Chimeric antigen receptors targeting tim-1

Abstract

The invention provides chimeric antigen receptors (CARs) that specifically bind to the T-cell immunoglobulin and mucin domain 1 (TIM-1) protein. The invention further relates to modified immune cells, e.g., T or NK cells, comprising such CARs, CAR-encoding nucleic acids, CAR-encoding vectors, and methods of making such compositions. The invention further relates to methods for therapeutic use of these CARs and modified immune cells for the treatment of a condition, disorder, or disease associated with cells expressing TIM-1 (e.g., cancer).

What is claimed is: 
     
         1 . An isolated chimeric antigen receptor (CAR) comprising (i) an antigen-binding domain that binds to TIM-1; (ii) a transmembrane domain; and (iii) at least one intracellular signaling domain. 
     
     
         2 . The isolated CAR according to  claim 1 , wherein the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof that binds to TIM-1. 
     
     
         3 . The isolated CAR according to  claim 2 , wherein the antibody or antigen-binding fragment thereof is selected from the group of: a monoclonal antibody; a monospecific antibody; a polyspecific antibody; a humanized antibody; a human antibody; a tetrameric antibody; a tetravalent antibody; a multispecific antibody; a single chain antibody; a domain-specific antibody; a single domain antibody; a domain-deleted antibody; an scFc fusion protein; a single-chain antibody; a chimeric antibody; a synthetic antibody; a recombinant antibody; a hybrid antibody; a mutated antibody; a CDR-grafted antibody; an antibody fragment comprising an Fab; an F(ab′)2 fragment; an Fab′ fragment; an Fv fragment; a single-chain Fv (scFv) fragment; an Fd fragment; a dAb fragment; a diabody; a nanobody; a bivalent nanobody; a shark variable IgNAR domain; a VHH antibody; a camelid antibody; and a minibody. 
     
     
         4 . The isolated CAR according to  claim 1 , wherein the antigen-binding domain comprises a human IgG antibody or antigen-binding fragment thereof, a Fab, or an scFv. 
     
     
         5 . The isolated CAR according to any one of  claims 1 - 4 , wherein the antigen-binding domain binds to the extracellular mucin domain region of TIM-1, optionally binding to the amino acid sequence LPRQNH (SEQ ID NO:97) or to the amino acid residues at positions 192-197 of TIM-1 (SEQ ID NO:315). 
     
     
         6 . The isolated CAR according to any one of  claims 1 - 5 , wherein the antigen-binding domain competes for binding to TIM-1 with an antibody comprising a VL chain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of the VL chain of Ab 1.29, Ab 2.70.2, or Ab 2.59.2 (SEQ ID NO: 203, 205, or 207), or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 253, 255, or 257; and comprising a VH chain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of the VH chain of Ab 1.29, Ab 2.70.2, or Ab 2.59.2 (SEQ ID NO: 202, 204, or 206), or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 252, 254, or 256. 
     
     
         7 . The isolated CAR according to any one of  claims 1 - 6 , wherein the antigen-binding domain comprises an antibody or antigen-binding fragment thereof that comprises a VL chain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of the VL chain of Ab 1.29, Ab 2.70.2, or Ab 2.59.2 (SEQ ID NO: 203, 205, or 207), or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 253, 255, or 257; and comprises a VH chain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of the VH chain of Ab 1.29, Ab 2.70.2, or Ab 2.59.2 (SEQ ID NO: 202, 204, or 206), or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 252, 254, or 256. 
     
     
         8 . The isolated CAR according to any one of  claims 1 - 7 , wherein the antigen-binding domain comprises a variable light (VL) chain linked to a variable heavy (VH) chain by a flexible linker. 
     
     
         9 . The isolated CAR according to  claim 8 , wherein the flexible linker is a G4S linker, optionally having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 201, or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 251. 
     
     
         10 . The isolated CAR according to any one of  claims 1 - 9 , wherein the antigen-binding domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of the scFv 129H, 129L, 272H, 272L, 2592H, or 2592L (SEQ ID NO: 208, 209, 210, 211, 212, or 213), or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 258, 259, 260, 261, 262, or 263. 
     
     
         11 . The isolated CAR according to any one of  claims 1 - 10 , wherein the antigen-binding domain comprises an antibody VL chain and an antibody VH chain, and wherein:
 (i) the VL chain is located at the N-terminus of the CAR (the VL chain is positioned N-terminal to the VH chain in the CAR); or   (ii) the VH chain is located at the N-terminus of the CAR (the VH chain is positioned N-terminal to the VL chain in the CAR).   
     
     
         12 . The isolated CAR according to any one of  claims 1 - 11 , wherein said antigen-binding domain is joined to said transmembrane domain by a linker, a spacer, or a hinge, optionally derived from one or more of the group of: CD28, CD8α, an immunoglobulin constant region or variant thereof, an immunoglobulin hinge region, an IgG4 hinge region, an immunoglobulin CH1/CL region, an Fc region, an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, and/or any combination thereof. 
     
     
         13 . The isolated CAR according to  claim 12 , wherein the linker, the spacer, or the hinge is derived from CD28, optionally having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of CD28H (SEQ ID NO: 214), or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 264. 
     
     
         14 . The isolated CAR according to any one of  claims 1 - 13 , wherein the antigen-binding domain is conjugated to a cytotoxic agent. 
     
     
         15 . The isolated CAR according to any one of  claims 1 - 14 , wherein the transmembrane domain comprises a transmembrane domain derived from a protein selected from the group consisting of CD28, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCR alpha, TCR beta, and CD3 zeta. 
     
     
         16 . The isolated CAR according to any one of  claims 1 - 15 , wherein the transmembrane domain is derived from CD28, optionally having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of CD28TM (SEQ ID NO: 215), or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 265. 
     
     
         17 . The isolated CAR according to any one of  claims 1 - 16 , wherein the intracellular signaling domain is an intracellular signaling domain of a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit, an IL-2 receptor subunit, CD3ζ, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, CD278 (ICOS), FcεRI, DAP10, and DAP12. 
     
     
         18 . The isolated CAR according to any one of  claims 1 - 17 , wherein the intracellular signaling domain is derived from CD3ζ, optionally having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of CD3ζCYP (SEQ ID NO: 219), or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 269. 
     
     
         19 . The isolated CAR according to any one of  claims 1 - 18 , further comprising one or more costimulatory domains derived from a protein selected from the group consisting of an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, a Toll ligand receptor, B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CD5, CD7, CD8alpha, CD8beta, CD11a, LFA-1 (CD11a/CD18), CD11b, CD11c, CD11d, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103, OX40 (CD134), 4-1BB (CD137), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), CEACAM1, CDS, CRTAM, DAP10, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG/Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE/RANKL, VLA1, VLA-6, and a ligand that specifically binds with CD83. 
     
     
         20 . The isolated CAR according to any one of  claims 1 - 19 , further comprising one or more costimulatory domains derived from a protein selected from the group consisting of CD28, 4-1BB (CD137), and DAP10, optionally having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of CD28CYP (SEQ ID NO: 217), BBCYP (SEQ ID NO: 216), or DAP (SEQ ID NO: 218), or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 266, 267, or 268. 
     
     
         21 . The isolated CAR according to any one of  claims 1 - 20 , wherein the transmembrane domain comprises a CD28 transmembrane domain comprising the amino acid sequence of CD28TM (SEQ ID NO: 215); the intracellular signaling domain comprises a CD3 intracellular signaling domain comprising the amino acid sequence of CD3CYP (SEQ ID NO: 219); and the antigen-binding domain comprises an scFv comprising the amino acid sequence of
 (i) 272H (SEQ ID NO: 210);   (ii) 2592H (SEQ ID NO: 212); or   (iii) 2592L (SEQ ID NO:213).   
     
     
         22 . The isolated CAR according to  claim 21 , wherein the antigen-binding domain is joined to the transmembrane domain via a CD28 hinge, optionally having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of CD28H (SEQ ID NO: 214), or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 264. 
     
     
         23 . The isolated CAR according to  claim 21  or  22 , further comprising one or more costimulatory domains derived from CD28, 4-1BB, or DAP10, optionally having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of CD28CYP (SEQ ID NO: 217), BBCYP (SEQ ID NO: 216), or DAP10CYP (SEQ ID NO: 218), or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 266, 267, or 268. 
     
     
         24 . An isolated CAR comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of
 (i) 272H-BB (SEQ ID NO: 222);   (ii) 272H-CD28 (SEQ ID NO: 223);   (iii) 272H-DAP10 (SEQ ID NO: 224);   (iv) 2592H-BB (SEQ ID NO: 226);   (v) 2592H-CD28 (SEQ ID NO: 227);   (vi) 2592H-DAP10 (SEQ ID NO: 228); or   (vii) 2592L-CD28 (SEQ ID NO: 229);   or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 272, 273, 274, 276, 277, 278, 279.   
     
     
         25 . An isolated nucleic acid sequence encoding a CAR according to any one of  claims 1 - 24 . 
     
     
         26 . A vector comprising the nucleic acid sequence according to  claim 25 . 
     
     
         27 . The vector according to  claim 26 , which is selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, a retrovirus vector, or an in vitro transcribed vector. 
     
     
         28 . The isolated nucleic acid sequence according to  claim 25  or the vector according to  claim 26  or  claim 27 , further comprising a sequence encoding:
 (i) a signal peptide, optionally having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 230, or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 280; 
 (ii) a T2A ribosome skip sequence, optionally having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 231, or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 281; 
 (iii) a selectable marker, optionally wherein the selectable marker is truncated CD19 (tCD19), optionally having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 232, or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence encoded by SEQ ID NO: 282; 
 (iv) a promoter 
 (v) a poly(A) tail; 
 (vi) a 3′UTR; 
 (vii) a suicide mechanism; 
 (viii) one or more signaling domains of a co-inhibitory receptor, such as CTLA-4 or PD-1 (“iCAR”); and/or 
 (ix) a substrate peptide cleaved in the presence of matrix metalloproteinases enriched within the tumor microenvironment (“masked CAR”). 
 
     
     
         29 . A recombinant or isolated cell comprising a CAR according to any one of  claims 1 - 24  or comprising a nucleic acid sequence or vector encoding a CAR according to any one of  claims 25 - 28 . 
     
     
         30 . The recombinant or isolated cell according to  claim 29  which is a primary human cell or other mammalian cell or is derived therefrom. 
     
     
         31 . The recombinant or isolated cell according to  claim 29  or  30 , which is an immune cell, optionally selected from a T lymphocyte, a B lymphocyte, a natural killer cell, an eosinophil, an NK/T cells, a macrophage, and a monocyte. 
     
     
         32 . The recombinant or isolated cell according to any one of  claims 29 - 31 , which is selected from a T cell, a CD4 +  T cell, a CD8 +  T cell, a naive T (TN) cell, an effector T (TEFF) cell, a memory T cell, a stem cell memory T (TSCM) cell, a central memory T (TCM) cell, an effector memory T (TEM) cell, a terminally differentiated effector memory T cell, a tumor-infiltrating lymphocyte (TIL), an immature T cell, a mature T cell, a helper T cell, a cytotoxic T cell, a mucosa-associated invariant T (MAIT) cell, a regulatory T (Treg) cell, a helper T cell, a TH1 cell, a TH2 cell, a TH3 cell, a TH17 cell, a TH9 cell, a TH22 cell, a follicular helper T cell, an alpha/beta T cell, a delta/gamma T cell, a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, a cytokine-induced killer (CIK) cell, and a lymphokine-activated killer (LAK) cell. 
     
     
         33 . The recombinant or isolated cell according to any one of  claims 30 - 33 , which is a T cell or a T cell progenitor cell. 
     
     
         34 . The recombinant or isolated cell according to any one of  claims 29 - 33 , which is further modified or selected to incorporate one or more of the following features:
 (i) to express another CAR, optionally an activating or inhibitory CAR;   (ii) to comprise a suicide gene;   (iii) to be specific for another antigen, optionally a tumor antigen;   (iv) to be a pp65CMV-specific T cell, a CMV-specific T cell, an EBV-specific T cell, a Varicella Virus-specific T cell, an Influenza Virus-specific T cell and/or an Adenovirus-specific T cell;   (v) to overexpress pro-survival signals;   (vi) to reverse anti-survival signals;   (vii) to overexpress Bcl-xL or BCL-2;   (viii) to suppress the expression or inhibit the function of cell death genes, including, but not limited to, Bak or Bax;   (ix) to overexpress hTERT;   (x) to eliminate Fas expression;   (xi) to express a TGFβ dominant negative receptor;   (xii) to reduce or eliminate expression of its endogenous TCR compared to a wildtype T cell;   (xiii) to evade immunosuppressive mediators; and/or   (xiv) to comprise a homing mechanism.   
     
     
         35 . The recombinant or isolated cell according to any one of  claims 29 - 34 , wherein the cell:
 (i) exhibits anti-tumor cytotoxicity when the CAR binds to TIM-1;   (ii) increases production of cytokines and/or chemokines upon exposure to TIM-1-expressing cells, optionally one or more of GM-CSF, IL-6, RANTES (CCL5), TNF-α, IL-4, IL-10, IL-13, and IFN-γ;   (iii) exhibits cytotoxic activity upon exposure to TIM-1-expressing cells, optionally measured via lactate dehydrogenase production;   (iv) is activated or stimulated to proliferate when the CAR binds to TIM-1.   
     
     
         36 . A pharmaceutical composition comprising a pharmaceutically effective amount of a CAR according to any one of  claims 1 - 24  or a recombinant or isolated cell according to any one of  claims 29 - 35  and a pharmaceutically acceptable carrier or excipient. 
     
     
         37 . The composition of  claim 36 , further comprising one or more additional agents that specifically bind to one or more tumor-associated antigens. 
     
     
         38 . A method for stimulating an immune cell-mediated response in a subject, the method comprising administering to a subject in need thereof an effective amount of an immune cell modified to express a CAR comprising (i) an antigen-binding domain that binds to TIM-1; (ii) a transmembrane domain; and (iii) at least one intracellular signaling domain, wherein the modified immune cell is activated or stimulated to proliferate when the CAR binds to TIM-1 thereby stimulating an immune cell-mediated response in the subject. 
     
     
         39 . A method for treating a disease, a disorder, or a condition associated with undesired proliferation of cells expressing TIM-1 in a subject, the method comprising administering to the subject in need thereof an effective amount of an immune cell genetically modified to express a CAR comprising (i) an antigen-binding domain that binds to TIM-1; (ii) a transmembrane domain; and (iii) at least one intracellular signaling domain, wherein the modified immune cell is activated or stimulated to proliferate when the CAR binds to TIM-1 thereby treating the disease, disorder, or condition associated with undesired proliferation of cells expressing TIM-1. 
     
     
         40 . The method according to  claim 38  or  39 , which is used in the treatment of a condition selected from cancer, autoimmunity, infection, and an inflammatory disorder. 
     
     
         41 . A method for treating a cancer in a subject, the method comprising administering to the subject in need thereof an effective amount of an immune cell modified to express a CAR comprising (i) an antigen-binding domain that binds to TIM-1, (ii) a transmembrane domain, and (iii) at least one intracellular signaling domain. 
     
     
         42 . The method according to any one of  claims 38 - 41 , wherein the modified immune cell induces an immune response as measured via increased production of cytokines and chemokines, optionally one or more of GM-CSF, IL-6, RANTES (CCL5), TNF-α, IL-4, IL-10, IL-13, and IFN-γ. 
     
     
         43 . The method according to any one of  claims 38 - 42 , wherein the modified immune cell is a T cell, optionally an autologous T cell or a donor-derived T cell. 
     
     
         44 . The method according to any one of  claims 38 - 43 , wherein the method is used to treat a cancer in a subject, and wherein:
 (i) the cancer is a solid tumor, optionally selected from a group consisting of carcinomas, melanomas, sarcomas, gliomas, and skin cancers;   (ii) the tumor cells express TIM-1;   (iii) the cancer is selected from a renal cell carcinoma, an ovarian clear cell carcinoma, and a lung carcinoma;   (iv) the method reduces tumor growth and/or tumor volume; and/or   (v) the treated subject is resistant to at least one chemotherapeutic agent.   
     
     
         45 . The method according to any one of  claims 38 - 44 , further comprising
 (i) administration of another therapy to the subject, optionally wherein the therapy is chemotherapy, radiotherapy, toxin-based therapy, radiochemical based therapy, or surgical therapy; and/or   (ii) administration in combination with another therapeutic agent, optionally wherein the therapeutic agent increases the efficacy of a cell expressing a CAR molecule and/or the therapeutic agent ameliorates one or more side effects associated with administration of a cell expressing a CAR molecule   
     
     
         46 . The method according to any one of  claims 38 - 45 , wherein the modified immune cell is a recombinant or isolated cell according to any one of  claims 29 - 35 , or wherein the immune cell expresses a CAR according to any one of  claims 1 - 24  or comprises a nucleic acid or vector encoding a CAR according to any one of  claims 25 - 28 . 
     
     
         47 . A method of expanding a population of or generating a persisting population of modified immune cells in a subject, the method comprising administering to the subject an immune cell expressing a CAR according to any one of  claims 1 - 24 , an immune cell comprising a nucleic acid or vector encoding a CAR according to any one of  claims 25 - 28 , or an immune cell which is a recombinant or isolated cell according to any one of  claims 29 - 35 , wherein the modified immune cells persist in the subject for at least one month after administration, optionally wherein the population of modified immune cells comprises at least one modified immune cell that was administered to the subject, a progeny of the modified immune cell that was administered to the subject, or a combination thereof, and/or a memory T cell. 
     
     
         48 . The method according to  claim 47 , wherein the population of cells persists in the subject for at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least eighteen months, at least two years, or at least three years after administration. 
     
     
         49 . A method of producing a CAR-expressing immune cell comprising introducing into an immune cell an isolated nucleic acid sequence or vector encoding a CAR according to any one of  claims 25 - 28 . 
     
     
         50 . The method according to  claim 49 , wherein the CAR-expressing immune cell:
 (i) is isolated based on expression of said CAR as determined via flow cytometry or immunofluorescence assays, optionally enriching for CD3 and tCD19 expression; and/or   (ii) is stimulated to grow via exposure to magnetic beads; soluble antibodies, optionally including OKT3; and/or cytokines, optionally including IL-2.