Enhanced chimeric antigen receptors and use thereof

Abstract

Provided herein are chimeric antigen receptors (CARs) comprising a truncated EGFRvIII (Ev3) in the hinge region of the CAR and/or a humanized scFv. Further provided herein are immune cells expressing the CARs as well as methods of their use in the treatment of immune disorders.

1 . A chimeric antigen receptor (CAR) comprising a truncated EGFRvIII (Ev3) in the hinge of said CAR, wherein said Ev3 hinge links an extracellular domain and at least one intracellular signaling domain. 
     
     
         2 . The CAR of  claim 1 , wherein the Ev3 hinge comprises a transmembrane domain and a non-functional ectodomain of EGFRvIII. 
     
     
         3 . The CAR of  claim 1 , wherein the Ev3 hinge does not comprise EGFRvIII endodomain. 
     
     
         4 . The CAR of  claim 2 , wherein the non-functional ectodomain of EGFRvIII has essentially no binding to epidermal growth factor (EGF). 
     
     
         5 . The CAR of  claim 1 , wherein the Ev3 hinge comprises a truncated domain 1, truncated domain 2, domain 3, and domain 4 of EGFR. 
     
     
         6 . The CAR of  claim 5 , wherein the domain 3 and domain 4 of EGFR are not truncated. 
     
     
         7 . The CAR of  claim 1 , wherein the Ev3 hinge comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2. 
     
     
         8 . The CAR of  claim 1 , wherein the Ev3 hinge comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2. 
     
     
         9 . The CAR of  claim 1 , wherein the Ev3 hinge comprises an amino acid sequence of SEQ ID NO:2. 
     
     
         10 . The CAR of  claim 1 , wherein the Ev3 hinge is encoded by a nucleotide sequence with at least 80% sequence identity to SEQ ID NO:1. 
     
     
         11 . The CAR of  claim 1 , wherein the Ev3 hinge is encoded by a nucleotide sequence with at least 95% sequence identity to SEQ ID NO:1. 
     
     
         12 . The CAR of  claim 1 , wherein the Ev3 hinge is encoded by a nucleotide sequence of SEQ ID NO:1. 
     
     
         13 . The CAR of  claim 1 , wherein the extracellular domain of the CAR comprises an antigen-binding domain selected from the group consisting of F(ab′)2, Fab′, Fab, Fv, and scFv. 
     
     
         14 . The CAR of  claim 13 , wherein the antigen-binding domain comprises a scFv. 
     
     
         15 . The CAR of  claim 14 , wherein the scFv is further defined as a humanized scFv. 
     
     
         16 . The CAR of  claim 13 , wherein the antigen binding region of the CAR binds one or more tumor associated antigens. 
     
     
         17 . The CAR of  claim 16 , wherein the one or more tumor associated antigens are selected from the group consisting of CD19, CD319 (CS1), ROR1, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL/DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, and CD99. 
     
     
         18 . The CAR of  claim 16 , wherein the one or more tumor-associated antigens are CD19, CD319, CD123, CD5, ROR1, CD33, CD99, CLL-1, and/or mesothelin. 
     
     
         19 . The CAR of  claim 13 , wherein the scFV does not comprise an EGFR binding domain. 
     
     
         20 . The CAR of  claim 1 , wherein the at least one signaling domain comprises CD3ζ, CD28, OX40/CD134, 4-1BB/CD137, FcεRIγ, ICOS/CD278, ILRB/CD122, IL-2RG/CD132, DAP12, CD70, CD40, or a combination thereof. 
     
     
         21 . The CAR of  claim 1 , wherein the at least one signaling domain comprises DAP12. 
     
     
         22 . The CAR of  claim 1 , wherein the CAR comprises IL-15. 
     
     
         23 . The CAR of  claim 1 , wherein the CAR comprises CD3ζ, CD28, DAP12, and IL-15. 
     
     
         24 . The CAR of  claim 1 , wherein the CAR further comprises a suicide gene. 
     
     
         25 . The CAR of  claim 24 , wherein the suicide gene is inducible caspase 9. 
     
     
         26 . An isolated antigen-specific humanized single chain variable fragment (scFv) comprising a light chain variable region (V L ) and a heavy chain variable region (V H ), wherein the scFv is:
 (a) CD19-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:7 and the V H  comprises an amino acid sequence of SEQ ID NO:8;   (b) CD123-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:13 and the V H  comprises an amino acid sequence of SEQ ID NO:14;   (c) mesothelin-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:19 and the V H  comprises an amino acid sequence of SEQ ID NO:20;   (d) ROR1-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:25 and the V H  comprises an amino acid sequence of SEQ ID NO:26;   (e) CD5-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:31 and the V H  comprises an amino acid sequence of SEQ ID NO:32;   (f) CLL-1-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:59 and the V H  comprises an amino acid sequence of SEQ ID NO:60;   (g) CD99-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:63 and the V H  comprises an amino acid sequence of SEQ ID NO:64 or   (h) a sequence with 90% sequence identity to framework regions of any one of (a)-(g).   
     
     
         27 . The humanized scFv of  claim 26 , wherein the scFv is CD19-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:7 and the V H  comprises an amino acid sequence of SEQ ID NO:8. 
     
     
         28 . The humanized scFv of  claim 27 , wherein the CD19-specific scFv comprises an amino acid sequence of SEQ ID NO:6. 
     
     
         29 . The humanized scFv of  claim 26 , wherein the scFv is CD123-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:13 and the V H  comprises an amino acid sequence of SEQ ID NO:14. 
     
     
         30 . The humanized scFv of  claim 29 , wherein the CD123-specific scFv comprises an amino acid sequence of SEQ ID NO:12. 
     
     
         31 . The humanized scFv of  claim 26 , wherein the scFv is mesothelin-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:19 and the V H  comprises an amino acid sequence of SEQ ID NO:20. 
     
     
         32 . The humanized scFv of  claim 31 , wherein the mesothelin-specific scFv comprises an amino acid sequence of SEQ ID NO:18. 
     
     
         33 . The humanized scFv of  claim 26 , wherein the scFv is ROR1-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:25 and the V H  comprises an amino acid sequence of SEQ ID NO:26. 
     
     
         34 . The humanized scFv of  claim 33 , wherein the ROR1-specific comprises an amino acid sequence of SEQ ID NO:24. 
     
     
         35 . The humanized scFv of  claim 26 , wherein the scFv is CD5-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:31 and the V H  comprises an amino acid sequence of SEQ ID NO:32. 
     
     
         36 . The humanized scFv of  claim 35 , wherein the CD5-specific comprises an amino acid sequence of SEQ ID NO:30. 
     
     
         37 . The humanized scFv of  claim 26 , wherein the scFv is CD99-specific and wherein the V L  comprises an amino acid sequence of SEQ ID NO:63 and the V H  comprises an amino acid sequence of SEQ ID NO:64. 
     
     
         38 . The humanized scFv of  claim 35 , wherein the CD99-specific comprises an amino acid sequence of SEQ ID NO:62. 
     
     
         39 . The humanized scFv of  claim 26 , wherein the scFv comprises an amino acid sequence with at least 95% sequence identity to framework regions an amino acid sequence of SEQ ID NO:6, 12, 18, 24, or 30. 
     
     
         40 . The humanized scFv of  claim 26 , wherein the scFv comprises an amino acid sequence with at least 98% sequence identity to framework regions an amino acid sequence of SEQ ID NO:6, 12, 18, 24, or 30. 
     
     
         41 . An isolated polynucleotide encoding an isolated humanized scFv of  claim 26 . 
     
     
         42 . The isolated polynucleotide of  claim 41 , wherein the polynucleotide comprises SEQ ID NO:3, 9, 15, 21, or 27. 
     
     
         43 . An expression vector comprising an isolated polynucleotide of  claim 41 . 
     
     
         44 . The vector of  claim 43 , wherein the vector is further defined as a viral vector. 
     
     
         45 . The CAR of  claim 15 , wherein the humanized scFv is a scFv according to  claim 26 . 
     
     
         46 . An isolated nucleic acid comprising a nucleotide sequence encoding the CAR according to  claim 1 . 
     
     
         47 . A host cell engineered to express a CAR comprising a humanized scFv according to  claim 26  and/or a truncated EGFRvIII (Ev3) in the hinge region of said CAR. 
     
     
         48 . The cell of  claim 47 , wherein said CAR is according to  claim 1 . 
     
     
         49 . The cell of  claim 47 , wherein the host cell is further defined as a CAR immune cell. 
     
     
         50 . The cell of  claim 49 , wherein the immune cell is a T cell, peripheral blood lymphocyte, NK cell, invariant NK cell, NKT cell, or stem cell. 
     
     
         51 . The cell of  claim 49 , wherein the immune cell is a T cell or a NK cell. 
     
     
         52 . The cell of  claim 50 , wherein the stem cell is a mesenchymal stem cell (MSC) or an induced pluripotent stem (iPS) cell. 
     
     
         53 . The cell of  claim 49 , wherein the immune cell is derived from an iPS cell. 
     
     
         54 . The cell of  claim 50 , wherein the ‘I’ cell is a CD8 +  T cell, CD4 +  T cell, or gamma-delta T cell. 
     
     
         55 . The cell of  claim 50 , wherein the T cell is a cytotoxic T lymphocyte (CTL). 
     
     
         56 . The cell of  claim 49 , wherein the immune cell is allogeneic. 
     
     
         57 . The cell of  claim 49 , wherein the immune cell is autologous. 
     
     
         58 . The cell of  claim 49 , wherein the immune cell is isolated from peripheral blood, cord blood, or bone marrow. 
     
     
         59 . The cell of  claim 49 , wherein the immune cell is isolated from cord blood. 
     
     
         60 . The cell of  claim 59 , wherein the cord blood is pooled from 2 or more individual cord blood units. 
     
     
         61 . The cell of  claim 47 , wherein DNA encoding the CAR is integrated into the genome of the cell. 
     
     
         62 . A pharmaceutical composition comprising a population of cells according to  claim 47 . 
     
     
         63 . A composition comprising a population of cells of  claim 47  for use in the treatment of an immune-related disorder 
     
     
         64 . A method of treating an immune-related disorder in a subject comprising administering an effective amount of cells of  claim 47  to the subject. 
     
     
         65 . The method of  claim 64 , wherein the immune-related disorder is a cancer, autoimmune disorder, graft versus host disease, allograft rejection, or inflammatory condition. 
     
     
         66 . The method of  claim 64 , wherein the immune-related disorder is an inflammatory condition and the immune cells have essentially no expression of glucocorticoid receptor. 
     
     
         67 . The method of  claim 64 , wherein the immune cells are autologous. 
     
     
         68 . The method of  claim 64 , wherein the immune cells are allogeneic. 
     
     
         69 . The method of  claim 64 , wherein the immune-related disorder is a cancer. 
     
     
         70 . The method of  claim 69 , wherein the cancer is a solid cancer or a hematologic malignancy. 
     
     
         71 . The method of  claim 64 , further comprising administering at least a second therapeutic agent. 
     
     
         72 . The method of  claim 71 , wherein the at least a second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiotherapy, or biotherapy. 
     
     
         73 . The method of  claim 71 , wherein the immune cells and/or the at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. 
     
     
         74 . The method of  claim 64 , further comprising administering an anti-EGFR antibody. 
     
     
         75 . The method of  claim 74 , wherein the anti-EGFR antibody is a monoclonal antibody. 
     
     
         76 . The method of  claim 75 , wherein the anti-EGFR antibody is cetuximab or panitumumab. 
     
     
         77 . The method of  claim 74 , wherein the anti-EGFR antibody selectively ablates Ev3-CAR immune cells through antibody-dependent cell-mediated cytotoxicity (ADCC). 
     
     
         78 . The method of  claim 74 , wherein the anti-EGFR antibody is fused to a detectable tag and/or a cytotoxic agent. 
     
     
         79 . The method of  claim 78 , further comprising imaging the detectable tag, thereby detecting the Ev3-CAR immune cells. 
     
     
         80 . The method of  claim 74 , wherein the anti-EGFR antibody is fused to a cytotoxic agent. 
     
     
         81 . The method of  claim 79 , wherein the cytotoxic agent induces activation of the suicide gene in the Ev3-CAR immune cells. 
     
     
         82 . The method of  claim 78 , wherein the cytotoxic agent results in the ablation of the Ev3-CAR immune cells.