxicity, cardiac toxicity, skin toxicity, muscle toxicity, and/or neurologic toxicity.
  • a subject receiving a modified immune cell of the present disclosure exhibits a decreased amount, type, and/or severity of toxicity (e.g, splenic necrosis, weight loss, anemia, thrombocytopenia, myelofibrosis, hematopoietic toxicity, pulmonary toxicity, hepatic toxicity, renal toxicity, cardiac toxicity, skin toxicity, muscle toxicity, and/or neurologic toxicity) as compared to a reference subject that receives a host cell or a compostion comprising a transcribed agent that targets ROR1 but not comprising a trigger polypeptide of the present disclosure that controls transcription of the agent that targets ROR.
  • toxicity e.g, splenic necrosis, weight loss, anemia, thrombocytopenia, myelofibrosis, hematopoietic toxicity, pulmonary toxicity, hepatic toxicity, renal toxicity, cardiac toxicity, skin toxicity, muscle toxicity, and/or neurologic toxicity
  • Table 1 An exemplary scoring system for evaluating histopathology is provided in Table 1. Table 1. Histopathology scoring system
  • Subjects that can be treated by the present invention are, in general, human and other primate subjects, such as monkeys and apes for veterinary medicine purposes.
  • the subject may be a human subject.
  • the subjects can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
  • Cells according to the present disclosure may be administered in a manner appropriate to the disease, condition, or disorder to be treated as determined by persons skilled in the medical art.
  • a cell comprising a cell as described herein is administered
  • compositions intravenously, intraperitoneally, intratumorally, into the bone marrow, into a lymph node, or into the cerebrospinal fluid.
  • An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as the age, size, gender, and condition of the patient; the type and severity of the disease, condition, or disorder; the particular form of the active ingredient; and the method of administration.
  • methods of the present disclosure comprise administering a host cell of the present disclosure.
  • the amount of cells in a host cell of the present disclosure comprises administering a host cell of the present disclosure.
  • composition is at least one cell (for example, one modified CD8 + T cell subpopulation; one modified CD4 + T cell subpopulation) or is more typically greater than 10 2 cells, for example, up to 10 6 , up to 10 7 , up to 10 8 cells, up to 10 9 cells, or more than 10 10 cells.
  • the cells are administered in a range from about 10 6 to about 10 10 cells/m 2 , preferably in a range of about 10 5 to about 10 9 cells/m 2 .
  • the number of cells will depend upon the ultimate use for which the composition is intended as well the type of cells included therein.
  • cells modified to contain a trigger polypeptide and a binding protein specific for a particular antigen will comprise a cell population containing at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells.
  • cells are generally in a volume of a liter or less, 500 mls or less, 250 mls or less, or 100 mls or less.
  • the density of the desired cells is typically greater than 10 4 cells/ml and generally is greater than 10 7 cells/ml, generally 10 8 cells/ml or greater.
  • the cells may be administered as a single infusion or in multiple infusions over a range of time.
  • a clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 cells.
  • Unit doses are also provided herein which comprise a host cell or host cell composition of this disclosure.
  • a unit dose comprises a host cell (/%., expressing a fusion protein and a binding protein) and an effector immune cell, wherein the host cell and the effector immune cell can each be a CD4 + T cell, a CD8 + T cell, or both.
  • a unit dose comprises (i) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells (i.e., has less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less then about 1% the population of naive T cells present in a unit dose as compared to a patient sample having a comparable number of PBMCs).
  • a unit dose comprises (i) a composition comprising at least about 50% modified or unmodified CD4 + T cells, combined with (ii) a
  • a unit dose comprises (i) a composition comprising at least about 60% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 60% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a unit dose comprises (i) a composition comprising at least about 70% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 70% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a unit dose comprises (i) a composition comprising at least about 80% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 80% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a unit dose comprises (i) a composition comprising at least about 85% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 85% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a unit dose comprises (i) a composition comprising at least about 90% modified or unmodified CD4 + T cells, combined with (ii) a composition comprising at least about 90% modified or unmodified CD8 + T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.
  • a unit dose comprises equal, or approximately equal numbers of modified or unmodified CD45RA CD3 + CD8 + and modified or unmodified CD45RA CD3 + CD4 + TM cells.
  • compositions that comprise fusion proteins or cells expressing the fusion proteins as disclosed herein and a
  • compositions comprising fusion proteins or host cells as disclosed herein further comprise a suitable infusion media.
  • suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), 5% dextrose in water, Ringer's lactate can be utilized.
  • An infusion medium can be supplemented with human serum albumin or other human serum components.
  • compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art.
  • An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (i.e., weight, mass, or body area), the type and severity of the patient's condition, the undesired type or level or activity of the tagged cells, the particular form of the active ingredient, and the method of
  • an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity).
  • a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder. Prophylactic benefit of the immunogenic compositions
  • administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.
  • Certain methods of treatment or prevention contemplated herein include administering a host cell (which may be autologous, allogeneic or syngeneic) comprising a desired polynucleotide as described herein that is stably integrated into the host cell (which may be autologous, allogeneic or syngeneic) comprising a desired polynucleotide as described herein that is stably integrated into the host cell (which may be autologous, allogeneic or syngeneic) comprising a desired polynucleotide as described herein that is stably integrated into the
  • a cellular composition may be generated ex vivo using autologous, allogeneic or syngeneic immune system cells (e.g T cells, antigen-presenting cells, natural killer cells) in order to administer a desired, fusion protein-expressing T-cell composition to a subject as an adoptive immunotherapy.
  • the host cell is a hematopoietic progenitor cell or a human immune cell.
  • the immune system cell is a CD4 + T cell, a CD8 + T cell, a CD4 CD8 double-negative T cell, a gd T cell, a natural killer cell, a dendritic cell, or any combination thereof.
  • the immune system cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.
  • the cell is a CD4 + T cell.
  • the cell is a CD8 + T cell.
  • administration of a composition refers to delivering the same to a subject, regardless of the route or mode of delivery. Administration may be effected continuously or intermittently, and parenterally. Administration may be for treating a subject already confirmed as having a recognized condition, disease or disease state, or for treating a subject susceptible to or at risk of developing such a condition, disease or disease state.
  • Co-administration with an adjunctive therapy may include simultaneous and/or sequential delivery of multiple agents in any order and on any dosing schedule (e.g ., fusion protein-expressing recombinant (i.e., engineered) host cells with one or more cytokines; immunosuppressive therapy such as calcineurin inhibitors,
  • corticosteroids corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof.
  • a plurality of doses of a recombinant host cell as described herein is administered to the subject, which may be administered at intervals between administrations of about two to about four or more weeks.
  • the subject being treated is further receiving immunosuppressive therapy, such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof.
  • immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof.
  • the subject being treated has received a non-myeloablative or a myeloablative hematopoietic cell transplant, wherein the treatment may be
  • An effective amount of a pharmaceutical composition refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein.
  • An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term "therapeutic amount” may be used in reference to treatment, whereas “prophylactically effective amount” may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative course.
  • a disease or disease-state e.g., recurrence
  • the level of a CTL immune response may be determined by any one of numerous immunological methods described herein and routinely practiced in the art.
  • the level of a CTL immune response may be determined prior to and following administration of any one of the herein described fusion proteins expressed by, for example, a T cell.
  • Cytotoxicity assays for determining CTL activity may be performed using any one of several techniques and methods routinely practiced in the art (see, e.g, Henkart et al., "Cytotoxic T-Lymphocytes" in Fundamental Immunology, Paul (ed.) (2003 Lippincott Williams & Wilkins, Philadelphia, PA), pages 1127-50, and references cited therein).
  • Antigen-specific T cell responses are typically determined by comparisons of observed T cell responses according to any of the herein described T cell functional parameters (e.g. , proliferation, cytokine release, CTL activity, altered cell surface marker phenotype, etc.) that may be made between T cells that are exposed to a cognate antigen in an appropriate context (e.g., the antigen used to prime or activate the T cells, when presented by immunocompatible antigen-presenting cells) and T cells from the same source population that are exposed instead to a structurally distinct or irrelevant control antigen.
  • a cognate antigen e.g., the antigen used to prime or activate the T cells, when presented by immunocompatible antigen-presenting cells
  • a response to the cognate antigen that is greater, with statistical significance, than the response to the control antigen signifies antigen-specificity.
  • a biological sample may be obtained from a subject for determining the presence and level of an immune response to a tagged protein or cell as described herein.
  • a "biological sample” as used herein may be a blood sample (from which serum or plasma may be prepared), biopsy specimen, body fluids (e.g, lung lavage, ascites, mucosal washings, synovial fluid), bone marrow, lymph nodes, tissue explant, organ culture, or any other tissue or cell preparation from the subject or a biological source.
  • Biological samples may also be obtained from the subject prior to receiving any immunogenic composition, which biological sample is useful as a control for establishing baseline (i.e., pre-immunization) data.
  • compositions described herein may be presented in unit- dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to preserve the stability of the formulation until.
  • a unit dose comprises a recombinant host cell as described herein at a dose of about 10 7 cells/m 2 to about 10 11 cells/m 2 .
  • the composition may also include sterile aqueous or oleaginous solution or suspension.
  • suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer’s solution, isotonic salt solution, l,3-butanediol, ethanol, propylene glycol or polythethylene glycols in mixtures with water.
  • Aqueous solutions or suspensions may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate.
  • any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of recombinant cells or active compound calculated to produce the desired effect in association with an appropriate pharmaceutical carrier.
  • an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide therapeutic or prophylactic benefit.
  • a response can be monitored by establishing an improved clinical outcome (e.g ., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to non-treated subjects.
  • Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome.
  • Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which are routine in the art and may be performed using samples obtained from a subject before and after treatment.
  • Methods according to this disclosure may further include administering one or more additional agents to treat the disease or disorder in a combination therapy.
  • a combination therapy comprises administering a fusion protein (or an engineered host cell expressing the same) with (concurrently, simultaneously, or sequentially) an immune checkpoint inhibitor.
  • a combination therapy comprises administering fusion protein of the present disclosure (or an engineered host cell expressing the same) with an agonist of a stimulatory immune checkpoint agent.
  • a combination therapy comprises administering a fusion protein of the present disclosure (or an engineered host cell expressing the same) with a secondary therapy, such as chemotherapeutic agent, a radiation therapy, a surgery, an antibody, or any combination thereof.
  • immune suppression agent refers to one or more cells, proteins, molecules, compounds or complexes providing inhibitory signals to assist in controlling or suppressing an immune response.
  • immune suppression agents include those molecules that partially or totally block immune stimulation; decrease, prevent or delay immune activation; or increase, activate, or up regulate immune suppression.
  • immunosuppression agents to target include PD-l, PD-L1, PD- L2, LAG3, CTLA4, B7-H3, B7-H4, CD244/2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2aR, immunosuppressive cytokines (e.g., IL-10, IL-4, IL-1RA, IL-35), IDO, arginase, VISTA, TIGIT, LAIR1, CEACAM-l, CEACAM-3, CEACAM-5, Treg cells, or any combination thereof.
  • immunosuppression agents to target include PD-l, PD-L1, PD- L2, LAG3, CTLA4, B7-H3, B7-H4, CD244/2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine
  • An immune suppression agent inhibitor may be a compound, an antibody, an antibody fragment or fusion polypeptide (e.g., Fc fusion, such as CTLA4-Fc or LAG3-Fc), an antisense molecule, a ribozyme or RNAi molecule, or a low molecular weight organic molecule.
  • a method may comprise administering an engineered host cell of the present disclosure with one or more inhibitor of any one of the following immune suppression components, singly or in any combination.
  • a modified immune cell is used in combination with a PD-l inhibitor, for example a PD-l -specific antibody or binding fragment thereof, such as pidilizumab, nivolumab (Keytruda, formerly MDX-1106), pembrolizumab (Opdivo, formerly MK-3475), MEDI0680 (formerly AMP-514), AMP-224, BMS-936558 or any combination thereof.
  • a PD-l inhibitor for example a PD-l -specific antibody or binding fragment thereof, such as pidilizumab, nivolumab (Keytruda, formerly MDX-1106), pembrolizumab (Opdivo, formerly MK-3475), MEDI0680 (formerly AMP-514), AMP-224, BMS-936558 or any combination thereof.
  • a modified immune cell of the present disclosure is used in combination with a PD-L1 specific antibody or binding fragment thereof, such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof.
  • a modified immune cell of the present disclosure is used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.
  • a modified immune cell is used in combination with an inhibitor of CTLA4.
  • a modified immune cell of the present disclosure is used in combination with a CTLA4 specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof.
  • a modified immune cell of the present disclosure is used in combination with a B7-H3 specific antibody or binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both. Additional antibodies that bind to B7- H3 are disclosed in PCT Publication Nos. WO 2011/109400A2 and WO
  • a B7-H4 antibody binding fragment may be a scFv or fusion protein thereof, as described in, for example, Dangaj et al, Cancer Res. 73:4820, 2013, as well as those described in U.S. Patent No. 9,574,000 and PCT Patent Publication Nos.
  • a modified immune cell of the present disclosure is used in combination with an inhibitor of CD244.
  • a modified immune cell of the present disclosure is used in combination with an inhibitor of BLTA, HVEM, CD 160, or any combination thereof.
  • Anti CD- 160 antibodies are described in, for example, PCT Publication No. WO 2010/084158.
  • a modified immune cell of the present disclosure is used in combination with an inhibitor of TIM3.
  • a modified immune cell of the present disclosure is used in combination with an inhibitor of Gal9. In certain embodiments, a modified immune cell of the present disclosure is used in combination with an inhibitor of adenosine signaling, such as a decoy adenosine receptor.
  • a modified immune cell of the present disclosure is used in combination with an inhibitor of A2aR.
  • a modified immune cell of the present disclosure is used in combination with an inhibitor of KIR, such as lirilumab (BMS-986015).
  • a modified immune cell of the present disclosure is used in combination with an inhibitor of an inhibitory cytokine (typically, a cytokine other than TGFP) or Treg development or activity.
  • an inhibitory cytokine typically, a cytokine other than TGFP
  • Treg development or activity typically, a cytokine other than TGFP
  • a modified immune cell of the present disclosure is used in combination with an IDO inhibitor, such as levo-l -methyl tryptophan, epacadostat (INCB024360; Liu et al., Blood 775:3520-30, 2010), ebselen (Terentis et al. , Biochem. 49 591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research l04th Annual Meeting 2013; Apr 6-10, 2013), 1- methyl-tryptophan (l-MT)-tira-pazamine, or any combination thereof.
  • an IDO inhibitor such as levo-l -methyl tryptophan, epacadostat (INCB024360; Liu et al., Blood 775:3520-30, 2010), ebselen (Terentis et al. , Biochem. 49 591-600, 2010), indoximod, NLG919 (Mau
  • a modified immune cell of the present disclosure is used in combination with an arginase inhibitor, such as N(omega)-Nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof.
  • an arginase inhibitor such as N(omega)-Nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof.
  • a modified immune cell of the present disclosure is used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).
  • a modified immune cell of the present disclosure is used in combination with an inhibitor of TIGIT such as, for example, COM902
  • CD155 an inhibitor of CD155, such as, for example, COM701 (Compugen), or both.
  • a modified immune cell of the present disclosure is used in combination with an inhibitor of PVRIG, PVRL2, or both.
  • Anti-PVRIG antibodies are described in, for example, PCT Publication No. WO 2016/134333.
  • Anti-PVRL2 antibodies are described in, for example, PCT Publication No.
  • a modified immune cell of the present disclosure is used in combination with a LAIR1 inhibitor.
  • a modified immune cell of the present disclosure is used in combination with an inhibitor of CEACAM-l, CEACAM-3, CEACAM-5, or any combination thereof.
  • a modified immune cell of the present disclosure is used in combination with an agent that increases the activity (i.e., is an agonist) of a stimulatory immune checkpoint molecule.
  • a modified immune cell of the present disclosure can be used in combination with a CD137 (4-1BB) agonist (such as, for example, urelumab), a CD 134 (OX-40) agonist (such as, for example, MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, a CD27 agonist (such as, for example, CDX-l 127), a CD28 agonist (such as, for example, TGN1412, CD80, or CD86), a CD40 agonist (such as, for example, CP-870,893, rhuCD40L, or SGN-40), a CD122 agonist (such as, for example, IL-2) an agonist of GITR (such as, for example, humanized monoclonal antibodies described in
  • a method may comprise administering a modified immune cell of the present disclosure with one or more agonist of a stimulatory immune checkpoint molecule, including any of the foregoing, singly or in any combination.
  • a combination therapy comprises a modified immune cell of the present disclosure and a secondary therapy comprising one or more of: an antibody or antigen binding-fragment thereof that is specific for a cancer antigen expressed by the non-inflamed solid tumor, a radiation treatment, a surgery, a chemotherapeutic agent, a cytokine, RNAi, a further adoptive cell therapy, or any combination thereof.
  • a combination therapy method comprises administering a modified immune cell and further administering a radiation treatment or a surgery.
  • Radiation therapy is well-known in the art and includes X-ray therapies, such as gamma-irradiation, and radiopharmaceutical therapies. Surgeries and surgical techniques appropriate to treating a given cancer or tumor in a subject are known to those of ordinary skill in the art.
  • a combination therapy method comprises administering a modified immune cell of the present disclosure and further administering a chemotherapeutic agent.
  • a chemotherapeutic agent includes, but is not limited to, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), and a DNA repair inhibitor.
  • Illustrative chemotherapeutic agents include, without limitation, the following groups: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busul
  • antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L- asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents;
  • antiproliferative/antimitotic alkylating agents such as nitrogen mustards
  • mycophenolate mofetil anti-angiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti- sense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-l l) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydro
  • Cytokines are used to manipulate host immune response towards anticancer activity. See, e.g., Floros & Tarhini, Semin. Oncol. 42 ⁇ 4):539-548, 2015. Cytokines useful for promoting immune anticancer or antitumor response include, for example, IFN-a, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, singly or in any combination with the cells of this disclosure.
  • a polynucleotide or vector is used to generate a modified immune cell that comprises, contains, and/or expresses the polynucleotide and/or the expression product of the polynucleotide or vector.
  • tCDl9 A truncated cell-surface murine CD 19 (tCDl9) was co-expressed with the CAR using a 2A ribosomal skip sequence to enable detection of tCDl9 + CAR-T cells by flow cytometry.
  • Adoptive transfer of ROR1 CAR-T cells or control T cells transduced with tCDl9 alone into C57BL/6 or BALB/c mice did not induce toxicity, but engraftment of donor T cells was low ( ⁇ 0.02% of live cells) (FIGS. 1B, 1C).
  • mice were pre-conditioned with sub-lethal radiation (500R) to deplete endogenous lymphocytes and increase engraftment of transferred T cells (Gattinoni et al., 2005), all mice receiving ROR1 CAR-T cells, but not control T cells, progressively lost weight and died within two weeks (FIG. 1D).
  • mice Analysis of organ histology from treated mice identified significant pathology in the spleen and bone marrow. Whereas spleen and bone marrow from control T cell- treated mice showed hyperplasia and recovery of all hematopoietic lineages following radiation, spleens from mice treated with ROR1 CAR-T cells showed diffuse necrosis with a paucity of nucleated cells by 14 days after T cell transfer (FIGS. 1 J and 1K; scoring as in FIG. 1L).
  • Femurs from ROR1 CAR-T cell-treated mice exhibited myelofibrosis at the epiphyseal ends of the bone and markedly reduced hematopoiesis, with an absence of erythroid cells and megakaryocytes consistent with the marked decline in RBC and platelet counts (FIGS. 1 J and 1K).
  • histologic changes in the lungs, pancreas, kidney, and small and large intestines were mild, and resolved to that observed with control T cells by 14 days (FIGS. 1M, 1N).
  • Lymphodepletion and cell dose are important factors determining engraftment and toxicity of adoptively transferred T cells in humans (Hay et al., 2017; Turtle et al., 20l6b).
  • mice were given 100R or 500R of radiation, or 100 mg/kg or 200 mg/kg of Cy, prior to adoptive T cell transfer.
  • mice preconditioned with the lower doses exhibited reduced weight loss and survived (FIGS. 2A, 2B).
  • Low-dose preconditioned mice had less robust CAR-T cell expansion in the peripheral blood, suggesting that more intense lymphodepletion resulted in toxicity by promoting greater CAR-T cell proliferation and accumulation (FIG. 2C).
  • Giving a two fold lower dose of ROR1 CAR-T cells to mice conditioned with 500R also induced intermediate weight loss, and all mice eventually recovered (FIG. 2D). Mice that received a lower CAR-T cell dose showed less expansion and faster contraction of CAR-T cells in the peripheral blood (FIG.
  • ROR1 CAR-T cells accumulated in spleen and bone marrow to levels comparable to those in mice that received chemotherapy or radiation, were PD-l + and Ki67 + , and eventually upregulated TIM-3, suggesting that some RORl + target cells were being recognized in those tissues in the absence of pre-conditioning (FIGS. 3C, 3D). Although weight loss and hematopoietic toxicity did not develop, splenic necrosis observed in WT irradiated mice receiving ROR1 CAR-T cells occurred in Rag2 /_ mice (FIGS. 3E, 3F).
  • ROR1 CAR-T ROR1 "knockout" mice were generated by crossing ROR 1 n/n mice to EIIa-Cre mice, which express Cre in the early mouse embryo, resulting in deletion of ROR1 in all tissues (Ho et al., 2012). WT or ROR1-KO mice were irradiated (500R) and received either control or ROR1 CAR-T cells.
  • ROR1-KO mice that received Rl 1 ROR1 CAR-T cells did not exhibit any of the toxicities observed in WT mice, including weight loss, anemia, thrombocytopenia, and splenic necrosis, indicating that toxicity was due to recognition of ROR1 (FIGS. 4A-4C).
  • hematopoiesis (Anthony and Link, 2014).
  • Different bone marrow stromal subsets from WT mice were sorted, and high levels of mRORl transcript were found in osteoblasts and mesenchymal stem cells (MSC), but not in endothelial cells or hematopoietic progenitors.
  • Transcript levels were further increased in MSC, but not in osteoblasts or endothelial cells, 48 hours after irradiation (500R) (FIG. 5A).
  • ROR1 CAR-T cells secreted high levels of IFNy upon co-culture with MSC derived from WT mice, but not after co-culture with MSC from ROR1-KO mice, providing direct evidence that WT MSC express sufficient ROR1 protein to activate CAR-T cells (FIG. 5B).
  • CFU assays performed on femurs from control- and CAR-T cell-treated mice revealed that MSC were drastically reduced in CAR-T cell-treated mice, demonstrating these cells were targeted in vivo (FIGS. 5C, 5D).
  • Pre-B cell progenitors which are known to express ROR1 (Hudecek et al., 2010), were also absent from ROR1 CAR-T cell-treated mice (FIG. 5C). By contrast, no differences in erythrocyte, megakaryocyte, or
  • CD45-Terl l9-VE-cadherin + endothelial cells and CD45 Terl l9 Tcf2l + perivascular stromal cells produce Scf and CXCL12 that support EMH in situations of stress to the bone marrow (Inra et al., 2015).
  • Microarray data indicated that mRORl was enriched in Scf-producing spleen stromal cells relative to unenriched spleen cells, suggesting that elimination of these cells by ROR1 CAR-T cells may be responsible for the observed splenic necrosis (Inra et al., 2015).
  • ROR1 CAR-T cells became activated and secreted IFNy upon co-culture with CD45 stromal, but not CD45 + hematopoietic, splenic cells, indicating recognition of a stromal population in the spleen (FIG. 5E).
  • Different stromal populations were sorted, and mRORl expression was analyzed by RT-PCR.
  • Most Tcf2l + stromal cells were uniformly PDGFRP + ; therefore sorting was performed for CD45 VE cadherin + splenic endothelial cells and CD45 PDGFRP + stromal cells from WT mice prior to and 48 hours after radiation.
  • mRORl transcripts were found in both splenic stromal subsets, but not in CD45 + hematopoietic cells from the spleen, with mRORl transcript levels selectively increased in CD45 PDGFRP + stromal cells 48 hours after irradiation (FIG. 5F). These data show that ROR1 CAR-T cells target splenic stromal cells and bone marrow MSC, resulting in splenic necrosis, failure of EMH, and the inability to recover hematopoiesis after cytotoxic therapy.
  • EPCAM-TRIGGER-INDUCIBLE ROR1 CAR-T CELLS SELECTIVELY TARGET EPCAM + ROR1 + TUMORS BUT NOT EPCAM ROR1 + NORMAL TISSUES Strategies that mitigate off-tumor toxicity to normal tissues without
  • a lentiviral vector was constructed that encodes a myc-tagged EpCAM-specific trigger polypeptide bearing a Gal4-VP64 intracellular transcription activation domain; a second lentiviral vector was generated in which the Rl 1 ROR1 4- 1 BB/E ⁇ 3z CAR transgene was placed under control of the UAS promoter that is activated by Gal4-VP64 released after engagement of the synthetic Notch receptor (FIG. 6B).
  • Truncated CD 19 was included in the inducible CAR cassette as a surrogate marker of CAR expression to verify lack of basal CAR expression and CAR induction after trigger polypeptide engagement.
  • a constitutively expressed blue fluorescent protein (BFP) was placed downstream of the inducible CAR transgene to identify transduced T cells.
  • Murine CD8 + T cells were co-transduced with both lentiviral vectors and co- transduction verified by myc and BFP expression (FIG. 6C).
  • T cells expressing the trigger polypeptide were enriched by FACS sorting BFP + T cells such that -30-60% of T cells were myc + BFP + and carried the full EpCAM-specific trigger polypeptide /UAS- ROR1-CAR circuit while 40-70% of cells were myc BFP + and incapable of binding EpCAM or inducing ROR1 CAR expression (FIG. 6C).
  • tCDl9 expression was not observed in the absence of trigger polypeptide engagement, indicating lack of leakiness of CAR expression (FIG. 6C).
  • BFP-sorted trigger polypeptide -expressing T cells, ROR1 CAR-T cells, and untransduced T cells were cultured with K562, K562-mRORl, 4T1, or 4Tl-mRORl cells to test recognition of ROR1 and EpCAM single- and dual-positive tumor cells.
  • ROR1 CAR-T cells killed both EpCAM + RORl + and EpCAM RORl + tumors in 6 hour and 24 hour co-cultures.
  • trigger polypeptide-expressing T cells only recognized tumor cells expressing both EpCAM and ROR1, and comparable lytic activity to ROR1 CAR-T cells required 24 hours of co-culture, consistent with previous reports showing 12-24 hours is required to fully upregulate CAR expression after engagement of the trigger polypeptide (FIGS. 6E, 6F) (see Roybal et al., 2016).
  • tCDl9 + T cells only produced ⁇ FNy when ROR1 antigen was also present (FIG. 6G).
  • Low target antigen expression is a tumor escape mechanism that may impair the ability of trigger polypeptide-expressing T cells to upregulate CAR expression.
  • EpCAM 111 and EpCAM low 4T1 tumor cells were sorted and co- cultured with EpCAM-trigger-inducible ROR1 CAR-T cells.
  • EpCAM 111 4T1 cells had >4-fold higher expression than EpCAM low tumor cells, both populations induced CAR expression on trigger polypeptide-expessing T cells with similar kinetics and to similar levels (FIGS. 7A, 7B). Consistent with their comparable CAR
  • trigger polypeptide-expressing T cells killed both 4T 1 -EpCAM hl -mROR l and 4Tl-EpCAM low -mRORl tumors equivalently well in vitro (FIG. 7C).
  • mice were inoculated with 4Tl-mRORl tumors in the mammary fat pad and, after tumors were palpable, treated with 200 mg/kg Cy and either untransduced T cells, ROR1 CAR-T cells, or BFP-sorted trigger polypeptide expressing T cells. Cyclophosphamide was used instead of irradiation, as it is more commonly used for lymphodepletion in the clinic.
  • ROR1 CAR-T cells induced rapid weight loss within one week of transfer and induced severe anemia, thrombocytopenia, and splenic necrosis (FIG. 7D-7F).
  • mice receiving T cells expressing the trigger polypeptide recovered weight, RBC and platelet counts with the same kinetics and showed similar splenic morphology as mice that received untransduced T cells.
  • trigger polypeptide-expressing T cells did not induce toxicity to normal RORl + tissues, they mediated tumor control comparable to that observed with ROR1 CAR-T cells, and had improved survival compared to mice receiving untransduced or ROR1 CAR-T cells, though mice receiving trigger polypeptide-expressing T cells died later due to tumor outgrowth (FIGS.
  • ROR1 CAR-T cells accumulated in spleen, bone marrow, and tumors, and expressed high levels of PD-l, consistent with local activation (FIG. 71). Although transferred trigger polypeptide-expressing T cells were detected in the spleen and bone marrow, at least lO-fold fewer of these cells expressed the ROR1 CAR (FIGS. 71, 7J). By contrast, in resected tumors, trigger polypeptide-expressing T cells expressing the ROR1 CAR were present in the same high numbers as ROR1 CAR-T cells, and expressed similar levels of the CAR and markers of activation and proliferation, including PD-l and Ki- 67 (FIGS. 71, 7J). Thus, trigger polypeptide -expressing T cells exhibited tumor- selective accumulation, CAR expression, and anti-tumor activity without causing toxicity to critical RORl + cells in bone marrow and spleen.
  • Trigger polypeptide-expressing T cells were safe and effective in the 4T1 breast tumor model, but a potential limitation of this modality is the absence of spatial proximity of dual-positive tumors to single-positive normal tissue.
  • Raji lymphoma cells were transfected to express human ROR1 and injected intravenously into immunodeficient NOD/SCID/yc (NSG) mice, which results in rapidly progressive metastatic disease in blood and bone marrow.
  • Primary human T cells were transduced with a vector encoding the ROR1 CAR targeting mouse and human ROR1 and including human 4- 1 BB/E ⁇ 3z signaling domains, and truncated human EGFR (tEGFR) as a transduction marker. Similar to results observed using immunocompetent B6 and BALB/c mice, treatment of irradiated non-tumor bearing NSG mice with primary human ROR1 CAR- T cells resulted in severe weight loss and anemia, and activated PD-l + CAR-T cells accumulated in the spleen and bone marrow (FIGS. 8A-8D).
  • tEGFR truncated human EGFR
  • B7-H3 is A CLINICALLY RELEVANT TRIGGER POLYPEPTIDE TARGET THAT RESTRICTS ROR1 CAR EXPRESSION TO TUMORS AND RESCUES TOXICITY TO
  • EpCAM-specific trigger polypeptide was used in the 4T1 model (Example 5) because EpCAM is not co-expressed on RORl + bone marrow or splenic stromal cells in mice. EpCAM is, however, co-expressed with ROR1 on some normal human epithelial tissues, suggesting that EpCAM may not be optimal for some clinical applications of a trigger polypeptide/CAR approach. Examination of other antigens that could be targeted by the trigger polypeptide receptor in humans identified B7-H3 as a promising candidate, as it is often co-expressed with ROR1 in human breast, lung, and ovarian cancers (FIG.
  • a trigger polypeptide specific for human B7-H3 was designed that regulated expression of the ROR1 CAR in primary human T cells (FIG. 9B), and induced selective IFNy production and lysis of B7-H3 + RORl + , but not B7-H3-RORl + , human tumor cells after 24 hours of co-culture (FIGS. 9C-9E).
  • mice received MDA-MB-231 human breast tumors that endogenously express ROR1 and B7-H3 by subcutaneous transplant.
  • Mice treated with human T cells constitutively expressing the ROR1 CAR that targets both hRORl + tumors and normal mRORl + stromal cells had severe weight loss, anemia, and thrombocytopenia, whereas mice treated with trigger polypeptide-expressing T cells showed no toxicity, with weight loss, RBC, and platelet levels comparable to mice treated with untransduced T cells (FIGS.
  • Trigger polypeptide-expressing T cells only upregulated ROR1 CAR expression at the tumor site, and these T cells were PD-l + and Ki67 + , consistent with local activation by tumor (FIGS. 91, 9J). Few T cells expressing the trigger polypeptide and the ROR1 CAR were detected in the spleen or bone marrow, and these were PDl low (FIGS. 91, 9J).
  • ROR1 CAR-T cells were present in tumor, spleen, and bone marrow, and were PD-l + and Ki67 + at all sites.
  • RBC red blood cell
  • PHT platelet
  • the aim of this study was to investigate the mechanism of ROR1 CAR-T cell- mediated toxicities and to test whether logic-gated CAR expression using trigger polypeptides derived from Notch receptors could be used to avert toxicity without impairing anti-tumor efficacy.
  • B6 EIIa-Cre B6 CD45.1, BALB/cByJ, BALB/cByJ CD45.1, BALB/cByJ Rag2 /_ , B6 EIIa-Cre, RORlf/f, and NOD/SCID/yc (NSG) mice were purchased from Jackson Laboratory. RORlf/f mice were backcrossed for three generations to B6 mice and subsequently crossed to EIIa-Cre mice. All mice were housed and bred at the FHCRC (Seattle, WA).
  • PBMC Peripheral blood mononuclear cells
  • CD8 + and/or CD4 + T cells were enriched from PBMC of normal donors using EasySep Human CD8 and CD4 T Cell Isolation kits (Stem Cell Technologies). Cell Lines
  • K562, 4T1, Raji, and MDA-MB-231 cell lines were obtained from the American Type Culture Collection and maintained in RPMI 1640 with 5% FBS, lOOU/ml penicillin/streptomycin and 2mM L-glutamine or RPMI 1640 with 10% FBS, lmM sodium pyruvate, lmM HEPES, lOOU/ml penicillin/streptomycin, and 50mM b- mercaptoethanol.
  • SK-N-DZ cells were generously provided by Michael Jensen (Seattle Children’s Hospital).
  • Lenti-X cells for lentiviral packaging were purchased from Clontech.
  • Plat-E cells for retroviral packaging were purchased from Cell Biolabs.
  • 4T1- mRORl, K562-mRORl, and Raji-hRORl cell lines were generated by retroviral transduction with full-length murine ROR1 cDNA (ETniProt: Q9Z139) or human ROR1 cDNA (UniProt: Q01973) and subsequent FACS sorting of ROR1+ cells to >95% purity.
  • MDA-MB-231 GFP-ffluc and Raji GFP-ffluc cell lines were generated by retroviral transduction with cDNA encoding GFP fused to firefly luciferase (ffluc) and subsequent FACS sorting of GFP + cells to >95% purity.
  • 4T 1 -EpCAM hl , 4T1- EpCAM low , 4T l -mROR l -EpCAM hi , and 4T 1 -mRORl -EpC AM low cell lines were generated by FACS sorting cells to >95% purity. All cells were tested bi-monthly for the absence of mycoplasma.
  • the mp7l retroviral vector was modified to encode either a murine ROR1- specific CAR or truncated murine CD 19 (mp7l-tCDl9, UniProt: P25918, amino acids [aa] 1-321) for transduction of control murine T cells (Engels et ak, 2003).
  • the CAR possessed a murine CD8a signal peptide (UniProt: P01731, aal-27), Rl l scFv, modified human IgG4 long spacer with 4/2NQ mutations (Hudecek et ak, 2015), murine CD28 transmembrane (UniProt: P31041, aal5 l-l77), murine 4-1BB (UniProt: P20334, aa2l 1-256), murine E03z (UniProt: P24161, aa52-l64), and was linked by a P2A ribosomal skip element to murine truncated tCDl9 (mp71 -R 1 1 -41 BB-E ⁇ 3z-R2A- tCDl9).
  • the HIV7 lentiviral vector was modified to encode a human ROR1 -specific CAR possessing a human GM-CSFR signal peptide (UniProt: P15509, aal-22), Rl 1 scFv, modified human IgG4 long spacer with 4/2NQ mutations (Hudecek et ak, 2015), human CD28 transmembrane (UniProt: P10747, aal53-l79), human 4-1BB (UniProt: Q07011, aa2l4-255), and human CD3C (UniProt: P20963-3, aa52-l63).
  • the CAR sequence was linked by a T2A ribosomal skip element to human truncated EGFR (tEGFR) (Wang et ah, 2011).
  • Trigger polypeptides and response elements were obtained from Addgene (Addgene plasmids #79123 and 79125).
  • Addgene Addgene plasmids #79123 and 79125.
  • G8.8 an EpCAM-specific monoclonal antibody, G8.8, was synthesized in VH-VL format with N-terminal murine CD8a signal peptide and a myc tag (EQKLISEEDL (SEQ ID NO:9)) (Bourquin et al., 2014).
  • B7-H3-specific monoclonal antibody BRCA69D
  • BRCA69D N-terminal human CD8a signal peptide (ETniProt: P01732, aal-2l) and myc tag (Loo et al., 2012).
  • EniProt P01732, aal-2l
  • myc tag Myc tag
  • Completed fragments were fused onto the synNotch-Gal4VP64 receptor backbone (Addgene plasmid #79125) in place of the CD 19-specific scFv.
  • Addgene plasmid #79123 was replaced with the previously described murine or human ROR1 CAR transgenes.
  • constitutive myc tag or BFP expression defined T cells transduced with the trigger polypeptide and response element, respectively.
  • tCDl9 or tEGFR expression identified cells actively expressing the Rl 1 ROR1 CAR.
  • constitutive CAR controls for trigger polypeptide the previously described murine or human ROR1 CARs were cloned into the pHR backbone under control of the PGK promoter. All cloning was performed using fusion PCR, Gibson assembly, and/or restriction enzyme digest. Plasmids were verified by restriction digest and capillary sequencing prior to use. Trigger polypeptide, response element, and CAR amino acid sequences are provided in herein.
  • Retrovirus was produced by transient transfection (Clontech) of Plat-E cells with the indicated MP71 vectors (Engels et al., 2003; Morita et al., 2000). 48 hours after transfection, viral supernatant was harvested and filtered through a 0.45-pm syringe filter (Millipore). 24-well non-tissue culture plates were coated with l2.5pg/ml RetroNectin (TaKaRa) according to the manufacturer’s protocol, and plates were loaded with lml filtered virus per well and centrifuged for 2 hours at 3000xg at 32°C.
  • Murine CD8 + and/or CD4 + T cells were enriched from spleens and peripheral lymph nodes of congenic CD45.1 B6 or BALB/c mice using untouched negative isolation kits (Stem Cell) and stimulated with 1 pg/ml each of plate-bound anti-CD3 and anti-CD28 (clone 145-2C11 and 37.51, respectively) for 24 hours in a 37°C, 5% C02 incubator in complete RPMI (RPM1 1640, 10% heat inactivated FBS, lmM sodium pyruvate, lmM HEPES, lOOU/ml penicillin/streptomycin, 50mM b-mercaptoethanol) supplemented with 50U/ml recombinant murine IL-2 (Peprotech).
  • RPM1 1640 10% heat inactivated FBS, lmM sodium pyruvate, lmM HEPES, lOOU/ml penicillin/streptomycin, 50mM b-
  • Murine T cells were harvested from anti-CD3/28-coated plates and resuspended to lxlO 6 cells/ml in complete RPMI supplemented with 50U/ml IL-2 and anti-CD3/28 mouse T-activator Dynabeads (ThermoFisher) at a bead to cell ratio of 1 : 1. Viral supernatant was aspirated from RetroNectin-coated plates, plates were rinsed with PBS, and lml (lxlO 6 ) T cells were added to each virus-coated well. Plates were then centrifuged at 800g for 30 mins at 32°C and returned to 37°C, 5% C02 incubators.
  • a second transduction was performed as described the next day by harvesting another batch of viral supernatant from Plat-E cells 72 hours after transfection. T cells were subsequently harvested, counted, and resuspended in complete RPMI with 50ng/ml IL-15 every 1-2 days after. 4-5 days after transduction, magnetic beads were removed and T cell transduction was measured by flow cytometry staining for tCDl9 and/or ROR1 CAR. Transduction rates between control and ROR1 CAR-T cells were normalized by diluting cultures with untransduced T cells cultured in parallel such that the proportion of transduced cells was the same between control and CAR-T cell populations, and control and CAR-treated mice received the same number of transduced and untransduced T cells.
  • lentivirus was produced by transient calcium phosphate transfection of the packaging cell line LentiX with the indicated pHR lentiviral vectors, psPAX2 (Addgene #12260), and rHIT123 ecotropic envelope (Soneoka et ak, 1995). Viral supernatant was harvested 48 and 72 hours after transfection and filtered through a 0.45-pm pore filter. Lentivirus was concentrated 40X by mixing filtered lentivirus with 40% polyethylene glycol (PEG, Sigma) at a PEG to virus ratio of 1 :3 for 1-2 hours at 4°C.
  • PEG polyethylene glycol
  • the virus/PEG mixture was then centrifuged at l500g for 45 minutes at 4°C, supernatant was aspirated, and the virus pellet was resuspended in 40X smaller volume of serum-free DMEM and snap-frozen in liquid nitrogen for long-term storage.
  • Frozen virus was used to transduce murine T cells using the RetroNectin-based transduction protocol described above. When co-transduction of two constructs was desired, frozen virus from both vectors was mixed 1 : 1 and added to the same RetroNectin-coated well.
  • BFP + trigger polypeptide-expressing T cells were FACS sorted to enrich for T cells capable of inducing expression of the ROR1 CAR before adoptive transfer into mice.
  • LentiX cells were transiently transfected with the indicated CAR vectors, psPAX2 (Addgene #12260), and pMD2.G (Addgene #12259) packaging plasmids.
  • psPAX2 Additional CD8 + and CD4 + T cells were activated with Dynabeads Human T-Activator CD3/28 (Therm oFisher) in fresh CTL medium (RPMI 1640 with 10% human serum, 2 mM L-glutamine, 25mM HEPES, penicillin/streptomycin
  • T cells were transduced by centrifugation at 800g for 90 min at 32°C with filtered lentiviral supernatant supplemented with 4.4pg/mL polybrene (Millipore). Viral supernatant was replaced 8 hours later with fresh CTL medium with 50U/ml human IL-2, and T cells were further expanded with half media changes every 48 hours. Dynabeads were removed on day 6.
  • T cells were FACS sorted to >95% purity with the following markers: untransduced T cells: tEGFR ; ROR1 CAR- T cells: tEGFR + ; trigger polypeptide T cells: myc + BFP + . Sorted T cells were subsequently expanded for 4-5 days in CTL medium with 50U/ml human IL-2 prior to in vitro assays or adoptive transfer. CD8 + and CD4 + T cells were cultured and transduced separately.
  • B6 or BALB/c mice were pre-conditioned with sublethal radiation (100R or 500R) or with intraperitoneal injection with cyclophosphamide (lOOmg/kg or
  • mice 200mg/kg and 5-6 hours later were injected intravenously by retro-orbital injection with lxlO 6 CD8 + tCDl9 + control or ROR1 CAR-T cells.
  • NSG mice were pre-conditioned with sublethal radiation (250R) and 5-6 hours later were injected intravenously by tail vein injection with 2xl0 6 CD8 + untransduced or CD8 + tEGFR + ROR1 CAR-T cells. Mice were weighed just prior to pre-conditioning and 1-2 times per week thereafter. Percent weight change was calculated as: (weight at time X - weight at time 0) / (weight at time 0). All mice in each experiment were sacrificed when any individual mice showed clinical signs of severe disease or 20 percent weight loss.
  • Cell suspensions were prepared from spleen and peripheral lymph nodes by tissue disruption with glass slides and filtered through a 40pm filter.
  • femurs and tibia were flushed with complete RPMI using a 27-gauge needle and cells were filtered with 40pm filter.
  • Liver, kidney, pancreas, lungs, and tumors were digested with lOmg/ml collagenase type IV (Worthington) for lhr at 37°C with gentle agitation and then filtered through a 40pm filter.
  • Cells were lysed with ACK lysing buffer (Gibco) and resuspended as single cell suspensions for downstream analysis.
  • ACK lysing buffer Gibco
  • bone marrow was isolated from femurs and tibia as described above, filtered through a 40pm filter, and ACK lysed. Cells were subsequently stained and FACS sorted for subpopulations using the following markers: LT-HSC (Lin-Scal + ckit + CDl50 + CD48 Flt3 ), pre-MEP (Lin- S ca 1 ckit + CD 150 + CD34 + F cyR 10 ), MEP (Lin-Scal ckit + CD34-FcYR-CDl50-), CMP (Lin Scal ckit + CDl50 CD34 + FcYR l0 ), pre-B cells (CD45 + Terl l9 B220 + ), where lineage- negative (Lin ) cells are defined as CD3 CD4 CD8 B220 CDl lb CDl lc Gr-l Terl 19 .
  • femurs and tibia were crushed with a mortar and pestle and digested with lmg/ml collagenase type I (Worthington) and 200U/ml DNase I (Stem Cell) for 1 hour at 37°C. Cells were filtered through a 40pm filter, ACK lysed, and depleted of CD45 + cells using anti-CD45 microbeads (Miltenyi).
  • Cells were subsequently stained and FACS sorted for subpopulations using the following markers: mesenchymal stem cells (CD45 Terl l9 PDGFRp + CD5 l + CD3 l ), endothelial cells (CD45 Terl l9 PDGFRp CD5 l CD3 l + ), and osteoblasts (CD45 Terl l9 PDGFRp CD5 l + CD3 l ).
  • mesenchymal stem cells CD45 Terl l9 PDGFRp + CD5 l + CD3 l
  • endothelial cells CD45 Terl l9 PDGFRp CD5 l CD3 l +
  • osteoblasts CD45 Terl l9 PDGFRp CD5 l + CD3 l .
  • spleens were cut into ⁇ lmm3 fragments using scissors and digested with lmg/ml collagenase type I (Worthington) and 200U/ml DNase I (Stem Cell) for 1 hour at
  • Bone marrow stromal cells were prepared from femurs and tibia of WT or ROR1-KO mice as described above, and MSCs were expanded in vitro using the MesenCult Expansion Kit (StemCell) by culturing in a hypoxic incubator (5% 02, 5% C02, 37°C) for 14 days.
  • a hypoxic incubator 5% 02, 5% C02, 37°C
  • spleens were digested as described above and separated into CD45 + and CD45 fractions using anti-CD45 microbeads (Miltenyi).
  • 50,000 in w/ra-expanded MSC or splenic cells were co-cultured with 50,000 CD8 + control or ROR1 CAR-T cells in 0.2 ml of complete RPMI in triplicate in 96-well EG-bottomed plates (Costar) at 37°C, 5% C02. After 48 hours, supernatant was harvested and frozen at -20°C for long-term storage and analyzed for IFNy expression using the Ready-Set-Go Mouse IFNy ELISA Kit (eBioscience).
  • Peripheral blood was collected by retro-orbital bleeds into serum separator tubes or EDTA FACS tubes.
  • Serum separator tubes were incubated at room temperature for 30 minutes to allow blood to clot and then centrifuged at l5,800xg for 5 mins. Serum was either frozen at -80°C for multiple cytokine immunoassay by the FHCRC Immune Monitoring Core (Luminex) or submitted to Phoenix Central Labs for serum chemistry analysis. Blood in EDTA FACS tubes was submitted to Phoenix Central Labs for complete blood count analysis with differential. Flow Cytometry
  • Biotinylated IgGl Rl 1 and biotinylated recombinant Fc-mRORl were used to measure mRORl and ROR1 CAR expression, respectively (Yang et al., 2011).
  • For intracellular staining cells were surface stained as described, washed and permeabilized for 20 minutes with
  • cytokine staining For intracellular cytokine staining following restimulation, 50,000 T cells were stimulated with 50,000 tumor cells or with 50ng/ml PMA and lpg/ml ionomycin in 0.2 ml complete RPMI in triplicate in 96-well EG-bottomed plates (Costar) at 37°C, 5% C02 for 6 hours or 24 hours. GolgiPlug (BD) was added to all wells according to the manufacturer’s protocol for the last 6 hours of culture before staining for flow cytometry as described above. Data were acquired on LSRII, Canto 2 or Symphony flow cytometers (BD Biosciences) and analyzed using FlowJo software (Treestar). Immunohistochemistry and Disease Scoring
  • Tissues were immersion fixed in 10% neutral buffered formalin, paraffin embedded, cut into 5 pm sections, and stained with hematoxylin and eosin by the FHCRC Experimental Histopathology Core. Sections were scored semi-quantitatively from 0 to 4 for inflammation and toxicity in a blinded fashion. Changes typically associated with each grade for various tissues are described in Figure 1L. For ROR1 and B7-H3 staining, formalin-fixed paraffin-embedded tissues were sectioned onto charged slides, baked for 1 hour at 60°C, de-paraffmized in xylene, and rehydrated in graded dilutions of ethanol to water.
  • Antigen retrieval was achieved in a Decloaking Chamber (Biocare Medical, Pacheco, CA) with a Tris-EDTA solution (lOmM Tris, 1 mM EDTA, 0.05% tween, pH 9.4) for 25 min at 1 l0°C before staining the slides on a Biocare intelliPATH system. Endogenous peroxidase was blocked with 3% H202 for 5 minutes followed by protein blocking with Background Punisher (Biocare Medical) for 5 minutes. ROR1 primary antibody (2ug/ml, mouse clone 6D4, Fred Hutchinson Cancer Research Center) was applied for 30 minutes then rinsed with TBS Automation Wash buffer (Biocare). OPAL Polymer HRP Mouse plus Rabbit secondary
  • Femurs and tibia were isolated from B6 or EIIa-Cre/RORlf/f mice and flushed using a 27 gauge needle to isolate bone marrow cells. Bone marrow cells were filtered using 40pm filters, ACK lysed, and depleted of CD4 + and CD8 + cells using anti-CD4 and anti-CD8 microbeads (Miltenyi Biotec). 2-4xl0 6 T cell-depleted bone marrow cells were injected intravenously into lethally irradiated (1000 Rad) B6 or EIIa-Cre/RORlf/f mice. Mice were bled retro-orbitally 8 weeks after transplant and analyzed by flow cytometry to confirm reconstitution of hematopoietic lineages before use in adoptive transfer experiments. CFU Assays
  • Femurs and tibia from control and ROR1 CAR-T cell-treated mice were harvested 9 days post-T cell transfer and submitted to ReachBio Inc. for CFET-GM, BFU-E, CFU-Mk, Pre-B CFC, and CFU-F assays.
  • Amplifications were performed for 50 cycles on an ABI Prism 7900 (Applied Biosystems) in a 20ul reaction consisting of Power SYBR Green PCR Master Mix (Applied Biosystems), 5ng of cDNA, and 500nM gene-specific forward and reverse primers: mRORl, 5’- CAAAACCCGTCAGAGGACAGA-3’ and 5’ AT GAAACGC AC AGCGGAAAG-3’ ; mActb, 5’-CTGTCCCTGTATGCCTCTG-3’ and 5’ - ATGTC ACGC ACGATTTCC-3’ .
  • the cycle threshold (Ct) was determined using SDS software (Applied Biosystems) and the level of gene expression calculated using the comparative Ct method (2( A Ct)).
  • Tumor cells were labeled with 51 Cr (PerkinElmer) for 1 hour at 37°C and washed with complete RPMI.
  • lxlO 3 51 Cr-labeled target cells were plated per well in triplicate and co-cultured with T-cells at various effector to target (E:T) ratios for 24 hours in a 37°C, 5% C02 incubator.
  • E:T effector to target
  • Supernatants were harvested for g-counting after 6 hours and 24 hours of incubation and specific lysis calculated by comparing counts to standardized wells where target cells were lysed with NP40-based soap solution.
  • mice subcutaneously into the 4th right mammary fat pad of 6-8 week old BALB/c female mice. After 7 days, mice were injected intraperitoneally with 200mg/kg
  • Trigger polypeptide T cells were enriched by untouched sorting for BFP+ T cells, as described above, such that -30% of sorted T cells were myc + BFP + and carried the full EpCAM- trigger polypeptide /UAS-ROR 1-CAR circuit.
  • Transduction rate of constitutive ROR1 CAR-T cells was equalized to -30% by diluting cultures with untransduced T cells cultured in parallel such ROR1 CAR- and trigger polypeptide -treated mice each received the same number of T cells capable of expressing the ROR1 CAR and the same number of total T cells. Tumor size was monitored using calipers, and tumor volume was calculated as (length in mm)*(width in mm) A 2.
  • mice 5xl0 5 Raji-hRORl-GFP-ffluc tumor cells were injected intravenously via tail vein into 8-12 week old NSG mice. After 7 days, mice were irradiated (250R) and 5-6 hours later injected intravenously with 2xl0 6 primary human CD8 + untransduced, CD8 + tEGFR + ROR1 CAR-T, or CD8 + myc + BFP + trigger polypeptide CD 19-inducible ROR1 CAR-T cells.
  • MDA-MB-23 l model 5xl0 5 MDA-MB-231 GFP-ffluc cells were injected subcutaneously in the right flank of 8-12 week old NSG mice.
  • mice were irradiated 250R and 5-6 hours later injected intravenously with 3xl0 6 CD8 + and 3xl0 6 CD4 + primary human untransduced, tEGFR + ROR1 CAR-T, or myc + BFP + trigger polypeptide B7-H3-inducible ROR1 CAR-T cells.
  • T cells were sorted to >95% purity and expanded in IL-2 for 4-5 days prior to injection. Tumor burden was monitored by IVIS bioluminescence imaging.
  • mice received intraperitoneal injections of luciferin substrate (Caliper Life Sciences) resuspended in PBS (15 pg/g body weight). Mice were anesthetized with isoflurane and imaged using an Xenogen IVIS Imaging System (Caliper) 10, 12 and 14 minutes after luciferin injection in small binning mode at an acquisition time of 1 s to 1 min to obtain unsaturated images.
  • luciferin substrate Caliper Life Sciences
  • Luciferase activity was analyzed using Living Image Software (Caliper) and the photon flux analyzed within regions of interest that encompassed the entire body of each individual mouse. Statistical Analysis

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Abstract

La présente invention concerne des compositions et des procédés pour moduler l'expression d'une protéine de liaison spécifique d'un antigène par une cellule hôte, telle qu'un lymphocyte T. Selon certains aspects, l'invention concerne des polypeptides de déclenchement qui peuvent moduler l'expression d'une protéine de liaison, telle qu'un CAR ou un TCR spécifique pour ROR1, lorsqu'un polypeptide de déclenchement exprimé en surface cellulaire se lie à un antigène, tel qu'un antigène B7-H3 ou un antigène EpCAM. Les compositions et les procédés selon la présente invention sont utiles dans le traitement de diverses maladies ou affections, tels que le cancer, et, dans certains modes de réalisation, peuvent améliorer de manière avantageuse l'expression spécifique d'une protéine de liaison de ROR1 à des cellules malades cibles.
PCT/US2019/042696 2018-07-20 2019-07-19 Compositions et procédés pour réguler l'expression de récepteurs spécifiques à l'antigène Ceased WO2020018964A1 (fr)

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WO2022011065A1 (fr) * 2020-07-07 2022-01-13 The Nemours Foundation Lymphocytes t alloréactifs et xénoréactifs activés par une tumeur et leur utilisation en immunothérapie contre le cancer
WO2022022745A1 (fr) * 2020-07-31 2022-02-03 南京北恒生物科技有限公司 Nouveau domaine de costimulation et utilisations de celui-ci
WO2022179620A1 (fr) * 2021-02-25 2022-09-01 克莱格医学有限公司 Cellule modifiée par cd94 et composition associée
WO2022178211A3 (fr) * 2021-02-19 2022-09-29 Memorial Sloan-Kettering Cancer Center Cellules immunitaires reprogrammées à axe mir200c-epcam pour une fonction antitumorale améliorée
CN116732161A (zh) * 2023-06-02 2023-09-12 杭州师范大学 Zcchc8作为分子标志物在制备诊断骨髓衰竭产品中的应用
WO2024040032A1 (fr) * 2022-08-15 2024-02-22 The Regents Of The University Of California Nouveaux récepteurs à domaines transmembranaires synthétiques pour une régulation améliorée de l'activation transcriptionnelle dépendante d'un ligand
US12144827B2 (en) 2021-02-25 2024-11-19 Lyell Immunopharma, Inc. ROR1 targeting chimeric antigen receptor
WO2025076422A1 (fr) * 2023-10-05 2025-04-10 The Regents Of The University Of California Expression régulée de protéine hybride card11-pik3r3
WO2024249770A3 (fr) * 2023-06-01 2025-05-30 University Of Southern California Contrôle spatio-temporel de la génomique et de l'épigénomique par ultrasons

Citations (76)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA15704A (fr) 1882-10-26 Jordan, Robert G. Perfectionnements aux roulettes des meubles
CA114536A (fr) 1908-09-18 1908-10-13 Adolphe Chalas Procede de recouvrement du nickel
US5283173A (en) 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
US5420032A (en) 1991-12-23 1995-05-30 Universitge Laval Homing endonuclease which originates from chlamydomonas eugametos and recognizes and cleaves a 15, 17 or 19 degenerate double stranded nucleotide sequence
US6291161B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertiore
US6291158B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertoire
US6410319B1 (en) 1998-10-20 2002-06-25 City Of Hope CD20-specific redirected T cells and their use in cellular immunotherapy of CD20+ malignancies
US20040002092A1 (en) 2002-03-15 2004-01-01 Sylvain Arnould Hybrid and single chain meganucleases and use thereof
US20040087025A1 (en) 1995-06-07 2004-05-06 The United States Of America As Represented By The Secretary Of The Navy Methods for transfecting T cells
US6833252B1 (en) 1992-05-05 2004-12-21 Institut Pasteur Nucleotide sequence encoding the enzyme I-SecI and the uses thereof
WO2005080428A2 (fr) 2004-02-13 2005-09-01 Micromet Ag Immunoglobulines anti-epcam
US20060153826A1 (en) 2003-01-28 2006-07-13 Sylvain Arnould Use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof
US20070117128A1 (en) 2005-10-18 2007-05-24 Smith James J Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity
WO2008116219A2 (fr) 2007-03-22 2008-09-25 Sloan-Kettering Institute For Cancer Research Utilisations de l'anticorps monoclonal 8h9
US7446191B2 (en) 2001-04-11 2008-11-04 City Of Hope DNA construct encoding CE7-specific chimeric T cell receptor
WO2008122551A9 (fr) 2007-04-04 2009-02-05 Sigma Tau Ind Farmaceuti Anticorps anti-epcam et ses utilisations
US7514537B2 (en) 2001-04-30 2009-04-07 City Of Hope Chimeric immunoreceptor useful in treating human gliomas
US20100065818A1 (en) 2008-02-22 2010-03-18 Kim Jae-Hyun Layers and patterns of nanowire or carbon nanotube using chemical self assembly and fabricating method in liquid crystal display device thereby
WO2010084158A1 (fr) 2009-01-21 2010-07-29 Monoclonal Antibodies Therapeutics Anticorps monoclonaux anti-cd 160 et leurs utilisations
WO2010142990A1 (fr) 2009-06-09 2010-12-16 Affitech Research As Anticorps anti-epcam
WO2011079283A1 (fr) 2009-12-23 2011-06-30 Bioalliance C.V. ANTICORPS ANTI-EpCAM QUI INDUISENT L'APOPTOSE DE CELLULES CANCÉREUSES ET LEURS PROCÉDÉS D'UTILISATION
US20110189141A1 (en) 2009-05-19 2011-08-04 Max-Delbrück-Centrum für Molekulare Medizin Multiple target t cell receptor
WO2011109400A2 (fr) 2010-03-04 2011-09-09 Macrogenics,Inc. Anticorps réagissant avec b7-h3, fragments immunologiquement actifs associés et utilisations associées
US20110243972A1 (en) 2005-01-13 2011-10-06 Johns Hopkins University Prostate stem cell antigen vaccines and uses thereof
US20110301073A1 (en) 2010-05-17 2011-12-08 Sangamo Biosciences, Inc. Novel DNA-binding proteins and uses thereof
US8119772B2 (en) 2006-09-29 2012-02-21 California Institute Of Technology MART-1 T cell receptors
US8212009B2 (en) 2005-10-28 2012-07-03 The Regents Of The University Of California Methods and compounds for lymphoma cell detection and isolation
WO2012147713A1 (fr) 2011-04-25 2012-11-01 第一三共株式会社 Anticorps anti-b7-h3
US20120294796A1 (en) 2010-03-04 2012-11-22 Macrogenics, Inc. Antibodies Reactive with B7-H3 and Uses Thereof
WO2013025779A1 (fr) 2011-08-15 2013-02-21 Amplimmune, Inc. Anticorps anti-b7-h4 et leurs utilisations
WO2013131001A1 (fr) 2012-03-02 2013-09-06 Academia Sinica Anticorps anti-molécules d'adhérence cellulaire épithéliale (epcam) et leurs procédés d'utilisation
US20130251642A1 (en) 2010-12-01 2013-09-26 The United States of America, as represented by the secretary, Department of Heath and Human Servi Chimeric rabbit/human ror1 antibodies
WO2014031174A1 (fr) 2012-08-24 2014-02-27 The Regents Of The University Of California Anticorps et vaccins utilisables en vue du traitement de cancers ror1 et de l'inhibition de la métastase
WO2014031687A1 (fr) 2012-08-20 2014-02-27 Jensen, Michael Procédé et compositions pour l'immunothérapie cellulaire
US20140068797A1 (en) 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US20140186843A1 (en) 2012-12-12 2014-07-03 Massachusetts Institute Of Technology Methods, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
US8822647B2 (en) 2008-08-26 2014-09-02 City Of Hope Method and compositions using a chimeric antigen receptor for enhanced anti-tumor effector functioning of T cells
WO2014159531A1 (fr) 2013-03-14 2014-10-02 The Board Of Regents Of The University Of Texas System Epcam de ciblage d'anticorps monoclonaux pour la détection de métastases ganglionnaires du cancer de la prostate
US20140308746A1 (en) 2010-04-16 2014-10-16 Children's Medical Center Corporation Sustained polypeptide expression from synthetic, modified rnas and uses thereof
WO2015048901A1 (fr) 2013-10-02 2015-04-09 Viventia Bio Inc. Anticorps anti-epcam et leurs procédés d'utilisation
WO2015071474A2 (fr) 2013-11-18 2015-05-21 Crispr Therapeutics Ag Système crips-cas, matériels et procédés
US9150647B2 (en) 2009-12-18 2015-10-06 Kancera Ab Biological inhibitors of ROR1 capable of inducing cell death
WO2015181267A1 (fr) 2014-05-29 2015-12-03 Spring Bioscience Corporation Anticorps anti-b7-h3 et leurs utilisations diagnostiques
US9217040B2 (en) 2011-01-14 2015-12-22 The Regents Of The University Of California Therapeutic antibodies against ROR-1 protein and methods for use of same
US9226952B2 (en) 2008-12-12 2016-01-05 The University Of Toledo Na/K-ATPase-derived peptide Src inhibitors and ouabain antagonists and uses thereof
US9228023B2 (en) 2010-10-01 2016-01-05 Oxford Biotherapeutics Ltd. Anti-ROR1 antibodies and methods of use for treatment of cancer
US9242014B2 (en) 2010-06-15 2016-01-26 The Regents Of The University Of California Receptor tyrosine kinase-like orphan receptor 1 (ROR1) single chain Fv antibody fragment conjugates and methods of use thereof
WO2016016344A1 (fr) * 2014-07-29 2016-02-04 Cellectis Récepteurs antigéniques chimériques spécifiques de ror1(ntrkr1), utiles dans l'immunothérapie anticancéreuse
WO2016033225A2 (fr) 2014-08-27 2016-03-03 Memorial Sloan Kettering Cancer Center Anticorps, compositions et leurs utilisations
WO2016040724A1 (fr) 2014-09-12 2016-03-17 Genentech, Inc. Anticorps anti-b7-h4 et immunoconjugués
WO2016044383A1 (fr) 2014-09-17 2016-03-24 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anticorps anti-cd276 (b7h3)
WO2016054638A1 (fr) 2014-10-03 2016-04-07 Dana-Farber Cancer Institute, Inc. Anticorps dirigés contre le récepteur du facteur de nécrose tumorale induit par glucocorticoïdes (gitr) et leurs procédés d'utilisation
US9316646B2 (en) 2009-04-23 2016-04-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-human ROR1 antibodies
WO2016094873A2 (fr) 2014-12-12 2016-06-16 Emergent Product Development Seattle, Llc Protéine de liaison au récepteur orphelin de type récepteur à tyrosine kinase et compositions et procédés associés
WO2016106004A1 (fr) 2014-12-23 2016-06-30 Full Spectrum Genetics, Inc. Nouveaux composés de liaison anti-b7h3 et leurs utilisations
WO2016134333A1 (fr) 2015-02-19 2016-08-25 Compugen Ltd. Anticorps anti-pvrig et méthodes d'utilisation
WO2016138034A1 (fr) 2015-02-24 2016-09-01 The Regents Of The University Of California Commutateurs transcriptionnels déclenchés par une liaison et procédés d'utilisation associés
WO2016187216A1 (fr) * 2015-05-18 2016-11-24 Bluebird Bio, Inc. Récepteurs antigéniques chimériques anti-ror1
WO2016187220A2 (fr) 2015-05-18 2016-11-24 Five Prime Therapeutics, Inc. Anticorps anti-ror1
WO2017021526A1 (fr) 2015-08-05 2017-02-09 Amgen Research (Munich) Gmbh Inhibiteurs de points de contrôle immunitaires destinés à être utilisés dans le traitement de cancers à diffusion hématogène
WO2017023704A1 (fr) 2015-07-31 2017-02-09 Sutro Biopharma, Inc. Anticorps anti-epcam, compositions comprenant des anticorps anti-epcam et procédés de fabrication et d'utilisation d'anticorps anti-epcam
US9574000B2 (en) 2012-12-19 2017-02-21 Medimmune, Llc Anti-human B7-H4 antibodies and their uses
WO2017072361A1 (fr) 2015-10-30 2017-05-04 Nbe-Therapeutics Ag Anticorps anti-ror1
WO2017127664A1 (fr) 2016-01-20 2017-07-27 The Scripps Research Institute Compositions d'anticorps anti-ror1 et procédés associés
WO2017180813A1 (fr) 2016-04-15 2017-10-19 Macrogenics, Inc. Nouvelles molécules de liaison à b7-h3, leurs conjugués anticorps-médicaments et leurs procédés d'utilisation
WO2017193059A1 (fr) * 2016-05-06 2017-11-09 The Regents Of The University Of California Systèmes et procédés de ciblage de cellules cancéreuses
WO2017214339A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-b7-h3 et conjugués anticorps-médicaments
WO2017214322A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-b7-h3 et conjugués anticorps-médicaments
WO2018129090A1 (fr) 2017-01-03 2018-07-12 Trellis Bioscience, Llc Anticorps humains natifs pour cibles de modulation de points de contrôle immunitaires tim-3 et b7-h3
WO2018161872A1 (fr) 2017-03-06 2018-09-13 江苏恒瑞医药股份有限公司 Anticorps anti-b7-h3, fragment de liaison à l'antigène de celui-ci, et utilisation pharmaceutique associée
WO2018177393A1 (fr) 2017-03-31 2018-10-04 江苏恒瑞医药股份有限公司 Anticorps b7-h3, fragment de liaison à l'antigène de celui-ci, et son utilisation médicale
WO2018197675A1 (fr) 2017-04-28 2018-11-01 Julius-Maximilians-Universität Würzburg Récepteurs antigéniques chimériques (car) spécifiques à ror1 ayant des domaines de ciblage humanisés
WO2019005636A2 (fr) 2017-06-25 2019-01-03 Systimmune, Inc. Anticorps anti-ror1 et leurs procédés de préparation et d'utilisation
WO2019008377A1 (fr) 2017-07-05 2019-01-10 Ucl Business Plc Anticorps ror1
WO2019122447A1 (fr) 2017-12-22 2019-06-27 Almac Discovery Limited Molécules de fixation à l'antigène spécifique de ror1

Patent Citations (81)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA15704A (fr) 1882-10-26 Jordan, Robert G. Perfectionnements aux roulettes des meubles
CA114536A (fr) 1908-09-18 1908-10-13 Adolphe Chalas Procede de recouvrement du nickel
US6291161B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertiore
US6291158B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertoire
US5283173A (en) 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
US5468614A (en) 1990-01-24 1995-11-21 The Research Foundation Of State University Of New York System to detect protein-protein interactions
US5420032A (en) 1991-12-23 1995-05-30 Universitge Laval Homing endonuclease which originates from chlamydomonas eugametos and recognizes and cleaves a 15, 17 or 19 degenerate double stranded nucleotide sequence
US6833252B1 (en) 1992-05-05 2004-12-21 Institut Pasteur Nucleotide sequence encoding the enzyme I-SecI and the uses thereof
US20040087025A1 (en) 1995-06-07 2004-05-06 The United States Of America As Represented By The Secretary Of The Navy Methods for transfecting T cells
US6410319B1 (en) 1998-10-20 2002-06-25 City Of Hope CD20-specific redirected T cells and their use in cellular immunotherapy of CD20+ malignancies
US7446191B2 (en) 2001-04-11 2008-11-04 City Of Hope DNA construct encoding CE7-specific chimeric T cell receptor
US7514537B2 (en) 2001-04-30 2009-04-07 City Of Hope Chimeric immunoreceptor useful in treating human gliomas
US20040002092A1 (en) 2002-03-15 2004-01-01 Sylvain Arnould Hybrid and single chain meganucleases and use thereof
US20060078552A1 (en) 2002-03-15 2006-04-13 Sylvain Arnould Hybrid and single chain meganucleases and use thereof
US20060206949A1 (en) 2003-01-28 2006-09-14 Sylvain Arnould Custom-made meganuclease and use thereof
US20060153826A1 (en) 2003-01-28 2006-07-13 Sylvain Arnould Use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof
WO2005080428A2 (fr) 2004-02-13 2005-09-01 Micromet Ag Immunoglobulines anti-epcam
US20110243972A1 (en) 2005-01-13 2011-10-06 Johns Hopkins University Prostate stem cell antigen vaccines and uses thereof
US20070117128A1 (en) 2005-10-18 2007-05-24 Smith James J Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity
US8212009B2 (en) 2005-10-28 2012-07-03 The Regents Of The University Of California Methods and compounds for lymphoma cell detection and isolation
US8119772B2 (en) 2006-09-29 2012-02-21 California Institute Of Technology MART-1 T cell receptors
WO2008116219A2 (fr) 2007-03-22 2008-09-25 Sloan-Kettering Institute For Cancer Research Utilisations de l'anticorps monoclonal 8h9
US20100092491A1 (en) 2007-04-04 2010-04-15 Anna Anastasi Anti-epcam antibody and uses thereof
WO2008122551A9 (fr) 2007-04-04 2009-02-05 Sigma Tau Ind Farmaceuti Anticorps anti-epcam et ses utilisations
US20100065818A1 (en) 2008-02-22 2010-03-18 Kim Jae-Hyun Layers and patterns of nanowire or carbon nanotube using chemical self assembly and fabricating method in liquid crystal display device thereby
US8822647B2 (en) 2008-08-26 2014-09-02 City Of Hope Method and compositions using a chimeric antigen receptor for enhanced anti-tumor effector functioning of T cells
US9226952B2 (en) 2008-12-12 2016-01-05 The University Of Toledo Na/K-ATPase-derived peptide Src inhibitors and ouabain antagonists and uses thereof
WO2010084158A1 (fr) 2009-01-21 2010-07-29 Monoclonal Antibodies Therapeutics Anticorps monoclonaux anti-cd 160 et leurs utilisations
US9316646B2 (en) 2009-04-23 2016-04-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-human ROR1 antibodies
US20110189141A1 (en) 2009-05-19 2011-08-04 Max-Delbrück-Centrum für Molekulare Medizin Multiple target t cell receptor
WO2010142990A1 (fr) 2009-06-09 2010-12-16 Affitech Research As Anticorps anti-epcam
US9150647B2 (en) 2009-12-18 2015-10-06 Kancera Ab Biological inhibitors of ROR1 capable of inducing cell death
WO2011079283A1 (fr) 2009-12-23 2011-06-30 Bioalliance C.V. ANTICORPS ANTI-EpCAM QUI INDUISENT L'APOPTOSE DE CELLULES CANCÉREUSES ET LEURS PROCÉDÉS D'UTILISATION
US20110165161A1 (en) 2009-12-23 2011-07-07 Shih-Yao Lin Anti-epcam antibodies that induce apoptosis of cancer cells and methods using same
US20120294796A1 (en) 2010-03-04 2012-11-22 Macrogenics, Inc. Antibodies Reactive with B7-H3 and Uses Thereof
WO2011109400A2 (fr) 2010-03-04 2011-09-09 Macrogenics,Inc. Anticorps réagissant avec b7-h3, fragments immunologiquement actifs associés et utilisations associées
US20140308746A1 (en) 2010-04-16 2014-10-16 Children's Medical Center Corporation Sustained polypeptide expression from synthetic, modified rnas and uses thereof
US20110301073A1 (en) 2010-05-17 2011-12-08 Sangamo Biosciences, Inc. Novel DNA-binding proteins and uses thereof
US9242014B2 (en) 2010-06-15 2016-01-26 The Regents Of The University Of California Receptor tyrosine kinase-like orphan receptor 1 (ROR1) single chain Fv antibody fragment conjugates and methods of use thereof
US9228023B2 (en) 2010-10-01 2016-01-05 Oxford Biotherapeutics Ltd. Anti-ROR1 antibodies and methods of use for treatment of cancer
US20130251642A1 (en) 2010-12-01 2013-09-26 The United States of America, as represented by the secretary, Department of Heath and Human Servi Chimeric rabbit/human ror1 antibodies
US9217040B2 (en) 2011-01-14 2015-12-22 The Regents Of The University Of California Therapeutic antibodies against ROR-1 protein and methods for use of same
WO2012147713A1 (fr) 2011-04-25 2012-11-01 第一三共株式会社 Anticorps anti-b7-h3
WO2013025779A1 (fr) 2011-08-15 2013-02-21 Amplimmune, Inc. Anticorps anti-b7-h4 et leurs utilisations
WO2013131001A1 (fr) 2012-03-02 2013-09-06 Academia Sinica Anticorps anti-molécules d'adhérence cellulaire épithéliale (epcam) et leurs procédés d'utilisation
US20140068797A1 (en) 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
WO2014031687A1 (fr) 2012-08-20 2014-02-27 Jensen, Michael Procédé et compositions pour l'immunothérapie cellulaire
WO2014031174A1 (fr) 2012-08-24 2014-02-27 The Regents Of The University Of California Anticorps et vaccins utilisables en vue du traitement de cancers ror1 et de l'inhibition de la métastase
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US20140186843A1 (en) 2012-12-12 2014-07-03 Massachusetts Institute Of Technology Methods, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
US9574000B2 (en) 2012-12-19 2017-02-21 Medimmune, Llc Anti-human B7-H4 antibodies and their uses
WO2014159531A1 (fr) 2013-03-14 2014-10-02 The Board Of Regents Of The University Of Texas System Epcam de ciblage d'anticorps monoclonaux pour la détection de métastases ganglionnaires du cancer de la prostate
WO2015048901A1 (fr) 2013-10-02 2015-04-09 Viventia Bio Inc. Anticorps anti-epcam et leurs procédés d'utilisation
WO2015071474A2 (fr) 2013-11-18 2015-05-21 Crispr Therapeutics Ag Système crips-cas, matériels et procédés
WO2015181267A1 (fr) 2014-05-29 2015-12-03 Spring Bioscience Corporation Anticorps anti-b7-h3 et leurs utilisations diagnostiques
WO2016016344A1 (fr) * 2014-07-29 2016-02-04 Cellectis Récepteurs antigéniques chimériques spécifiques de ror1(ntrkr1), utiles dans l'immunothérapie anticancéreuse
WO2016033225A2 (fr) 2014-08-27 2016-03-03 Memorial Sloan Kettering Cancer Center Anticorps, compositions et leurs utilisations
WO2016040724A1 (fr) 2014-09-12 2016-03-17 Genentech, Inc. Anticorps anti-b7-h4 et immunoconjugués
WO2016044383A1 (fr) 2014-09-17 2016-03-24 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anticorps anti-cd276 (b7h3)
WO2016054638A1 (fr) 2014-10-03 2016-04-07 Dana-Farber Cancer Institute, Inc. Anticorps dirigés contre le récepteur du facteur de nécrose tumorale induit par glucocorticoïdes (gitr) et leurs procédés d'utilisation
WO2016094873A2 (fr) 2014-12-12 2016-06-16 Emergent Product Development Seattle, Llc Protéine de liaison au récepteur orphelin de type récepteur à tyrosine kinase et compositions et procédés associés
WO2016106004A1 (fr) 2014-12-23 2016-06-30 Full Spectrum Genetics, Inc. Nouveaux composés de liaison anti-b7h3 et leurs utilisations
WO2016134333A1 (fr) 2015-02-19 2016-08-25 Compugen Ltd. Anticorps anti-pvrig et méthodes d'utilisation
WO2016138034A1 (fr) 2015-02-24 2016-09-01 The Regents Of The University Of California Commutateurs transcriptionnels déclenchés par une liaison et procédés d'utilisation associés
WO2016187216A1 (fr) * 2015-05-18 2016-11-24 Bluebird Bio, Inc. Récepteurs antigéniques chimériques anti-ror1
WO2016187220A2 (fr) 2015-05-18 2016-11-24 Five Prime Therapeutics, Inc. Anticorps anti-ror1
WO2017023704A1 (fr) 2015-07-31 2017-02-09 Sutro Biopharma, Inc. Anticorps anti-epcam, compositions comprenant des anticorps anti-epcam et procédés de fabrication et d'utilisation d'anticorps anti-epcam
WO2017021526A1 (fr) 2015-08-05 2017-02-09 Amgen Research (Munich) Gmbh Inhibiteurs de points de contrôle immunitaires destinés à être utilisés dans le traitement de cancers à diffusion hématogène
WO2017072361A1 (fr) 2015-10-30 2017-05-04 Nbe-Therapeutics Ag Anticorps anti-ror1
WO2017127664A1 (fr) 2016-01-20 2017-07-27 The Scripps Research Institute Compositions d'anticorps anti-ror1 et procédés associés
WO2017180813A1 (fr) 2016-04-15 2017-10-19 Macrogenics, Inc. Nouvelles molécules de liaison à b7-h3, leurs conjugués anticorps-médicaments et leurs procédés d'utilisation
WO2017193059A1 (fr) * 2016-05-06 2017-11-09 The Regents Of The University Of California Systèmes et procédés de ciblage de cellules cancéreuses
WO2017214339A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-b7-h3 et conjugués anticorps-médicaments
WO2017214322A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-b7-h3 et conjugués anticorps-médicaments
WO2018129090A1 (fr) 2017-01-03 2018-07-12 Trellis Bioscience, Llc Anticorps humains natifs pour cibles de modulation de points de contrôle immunitaires tim-3 et b7-h3
WO2018161872A1 (fr) 2017-03-06 2018-09-13 江苏恒瑞医药股份有限公司 Anticorps anti-b7-h3, fragment de liaison à l'antigène de celui-ci, et utilisation pharmaceutique associée
WO2018177393A1 (fr) 2017-03-31 2018-10-04 江苏恒瑞医药股份有限公司 Anticorps b7-h3, fragment de liaison à l'antigène de celui-ci, et son utilisation médicale
WO2018197675A1 (fr) 2017-04-28 2018-11-01 Julius-Maximilians-Universität Würzburg Récepteurs antigéniques chimériques (car) spécifiques à ror1 ayant des domaines de ciblage humanisés
WO2019005636A2 (fr) 2017-06-25 2019-01-03 Systimmune, Inc. Anticorps anti-ror1 et leurs procédés de préparation et d'utilisation
WO2019008377A1 (fr) 2017-07-05 2019-01-10 Ucl Business Plc Anticorps ror1
WO2019122447A1 (fr) 2017-12-22 2019-06-27 Almac Discovery Limited Molécules de fixation à l'antigène spécifique de ror1

Non-Patent Citations (103)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. AAC50291.1
"Molecular Cell Biology", 2000, W.H. FREEMAN
"UniProt", Database accession no. P20963-3
ALDER ET AL., NATURE IMMUNOL., vol. 9, 2008, pages 319
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3379 - 3388
ARGAST ET AL., J. MOL. BIOL., vol. 280, 1998, pages 345 - 353
ASHWINI BALAKRISHNAN ET AL: "Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues", CLINICAL CANCER RESEARCH, vol. 23, no. 12, 16 November 2016 (2016-11-16), US, pages 3061 - 3071, XP055634726, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-16-2083 *
ASHWORTH ET AL., NATURE, vol. 441, 2006, pages 656 - 659
BARAL ET AL., NATURE MED., vol. 12, 2006, pages 580
BARTHELEMY ET AL., J. BIOL. CHEM., vol. 283, 2008, pages 3639
BEAVIL ET AL., PROC. NAT'L. ACAD. SCI. USA, vol. 89, 1992, pages 153
BERGER ET AL., CANCER IMMUNOL. RES., vol. 3, no. 2, 2015, pages 206
BORQUIN ET AL., J. NAT'L CANCER INST., vol. 107, 2014, pages 364
BOWERMAN ET AL., MOL. IMMUNOL., vol. 46, no. 15, 2009, pages 3000
BOYINGTON ET AL., IMMUNITY, vol. 10, 1999, pages 15
BRENTJENS ET AL., CLIN. CANCER RES., vol. 13, 2007, pages 5426
BRENTJENS ET AL., SCI TRANSL MED, vol. 5, no. 177, 2013, pages 177ra38
CHEVALIER ET AL., MOLEC. CELL, vol. 10, 2002, pages 895 - 905
CHOTHIA ET AL., EMBO J., vol. 7, 1988, pages 3745
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
COFFIN, J. M. ET AL.: "Fundamental Virology", 1996, LIPPINCOTT-RAVEN PUBLISHERS, article "Retroviridae: The viruses and their replication"
COIFFIER ET AL., N ENGL J MED, vol. 346, no. 4, 2002, pages 235 - 42
COLEGAO, CELL. MOL. IMMUNOL., vol. 1, 2004, pages 81 - 88
CORTEZ-RETAMOZO ET AL., CANCER RES., vol. 64, 2004, pages 2853
CREIGHTON: "Proteins", 1984, W.H. FREEMAN AND COMPANY
DANGAJ ET AL., CANCER RES., vol. 73, 2013, pages 4820
DESJARLAIS ET AL., PROC. NATL. ACAD. SCI., vol. 90, 1993, pages 2256 - 2260
DOSSETT ET AL., MOL. THER., vol. 17, 2009, pages 742
DUJON ET AL., GENE, vol. 82, 1989, pages 115 - 118
E. PICARDA ET AL: "Molecular Pathways: Targeting B7-H3 (CD276) for Human Cancer Immunotherapy", CLINICAL CANCER RESEARCH, vol. 22, no. 14, 20 May 2016 (2016-05-20), US, pages 3425 - 3431, XP055396638, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-15-2428 *
ENGELS ET AL., HUM. GENE THER., vol. 14, 2003, pages 1155
EPINAT ET AL., NUCLEIC ACIDS RES., vol. 31, 2003, pages 2952 - 62
FAUCI ET AL., GYNECOL ONCOL., vol. 132, 2014, pages 203 - 210
FIGDOR ET AL., NAT. REV. IMMUNOL., vol. 2, 2002, pages 11
FLOROSTARHINI, SEMIN. ONCOL., vol. 42, no. 4, 2015, pages 539 - 548
FRECHA ET AL., MOL. THER., vol. 18, 2010, pages 1748
GAOJAKOBSEN, IMMUNOL. TODAY, vol. 27, 2000, pages 630 - 636
GEURTS ET AL., MOL. THER., vol. 8, 2003, pages 108
GHAHROUDI ET AL., FEBS LETTERS, vol. 414, 1997, pages 521
GIMBLE ET AL., J. MOL. BIOL., vol. 263, 1996, pages 163 - 180
GUEST ET AL., J. IMMUNOTHER., vol. 28, 2005, pages 203 - 11
HAMERS-CASTERMAN ET AL., NATURE, vol. 363, 1993, pages 446
HARRISKRANZ, TRENDS PHARMACOL. SCI., vol. 37, 2016, pages 220
HENKART ET AL.: "Fundamental Immunology", 2003, LIPPINCOTT WILLIAMS & WILKINS, PHILADELPHIA, PA, article "Cytotoxic T-Lymphocytes", pages: 1127 - 50
HERRIN ET AL., PROC. NAT'L. ACAD. SCI. (USA), vol. 105, 2008, pages 2040
HONEGGERPLIICKTHUN, J. MOL. BIO., vol. 309, 2001, pages 657 - 670
HUDECEK ET AL., BLOOD, vol. 115, 2010, pages 3520 - 30
HUDECEK ET AL., CLIN. CANCER RES., vol. 19, no. 12, 2013, pages 3153
JAMES ET AL., J IMMUNOL, vol. 180, no. 10, 2008, pages 7028 - 3 8
JANEWAY ET AL.: "Current Biology Publications", vol. 33, 1997, article "Immunobiology: The Immune System in Health and Disease", pages: 4
JASIN, TRENDS GENET., vol. 12, 1996, pages 224 - 228
JESPERS ET AL., NATURE BIOTECHNOL., vol. 22, 2004, pages 1161
JINEK ET AL., SCIENCE, vol. 337, 2012, pages 816 - 21
JOLLY, D J., EMERGING VIRAL VECTORS., 1999, pages 209 - 40
JORES ET AL., PROC. NAT'1 ACAD. SCI. U.S.A., vol. 87, 1990, pages 9138
KABAT ET AL.: "US Dept. Health and Human Services", 1991, PUBLIC HEALTH SERVICE NATIONAL INSTITUTES OF HEALTH, article "Sequences of Proteins of Immunological Interest"
KALOS ET AL., SCI TRANSL MED, vol. 3, 2011, pages 95
KHANZHENG, NUCL. ACIDS RES., 2016, pages D164 - D171
KIM ET AL., PLOS ONE, vol. 6, 2011, pages el8556
KOCHENDERFER ET AL., J CLIN ONCOL, vol. 33, no. 6, 2015, pages 540 - 9
KRISKY ET AL., GENE THER., vol. 5, 1998, pages 1517
KUBALL ET AL., BLOOD, vol. 109, 2007, pages 2331
LEE ET AL., LANCET, vol. 385, no. 9967, 2015, pages 517 - 28
LEEN ET AL., ANN. REV. IMMUNOL., vol. 25, 2007, pages 243
LEFRANC ET AL., DEV. COMP. IMMUNOL., vol. 27, 2003, pages 55
LOO ET AL., CLIN. CANCER RES., vol. 18, no. 14, 2012, pages 3834 - 45
MATES ET AL., NAT. GENET., vol. 41, 2009, pages 753
MAUTINO ET AL., AMERICAN ASSOCIATION FOR CANCER RESEARCH 104TH ANNUAL MEETING, 6 April 2013 (2013-04-06)
NGUYEN ET AL., IMMUNOGENETICS, vol. 54, 2002, pages 39
PAQUES ET AL., CURR. GENE THER., vol. 7, 2007, pages 49 - 66
PAREDES-MOSCOSSO ET AL., BLOOD, vol. 128, 2016, pages 2052
PERLER ET AL., NUCLEIC ACIDS RES., vol. 22, 1994, pages 1125 - 1127
PHILIP ET AL., BLOOD, vol. 124, 2014, pages 1277 - 1287
PORTER ET AL., SCI TRANSL MED, vol. 7, no. 303, 2015
PORTEUS ET AL., NAT. BIOTECHNOL., vol. 23, 2005, pages 967 - 73
REN ET AL., CLIN. CANCER RES., vol. 23, no. 9, 2017, pages 2255 - 2266
ROUX ET AL., PROC. NAT'L. ACAD. SCI. (USA), vol. 95, 1998, pages 11804
ROYBAL KOLE T ET AL: "Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits", CELL, ELSEVIER, AMSTERDAM, NL, vol. 164, no. 4, 28 January 2016 (2016-01-28), pages 770 - 779, XP029416808, ISSN: 0092-8674, DOI: 10.1016/J.CELL.2016.01.011 *
SADELAIN ET AL., CANCER DISCOV., vol. 3, 2013, pages 388
SAVOLDO ET AL., J CLIN INVEST, vol. 121, no. 5, 2011, pages 1822 - 6
SAZINSKY ET AL., PNAS, vol. 105, no. 51, 2008, pages 20167
SCATCHARD ET AL., ANN. N.Y. ACAD. SCI., vol. 51, 1949, pages 660
SCHMITT ET AL., HUM. GEN., vol. 20, 2009, pages 1240
SCHOLTEN ET AL., CLIN. IMMUNOL., vol. 119, 2006, pages 135
SRIVASTAVA SHIVANI ET AL: "Logic-Gated ROR1 Chimeric Antigen Receptor Expression Rescues T Cell-Mediated Toxicity to Normal Tissues and Enables Selective Tumor Targeting", CANCER CELL, vol. 35, no. 3, 2 October 2019 (2019-10-02), pages 489, XP085638448, ISSN: 1535-6108, DOI: 10.1016/J.CCELL.2019.02.003 *
STONE ET AL., CANCER IMMUNOL. IMMUNOTHER., vol. 63, 2014, pages 1163
SUSSMAN ET AL., J. MOL. BIOL., vol. 342, 2004, pages 31 - 41
TERENTIS ET AL., BIOCHEM., vol. 49, 2010, pages 591 - 600
TILL ET AL., BLOOD, vol. 112, no. 6, 2008, pages 2261 - 71
TORIKAI ET AL., BLOOD, vol. 119, no. 24, 2012, pages 5697 - 50
TORIKAI ET AL., BLOOD, vol. 122, no. 7, 2013, pages 1341 - 74
TORIKAI ET AL., NATURE SCI. REP., vol. 6, 2016, pages 21757
VERHOEYEN ET AL., METHODS MOL. BIOL., vol. 506, 2009, pages 97
VINCKE ET AL., J. BIOL. CHEM., vol. 284, 2009, pages 3273
WALCHLI ET AL., PLOS ONE, vol. 6, 2011, pages 327930
WALSENG ET AL., SCIENTIFIC REPORTS, vol. 7, 2017, pages 10713
WANG ET AL., BLOOD, vol. 118, 2011, pages 1255
WANG ET AL., HUM. GENE THER., vol. 18, 2007, pages 712
WILSON, SCIENCE, vol. 295, 2002, pages 2103
WOLFE ET AL., J. MOL. BIOL., vol. 285, 1999, pages 1917 - 1934
WOLFF ET AL., CANCER RES., vol. 53, 1993, pages 2560
XIE ET AL., PLOS ONE, vol. 9, 2014, pages el00448
ZHAO ET AL., J. IMMUNOL., vol. 174, 2005, pages 4415

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