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WO2025090749A1 - Récepteurs antigéniques chimériques ciblant une répétition riche en leucine 15 (lrrc15) - Google Patents

Récepteurs antigéniques chimériques ciblant une répétition riche en leucine 15 (lrrc15) Download PDF

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Publication number
WO2025090749A1
WO2025090749A1 PCT/US2024/052791 US2024052791W WO2025090749A1 WO 2025090749 A1 WO2025090749 A1 WO 2025090749A1 US 2024052791 W US2024052791 W US 2024052791W WO 2025090749 A1 WO2025090749 A1 WO 2025090749A1
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seq
amino acid
cell
polynucleotide
acid sequence
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Inventor
Christopher DERENZO
Stephen GOTTSCHALK
Jinghui Zhang
Timothy Isham SHAW
Liqing TIAN
Phuong Nguyen
Carla O'REILLY
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St Jude Childrens Research Hospital
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St Jude Childrens Research Hospital
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    • 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
    • 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
    • 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
    • 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
    • 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)

Definitions

  • the application relates to chimeric antigen receptors (CARs), particularly CARs targeting leucine rich repeat containing 15 (LRRC15), and their uses in tumor immunotherapy (e.g., adoptive cell therapy).
  • CARs chimeric antigen receptors
  • LRRC15 leucine rich repeat containing 15
  • the application further relates to therapeutic cells that express such CARs and methods for treating patients using the CAR-expressing therapeutic cells.
  • TME tumor microenvironment
  • ECM extracellular matrix
  • CAR chimeric antigen receptor
  • HLA human leukocyte antigen
  • LRRC15 is a member of the leucine-rich repeat superfamily that is overexpressed in various cancers and associated with higher tumor grade and aggression (Hingorani, et al., Mol Cancer Ther. 2021 Mar; 20(3): 535-540).
  • CARs chimeric antigen receptors
  • therapeutic cells that express such CARs which target antigens critical to tumor-stromal interactions, e.g., leucine-rich repeat-containing 15 (LRRC15), to improve drug delivery in the treatment of various cancer types, including solid tumors.
  • LRRC15 leucine-rich repeat-containing 15
  • a polynucleotide encoding a chimeric antigen receptor comprising: a) an extracellular target-binding domain comprising a binding moiety which binds to leucine rich repeat containing 15 (LRRC15); b) a transmembrane domain; and c) a cytoplasmic domain comprising a signaling domain.
  • CAR chimeric antigen receptor
  • the binding moiety is an antibody, or a fragment thereof, or a peptide that binds to LRRC15.
  • the anti-LRRC15 antibody fragment is a single chain variable fragment (scFv), Fab, Fab', F(ab')2, Fv fragment, disulfide-linked Fv (dsFv), diabody, VHH, VNAR, single-domain antibody (sdAb), nanobody, dAb fragment, Fd 1 fragment, or Fd fragment.
  • the anti-LRRC15 scFv comprises a heavy chain complementarity determining region 1 (HCDR1), a HCDR2, and a HCDR3 contained within a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 80% identity thereof; and/or a light chain complementarity determining region 1 (LCDR1), a LCDR2, and a LCDR3 contained within a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 80% identity thereof.
  • HCDR1 heavy chain complementarity determining region 1
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the anti-LRRC15 scFv comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 49, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 51; and/or a LCDR1 comprising the amino acid sequence of SEQ ID NO: 52, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 54.
  • the anti-LRRC15 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 80% identity thereof.
  • the nucleotide sequence encoding the anti- LRRC15 scFv VH comprises the sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a nucleotide sequence having at least 80% identity thereof.
  • the anti-LRRC15 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 80% identity thereof.
  • the nucleotide sequence encoding the anti-LRRC15 scFv VL comprises the sequence of SEQ ID NO: 11 or SEQ ID NO: 12, or a nucleotide sequence having at least 80% identity thereof.
  • the anti-LRRC 15 scFv further comprises a linker between the VH and the VL.
  • the linker sequence comprises the amino acid sequence GGGGSGGGGSGGGGS ((G 4 S) 3 ; SEQ ID NO: 7), GGGGS (SEQ ID NO: 55), (G 4 S) 2 (SEQ ID NO: 56), (G 4 S) 4 (SEQ ID NO: 57), KESGSVSSEQLAQFRSLD (SEQ ID NO: 58), EGKSSGSGSESKST (SEQ ID NO: 59), EGKSSGSGSESKSTQ (SEQ ID NO: 60), GSTSGSGKSSEGKG (SEQ ID NO: 61), SSADDAKKDDAKKDDAKKDDAKKDG (SEQ ID NO: 62), EGKSSGSGSESKVD (SEQ ID NO: 63), ESGSVSSEELAFRSLD (SEQ ID NO: 64), or an amino acid sequence having at least 80% identity thereof.
  • the linker sequence comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 80% identity thereof.
  • the nucleotide encoding the linker sequence comprises the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or a nucleotide sequence having at least 80% identity thereof.
  • the anti-LRRC 15 scFv comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence having at least 80% identity thereof.
  • the nucleotide sequence encoding the anti-LRRC 15 scFv comprises the sequence of SEQ ID NO: 29 or SEQID NO: 30, or a nucleotide sequence having at least 80% identity thereof.
  • the transmembrane domain is derived from CD8a, CD28, CD8, CD4, CD3 ⁇ , CD40, CD134 (OX-40), or CD7.
  • the transmembrane domain is derived from CD8a.
  • the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 80% sequence identity thereof.
  • the nucleotide sequence encoding the CD8a transmembrane domain comprises the sequence of SEQ ID NO: 19, or a nucleotide sequence having at least 80% identity thereof.
  • the transmembrane domain is derived from CD28.
  • the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 80% sequence identity thereof.
  • the nucleotide sequence encoding the CD28 transmembrane domain comprises the sequence of SEQ ID NO: 21, or a nucleotide sequence having at least 80% identity thereof.
  • the extracellular target-binding domain further comprises a hinge domain between the LRRC 15 -binding moiety and the transmembrane domain.
  • the hinge domain is derived from CD8ot stalk, CD28, or IgGl, IgG2, IgG3, IgG4, CD28, or CD8a.
  • the hinge domain is derived from CD8a stalk, CD28, or IgGl. In some embodiments, the hinge domain is derived from CD8a stalk. In some embodiments, the CD8a stalk hinge domain comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the CD8a stalk hinge domain comprises the sequence of SEQ ID NO: 14 or SEQ ID NO: 15, or a nucleotide sequence having at least 80% identity thereof. In some embodiments, the hinge domain is derived from CD28. In some embodiments, the CD28 hinge domain comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 80% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the CD28 hinge domain comprises the sequence of SEQ ID NO: 17, or a nucleotide sequence having at least 80% identity thereof.
  • the hinge domain is derived from CD8a stalk, CD28, or IgGl, and the transmembrane domain is derived from CD8a, CD28, CD8, CD4, CD3i ⁇ , CD40, CD134 (OX-40), or CD7.
  • the hinge domain is derived from CD8a stalk and the transmembrane domain is derived from CD8a.
  • the CD8a stalk hinge domain and the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence having at least 80% identity thereof.
  • the nucleotide sequence encoding the CD8a stalk hinge domain and the CD8a transmembrane domain comprises the sequence of SEQ ID NO: 35, or a nucleotide sequence having at least 80% identity thereof.
  • the hinge domain is derived from CD8a stalk and the transmembrane domain is derived from CD28.
  • the CD8a stalk hinge domain and the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence having at least 80% identity thereof.
  • the nucleotide sequence encoding the CD8a stalk hinge domain and the CD28 transmembrane domain comprises the sequence of SEQ ID NO: 37 or a nucleotide sequence having at least 80% identity thereof.
  • the hinge domain is derived from CD28 and the transmembrane domain is derived from CD28.
  • the CD28 hinge domain and the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence having at least 80% identity thereof.
  • the nucleotide sequence encoding the CD28 hinge domain and the CD28 transmembrane domain comprises the sequence of SEQ ID NO: 39 or a nucleotide sequence having at least 80% identity thereof.
  • the extracellular target-binding domain further comprises a leader sequence.
  • the leader sequence is derived from CD8a or human immunoglobulin heavy chain variable region.
  • the leader sequence comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80% identity thereof.
  • the nucleotide sequence encoding the leader sequence comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or a nucleotide sequence having at least 80% identity thereof.
  • the extracellular binding domain comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least 80% identity thereof.
  • the nucleotide sequence encoding the extracellular binding domain comprises the sequence of SEQ ID NO: 32 or SEQ ID NO: 33, or a nucleotide sequence having at least 80% identity thereof.
  • the signaling domain is derived from CD3 ⁇ , CD38, CD3s, CD3y, ZAP70, DAP10, DAP12, Fc epsilon receptor I y chain (FCER1G), CD226, or CD79A.
  • the signaling domain is derived from CD3( ⁇ .
  • the CD3 ⁇ signaling domain comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 80% identity thereof.
  • the nucleotide sequence encoding the CD3( ⁇ signaling domain comprises the sequence of SEQ ID NO: 26 or SEQ ID NO: 27, or a nucleotide sequence having at least 80% identity thereof.
  • the cytoplasmic domain further comprises one or more costimulatory domains.
  • the one or more costimulatory domains are derived from CD28, 4-1BB, 0x40, CD27, CD40, CD134, CD226, CD79A, ICOS, or MyD88, or any combination thereof.
  • the costimulatory domain is derived from CD28.
  • the CD28 costimulatory domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 80% identity thereof.
  • the nucleotide sequence encoding the CD28 costimulatory domain comprises the sequence of SEQ ID NO: 23 or SEQ ID NO: 24, or a nucleotide sequence having at least 80% identity thereof.
  • the cytoplasmic domain comprises the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence having at least 80% identity thereof.
  • the nucleotide sequence encoding the cytoplasmic domain comprises the sequence of SEQ ID NO: 41 or SEQ ID NO: 42, or a nucleotide sequence having at least 80% identity thereof.
  • the polynucleotide further encodes at least one additional polypeptide.
  • the sequence encoding the CAR is operably linked to the sequence encoding the at least one additional polypeptide via a sequence encoding a self-cleaving peptide and/or an internal ribosomal entry site (IRES).
  • the self-cleaving peptide is a 2A peptide.
  • the 2A peptide is T2A, P2A, E2A, or F2A peptide.
  • the 2A peptide is a T2A peptide.
  • the T2A peptide comprises the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 80% sequence identity thereof.
  • the sequence encoding the T2A peptide comprises the nucleotide sequence of SEQ ID NO: 71, or a nucleotide sequence having at least 80% sequence identity thereof.
  • the at least one polypeptide is a transduced host cell selection marker, an in vivo tracking marker, a cytokine, or a safety switch gene.
  • the CAR comprises the amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 45, or SEQ ID NO: 47, or an amino acid sequence having at least 80% identity thereof.
  • the sequence encoding the CAR comprises the nucleotide sequence of SEQ ID NO: 44, SEQ ID NO: 46, or SEQ ID NO: 48, or a nucleotide sequence having at least 80% identity thereof.
  • the polynucleotide of any one of those described above is a DNA molecule. In various embodiments, the polynucleotide of any one of those described above is an RNA molecule.
  • the vector is a viral vector.
  • the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, a baculoviral vector, or a vaccinia virus vector.
  • the viral vector is a lentiviral vector.
  • the vector is a non-viral vector.
  • the non- viral vector can be, but is not limited to, a minicircle plasmid, a Sleeping Beauty transposon, a PiggyBac transposon, or a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR) based gene editing.
  • HDR homology directed repair
  • CAR chimeric antigen receptor
  • an isolated host cell comprising the polynucleotide of any one of those described above or the recombinant vector of any one of those described above.
  • an isolated host cell comprising a CAR described above.
  • the host cell is an immune cell.
  • the host cell is a T-cell, a nature killer (NK) cell, or a macrophage.
  • the host cell is a T cell.
  • the host cell is a CD8+ T-cell, a CD4+ T-cell, a cytotoxic T-cell, an aP T-cell receptor (TCR) T-cell, an invariant natural killer T (iNKT) cell, a yo T-cell, a memory T-cell including memory stem T-cell (TSCM), a naive T-cell, an effector T-cell, a T-helper cell, and a regulatory T-cell (Treg).
  • the host cell is a NK cell.
  • the host cell has been activated and/or expanded ex vivo.
  • the host cell is an allogeneic cell. In some embodiments, the host cell is an autologous cell. In some embodiments, the host cell is isolated from a subject having a tumor, wherein one or more cells of the tumor express LRRC15. In some embodiments, the tumor is a solid tumor. In some embodiments, the solid tumor is a stromal-postive/cancer-postive tumor, stromal-positive/cancer-negative tumor, or stromal-negative/cancer-positive tumor. In some embodiments, the host cell is derived from a blood sample, a marrow sample, a tissue sample, or a tumor sample.
  • the tumor described herein is selected from osteosarcoma, soft tissue sarcoma, brain tumor, ovarian cancer, rhabdomyosarcoma, Ewing’s sarcoma and other Ewing’s sarcoma family of tumors, liposarcoma, fibrosarcoma, leiomyosarcoma, epithelioid sarcoma, vascular sarcoma, synovial sarcoma, Kaposi's sarcoma, uterine sarcoma, malignant fibrous histiocytoma, neuroblastoma, ganglioneuroblastoma, desmoplastic small round cell tumor, malignant peripheral nerve sheath tumor, undifferentiated pleiomorphic sarcoma, undifferentiated spindle cell sarcoma, chondrosarcoma, mesenchymoma, adrenocortical carcinoma, hepatoblasto
  • the solid tumor is osteosarcoma, glioblastoma, melanoma, prostate cancer, cervical cancer, soft tissue sarcoma, desmoplastic small round cell tumor, nonsmall cell lung cancer, lung adenocarcinoma, breast cancer, head and neck cancer, pancreatic cancer, ovarian cancer, or a brain tumor.
  • composition comprising the host cell described above and a pharmaceutically acceptable carrier and/or excipient.
  • a method of generating the isolated host cell described above comprising genetically modifying the host cell with the polynucleotide described above or the recombinant vector described above.
  • the vector is a viral vector and the genetic modification is conducted by a transduction using said vector.
  • the genetic modification is conducted ex vivo.
  • the method further comprises activation and/or expansion of the host cell ex vivo before, after, and/or during said genetic modification.
  • a method for killing a tumor cell expressing LRRC15 comprising contacting said cell with the host cell(s) described above or the pharmaceutical composition described above.
  • a method for treating a tumor in a subject in need thereof, wherein one or more cells of the tumor express LRRC15 comprising administering to the subject a therapeutically effective amount of the host cell(s) described above, or the pharmaceutical composition described above.
  • the tumor is a solid tumor.
  • the solid tumor is a stromal-postive/cancer-postive tumor, stromal- positive/cancer-negative tumor, or stromal-negative/cancer-positive tumor.
  • the solid tumor is selected from osteosarcoma, glioblastoma, melanoma, prostate cancer, cervical cancer, soft tissue sarcoma, desmoplastic small round cell tumor, non-small cell lung cancer, lung adenocarcinoma, breast cancer, head and neck cancer, pancreatic cancer, ovarian cancer, and a brain tumor.
  • the method further comprises: a) isolating T-cells, NK cells, iNKT cells, or macrophages from the subject or generating T-cells, NK cells, iNKT cells or macrophages from stem cells; b) genetically modifying said T-cells, NK cells, iNKT cells, macrophages, or stem cells ex vivo with the polynucleotide of any one of those described above or the vector of any one of those described above; c) optionally, expanding and/or activating said T-cells, NK cells, iNKT cells or macrophages before, after, or during step b); and d) introducing the genetically modified T-cells, NK cells, iNKT cells or macrophages into the subj ect.
  • the stem cells comprise induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • Figs. 1A-1E show transduction and in vitro activity of LRRC15-CAR T cells.
  • Fig. 1A shows a schematic representation of LRRC15- and control-CAR (Ctrl) constructs.
  • Fig. 1C shows LRRC15 surface expression on indicated tumor cells.
  • Fig. 1A shows a schematic representation of LRRC15- and control-CAR (Ctrl) constructs.
  • Fig. 1C shows LRRC15 surface expression on indicated tumor cells.
  • Fig. ID shows Ctrl- or LRRC15-CAR T cell interferon gamma (IFNy) (left) or IL2 secretion (right) 24 hours post-coculture with LRRC 15-negative (A549) or LRRC 15 -positive (LM7, U87) cells.
  • Fig. IE shows Ctrl- or LRRC15-CAR T cell cytotoxicity 24 hours post-coculture with LM7 (left) or U87 cells (right). Data represent mean with SEM (Figs. IB, ID and IE).
  • Figs. 2A-2J show in vivo anti-osteosarcoma activity of LRRC15-CAR T cells.
  • Fig. 2B and Fig. 2C show individual and summary tumor burden post tumor injection determined by bioluminescence imaging, respectively.
  • Fig. 2D shows survival post tumor injection for the locoregional model.
  • Fig. 2F shows individual (left) and summary tumor burden (right) post tumor rechallenge. Fig.
  • FIG. 2G shows a schema of the intravenous lung osteosarcoma model.
  • Fig. 2H and Fig. 21 show individual and summary tumor burden post T cell injection determined by bioluminescence imaging, respectively.
  • Fig. 2 J shows survival post T cell injection for the lung osteosarcoma model. Data represent mean with SEM (Figs. 2C, 2F and 21).
  • Figs. 3A-3K show antitumor activity of murine LRRC15-CAR T cells in syngeneic osteosarcoma models.
  • Fig. 3A shows a schematic representation of murine LRRC15- (mLRRC15) and control -CAR (mCtrl) constructs.
  • Fig. 3C shows mCtrl- or mLRRC15-CAR T cell IFNy secretion 24 hours post-coculture with mLRRC 15 -negative (F420) or mLRRC 15 -positive (F420.mLRRC15) cells.
  • Fig. 3E and Fig. 3F show individual and summary tumor burden, respectively.
  • Fig. 3G shows survival post T cell injection for the s.c. model.
  • Fig. 31 and Fig. 3J show individual and summary tumor burden, respectively.
  • Fig. 3K shows survival post T cell injection for the murine locoregional model. Data represent mean with SEM (Figs. 3B, 3C, 3F, and 3 J). ** p ⁇ 0.01, ns, non-significant by unpaired t test (Figs.
  • Figs. 4A-4E show LRRC15-CAR T cell transduction and phenotype.
  • FIG. 4A Vector copy number (VCN),
  • FIG. 4B fold expansion
  • Fig. 4C CD4/CD8 and
  • Fig. 4D memory phenotypes 7 post-transduction.
  • Fig. 4E shows Ctrl- or LRRC15-CAR T cell cytotoxicity 24 hours post-coculture with LRRC 15 -negative A549 cells. ****p ⁇ 0.0001 by one-way analysis of variance with Dunnett’s multiple comparisons test (Fig. 4A).
  • Figs. 5A-5D show antitumor activity and enhanced survival exhibited by LRRC 15-CAR T cells in a lung osteosarcoma model.
  • Fig. 5A shows a schema of an in vivo study: 2xl0 6 LM7.GFP.ffLuc cells were injected intravenously (i.v.) followed by 3xl0 6 Ctrl- or LRRC15-CAR T cells i.v. 28 days later.
  • Fig. 5D shows survival post T cell injection.
  • mice in the LI group, one in the L2 group, and five in the L3 group were euthanized due to signs of GvHD and were not censored in the survival analysis.
  • Figs. 6A-6D show antitumor activity and enhanced survival exhibited by LRRC 15-CAR T cells in an orthotopic high grade glioma model.
  • Fig. 6B and Fig. 6C show individual and summary tumor burden post tumor cell injection determined by bioluminescence imaging, respectively.
  • Figs. 7A and 7B show an amino acid sequence and a nucleotide sequence of Ll-CAR: huM25.CD8aH.CD8aTM.CD28.z, respectively.
  • Figs. 8A and 8B show an amino acid sequence and a nucleotide sequence of L2-CAR: huM25.CD8aH.CD28TM.CD28.z, respectively.
  • Figure discloses SEQ ID NOs: 45-46, respectively, in order of appearance.
  • Figs. 9A and 9B show an amino acid sequence and a nucleotide sequence of L3-CAR: huM25.CD28H.CD28TM.CD28.z, respectively.
  • Figure discloses SEQ ID NOs: 47-48, respectively, in order of appearance.
  • Fig. 10 shows amino acid sequences of CDR regions of VH and VL chains of the huM25 antibody.
  • Figure discloses SEQ ID NOs: 49-54, respectively, in order of appearance.
  • the present disclosure provides, among other things, chimeric antigen receptors (CARs) that target leucine rich repeat containing 15 (LRRC 15).
  • LRRC 15 can be located on the target tumor cells and/or in cancer-associated fibroblasts within the tumor micro-environment (TME).
  • TAE tumor micro-environment
  • the present disclosure further provides T-cells or other lymphocytes expressing the anti-LRRC15 CARs disclosed herein.
  • CAR-expressing cells targeting LRRC 15 could potentially target a broad range of solid tumors including sarcomas (e.g., osteosarcoma), brain tumors (e.g. high grade glioma), melanomas, or benign or malignant mesenchymal tumors.
  • LRRC 15 is expressed in pediatric and adult tumors.
  • CAR T-cells Compared to antibodydrug conjugates, CAR T-cells have the ability to self replicate and extravasate into tumor tissues, which are both advantages.
  • CAR-expressing cells targeting the LRRC 15 have the potential to reduce the risk of “on target/off cancer” toxicity because LRRC 15 has low expression on normal tissues.
  • CARs are primarily comprised of 1) an antigen-binding moiety such as, but not limited to, a single-chain variable fragment (scFv) derived from an antigen-specific monoclonal antibody, and 2) a lymphocyte activation domain such as, but not limited to, the ⁇ -chain from the T-cell receptor CD3. These two regions are fused together via a transmembrane domain.
  • a hinge domain is usually required to provide more flexibility and accessibility between the antigen-binding moiety and the transmembrane domain.
  • CAR constructs may also include co-stimulatory polypeptides to boost the CAR-induced immune response.
  • the most commonly used co-stimulating molecules include CD28 and 4-1BB, which promote both T-cell proliferation and cell survival.
  • Another example of a co-stimulatory domain is a MyD88/CD40 molecule that can be used with or without the use of a separate dimerization agent.
  • Additional CAR constructs may also include three or more signaling domains (e.g., CD3( ⁇ , CD28, and 4-1BB), which further improves lymphocyte cell survival and efficacy.
  • chimeric antigen receptor or “CAR” as used herein is defined as a cell-surface receptor comprising an extracellular target-binding domain, a transmembrane domain, and a cytoplasmic domain comprising a lymphocyte activation domain and, optionally, at least one co- stimulatory signaling domain, all in a combination that is not naturally found together on a single protein. This particularly includes receptors wherein the extracellular domain and the cytoplasmic domain are not naturally found together on a single receptor protein.
  • the CARs of the present disclosure can be used with lymphocytes such as T-cells and natural killer (NK) cells.
  • T cell includes thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • a T- cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell.
  • the T- cell can be a helper T-cell (HTL; CD4+ T-cell) CD4+ T-cell, a cytotoxic T-cell (CTL; CD8+ T- cell), a tumor infdtrating cytotoxic T-cell (TIL; CD8+ T-cell), CD4+CD8+ T-cell, or any other subset of T-cells.
  • HTL helper T-cell
  • CTL cytotoxic T-cell
  • TIL tumor infdtrating cytotoxic T-cell
  • CD4+CD8+ T-cell CD4+CD8+ T-cell
  • Other illustrative populations of T-cells suitable for use in particular embodiments include naive T-cells and memory T-cells.
  • NKT cells refer to a specialized population of T-cells that express a semi-invariant aP T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1.
  • NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+, and CD8- cells.
  • the TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or deleterious effects due to their abilities to produce cytokines that promote either inflammation or immune tolerance.
  • gamma-delta T-cells (y5 T-cells),” which refer to a specialized population that to a small subset of T-cells possessing a distinct TCR on their surface, and unlike the majority of T- cells in which the TCR is composed of two glycoprotein chains designated a- and P-TCR chains, the TCR in y8 T-cells is made up of a y-chain and a 8-chain. y8 T-cells can play a role in immunosurveillance and immunoregulation, and were found to be an important source of IL-17 and to induce robust CD8+ cytotoxic T-cell response.
  • Tregs refer to T-cells that suppress an abnormal or excessive immune response and play a role in immune tolerance.
  • Tregs cells are typically transcription factor Foxp3 -positive CD4+ T- cells and can also include transcription factor Foxp3 -negative regulatory T-cells that are IL-10- producing CD4+ T-cells.
  • NK cell refers to a differentiated lymphocyte with a CD 16+ CD56+ and/or CD57+ TCR- phenotype. NK cells are characterized by their ability to bind to and kill cells that fail to express “self’ MHC/HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response.
  • the term “antigen” refers to any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) capable of being bound by a T-cell receptor.
  • An antigen is also able to provoke an immune response.
  • An example of an immune response may involve, without limitation, antibody production, or the activation of specific immunologically competent cells, or both.
  • an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be synthesized or can be derived from a biological sample, or can be a macromolecule besides a polypeptide.
  • Such abiological sample can include, but is not limited to, a tissue sample, a tumor sample, a cell or a fluid with other biological components, organisms, subunits of proteins/antigens, or killed or inactivated whole cells or lysates.
  • an antigen described herein is leucine rich repeat containing 15 (LRRC15).
  • antigen-binding moiety refers to a target-specific binding element that may be any ligand that binds to an antigen of interest (e.g., LRRC15), or a polypeptide or fragment thereof, wherein the ligand is either naturally-derived or synthetic.
  • antigen-binding moieties include, but are not limited to, antibodies; polypeptides derived from antibodies, such as, for example, single chain variable fragments (scFv), Fab, Fab', F(ab')2, and Fv fragments; polypeptides derived from T-cell receptors (TCRs), such as, for example, TCR variable domains; secreted factors (e.g., cytokines, growth factors) that can be artificially fused to signaling domains (e.g., “zytokines”); and any ligand or receptor fragment (e.g., CD27, NKG2D) that binds to the antigen of interest.
  • TCRs T-cell receptors
  • secreted factors e.g., cytokines, growth factors
  • signaling domains e.g., “zytokines”
  • any ligand or receptor fragment e.g., CD27, NKG2D
  • Combinatorial libraries can also be used to identify peptides
  • antibody and “antibodies” refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, diabodies and anti -idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antigen-specific TCR), and epitope-binding fragments of any of the above.
  • the terms “antibody” and “antibodies” also refer to covalent diabodies such as those disclosed in U.S. Pat. Appl. Pub.
  • Antibodies useful as a TCR-binding molecule include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl and IgA2) or subclass.
  • the term “host cell” means any cell that contains a heterologous nucleic acid.
  • the heterologous nucleic acid can be a vector (e.g., an expression vector).
  • a host cell can be a cell from any organism that is selected, modified, transformed, grown, used, or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein, or an enzyme.
  • the host cell may be selected based on the vector backbone and the desired result.
  • a plasmid or cosmid can be introduced into a prokaryote host cell for replication of several types of vectors.
  • Bacterial cells such as, but not limited to, DH5a, JM109, KCB, SURE® Competent Cells, and SOLOP ACK Gold Cells, can be used as host cells for vector replication and/or expression. Additionally, bacterial cells such as E. coli LE392 can be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500 and YPH501), insects and mammals. Examples of mammalian eukaryotic host cells include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12 cells.
  • the host cell is autologous. In certain embodiments, the host cell is allogenic.
  • Host cells of the present disclosure include T-cells and NK cells that contain DNA or RNA sequences encoding a CAR and expressing the CAR on the cell surface. Such host cells may be used for enhancing T-cell or NK cell activity, treatment of tumors, and treatment of autoimmune disease.
  • activation or “stimulation” mean to induce a change in the biologic state by which the cells (e.g., T-cells and NK cells) express activation markers, produce cytokines, proliferate, and/or become cytotoxic to target cells. All of these changes can be produced by primary stimulatory signals. Co-stimulatory signals can amplify the magnitude of the primary signals and suppress cell death following initial stimulation, thereby resulting in a more durable activation state and thus a higher cytotoxic capacity.
  • a “co-stimulatory signal” refers to a signal, which, in combination with a primary signal such as TCR/CD3 ligation, leads to T-cell and/or NK cell proliferation and/or upregulation or downregulation of key molecules.
  • proliferation refers to an increase in cell division, either symmetric or asymmetric division of cells.
  • expansion refers to the outcome of cell division and cell death.
  • the term “differentiation” refers to a method of decreasing the potency or proliferation of a cell or moving the cell to a more developmentally restricted state.
  • express and “expression” mean allowing or causing the information in a gene or DNA sequence to become produced, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence.
  • a DNA sequence is expressed in or by a cell to form an “expression product” such as a protein.
  • the expression product itself e.g., the resulting protein, may also be said to be “expressed” by the cell.
  • An expression product can be characterized as intracellular, extracellular, or transmembrane.
  • transfection means the introduction of a “foreign” (z.e., extrinsic, or extracellular) nucleic acid into a cell using recombinant DNA technology.
  • genetic modification means the introduction of a “foreign” (i.e., extrinsic, or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence.
  • the introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence, and may include regulatory or control sequences operably linked to a polynucleotide encoding an expression product of interest (e g., a CAR), such as start, stop, promoter, signal, secretion, or other sequences which can be used by a cell's genetic machinery.
  • the gene or sequence may include nonfunctional sequences or sequences with no known function.
  • a host cell that receives and expresses introduced DNA or RNA has been “genetically engineered.”
  • the DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or from a different genus or species.
  • transduction means the introduction of a foreign nucleic acid into a cell using a viral vector.
  • genetically modified or “genetically engineered” refer to the addition of extra genetic material in the form of DNA or RNA into a cell.
  • the term “derivative” or “variant” in the context of proteins or polypeptides refer to: (a) a polypeptide that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the polypeptide it is a derivative or variant of; (b) a polypeptide encoded by a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a nucleotide sequence encoding the polypeptide it is a derivative or variant of; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and/or substitutions) relative to the
  • Percent sequence identity can be determined using any method known to one of skill in the art. In a specific embodiment, the percent identity is determined using the “Best Fit” or “Gap” program of the Sequence Analysis Software Package (Version 10; Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, Madison, Wisconsin). Information regarding hybridization conditions (e.g., high, moderate, and typical stringency conditions) have been described, see, e.g., U.S. Patent Application Publication No. US 2005/0048549 (e.g., paragraphs 72-73), the content of which is incorporated herein by reference in its entirety.
  • vector means the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to genetically modify the host cell and promote expression (e.g., transcription and translation) of the introduced sequence.
  • Vectors include plasmids, synthesized RNA and DNA molecules, phages, viruses, etc.
  • the vector is a viral vector such as, but not limited to, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, a baculoviral vector, and a vaccinia virus vector.
  • the viral vector is a lentiviral vector.
  • promoter refers to any cis-acting genetic element that controls some aspect of the expression of nucleic acid sequences.
  • the term “promoter” comprises essentially the minimal sequences required to initiate transcription.
  • the term “promoter” includes the sequences to start transcription, and in addition, also include sequences that can upregulate or downregulate transcription, commonly termed “enhancer elements” and “repressor elements,” respectively.
  • operatively linked when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed in functional relationships with each other.
  • an operatively linked promoter, enhancer element(s), open reading frame, 5' and 3' UTR, and terminator sequences can result in the accurate production of a nucleic acid molecule (e.g., RNA).
  • operatively linked nucleic acid elements result in the transcription of an open reading frame and, ultimately, the production of a polypeptide (i.e., expression of the open reading frame).
  • an operatively linked peptide is one in which the functional domains are placed at appropriate distances from each other to impart the intended function of each domain.
  • the terms “enhance” or “promote” or “increase” or “expand” or “improve” refer generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule/composition.
  • a measurable physiological response may include an increase in T-cell expansion, activation, effector function, persistence, and/or tumor cell killing ability, among others apparent from the understanding in the art and the description herein.
  • an “increased” or “enhanced” amount can be a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or a control composition.
  • a “decrease” or “lower” or “lessen” or “reduce” or “abate” is generally meant the ability of a composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule/composition.
  • a “decrease” or “reduced” amount can be a “statistically significant” amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle, a control composition, or the response in a particular cell lineage.
  • the terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and/or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms.
  • the benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
  • the term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof.
  • the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode or route of administration, and the like.
  • compositions described herein refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal e.g., a human).
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
  • protein encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins including, without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).
  • modified proteins e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.
  • nucleic acid encompass both DNA and RNA unless specified otherwise.
  • nucleic acid sequence or “nucleotide sequence” is meant the nucleic acid sequence encoding an amino acid, the term may also refer to the nucleic acid sequence including the portion coding for any amino acids added as an artifact of cloning, including any amino acids coded for by linkers.
  • patient refers to mammals including, without limitation, human and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models.
  • subject is a human.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • the carrier can be a solid dosage form carrier including, but not limited to, one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant.
  • Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin. [0088] Singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.
  • the term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range.
  • the allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.
  • John Wiley and Sons, Inc. Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are explained, e.g., in U.S. Patent No. 7,912,698 and U.S. Patent Appl. Pub. Nos. 2011/0202322 and 2011/0307437.
  • the present disclosure provides chimeric antigen receptors (CARs) that target leucine rich repeat containing 15 (LRRC1 ).
  • LRRC15 can be expressed, e.g., by target tumor cells and/or cancer-associated fibroblasts within the tumor micro-environment (TME).
  • the present disclosure provides a polynucleotide encoding a CAR comprising: a) an extracellular target-binding domain comprising a binding moiety which binds to LRRC15; b) a transmembrane domain; and c) a cytoplasmic domain comprising a signaling domain.
  • the present disclosure provides a CAR polypeptide comprising: a) an extracellular target-binding domain comprising a binding moiety which binds to LRRC15; b) a transmembrane domain; and c) a cytoplasmic domain comprising a signaling domain.
  • CARs of the present disclosure comprise an extracellular targetbinding domain, wherein the extracellular target-binding domain comprises an antigen -binding moiety.
  • antigen-binding moiety depends upon the type and number of antigens that define the surface of a target cell.
  • the antigen-binding moiety may be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state.
  • the CARs of the present disclosure can be genetically modified to target a tumor antigen of interest by way of engineering a desired antigen-binding moiety that specifically binds to an antigen (e.g., on a tumor cell).
  • the antigen that is targeted by the extracellular target-binding domain is leucine rich repeat containing 15 (LRRC15).
  • the antigen-binding moiety can be monomeric or multimeric (e.g., homodimeric or heterodimeric), or associated with multiple proteins in a non-covalent complex.
  • the antigen-binding moiety comprises an antigen-binding peptide, polypeptide, or functional variant thereof, that binds to an antigen.
  • the antigen-binding polypeptide is an antibody or an antibody fragment that binds to an antigen.
  • Antigen-binding moi eties may comprise antibodies and/or antibody fragments such as monoclonal antibodies, multispecific antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (dsFv), intrabodies, minibodies, single domain antibody variable domains, nanobodies (VHHs), diabodies and anti -idiotypic (anti- id) antibodies (including, e.g., anti -Id antibodies to antigen-specific TCR), and epitope-binding fragments of any of the above.
  • Antibodies and/or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.
  • the antigen-binding moiety is a single-chain Fv (scFv).
  • the scFv comprises a linker between the VH and VL.
  • linker sequences that may be used in the scFvs described herein include GGGGSGGGGSGGGGS ((G 4 S) 3 ; SEQ ID NO: 7), GGGGS (SEQ ID NO: 55), (G 4 S) 2 (SEQ ID NO: 56), (G 4 S) 4 (SEQ ID NO: 57), KESGSVSSEQLAQFRSLD (SEQ ID NO: 58), EGKSSGSGSESKST (SEQ ID NO: 59), EGKSSGSGSESKSTQ (SEQ ID NO: 60), GSTSGSGKSSEGKG (SEQ ID NO: 61), SSADDAKKDDAKKDDAKKDDAKKDG (SEQ ID NO: 62), EGKSSGSGSESKVD (SEQ ID NO:
  • the linker sequence comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 7), or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 7.
  • the nucleotide sequence that encodes the linker sequence comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 7.
  • the nucleotide sequence that encodes the linker sequence comprises the nucleotide sequence set forth in SEQ ID NO: 8 or 9, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 8 or 9.
  • the linker sequence comprises the amino acid sequence set forth in SEQ ID NO: 7.
  • the nucleotide sequence that encodes the linker sequence comprises the nucleotide sequence set forth in SEQ ID NO: 8 or 9.
  • the antigen-binding moiety comprises a polypeptide or functional variant thereof that binds to LRRC15.
  • the antigen-binding moiety is an antibody or an antibody fragment that binds to LRRC15.
  • the antigen-binding moiety is a single chain variable fragment (scFv) that binds to LRRC15 (anti- LRRC15 scFv).
  • the anti-LRRC15 scFv is derived from an mAb specific for the leucine rich repeat containing 15 (LRRC15).
  • the anti-LRRC15 scFv is derived from a LRRC15 specific mAb huM25 (huM25 scFv), huAD208.4.1 (huAD208.4.1 scFv), huAD208.12.1 (huAD208.12.1 scFv), huAD208.14.1(huAD208.14.1 scFv), hu!39.10 (hul3940 scFv), muAD210.40.9 (muAD210.40.9 scFv), or muAD209.9 1 (muAD209.9.1 scFv), or an antibody that competes for binding huLRRC 15 with such an antibody; or a functional variant thereof.
  • Antibodies huM25, huAD208.4.1, huAD208.12. 1, huAD208.14.1, hul39.10, muAD210.40.9 and muAD209.9.1 are antibodies specific for LRRC15 described in U.S. Patent No. 10,188,660, which is herein incorporated by reference in its entirety for all purposes.
  • huM25 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 49 (SYWIE); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 50 (EILPGSDTTNYNEKFKD); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 51 (DRGNYRAWFGY).
  • CDRs complementarity determining regions
  • huM25 scFv comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 52 (RASQDISNYLN); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 53 (YTSRLHS); and a light chain CDR3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 54 (QQGEALPWT).
  • VL light chain variable region
  • CDRs complementarity determining regions
  • huM25 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 49 (SYWIE); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 50 (EILPGSDTTNYNEKFKD); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 51 (DRGNYRAWFGY); and comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 52 (RASQDISNYLN); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 53 (YTSRLHS); and a light chain CDR3 (LCDRs): a light chain C
  • huM25 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 4, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 4.
  • VH heavy chain variable domain
  • the nucleotide sequence that encodes the VH of huM25 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 4, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 4.
  • the nucleotide sequence that encodes the VH of huM25 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 6, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 5 or 6.
  • the VH of huM25 scFv comprises the amino acid sequence set forth in SEQ ID NO: 4.
  • the nucleotide sequence that encodes the VH of huM25 scFV comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 6.
  • huM25 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 10, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 10.
  • VL light chain variable domain
  • the nucleotide sequence that encodes the VL of huM25 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 10, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 10.
  • the nucleotide sequence that encodes the VL of huM25 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 11 or 12, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 11 or 12.
  • the VL of huM25scFv comprises the amino acid sequence set forth in SEQ ID NO: 10.
  • the nucleotide sequence that encodes the VL of huM25 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 11 or 12.
  • huM25 scFv comprises the amino acid sequence set forth in SEQ ID NO: 28, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 28.
  • the nucleotide sequence that encodes huM25 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 28.
  • the nucleotide sequence that encodes huM25 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 29 or 30, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 29 or 30.
  • huM25 scFv comprises the amino acid sequence set forth in SEQ ID NO: 28.
  • the nucleotide sequence that encodes huM25 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 29 or 30.
  • huAD208.4.1 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 86 (DYYIH); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 87 (LVYPYIGGTNYNQKFKG); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 88 (GDNKYDAMDY).
  • VH heavy chain variable region
  • huAD208.4.1 scFv comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 89 (RASQSVSTSSYSYMH); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 90 (YASSLES); and a light chain CDR3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 91 (EQSWEIRT).
  • VL light chain variable region
  • CDRs complementarity determining regions
  • huAD208.4.1 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 86 (DYYIH); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 87 (LVYPYIGGTNYNQKFKG); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 88 (GDNKYDAMDY); and comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 89 (RASQSVSTSSYSYMH); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 90 (YASSLES); and a light chain CDRs
  • huAD208.4.1 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 92, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 92.
  • VH heavy chain variable domain
  • the nucleotide sequence that encodes the VH of huAD208.4.1 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 92, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 92.
  • the VH of huAD208.4.1 scFv comprises the amino acid sequence set forth in SEQ ID NO: 92.
  • huAD208.4.1 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 93, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 93.
  • VL light chain variable domain
  • the nucleotide sequence that encodes the VL of huAD208.4.1 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 93, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 93.
  • the VL of huAD208.4.1scFv comprises the amino acid sequence set forth in SEQ ID NO: 93.
  • huAD208.12.1 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 94 (NYWMH); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 95 (MIHPNSGSTKHNEKFRG); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 96 (SDFGNYRWYFDV).
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • huAD208.12.1 scFv comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 97 (RASQSSSNNLH); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 98 (YVSQSIS); and a light chain CDR3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 99 (QQSNSWPFT).
  • VL light chain variable region
  • CDRs complementarity determining regions
  • huAD208.12.1 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 94 (NYWMH); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 95 (MIHPNSGSTKHNEKFRG); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 96 (SDFGNYRWYFDV); and comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 97 (RASQSSSNNLH); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 98 (YVSQSIS); and a light chain CDRs
  • huAD208.12.1 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 100, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 100.
  • the nucleotide sequence that encodes the VH of huAD208.12.1 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:
  • the VH of huAD208.12.1 scFv comprises the amino acid sequence set forth in SEQ ID NO: 100.
  • huAD208.12.1 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 101, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 101.
  • the nucleotide sequence that encodes the VL of huAD208.12.1 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:
  • the VL of huAD208.12.1scFv comprises the amino acid sequence set forth in SEQ ID NO: 101.
  • huAD208.14.1 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 102 (DYYIH); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 103 (LVYPYIGGSSYNQQFKG); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 104 (GDNNYDAMDY).
  • CDRs complementarity determining regions
  • huAD208.14.1 scFv comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 105 (RASQSVSTSTYNYMH); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 106 (YASNLES); and a light chain CDR3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 107 (HHTWEIRT).
  • VL light chain variable region
  • CDRs complementarity determining regions
  • huAD208.14.1 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 102 (DYYIH); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 103 (LVYPYIGGSSYNQQFKG); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 104 (GDNNYDAMDY); and comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 105 (RASQSVSTSTYNYMH); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 106 (YASNLES); and
  • huAD208.14.1 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 108, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 108.
  • the nucleotide sequence that encodes the VH of huAD208.14.1 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:
  • the VH of huAD208.14.1 scFv comprises the amino acid sequence set forth in SEQ ID NO: 108.
  • huAD208.14.1 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 109, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 109.
  • the nucleotide sequence that encodes the VL of huAD208.14.1 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:
  • the VL of huAD208.14.1scFv comprises the amino acid sequence set forth in SEQ ID NO: 109.
  • hu!39.10 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 1 10 (SYGVH); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 111 (VIWAGGSTNYNSALMS); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 112 (HMITEDYYGMDY).
  • VH heavy chain variable region
  • CDR1 heavy chain CDR1
  • HCDR2 HCDR2
  • HCDR3 heavy chain CDR3
  • hul39.10 scFv comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 113 (KSSQSLLNSRTRKNYLA); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 114 (WASTRES); and a light chain CDR3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 115 (KQSYNLPT).
  • VL light chain variable region
  • CDRs complementarity determining regions
  • hul39.10 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 110 (SYGVH); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 111 (VIWAGGSTNYNSALMS); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 112 (HMITEDYYGMDY); and comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 113 (KSSQSLLNSRTRKNYLA); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 114 (WASTRES); and a light chain CDR
  • hul39.10 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 116, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 116.
  • VH heavy chain variable domain
  • the nucleotide sequence that encodes the VH of hul39.10 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 116, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 116.
  • the VH of hul39.10 scFv comprises the amino acid sequence set forth in SEQ ID NO: 116.
  • hu!39.10 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 117, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 117.
  • VL light chain variable domain
  • the nucleotide sequence that encodes the VL of hul39.10 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 117, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 117.
  • the VL of hul39.10scFv comprises the amino acid sequence set forth in SEQ ID NO: 117.
  • muAD210.40.9 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 118 (NYWLG); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 119 (DIYPGGGNTYYNEKLKG); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 120 (WGDKKGNYFAY).
  • CDRs complementarity determining regions
  • muAD210.40.9 scFv comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 121 (TASSSVYSSYLH); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 122 (STSNLAS); and a light chain CDR3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 123 (HQYHRSPT).
  • VL light chain variable region
  • CDRs complementarity determining regions
  • muAD210.40.9 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 118 (NYWLG); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 119 (DIYPGGGNTYYNEKLKG); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 120 (WGDKKGNYFAY); and comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 121 (TASSSVYSSYLH); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 122 (STSNLAS); and a light chain CDR
  • muAD210.40.9 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 124, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 124.
  • the nucleotide sequence that encodes the VH of muAD210.40.9 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:
  • the VH of muAD210.40.9 scFv comprises the amino acid sequence set forth in SEQ ID NO: 124.
  • muAD210.40.9 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 125, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 125.
  • the nucleotide sequence that encodes the VL of muAD210.40.9 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:
  • the VL of muAD210.40.9scFv comprises the amino acid sequence set forth in SEQ ID NO: 125.
  • muAD209.9.1 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 126 (NFGMN); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 127 (WINLYTGEPTFADDFKG); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 128 (KGETYYRYDGFAY).
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • muAD209.9.1 scFv comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 129 (RSSKSLLHSNGNTHLY); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 130 (RMSNLAS); and a light chain CDR3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 131 (MQLLEYPYT).
  • VL light chain variable region
  • CDRs complementarity determining regions
  • muAD209.9.1 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 126 (NFGMN); a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 127 (WINLYTGEPTFADDFKG); and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 128 (KGETYYRYDGFAY); and comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 129 (RSSKSLLHSNGNTHLY); a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 130 (RMSNLAS); and a light chain CDR3
  • muAD209.9.1 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 132, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 132.
  • the nucleotide sequence that encodes the VH of muAD209.9.1 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:
  • the VH of muAD209.9.1 scFv comprises the amino acid sequence set forth in SEQ ID NO: 132.
  • muAD209.9. 1 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 133, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 133.
  • the nucleotide sequence that encodes the VL of muAD209.9.1 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:
  • the VL of muAD209.9.1scFv comprises the amino acid sequence set forth in SEQ ID NO: 133.
  • the CAR of the present disclosure comprises a leader sequence.
  • the leader sequence may be positioned amino-terminal to the extracellular target-binding domain.
  • the leader sequence may be optionally cleaved from the antigen-binding moiety during cellular processing and localization of the CAR to the cellular membrane.
  • the leader sequence may be derived from human immunoglobulin heavy chain variable region.
  • the leader sequence comprises the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 1.
  • the nucleotide sequence encoding the leader sequence comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least
  • the nucleotide sequence encoding the leader sequence comprises the sequence set forth in SEQ ID NO: 2 or 3, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least
  • the leader sequence comprises the amino acid sequence of SEQ ID NO: 1.
  • the nucleotide sequence encoding the leader sequence comprises the nucleotide sequence set forth in SEQ ID NO: 2 or 3.
  • the CAR further comprises a hinge domain between the extracellular antigen-binding domain and the transmembrane domain, wherein the antigen-binding moiety, linker, and the transmembrane domain are in frame with each other.
  • a hinge domain can comprise any oligo- or polypeptide that functions to link the antigenbinding moiety to the transmembrane domain.
  • a hinge domain can be used to provide more flexibility and accessibility for the antigen-binding moiety.
  • a hinge domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • a hinge domain may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8a stalk, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the hinge domain may be a synthetic sequence that corresponds to a naturally occurring linker region sequence, or may be an entirely synthetic linker region sequence.
  • Non-limiting examples of hinge domains which may be used in accordance with the disclosure include a part of human CD8a chain, partial extracellular domain of CD28, FcyRllla receptor, IgG, IgM, IgA, IgD, IgE, an Ig hinge, or functional fragment thereof.
  • additional linking amino acids are added to the linker region to ensure that the antigen-binding moiety is an optimal distance from the transmembrane domain.
  • the linker when the hinge domain is derived from an Ig, the linker may be mutated to prevent Fc receptor binding.
  • the hinge domain may be derived from CD8a, CD28, or an immunoglobulin (IgG).
  • IgG hinge may be from IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof.
  • the hinge domain may be derived from CD8a stalk, CD28, or IgGl.
  • the linker domain comprises an immunoglobulin IgG hinge or functional fragment thereof.
  • the IgG hinge is from IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof.
  • the linker domain comprises the CHI, CH2, CH3 and/or hinge region of the immunoglobulin.
  • the linker domain comprises the core hinge region of the immunoglobulin.
  • core hinge can be used interchangeably with the term “short hinge” (a.k.a “SH”).
  • linker domains are the core immunoglobulin hinge regions listed in Table 1 (see also Wypych et al., JBC 2008 283(23): 16194-16205, which is incorporated herein by reference in its entirety for all purposes).
  • the linker domain is a fragment of the immunoglobulin hinge. Table 1. Amino Acid Sequence of Short Hinge Regions of IgG Immunoglobulins
  • the hinge domain comprises an IgGl hinge, or a variant thereof. In certain embodiments, the hinge domain comprises the short hinge structure of IgGl, IgG2, IgG3, or IgG4 or a variant thereof.
  • hinge domain comprises a short hinge region and comprises the amino acid sequence set forth in SEQ ID NO: 65, 66, 67, 68 or 69, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 65, 66, 67, 68 or 69.
  • the nucleotide sequence encoding the hinge comprising the short hinge region comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 65, 66, 67, 68 or 69, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 65, 66, 67, 68 or 69.
  • hinge domain comprises a short hinge region and comprises the amino acid sequence set forth in SEQ ID NO: 65, 66, 67, 68 or 69.
  • the hinge domain is derived from CD8a stalk or complete or partial sequences of the CD8a stalk, which are also called CD8a hinge.
  • the hinge domain derived from CD8a stalk comprises the amino acid sequence set forth in SEQ ID NO: 13, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 13.
  • the nucleotide sequence that encodes the CD8a stalk hinge domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 13, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 13.
  • the nucleotide sequence that encodes the CD8a stalk hinge domain comprises the nucleotide sequence set forth in SEQ ID NO: 14 or 15, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 14 or 15.
  • the CD8a stalk hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 13.
  • the nucleotide sequence that encodes the CD8a stalk hinge domain comprises the nucleotide sequence set forth in SEQ ID NO: 14 or 15.
  • the hinge domain is derived from CD28.
  • the hinge domain derived from CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 16, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 16.
  • the nucleotide sequence that encodes the CD28 hinge domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 16.
  • the nucleotide sequence that encodes the CD28 hinge domain comprises the nucleotide sequence set forth in SEQ ID NO: 17, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 17.
  • the CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 16.
  • the nucleotide sequence that encodes the CD28 hinge domain comprises the nucleotide sequence set forth in SEQ ID NO: 17.
  • the hinge domain in addition to the sequences described above, can comprise additional linker amino acids to allow for extra flexibility and/or accessibility.
  • the CARs of the present disclosure comprise a transmembrane domain, fused in frame between the extracellular target-binding domain and the cytoplasmic domain.
  • the transmembrane domain may be derived from the protein contributing to the extracellular target-binding domain, the protein contributing the signaling or co-signaling domain, or by a totally different protein.
  • the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to minimize interactions with other members of the CAR complex.
  • the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to avoid-binding of proteins naturally associated with the transmembrane domain.
  • the transmembrane domain includes additional amino acids to allow for flexibility and/or optimal distance between the domains connected to the transmembrane domain.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • Non-limiting examples of transmembrane domains of particular use in this disclosure may be derived from (i.e. comprise at least the transmembrane region(s) of) the a, P or chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154.
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and/or valine can be found at each end of a synthetic transmembrane domain.
  • transmembrane domain of the , q or FcsRly chains which contain a cysteine residue capable of disulfide bonding so that the resulting chimeric protein will be able to form disulfide linked dimers with itself, or with unmodified versions of the ⁇ , or FcsRly chains or related proteins.
  • the transmembrane domain will be selected or modified by amino acid substitution to avoid-binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • in order to retain physical association with other members of the receptor complex.
  • the transmembrane domain in the CAR of the disclosure is derived from the CD28 transmembrane domain.
  • the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 20, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 20.
  • the nucleotide sequence that encodes the CD28 transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 20.
  • the nucleotide sequence that encodes the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 21, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 21.
  • the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 20.
  • the nucleotide sequence that encodes the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 21.
  • the transmembrane domain in the CAR of the disclosure is derived from the CD8a transmembrane domain.
  • the CD8a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 18.
  • the nucleotide sequence that encodes the CD8a transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 18.
  • the nucleotide sequence that encodes the CD8a transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 19, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 19.
  • the CD8a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 18.
  • the nucleotide sequence that encodes the CD8u transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 19.
  • the CARs of the present disclosure comprise a hinge domain and a transmembrane domain.
  • CARs of the present disclosure comprise the hinge domain derived from CD8a stalk, CD28, or IgGl and the transmembrane domain derived from CD8a, CD28, CD8, CD4, CD3 ⁇ , CD40, CD134 (OX-40), or CD7.
  • the CARs of the present disclosure comprise the hinge domain derived from CD8a stalk and the transmembrane domain derived from CD8a.
  • the CD8a hinge domain and the CD8a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 34, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 34.
  • the nucleotide sequence that encodes the CD8a hinge domain and the CD8a transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 34, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 34.
  • the nucleotide sequence that encodes the CD8a hinge domain and the CD8a transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 35, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 35.
  • the CD8a hinge domain and the CD8a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 34.
  • the nucleotide sequence that encodes the CD8a hinge domain and the CD8u transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 35.
  • the CARs of the present disclosure comprise the hinge domain derived from CD8a stalk and the transmembrane domain derived from CD28.
  • the CD8a hinge domain and the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 36, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 36.
  • the nucleotide sequence that encodes the CD8a hinge domain and the CD28 transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 36, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 36.
  • the nucleotide sequence that encodes the CD8a hinge domain and the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 37, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 37.
  • the CD8a hinge domain and the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 36.
  • the nucleotide sequence that encodes the CD8a hinge domain and the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 37.
  • the CARs of the present disclosure comprise the hinge domain derived from CD28 and the transmembrane domain derived from CD28.
  • the CD28 hinge domain and the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 38, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 38.
  • the nucleotide sequence that encodes the CD28 hinge domain and the CD28 transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 38, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 38.
  • the nucleotide sequence that encodes the CD28 hinge domain and the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 39.
  • the CD28 hinge domain and the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 38.
  • the nucleotide sequence that encodes the CD28 hinge domain and the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 39.
  • CARs of the present disclosure comprise a cytoplasmic domain, which comprises one or more costimulatory domains and one or more signaling domains.
  • the cytoplasmic domain which comprises one or more costimulatory domains and one or more signaling domains, is responsible for activation of at least one of the normal effector functions of the lymphocyte in which the CAR has been placed in.
  • effector function refers to a specialized function of a cell. Effector function of a T-cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • the term “signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire signaling domain is present, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
  • intracellular signaling domain is thus meant to include any truncated portion of the signaling domain sufficient to transduce the effector function signal.
  • Non-limiting examples of signaling domains which can be used in the CARs of the present disclosure include, e.g., signaling domains derived from CD3( ⁇ , CD38, CD3s, CD3y, ZAP70, DAP10, DAP12, Fc epsilon receptor I y chain (FCER1G), CD226, CD79A, FcR 0, CD5, CD22, CD66d, or CD79B.
  • the CAR of the present disclosure comprises a signaling domain derived from CD3( ⁇ .
  • the lymphocyte activation domain in the CAR of the disclosure is designed to comprise a signaling domain derived from CD3( ⁇ .
  • the CD3 signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 25 or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 25.
  • the nucleotide sequence that encodes the CD3i ⁇ signaling domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 25.
  • the nucleotide sequence that encodes the CD3 ⁇ signaling domain comprises the nucleotide sequence set forth in SEQ ID NO: 26 or 27, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 26 or 27.
  • the CD3( ⁇ signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 25.
  • the nucleotide sequence that encodes the CD3 ⁇ signaling domain comprises the nucleotide sequence set forth in SEQ ID NO: 26 or 27.
  • Non-limiting examples of costimulatory domains which can be used in the CARs of the present disclosure include, those derived from CD28, 4-lBB(CD137), 0x40, CD27, CD40, CD134 (OX-40), CD226, CD79A, ICOS, MyD88, BTLA, CD27, CD30, GITR, HVEM, IL-2RP, or the STAT3 -binding YXXQ, or any combination thereof.
  • the CAR of the present disclosure comprises one costimulatory domain.
  • the CAR of the present disclosure comprises a costimulatory domain derived from CD28.
  • the CAR of the present disclosure comprises two or more costimulatory domains. In certain embodiments, the CAR of the present disclosure comprises two, three, four, five, six or more costimulatory domains. For example, the CAR of the present disclosure may comprise a costimulatory domain derived from 4-1BB and a costimulatory domain derived from CD28.
  • the CARs of the present disclosure comprise a cytoplasmic domain, which comprises a signaling domain, a MyD88 polypeptide or functional fragment thereof, and a CD40 cytoplasmic polypeptide region or a functional fragment thereof.
  • the CAR lacks the CD40 transmembrane and/or CD40 extracellular domains.
  • the CAR includes the CD40 transmembrane domain.
  • the CAR includes the CD40 transmembrane domain and a portion of the CD40 extracellular domain, wherein the CD40 extracellular domain does not interact with natural or synthetic ligands of CD40.
  • the signaling domain is separated from the MyD88 polypeptide or functional fragment thereof and/or the CD40 cytoplasmic polypeptide region or a functional fragment thereof.
  • the lymphocyte activation domain is separated from the MyD88 polypeptide or functional fragment thereof and/or the CD40 cytoplasmic polypeptide region or a functional fragment thereof by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.
  • the signaling domain(s) and costimulatory domain(s) can be in any order.
  • the signaling domain is upstream of the costimulatory domains.
  • the signaling domain is downstream from the costimulatory domains. In the cases where two or more costimulatory domains are included, the order of the costimulatory domains could be switched.
  • the costimulatory domain derived from CD28 comprises the amino acid sequence set forth in SEQ ID NO: 22, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 22.
  • the nucleotide sequence that encodes the CD28 costimulatory domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 22.
  • the nucleotide sequence that encodes the CD28 costimulatory domain comprises the nucleotide sequence set forth in SEQ ID NO: 23 or 24, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 23 or 24.
  • the CD28 costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 22.
  • the nucleotide sequence that encodes the CD28 costimulatory domain comprises the nucleotide sequence set forth in SEQ ID NO: 23 or 24.
  • the CAR may further comprise at least one additional gene that encodes an additional peptide.
  • additional genes can include a transduced host cell selection marker, an in vivo tracking marker, a cytokine, a suicide gene, or some other functional gene.
  • the functional additional gene can induce the expression of another molecule.
  • the functional additional gene can increase the safety of the CAR.
  • the CAR construct may comprise an additional gene which is truncated CD19 (tCD19).
  • tCD19 can be used as a tag. Expression of tCD19 may also help determine transduction efficiency.
  • additional genes include genes that encode polypeptides with a biological function; examples include, but are not limited to, cytokines, chimeric cytokine receptors, dominant negative receptors, safety switches (CD20, truncated EGFR or HER2, inducible caspase 9 molecules).
  • the CAR construct may comprise an additional gene which is a synNotch receptor. Once activated, the synNotch receptor can induce the expression of a target gene (e g., a second CAR and/or bispecific molecule).
  • the CAR comprises at least one additional gene (i.e., a second gene). In certain embodiments, the CAR comprises one second gene.
  • the CAR comprises two additional genes (i.e., a third gene). In yet another embodiment, the CAR comprises three additional genes (i.e., a fourth gene). In certain embodiments, the additional genes are separated from each other and the CAR construct. For example, they may be separated by 2A sequences and/or an internal ribosomal entry sites (IRES). In certain examples, the CAR can be at any position of the polynucleotide chain (for example construct A: CAR, second gene, third gene, fourth gene; construct B: second gene, CAR, third gene, fourth gene; etc.)
  • Non-limiting examples of classes of additional genes that can be used to increase the effector function of CAR containing host cells include (a) secretable cytokines (e.g., but not limited to, IL-7, IL-12, IL-15, IL-18), (b) membrane bound cytokines (e.g., but not limited to, IL- 15), (c) chimeric cytokine receptors (e.g., but not limited to, IL-2/IL-7, IL-4/IL-7), (d) constitutive active cytokine receptors (e.g., but not limited to, C7R), (e) dominant negative receptors (DNR; e.g., but not limited to TGFRII DNR), (f) ligands of costimulatory molecules (e.g., but not limited to, CD80, 4-1BBL), (g) nuclear factor of activated T-cells (NFATs) (e.g., NFATcl, NFATc2, NFATc
  • the additional gene sequence may be derived from tCD19.
  • the tCD19 sequence comprises the amino acid sequence set forth in SEQ ID NO: 72 or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 72.
  • the nucleotide sequence encoding the tCD19 sequence comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:72, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 72.
  • the nucleotide sequence encoding the tCD19 sequence comprises the sequence set forth in SEQ ID NO: 73 or 74, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 73 or 74.
  • the tCD19 sequence comprises the amino acid sequence of SEQ ID NO: 72.
  • the nucleotide sequence encoding the tCD19 sequence comprises the nucleotide sequence set forth in SEQ ID NO: 73 or 74.
  • the additional gene may be regulated by an NFAT dependentpromoter.
  • Activation of the T-cell or other lymphocyte leads to activation of the transcription factor NFAT resulting in the induction of the expression of the protein encoded by the gene linked with the NFAT dependent promoter.
  • One or more members of the NFAT family i.e., NFATcl, NFATc2, NFATc3, NFATc4, and NFAT5
  • NFAT-dependent promoters and enhancers tend to have three to five NFAT binding sites.
  • the functional additional gene can be a suicide gene.
  • a suicide gene is a recombinant gene that will cause the host cell that the gene is expressed in to undergo programmed cell death or antibody mediated clearance at a desired time.
  • Suicide genes can function to increase the safety of the CAR.
  • the additional gene is an inducible suicide gene.
  • Non-limiting examples of suicide genes include i) molecules that are expressed on the cell surface and can be targeted with a clinical grade monoclonal antibody including CD20, EGFR or a fragment thereof, HER2 or a fragment thereof, and ii) inducible suicide genes (e.g., but not limited to inducible caspase 9 (see Straathof et al. (2005) Blood. 105(11): 4247-4254; US Publ. No. 2011/0286980, each of which is incorporated herein by reference in its entirety for all purposes)).
  • CARs of the present disclosure may be regulated by a safety switch.
  • the term “safety switch” refers to any mechanism that is capable of removing or inhibiting the effect of a CAR from a system (e g., a culture or a subject). Safety switches can function to increase the safety of the CAR.
  • the function of the safety switch may be inducible.
  • safety switches include (a) molecules that are expressed on the cell surface and can be targeted with a clinical grade monoclonal antibody including CD20, EGFR or a fragment thereof, HER2 or a fragment thereof, and (b) inducible suicide genes (e.g., but not limited to, herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase 9 (see Straathof et al. (2005) Blood. 105(11): 4247-4254; US Publ. No. 2011/0286980, each of which is incorporated herein by reference in its entirety for all purposes).
  • HSV-TK herpes simplex virus thymidine kinase
  • caspase 9 see Straathof et al. (2005) Blood. 105(11): 4247-4254; US Publ. No. 2011/0286980, each of which is incorporated herein by reference in its entirety for all purposes
  • the safety switch is a CD20 polypeptide.
  • Expression of human CD20 on the cell surface presents an attractive strategy for a safety switch.
  • Cells that express CD20 can be rapidly eliminated with the FDA approved monoclonal antibody rituximab through complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (see e.g., Griffioen, M., et al. Haematologica 94, 1316-1320 (2009), which is incorporated herein by reference in its entirety for all purposes).
  • Rituximab is an anti-CD20 monoclonal antibody that has been FDA approved for Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin’s Lymphoma (NHL), among others (Storz, U. MAbs 6, 820-837 (2014), which is incorporated herein by reference in its entirety for all purposes).
  • the CD20 safety switch is non-immunogenic and can function as a reporter/selection marker in addition to a safety switch (Bonifant, C.L., et al. Mol Ther 24, 1615-1626 (2016); van Loenen, M.M., et al. Gene Ther 20, 861-867 (2013); each of which is incorporated herein by reference in its entirety for all purposes).
  • sequence encoding an additional gene is operably linked to the sequence encoding CAR via a sequence encoding a self-cleaving peptide and/or an Internal Ribosome Entry Site (IRES) as disclosed herein.
  • IRS Internal Ribosome Entry Site
  • Non-limiting examples of self-cleaving peptide sequences includes Thoseaasigna virus 2A (T2A; AEGRGSLLTCGDVEENPGP, SEQ ID NO: 75, EGRGSLLTCGDVEENPGP, SEQ ID NO: 70, or GSGEGRGSLLTCGDVEENPGP, SEQ ID NO: 76); the foot and mouth disease virus (FMDV) 2A sequence (F2A; GSGSRVTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAP VKQLLNFDLLKLAGDVESNPGP, SEQ ID NO: 77), Sponge (Amphimedon queenslandica) 2A sequence (LLCFLLLLLSGDVELNPGP, SEQ ID NO: 78; or HHFMFLLLLLAGDIELNPGP, SEQ ID NO: 79); acorn worm 2A sequence (Saccoglossus kowalevskii) (WFLVLLSFILSGDIEV NPGP, SEQ ID NO: 80); am
  • an Internal Ribosome Entry Site may be used to link the CAR and the additional gene.
  • IRES is an RNA element that allows for translation initiation in a capindependent manner. IRES can link two coding sequences in one bicistronic vector and allow the translation of both proteins in cells.
  • the self-cleaving 2A peptide is a T2A peptide and comprises the amino acid sequence set forth in SEQ ID NO: 70.
  • the sequence encoding the T2A peptide comprises the nucleotide sequence SEQ ID NO: 71.
  • the host cells can be genetically modified to express not only CARs as disclosed herein but to also express fusion protein with signaling activity (e.g., costimulation, T-cell activation). These fusion proteins can improve host cell activation and/or responsiveness. In certain embodiments, the fusion protein can enhance the host cell’s response to the target antigen. In certain embodiments, the fusion protein can impart resistance to suppression signals.
  • fusion proteins can comprise portions of CD4, CD8a, CD28, portions of a T-cell receptor, or an antigen-binding moiety (e.g., scFv) linked to a MyD88, CD40, and/or other signaling molecules.
  • an antigen-binding moiety e.g., scFv
  • the fusion protein comprises an extracellular target-binding domain (as disclosed above), a transmembrane domain (as described above) and a cytoplasmic domain, wherein the cytoplasmic domain comprises at least one co-stimulatory protein (as described above).
  • the co-stimulatory fusion protein does not comprise a lymphocyte activation domain (e.g., CD3Q.
  • the at least one co- stimulatory protein can be a MyD88 polypeptide or functional fragment thereof, and/or a CD40 cytoplasmic polypeptide region or a functional fragment thereof.
  • the fusion protein comprises an extracellular domain (such as, but not limited to CD19, CD34), a transmembrane domain (as described above) and a cytoplasmic domain, wherein the cytoplasmic domain comprises at least one co-stimulatory protein (as described above).
  • the fusion protein does not comprise a lymphocyte activation domain (e.g., CD3Q.
  • the at least one portion of the fusion protein can be a MyD88 polypeptide or functional fragment thereof, and/or a CD40 cytoplasmic polypeptide region or a functional fragment thereof.
  • Non-limiting examples of fusion proteins include, but are not limited to, the constructs in the publication of WO2019222579 and WO2016073875, which are incorporated herein by reference in its entirety for all purposes.
  • the fusion proteins are introduced into the host cell on a separate vector from the CAR. In certain embodiments, the fusion proteins are introduced into the host cell on the same vector as the CAR. In certain embodiments, the fusion proteins are introduced into the host cell on the same vector as the CAR but separated by a separation sequence such as 2A.
  • a separation sequence such as 2A.
  • an anti-LRRC15 CAR of the disclosure comprises an extracellular binding domain comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 31.
  • the extracellular binding domain of an anti- LRRC15 CAR of the disclosure is encoded by a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 31.
  • the nucleotide sequence that encodes the extracellular binding domain of an anti-LRRC15 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 32 or 33, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 32 or 33.
  • an anti-LRRC15 CAR of the disclosure comprises an extracellular binding domain comprising the amino acid sequence set forth in SEQ ID NO: 31.
  • the nucleotide sequence that encodes the extracellular binding domain of an anti-LRRC15 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 32 or 33.
  • an anti-LRRC15 CAR of the disclosure comprises a cytoplasmic domain comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 40.
  • the cytoplasmic domain of an anti-LRRC15 CAR of the disclosure is encoded by a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 40, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 40.
  • the nucleotide sequence that encodes the cytoplasmic domain of an anti-LRRC15 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 41 or 42, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 41 or 42.
  • an anti-LRRC15 CAR of the disclosure comprises a cytoplasmic domain comprising the amino acid sequence set forth in SEQ ID NO: 40.
  • the nucleotide sequence that encodes the cytoplasmic domain of an anti-LRRC 15 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 41 or 42.
  • an anti-LRRC15 CAR of the disclosure is encoded by a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 43, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 43.
  • the nucleotide sequence that encodes an anti-LRRC15 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 44, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 44.
  • an anti-LRRC15 CAR of the disclosure comprises an amino acid sequence set forth in SEQ ID NO: 43.
  • the nucleotide sequence that encodes an anti-LRRC15 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 44.
  • an anti-LRRC15 CAR of the disclosure comprises the amino acid sequence of SEQ ID NO: 45, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 45.
  • an anti-LRRC15 CAR of the disclosure is encoded by a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 45, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 45.
  • the nucleotide sequence that encodes an anti-LRRC15 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 46, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 46.
  • an anti-LRRC15 CAR of the disclosure comprises an amino acid sequence set forth in SEQ ID NO: 45.
  • the nucleotide sequence that encodes an anti-LRRC15 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 46.
  • an anti-LRRC15 CAR of the disclosure comprises the amino acid sequence of SEQ ID NO: 47, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 47.
  • an anti-LRRC15 CAR of the disclosure is encoded by a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 47, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 47.
  • the nucleotide sequence that encodes an anti-LRRC15 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 48, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 48.
  • an anti-LRRC15 CAR of the disclosure comprises an amino acid sequence set forth in SEQ ID NO: 47.
  • the nucleotide sequence that encodes an anti-LRRC15 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 48.
  • the polynucleotide encoding a CAR is a DNA molecule. In various embodiments, the polynucleotide encoding a CAR is an RNA molecule.
  • the present disclosure provides CAR polypeptides encoded by a polynucleotide described above.
  • the present disclosure provides recombinant vectors comprising a polynucleotide encoding a CAR comprising polynucleotides encoding the proteins disclosed above.
  • the polynucleotide is operatively linked to at least one regulatory element for expression of the chimeric antigen receptor.
  • recombinant vectors of the disclosure comprise the nucleotide sequence of SEQ ID NO: 44, 46, or 48, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 44, 46, or 48.
  • recombinant vectors comprise a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 44, 46, or 48, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 44, 46, or 48.
  • the recombinant vector comprises a polynucleotide encoding a CAR, wherein the polynucleotide is operatively linked to at least one additional gene.
  • the additional gene is a tCD19.
  • the vector is a viral vector.
  • the viral vector can be, but is not limited to, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, a baculoviral vector, and a vaccinia virus vector.
  • the viral vector is a lentiviral vector.
  • the vector is a non-viral vector.
  • the viral vector may be a plasmid or a transposon (such as a PiggyBac or a Sleeping Beauty transposon).
  • the non-viral vector can be, but is not limited to, a minicircle plasmid, a Sleeping Beauty transposon, a PiggyBac transposon, or a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR) based gene editing.
  • HDR homology directed repair
  • the polynucleotide encoding the CAR is operably linked to at least a regulatory element.
  • the regulatory element can be capable of mediating expression of the CAR in the host cell. Regulatory elements include, but are not limited to, promoters, enhancers, initiation sites, polyadenylation (poly A) tails, IRES elements, response elements, and termination signals.
  • the regulatory element regulates CAR expression.
  • the regulatory element increased the expression of the CAR.
  • the regulatory element increased the expression of the CAR once the host cell is activated.
  • the regulatory element decreases expression of the CAR.
  • the regulatory element decreases expression of the CAR once the host cell is activated.
  • the present disclosure provides an isolated host cell comprising a polynucleotide or a recombinant vector described herein. In one aspect, the present disclosure provides an isolated host cell comprising a CAR described herein. In some embodiments, the CAR targets leucine rich repeat containing 15 (LRRC15).
  • LRRC15 leucine rich repeat containing 15
  • the host cell is an immune cell.
  • the immune cell may be a T- cell, a natural killer (NK) cell, or a macrophage.
  • the host cell is a T-cell.
  • T-cells may include, but are not limited to, thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • a T-cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell.
  • the T-cell can be a helper T-cell (HTL; CD4+ T-cell) CD4+ T-cell, a cytotoxic T-cell (CTL; CD8+ T-cell), a tumor infdtrating cytotoxic T-cell (TIL; CD8+ T-cell), CD4+ CD8+ T-cell, or any other subset of T-cells.
  • HTL helper T-cell
  • CTL cytotoxic T-cell
  • TIL tumor infdtrating cytotoxic T-cell
  • CD4+ CD8+ T-cell CD4+ CD8+ T-cell
  • Other illustrative populations of T-cells suitable for use in particular embodiments include naive T-cells memory T-cells, and NKT cells.
  • the T-cell is selected from a CD8+ T-cell, a CD4+ T-cell, a cytotoxic T-cell, an a.p T-cell receptor (TCR) T-cell, an invariant natural killer T (iNKT) cell, a natural killer T (NKT) cell, a y6 T-cell, a memory T-cell, a memory T-cell, a memory stem T-cell (TSCM), a naive T-cell, an effector T-cell, a T-helper cell, and a regulatory T-cell (Treg).
  • TCR T-cell receptor
  • iNKT invariant natural killer T
  • NKT natural killer T
  • y6 T-cell a memory T-cell
  • a memory T-cell a memory T-cell
  • TSCM memory stem T-cell
  • Reg regulatory T-cell
  • the host cell is a NK cell.
  • NK cell refers to a differentiated lymphocyte with a CD3- CD16+, CD3- CD56+, CD16+ CD56+ and/or CD57+ TCR- phenotype.
  • the host cell has been activated and/or expanded ex vivo.
  • the host cell is an allogeneic cell. In various embodiments, the host cell is an autologous cell.
  • the host cell is isolated from a subject having a tumor, wherein the one or more cells of the tumor express LRRC 15.
  • the tumor can be found within, but not limited to, bone, brain, breast tissue, prostate tissue, bladder tissue, oral and/or dental tissue, head and/or neck tissue, colorectal tissue, lung tissue, skin, and lymph nodes.
  • the tumor is a cancer.
  • the cancer can be, but not limited to, breast cancer, cervical cancer, pancreatic cancer, prostate cancer, bladder cancer, oral squamous cell carcinoma, ovarian cancer, head and/or neck squamous cell carcinoma, colorectal cancer, lung cancer, brain tumors, melanoma, bone cancer, and/or pediatric solid tumors.
  • the tumor is a solid tumor.
  • the solid tumor can be a stromal-postive/cancer-postive tumor, stromal-positive/cancer-negative tumor, or stromal-negative/cancer-positive tumor.
  • solid tumors include, without limitation, osteosarcoma, glioblastoma, melanoma, prostate cancer, cervical cancer, soft tissue sarcoma, desmoplastic small round cell tumor, non-small cell lung cancer, lung adenocarcinoma, breast cancer, head and neck cancer, pancreatic cancer, ovarian cancer, and a brain tumor.
  • the host cell is isolated from a subject having a cancer, wherein one or more cells of the cancer cells express a LRRC15.
  • the cancer is a solid tumor.
  • cancers that express LRRC15 include adult solid tumors and brain tumors, adrenal gland tumors, adrenocortical carcinoma, anal cancer, atypical teratoid rhabdoid tumor, bile duct cancer, bladder cancer, bone cancer, bowel cancer, brain tumors, breast cancer, cancer of unknown primary, cancer spread to bone, cancer spread to brain, cancer spread to liver, cancer spread to lung, carcinoid, cervical cancer, children's cancers, chondrosarcoma, colorectal cancer, colorectal cancer, craniopharyngioma, desmoplastic small round cell tumor, diffuse intrinsic pontine glioma and other brain tumors, ear cancer, endometrial cancer, ependymoma, epithelioid
  • the tumor described herein is selected from osteosarcoma, soft tissue sarcoma, brain tumor, ovarian cancer, rhabdomyosarcoma, Ewing’s sarcoma and other Ewing’s sarcoma family of tumors, liposarcoma, fibrosarcoma, leiomyosarcoma, epithelioid sarcoma, vascular sarcoma, synovial sarcoma, Kaposi's sarcoma, uterine sarcoma, malignant fibrous histiocytoma, neuroblastoma, ganglioneuroblastoma, desmoplastic small round cell tumor, malignant peripheral nerve sheath tumor, undifferentiated pleiomorphic sarcoma, undifferentiated spindle cell sarcoma, chondrosarcoma, mesenchymoma, adrenocortical carcinoma, hepatoblasto
  • the host cell is derived from a blood sample, a marrow sample, a tissue sample, or a tumor sample.
  • the present disclosure provides a method of generating an isolated host cell described herein.
  • the method includes genetically modifying the host cell with a polynucleotide encoding a CAR and optionally an additional gene (e.g., tCD19).
  • the host cell is genetically modified with a recombinant vector described herein.
  • the vector is a viral vector and the genetic modification is conducted by a transduction using the viral vector.
  • the genetically modifying step may be conducted in vivo or ex vivo. In some embodiments, the genetically modifying step is conducted ex vivo.
  • the method may further include activation and/or expansion of the host cell ex vivo before, after and/or during the genetic modification.
  • the host cells may be autologous/autogeneic (“self’) or non-autologous (“non- self,” e.g., allogeneic, syngeneic, or xenogeneic).
  • the host cells are obtained from a mammalian subject.
  • the host cells are obtained from a primate subject.
  • the host cells are obtained from a human subject.
  • Lymphocytes can be obtained from sources such as, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Lymphocytes may also be generated by differentiation of stem cells. In certain embodiments, lymphocytes can be obtained from blood collected from a subject using techniques generally known to the skilled person, such as sedimentation, e.g., FICOLLTM separation.
  • cells from the circulating blood of a subject are obtained by apheresis.
  • An apheresis device typically contains lymphocytes, including T-cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing.
  • the cells can be washed with PBS or with another suitable solution that lacks calcium, magnesium, and most, if not all other, divalent cations.
  • a washing step may be accomplished by methods known to those in the art, such as, but not limited to, using a semiautomated flowthrough centrifuge (e.g., Cobe 2991 cell processor, or the Baxter CytoMate).
  • a semiautomated flowthrough centrifuge e.g., Cobe 2991 cell processor, or the Baxter CytoMate.
  • the cells may be resuspended in a variety of biocompatible buffers, cell culture medias, or other saline solution with or without buffer.
  • host cells can be isolated from peripheral blood mononuclear cells (PBMCs) by lysing the red blood cells and depleting the monocytes.
  • PBMCs peripheral blood mononuclear cells
  • the cells can be sorted by centrifugation through a PERCOLLTM gradient.
  • both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T-cell subpopulations either before or after activation, expansion, and/or genetic modification.
  • T lymphocytes can be enriched.
  • a specific subpopulation of T lymphocytes expressing one or more markers such as, but not limited to, CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD27, CD28, CD34, CD36, CD45RA, CD45RO, CD56, CD62, CD62L, CD122, CD123, CD127, CD235a, CCR7, HLA-DR or a combination thereof using either positive or negative selection techniques.
  • the T lymphocytes for use in the compositions of the disclosure do not express or do not substantially express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.
  • NK cells can be enriched.
  • a specific subpopulation of T lymphocytes expressing one or more markers such as, but not limited to, CD2, CD 16, CD56, CD57, CD94, CD 122 or a combination thereof using either positive or negative selection techniques.
  • a method of producing host cells for administration to a subject comprises stimulating the host cells to become activated in the presence of one or more stimulatory signals or agents (e.g., compound, small molecule, e.g., small organic molecule, nucleic acid, polypeptide, or a fragment, isoform, variant, analog, or derivative thereof).
  • a method of producing host cells for administration to a subject comprises stimulating the host cells to become activated and to proliferate in the presence of one or more stimulatory signals or agents.
  • Host cells e.g., T lymphocytes, NK cells, and macrophages
  • T lymphocytes can be activated by inducing a change in their biologic state by which the cells express activation markers, produce cytokines, proliferate and/or become cytotoxic to target cells. All these changes can be produced by primary stimulatory signals.
  • Co-stimulatory signals amplify the magnitude of the primary signals and suppress cell death following initial stimulation resulting in a more durable activation state and thus a higher cytotoxic capacity.
  • T cells can be activated generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety.
  • the T-cell based host cells can be activated by binding to an agent that activates CD3( ⁇ .
  • a CD2-binding agent may be used to provide a primary stimulation signal to the T-cells.
  • CD2 agents include CD2 ligands and anti-CD2 antibodies, e.g., the T1 1.3 antibody in combination with the T1 1.1 or T1 1.2 antibody (Meuer, S. C. et al. (1984) Cell 36:897-906) and the 9.6 antibody (which recognizes the same epitope as TI 1.1) in combination with the 9-1 antibody (Yang, S. Y. et al. (1986) J. Immunol. 137: 1097-1100).
  • Other antibodies which bind to the same epitopes as any of the above described antibodies can also be used.
  • the host cells are activated by administering phorbol myristate acetate (PMA) and ionomycine.
  • the host cells are activated by administering an appropriate antigen that induces activation and then expansion.
  • PMA, ionomycin, and/or appropriate antigen are administered with CD3 induce activation and/or expansion.
  • the activating agents used in the present disclosure includes, but is not limited to, an antibody, a fragment thereof and a proteinaceous binding molecule with antibody-like functions.
  • Examples of (recombinant) antibody fragments are Fab fragments, Fv fragments, singlechain Fv fragments (scFv), a divalent antibody fragment such as an (Fab)2 '-fragment, diabodies, triabodies (Iliades, P., et al., FEBS Lett (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94) and other domain antibodies (Holt, L.
  • the divalent antibody fragment may be an (Fab)2'- fragment, or a divalent single-chain Fv fragment while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, a Fv fragment, and a single-chain Fv fragment (scFv).
  • one or more binding sites of the CD3( ⁇ agents may be a bivalent proteinaceous artificial binding molecule such as a dimeric lipocalin mutein (i.e., duocalin).
  • the receptor binding reagent may have a single second binding site, (z.e., monovalent).
  • monovalent agents include, but are not limited to, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties or an MHC molecule.
  • monovalent antibody fragments include, but are not limited to a Fab fragment, a Fv fragment, and a single-chain Fv fragment (scFv), including a divalent single-chain Fv fragment.
  • the agent that specifically binds CD3 includes, but is not limited to, an anti-CD3- antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3-antibody, and a proteinaceous CD3-binding molecule with antibody-like binding properties.
  • a proteinaceous CD3-binding molecule with antibody-like binding properties can be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on the ankyrin scaffold, a protein based on the crystalline scaffold, an adnectin, and an avimer. It also can be coupled to a bead.
  • the activating agent e.g., CD3 -binding agents
  • the activating agent can be present in a concentration of about 0.1 to about 10 pg/ml.
  • the activating agent e.g., CD3-binding agents
  • the activating agent e.g., CD3-binding agents
  • the activating agent is administered at a concentration of about 0.1 pg/ml, about 0.2 pg/ml, about 0.3 pg/ml, about 0.4 pg/ml, about 0.5 pg/ml, about 0.6 pg/ml, about 0.7 pg/ml, about 0.8 pM, about 0.9 pg/ml, about 1 pg/ml, about 2 pg/ml, about 3 pg/ml, about 4 pM, about 5 pg/ml, about 6 pg/ml, about 7 pg/ml, about 8 pg/ml, about 9 pg/ml, or about 10 pg/ml.
  • the CD3-binding agents can be present in a concentration of 1 pg/ml.
  • NK cells can be activated generally using methods as described, for example, in U.S. Patents 7,803,376, 6,949,520, 6,693,086, 8,834,900, 9,404,083, 9,464,274, 7,435,596, 8,026,097, 8,877,182; U.S. Patent Applications US2004/0058445, US2007/0160578, US2013/0011376, US2015/0118207, US2015/0037887; and PCT Patent Application WO2016/122147, each of which is incorporated herein by reference in its entirety.
  • the NK based host cells can be activated by, for example and not limitation, inhibition of inhibitory receptors on NK cells (e.g., KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E or LILRB5 receptor).
  • inhibitory receptors on NK cells e.g., KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E or LILRB5 receptor.
  • the NK based host cells can be activated by, for example and not limitation, feeder cells (e.g., native K562 cells or K562 cells that are genetically modified to express 4-1BBL and cytokines such as IL 15 or IL21).
  • feeder cells e.g., native K562 cells or K562 cells that are genetically modified to express 4-1BBL and cytokines such as IL 15 or IL21.
  • interferons or macrophage-derived cytokines can be used to activate NK cells.
  • interferons include but are not limited to interferon alpha and interferon gamma
  • cytokines include but are not limited to IL- 15, IL-2, IL-21.
  • the NK activating agent can be present in a concentration of about 0.1 to about 10 pg/ml. In certain embodiments, the NK activating agent can be present in a concentration of about 0.2 pg/ml to about 9 pg/ml, about 0.3 pg/ml to about 8 pg/ml, about 0.4 pg/ml to about 7 pg/ml, about 0.5 pg/ml to about 6 pg/ml, about 0.6 pg/ml to about 5 pg/ml, about 0.7 pg/ml to about 4 pg/ml, about 0.8 pg/ml to about 3 pg/ml, or about 0.9 pg/ml to about 2 pg/ml.
  • the activating agent is attached to a solid support such as, but not limited to, a bead, an absorbent polymer present in culture plate or well or other matrices such as, but not limited to, Sepharose or glass; may be expressed (such as in native or recombinant forms) on cell surface of natural or recombinant cell line by means known to those skilled in the art.
  • the host cells are genetically modified to express a CAR described above.
  • the host cells can be genetically modified after stimulation/activation.
  • the host cells are modified within 12 hours, 16 hours, 24 hours, 36 hours, or 48 hours of stimulation/activation.
  • the cells are modified within 16 to 24 hours after stimulation/activation.
  • the host cells are modified within 24 hours.
  • the CAR polynucleotide construct In order to genetically modify the host cell to express the CAR, the CAR polynucleotide construct must be transferred into the host cell. Polynucleotide transfer may be via viral or non- viral gene methods. Suitable methods for polynucleotide delivery for use with the current methods include any method known by those of skill in the art, by which a polynucleotide can be introduced into an organelle, cell, tissue, or organism.
  • polynucleotides are transferred to the cell in a non-viral vector.
  • the non-viral vector is a transposon.
  • Exemplary transposons hat can be used in the present disclosure include, but are not limited to, a sleeping beauty transposon and a PiggyBac transposon.
  • the non-viral vector is a minicircle plasmid or a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR) based gene editing.
  • HDR homology directed repair
  • Nucleic acid vaccines can be used to transfer CAR polynucleotides into the host cells.
  • Such vaccines include, but are not limited to non-viral polynucleotide vectors, “naked” DNA and RNA, and viral vectors. Methods of genetically modifying cells with these vaccines, and for optimizing the expression of genes included in these vaccines are known to those of skill in the art.
  • the host cells can be genetically modified by methods ordinarily used by one of skill in the art.
  • the host cells can be transduced via retroviral transduction.
  • References describing retroviral transduction of genes are Anderson et al., U.S. Pat. No. 5,399,346; Mann et al., Cell 33: 153 (1983); Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol. 62: 1120 (1988); Temin et al., U.S. Pat. No.
  • One method of genetic modification includes ex vivo modification.
  • Various methods are available for transfecting cells and tissues removed from a subject via ex vivo modification.
  • retroviral gene transfer in vitro can be used to genetically modified cells removed from the subject and the cell transferred back into the subject. See e.g., Wilson et al., Science, 244: 1344- 1346, 1989 and Nabel et al., Science, 244(4910): 1342-1344, 1989, both of which are incorporated herein by reference in their entity.
  • the host cells may be removed from the subject and transfected ex vivo using the polynucleotides (e.g., expression vectors) of the disclosure.
  • the host cells obtained from the subject can be transfected or transduced with the polynucleotides (e.g., expression vectors) of the disclosure and then administered back to the subject.
  • a cell or a polynucleotide or viral vector may be delivered to a cell, tissue, or organism via one or more injections (e.g., a needle injection).
  • Non-limiting methods of injection include injection of a composition (e.g., a saline based composition).
  • Polynucleotides can also be introduced by direct microinjection.
  • Non-limiting sites of injection include, subcutaneous, intradermal, intramuscular, intranodal (allows for direct delivery of antigen to lymphoid tissues), intravenous, intraprostatic, intratumor, intralymphatic (allows direct administration of DCs) and intraperitoneal. It is understood that proper site of injection preparation is necessary (e.g., shaving of the site of injection to observe proper needle placement).
  • Electroporation is another method of polynucleotide delivery. See e.g., Potter et al., (1984) Proc. Nat'l Acad. Sci. USA, 81, 7161-7165 and Tur-Kaspa et al., (1986) Mol. Cell Biol., 6, 716-718, both of which are incorporated herein in their entirety for all purposes. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. In certain embodiments, cell wall-degrading enzymes, such as pectin-degrading enzymes, can be employed to render the host cells more susceptible to genetic modification by electroporation than untreated cells. See e.g., U.S. Pat. No. 5,384,253, incorporated herein by reference in its entirety for all purposes.
  • In vivo electroporation involves a basic injection technique in which a vector is injected intradermally in a subject. Electrodes then apply electrical pulses to the intradermal site causing the cells localized there (e.g., resident dermal dendritic cells), to take up the vector. These tumor antigen-expressing dendritic cells activated by local inflammation can then migrate to lymphnodes.
  • Methods of electroporation for use with this disclosure include, for example, Sardesai, N. Y., and Weiner, D. B., Current Opinion in Immunotherapy 23:421-9 (2011) and Ferraro, B. et al., Human Vaccines 7: 120-127 (2011), both of which are hereby incorporated by reference herein in their entirety for all purposes.
  • Additional methods of polynucleotide transfer include liposome-mediated transfection (e.g., polynucleotide entrapped in a lipid complex suspended in an excess of aqueous solution. See e.g., Ghosh and Bachhawat, (1991) In: Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, pp. 87-104). Also contemplated is a polynucleotide complexed with Lipofectamine, or Superfect); DEAE-dextran (e.g., a polynucleotide is delivered into a cell using DEAE-dextran followed by polyethylene glycol.
  • liposome-mediated transfection e.g., polynucleotide entrapped in a lipid complex suspended in an excess of aqueous solution. See e.g., Ghosh and Bachhawat, (1991) In: Liver Diseases, Targeted Diagnosis and Therapy
  • microprojectile bombardment e.g., one or more particles may be coated with at least one polynucleotide and delivered into cells by a propelling force.
  • microprojectile bombardment e.g., one or more particles may be coated with at least one polynucleotide and delivered into cells by a propelling force.
  • host cells are genetically modified using gene editing with homology-directed repair (HDR).
  • HDR homology-directed repair
  • HDR is a mechanism used by cells to repair double strand DNA breaks.
  • a donor polynucleotide with homology to the site of the double strand DNA break is used as a template to repair the cleaved DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to the DNA.
  • new nucleic acid material may be inserted or copied into a target DNA cleavage site.
  • Double strand DNA breaks in host cells may be induced by a site-specific nuclease.
  • site-specific nuclease refers to a nuclease capable of specifically recognizing and cleaving a nucleic acid (DNA or RNA) sequence.
  • Suitable site-specific nucleases for use in the present disclosure include, but are not limited to, RNA-guided endonuclease (e.g., CRISPR-associated (Cas) proteins), zinc finger nuclease, a TALEN nuclease, or mega-TALEN nuclease.
  • a site-specific nuclease e.g., a Cas9 + guide RNA
  • a donor polynucleotide encoding a CAR of the present disclosure is introduced to a host cell, along with a donor polynucleotide encoding a CAR of the present disclosure and optionally an additional protein (e.g., tCD19).
  • an additional protein e.g., tCD19
  • T- cells may be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5, or 6 months or more with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion.
  • Agents that can be used for the expansion of T-cells can include interleukins, such as IL- 2, IL-7, IL-15, or IL-21 (see for example Cornish et al. 2006, Blood. 108(2):600-8, Bazdar and Sieg, 2007, Journal of Virology, 2007, 81(22):12670-12674, Battalia et al, 2013, Immunology, 139(1): 109-120).
  • interleukins such as IL- 2, IL-7, IL-15, or IL-21
  • Other illustrative examples for agents that may be used for the expansion of T- cells are agents that bind to CD8, CD45 or CD90, such as aCD8, aCD45 or aCD90 antibodies.
  • T-cell population including antigen-specific T-cells, T helper cells, cytotoxic T-cells, memory T-cell (an illustrative example of memory T-cells are CD62L
  • Additional agents that can be used to expand T lymphocytes includes methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety.
  • the agent(s) used for expansion are administered at about 20 units/ml to about 200 units/ml. In certain embodiments, the agent(s) used for expansion (e.g., IL-2) are administered at about 25 units/ml to about 190 units/ml, about 30 units/ml to about 180 units/ml, about 35 units/ml to about 170 units/ml, about 40 units/ml to about 160 units/ml, about 45 units/ml to about 150 units/ml, about 50 units/ml to about 140 units/ml, about 55 units/ml to about 130 units/ml, about 60 units/ml to about 120 units/ml, about 65 units/ml to about 110 units/ml, about 70 units/ml to about 100 units/ml, about 75 units/ml to about 95 units/ml, or about 80 units/ml to about 90 units/ml.
  • the agent(s) used for expansion are administered at about 20 units/ml, about 25 units/ml, about 30 units/ml, 35 units/ml, 40 units/ml, 45 units/ml, about 50 units/ml, about 55 units/ml, about 60 units/ml, about 65 units/ml, about 70 units/ml, about 75 units/ml, about 80 units/ml, about 85 units/ml, about 90 units/ml, about 95 units/ml, about 100 units/ml, about 105 units/ml, about 110 units/ml, about 115 units/ml, about 120 units/ml, about 125 units/ml, about 130 units/ml, about 135 units/ml, about 140 units/ml, about 145 units/ml, about 150 units/ml, about 155 units/ml, about 160 units/ml, about 165 units/ml, about 170 units/ml, about 175 units/ml
  • the agent(s) used for expansion are administered at about 5 mg/ml to about 10 ng/ml. In certain embodiments, the agent(s) used for expansion (e.g., IL -2) are administered at about 5.5 ng/ml to about 9.5 ng/ml, about 6 ng/ml to about 9 ng/ml, about 6.5 ng/ml to about 8.5 ng/ml, or about 7 ng/ml to about 8 ng/ml.
  • the agent(s) used for expansion are administered at about 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9, ng/ml, or 10 ng/ml.
  • NK cells may be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5, or 6 months or more with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion.
  • Agents that can be used for the expansion of natural killer cells can include agents that bind to CD 16 or CD56, such as for example uCD16 or aCD56 antibodies.
  • the binding agent includes antibodies (see for example Hoshino et al, Blood. 1991 Dec. 15; 78(12):3232-40 ).
  • Other agents that may be used for expansion of NK cells may be IL-15 (see for example Vitale et al. 2002. The Anatomical Record. 266:87-92, which is hereby incorporated by reference in its entirety for all purposes).
  • Conditions appropriate for T-cell culture include an appropriate media (e.g., Minimal Essential Media (MEM), RPMI Media 1640, Lonza RPMI 1640, Advanced RPMI, Clicks, AIM- V, DMEM, a-MEM, F-12, TexMACS, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion).
  • MEM Minimal Essential Media
  • RPMI Media 1640 e.g., Lonza RPMI 1640, Advanced RPMI
  • Clicks e.g., AIM- V, DMEM, a-MEM, F-12, TexMACS, X-Vivo 15, and X-Vivo 20
  • Optimizer e.g., Optimizer, with added amino acids, sodium pyruv
  • Examples of other additives for host cell expansion include, but are not limited to, surfactant, piasmanate, pH buffers such as HEPES, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol, Antibiotics (e.g., penicillin and streptomycin), are included only in experimental cultures, not in cultures of cells that are to be infused into a subject.
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).
  • host cells of the present disclosure may be modified such that the expression of an endogenous TCR, MHC molecule, or other immunogenic molecule is decreased or eliminated.
  • allogeneic cells When allogeneic cells are used, rejection of the therapeutic cells may be a concern as it may cause serious complications such as the graft-versus-host disease (GvHD).
  • immunogenic molecules e g., endogenous TCRs and/or MHC molecules
  • endogenous TCRs and/or MHC molecules are typically expressed on the cell surface and are involved in self vs nonself discrimination. Decreasing or eliminating the expression of such molecules may reduce or eliminate the ability of the therapeutic cells to cause GvHD.
  • expression of an endogenous TCR in the host cells is decreased or eliminated.
  • expression of an endogenous TCR (e.g., 0 TCR) in the host cells is decreased or eliminated.
  • Expression of the endogenous TCR may be decreased or eliminated by disrupting the TRAC locus, TCR beta constant locus, and/or CD3 locus.
  • expression of an endogenous TCR may be decreased or eliminated by disrupting one or more of the TRAC, TRBC1, TRBC2, CD3E, CD3G, and/or CD3D locus.
  • Modified MHC molecule may be an MHC class I or class II molecule.
  • expression of an endogenous MHC molecule may be decreased or eliminated by disrupting one or more of the MHC, [32M, TAPI, TAP2, CIITA, RFX5, RFXAP and/or RFXANK locus.
  • Expression of the endogenous TCR, an MHC molecule, and/or any other immunogenic molecule in the host cell can be disrupted using genome editing techniques such as Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Meganucleases. These genome editing methods may disrupt a target gene by entirely knocking out all of its output or partially knocking down its expression. In a particular embodiment, expression of the endogenous TCR, an MHC molecule and/or any other immunogenic molecule in the host cell is disrupted using the CRISPR/Cas technique.
  • compositions comprise one or more polypeptides of the CARs and other related molecules (e.g., second CAR or bispecific molecule), polynucleotides, vectors comprising same, and cell compositions, as disclosed herein.
  • Compositions of the present disclosure include, but are not limited to pharmaceutical compositions.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polynucleotide or a recombinant vector described herein, and a pharmaceutically accepted carrier and/or excipient.
  • the present disclosure provides pharmaceutical composition comprising the CAR-modified host cells described herein and a pharmaceutically acceptable carrier and/or excipient.
  • the host cells are modified with a LRRC 15 -binding CAR.
  • the present disclosure provides pharmaceutical composition comprising host cells modified with a LRRC15-binding CAR and a pharmaceutically acceptable carrier and/or excipient.
  • Examples of pharmaceutical carriers include but are not limited to sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • compositions comprising CAR-modified host cells disclosed herein may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids such as glycine
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • compositions comprising CAR-modified host cells disclosed herein may comprise one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which
  • the compositions are formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, intratumoral, intraventricular, intrapleural or intramuscular administration.
  • parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • An injectable pharmaceutical composition is preferably sterile.
  • the composition is reconstituted from a lyophilized preparation prior to administration.
  • the CAR-modified host cells may be mixed with substances that adhere or penetrate then prior to their administration, e.g., but not limited to, nanoparticles.
  • the present disclosure provides a method for treating a tumor in a subject in need thereof, wherein one or more cells of the tumor express LRRC15.
  • a therapeutically effective amount of the CAR-modified host cells described herein or the pharmaceutical composition comprising the host cells is administered to the subject.
  • tumor refers to a benign or malignant abnormal growth of tissue.
  • the term “tumor” includes cancer.
  • tumors are, but not limited to, the soft tissue tumors (e.g., lymphomas), and tumors of the blood and blood-forming organs (e.g., leukemias), and solid tumors, which is one that grows in an anatomical site outside the bloodstream (e.g., carcinomas).
  • cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (e.g., osteosarcoma or rhabdomyosarcoma), and leukemia or lymphoid malignancies.
  • cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), adenosquamous cell carcinoma, lung cancer (e.g., including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (e.g., including gastrointestinal cancer, pancreatic cancer), cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, primary or metastatic melanoma, multiple myeloma and B-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin
  • tumors can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, ⁇ on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); The Merck Manual of Diagnosis and Therapy, 20th Edition, ⁇ on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2018 (ISBN 978-0-911-91042-1) (2018 digital online edition at internet website of Merck Manuals); and SEER Program Coding and Staging Manual 2016, each of which is incorporated by reference in its entirety for all purposes.
  • host cells modified with a LRRC 15 -binding CAR, or pharmaceutical compositions thereof can be used in a method for killing a tumor cell expressing LRRC15.
  • the tumor cell is in a subject in need thereof.
  • host cells modified with a LRRC 15 -binding CAR, or pharmaceutical compositions thereof are administered to a subject to treat a tumor expressing LRRC 15.
  • tumors expressing LRRC 15 include adult solid tumors and brain tumors, adrenal gland tumors, adrenocortical carcinoma, anal cancer, atypical teratoid rhabdoid tumor, bile duct cancer, bladder cancer, bone cancer, bowel cancer, brain tumors, breast cancer, cancer of unknown primary, cancer spread to bone, cancer spread to brain, cancer spread to liver, cancer spread to lung, carcinoid, cervical cancer, children's cancers, chondrosarcoma, colorectal cancer, colorectal cancer, craniopharyngioma, desmoplastic small round cell tumor, diffuse intrinsic pontine glioma and other brain tumors, ear cancer, endometrial cancer, ependymoma, epithelioid sarcoma
  • the cancer described herein selected from osteosarcoma, soft tissue sarcoma, brain tumor, ovarian cancer, rhabdomyosarcoma, Ewing’s sarcoma and other Ewing’s sarcoma family of tumors, liposarcoma, fibrosarcoma, leiomyosarcoma, epithelioid sarcoma, vascular sarcoma, synovial sarcoma, Kaposi's sarcoma, uterine sarcoma, malignant fibrous histiocytoma, neuroblastoma, ganglioneuroblastoma, desmoplastic small round cell tumor, malignant peripheral nerve sheath tumor, undifferentiated pleiomorphic sarcoma, undifferentiated spindle cell sarcoma, chondrosarcoma, mesenchymoma, adrenocortical carcinoma, hepatoblasto
  • the tumor is a solid tumor.
  • the solid tumor can be a stromal- postive/cancer-postive tumor, stromal-negative/cancer-positive tumor, or stromal- negative/cancer-positive tumor.
  • the solid tumor is selected from osteosarcoma, glioblastoma, melanoma, prostate cancer, cervical cancer, soft tissue sarcoma, desmoplastic small round cell tumor, non-small cell lung cancer, lung adenocarcinoma, breast cancer, head and neck cancer, pancreatic cancer, ovarian cancer, and a brain tumor.
  • the method may further include administering an anti-CD20 antibody to the subject for removal of the isolated host cells.
  • the anti-CD20 antibody is administered in an amount effective for sufficient removal of the isolated host cells from the subject.
  • the anti-CD20 antibody is administered in an amount effective for removal of more than 50% of the isolated host cells from the subject.
  • the anti-CD20 antibody may be administered in an amount effective for removal of more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, or about 100% of the isolated host cells from the subject.
  • the anti-CD20 antibody may be administered in an amount effective for removal of about 50% to about 70%, about 60% to about 80%, about 70% to about 90%, or about 80% to about 100% of the isolated host cells from the subject.
  • Non-limiting examples of anti-CD20 antibodies that can be used for removal the isolated host cells include Rituximab, Ibritumomab tiuxetan, Tositumomab, Ofatumumab, Ocrelizumab, TRU-015, Veltuzumab, AME-133v, PRO131921, and Obinutuzumab.
  • the anti-CD20 antibody is Rituximab.
  • the therapeutic method of the present disclosure includes one or more of the following steps: (a) isolating immune cells from the subject or donor; (b) modifying the immune cells ex vivo with a polynucleotide encoding a CAR and optionally an additional protein, a second CAR and/or a bispecific molecule, or a recombinant vector comprising the same; (c) optionally, expanding and/or activating the modified immune cells before, after and/or during step (b); (d) introducing a therapeutically effective amount of the modified immune cells into the subject, and (e) in cases when the modified immune cells comprise the CD20 suicide switch, optionally, administering an anti-CD20 antibody to the subject, wherein the anti-CD20 antibody is administered in an amounts effective for removal of the modified immune cells from the subject.
  • the immune cells may be T-cells and/or NK cells.
  • the method of treating a tumor in a subject in need thereof, wherein one or more cells of the tumor express LRRC15 includes one or more of the following steps: (a) isolating T cells, NK cells, iNKT cells, or macrophages from the subject or generating T-cells, NK cells, iNKT cells, or macrophages from stem cells; (b) genetically modifying the T cells, NK cells, iNKT cells, macrophages ex vivo with the polynucleotide of any one of those described above or the vector of any one of those described above; (c) optionally, expanding and/or activating the T cells, NK cells, iNKT cells, or macrophages before, after, or during step (b); and (d) introducing the genetically modified T cells, NK cells, iNKT cells, macrophages into the subject.
  • the stem cells comprise induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • the modified host cell is an autologous cell. In some embodiments, the modified host cell is an allogeneic cell. In cases where the host cell is isolated from a donor, the method may further include a method to prevent graft-versus-host disease (GvHD) and the host cell rejection.
  • GvHD graft-versus-host disease
  • the composition is administered in a therapeutically effective amount.
  • the dosages of the composition administered in the methods of the disclosure will vary widely, depending upon the subject’s physical parameters, the frequency of administration, the manner of administration, the clearance rate, and the like.
  • the initial dose may be larger, and might be followed by smaller maintenance doses.
  • the dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level. It is contemplated that a variety of doses will be effective to achieve in vivo persistence of modified host cells. It is also contemplated that a variety of doses will be effective to improve in vivo effector function of modified host cells.
  • composition comprising the modified host cells manufactured by the methods described herein may be administered at a dosage of 10 2 to 10 10 cells/kg body weight, 10 5 to 10 9 cells/kg body weight, 1 C to 10 8 cells/kg body weight, 1 C to 10 7 cells/kg body weight, 10 7 to 10 9 cells/kg body weight, or 10 7 to 10 8 cells/kg body weight, including all integer values within those ranges.
  • the number of modified host cells will depend on the therapeutic use for which the composition is intended for.
  • Modified host cells may be administered multiple times at dosages listed above.
  • the modified host cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy.
  • compositions and methods described in the present disclosure may be utilized in conjunction with other types of therapy for tumors, such as chemotherapy, surgery, radiation, gene therapy, and so forth.
  • compositions and methods of the present disclosure can be utilized with other therapeutic methods/agents suitable for the same or similar diseases/disorders.
  • Such other therapeutic methods/agents can be co-administered (simultaneously or sequentially) to generate additive or synergistic effects.
  • Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.
  • the method further comprises administering to the subject one or more additional compounds selected from the group consisting of immuno-suppressives, biologicals, probiotics, prebiotics, and cytokines (e.g., IFN or IL-2).
  • additional compounds selected from the group consisting of immuno-suppressives, biologicals, probiotics, prebiotics, and cytokines (e.g., IFN or IL-2).
  • the disclosure can be combined with other therapies that block inflammation (e.g., via blockage of IL1, INFa/p, IL6, TNF, IL23, etc.).
  • compositions of the disclosure can be combined with other immunomodulatory treatments such as, e.g., therapeutic vaccines (including but not limited to GV AX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.) or activators (including but not limited to agents that enhance 4- IBB, 0X40, etc.).
  • therapeutic vaccines including but not limited to GV AX, DC-based vaccines, etc.
  • checkpoint inhibitors including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.
  • activators including but not limited to agents that enhance 4- IBB, 0X40, etc.
  • the methods of the disclosure can be also combined with other treatments that possess the ability to modulate NKT function or stability, including but not limited to CD Id, CD Id-fusion proteins, CD Id dimers or larger polymers of CD Id either unloaded or loaded with antigens, CD 1 d-chimeric antigen receptors (CDld-CAR), or any other of the five known CD1 isomers existing in humans (CD la, CD lb, CDlc, CDle).
  • CD la, CD lb, CDlc, CDle CD 1 d-chimeric antigen receptors
  • the methods of the disclosure can also be combined with other treatments such as midostaurin, enasidenib, or a combination thereof.
  • compositions of the disclosure can be used in combination with conventional therapies, such as, e.g., surgery, radiotherapy, chemotherapy, or combinations thereof, depending on type of the tumor, patient condition, other health issues, and a variety of factors.
  • conventional therapies such as, e.g., surgery, radiotherapy, chemotherapy, or combinations thereof, depending on type of the tumor, patient condition, other health issues, and a variety of factors.
  • other therapeutic agents useful for combination tumor therapy with the inhibitors of the disclosure include anti-angiogenic agents.
  • TNP- 470 platelet factor 4
  • thrombospondin- 1 tissue inhibitors of metalloproteases (TEMPI and TIMP2)
  • prolactin (16-Kd fragment) angiostatin (38-Kd fragment of plasminogen)
  • endostatin bFGF soluble receptor
  • transforming growth factor beta interferon alpha
  • soluble KDR and FLT- 1 receptors placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000).
  • the modified host cells of the disclosure can be used in combination with a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti- VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab or ranibizumab).
  • a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti- VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab or ranibizumab).
  • Non-limiting examples of chemotherapeutic compounds which can be used in combination treatments of the present disclosure include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, azacitidine, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramnustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouraci
  • chemotherapeutic compounds may be categorized by their mechanism of action into, for example, following groups: anti-metabolites/anti-tumor 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- chlorodeoxy adenosine (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, anthrofipramine,
  • the subject is a human.
  • the subject may be a juvenile or an adult, of any age or sex.
  • Example 1 Construction of LRRC15-CAR T cells that specifically recognize and kill LRRC 15-positive tumors
  • LVs lentiviral vectors
  • mAb humanized monoclonal antibody
  • Fig. 1A CD28( ⁇ endodomain
  • LRRC15-CAR T cells were specific for LRRC15 as judged by greater cytokine secretion in response to positive versus negative targets (Fig. ID). Cytotoxicity against osteosarcoma (LM7) was measured by an impedance based assay (Xcelligence), and against high grade glioma (U87) via an imaging based assay (Incucyte). All functional LRRC15-CAR T cells were cytotoxic against LM7 and U87, with only background activity against LRRC15 negative A549 cells (Fig. IE and Fig. 4E).
  • Ctrl-CAR T cells did not produce substantial cytokines or kill LRRC15 positive targets (Figs. 1D-1E). Furthermore, limited differences in cytokine secretion or tumor cell killing were noted between L1-, L2-, and L3-CAR T cells (Figs. 1D-1E).
  • ffluc expressing LM7 cells were injected i.v. and allowed to establish for 28 days prior to i.v. injection of LRRC15-CAR or Ctrl-CAR T cells (Fig. 2FT). Consistent with the locoregional model, LRRC15-CAR T cells had potent antitumor activity and enhanced survival compared to Ctrl-CAR T cells in this clinically relevant orthotopic lung osteosarcoma model (Figs. 2I-2K). To repeat this with longer follow up, mice were treated in the same conditions (Fig. 5A) and followed for 70 days post T cell injection.
  • LRRC15-CAR T cells again had significant antitumor activity leading to enhanced survival up to 10 weeks post treatment (Figs. 5B-5D).
  • LRRC15-CAR T cell antitumor activity in vivo was confirmed in the orthotopic U87 HGG model (Figs. 6A-6D).
  • a murine LRRC 15-CAR (mLRRCl 5-CAR) derived from the huM25 scFv and containing murine CD28 hinge, TM and costimulatory domains, and murine CD3(j were generated (Fig. 3A).
  • These CARs successfully transduced murine T cells (Fig. 3B), and mLRRCl 5-CAR T cells specifically recognized murine F420 osteosarcoma cells modified to overexpress murine LRRC 15 (F420.mLRRC15) by IFNy assay (Fig. 3C).
  • mLRRCl 5-CAR T cells antitumor activity of mLRRCl 5-CAR T cells was evaluated in syngeneic, immunocompetent in vivo models. To evaluate this, both subcutaneous and locoregional murine osteosarcoma models were employed. For the subcutaneous model, F420.mLRRC15 cells injected s.c., followed by a single dose of cyclophosphamide and 1 x 10 7 T cells i.v. (Fig. 3D). mLRRCl 5-CAR T cells had significantly greater antitumor activity compared to Ctrl-CAR T cells, leading to a survival advantage.
  • mLRRC15-CAR T cells were injected i.p, followed by a single dose of 1 x 10 7 T cells 7 days later.
  • mLRRCl 5-CAR T cells again significantly slowed tumor growth and enhanced survival compared to controls.
  • these findings demonstrated that mLRR15- CAR T cells have antitumor activity in syngeneic osteosarcoma models.

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Abstract

L'invention concerne des récepteurs antigéniques chimériques (CAR) qui ciblent une répétition riche en leucine 15 (LRRC15), et leurs utilisations dans l'immunothérapie antitumorale. L'invention concerne également des polynucléotides et des vecteurs qui codent pour les CAR, ainsi que des cellules hôtes comprenant les CAR. L'invention concerne également des procédés de préparation de cellules hôtes comprenant les CAR et des procédés de traitement de patients à l'aide des cellules hôtes modifiées.
PCT/US2024/052791 2023-10-25 2024-10-24 Récepteurs antigéniques chimériques ciblant une répétition riche en leucine 15 (lrrc15) Pending WO2025090749A1 (fr)

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