MUC16 chimeric antigen receptor

Abstract

The present disclosure provides chimeric antigen receptors (CARs) that target MUC16, genetically modified immune effector cells, and uses of these compositions to treat cancer.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 298,141, filed January 10, 2022. The entire contents of the aforementioned application are expressly incorporated herein by reference. The present invention relates to chimeric antigen receptors (CARs) comprising anti-MUC16 (mucin 16, cell surface associated) antibodies or antigen-binding fragments thereof, immune effector cells genetically modified to express these CARs, and the use of these compositions to effectively treat solid tumors. [Background technology]

[0002] Mucin 16 (MUC16), also known as cancer antigen 125 (CA-125), is a single transmembrane domain that is overexpressed in solid tumors, such as ovarian cancer. Expression of MUC16 on cancer cells has been shown to protect tumor cells from the immune system (Felder, M. et al. 2014, Molecular Cancer, 13:129). Antibodies that target MUC16 (e.g., oregovomab and abugovomab) have been investigated, but they have had limited clinical success. Therefore, additional therapies that target this antigen are needed. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Felder,M.et al.2014,Molecular Cancer,13:129 Summary of the Invention

[0004] The present disclosure is based, at least in part, on the discovery of chimeric antigen receptors (CARs) specific for MUC16 polypeptides. Specifically, the CARs described herein are specific for fragments or portions of MUC16 present on the surface of cells following proteolytic cleavage of the full-length MUC16 polypeptide. As demonstrated herein, the disclosed anti-MUC16 CARs include surprising and unexpected properties. As discussed in more detail in the Examples, the CARs of the present disclosure exhibit strong antigen-dependent activity as assessed by IFN-gamma release, high transduction efficiency, and high expression (see, e.g., Example 2). The anti-MUC16 CARs disclosed herein also exhibit antigen-dependent cytotoxicity in a dose-dependent manner, e.g., with at least 60%, at least 65%, or at least 70% cytotoxicity (Example 3). Additionally, certain anti-MUC16 CARs disclosed herein exhibit increased expression of markers such as CD62L and CD45RA (Example 4), pointing to a more naive-like phenotype with a greater potential for persistence in vivo. Finally, certain anti-MUC16 CARs tested herein surprisingly demonstrated superior anti-tumor activity in vivo, including a reduction in the number of cancer cells expressing MUC16 in vivo (Examples 6 and 7).

[0005] Thus, in some aspects, the disclosure provides a chimeric antigen receptor (CAR) comprising: an extracellular domain comprising an anti-MUC16 antibody or antigen-binding fragment thereof that binds to one or more epitopes of a human MUC16 polypeptide; a transmembrane domain, one or more intracellular costimulatory signaling domains, and a primary signaling domain. In some aspects, the MUC16 antibody or antigen-binding fragment thereof binds to a membrane proximal fragment of MUC16 that remains on the cell surface following proteolytic cleavage of the full-length MUC16 polypeptide. In one aspect, the fragment of MUC16 that remains on the cell surface following proteolytic cleavage comprises SEQ ID NO: 151. In some aspects, the full-length MUC16 polypeptide comprises 16 sea urchin sperm, enterokinase and agrin (SEA) domains numbered 1-16 from the N-terminus to the C-terminus, and the fragment of MUC16 comprises SEA domains 12-16.

[0006] In any of the foregoing or related aspects, the anti-MUC16 antibody or antigen-binding fragment thereof that binds to a human MUC16 polypeptide is selected from the group consisting of a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single chain Fv protein ("scFv"), a bis-scFv, (scFv)2, a camelid Ig, a VHH, an Ig NAR, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody) or fragment thereof. In some aspects, the anti-MUC16 antibody or antigen-binding fragment thereof that binds to a human MUC16 polypeptide is a scFv.

[0007] In any of the foregoing or related aspects, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises CDRL1, CDRL2, and CDRL3 regions within the variable light chain amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises CDRL1, CDRL2, and CDRL3 regions within the variable light chain amino acid sequence set forth in any one of SEQ ID NOs: 47, 59, 71, 83, 95, 107, 119, 133, or 145. In some aspects, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises CDRH1, CDRH2, and CDRH3 regions within the variable heavy chain amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises CDRH1, CDRH2, and CDRH3 regions within the variable heavy chain amino acid sequence set forth in any one of SEQ ID NOs: 48, 60, 72, 84, 96, 108, 120, 134, or 146. In some embodiments, the anti-MUC16 antibody or antigen-binding fragment thereof comprises one or more CDRs set forth in any one of SEQ ID NOs: 1-3. In some embodiments, the anti-MUC16 antibody or antigen-binding fragment thereof comprises one or more CDRs set forth in any one of (a) SEQ ID NOs: 41-43, (b) SEQ ID NOs: 53-55, (c) SEQ ID NOs: 65-67, (d) SEQ ID NOs: 77-79, (e) SEQ ID NOs: 89-91, (f) SEQ ID NOs: 101-103, (g) SEQ ID NOs: 113-115, (h) SEQ ID NOs: 127-129, or (i) SEQ ID NOs: 139-141. In some embodiments, the anti-MUC16 antibody or antigen-binding fragment thereof comprises one or more CDRs set forth in any one of SEQ ID NOs: 4-6. In some embodiments, the anti-MUC16 antibody or antigen-binding fragment thereof comprises the variable light chain amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises the variable heavy chain amino acid sequence shown in SEQ ID NO:8.In some aspects, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises one or more CDRs set forth in any one of (a) SEQ ID NOs: 44-46, (b) SEQ ID NOs: 56-58, (c) SEQ ID NOs: 68-70, (d) SEQ ID NOs: 80-82, (e) SEQ ID NOs: 92-94, (f) SEQ ID NOs: 104-106, (g) SEQ ID NOs: 116-118, (h) SEQ ID NOs: 130-132, or (i) SEQ ID NOs: 142-144.

[0008] In any of the foregoing or related embodiments, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises a variable light chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 7, or comprises SEQ ID NO: 7. In some embodiments, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises a variable light chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any one of SEQ ID NOs: 47, 59, 71, 83, 95, 107, 119, 133, or 145, or comprises any one of them. In some aspects, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises a variable heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 8, or comprises SEQ ID NO: 8. In some aspects, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises a variable heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any one of SEQ ID NOs: 48, 60, 72, 84, 96, 108, 120, 134, or 146, or comprises any one of them.

[0009] In any of the foregoing or related aspects, the transmembrane domain is from a polypeptide selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. In some aspects, the transmembrane domain is from a polypeptide selected from the group consisting of CD8α, CD4, CD45, PD1, and CD154. In some aspects, the transmembrane domain is from CD8α. In some aspects, the transmembrane domain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, or comprises, the sequence of SEQ ID NO:154. In one embodiment, the transmembrane domain comprises a sequence having at least 95% identity to SEQ ID NO: 154. In one embodiment, the transmembrane domain comprises SEQ ID NO:154.

[0010] In any of the foregoing or related aspects, the one or more costimulatory signaling domains are from a costimulatory molecule selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In some aspects, the one or more costimulatory signaling domains are from a costimulatory molecule selected from the group consisting of CD28, CD134, and CD137 (4-1BB). In some aspects, the one or more costimulatory signaling domains are from CD137(4-1BB). In some aspects, the costimulatory signaling domain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, or comprises, the sequence of SEQ ID NO: 155. In one embodiment, the costimulatory signaling domain comprises a sequence having at least 95% identity to, SEQ ID NO: 155. In one embodiment, the costimulatory signaling domain comprises SEQ ID NO: 155.

[0011] In any of the foregoing or related aspects, the primary signaling domain is from CD3zeta. In some aspects, the primary signaling domain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, or comprises, the sequence of SEQ ID NO: 156. In one embodiment, the primary signaling domain comprises a sequence having at least 95% identity to SEQ ID NO: 156. In one embodiment, the primary signaling domain comprises SEQ ID NO: 156.

[0012] In any of the foregoing or related aspects, the CAR comprises a hinge region polypeptide. In some aspects, the hinge region polypeptide comprises the hinge region of CD8α. In some aspects, the hinge region polypeptide comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 153, or comprises the sequence of SEQ ID NO: 153. In one embodiment, the hinge region polypeptide comprises a sequence having at least 95% identity to SEQ ID NO: 153. In one embodiment, the hinge region polypeptide comprises SEQ ID NO: 153.

[0013] In any of the foregoing or related aspects, the CAR comprises a signal peptide. In some aspects, the signal peptide comprises an IgG1 heavy chain signal polypeptide, a granulocyte macrophage colony stimulating factor receptor 2 (GM-CSFR2) signal polypeptide, an Igκ signal polypeptide, or a CD8α signal polypeptide. In some aspects, the signal polypeptide comprises a CD8α signal polypeptide. In some aspects, the signal polypeptide comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 152, or comprises the sequence of SEQ ID NO: 152. In one embodiment, the signal polypeptide comprises a sequence having at least 95% identity to SEQ ID NO: 152. In one embodiment, the signal polypeptide comprises SEQ ID NO: 152.

[0014] In any of the foregoing or related aspects, the CAR further comprises a first polypeptide linker between the variable heavy domain and the variable light domain. In some aspects, the polypeptide linker between the variable heavy domain and the variable light domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14-25. In certain embodiments, the polypeptide linker between the variable heavy domain and the variable light domain comprises the 3xG4S amino acid linker set forth in SEQ ID NO:24.

[0015] In any of the foregoing or related embodiments, the CAR further comprises a second polypeptide linker between the transmembrane domain and the one or more intracellular costimulatory signaling domains. In some embodiments, the polypeptide linker between the transmembrane domain and the one or more intracellular costimulatory signaling domains comprises a sequence of LYC.

[0016] In any of the foregoing or related embodiments, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises CDRL1, CDRL2, and CDRL3 regions within the variable light chain amino acid sequence set forth in SEQ ID NO:7, and CDRH1, CDRH2, and CDRH3 regions within the variable heavy chain amino acid sequence set forth in SEQ ID NO:8; the transmembrane domain is from a polypeptide selected from the group consisting of CD8α, CD4, CD45, PD1, and CD154; the one or more costimulatory signaling domains are from a costimulatory molecule selected from the group consisting of CD28, CD134, and CD137 (4-1BB); and the primary signaling domain is from CD3ζ.

[0017] In any of the foregoing or related embodiments, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises CDRL1, CDRL2, and CDRL3 regions within the variable light chain amino acid sequence set forth in SEQ ID NO:7, and CDRH1, CDRH2, and CDRH3 regions within the variable heavy chain amino acid sequence set forth in SEQ ID NO:8; the transmembrane domain is from CD8α; the one or more costimulatory signaling domains are from CD137 (4-1BB); and the primary signaling domain is from CD3ζ.

[0018] In any of the foregoing or related aspects, the CAR comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, or including, SEQ ID NO:9 or SEQ ID NO:11. In some aspects, the CAR comprises the amino acid sequence of SEQ ID NO:9. In some aspects, the CAR comprises the amino acid sequence of SEQ ID NO:11. In some embodiments, the CAR is identical to any one of SEQ ID NOs: 49, 50, 51, 52, 61, 62, 63, 64, 73, 74, 75, 76, 85, 86, 87, 88, 97, 98, 99, 100, 109, 110, 111, 112, 121, 122, 123, 124, 125, 126, 135, 136, 137, 138, 147, 148, 149, or 150 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 109, 110, 111, 112, 121, 122, 123, 124, 125, 126, 135, 136, 137, 138, 147, 148, 149, or 150. and / or comprising an amino acid sequence having 96%, 97%, 98% or 99% identity to, or comprising any one of SEQ ID NOs: 49, 50, 51, 52, 61, 62, 63, 64, 73, 74, 75, 76, 85, 86, 87, 88, 97, 98, 99, 100, 109, 110, 111, 112, 121, 122, 123, 124, 125, 126, 135, 136, 137, 138, 147, 148, 149, or 150.

[0019] In some aspects, the disclosure provides CARs that compete with a CAR disclosed herein for binding to one or more epitopes of a human MUC16 polypeptide.

[0020] In some aspects, the disclosure provides a polynucleotide encoding a CAR described herein. In some aspects, the polynucleotide sequence encoding a CAR is set forth in SEQ ID NO: 10 or SEQ ID NO: 12. In some aspects, the disclosure provides a polynucleotide comprising SEQ ID NO: 10. In some aspects, the disclosure provides a polynucleotide comprising SEQ ID NO: 12. In some aspects, the polynucleotide sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, or 96% identical to any one of SEQ ID NOs: 9, 11, 49, 50, 51, 52, 61, 62, 63, 64, 73, 74, 75, 76, 85, 86, 87, 88, 97, 98, 99, 100, 109, 110, 111, 112, 121, 122, 123, 124, 125, 126, 135, 136, 137, 138, 147, 148, 149, or 150. 9, 11, 49, 50, 51, 52, 61, 62, 63, 64, 73, 74, 75, 76, 85, 86, 87, 88, 97, 98, 99, 100, 109, 110, 111, 112, 121, 122, 123, 124, 125, 126, 135, 136, 137, 138, 147, 148, 149, or 150.

[0021] In some aspects, the disclosure provides a vector comprising a polynucleotide described herein. In some aspects, the vector is an expression vector. In some aspects, the vector is an episomal vector. In some aspects, the vector is a viral vector. In some aspects, the vector is a retroviral vector. In some aspects, the vector is a lentiviral vector. In some aspects, the lentiviral vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), Visna-Maedi virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In some aspects, the vector is an adeno-associated virus vector.

[0022] In any of the foregoing or related embodiments, the vector comprises a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract / DNA flap (cPPT / FLAP), a retroviral export element; a promoter operably linked to a polynucleotide described herein; and a right (3') retroviral LTR. In some embodiments, the vector comprises a heterologous polyadenylation sequence. In some embodiments, the promoter of the 5'LTR is replaced with a heterologous promoter. In some embodiments, the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, or a simian virus 40 (SV40) promoter. In some embodiments, the 5'LTR or the 3'LTR is a lentiviral LTR. In some embodiments, the 3'LTR comprises one or more modifications. In some embodiments, the 3'LTR comprises one or more deletions. In some embodiments, the 3'LTR is a self-inactivating (SIN) LTR. In some aspects, the promoter operably linked to the polynucleotides described herein is selected from the group consisting of cytomegalovirus immediate early gene promoter (CMV), elongation factor 1 alpha promoter (EF1-α), phosphoglycerate kinase-1 promoter (PGK), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer / chicken beta-actin promoter (CAG), polyoma enhancer / herpes simplex thymidine kinase promoter (MC1), beta actin promoter (β-ACT), simian virus 40 promoter (SV40), and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site replacement (MND) promoter. In some aspects, the promoter operably linked to the polynucleotides described herein is an MND promoter.

[0023] In some aspects, the disclosure provides a cell comprising a vector described herein. In some aspects, the disclosure provides a cell comprising a CAR described herein. In some aspects, the cell is an immune effector cell. In some aspects, the immune effector cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell. In some aspects, the cell is a T cell. In some aspects, the cell is an αβ T cell, a γδ T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell.

[0024] In some aspects, the disclosure provides compositions comprising the cells described herein. In some aspects, the disclosure provides pharmaceutical compositions comprising the cells described herein and a physiologically acceptable excipient.

[0025] In some aspects, the disclosure provides a method of generating an immune effector cell comprising a CAR described herein, comprising introducing into the immune effector cell a polynucleotide or vector described herein.

[0026] In some aspects, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition described herein. In some aspects, the cancer is a solid cancer, and optionally the solid cancer expresses MUC16. In some aspects, the solid cancer is selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, cervical cancer, mesothelioma, esophageal cancer, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, fallopian tube cancer, colorectal cancer, bile duct cancer, breast cancer, and bladder cancer. In some aspects, the solid cancer is selected from the group consisting of ovarian cancer, endometrial cancer, cervical cancer, mesothelioma, NSCLC, and SCLC. In some aspects, the solid cancer is ovarian cancer.

[0027] In any of the above or related aspects, the composition is administered in combination with an additional therapeutic agent.

[0028] In some aspects, the disclosure provides a method of treating cancer in a subject that has received or is receiving a therapeutic agent, comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0029] In any of the above or related aspects, the composition and the therapeutic agent are administered sequentially or simultaneously.

[0030] In any of the foregoing or related embodiments, the therapeutic agent is an immune checkpoint inhibitor, an oncolytic virus, or a costimulatory antibody. In some embodiments, the immune checkpoint inhibitor binds to a checkpoint protein or corresponding ligand selected from the group consisting of programmed cell death protein 1 (PD-1, PDCD1), lymphocyte activation gene 3 protein (LAG-3), T-cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T-lymphocyte antigen 4 (CTLA-4), band T-lymphocyte attenuation factor (BTLA), glucocorticoid-inducible tumor necrosis factor receptor (GITR), T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V-domain Ig suppressor of T-cell activation (VISTA), killer cell immunoglobulin-like receptor (KIR), ICOS, ICOSL, OX40, B7-H3, B7-H4, CD47, 4-1BB, CD27, and CD70. In some aspects, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In some aspects, the PD-1 inhibitor or PD-L1 inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some aspects, the PD-1 inhibitor is selected from the group consisting of: nivolumab, pembrolizumab, atezolizumab, and cemiplimab.

[0031] In any of the foregoing or related aspects, the costimulatory antibody is a bispecific antibody. In some aspects, the bispecific antibody binds a tumor associated antigen (TAA) and CD3. In some aspects, the bispecific antibody binds a TAA and CD28. In some aspects, the TAA is selected from the group consisting of AFP, ALK, BAGE protein, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase 8, CCR5, CD40, CDK4, CEA, CTLA4, cyclin B1, CYP1B1, ErbB3, ErbB4, ETV6-AML, Fra-1, FOLR1, GAGE Proteins (e.g. GAGE-1, -2), GD2, GloboH, glypican 3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MART-1, ML-IAP, Muc1, Muc2, Muc3, Mu c4, Muc5, Muc16, MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PRLR, RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, Stea p-1, Steap-2, survivin, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase, uroplakin 3, alpha folate receptor (FRα), αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CCR1, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD135 (also fmc-like tyrosine kinase 3;FLT3), CD138, CD171, carcinoembryonic antigen (CEA), claudin 6 (CLDN6), C-type lectin-like molecule 1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), fibroblast activation protein The EGFR family including protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican 3 (GPC3), ErbB2 (HER2), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), lambda, Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), leukocyte immunoglobulin-like receptor subfamily B member 2 (LIL RB2); melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid; placenta specific 1 (PLAC1), antigen preferentially expressed in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), receptor tyrosine kinase (TKI) The TAA is selected from the group consisting of rosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, X-breakpoint 2 (SSX2), tumor associated glycoprotein 72 (TAG72), T-cell immunoglobulin and mucin domain containing 3 (TIM-3), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 related (TEM7R), trophoblast glycoprotein (TPBG), NKG2D ligand, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1). In some embodiments, the TAA is MSLN. In some embodiments, the TAA is alpha folate receptor (FRα). In some embodiments, the TAA is MUC16.;

[0032] In any of the foregoing or related aspects, the therapeutic agent is a vascular endothelial growth factor (VEGF) inhibitor. In one embodiment, the VEGF inhibitor is bevacizumab. In one embodiment, the therapeutic agent is a cytokine. In one embodiment, the cytokine is IL-2 or masked IL-2. In one embodiment, the therapeutic agent is an oncolytic virus. In some embodiments, the oncolytic virus is selected from the group consisting of VSV (Voyager V1), HSV, adenovirus, Maraba virus, measles virus, NDV, picornavirus, reovirus, or vaccinia virus. In one embodiment, the oncolytic virus is Voyager V1. [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 is a schematic diagram of the MUC16 polypeptide. [Diagram 2] FIG. 2 is a graph showing vector copy numbers (VCN) from healthy donor T cells transduced with each of the indicated MUC16 CAR vectors. [Diagram 3] FIG. 3 is a graph showing CAR expression on healthy donor T cells transduced with each of the indicated MUC16 CAR vectors. [Figure 4A] Figure 4A is a graph showing IFNγ release from MUC16 CAR T cells. [Figure 4B] Figure 4B is a graph showing IFNγ release from MUC16 CAR T cells co-cultured in the presence of MUC16-expressing OVCAR3 cells. [Figure 4C] Figure 4C is a graph showing IFNγ release from MUC16 CAR T cells co-cultured in the presence of Jurkat cells that do not express MUC16. [Figure 4D] Figure 4D is a graph showing IFNγ release from MUC16 CAR T cells co-cultured in the presence of PANC-1 cells, which do not express MUC16. [Diagram 5]Figure 5 shows IFNγ release from MUC16 CAR T cells co-cultured in the presence of antigen-negative tumor cell lines (Jurkat, PANC-1, HUH7, K562, A549, and RD). [Figure 6A] FIG. 6A shows MUC16 ectodomain expression in RD cells stably expressing high, intermediate, or low levels of MUC16 ectodomain. [Figure 6B] Figure 6B shows IFNγ release from MUC16 CAR T cells co-cultured with RD cells stably expressing high, intermediate, or low levels of the MUC16 ectodomain. [Figure 7A] FIG. 7A shows MUC16 nub mRNA copy numbers in K562 tumor cells electroporated with titrating amounts of MUC16 nub mRNA. [Figure 7B] Figure 7B shows IFNγ release from MUC16 CAR T cells co-cultured with titrating amounts of electroporated MUC16 nub mRNA-expressing K562 tumor cells. [Figure 8A] Figure 8A shows the cytotoxicity of MUC16 CAR T cells co-cultured with RD cells stably expressing high, intermediate, or low levels of the MUC16 ectodomain at effector to T cell (E:T) ratios of 10:1, 5:1, and 2.5:1. [Figure 8B] Figure 8B shows the cytotoxicity of MUC16 CAR T cells co-cultured with RD cells stably expressing high, intermediate, or low levels of the MUC16 ectodomain at an effector to T cell (E:T) ratio of 2.5:1. [Figure 9] Figure 9 shows analysis of phenotypic markers via flow cytometry of MUC16 CAR T cells. [Figure 10A] Figures 10A and 10B provide graphs showing IFNγ production by anti-MUC16 CAR-expressing cells cultured in the presence of MUC16-nub ectodomain (Figure 10A) or CA125 (Figure 10B). [Figure 10B] Same as above. [Figure 11]Figure 11 is a graph showing the decline of MUC16 and luciferase expressing OVCAR3.FP tumor cells as indicated by a decline in luciferase expression. Mice treated with anti-MUC16 CAR T cells 14 days after inoculation of the mice with OVCAR3.FP cells showed a decline in luciferase. Mice were re-challenged with OVCAR3.FP tumor cells 28 days after administration of anti-MUC16 CAR T cells and tumor cells declined, indicating persistence of anti-MUC16 CAR T cells. Controls were untransduced cells (UTD), untreated mice (OVCAR3.FP), and cells transduced with vehicle only (vehicle). [Figure 12A] Figures 12A, 12B, and 12C show the anti-tumor activity of MUC16 CAR T cells transduced with the indicated anti-MUC16 CAR constructs in NGS mice inoculated with OVCAR3 tumor cells. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 13A] Figures 13A, 13B, and 13C show the anti-tumor activity of MUC16 CAR T cells transduced with the indicated anti-MUC16 CAR constructs in the OVCAR3.FP IP tumor model. [Figure 13B] Same as above. [Figure 13C] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Brief description of sequence identifiers SEQ ID NOs:1-3 show the amino acid sequences of exemplary light chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NOs:4-6 show the amino acid sequences of exemplary heavy chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NO:7 shows the amino acid sequence of an exemplary light chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO:8 shows the amino acid sequence of an exemplary heavy chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO:9 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 10 shows the nucleotide sequence of an exemplary anti-MUC16 CAR lacking a signal peptide as contemplated herein. SEQ ID NO:11 shows the amino acid sequence of an exemplary anti-MUC16 CAR contemplated herein. SEQ ID NO: 12 shows the nucleotide sequence of an exemplary anti-MUC16 CAR contemplated herein. SEQ ID NO: 13 shows the amino acid sequence of human MUC16. SEQ ID NOs:14-25 show the amino acid sequences of exemplary linkers suitable for use in the anti-MUC16 CARs contemplated herein. SEQ ID NOs: 26 to 35 show the amino acid sequences of exemplary 2A cleavage sites. SEQ ID NOs:36 to 38 show the amino acid sequences of exemplary cleavage sites. SEQ ID NO: 39 shows the amino acid sequence of a proteolytic cleavage fragment of human MUC16 containing the myc-myc-his tag. SEQ ID NO: 40 shows the amino acid sequence of human MUC16 lacking the nub ectodomain antigen (CA125). SEQ ID NOs:41-43 show the amino acid sequences of exemplary light chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NOs:44-46 show the amino acid sequences of exemplary heavy chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NO: 47 shows the amino acid sequence of an exemplary light chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 48 shows the amino acid sequence of an exemplary heavy chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 49 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO:50 shows the amino acid sequence of an exemplary anti-MUC16 CAR including the signal peptide contemplated herein. SEQ ID NO:51 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO:52 shows the amino acid sequence of an exemplary anti-MUC16 CAR including the signal peptide contemplated herein. SEQ ID NOs:53-55 show the amino acid sequences of exemplary light chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NOs:56-58 show the amino acid sequences of exemplary heavy chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NO:59 shows the amino acid sequence of an exemplary light chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO:60 shows the amino acid sequence of an exemplary heavy chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 61 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 62 shows the amino acid sequence of an exemplary anti-MUC16 CAR including the signal peptide contemplated herein. SEQ ID NO: 63 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO:64 shows the amino acid sequence of an exemplary anti-MUC16 CAR including the signal peptide contemplated herein. SEQ ID NOs:65-67 show the amino acid sequences of exemplary light chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NOs:68-70 show the amino acid sequences of exemplary heavy chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NO:71 shows the amino acid sequence of an exemplary light chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 72 shows the amino acid sequence of an exemplary heavy chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 73 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 74 shows the amino acid sequence of an exemplary anti-MUC16 CAR including the signal peptide contemplated herein. SEQ ID NO: 75 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 76 shows the amino acid sequence of an exemplary anti-MUC16 CAR including a signal peptide as contemplated herein. SEQ ID NOs:77-79 show the amino acid sequences of exemplary light chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NOs:80-82 show the amino acid sequences of exemplary heavy chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NO: 83 shows the amino acid sequence of an exemplary light chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO:84 shows the amino acid sequence of an exemplary heavy chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 85 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 86 shows the amino acid sequence of an exemplary anti-MUC16 CAR including a signal peptide as contemplated herein. SEQ ID NO: 87 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 88 shows the amino acid sequence of an exemplary anti-MUC16 CAR including a signal peptide as contemplated herein. SEQ ID NOs:89-91 show the amino acid sequences of exemplary light chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NOs:92-94 show the amino acid sequences of exemplary heavy chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NO: 95 shows the amino acid sequence of an exemplary light chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 96 shows the amino acid sequence of an exemplary heavy chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 97 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 98 shows the amino acid sequence of an exemplary anti-MUC16 CAR including a signal peptide as contemplated herein. SEQ ID NO: 99 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 100 shows the amino acid sequence of an exemplary anti-MUC16 CAR including the signal peptide contemplated herein. SEQ ID NOs:101-103 show the amino acid sequences of exemplary light chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NOs:104-106 show the amino acid sequences of exemplary heavy chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NO: 107 shows the amino acid sequence of an exemplary light chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 108 shows the amino acid sequence of an exemplary heavy chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 109 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 110 shows the amino acid sequence of an exemplary anti-MUC16 CAR including the signal peptide contemplated herein. SEQ ID NO: 111 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 112 shows the amino acid sequence of an exemplary anti-MUC16 CAR including a signal peptide as contemplated herein. SEQ ID NOs:113-115 show the amino acid sequences of exemplary light chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NOs:116-118 show the amino acid sequences of exemplary heavy chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NO: 119 shows the amino acid sequence of an exemplary light chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 120 shows the amino acid sequence of an exemplary heavy chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 121 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 122 shows the amino acid sequence of an exemplary anti-MUC16 CAR including a signal peptide as contemplated herein. SEQ ID NO: 123 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 124 shows the amino acid sequence of an exemplary anti-MUC16 CAR including a signal peptide as contemplated herein. SEQ ID NO: 125 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 126 shows the amino acid sequence of an exemplary anti-MUC16 CAR including a signal peptide as contemplated herein. SEQ ID NOs:127-129 show the amino acid sequences of exemplary light chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NOs:130-132 show the amino acid sequences of exemplary heavy chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NO: 133 shows the amino acid sequence of an exemplary light chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 134 shows the amino acid sequence of an exemplary heavy chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 135 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 136 shows the amino acid sequence of an exemplary anti-MUC16 CAR including a signal peptide as contemplated herein. SEQ ID NO: 137 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 138 shows the amino acid sequence of an exemplary anti-MUC16 CAR including a signal peptide as contemplated herein. SEQ ID NOs:139-141 show the amino acid sequences of exemplary light chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NOs:142-144 show the amino acid sequences of exemplary heavy chain CDRs of anti-MUC16 scFvs for the anti-MUC16 CARs contemplated herein. SEQ ID NO: 145 shows the amino acid sequence of an exemplary light chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 146 shows the amino acid sequence of an exemplary heavy chain variable region of an anti-MUC16 scFv for an anti-MUC16 CAR contemplated herein. SEQ ID NO: 147 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 148 shows the amino acid sequence of an exemplary anti-MUC16 CAR including a signal peptide as contemplated herein. SEQ ID NO: 149 shows the amino acid sequence of an exemplary anti-MUC16 CAR lacking the signal peptide contemplated herein. SEQ ID NO: 150 shows the amino acid sequence of an exemplary anti-MUC16 CAR including the signal peptide contemplated herein. SEQ ID NO: 151 shows the amino acid sequence of a proteolytic cleavage fragment of human MUC16 lacking the myc-myc-his tag. SEQ ID NO: 152 shows the amino acid sequence of an exemplary signal peptide contemplated herein. SEQ ID NO:153 shows the amino acid sequence of an exemplary hinge region peptide contemplated herein. SEQ ID NO:154 shows the amino acid sequence of an exemplary transmembrane domain peptide contemplated herein. SEQ ID NO: 155 shows the amino acid sequence of an exemplary costimulatory peptide contemplated herein. SEQ ID NO:156 shows the amino acid sequence of an exemplary signaling region peptide contemplated herein.

[0035] Detailed Description A. Overview In some embodiments, the present disclosure provides a chimeric antigen receptor (CAR) comprising a binding domain for MUC16. MUC16 is also known as cancer antigen 125, carcinoma antigen 125, carbohydrate antigen 125, or CA-125, and is a highly glycosylated integral membrane glycoprotein. MUC16 is overexpressed in cancers including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma-tumor-forming type, adenocarcinoma of the cervix, and adenocarcinoma of the gastric tract, as well as in diseases and conditions including inflammatory bowel disease, liver cirrhosis, heart failure, peritoneal infection, and abdominal surgery (Haridas, D. et al., 2014, FASEB J., 28:4183-4199), and expression of MUC16 on cancer cells has been shown to protect cancer cells from the immune system (Felder, M. et al., 2014, Molecular Cancer, 13:129). In some embodiments, immune effector cells expressing a CAR that comprises a binding domain for MUC16 surprisingly exhibit increased immune responses and cytotoxicity when co-cultured with cancer cells compared to non-transduced effector cells.

[0036] MUC16 contains three major domains: an extracellular N-terminal domain, a large tandem repeat domain interspersed with sea urchin sperm, enterokinase, and agrin (SEA) domains, and a single transmembrane domain consisting of a carboxyl-terminal domain that includes a segment of the transmembrane region and a short cytoplasmic tail. FIG. 1 provides a schematic diagram showing the domains of MUC16. Upon proteolytic cleavage of MUC16, most of the extracellular portion is shed into the bloodstream. Fragments of MUC16 remain on the surface of cells. In some embodiments, MUC16 contains 16 SEA domains at the C-terminus of the polypeptide, numbered 1-16 from the N-terminus to the C-terminus. In some embodiments, the MUC16 fragments remaining on the surface of cells contain SEA domains 12-16. In some embodiments, the CARs described herein selectively bind to SEA domains 12-16 of MUC16. In some embodiments, the CARs described herein do not bind to SEA domains 1-11 of MUC16.

[0037] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may generally be performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology, as cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning:A Practical Approach,vol.I&II(IRL Press,Oxford Univ.Press USA,1985);Current Protocols in Immunology(Edited by:John E.Coligan,Ada M.Kruisbeek,David H.Margulies,Ethan M.Shevach,Warren Strober 2001 John Wiley&Sons,NY,NY);Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK;Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991);Oligonucleotide Synthesis(N.Gait,Ed.,1984);Nucleic Acid The Hybridization(B.Hames&S.Higgins,Eds.,1985);Transcription and Translation(B.Hames&S.Higgins,Eds.,1984);Animal Cell Culture(R.Freshney,Ed.,1986);Perbal,A Practical Guide to Molecular Cloning(1984);Next-Generation Genome Sequencing(Janitz,2008 Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010 Humana Press);Immobilized Cells And Enzymes(IRL Press,1986);the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.);Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as research articles in academic journals such as Advances in Immunology.

[0038] B. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of the compositions, methods, and materials are described herein. For purposes of this disclosure, the following terms are defined below.

[0039] The articles "a", "an" and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0040] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives.

[0041] The term "and / or" should be understood to mean either one or both of the alternatives.

[0042] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0043] In some embodiments, ranges, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refer to each of the numbers encompassed by the range. For example, in one non-limiting and merely illustrative embodiment, the range "1 to 5" is equivalent to the expressions 1, 2, 3, 4, 5, or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0044] As used herein, the term "substantially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more compared to the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length of a reference. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces about the same effect, e.g., a physiological effect, as the amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length of a reference.

[0045] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. By "consisting of" it is meant to include and be limited to everything that follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are required or mandatory, and that no other elements will be present. "Consisting essentially of" means to include any elements listed after the phrase, and any elements limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or mandatory, but that there are no other elements that materially affect the activity or action of the recited elements.

[0046] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. As such, appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be understood that the positive recitation of a feature in an embodiment serves as a basis for the exclusion of that feature in certain embodiments.

[0047] C. Chimeric antigen receptor In some embodiments, improved engineered receptors are provided that redirect the cytotoxicity of immune effector cells against MUC16-expressing cells. These engineered receptors are referred to herein as chimeric antigen receptors (CARs). CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., MUC16) with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific anti-MUC16 cellular immune activity. As used herein, the term "chimeric" describes being made up of different protein or DNA portions from different sources.

[0048] In some embodiments, the CARs contemplated herein comprise an extracellular domain that binds to MUC16 (also referred to as a binding domain or an antigen-specific binding domain), a transmembrane domain, and an intracellular signaling domain. Association of the anti-MUC16 antigen-binding domain of the CAR with MUC16 on the surface of the target cell results in clustering of the CAR and transmits an activating stimulus to the CAR-containing cell. A key feature of CARs is their ability to redirect the specificity of immune effector cells, thereby triggering the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of target antigen-expressing cells in a major histocompatibility complex (MHC)-independent manner, eliciting the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific coreceptors.

[0049] In some embodiments, the CAR comprises an extracellular binding domain comprising a human anti-MUC16 specific binding domain; a transmembrane domain; one or more intracellular costimulatory signaling domains; and a primary signaling domain.

[0050] In some embodiments, the CAR comprises an extracellular binding domain comprising a human anti-MUC16 antibody, or an antigen-binding fragment thereof; one or more hinge or spacer domains; a transmembrane domain; one or more intracellular costimulatory signaling domains; and a primary signaling domain.

[0051] 1. Binding Domain In some embodiments, a CAR contemplated herein comprises an extracellular binding domain comprising an anti-MUC16 antibody or antigen-binding fragment thereof that specifically binds to a human MUC16 polypeptide expressed on a target cell (e.g., a cancer cell). The term "MUC16" as used herein refers to the human MUC16 protein unless specified as being from a non-human species (e.g., "mouse MUC16", "monkey MUC16", etc.). MUC16 comprises three major domains: an extracellular N-terminal domain, a large tandem repeat domain interspersed with sea urchin sperm, enterokinase, agrin (SEA) domains, and a carboxyl-terminal domain that includes a segment of a transmembrane region and a short cytoplasmic tail. The human MUC16 protein has the amino acid sequence set forth in SEQ ID NO: 13 and / or has the amino acid sequence set forth in NCBI Accession No. NP_078966. The human MUC16 membrane proximal domain (P13810-P14451) with a myc-myc-his tag (MUC16 "nub") is set forth as SEQ ID NO: 39. The human MUC16 membrane proximal domain (P13810-P14451) lacking the myc-myc-his tag (MUC16 "nub") is set forth as SEQ ID NO: 151. The term "CA125," as used herein, refers to a human MUC16 polypeptide lacking the MUC16 membrane proximal domain (MUC16 "nub") set forth as SEQ ID NO: 40.

[0052] As used herein, the terms "MUC16-nub," "nub," "MUC16 ectodomain," "MUC16-nub ectodomain," and "ectodomain" are used interchangeably and refer to the membrane-proximal MUC16 polypeptide domain or fragment that remains on the cell surface following cleavage. In some embodiments, the MUC16-nub ectodomain comprises SEA domains 12-16.

[0053] As used herein, the terms "binding domain," "extracellular domain," "extracellular binding domain," "antigen-specific binding domain," and "extracellular antigen-specific binding domain" are used interchangeably and provide a CAR with the ability to specifically bind to a target antigen of interest, e.g., MUC16. The binding domain can be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0054] As used herein, the terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targets" describe binding of an anti-MUC16 antibody or antigen-binding fragment thereof (or a CAR comprising the same) to MUC16 with a binding affinity greater than background binding. The binding domain (or a CAR comprising the binding domain or a fusion protein comprising the binding domain) may be used to target MUC16 with a binding affinity greater than background binding, for example, about 10 5 M -1 Affinity greater than or equal to K a (i.e., the equilibrium association constant for a particular binding interaction with units of 1 / M). In certain embodiments, the binding domain (or fusion protein) "specifically binds" to MUC16 if it binds or associates with MUC16 with a binding affinity of about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 A "high affinity" binding domain (or single-chain fusion protein thereof) binds to a target with a Ka greater than or equal to that of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 1011 M -1 , at least 10 12 M -1 , at least 10 13 M -1 or greater a It refers to a binding domain having the following structure:

[0055] Alternatively, the affinity may be expressed in M ​​units (e.g., 10 -5 M~10 -13 The equilibrium dissociation constant (K d The affinity of the binding domain polypeptides and CAR proteins according to the present disclosure can be readily determined using conventional techniques, for example, by competitive ELISA (enzyme-linked immunosorbent assay), or binding association or displacement assays using labeled ligands, or using surface plasmon resonance instruments available from Biacore, Piscataway, NJ, such as the Biacore T100, or optical biosensor technology available from Corning and Perkin Elmer, such as the EPIC system or EnSpire, respectively (see also, for example, Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173; 5,468,614, or equivalents).

[0056] In some embodiments, the affinity of specific binding is about 2-fold greater than background binding, about 5-fold greater than background binding, about 10-fold greater than background binding, about 20-fold greater than background binding, about 50-fold greater than background binding, about 100-fold greater than background binding, or about 1000-fold greater than background binding, or more.

[0057] In some embodiments, the extracellular binding domain of the CAR comprises an antibody or an antigen-binding fragment thereof. "Antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen, such as a peptide, lipid, polysaccharide, or nucleic acid that comprises an antigenic determinant, such as one that is recognized by an immune cell.

[0058] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, including compositions that are injected or absorbed into an animal (e.g., compositions that include a cancer-specific protein, etc.). An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens. In some embodiments, the target antigen is an epitope of a MUC16 polypeptide.

[0059] "Epitope" or "antigenic determinant" refers to a region of an antigen to which a binding agent binds. Epitopes can be formed from both contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically comprises at least 3, more usually at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

[0060] Antibodies include antigen-binding fragments thereof, such as camelid Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain Fv proteins ("scFv"), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins ("dsFv"), and single domain antibodies (sdAb, nanobodies), as well as the portion of a full length antibody that is responsible for antigen binding. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.

[0061] As used herein, the expression "bispecific antibody" refers to an antibody comprising at least a first antigen-binding domain and a second antigen-binding domain, each antigen-binding domain in a bispecific antibody comprising at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs.

[0062] As will be understood by those of skill in the art, and as described elsewhere herein, a complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y consists of the second and third constant regions of the two heavy chains (and for IgE and IgM, the fourth constant region) joined together, with disulfide bonds (interchain) formed at the hinge. Heavy chains gamma, alpha and delta have a constant region made up of three tandem (in a row) Ig domains, a hinge region for additional flexibility; heavy chains mu and epsilon have a constant region made up of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and "CH3 domain", respectively. Each arm of the Y contains the variable region and first constant region of a single heavy chain bound to the variable and constant region of a single light chain. The light and heavy chain variable regions are responsible for antigen binding.

[0063] Light and heavy chain variable regions also contain a "framework" region interrupted by three hypervariable regions, called "complementarity determining regions" or "CDRs." CDRs can be defined or identified by conventional methods, such as by sequence according to Kabat et al. (Wu, TT and Kabat, EA, J Exp Med. 132(2):211-50, (1970); Borden, P. and Kabat EA, PNAS, 84:2440-2443 (1987); (see Kabat et al., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, 1991, which are hereby incorporated by reference) or structure according to Chothia et al. (Chothia, C. and Lesk, AM, J Mol. Biol., 196(4):901-917 (1987); Chothia, C. et al, Nature, 342:877-883 (1989)).

[0064] The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework regions of an antibody are the combined framework regions of the constituent light and heavy chains, and serve to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, are numbered consecutively from the N-terminus, and are typically identified by the chain in which the particular CDR is located. Thus, the CDRs located in the variable domain of the heavy chain of an antibody are referred to as CDRH1, CDRH2, and CDRH3, whereas the CDRs located in the variable domain of the light chain of an antibody are referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although it is the CDRs that differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs). Illustrative examples of light chain CDRs that are suitable for constructing an anti-MUC16 CAR contemplated herein include, but are not limited to, the CDR sequences shown in SEQ ID NOs: 1-3. Illustrative examples of heavy chain CDRs that are suitable for constructing an anti-MUC16 CAR contemplated herein include, but are not limited to, the CDR sequences shown in SEQ ID NOs: 4-6.

[0065] Illustrative examples of rules for predicting light chain CDRs include: CDR-L1 starts at about residue 24, is preceded by a Cys, is about 10-17 residues, and is followed by Trp (typically Trp-Tyr-Gln, but also Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu); CDR-L2 starts about 16 residues after the end of CDR-L1, is generally preceded by Ile-Tyr, but also Val-Tyr, Ile-Lys, Ile-Phe, and is 7 residues; and CDR-L3 starts about 33 residues after the end of CDR-L2, is preceded by a Cys, is 7-11 residues, and is followed by Phe-Gly-XXX-Gly (XXX is any amino acid).

[0066] Illustrative examples of rules for predicting heavy chain CDRs include the following: CDRH1 starts at about residue 26, is preceded by Cys-XXX-XXX-XXX, is 10-12 residues long, followed by Trp (typically Trp-Val, but also Trp-Ile, Trp-Ala); CDR-H2 starts about 15 residues after the end of CDR-H1, is generally preceded by Leu-Glu-Trp-Ile-Gly, or a number of variations, is 16-19 residues long, followed by Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala; CDR-H3 starts about 33 residues after the end of CDR-H2, is preceded by Cys-XXX-XXX (typically Cys-Ala-Arg), is 3-25 residues long, followed by Trp-Gly-XXX-Gly.

[0067] In some embodiments, the light chain CDRs and the heavy chain CDRs are determined according to the Kabat method. In some embodiments, the light chain CDRs and the heavy chain CDR2 and CDR3 are determined according to the Kabat method, and the heavy chain CDR1 is determined according to the AbM method, which is included between the Kabat method and the Clothia method, see, for example, Whitelegg N&Rees AR, Protein Eng. 2000 Dec; 13(12): 819-24 and Methods Mol Biol. 2004; 248: 51-91. Programs for predicting CDRs are publicly available, for example, in AbYsis (www.bioinf.org.uk / abysis / ).

[0068] References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment disclosed herein. References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment disclosed herein.

[0069] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for example, by creating hybrid antibody-forming cells from the fusion of a myeloma cell with an immune spleen cell. Monoclonal antibodies include humanized monoclonal antibodies.

[0070] A "chimeric antibody" has framework residues derived from one species, such as human, and CDRs (which generally confer antigen binding) derived from another species, such as mouse. In particularly preferred embodiments, a CAR contemplated herein comprises an antigen-specific binding domain that is a chimeric antibody or an antigen-binding fragment thereof.

[0071] A "humanized" antibody is an immunoglobulin that contains a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor."

[0072] In certain embodiments, anti-MUC16 antibodies or antigen-binding fragments thereof include, but are not limited to, camelid Ig (Camelidae antibody (VHH)), Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single chain Fv antibody ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein ("dsFv"), and single domain antibody (sdAb, nanobody).

[0073] As used herein, "Camel Ig" or "Camelidae VHH" refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-Nolte, et al, FASEB J., 21:3490-3498 (2007)). "Heavy chain antibody" or "Camelidae antibody" refers to an antibody that contains two VH domains and no light chains (Riechmann L. et al, J. Immunol. Methods 231:25-38 (1999); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079).

[0074] "IgNAR" for "immunoglobulin novel antigen receptor" refers to a class of antibodies from the shark immune repertoire consisting of a homodimer of one variable novel antigen receptor (VNAR) domain and five constant novel antigen receptor (CNAR) domains. IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds and are highly stable and have efficient binding properties. The inherent stability can be attributed to both (i) the underlying Ig scaffold, which displays a significant number of charged and hydrophilic surface-exposed residues compared to traditional antibody VH and VL domains found in mouse antibodies, and (ii) structural properties in the complementarity determining region (CDR) loops, including inter-loop disulfide bridges, and stabilizing patterns of intra-loop hydrogen bonds.

[0075] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0076] An "Fv" is the smallest antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain are covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimeric" structure similar to that in two two-chain Fv (scFv) species. In this configuration, the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or even half of an Fv containing only the three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0077] Fab fragments contain the heavy and light chain variable domains, and also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

[0078] The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0079] A "single domain antibody" or "sdAb" or "nanobody" refers to an antibody fragment consisting of the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL domain) (Holt, L., et al, Trends in Biotechnology, 21(11):484-490).

[0080] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, which domains are present in a single polypeptide chain and in any orientation (e.g., VL-VH or VH-VL). Generally, an scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Eds. Rosenburg and Moore, (Springer-Verlag, New York, 1994), pp. 269-315.

[0081] In some embodiments, the antigen-specific binding domain is an scFv. Single-chain antibodies may be cloned from the V-region genes of hybridomas specific for the desired target. The production of such hybridomas has become routine. Techniques that can be used to clone the variable heavy chain (VH) and variable light chain (VL) have been described, for example, in Orlandi et al., PNAS, 1989;86:3833-3837.

[0082] In some embodiments, the antigen-specific binding domain is an scFv that binds to a human MUC16 polypeptide. An illustrative example of a variable heavy chain that is suitable for constructing an anti-MUC16 CAR contemplated herein is the amino acid sequence shown in SEQ ID NO: 8. An illustrative example of a variable light chain that is suitable for constructing an anti-MUC16 CAR contemplated herein is the amino acid sequence shown in SEQ ID NO: 7.

[0083] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:8 and a variable light chain comprising the amino acid sequence of SEQ ID NO:7.

[0084] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:48 and a variable light chain comprising the amino acid sequence of SEQ ID NO:47.

[0085] In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:60.

[0086] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 59. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 59. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 60 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 59.

[0087] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 72 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 71.

[0088] In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:84.

[0089] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 83. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 83.

[0090] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 96 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0091] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 108. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 107. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 108 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 107.

[0092] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 120. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 119. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 120 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 119.

[0093] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 134. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 133. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 134 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 133.

[0094] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 146. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 146. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 145. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises a variable light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 145. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 146 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 145.

[0095] The MUC16-specific binding domains provided herein also comprise one, two, three, four, five, or six CDRs. Such CDRs can be non-human or modified non-human CDRs selected from CDRL1, CDRL2, and CDRL3 of the light chain and CDRH1, CDRH2, and CDRH3 of the heavy chain. In some embodiments, the MUC16-specific binding domain comprises (a) a light chain variable region comprising light chain CDRL1, light chain CDRL2, and light chain CDRL3, and (b) a heavy chain variable region comprising heavy chain CDRH1, heavy chain CDRH2, and heavy chain CDRH3.

[0096] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 1-3. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the heavy chain CDRs of SEQ ID NOs: 4-6. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 1-3 and the heavy chain CDRs of SEQ ID NOs: 4-6.

[0097] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 41-43. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the heavy chain CDRs of SEQ ID NOs: 44-46. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 41-43 and the heavy chain CDRs of SEQ ID NOs: 44-46.

[0098] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 53-55. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the heavy chain CDRs of SEQ ID NOs: 56-58. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 53-55 and the heavy chain CDRs of SEQ ID NOs: 56-58.

[0099] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 65-67. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the heavy chain CDRs of SEQ ID NOs: 68-70. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 65-67 and the heavy chain CDRs of SEQ ID NOs: 68-70.

[0100] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 77-79. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the heavy chain CDRs of SEQ ID NOs: 80-82. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 77-79 and the heavy chain CDRs of SEQ ID NOs: 80-82.

[0101] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 89-91. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the heavy chain CDRs of SEQ ID NOs: 92-94. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 89-91 and the heavy chain CDRs of SEQ ID NOs: 92-94.

[0102] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 101-103. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the heavy chain CDRs of SEQ ID NOs: 104-106. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 101-103 and the heavy chain CDRs of SEQ ID NOs: 104-106.

[0103] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 113-115. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the heavy chain CDRs of SEQ ID NOs: 116-118. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 113-115 and the heavy chain CDRs of SEQ ID NOs: 116-118.

[0104] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 127-129. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the heavy chain CDRs of SEQ ID NOs: 130-132. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 127-129 and the heavy chain CDRs of SEQ ID NOs: 130-132.

[0105] In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 139-141. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the heavy chain CDRs of SEQ ID NOs: 142-144. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR comprises the light chain CDRs of SEQ ID NOs: 139-141 and the heavy chain CDRs of SEQ ID NOs: 142-144.

[0106] Additional anti-MUC16 antibodies and antigen-binding fragments thereof are known in the art. See, e.g., WO2018 / 058003, published March 29, 2018, the entire contents of each of which are expressly incorporated herein by reference in their entirety.

[0107] In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR binds to human MUC16 within one or more of the five membrane proximal SEA domains of human MUC16. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR binds to SEA domains 12-16, numbered from N-terminus to C-terminus, of human MUC16. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR binds to the SEA domain of human MUC16, as depicted at residues 13791-14451 of SEQ ID NO: 13. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR binds within residues 13810-14451 of SEQ ID NO: 13. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR does not bind to SEA domains 1-11, numbered from N-terminus to C-terminus, of human MUC16. In some embodiments, the antigen-specific binding domain of the anti-MUC16 CAR does not bind within residues 1-13790 of SEQ ID NO: 13. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR does not bind within residues 1-13809 of SEQ ID NO: 13. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR binds within SEQ ID NO: 39. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR binds within SEQ ID NO: 151. In some embodiments, the antigen-specific binding domain of an anti-MUC16 CAR does not bind within SEQ ID NO: 40.

[0108] 2. Linker In some embodiments, the CARs contemplated herein may include linker residues between the various domains, e.g., may be added for proper spacing and conformation of the molecule. In some embodiments, the linker is a variable region linking sequence. A "sequence that links the variable regions" is a V H and V L and providing a spacer function compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains the same specific binding affinity for the same target molecule as an antibody comprising the same light chain variable region and heavy chain variable region. In other embodiments, the linker is a linking sequence between the transmembrane domain and one or more intracellular domains.

[0109] CARs contemplated herein can include one, two, three, four, or five or more linkers. In some embodiments, the length of the linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids in length.

[0110] An illustrative example of a linker is a glycine polymer (G) n ; glycine-serine polymer (G 1-5 S 1-5 ) n , where n is an integer of at least 1, 2, 3, 4, or 5; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore can function as neutral tethers between domains of fusion proteins, such as the CARs described herein. Glycine has access to significantly more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). One of skill in the art will recognize that the design of CARs can include linkers that are fully or partially flexible, allowing the linker to include a flexible linker as well as one or more moieties that impart less flexible structure to provide the desired CAR structure.

[0111] Other exemplary linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 14); TGEKP (SEQ ID NO: 15) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 16) (Pomerantz et al. 1995, supra); (GGGGS) n, where =1, 2, 3, 4 or 5 (SEQ ID NO:17) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQ ID NO:18) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSSEQLAQFRSLD (SEQ ID NO:19) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO:20); LQRDGERP (SEQ ID NO:21); LRQKDGGGSERP (SEQ ID NO:22); LRQKd(GGGS)2ERP (SEQ ID NO:23). In some embodiments, the linker is GGGGSGGGSGGGGGS (SEQ ID NO:24). Alternatively, flexible linkers can be rationally designed using computer programs capable of modeling both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994), or by phage display methods. In some embodiments, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO:25) (Cooper et al., Blood, 101(4):1637-1644 (2003)). In one embodiment, the linker comprises the sequence of LYC.

[0112] 3. Spacer domain In some embodiments, the binding domain of the CAR is followed by one or more "spacer domains," which refer to regions that move the antigen binding domain away from the effector cell surface, allowing for proper cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). Spacer domains can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region, or a modified immunoglobulin hinge region.

[0113] In some embodiments, the spacer domain comprises the CH2 and CH3 domains of IgG1 or IgG4.

[0114] 4. Hinge domain In some embodiments, the binding domain of a CAR is followed by one or more "hinge domains" or "hinge regions", which play a role in positioning the antigen binding domain away from the effector cell surface to allow proper cell / cell contact, antigen binding and activation. In some embodiments, a CAR comprises one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0115] A "modified hinge region" refers to (a) a native hinge region with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions); (b) a portion of a native hinge region that is at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) in length and with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions); or (c) a portion of a native hinge region that includes the core hinge region (which can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length). In some embodiments, one or more cysteine ​​residues in the native immunoglobulin hinge region may be replaced by one or more other amino acid residues (e.g., one or more serine residues). The modified immunoglobulin hinge region may alternatively or additionally have a proline residue of the wild-type immunoglobulin hinge region replaced by another amino acid residue (e.g., a serine residue).

[0116] Other exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8α, CD4, CD28, and CD7, which may be wild-type hinge regions from these molecules or may be modified. In one embodiment, the hinge domain comprises a CD28 hinge region. In another embodiment, the hinge domain comprises a CD4 hinge region. In another embodiment, the hinge domain comprises a CD8α hinge region.

[0117] In various embodiments, the modified hinge region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to a suitable hinge domain / region contemplated herein and / or known in the art. In some embodiments, modified hinge regions comprising hinge sequences contemplated herein have four or fewer, three or fewer, or two or fewer amino acid substitutions and / or deletions.

[0118] In certain embodiments, one or more cysteine ​​residues in the native hinge region / domain may be replaced by one or more other amino acid residues to produce a modified hinge domain. In some embodiments, the modified hinge domain comprises one or more cysteine ​​residues substituted with serine or alanine. In other embodiments, the modified hinge domain comprises one or more cysteine ​​residues substituted with serine. In other embodiments, the modified hinge domain comprises one or more cysteine ​​residues substituted with alanine.

[0119] In certain embodiments, the modified hinge region comprises a substitution of a proline residue with another amino acid residue (eg, a serine residue).

[0120] In some aspects, the hinge region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, or comprises the sequence of SEQ ID NO: 153. In one embodiment, the hinge region polypeptide comprises a sequence having at least 95% identity to SEQ ID NO: 153. In one embodiment, the hinge region polypeptide comprises SEQ ID NO: 153.

[0121] 5. Transmembrane (TM) domain In some embodiments, the CARs contemplated herein comprise a transmembrane domain. A "transmembrane domain" or "TM domain" is the portion of a CAR that fuses the extracellular binding moiety and the intracellular signaling domain and anchors the CAR to the cell membrane of an immune effector cell. The TM domain can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The TM domain can be derived from (i.e., include at least the transmembrane regions of) the alpha, beta, or zeta chain of the T cell receptor, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. In certain embodiments, the TM domain is synthetic and comprises primarily hydrophobic residues such as leucine and valine.

[0122] In some aspects, the CARs contemplated herein comprise a TM domain derived from CD8α. In some embodiments, the transmembrane domain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 154, or comprises the sequence of SEQ ID NO: 154. In one embodiment, the transmembrane domain comprises a sequence having at least 95% identity to SEQ ID NO: 154. In one embodiment, the transmembrane domain comprises SEQ ID NO: 154.

[0123] In some embodiments, the CAR contemplated herein comprises a TM domain derived from CD8α and a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, linking the TM domain and the intracellular signaling domain of the CAR. In some embodiments, the linker is a glycine-serine based linker. In other embodiments, the linker is a LYC linker.

[0124] In another embodiment, a CAR contemplated herein comprises a TM domain derived from CD28. In another embodiment, a CAR contemplated herein comprises a TM domain derived from CD4.

[0125] 6. Intracellular signaling domains In some embodiments, a CAR contemplated herein comprises an intracellular signaling domain. An "intracellular signaling domain" refers to a portion of a CAR that is involved in transmitting the message of an effective MUC16 CAR that binds to a human MUC16 polypeptide to the interior of an immune effector cell to elicit effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity (including release of cytotoxic factors toward a CAR-bound target cell), or other cellular responses elicited by antigens that bind to the extracellular CAR domain.

[0126] The term "effector function" refers to a specialized function of an immune effector cell. The effector function of a T cell may be, for example, cytolytic activity or help or activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs a cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, so long as it transduces an effector function signal. The term "intracellular signaling domain" is meant to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

[0127] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains, which initiate antigen-dependent primary activation through the TCR (e.g., the TCR / CD3 complex), and costimulatory signaling domains, which act in an antigen-dependent manner to provide secondary or costimulatory signals. In preferred embodiments, the CARs contemplated herein comprise an intracellular signaling domain that includes one or more "costimulatory signaling domains" and "primary signaling domains."

[0128] A primary signaling domain regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0129] Illustrative examples of ITAMs comprising primary signaling domains of particular use in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In some embodiments, a CAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling domain and the costimulatory signaling domain can be linked in tandem in any order to the carboxyl terminus of the transmembrane domain.

[0130] The CARs contemplated herein comprise one or more costimulatory signaling domains to enhance the efficacy and proliferation of T cells expressing the CAR receptor. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that, upon binding to an antigen, provides a second signal required for efficient activation and function of T lymphocytes. Illustrative examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In some embodiments, the CAR comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3zeta primary signaling domain.

[0131] In some embodiments, the CAR comprises CD28 and CD137 costimulatory signaling domains and a CD3zeta primary signaling domain.

[0132] In some embodiments, the CAR comprises CD28 and CD134 costimulatory signaling domains and a CD3zeta primary signaling domain.

[0133] In some embodiments, the CAR comprises CD137 and CD134 costimulatory signaling domains and a CD3zeta primary signaling domain.

[0134] In some embodiments, the CAR comprises a CD134 costimulatory signaling domain and a CD3ζ primary signaling domain.

[0135] In some embodiments, the CAR comprises a CD28 costimulatory signaling domain and a CD3 zeta primary signaling domain.

[0136] In some embodiments, the CAR comprises a CD137 costimulatory signaling domain and a CD3zeta primary signaling domain.

[0137] In some aspects, the one or more costimulatory signaling domains are from CD137(4-1BB). In some aspects, the costimulatory signaling domain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, or comprises, the sequence of SEQ ID NO: 155. In one embodiment, the costimulatory signaling domain comprises a sequence having at least 95% identity to, SEQ ID NO: 155. In one embodiment, the costimulatory signaling domain comprises SEQ ID NO: 155.

[0138] In any of the foregoing or related aspects, the primary signaling domain is from CD3zeta. In some aspects, the primary signaling domain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, or comprises, the sequence of SEQ ID NO: 156. In one embodiment, the primary signaling domain comprises a sequence having at least 95% identity to SEQ ID NO: 156. In one embodiment, the primary signaling domain comprises SEQ ID NO: 156.

[0139] D. Exemplary Chimeric Antigen Receptors In some embodiments, the CAR comprises an anti-MUC16 scFv that binds to a MUC16 polypeptide; a transmembrane domain derived from a polypeptide selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1; and a CARD1 1, one or more intracellular costimulatory signaling domains from a costimulatory molecule selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70; and a primary signaling domain from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD79b, and CD66d.

[0140] In some embodiments, the CAR comprises an anti-MUC16 scFv that binds a MUC16 polypeptide; a hinge domain selected from the group consisting of IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, and CD8α hinge; an alpha, beta, or zeta chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. and one or more intracellular costimulatory signaling domains from a costimulatory molecule selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70; and a primary signaling domain from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD79b, and CD66d.

[0141] In some embodiments, the CAR comprises an anti-MUC16 scFv that binds to a MUC16 polypeptide; a hinge domain selected from the group consisting of IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, and CD8α hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1; a TM domain that is associated with the intracellular signaling domain of the CAR; and short oligo- or polypeptide linkers, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, linked to the IL-11, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49 ...9, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, one or more intracellular costimulatory signaling domains from a costimulatory molecule selected from the group consisting of SLP76, TRIM, and ZAP70; and a primary signaling domain from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0142] In some embodiments, the CAR comprises an anti-MUC16 scFv that binds a MUC16 polypeptide; a hinge domain comprising a CD8α polypeptide; a CD8α transmembrane domain, optionally comprising a polypeptide linker of about 3 to about 10 amino acids; a CD137 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0143] In some embodiments, the CAR comprises an anti-MUC16 scFv that binds within SEQ ID NO: 39 or 151; a transmembrane domain derived from a polypeptide selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1; and a CARD1 1, one or more intracellular costimulatory signaling domains from a costimulatory molecule selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70; and a primary signaling domain from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD79b, and CD66d.

[0144] In some embodiments, the CAR comprises an anti-MUC16 scFv that binds within SEQ ID NO: 39 or 151; a hinge domain selected from the group consisting of IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, and CD8α hinge; an alpha, beta, or zeta chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. and one or more intracellular costimulatory signaling domains from a costimulatory molecule selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70; and a primary signaling domain from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD79b, and CD66d.

[0145] In some embodiments, the CAR comprises an anti-MUC16 scFv that binds within SEQ ID NO: 39 or 151; a hinge domain selected from the group consisting of IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, and CD8α hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1; and optionally a TM domain that is coupled to an intracellular signaling domain of the CAR. short oligo- or polypeptide linkers, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, linking the CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C one or more intracellular costimulatory signaling domains from a costimulatory molecule selected from the group consisting of SLP76, TRIM, and ZAP70; and a primary signaling domain from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0146] In some embodiments, the CAR comprises an anti-MUC16 scFv binding within SEQ ID NO:39 or SEQ ID NO:151; a hinge domain comprising a CD8α polypeptide; a CD8α transmembrane domain, optionally comprising a polypeptide linker of about 3 to about 10 amino acids, such as LYC; a CD137 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0147] In some embodiments, the CAR comprises an anti-MUC16 scFv comprising the light chain CDRs set forth in SEQ ID NOs: 1-3 and the heavy chain CDRs set forth in SEQ ID NOs: 4-6; a hinge domain comprising a CD8α polypeptide; a CD8α transmembrane domain, optionally comprising a polypeptide linker of about 3 to about 10 amino acids; a CD137 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0148] In some embodiments, the CAR comprises: (a) a light chain CDR set forth in SEQ ID NOs: 41 to 43 and a heavy chain CDR set forth in SEQ ID NOs: 44 to 46; (b) a light chain CDR set forth in SEQ ID NOs: 53 to 55 and a heavy chain CDR set forth in SEQ ID NOs: 56 to 58; (c) a light chain CDR set forth in SEQ ID NOs: 65 to 67 and a heavy chain CDR set forth in SEQ ID NOs: 68 to 70; (d) a light chain CDR set forth in SEQ ID NOs: 77 to 79 and a heavy chain CDR set forth in SEQ ID NOs: 80 to 82; (e) a light chain CDR set forth in SEQ ID NOs: 89 to 91 and a heavy chain CDR set forth in SEQ ID NOs: (f) heavy chain CDRs set forth in SEQ ID NOs: 101-103 and heavy chain CDRs set forth in SEQ ID NOs: 104-106; (g) light chain CDRs set forth in SEQ ID NOs: 113-115 and heavy chain CDRs set forth in SEQ ID NOs: 116-118; (h) light chain CDRs set forth in SEQ ID NOs: 127-129 and heavy chain CDRs set forth in SEQ ID NOs: 130-132; or (i) light chain CDRs set forth in SEQ ID NOs: 139-141 and heavy chain CDRs set forth in SEQ ID NOs: 142-144; a hinge domain comprising a CD8α polypeptide;

[0149] In some embodiments, the CD8α signal polypeptide, CAR, comprises an anti-MUC16 scFv comprising the light chain CDRs set forth in SEQ ID NOs: 1-3 and the heavy chain CDRs set forth in SEQ ID NOs: 4-6; a hinge domain comprising a CD8α polypeptide; a CD8α transmembrane domain, optionally comprising a polypeptide linker of about 3 to about 10 amino acids, such as LYC; a CD137 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0150] In some embodiments, the CD8α signal polypeptide, CAR, comprises: (a) a light chain CDR set forth in SEQ ID NOs: 41-43 and a heavy chain CDR set forth in SEQ ID NOs: 44-46; (b) a light chain CDR set forth in SEQ ID NOs: 53-55 and a heavy chain CDR set forth in SEQ ID NOs: 56-58; (c) a light chain CDR set forth in SEQ ID NOs: 65-67 and a heavy chain CDR set forth in SEQ ID NOs: 68-70; (d) a light chain CDR set forth in SEQ ID NOs: 77-79 and a heavy chain CDR set forth in SEQ ID NOs: 80-82; (e) a light chain CDR set forth in SEQ ID NOs: 89-91 and a heavy chain CDR set forth in SEQ ID NOs: 90-92; (f) light chain CDRs set forth in SEQ ID NOs: 101 to 103 and heavy chain CDRs set forth in SEQ ID NOs: 104 to 106; (g) light chain CDRs set forth in SEQ ID NOs: 113 to 115 and heavy chain CDRs set forth in SEQ ID NOs: 116 to 118; (h) light chain CDRs set forth in SEQ ID NOs: 127 to 129 and heavy chain CDRs set forth in SEQ ID NOs: 130 to 132; or (i) anti-MUC16 scFv comprising a CD8α polypeptide; and a hinge domain comprising a CD8α polypeptide; optionally a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids, such as LYC; a CD137 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0151] In some embodiments, the CAR comprises an anti-MUC16 scFv comprising a variable light chain set forth in SEQ ID NO:7 and a variable heavy chain set forth in SEQ ID NO:8; a hinge domain comprising a CD8α polypeptide; a CD8α transmembrane domain, optionally comprising a polypeptide linker of about 3 to about 10 amino acids, such as LYC; a CD137 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0152] In some embodiments, the CAR comprises an anti-MUC16 antibody comprising: (a) a variable light chain set forth in SEQ ID NO: 47 and a variable heavy chain set forth in SEQ ID NO: 48; (b) a variable light chain set forth in SEQ ID NO: 59 and a variable heavy chain set forth in SEQ ID NO: 60; (c) a variable light chain set forth in SEQ ID NO: 71 and a variable heavy chain set forth in SEQ ID NO: 72; (d) a variable light chain set forth in SEQ ID NO: 83 and a variable heavy chain set forth in SEQ ID NO: 84; (e) a variable light chain set forth in SEQ ID NO: 95 and a variable heavy chain set forth in SEQ ID NO: 96; (f) a variable light chain set forth in SEQ ID NO: 107 and a variable heavy chain set forth in SEQ ID NO: 108; (g) a variable light chain set forth in SEQ ID NO: 119 and a variable heavy chain set forth in SEQ ID NO: 120; (h) a variable light chain set forth in SEQ ID NO: 133 and a variable heavy chain set forth in SEQ ID NO: 134; or (i) a variable light chain set forth in SEQ ID NO: 145 and a variable heavy chain set forth in SEQ ID NO: 146. scFv; and a hinge domain comprising a CD8α polypeptide; a CD8α transmembrane domain, optionally comprising a polypeptide linker of about 3 to about 10 amino acids, such as LYC; a CD137 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0153] In some embodiments, the CD8α signal polypeptide, CAR, comprises an anti-MUC16 scFv comprising a variable light chain set forth in SEQ ID NO:7 and a variable heavy chain set forth in SEQ ID NO:8; a hinge domain comprising a CD8α polypeptide; a CD8α transmembrane domain, optionally comprising a polypeptide linker of about 3 to about 10 amino acids, such as LYC; a CD137 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0154] In some embodiments, the CD8α signal polypeptide, CAR comprises: (a) a variable light chain set forth in SEQ ID NO: 47 and a variable heavy chain set forth in SEQ ID NO: 48; (b) a variable light chain set forth in SEQ ID NO: 59 and a variable heavy chain set forth in SEQ ID NO: 60; (c) a variable light chain set forth in SEQ ID NO: 71 and a variable heavy chain set forth in SEQ ID NO: 72; (d) a variable light chain set forth in SEQ ID NO: 83 and a variable heavy chain set forth in SEQ ID NO: 84; (e) a variable light chain set forth in SEQ ID NO: 95 and a variable heavy chain set forth in SEQ ID NO: 96; (f) a variable light chain set forth in SEQ ID NO: 107 and a variable heavy chain set forth in SEQ ID NO: 108; (g) a variable light chain set forth in SEQ ID NO: 119 and a variable heavy chain set forth in SEQ ID NO: 120; (h) a variable light chain set forth in SEQ ID NO: 133 and a variable heavy chain set forth in SEQ ID NO: 134; or (i) a variable light chain set forth in SEQ ID NO: 145 and a variable heavy chain set forth in SEQ ID NO: 146. scFv; and, optionally, a CD8α transmembrane domain, optionally including a polypeptide linker of about 3 to about 10 amino acids, such as LYC; a CD137 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0155] In some embodiments, the CAR comprises an amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 9. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 9. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9.

[0156] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 49. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 49. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 49.

[0157] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 51. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 51. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 51.

[0158] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 61. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 61. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 61. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 61.

[0159] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 63. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 63. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 63.

[0160] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 73. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 73. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 73.

[0161] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 75. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 75. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 75. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 75.

[0162] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 85. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 85. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 85.

[0163] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 87. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 87. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 87.

[0164] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 97. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 97. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 97.

[0165] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 99. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 99. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 99. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 99.

[0166] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 109. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 109. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 109.

[0167] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 111. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 111. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 111.

[0168] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 121. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 121. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 121. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 121.

[0169] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 123. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 123. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 123. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 123.

[0170] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 125. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 125. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 125. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 125.

[0171] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 135. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 135. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 135. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 135.

[0172] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 137. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 137. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 137. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 137.

[0173] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 147. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 147. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 147. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 147.

[0174] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 149. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 149. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 149. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 149.

[0175] In some embodiments, the CAR comprises an amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 11. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 11. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11.

[0176] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 50. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 50. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 50.

[0177] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 52. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 52. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 52.

[0178] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 62. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 62. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 62.

[0179] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 64. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 64. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 64. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 64.

[0180] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 74. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 74. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 74. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 74.

[0181] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 76. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 76. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 76. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 76.

[0182] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 86. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 86. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 86.

[0183] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 88. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 88. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 88. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 88.

[0184] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 98. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 98. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 98.

[0185] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 100. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 100. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 100. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 100.

[0186] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 110. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 110. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 110.

[0187] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 112. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 112. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 112. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 112.

[0188] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 122. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 122. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 122. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 122.

[0189] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 124. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 124. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 124. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 124.

[0190] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 126. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 126. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 126. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 126.

[0191] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 136. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 136. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 136.

[0192] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 138. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 138. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 138. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 138.

[0193] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 148. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 148. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 148. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 148.

[0194] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 150. In some embodiments, the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 150. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 150. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 150.

[0195] Furthermore, the CAR designs contemplated herein allow for improved proliferation, long-term persistence, and acceptable cytotoxicity in T cells expressing a CAR compared to unmodified T cells or T cells modified to express other CARs.

[0196] E. Polypeptides The present disclosure contemplates, in part, CAR polypeptides and fragments thereof, cells and compositions comprising the same, and vectors expressing the polypeptides. In some embodiments, a polypeptide is provided that comprises one or more CARs as set forth in SEQ ID NO:9. In some embodiments, a polypeptide is provided that comprises one or more CARs as set forth in SEQ ID NO:11.

[0197] "Polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably in their conventional sense, i.e., amino acid sequence, unless specified to the contrary. Polypeptides are not limited to a particular length, e.g., they may include full-length protein sequences or fragments of full-length proteins, and may include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like, as well as other modifications, both natural and non-natural, known in the art. In various embodiments, the CAR polypeptides contemplated herein include a signal (or leader) polypeptide sequence at the N-terminus of the protein, which directs translocation of the protein during or after translation. Illustrative examples of suitable signal polypeptide sequences useful in the CARs disclosed herein include, but are not limited to, an IgG1 heavy chain signal polypeptide sequence, a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal polypeptide sequence, an Igκ signal polypeptide sequence, or a CD8α signal polypeptide sequence. Polypeptides may be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides contemplated herein specifically include the CARs of the present disclosure, or sequences having deletions, additions, and / or substitutions from one or more amino acids of the CARs disclosed herein.

[0198] "Isolated peptide" or "isolated polypeptide" and the like, as used herein, refers to the in vitro isolation and / or purification of a peptide or polypeptide molecule from the cellular environment and from association with other components of a cell, i.e., it is not significantly associated with in vivo substances. Similarly, an "isolated cell" refers to a cell obtained from an in vivo tissue or organ and that is substantially free of extracellular matrix.

[0199] Polypeptides include "polypeptide variants." Polypeptide variants may differ from naturally occurring polypeptides by one or more substitutions, deletions, additions, and / or insertions. Such variants may be natural or synthetically generated, for example, by modifying one or more of the above polypeptide sequences. For example, in certain embodiments, it may be desirable to improve the binding affinity and / or other biological properties of the CAR by introducing one or more substitutions, deletions, additions, and / or insertions into the binding domain, hinge, TM domain, costimulatory signaling domain, or primary signaling domain of the CAR polypeptide. Preferably, the polypeptides of the invention include polypeptides having at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% amino acid identity thereto.

[0200] Polypeptides include "polypeptide fragments." A polypeptide fragment refers to a polypeptide that may be monomeric or multimeric, having an amino-terminal deletion, a carboxyl-terminal deletion, and / or internal deletions or substitutions of a naturally occurring or recombinantly produced polypeptide. In certain embodiments, a polypeptide fragment may comprise an amino acid chain at least 5 to about 500 amino acids in length. In certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Particularly useful polypeptide fragments include functional domains, including antigen binding domains or fragments of antibodies. For murine anti-BCMA antibodies, useful fragments include, but are not limited to: CDR regions, CDR3 regions of the heavy or light chain; heavy or light chain variable regions; portions of the antibody chain or variable region comprising two CDRs; and the like.

[0201] The polypeptides may also be fused in frame or attached to linkers or other sequences for ease of synthesis, purification, or identification of the polypeptide (e.g., poly-His) or to enhance binding of the polypeptide to a solid support.

[0202] As described above, the polypeptides of the present invention can be modified in various ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be generated by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al. (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, JD et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and references cited therein. Guidance regarding suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0203] In some embodiments, the variants include conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid having similar properties, and one skilled in the art of peptide chemistry would expect the secondary structure and hydrophobicity of the polypeptide to be substantially unchanged. Modifications can be made in the structure of the polynucleotides and polypeptides of the present invention, while still obtaining functional molecules encoding variants or derivative polypeptides with desirable characteristics. When it is desired to modify the amino acid sequence of a polypeptide to create an equivalent or even improved variant polypeptide, one skilled in the art can change one or more of the codons of the encoding DNA sequence, for example, according to Table 1. [Table 1]

[0204] Guidance in determining which amino acid residues can be substituted, inserted, or deleted without losing biological activity can be found using computer programs well known in the art, such as DNASTARTM software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similar charged or uncharged amino acids. Conservative amino acid changes include substitutions of one of a family of amino acids that are related at their side chains. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In peptides or proteins, suitable conservative substitutions of amino acids are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Exemplary conservative substitutions are described in U.S. Provisional Patent Application No. 61 / 241,647, the disclosure of which is incorporated herein by reference.

[0205] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values ​​are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0206] It is known in the art that certain amino acids can be substituted with other amino acids having similar hydropathic indexes or scores, while still resulting in a protein with similar biological activity, i.e., still obtaining a biologically functional equivalent protein. In making such changes, substitution of amino acids with hydropathic indexes within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity.

[0207] As detailed in US Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular an immunologically equivalent, protein. In such changes, substitution of amino acids whose hydrophilicity values ​​are within ±2 are preferred, with those within ±1 being particularly preferred, and those within ±0.5 being even more particularly preferred.

[0208] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0209] Polypeptide variants further include glycosylated forms, aggregate conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., pegylated molecules). Covalent variants can be prepared by linking functionalities to groups found in the amino acid chain or at the N- or C-terminal residues, as known in the art. Variants also include allelic variants, species variants, and muteins. Truncation or deletion of regions that do not affect the functional activity of the protein are also variants.

[0210] In some embodiments, where expression of two or more polypeptides is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence, as discussed elsewhere herein, hi another embodiment, two or more polypeptides may be expressed as a fusion protein comprising one or more self-cleaving polypeptide sequences.

[0211] Polypeptides of the present disclosure include fusion polypeptides. In some embodiments, fusion polypeptides and polynucleotides encoding the fusion polypeptides are provided, such as CARs. Fusion polypeptides and fusion proteins refer to polypeptides having at least two, three, four, five, six, seven, eight, nine, or ten or more polypeptide segments. Fusion polypeptides are typically linked C-terminus to N-terminus, but they may also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein may be in any order or specified order. Fusion polypeptides or fusion proteins may also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired transcriptional activity of the fusion polypeptide is maintained. Fusion polypeptides may be made by chemical synthesis methods or by chemical conjugation between two moieties, or may be generally prepared using other standard methods. The linked DNA sequences comprising the fusion polypeptide are operably linked to appropriate transcriptional or translational control elements as discussed elsewhere herein.

[0212] In one embodiment, the fusion polypeptide contains sequences (expression enhancers) that assist in expressing the protein at higher yields than the native recombinant protein. Other fusion partners may be selected to increase the solubility of the protein, or to allow the protein to be targeted to a desired intracellular compartment, or to facilitate transport of the fusion protein through the cell membrane.

[0213] The fusion polypeptide may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein. It should be noted that the polypeptide site can be placed in any linker peptide sequence. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).

[0214] Suitable protease cleavage sites and autocleaving peptides are known to those of skill in the art (see, e.g., Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Waika virus) 3C-like protease, PYVF (Parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to its high cleavage stringency, a TEV (Tobacco Etch Virus) protease cleavage site is preferred in one embodiment, e.g., EXXYXQ(G / S) (SEQ ID NO:36), e.g., ENLYFQG (SEQ ID NO:37) and ENLYFQS (SEQ ID NO:38), where X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S).

[0215] In certain embodiments, the polypeptide cleavage signal is a viral auto-cleaving peptide.

[0216] In some embodiments, the self-cleaving peptide comprises a polypeptide sequence obtained from aphthovirus, potyvirus, and cardiovirus 2A peptides, FMDV (foot and mouth disease virus), equine rhinitis A virus, Thosea asigna virus, and porcine teschovirus.

[0217] In some embodiments, the self-cleaving polypeptide site comprises a 2A or a 2A-like site, sequence or domain (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041). Illustrative examples of 2A sites are provided in Table 2. [Table 2]

[0218] In a preferred embodiment, the polypeptides contemplated herein include a CAR polypeptide.

[0219] F. Polynucleotides In some embodiments, a polynucleotide encoding one or more CAR polypeptides is provided, e.g., SEQ ID NOs: 10 and 12. As used herein, the term "polynucleotide" or "nucleic acid" refers to pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive strand RNA (RNA(+)), negative strand RNA (RNA(-)), genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. A polynucleotide refers to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide, as well as all intermediate lengths. It will be readily understood that "intermediate lengths" in this context means any length between the recited values, e.g., 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc. Preferably, polynucleotides of the invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), and typically variants maintain at least one biological activity of the reference sequence. In various illustrative embodiments, the present disclosure contemplates, in part, polynucleotides including expression vectors, viral vectors, and transfer plasmids, as well as compositions and cells comprising the same.

[0220] In some embodiments, polynucleotides encoding at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500, 1750, or 2000 or more consecutive amino acid residues of a polypeptide of the invention, as well as all intermediate lengths, are provided by the present disclosure. It will be readily understood that "intermediate length" in this context means any length between the recited values, e.g., 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

[0221] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence or hybridizes to a reference sequence under stringent conditions as defined hereinafter. These terms include polynucleotides in which one or more nucleotides have been added or deleted or replaced with different nucleotides compared to the reference polynucleotide. In this regard, it is well understood in the art that certain alterations or modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, whereby the altered or modified polynucleotide retains the biological function or activity of the reference polynucleotide. The term "polynucleotide fragment" refers to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, " refers to a polynucleotide that is 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more nucleotides in length.

[0222] "Sequence identity," or phrases such as "a sequence 50% identical to," as used herein, refers to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window. Thus, a "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which identical nucleobases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying this result by 100 to calculate the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, and typically the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0223] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" is at least 12, but frequently 15-18, and often at least 25 monomeric units, nucleotides, and amino acid residues in length. Two polynucleotides may each contain (1) sequences that are similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence), and (2) sequences that diverge between the two polynucleotides, and sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least six contiguous positions, usually about 50 to about 100, more commonly about 100 to about 150, and a sequence is compared to the reference sequence over the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may contain no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence (not including additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning the comparison window may be performed by computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA) in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, or by inspection and best alignment (i.e., resulting in the highest homology over the comparison window) generated by any of the various methods selected. See, for example, the BLAST family of programs disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389.A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, Unit 19.3.

[0224] In some embodiments, the anti-MUC16 CAR is encoded by a nucleotide sequence as set forth in SEQ ID NO: 10. In some embodiments, the anti-MUC16 CAR is encoded by a nucleotide sequence having at least 90% identity to SEQ ID NO: 10. In some embodiments, the anti-MUC16 CAR is encoded by a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10. In some embodiments, the anti-MUC16 CAR comprising the amino acid sequence of SEQ ID NO: 9 is encoded by a nucleotide sequence having at least 90% identity to SEQ ID NO: 10.

[0225] In some embodiments, the anti-MUC16 CAR is encoded by a nucleotide sequence as set forth in SEQ ID NO: 12. In some embodiments, the anti-MUC16 CAR is encoded by a nucleotide sequence having at least 90% identity to SEQ ID NO: 12. In some embodiments, the anti-MUC16 CAR is encoded by a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12. In some embodiments, the anti-MUC16 CAR comprising the amino acid sequence of SEQ ID NO: 11 is encoded by a nucleotide sequence having at least 90% identity to SEQ ID NO: 12.

[0226] In some embodiments, disclosed herein is a nucleotide sequence encoding an anti-MUC16 CAR comprising the sequence of any one of SEQ ID NOs: 49, 50, 51, 52, 61, 62, 63, 64, 73, 74, 75, 76, 85, 86, 87, 88, 97, 98, 99, 100, 109, 110, 111, 112, 121, 122, 123, 124, 125, 126, 135, 136, 137, 138, 147, 148, 149, or 150. In some embodiments, disclosed herein are nucleotide sequences encoding anti-MUC16 CARs having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 49, 50, 51, 52, 61, 62, 63, 64, 73, 74, 75, 76, 85, 86, 87, 88, 97, 98, 99, 100, 109, 110, 111, 112, 121, 122, 123, 124, 125, 126, 135, 136, 137, 138, 147, 148, 149, or 150.

[0227] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that naturally flank it, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment. An "isolated polynucleotide" also refers to a complementary DNA (cDNA), recombinant DNA, or other polynucleotide that is not found in nature and has been produced by the hand of man. In certain embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0228] In various embodiments, the polynucleotide comprises an mRNA that encodes a polypeptide contemplated herein, hi certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0229] In certain embodiments, a polynucleotide may be codon optimized. As used herein, the term "codon optimization" refers to the substitution of codons in a polynucleotide encoding a polypeptide to increase expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias between two or more organisms or genes or a synthetically constructed bias table; (ii) variation in the degree of codon bias within an organism, gene or set of genes; (iii) systematic variation of a codon with its context; (iv) variation of a codon with its decoding tRNA; (v) variation of a codon with the GC % either in the entire triplet or in one position of the triplet; (vi) variation in similarity to a reference sequence, such as a natural sequence; (vii) variation in the codon frequency cutoff; (viii) structural properties of the mRNA transcribed from the DNA sequence; (ix) prior knowledge of the function of the DNA sequence on which the design of the codon substitution set is based; (x) synthetic variation of the codon set for each amino acid; and / or (xi) isolated removal of incorrect translation start positions.

[0230] Terms describing the orientation of a polynucleotide include 5' (usually the end of a polynucleotide having a free phosphate group) and 3' (usually the end of a polynucleotide having a free hydroxyl (OH) group). A polynucleotide sequence may be annotated in a 5' to 3' orientation or a 3' to 5' orientation. For DNA and mRNA, the 5' to 3' strand is designated the "sense", "plus" or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is designated as the "template", "antisense", "minus" or "non-coding" strand. As used herein, the term "reverse" refers to a 5' to 3' sequence written in a 3' to 5' direction or a 3' to 5' sequence written in a 5' to 3' direction.

[0231] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the complement of the DNA sequence 5'AGTCATG3' is 3'TCAGTAC5'. The latter sequence is often written as a reverse complement, with the 5' end on the left and the 3' end on the right, 5'CATGACT 3'. A sequence equivalent to its reverse complement is said to be a palindromic sequence. Complementarity can be "partial," where only a portion of the nucleic acid bases match according to the base-pairing rules. Alternatively, there is "complete" or "total" complementarity between the nucleic acids.

[0232] Furthermore, those skilled in the art will recognize that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that code for the polypeptides described herein or variant fragments thereof. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention, e.g., polynucleotides that are optimized for human and / or primate codon preferences. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein may also be used. An allele is an endogenous gene that is altered as a result of one or more mutations, such as deletions, additions, and / or substitutions of nucleotides.

[0233] The term "nucleic acid cassette" or "expression cassette" as used herein refers to a genetic sequence within a vector capable of expressing an RNA and subsequently a protein. The nucleic acid cassette comprises a gene of interest, e.g., a polynucleotide of interest. The nucleic acid cassette comprises one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, and a gene of interest, e.g., a polynucleotide of interest. The nucleic acid cassette is positionally and sequentially oriented within the vector, such that the nucleic acid in the cassette can be transcribed into RNA, translated into a protein or polypeptide, if necessary, subjected to appropriate post-translational modifications required for activity in a transformed cell, and targeted to an appropriate intracellular compartment for transfer to an appropriate compartment for biological activity or secreted into an extracellular compartment. In some embodiments, the cassette has its 3' and 5' ends adapted for easy insertion into a vector, e.g., it has a restriction endonuclease site at each end. In some embodiments, the nucleic acid cassette comprises a sequence of a chimeric antigen receptor. The cassette can be excised and inserted as a single unit into a plasmid or viral vector.

[0234] In some embodiments, the polynucleotide comprises at least one polynucleotide of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide (i.e., a polypeptide of interest) that is inserted into an expression vector that is desired to be expressed. The vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotides of interest. In some embodiments, the polynucleotide of interest encodes a polypeptide that provides a therapeutic effect in the treatment or prevention of a disease or disorder. Polynucleotides of interest, and the polypeptides encoded therefrom, include both polynucleotides that encode wild-type polypeptides, as well as functional variants and fragments. In certain embodiments, functional variants have at least 80%, at least 90%, at least 95%, or at least 99% identity to the corresponding wild-type reference polynucleotide or polypeptide sequence. In some embodiments, functional variants or fragments have at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the biological activity of the corresponding wild-type polypeptide.

[0235] The polynucleotides of the present disclosure, regardless of the length of the coding sequence itself, may be combined with other DNA sequences disclosed elsewhere herein or known in the art, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, etc., such that their overall length may vary greatly. Thus, it is contemplated that polynucleotide fragments of almost any length may be used, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0236] In some embodiments, the polynucleotide, or the cell harboring the polynucleotide, utilizes a suicide gene, including an inducible suicide gene, to reduce the risk of direct toxicity and / or uncontrolled amplification. In some embodiments, the suicide gene is not immunogenic to the host or the cell harboring the polynucleotide. Particular examples of suicide genes that may be used are caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 may be activated using a specific chemical inducer of dimerization (CID).

[0237] In some embodiments, the vector comprises a gene segment that renders the immune effector cells, e.g., T cells, of the invention susceptible to negative selection in vivo. By "negative selection" is meant that the infused cells can be eliminated as a result of a change in the in vivo condition of the individual. A negatively selectable phenotype can result from the insertion of a gene that confers sensitivity to an administered drug, e.g., a compound. Negatively selectable genes are known in the art and include, among others: the herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which confers sensitivity to ganciclovir (Wigler et al., Cell 11:223, 1977); the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0238] In some embodiments, the genetically modified immune effector cells, such as T cells, comprise a polynucleotide that further comprises a positive marker that allows for the selection of cells of a negatively selectable phenotype in vitro. A positively selectable marker may be a gene that expresses a dominant phenotype when introduced into a host cell, allowing for the positive selection of cells carrying the gene. Genes of this type are known in the art and include, among others, the hygromycin-B phosphotransferase gene (hph), which confers resistance to hygromycin B, the aminoglycoside phosphotransferase gene (neo or aph) from Tn5, which encodes resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multidrug resistance (MDR) gene.

[0239] In some embodiments, the positive selectable marker and the negative selectable element are linked such that loss of the negative selectable element is necessarily accompanied by loss of the positive selectable marker. In some embodiments, the positive and negative selectable markers are fused such that loss of one obligately leads to loss of the other. An example of a fusion polynucleotide that results as an expression product a polypeptide that confers both the desired positive and negative selection properties described above is the hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene results in a polypeptide that confers hygromycin B resistance for positive selection in vitro and ganciclovir sensitivity for negative selection in vivo. See Lupton SD, et al, Mol. and Cell. Biology 1 1:3374- 3378, 1991. In some embodiments, the polynucleotide encoding the CAR is in a retroviral vector that contains a fusion gene, particularly one that confers hygromycin B resistance for positive selection in vitro and ganciclovir sensitivity for negative selection in vivo, such as the HyTK retroviral vector described in Lupton, SD et al. (1991), supra. See also PCT US91 / 08442 and PCT / US94 / 05601 publications by SD Lupton, which describe the use of bifunctional, selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker.

[0240] In some embodiments, the positive selectable marker is derived from a gene selected from the group consisting of hph, nco, and gpt, and the negative selectable marker is derived from a gene selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT, and gpt. In some embodiments, the marker is a bifunctional, selectable fusion gene, and the positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from a cytosine deaminase or TK gene or selectable marker. Inducible suicide genes

[0241] G. Vector Polynucleotides may be prepared, manipulated and / or expressed using any of a variety of established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding that polypeptide may be inserted into a suitable vector.

[0242] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in a cell, or may contain sequences sufficient to permit integration into the host cell DNA.

[0243] Exemplary vectors include, but are not limited to, autonomously replicating sequences, mRNA, plasmids (e.g., DNA or RNA plasmids), transposons, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). Examples of expression vectors are pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In some embodiments, the coding sequences of the chimeric proteins disclosed herein can be ligated into such expression vectors for expression of the chimeric proteins in mammalian cells.

[0244] In some embodiments, the vector is an episomal vector, or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without integration into the host chromosomal DNA, and without gradual loss from dividing host cells, also meaning that the vector replicates extrachromosomally or episomally. The vector is engineered to carry a sequence encoding an origin of DNA replication or "ori" from lymphotrophic herpesvirus or gammaherpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, specifically, a lymphotrophic herpesvirus or gammaherpesvirus replication origin corresponding to oriP of EBV. In certain aspects, the lymphotrophic herpesvirus can be Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), herpesvirus saimiri (HS), or Marek's disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gamma-herpesviruses.Typically, host cells contain viral replication transactivator proteins that activate replication.

[0245] "Control elements," "regulatory sequences" present in an expression vector are the untranslated regions of the vector - origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, polyadenylation sequences, 5' and 3' untranslated regions - which interact with host cellular proteins to effect transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation factors may be used, including ubiquitous and inducible promoters.

[0246] In some embodiments, vectors, including but not limited to expression vectors and viral vectors, will contain exogenous, endogenous, or heterologous control sequences, such as promoters and / or enhancers. An "endogenous" control sequence is a sequence that is naturally linked to a given gene in the genome. An "exogenous" control sequence is one that is placed in juxtaposition to a gene using genetic engineering (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A "heterologous" control sequence is an exogenous sequence that originates from a different species than the cell being genetically engineered.

[0247] As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In some embodiments, a promoter operative in a mammalian cell contains an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated, and / or another sequence, a CNCAAT region, found 70-80 bases upstream from the start of transcription, where N can be any nucleotide.

[0248] The term "enhancer" refers to a DNA segment that contains a sequence that can provide enhanced transcription, and in some cases can function regardless of orientation relative to another control sequence. Enhancers can function cooperatively or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment that contains a sequence that can provide both promoter and enhancer functions.

[0249] The term "operably linked" refers to a juxtaposition where the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0250] As used herein, the term "structural expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that allows for continuous or sequential transcription of an operably linked sequence. A structural expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that allows for expression in a variety of cell and tissue types, or it may be a "cell-specific," "cell type-specific," "cell line-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that allows for expression in restricted cell and tissue types, respectively.

[0251] Illustrative ubiquitous expression control sequences suitable for use in certain embodiments of the present invention include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the viral simian virus 40 (SV40) (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5 and P11 promoters from vaccinia virus, the elongation factor 1 (EF) promoter, the HIV-1 (HIV-2 ... EF1a promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), beta-kinesin (beta-KIN), human ROSA26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deleted type, dl587rev primer binding site substitution (MND) promoter (Challita et al., J Virol. 69(2):748-55 (1995)).

[0252] In some embodiments, the vector comprises an MND promoter. In some embodiments, the vector comprises an EF1a promoter that includes the first intron of the human EF1a gene. In some embodiments, the vector comprises an EF1a promoter that lacks the first intron of the human EF1a gene.

[0253] In certain embodiments, it may be desirable to express a polynucleotide comprising a CAR from a T cell specific promoter.

[0254] As used herein, "conditional expression" may refer to any type of conditional expression, including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological or disease state, and the like. This definition is not intended to exclude cell type-specific or tissue-specific expression. In some embodiments, the conditional expression of a polynucleotide of interest, e.g., expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes an increase or decrease in expression of a polynucleotide encoded by the polynucleotide of interest.

[0255] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid receptors or estrogen receptors (inducible by treatment with the corresponding hormones), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0256] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments of the invention, the vector contains at least one (typically two) sites for recombination mediated by a site-specific recombinase. As used herein, the term "recombinase" or "site-specific recombinase" includes excision or integration proteins, enzymes, cofactors, or associated proteins involved in a recombination reaction that includes one or more recombination sites (e.g., two, three, four, five, seven, 10, 12, 15, 20, 30, 50, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins including the recombination protein sequence or fragments thereof), fragments, and variants thereof. Illustrative examples of suitable recombinases include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0257] A vector may contain one or more recombination sites for any of a wide variety of site-specific recombinases. It should be understood that the target sites for a site-specific recombinase are in addition to any sites required for integration of the vector, e.g., a retroviral or lentiviral vector. As used herein, the terms "recombination sequence," "recombination site," or "site-specific recombination site" refer to a specific nucleic acid sequence that a recombinase recognizes and binds to.

[0258] For example, one recombination site for Cre recombinase is loxP, a 34 base pair sequence that contains two 13 base pair inverted repeats (which serve as recombinase binding sites) flanking an 8 base pair core sequence (see Figure 1 in Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include, but are not limited to, lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).

[0259] Suitable recognition sites for FLP recombinase include, but are not limited to, FRT (McLeod, et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), FRT(RE) (Senecoff et al., 1988).

[0260] Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme lambda integrase, e.g., phi-c31. φC31 SSR mediates recombination only between the heterotypic sites attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). attB and attP are named for the attachment sites of phage integrase on the bacterial and phage genomes, respectively, but both contain imperfect inverted repeats that are likely bound by φC31 homodimers (Groth et al., 2000). The product sites, attL and attR, are also effectively inactive to φC31-mediated recombination (Belteki et al., 2003), rendering the reaction irreversible. To catalyze insertion, it has been found that attB-bearing DNA inserts into genomic attP sites more readily than attP sites insert into genomic attB sites (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, a typical strategy positions, by homologous recombination, an attP-bearing "docking site" in a defined locus, which then partners with an attB-bearing incoming sequence for insertion.

[0261] As used herein, "internal ribosome entry site" or "IRES" refers to an element that facilitates direct entry of an internal ribosome into the initiation codon, such as ATG, of a cistron (protein coding region), leading to cap-independent translation of a gene. See, e.g., Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. In some embodiments, a vector comprises one or more polynucleotides of interest that encode one or more polypeptides. In some embodiments, to achieve efficient translation of each of the multiple polypeptides, the polynucleotide sequences can be separated by one or more IRES sequences or polynucleotide sequences that encode self-cleaving polypeptides.

[0262] As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly promotes initial binding of mRNA to the small ribosomal subunit and increases translation. The consensus Kozak sequence is (GCC)RCCATGG, where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48). In some embodiments, the vector has a consensus Kozak sequence and contains a polynucleotide encoding a desired polypeptide, e.g., a CAR.

[0263] 1. Viral Vectors In some embodiments, cells (e.g., immune effector cells) are transduced with a viral vector, e.g., a lentiviral vector, encoding a CAR. For example, immune effector cells are transduced with a vector encoding a CAR comprising an anti-MUC16 antibody or an antigen-binding fragment thereof that binds to a MUC16 polypeptide, and with an intracellular signaling domain of CD3zeta, CD28, 4-1BB, Ox40, or any combination thereof. In this way, these transduced cells can elicit a CAR-mediated cytotoxic response.

[0264] Illustrative examples of viral vector systems suitable for use in certain embodiments contemplated herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0265] Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. High titers and high levels of expression have been obtained using such vectors. The vectors can be produced in large quantities in a relatively simple system. Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and / or E3 genes, and the replication-deficient vectors are then propagated in human 293 cells that supply the deleted gene functions in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing cells, differentiated cells, such as those found in the liver, kidney, and muscle. Traditional Ad vectors have a large carrying capacity.

[0266] The creation and amplification of current replication-deficient adenoviral vectors may utilize a unique helper cell line called 293. This cell line was transformed with Ad5 DNA fragments from human embryonic kidney cells and constitutively expresses E1 protein (Graham et al., 1977). Because the E3 region is dispensable for the adenoviral genome (Jones & Shenk, 1978), current adenoviral vectors utilize 293 cells to deliver foreign DNA in either the E1, D3, or both regions (Graham & Prevec, 1991). Adenoviral vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Tests of recombinant adenoviruses administered to various tissues include tracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). Examples of the use of Ad vectors in clinical trials include polynucleotide therapy for antitumor immunization using intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0267] In some embodiments, one or more polynucleotides encoding a polycistronic message encoding an anti-MUC16 CAR are introduced into an immune effector cell, e.g., a T cell, by transducing the cell with a recombinant adeno-associated virus (rAAV) comprising the one or more polynucleotides.

[0268] AAV is a small (about 26 nm), replication-deficient, mostly episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can integrate its genome into the genome of a host cell. Recombinant AAV (rAAV) typically consists, at a minimum, of a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequences are about 145 bp long. In some embodiments, rAAV comprises the ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0269] In some embodiments, chimeric rAAVs are used. The ITR sequences are isolated from one AAV serotype, and the capsid sequences are isolated from another AAV serotype. For example, a rAAV that includes ITR sequences from AAV2 and capsid sequences from AAV6 is referred to as AAV2 / AAV6. In certain embodiments, a rAAV vector can include ITRs from AAV2 and capsid proteins from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In some embodiments, a rAAV includes ITR sequences from AAV2 and capsid sequences from AAV6. In some embodiments, a rAAV includes ITR sequences from AAV2 and capsid sequences from AAV2.

[0270] In some embodiments, methods of engineering and selection may be performed on AAV capsids to increase the probability that they will transduce cells of interest.

[0271] The construction, production, and purification of rAAV vectors are disclosed, for example, in U.S. Pat. Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated by reference herein in its entirety.

[0272] In various embodiments, one or more polynucleotides encoding a CAR are introduced into the immune effector cells by transducing the cells with a herpes simplex virus, e.g., HSV-1, HSV-2, comprising one or more polynucleotides. In some embodiments, one or more polynucleotides encoding a polycistronic message encoding a CAR are introduced into the immune effector cells by transducing the cells with a herpes simplex virus, e.g., HSV-1, HSV-2, comprising one or more polynucleotides.

[0273] Mature HSV virions consist of an enveloped icosahedral capsid that contains a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb. In one embodiment, the HSV-based viral vector is defective in one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication-defective. Most replication-defective HSV vectors contain deletions to remove one or more immediate early, early, or late HSV genes to prevent replication. For example, the HSV vector may be defective in an immediate early gene selected from the group consisting of ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantage of HSV vectors is their ability to enter latency, which can result in long-term DNA expression, and their large viral DNA genome, which can accommodate up to 25 kb of exogenous DNA. HSV-based vectors are described, for example, in U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO91 / 02788, WO96 / 04394, WO98 / 15637, and WO99 / 06583, which are incorporated by reference herein in their entireties.

[0274] Retroviruses are common tools for gene delivery (Miller, 2000, Nature. 357:455-460). In a particular embodiment, retroviruses are used to deliver polynucleotides encoding chimeric antigen receptors (CARs) to cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Once a virus is integrated into the host genome, it is referred to as a provirus. The provirus serves as a template for RNA polymerase II to direct the expression of RNA molecules that code for structural proteins and enzymes required to produce new viral particles.

[0275] Illustrative retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), as well as lentiviruses.

[0276] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visna-Maedi virus (VMV); Caprine arthritis encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred. In some embodiments, lentiviruses are used to deliver a polynucleotide comprising a CAR to a cell.

[0277] Retroviral vectors, and more specifically lentiviral vectors, may be used to carry out certain embodiments of the present disclosure. Thus, the terms "retrovirus" or "retroviral vector," as used herein, are meant to include "lentivirus" and "lentiviral vector," respectively.

[0278] As will be apparent to those of skill in the art, the term "viral vector" is used broadly to refer to either a nucleic acid molecule (e.g., a transfer plasmid) that typically contains virally derived nucleic acid elements that facilitate the transfer or integration of the nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid transfer. Viral particles typically contain, in addition to the nucleic acid, various viral components, and sometimes also host cell components.

[0279] The term "viral vector" may refer to either a virus or viral particle capable of transferring a nucleic acid into a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, that are primarily derived from a lentivirus, including LTRs. The term "hybrid vector" refers to a vector, LTR, or other nucleic acid that contains both retroviral sequences, e.g., lentiviral sequences, and non-lentiviral sequences. In one embodiment, a hybrid vector refers to a vector or transfer plasmid that contains retroviral sequences, e.g., lentiviral sequences, for reverse transcription, replication, integration, and / or packaging.

[0280] In some embodiments, the terms "lentiviral vector", "lentiviral expression vector" may be used to refer to lentiviral transfer plasmids and / or infectious lentiviral particles. When reference is made herein to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it should be understood that the sequences of these elements are present in RNA form in lentiviral particles and in DNA form in DNA plasmids.

[0281] At each end of the provirus there is a structure called a "long terminal repeat" or "LTR". The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA, which in the context of their native sequence are direct repeats and include the U3, R and U5 regions. LTRs generally provide essential functions for retroviral gene expression (e.g., promotion, initiation and polyadenylation of gene transcripts) and viral replication. LTRs contain a number of regulatory signals, including transcriptional regulatory elements, polyadenylation signals and sequences required for replication and integration of the viral genome. Viral LTRs are divided into three regions called U3, R and U5. The U3 region contains enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. The LTRs are composed of the U3, R and U5 regions and are present at both the 5' and 3' ends of the viral genome. Adjacent to the 5'LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and efficient packaging of viral RNA into particles (Psi site).

[0282] As used herein, the term "packaging signal" or "packaging sequence" refers to sequences located within the retroviral genome that are required for the insertion of viral RNA into the viral capsid or viral particle. See, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109. Some retroviral vectors use a minimal packaging signal (also referred to as a psi [ψ] sequence) that is required for encapsidation of the viral genome. Thus, as used herein, the terms "packaging sequence", "packaging signal", "psi" and the symbol "ψ" are used in reference to the non-coding sequences required for encapsidation of the viral RNA strand during viral particle formation.

[0283] In various embodiments, the vector comprises a modified 5'LTR and / or 3'LTR. Either or both of the LTRs may comprise one or more modifications, including, but not limited to, one or more deletions, insertions, or substitutions. Modifications of the 3'LTR are often made to improve the safety of lentiviral or retroviral systems by making the virus replication-deficient. As used herein, the term "replication-deficient" refers to a virus that is not capable of complete, efficient replication such that infectious virions are not produced (e.g., progeny of a replication-deficient lentivirus). The term "replication-competent" refers to a wild-type or mutant virus that is capable of replication such that viral replication of the virus is capable of producing infectious virions (e.g., progeny of a replication-competent lentivirus).

[0284] "Self-inactivating" (SIN) vector refers to a vector lacking replication capability, e.g., a retroviral or lentiviral vector in which the right (3') LTR enhancer-promoter region, known as the U3 region, is modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the right (3') LTR U3 region is used as a template for the left (5') LTR U3 region during viral replication, and thus viral transcripts cannot be made without the U3 enhancer-promoter. In a further embodiment of the invention, the 3'LTR is modified such that the U5 region is replaced, e.g., with an ideal poly(A) sequence. It should be noted that modifications to the LTR, such as modifications to the 3'LTR, 5'LTR, or both the 3'LTR and 5'LTR, are also included in the present invention.

[0285] Additional safety enhancement is provided by replacing the U3 region of the 5'LTR with a heterologous promoter that induces transcription of the viral genome during the production of viral particles. Examples of heterologous promoters that can be used include, for example, promoters of simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase). Typical promoters can drive high levels of transcription in a Tat-independent manner. This replacement reduces the possibility of recombination that produces replication-competent virus because of the absence of a complete U3 sequence in the virus production system. In certain embodiments, heterologous promoters have the added advantage of controlling the manner in which the viral genome is transcribed. For example, the heterologous promoter can be inducible, such that transcription of all or part of the viral genome occurs only in the presence of an inducer. Inducers include, but are not limited to, one or more chemical compounds or physiological conditions, such as the temperature or pH, at which the host cells are cultured.

[0286] In some embodiments, the viral vector comprises a TAR element. The term "TAR" refers to a "transactivation response" genetic element located in the R region of lentivirus (e.g., HIV) LTR. This element interacts with the lentivirus transactivator (tat) genetic element to enhance viral replication. However, this element is not required in embodiments in which the U3 region of the 5'LTR is replaced by a heterologous promoter.

[0287] "R region" refers to the region within a retroviral LTR that begins at the start of the capping group (i.e., the start of transcription) and ends just before the start of the polyA tract. The R region is also defined as being adjacent to the U3 and U5 regions. The R region plays a role in allowing the transfer of nascent DNA from one end of the genome to the other during reverse transcription.

[0288] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence comprises the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, such as HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, et al., 2000, Cell, 101:173. During HIV-1 reverse transcription, the central initiation of positive-strand DNA at the central polypurine tract (cPPT) and the central termination at the central termination sequence (CTS) lead to the formation of a triple-stranded DNA structure: the HIV-1 central DNA flap. Without wishing to be bound by any theory, the DNA flap may act as a cis-acting determinant of lentiviral genome nuclear import and / or increase viral titer. In certain embodiments, the retroviral or lentiviral vector backbone comprises one or more FLAP elements upstream or downstream of the heterologous gene of interest in the vector. For example, in certain embodiments, the transfer plasmid comprises a FLAP element. In one embodiment, the vector of the invention comprises a FLAP element isolated from HIV-1. In another embodiment, the lentiviral vector comprises a FLAP element with one or more mutations in the cPPT and / or CTS element. In yet another embodiment, the lentiviral vector comprises either a cPPT or CTS element. In yet another embodiment, the lentiviral vector does not comprise a cPPT or CTS element.

[0289] In some embodiments, the retroviral or lentiviral transfer vector comprises one or more export elements. The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the rev response element (RRE) of human immunodeficiency virus (HIV) (see, e.g., Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423) and the post-transcriptional regulatory element (HPRE) of hepatitis B virus. Generally, RNA export elements are located within the 3'UTR of a gene and can be inserted as one or multiple copies.

[0290] In some embodiments, the expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally transcription termination signals into the vector. A variety of post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids in proteins, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional regulatory element (HPRE) present in Hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766). In some embodiments, the vector comprises a post-transcriptional regulatory element, such as a WPRE or HPRE.

[0291] In some embodiments, the vector lacks or does not include a post-transcriptional regulatory element (PTE), such as a WPRE or HPRE. Because in some cases, these elements increase the risk of cell transformation and / or do not substantially or significantly increase the amount of mRNA transcripts or increase mRNA stability. Thus, in some embodiments, the vector lacks or does not include a PTE. In some embodiments, the vector lacks or does not include a WPRE or HPRE as an additional safety measure.

[0292] Factors that induce efficient termination and polyadenylation of heterologous nucleic acid transcripts increase expression of heterologous genes. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence 3' of the polynucleotide encoding the expressed polypeptide. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to increased translation efficiency. Efficient polyadenylation of recombinant transcripts is desirable because transcripts lacking a polyA tail are unstable and rapidly degraded. Cleavage and polyadenylation are directed by poly(A) sequences in the RNA. The core poly(A) sequence of mammalian pre-mRNA has two recognition elements that flank the cleavage polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is located 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled with the addition of up to 250 adenosines to the 5' cleavage product. Illustrative examples of polyA signals that can be used in the vectors of the invention include ideal polyA sequences (e.g., AATAAA, ATTAAA, AGGTAAA), bovine growth hormone polyA sequence (BGHpA), rabbit β-globin polyA sequence (rβgpA), or another suitable heterologous or endogenous polyA sequence known in the art.

[0293] In some embodiments, the retroviral or lentiviral vector further comprises one or more insulator elements. The insulator elements may contribute to protecting the lentiviral expression sequence, e.g., therapeutic polypeptide, from integration site effects mediated by cis-acting elements present in genomic DNA, which may lead to deregulated expression of the transferred sequence (i.e., position effects; see, e.g., Burgess-Beusse et al., 2002, Proc. Natl. Acad. Sci., USA, 99:16433; and Zhan et al., 2001, Hum. Genet., 109:471). In some embodiments, the transferred vector comprises one or more insulator elements in the 3'LTR, and upon integration of the provirus into the host genome, the provirus comprises one or more insulators in both the 5'LTR or 3'LTR due to the duplication of the 3'LTR. Suitable insulators for use in the present invention include, but are not limited to, the chicken β-globin insulator (see Chung et al., 1993. Cell 74:505; Chung et al., 1997. PNAS 94:575; and Bell et al., 1999. Cell 98:387, incorporated herein by reference). Examples of insulator elements include, but are not limited to, insulators from the β-globin locus, such as chicken HS4.

[0294] In some embodiments, most or all of the viral vector backbone sequence is derived from a lentivirus, such as HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used or combined, and many substitutions and modifications in a particular lentiviral sequence can be accommodated without compromising the ability of the transfer vector to perform the functions described herein. In addition, a variety of lentiviral vectors are known in the art. See Naldini et al. (1996a, 1996b, and 1998); Zufferey et al. (1997); Dull et al., 1998, U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136. Many of them can be adapted to produce viral vectors or transfer plasmids.

[0295] In some embodiments, the vector comprises a promoter operably linked to a polynucleotide encoding a CAR polypeptide. The vector may have one or more LTRs, any of which may contain one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vector may further comprise one of a number of accessory elements to increase transduction efficiency (e.g., cPPT / FLAP), viral packaging (e.g., Psi (Ψ) packaging signal, RRE), and / or other elements that increase therapeutic gene expression (e.g., poly(A) sequences), and may optionally include a WPRE or HPRE.

[0296] In various embodiments, the vector is an integrative viral vector.

[0297] In various other embodiments, the vector is an episomal vector or a non-integrating viral vector.

[0298] In various embodiments, the vectors contemplated herein include non-integrating or integration-defective retroviruses. In one embodiment, "integration-defective" retroviruses or lentiviruses refer to retroviruses or lentiviruses that have an integrase that lacks the ability to integrate the viral genome into the genome of a host cell. In various embodiments, the integrase protein is mutated to specifically reduce its integrase activity. Integration-defective lentivirus vectors are obtained by modifying the pol gene that codes for the integrase protein, resulting in a mutated pol gene that codes for an integration-defective integrase. Such integration-defective viral vectors are described in patent application WO2006 / 010834, which is incorporated herein by reference in its entirety.

[0299] Illustrative mutations in the HIV-1 pol gene suitable for reducing integrase activity include, but are not limited to, H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159 E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A, and K264H.

[0300] Illustrative mutations in the HIV-1 pol gene suitable for reducing integrase activity include, but are not limited to, the following: D64E, D64V, E92K, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, W235F, and W235E.

[0301] In certain embodiments, the integrase comprises a mutation at one or more of amino acids D64, D116, or E152. In one embodiment, the integrase comprises a mutation at amino acids D64, D116, and E152. In certain embodiments, the defective HIV-1 integrase comprises a D64V mutation.

[0302] "Host cells" include cells that have been electroporated, transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or polynucleotide of the invention. Host cells may include packaging cells, producer cells, and cells infected with a viral vector. In certain embodiments, host cells infected with a viral vector of the invention are administered to a subject in need of treatment. In certain embodiments, the term "target cell" is used interchangeably with host cell and refers to a transfected, infected, or transduced cell of a desired cell type. In a preferred embodiment, the target cell is a T cell.

[0303] In certain embodiments, viral vectors comprising contemplated polynucleotides may be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial infusion) or local application, as described below. Alternatively, vectors may be delivered ex vivo to cells, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsies, etc.) or hematopoietic stem cells of a universal donor, followed by reimplantation of the cells into the patient.

[0304] In one embodiment, a viral vector containing a polynucleotide encoding a CAR is directly administered to an organism for in vivo cell transduction. Administration is by any route that is normally used to introduce molecules into blood or tissue cells for final contact, including but not limited to injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and although a particular composition can be administered using more than one route, a particular route can often provide a more immediate and more effective response than another route.

[0305] Large-scale viral particle production is often necessary to achieve reasonable viral titers. Viral particles are produced by transfecting transfer vectors into packaging cell lines that contain viral structural and / or accessory genes, such as the gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes, or other retroviral genes.

[0306] As used herein, the term "packaging vector" refers to an expression vector or viral vector lacking a packaging signal and comprising a polynucleotide encoding one, two, three, four or more viral structural and / or accessory genes. Typically, the packaging vector is contained in a packaging cell and introduced into the cell via transfection, transduction or infection. Methods for transfection, transduction or infection are well known by those skilled in the art. The retroviral / lentiviral transfer vector of the present invention can be introduced into a packaging cell line via transfection, transduction or infection to generate a producer cell or cell line. The packaging vector of the present invention can be introduced into a human cell or cell line by standard methods including, for example, calcium phosphate transfection, lipofection or electroporation. In some embodiments, the packaging vector is introduced into cells along with a dominant selectable marker, such as neomycin, hygromycin, puromycin, blastocydin, zeocin, thymidine kinase, DHFR, Gln synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. The selectable marker gene can be physically linked by the packaging vector, for example to a gene encoded by an IRES or a self-cleaving viral peptide.

[0307] The viral envelope protein (env) determines the range of host cells that can ultimately be infected and transformed by the recombinant retroviruses produced from the cell line. In the case of lentiviruses, such as HIV-1, HIV-2, SIV, FIV, and EIV, the env proteins include gp41 and gp120. Preferably, the viral env protein expressed by the packaging cells of the invention is encoded on a separate vector from the viral gag and pol genes, as previously described.

[0308] Illustrative examples of retrovirus-derived env genes that can be used in the present invention include, but are not limited to, MLV envelope, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (fowl disease virus), and influenza virus envelope. Similarly, genes encoding envelopes from RNA viruses (e.g., the RNA virus families Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Labodoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Birnaviridae, Retroviridae) as well as from DNA viruses (the families Hepadnaviridae, Cycloviridae, Parvoviridae, Papovaviridae, Adnoviridae, Herpesviridae, Poxyviridae, and Iridoviridae) can be utilized. Representative examples include FeLV, VEE, HFVW, WDSV, SFV, rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10, and EIAV.

[0309] In other embodiments, envelope proteins for pseudotyping the viruses of the invention include, but are not limited to, any of the following viruses: influenza A, such as H1N1, H1N2, H3N2, and H5N1 (avian influenza), influenza B, influenza C virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any virus of the Norwalk virus group, enteric adenovirus, parvovirus, dengue virus, entero ... Fever viruses, Monkeypox, Mononegaviruses, Lyssaviruses such as Rabies, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 and 2 and Australian bat virus, Ephemeroviruses, Vesiculoviruses, Vesicular stomatitis virus (VSV), Herpes viruses such as Herpes simplex virus types 1 and 2, Varicella zoster, Cytomegalovirus, Epstein-Barr virus (EBV), Human herpes virus (HHV), Human herpes Virus types 6 and 8, human immunodeficiency virus (HIV), papillomaviruses, murine gammaherpesviruses such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, sciatic-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, Lymphocytic choriomeningitis virus (LCMV), Bunyaviruses such as Crimean-Congo hemorrhagic fever virus, Hantaviruses, hemorrhagic fever with renal syndrome virus, Rift Valley fever virus, Ebola hemorrhagic fever and Marsh syndrome. These include Filoviridae (Filoviruses), including Burg hemorrhagic fever, Flaviviridae, including Kyasanur Forest disease virus, Omsk hemorrhagic fever virus, viruses causing tick-borne encephalitis, and Paramyxoviridae, such as Hendra virus and Nipah virus, Variola major and Variola minor (Smallpox), Alphaviruses, such as Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, any encephalitis causing virus.

[0310] In one embodiment, the invention provides packaging cells that produce recombinant retroviruses, eg, lentiviruses pseudotyped with SVV-G glycoprotein.

[0311] As used herein, the term "pseudotype" or "pseudotyping" refers to a virus whose viral envelope protein has been replaced with the envelope of another virus having favorable properties. For example, HIV envelope protein (encoded by the env gene) normally targets the virus to CD4+ presenting cells, but pseudotyping HIV with the Vesicular Stomatitis Virus G-protein (VSV-G) envelope protein allows HIV to infect a broad range of cells. In a preferred embodiment of the invention, lentiviral envelope protein is pseudotyped with VSV-G. In one embodiment, the invention provides packaging cells that produce recombinant retroviruses, e.g., lentiviruses, pseudotyped with VSV-G envelope glycoprotein.

[0312] As used herein, the term "packaging cell line" is used in reference to a cell line that does not contain a packaging signal but stably or transiently expresses viral structural proteins and replicative enzymes (e.g., gag, pol, and env) that are necessary for correct packaging of viral particles. Any suitable cell line can be used to prepare the packaging cells of the present invention. Generally, the cells are mammalian cells. In certain embodiments, the cells used to produce the packaging cell line are human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In a preferred embodiment, the packaging cells are 293 cells, 293T cells, or A549 cells. In another preferred embodiment, the cells are A549 cells.

[0313] As used herein, the term "producer cell line" refers to a cell line capable of producing recombinant retroviral particles, including a packaging cell line and a transfer vector construct containing a packaging signal. The production of infectious viral particles and viral stock solutions may be performed using conventional techniques. Methods for preparing viral stock solutions are known in the art and are exemplified, for example, by Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and NRL Andau et al. (1992) J. Virol. 66:5110-5113. Infectious viral particles may be harvested from packaging cells using conventional techniques. For example, infectious particles may be harvested by cell lysis or harvesting the cell culture supernatant, as known in the art. Optionally, the harvested viral particles may be purified, if desired. Suitable purification techniques are well known to those skilled in the art.

[0314] Delivery of a gene or other polynucleotide sequence using a retroviral or lentiviral vector by viral infection rather than by transfection is referred to as "transduction." In one embodiment, a retroviral vector is introduced into a cell through infection and proviral integration. In certain embodiments, a target cell, e.g., a T cell, is "transduced" when it contains a gene or other polynucleotide sequence that is delivered to the cell by infection with a viral or retroviral vector. In certain embodiments, the transduced cell contains in its cellular genome one or more genes or other polynucleotide sequences delivered by a retroviral or lentiviral vector.

[0315] In certain embodiments, host cells transduced with a viral vector of the invention expressing one or more polypeptides are administered to a subject to treat and / or prevent disease. Other methods relating to the use of viral vectors in gene therapy may be utilized in accordance with certain embodiments of the present invention, for example, see Kay, MA (1997) Chest 111(6 Supp.):138S-142S; Ferry, N. and Heard, JM (1998) Hum. Gene Ther. 9:1975-81; Shiratory, Y. et al. (1999) Liver 19:265-74; Oka, K. et al. (2000) Curr. Opin. Lipidol. 11:179-86; Thule, PM and Liu, JM (2000) Gene Ther. 7:1744-52; Yang, NS (1992) Crit. Rev. Biotechnol. 12:335-56; Alt, M. (1995) J. Hepatol. 23:746-58; Brody, SL and Crystal, RG (1994) Ann. NY Acad. Sci. 716:90-101; Strayer, DS (1999) Expert Opin. Investig. Drugs 8:2159-2172; Smith-Arica, JR and Bartlett, JS (2001) Curr. Cardiol. Rep. 3:43-49; and Lee, HC et al. (2000) Nature 408:483-8.

[0316] H. Genetically Modified Cells In some embodiments, the disclosure provides cells that are genetically modified to express a CAR as contemplated herein. In some embodiments, the genetically modified cells are for use in the treatment of cancer (e.g., cancers that express MUC16). As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cell," "modified cell," and "redirected cell" are used interchangeably. As used herein, the term "gene therapy" refers to the introduction of extra genetic material in the form of DNA or RNA to the total genetic material in a cell to restore, correct, or modify the expression of a gene, or to introduce extra genetic material in the form of DNA or RNA for the purpose of expressing a therapeutic polypeptide, such as a CAR.

[0317] In some embodiments, the CARs contemplated herein are introduced and expressed in immune effector cells to redirect their specificity to a target antigen of interest, e.g., a MUC16 polypeptide. An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC, etc.).

[0318] Immune effector cells of the invention can be autologous ("self") or non-self ("non-self", e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "autologous" refers to cells derived from the same subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from a compared cell. As used herein, "syngeneic" refers to cells of a different subject that are genetically identical to a compared cell. As used herein, "xenogeneic" refers to cells of a different species from a compared cell. In a preferred embodiment, the cells of the invention are allogeneic.

[0319] Exemplary immune effector cells for use with the CARs contemplated herein include T lymphocytes. The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be T helper cells (HT1; CD4 + T cell) CD4 + T cells, cytotoxic T cells (CTL:CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - The population of T cells may be T cells, or any other subset of T cells. Other illustrative populations of T cells suitable for use in certain embodiments include naive T cells (TN), memory T cells, T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), and effector T cells (TEFF).

[0320] As can be understood by those skilled in the art, other cells can also be used as immune effector cells with CARs as described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include precursors of effector cells, and such precursor cells can be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in some embodiments, immune effector cells include precursors of immune effector cells, such as hematopoietic stem cells (HSCs) contained within the CD34+ population of cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, which differentiate into mature immune effector cells upon administration in a subject, or can be induced in vitro to differentiate into mature immune effector cells.

[0321] As used herein, immune effector cells engineered to contain a MUC16-specific CAR may be referred to as "MUC16-specific, redirected immune effector cells."

[0322] The term "CD34+ cells" as used herein refers to cells that express the CD34 protein on their cell surface. "CD34" as used herein refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in T cell entry into lymph nodes. CD34+ cell populations contain hematopoietic stem cells (HSCs), which upon administration to a patient differentiate and contribute to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineage.

[0323] In some embodiments, the present disclosure provides methods for generating immune effector cells that express a CAR as contemplated herein. In some embodiments, the methods include transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more CARs as described herein. In some embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells may then be directly re-administered to the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express a CAR. In this regard, the immune effector cells may be cultured before and / or after being genetically modified (i.e., transduced or transfected to express a CAR as contemplated herein).

[0324] In some embodiments, a source of cells is obtained from a subject prior to the in vitro manipulation or genetic modification of immune effector cells described herein. In some embodiments, the CAR-modified immune effector cells include T cells. T cells can be obtained from many sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells can be obtained from a blood unit collected from a subject using any number of techniques known to those skilled in the art, such as sedimentation, such as FICOLL™ separation. In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis may be washed to remove the plasma fraction and the cells placed in a suitable buffer or medium for further processing. The cells can be washed with PBS or another suitable solution lacking calcium, magnesium, and most, if not all, other divalent cations. As will be appreciated by those skilled in the art, the washing step can be accomplished by methods known to those skilled in the art, such as by using a semi-automated flow-through centrifuge, such as a Cobe 2991 cell processor, Baxter CytoMate, or similar. After washing, the cells can be resuspended in various biocompatible buffers or other saline solutions, with or without buffer. In some embodiments, undesirable components of the apheresis sample can be removed in cells resuspended directly in culture medium.

[0325] In some embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, e.g., by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells expressing one or more of the following markers: CD3, CD28, CD4, CD8, CD45RA, and CD45RO can be further isolated by positive or negative selection techniques. In one embodiment, specific subpopulations of T cells expressing CD3, CD28, CD4, CD8, CD45RA, and CD45RO can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies directed against surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, negative selection can be used to enrich for T cell populations expressing CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate cell populations of interest for use in the present invention.

[0326] PBMCs may be directly genetically modified to express a CAR using the methods contemplated herein. In some embodiments, following isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0327] CD8 + The cells can be obtained using standard methods. In some embodiments, the CD8 + The cells are CD8 +The cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each of these types of cells.

[0328] In some embodiments, naive CD8 + T lymphocytes are characterized by the expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

[0329] In some embodiments, the memory T cells are CD8 + CD62L on peripheral blood lymphocytes + and CD62L - After staining PBMCs with anti-CD8 and anti-CD62L antibodies, CD62L - CD8 + Fractions and CD62L + CD8 + In some embodiments, expression of phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and are negative for granzyme B. In some embodiments, central memory T cells are CD45RO + , CD62L + , CD8 + T cells.

[0330] In some embodiments, the effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin.

[0331] In some embodiments, CD4 + The T cells can be further sorted into subpopulations. For example, CD4 + T helper cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations that have cell surface antigens. CD4 + Lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO - , CD45RA + , CD62L + CD4 + In some embodiments, the central memory CD4 + The cells are CD62L positive and CD45RO positive. In some embodiments, effector CD4 + The cells are CD62L and CD45RO negative.

[0332] Immune effector cells, such as T cells, can be genetically modified after isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated, in the case of progenitor cells) in vitro prior to being genetically modified. In some embodiments, immune effector cells, such as T cells, are genetically modified with a chimeric antigen receptor contemplated herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR) and then activated and expanded in vitro. In various embodiments, T cells can be activated and expanded before or after genetic modification to express a CAR using, for example, the methods described in U.S. Patent Nos. 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; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0333] Generally, T cells are expanded by contacting them with a surface to which is attached an agent that stimulates CD3 TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. T cell populations can be stimulated by contact with an anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. Costimulation of accessory molecules on the surface of the T cells is also contemplated.

[0334] In some embodiments, PBMCs or isolated T cells are contacted with stimulatory and costimulatory agents, such as anti-CD3 and anti-CD28 antibodies, typically attached to beads or other surfaces, in culture medium containing suitable cytokines, such as IL-2, IL-7, and / or IL-15. + T cells or CD8 + To stimulate the proliferation of either T cells, anti-CD3 and anti-CD28 antibodies. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diacione, Besancon, France), and can be used as well as other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999). Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as "alternative" antigen presenting cells (APCs). In other embodiments, T cells can be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in US6040177; US5827642; and WO2012129514.

[0335] In some embodiments, artificial APCs (aAPCs) are generated by engineering K562, U937, 721.221, T2, and C1R cells to direct stable expression and secretion of various costimulatory molecules and cytokines. In certain embodiments, K32 aAPCs or U32 aAPCs are used to direct the presentation of one or more antibody-based stimulatory molecules on the aAPC cell surface. Expression of various combinations of genes on aAPCs allows precise determination of human T cell activation requirements, allowing aAPCs to be tailored for optimal expansion of T cell subsets with specific growth requirements and distinct functions. In contrast to the use of natural APCs, aAPCs support ex vivo growth and long-term expansion of functional human CD8 T cells without the requirement for the addition of exogenous cytokines. T cell populations can be expanded with aAPCs expressing various costimulatory molecules, including but not limited to CD137L (4-1BBL), CD134L (OX40L), and / or CD80 or CD86. Finally, aAPCs provide an efficient platform for expanding genetically modified T cells and maintaining CD28 expression on CD8 T cells. The aAPCs provided in WO03 / 057171 and US2003 / 0147869 are hereby incorporated by reference in their entireties.

[0336] In some embodiments, CD34 + The cells are transduced with a nucleic acid construct as contemplated herein. In some embodiments, the transduced CD34 + The cells differentiate in vivo into mature immune effector cells following administration into a subject, typically the subject from which the cells were originally isolated. In some embodiments, the CD34 + Cells may be stimulated in vitro after being genetically modified with a CAR described herein, prior to exposure to, or with, one or more of the following cytokines: Flt-3 Ligand (FLT3), Stem Cell Factor (SCF), Megakaryocyte Growth and Differentiation Factor (TPO), IL-3, and IL-6, according to previously reported methods (Asheuer et al., 2004; Imren, et al., 2004).

[0337] In some embodiments, the present disclosure provides a population of modified immune effector cells for the treatment of cancer, the modified immune effector cells comprising a CAR disclosed herein. For example, the population of modified immune effector cells is prepared from peripheral blood mononuclear cells (PBMCs) obtained from a patient (autologous donor) diagnosed with a cancer described herein. The PBMCs form a heterogeneous population of T lymphocytes that may be CD4+, CD8+, or CD4+ and CD8+.

[0338] PBMCs may also contain other cytotoxic lymphocytes, such as NK cells or NKT cells. An expression vector carrying the coding sequence of a CAR contemplated herein can be introduced into a population of human donor T cells, NK cells, or NKT cells. Successfully transduced T cells carrying the expression vector can be isolated and sorted for CD3 positive T cells using flow cytometry, and then further expanded to increase the number of T cells expressing these CAR proteins in addition to cell activation using anti-CD3 and / or anti-CD28 antibodies and IL-2 or any other method known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of T cells expressing CAR proteins T cells for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or expansion of T cells is performed in the absence of non-human animal derived products, such as fetal calf serum and fetal bovine serum. Because a heterogeneous population of PBMCs are genetically modified, the resulting transduced cells are a heterogeneous population of modified cells that contain a MUC16 targeting CAR as contemplated herein.

[0339] In some embodiments, for example, a mixture of one, two, three, four, five, or more different expression vectors can be used in genetically modifying a donor population of immune effector cells, with each vector encoding a different chimeric antigen receptor protein as contemplated herein. The resulting modified immune effector cells will form a mixed population of modified cells, with a proportion of modified cells expressing two or more different CAR proteins.

[0340] IT cell manufacturing method In some embodiments, the present disclosure provides methods for producing cells engineered to express the anti-MUC16 CAR described herein. The cells produced by the methods contemplated herein provide improved adoptive immunotherapy compositions. Without wishing to be bound by any particular theory, it is believed that the cell compositions produced by the methods contemplated herein possess superior properties, including increased survival rate, proliferation in the relative absence of differentiation, and persistence in vivo.

[0341] In some embodiments, the disclosure provides methods for producing T cells engineered to express an anti-MUC16 CAR described herein. In some embodiments, the method of producing T cells comprises contacting the cells with one or more agents that modulate the PI3K cell signaling pathway. In some embodiments, the method of producing T cells comprises contacting the cells with one or more agents that modulate the PI3K / Akt / mTOR cell signaling pathway. In some embodiments, the T cells may be obtained from any source and contacted with the agents during the activation and / or expansion stages of the production process. The resulting T cell composition is enriched in developmentally potent T cells that have the ability to expand and express one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, and CD38. In some embodiments, a population of cells comprising T cells treated with one or more PI3K inhibitors is enriched for a population of CD8+ T cells that co-express one or more, or all of the following biomarkers: CD62L, CD127, CD197, and CD38.

[0342] In some embodiments, modified T cells are produced that include maintained levels of proliferation and reduced differentiation, hi some embodiments, the T cells are produced by stimulating activation and proliferation of the T cells in the presence of one or more stimulatory signals and an agent that is an inhibitor of the PI3K cell signaling pathway.

[0343] To achieve a sufficient therapeutic dose of a T cell composition, the T cells are often subjected to one or more rounds of stimulation, activation, and / or expansion. T cells can generally be activated and expanded using methods described in, for example, U.S. Patent Nos. 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 by reference in its entirety.

[0344] The T cells can then be modified to express the anti-MUC16 CAR. In some embodiments, the T cells are modified by transducing the T cells with a viral vector comprising an anti-MUC16 CAR as contemplated herein. In some embodiments, the T cells are modified prior to stimulation and activation in the presence of an inhibitor of the PI3K cell signaling pathway. In some embodiments, the T cells are modified after stimulation and activation in the presence of an inhibitor of the PI3K cell signaling pathway. In some embodiments, the T cells are modified within 12 hours, 24 hours, 36 hours, or 48 hours of stimulation and activation in the presence of an inhibitor of the PI3K cell signaling pathway.

[0345] After the T cells are activated, the cells are expanded in culture. The 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, and for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion.

[0346] In some embodiments, the T cell compositions are produced in the presence of one or more inhibitors of the PI3K pathway. The inhibitors may target one or more activities or a single activity within the pathway. Without wishing to be bound by any particular theory, treating or contacting T cells with one or more inhibitors of the PI3K pathway during the stimulation, activation, and / or expansion phases of the production process preferentially expands young T cells, thereby producing a superior therapeutic T cell composition.

[0347] In some embodiments, methods are provided for increasing the proliferation of T cells expressing an engineered T cell receptor. Such methods may include, for example, harvesting a source of T cells from a subject, stimulating and activating the T cells in the presence of one or more inhibitors of the PI3K pathway, modifying the T cells to express an anti-MUC16 CAR, and expanding the T cells in culture.

[0348] In some embodiments, the genetically modified T cells are expanded by contact with an agent that stimulates a CD3 TCR complex-associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cell.

[0349] In some embodiments, PBMCs or isolated T cells are contacted with stimulatory and costimulatory agents, such as soluble anti-CD3 and anti-CD28 antibodies, or antibodies attached to beads or other surfaces, in culture medium with suitable cytokines, such as IL-2, IL-7, and / or IL-15.

[0350] In some embodiments, PBMCs or isolated T cells are contacted with stimulatory and costimulatory agents, such as soluble anti-CD3 and anti-CD28 antibodies, or antibodies attached to beads or other surfaces, in culture medium with suitable cytokines, such as IL-2, IL-7, and / or IL-15, and / or a PI3K inhibitor.

[0351] In some embodiments, a method for producing a population of T cells enriched for expression of one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, and CD38. In some embodiments, the young T cells comprise one or more, or all of the following biological markers: CD62L, CD127, CD197, and CD38. In one embodiment, young T cells are provided that lack expression of CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

[0352] In some embodiments, peripheral blood mononuclear cells (PBMCs) are used as a source of T cells in the T cell manufacturing methods contemplated herein. PBMCs are CD4 + , CD8 + , or CD4 + and CD8 + The TCR or CAR may form a heterogeneous population of T lymphocytes that may be competent and may include other mononuclear cells, such as monocytes, B cells, NK cells, and NKT cells. An expression vector comprising a polynucleotide encoding an engineered TCR or CAR as contemplated herein can be introduced into a population of human donor T cells, NK cells, or NKT cells. Successfully transduced T cells carrying the expression vector can be sorted using flow cytometry to isolate CD3 positive T cells, and then further expanded to increase the number of modified T cells in addition to cell activation using anti-CD3 and or anti-CD28 antibodies and IL-2, IL-7, and / or IL-15 or any other method known in the art as described elsewhere herein.

[0353] As used herein, the term "PI3K inhibitor" refers to a nucleic acid, peptide, or small organic molecule that binds to and inhibits at least one activity of PI3K. PI3K proteins can be divided into three classes: class 1 PI3K, class 2 PI3K, and class 3 PI3K. Class 1 PI3K exists as a heterodimer consisting of one of four p110 catalytic subunits (p110α, p110β, p110δ, and p110γ) and one of two regulatory subunit families. PI3K inhibitors preferably target class 1 PI3K inhibitors. In one embodiment, the PI3K inhibitors exhibit selectivity for one or more isoforms of class 1 PI3K inhibitors (i.e., selectivity for p110α, p110β, p110δ, and p110γ or p110α, p110β, p110δ, and p110γ). In another aspect, the PI3K inhibitors do not exhibit isoform selectivity and are considered “pan-PI3K inhibitors.” In one embodiment, the PI3K inhibitor competes for binding with ATP to the PI3K catalytic domain.

[0354] In some embodiments, PI3K inhibitors can target, for example, PI3K and additional proteins in the PI3K-AKT-mTOR pathway. In some embodiments, PI3K inhibitors that target both mTOR and PI3K can be referred to as either mTOR inhibitors or PI3K inhibitors. PI3K inhibitors that target only PI3K can be referred to as selective PI3K inhibitors. In some embodiments, selective PI3K inhibitors can be understood to refer to agents that exhibit a 50% inhibitory concentration for PI3K that is at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 1000 times, or more lower than the IC50 of the inhibitor for mTOR and / or other proteins in the pathway.

[0355] In some embodiments, exemplary PI3K inhibitors inhibit PI3K with an IC50 (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50 μM, 25 μM, 10 μM, 1 μM or less. In one embodiment, the PI3K inhibitor inhibits PI3K with an IC50 of about 2 nM to about 100 nM, more preferably about 2 nM to about 50 nM, even more preferably about 2 nM to about 15 nM.

[0356] Illustrative examples of PI3K inhibitors suitable for use in the T cell manufacturing methods contemplated in certain embodiments include, but are not limited to, BKM120 (Class 1 PI3K inhibitor, Novartis), XL147 (Class 1 PI3K inhibitor, Exelixis), (pan-PI3K inhibitor, GlaxoSmithKline), and PX-866 (Class 1 PI3K inhibitor, p110α, p110β, and p110γ isoforms, Oncothyreon).

[0357] Other illustrative examples of selective PI3K inhibitors include, but are not limited to, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, ZSTK474, and IPI-145. Further illustrative examples of pan-PI3K inhibitors include, but are not limited to, BEZ235, LY294002, GSK1059615, TG100713, and GDC-0941. In some embodiments, the PI3K inhibitor is ZSTK474.

[0358] In some embodiments, methods are provided for increasing the proliferation of T cells expressing an engineered T cell receptor. Such methods may include, for example, harvesting a source of T cells from a subject, stimulating and activating the T cells, modifying the T cells to express a CAR, and expanding the T cells in culture, where the T cells are produced in the presence of one or more PI3K inhibitors at any one of a number of steps in the production process.

[0359] The manufacturing methods contemplated herein may further include cryopreservation of modified T cells for storage and / or preparation for use in human subjects. T cells are cryopreserved such that upon thawing, the cells remain viable. When necessary, cryopreserved transformed immune effector cells can be thawed, grown, and expanded for more such cells. As used herein, "cryopreservation" refers to the preservation of cells by cooling to subzero temperatures, such as (typically) 77K or -196°C (the boiling point of liquid nitrogen). Cryopreservatives are often used at subzero temperatures to prevent the preserved cells from damage due to freezing at low temperatures or heating to room temperature. Cryopreservatives and optimal cooling rates can protect against cell damage. Cryoprotectants that can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183: 1394-1395; Ashwood-Smith, Nature, 1961; 190: 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. NY Acad. Sci., 1960; 85: 576), and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). The preferred cooling rate is 1°-3°C / min. After at least two hours, the T cells will reach a temperature of -80°C and can be placed directly into liquid nitrogen (-196°C) for permanent storage, such as in a long-term cryogenic storage vessel.

[0360] J. Compositions and Formulations In some embodiments, the present disclosure provides compositions comprising one or more polypeptides, polynucleotides, vectors comprising the same, genetically modified immune effector cells, etc., as contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated in a pharma- ceutical or physiologically acceptable solution for administration to a cell or animal, alone or in combination with one or more other therapeutic modalities. It will also be understood that, if desired, the compositions of the present disclosure may also be administered in combination with other agents, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharma- ceutical active agents. Indeed, there is also no limit to other components that may be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0361] The phrase "pharmaceutical acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0362] As used herein, a "pharmaceutical or physiologically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine. Exemplary pharma- ceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicones, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, phosphate buffers, and any other compatible substance used in pharmaceutical formulations.

[0363] In some embodiments, the composition comprises an amount of CAR-expressing immune effector cells as contemplated herein. As used herein, the term "amount" refers to an "effective amount" or "effective amount" of genetically modified therapeutic cells, such as T cells, to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0364] A "prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve a desired prophylactic result. Typically, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease.

[0365] A "therapeutically effective amount" of genetically modified therapeutic cells may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject).

[0366] Generally, the pharmaceutical compositions comprising the T cells described herein are 2 ~10 10 Cells / kg body weight, preferably 10 5 ~10 6 It can be said that a dosage of cells / kg body weight can be administered, including all integer values ​​within the range. The number of cells will depend on the end use for which the composition is intended, as well as the type of cells contained therein. For the uses presented herein, the cells will generally be in a volume of liters or less, which can be 500 ml or less, or even 250 ml or 100 ml or less. Thus, the desired cell density is typically 10 6 More than 10 cells / ml, typically 10 7 More than 10 cells / ml, typically 10 8 The clinically relevant immune cell count is 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 1010 , 10 11 or 10 12 In some aspects of the invention, a smaller number of cells may be injected, such as up to 10 cells, particularly since all injected cells are redirected to a specific target antigen. 6 / kilogram (10 per patient) 6 ~10 11 ) in the range of 100-250 mg / kg / day. The CAR-expressing cell composition may be administered multiple times at dosages within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy. If desired, treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance induction of an immune response.

[0367] In general, compositions comprising cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases occurring in immunocompromised individuals. In particular, compositions comprising CAR-modified T cells as contemplated herein are used in the treatment of cancers expressing MUC16. The CAR-modified T cells of the present invention may be administered as a pharmaceutical composition alone or in combination with carriers, diluents, excipients, and / or other components, such as IL-2 or other cytokines or other cell populations. In some embodiments, pharmaceutical compositions as contemplated herein include an amount of genetically modified T cells in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients.

[0368] The pharmaceutical compositions of the invention, including CAR-expressing immune effector cell populations, such as T cells, may include buffers, such as neutral buffered saline, phosphate buffered saline, and the like; sugars, such as glucose, mannose, sucrose, or dextran, mannitol, and the like; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, the compositions are formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0369] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more of the following: sterile diluents, such as water for injection, saline, preferably saline, Ringer's solution, isotonic saline, fixed oils, such as synthetic mono- or diglycerides that may serve as solvents or suspending media, polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium sulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, and agents for adjusting isotonicity, such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Pharmaceutical compositions for injection are preferably sterile.

[0370] In some embodiments, the T cell composition contemplated herein is formulated in a pharmaceutically acceptable cell culture medium.Such a composition is suitable for administration to human subjects.In some embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0371] Serum-free media have several advantages over serum-containing media, including a simpler and more defined composition, reduced levels of contamination, elimination of potential sources of infectious pathogens, and lower cost. In some embodiments, serum-free media can be animal-free and, optionally, protein-free. Optionally, media can contain biopharmaceutical acceptable recombinant proteins. "Animal-free" media refers to media whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, whose nutrients are derived from synthetic, plant, or microbial sources. "Protein-free" media, in contrast, are defined as being substantially protein-free.

[0372] Illustrative examples of serum-free media for use in certain compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0373] In some embodiments, compositions comprising immune effector cells contemplated herein are formulated in a solution comprising PlasmaLyte A.

[0374] In some embodiments, compositions comprising immune effector cells contemplated herein are formulated in a solution comprising a cryopreservation medium. For example, a cryopreservation medium comprising a cryopreservative may be used to maintain high cell activity after thawing. Illustrative examples of cryopreservation media used in certain compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0375] In some embodiments, compositions comprising immune effector cells contemplated herein are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10.

[0376] K.Kit In some embodiments, the disclosure provides a kit comprising a population of immune effector cells expressing an anti-MUC16 CAR, or a pharmaceutical composition thereof. In some embodiments, the kit comprises instructions for administering the population of immune effector cells or a pharmaceutical composition thereof to a subject in need thereof. In some embodiments, the subject in need thereof has cancer cells expressing MUC16.

[0377] In some embodiments, the disclosure provides kits comprising a population of immune effector cells expressing an anti-MUC16 CAR, or a pharmaceutical composition thereof, and instructions for administering the cells or compositions to a subject in need thereof who has received or is receiving an immune checkpoint inhibitor. In some embodiments, the disclosure provides kits comprising a population of immune effector cells expressing an anti-MUC16 CAR, or a pharmaceutical composition thereof, and instructions for administering the cells or compositions to a subject in need thereof who has received or is receiving nivolumab, pembrolizumab, atezolizumab, or cemiplimab.

[0378] In some embodiments, the disclosure provides a kit comprising a population of immune effector cells expressing an anti-MUC16 CAR, or a pharmaceutical composition thereof, and instructions for administering the cells or composition to a subject in need thereof who has received or is receiving a bispecific antibody comprising a first binding domain specific for a tumor associated antigen and a second binding domain specific for CD3. In some embodiments, the disclosure provides a kit comprising a population of immune effector cells expressing an anti-MUC16 CAR, or a pharmaceutical composition thereof, and instructions for administering the cells or composition to a subject in need thereof who has received or is receiving a bispecific antibody comprising a first binding domain specific for a tumor associated antigen and a second binding domain specific for CD28.

[0379] L. Treatment method The genetically modified immune effector cells expressing the CARs contemplated herein provide improved methods of adoptive immunotherapy for use in the prevention, treatment, and amelioration of cancer, or for preventing, treating, or ameliorating at least one symptom associated with cancer.

[0380] In some embodiments, the genetically modified immune effector cells contemplated herein provide improved methods of adoptive immunotherapy for use in increasing cytotoxicity in cancer cells in a subject or in reducing the number of cancer cells in a subject.

[0381] In some embodiments, the specificity of primary immune effector cells is redirected to cells expressing a particular antigen, e.g., MUC16, by genetically modifying the primary immune effector cells with an anti-MUC16 CAR, as contemplated herein. In some embodiments, a viral vector is used to genetically modify the immune effector cells with a specific polynucleotide encoding the anti-MUC16 CAR.

[0382] In some embodiments, a type of cell therapy is provided in which T cells are genetically modified to express anti-MUC16 CAR that targets MUC16-expressing cancer cells, and the T cells are infused into recipients that need it.The infused cells can kill disease-causing cells in recipients.Unlike antibody therapy, T cell therapy can replicate in vivo, resulting in long-term persistence that can lead to sustained cancer therapy.

[0383] In some embodiments, T cells expressing anti-MUC16 CARs can undergo robust in vivo T cell proliferation and persist for extended periods of time, hi some embodiments, T cells expressing anti-MUC16 CARs evolve into specific memory T cells or stem cell memory T cells that can be reactivated to inhibit any additional tumor formation or growth.

[0384] Illustrative examples of conditions that can be treated, prevented, or ameliorated using immune effector cells expressing anti-MUC16 CAR are contemplated in certain embodiments. In some embodiments, the disclosure provides a method for treating cancer, comprising administering to a subject in need thereof an anti-MUC16 CAR-expressing immune effector cell or a composition comprising the same. In some embodiments, the cancer is selected from ovarian cancer, endometrial cancer, cervical cancer, fallopian tube cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, mesothelioma, intrahepatic cholangiocarcinoma mass-forming type, adenocarcinoma of the uterine cervix, and adenocarcinoma of the gastric tract.

[0385] In some embodiments, methods are provided that include administering a therapeutically effective amount of immune effector cells expressing an anti-MUC16 CAR or a composition comprising the same to a patient in need thereof, alone or in combination with one or more therapeutic agents. In some embodiments, the cells are used in the treatment of a patient at risk of developing a condition associated with cancer cells. Thus, in certain embodiments, a method for treating, preventing, or ameliorating at least one symptom of cancer is provided that includes administering a therapeutically effective amount of modified T cells expressing an anti-MUC16 CAR to a subject in need thereof.

[0386] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits symptoms of a disease, disorder, or condition that can be treated with the gene therapy vectors, cell-based therapeutics, and methods contemplated elsewhere herein. In some embodiments, a subject includes any animal that exhibits symptoms of a cancer-related disease, disorder, or condition that can be treated with the gene therapy vectors, cell-based therapeutics, and methods contemplated elsewhere herein. Suitable subjects (e.g., patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, domestic animals, or pets (e.g., cats or dogs). Non-human primates, preferably human patients, are also included. Exemplary subjects include human patients diagnosed with or at risk for cancer or who have cancer.

[0387] As used herein, the term "patient" refers to a subject who has been diagnosed with a particular disease, disorder, or condition that can be treated using the gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein.

[0388] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally include either a reduction in the disease or condition, or a slowing of the progression of the disease or condition. "Treatment" does not necessarily indicate a complete elimination or cure of the disease or condition, or its associated symptoms.

[0389] As used herein, similar words such as "prevent" and, for example, "prevented," "preventing," etc., refer to an approach to prevent, inhibit, or reduce the likelihood of the onset or recurrence of a disease or condition. They also refer to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0390] As used herein, "ameliorating at least one symptom of" refers to a decrease in one or more symptoms of the disease or condition for which a subject is being treated. In some embodiments, the disease or condition being treated is cancer, and the one or more symptoms that are ameliorated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, swollen or painful abdomen (due to distended abdominal organs), bone or joint pain, bone fractures, unplanned weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (due to impaired kidney function).

[0391] By "enhance" or "promote," or "increase" or "expand," generally refers to the ability of the compositions contemplated herein, e.g., genetically modified T cells expressing an anti-MUC16 CAR, to produce, induce, or cause a greater physiological response (i.e., downstream effect) compared to the response caused by either vehicle or a control molecule / composition. Measurable physiological responses include, among others, an increase in T cell proliferation, activation, persistence, and / or an increase in cancer cell killing capacity, as will be apparent from an understanding of the art and the descriptions herein. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more (e.g., 500-fold, 1000-fold) (including all integers and decimals in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response produced by a solvent or control composition.

[0392] By "reduce" or "lower" or "lower" or "reduce" or "attenuate" generally refers to the ability of the compositions contemplated herein to produce, induce, or cause a physiological response (i.e., downstream effect) that is smaller than the response caused by a solvent or control molecule / composition. The amount of "reduction" or "reduction" is typically a "statistically significant" amount and can include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more (e.g., 500-fold, 1000-fold) (including all integers and decimals in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by a solvent, a control composition, or a response in a particular cell line.

[0393] "Maintain" or "preserve" or "maintain" or "no change" or "no substantial change" or "no substantial decrease" generally refers to the ability of the compositions contemplated herein to produce, induce, or cause a similar physiological response (i.e., downstream effect) in a cell compared to a response caused by a solvent, a control molecule / composition, or a response in a particular cell line. A comparable response is one that is not significantly or measurably different from the reference response.

[0394] In some embodiments, a method of treating cancer in a subject in need thereof includes administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising a genetically modified immune effector cell, e.g., as contemplated herein. The amount and frequency of administration will be determined by factors such as the condition of the patient, the type and severity of the patient's disease, although suitable dosages may be determined through clinical trials.

[0395] In some embodiments, the amount of immune effector cells, e.g., T cells, expressing an anti-MUC16 CAR in the composition administered to a subject is at least 0.1×10 5 Cells, at least 0.5 x 10 5 Cells, at least 1 x 10 5 Cells, at least 5 x 10 5 Cells, at least 1 x 10 6 Cells, at least 0.5 x 10 7 Cells, at least 1 x 10 7 Cells, at least 0.5 x 10 8 Cells, at least 1 x 10 8 Cells, at least 0.5 x 10 9 Cells, at least 1 x 10 9 Cells, at least 2 x 10 9 Cells, at least 3 x 10 9 Cells, at least 4 x 10 9 Cells, at least 5 x 10 9 Cells or at least 1 x 10 10 It is a cell.

[0396] In some embodiments, about 1×107 T cells ~ approx. 1 x 10 9 T cells, approximately 2 x 10 7 T cells ~ approx. 0.9×10 9 T cells, approximately 3 x 10 7 T cells ~ approx. 0.8×10 9 T cells, approximately 4 x 10 7 T cells ~ approx. 0.7×10 9 T cells, approximately 5 x 10 7 T cells ~ approx. 0.6×10 9 T cells, or approximately 5 × 10 7 T cells ~ approx. 0.5×10 9 The T cells are administered to a subject.

[0397] In some embodiments, the amount of immune effector cells, e.g., T cells, expressing an anti-MUC16 CAR in the composition administered to a subject is at least 0.1×10 4 At least 0.5 x 10 cells / kg body weight 4 At least 1 x 10 cells / kg body weight 4 Cells / kg body weight, at least 5 × 10 4 At least 1 x 10 cells / kg body weight 5 At least 0.5 x 10 cells / kg body weight 6 At least 1 x 10 cells / kg body weight 6 At least 0.5 x 10 cells / kg body weight 7 At least 1 x 10 cells / kg body weight 7 At least 0.5 x 10 cells / kg body weight 8 Cells / kg body weight, at least 1 x 10 8 Cells / kg body weight, at least 2 x 10 8 Cells / kg body weight, at least 3 x 10 8 Cells / kg body weight, at least 4 x 10 8 Cells / kg body weight, at least 5 × 10 8 cells / kg body weight or at least 1 x 10 9 cells / kg body weight.

[0398] In some embodiments, about 1×10 6 T cells / kg body weight ~ approx. 1×10 8 T cells / kg body weight, approximately 2×10 6T cells / kg body weight ~ approx. 0.9×10 8 T cells / kg body weight, approximately 3×10 6 T cells / kg body weight ~ approx. 0.8×10 8 T cells / kg body weight, approximately 4×10 6 T cells / kg body weight ~ approx. 0.7×10 8 T cells / kg body weight, approximately 5×10 6 T cells / kg body weight ~ approx. 0.6×10 8 T cells / kg body weight, or approximately 5 × 10 6 T cells / kg body weight ~ approx. 0.5×10 8 T cells / kg body weight are administered to the subject.

[0399] Those skilled in the art will recognize that multiple administrations of the compositions contemplated herein may be required to achieve the desired therapeutic effect. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or more.

[0400] In some embodiments, it may be desirable to administer activated immune effector cells to a subject, then subsequently draw blood again (or perform an apheresis), activate the immune effector cells derived therefrom, and reinfuse the patient with these activated and expanded immune effector cells. This process may be performed multiple times, every few weeks. In some embodiments, immune effector cells may be activated from a blood draw of 10cc to 400cc. In some embodiments, immune effector cells are activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more. Without being bound by theory, the use of a multiple blood draw / multiple reinfusion protocol may be useful for selecting specific populations of immune effector cells.

[0401] Administration of the compositions contemplated herein may be in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, transplantation, or implantation. In some embodiments, the compositions are administered parenterally. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In some embodiments, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0402] In some embodiments, a subject in need thereof is administered an effective amount of the composition to increase a cellular immune response to MUC16-expressing cells or tumors in the subject. The immune response may include a cellular immune response mediated by cytotoxic T cells capable of killing infected cells, regulatory T cells, and helper T cell responses. A humoral immune response may also be elicited, mediated primarily by helper T cells capable of activating B cells, thus leading to antibody production. Various techniques may be used to analyze the type of immune response induced by the composition, which are well described in the art, for example, in Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0403] In some embodiments, a method is provided for treating a subject diagnosed with cancer, comprising removing immune effector cells from the subject, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding an anti-MUC16 CAR, thereby producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In some embodiments, the immune effector cells comprise T cells.

[0404] In some embodiments, a method is provided for stimulating an immune effector cell-mediated immunomodulatory response against a target cell population in a subject, comprising administering to the subject a population of immune effector cells expressing a nucleic acid construct encoding an anti-MUC16 CAR.

[0405] Methods for administering the cell compositions contemplated in certain embodiments include any method effective to result in the reintroduction of ex vivo genetically modified immune effector cells that directly express an anti-MUC16 CAR in a subject, or genetically modified precursor cells of immune effector cells that differentiate into mature immune effector cells expressing a CAR upon introduction into a subject. One method includes transducing peripheral blood T cells ex vivo with a nucleic acid construct contemplated herein and returning the transduced cells to the subject.

[0406] 1. Combination treatment In some embodiments, the compositions contemplated herein include an effective amount of CAR-expressing immune effector cells, for example, in combination therapy with one or more therapeutic agents. As used herein, "combination therapy" includes administration of each agent or treatment in a sequential manner in a regimen that provides a beneficial effect of the combination, as well as co-administration of these agents or treatments in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of these active agents or in multiple separate capsules for each agent. Combination therapy also includes combinations in which the individual elements may be administered at different times and / or by different routes, but which act in combination to provide a beneficial effect due to the synergistic or pharmacokinetic and pharmacodynamic effects of each agent or tumor treatment approach of the combination therapy.

[0407] Thus, the CAR-expressing immune effector cell composition may be administered in combination with other known cancer therapies, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, etc. The composition may be administered in combination with an antibiotic. Such therapeutic agents may be accepted in the art as standard of care for, for example, a particular disease state described herein, such as a particular cancer. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, oncolytic viruses, or other active and adjunctive agents.

[0408] In some embodiments, the compositions comprising the CAR-expressing immune effector cells disclosed herein may be administered in combination with any number of chemotherapeutic agents. Illustrative examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, methyldopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaolamide, and trimethylololamine regimen. resume); nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trophosphamide, uracil mustard, etc.; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, etc.; antibiotics such as aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, Dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin, rodorubicin, streptoniclin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, etc.; antimetabolites such as methotrexate and 5-fluorouracil (5-FU), etc.; folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate, etc.; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmophor, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU, etc.; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.; antiadrenal agents such as aminoglutethimide, mitotane, trilostane, etc.; folic acid supplements such as florinic acid (florinic acid) acid);aceglatone;aldophosphamide glycosides;aminolevulinic acid;amsacrine;bestravcil;bisantrene;edatraxate;defofamine;demecolcine;diaziquone;elformitin;elliptinium acetate;ethoglucide;gallium nitrate;hydroxyurea;lentinan;lonidamine;mitoguazone;mitoxantrone;mopidamol;nitracrine;pentostatin;phenamet;pirarubicin;podophyllic acid;2-ethylhydrazone dode; procarbazine; PSK®; razoxane; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastone; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS 2000; difluoromethylomithine (DMFO);Retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin), ONTAK™ (denileukin diftitox), esperamycin, capecitabine, and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on cancer, such as antiestrogens including tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onapristone, and toremifene (Fareston), and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0409] A variety of other therapeutic agents may be used in conjunction with the compositions described herein. In some embodiments, the compositions comprising CAR-expressing immune effector cells are administered with an anti-inflammatory agent. Anti-inflammatory agents or anti-inflammatory drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide, and nonsteroidal anti-inflammatory agents (NSAIDs), including mycophenolate.

[0410] Other exemplary NSAIDs are selected from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib), and sialic acid salts. Exemplary analgesics are selected from the group consisting of acetaminophen, oxycodone, tramadol, proporxyphene hydrochloride. Exemplary glucocorticoids are selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0411] Illustrative examples of suitable therapeutic antibodies for combination with the CAR-modified T cells contemplated herein include, but are not limited to, bavituximab, bevacizumab (Avastin), bivatuzumab, blinatumomab, conatumumab, daratumumab, durigotumab, dacetuzumab, dalotuzumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, ocaratuzumab, ofatumumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0412] In some embodiments, the compositions described herein are administered in combination with a cytokine. As used herein, "cytokine" refers to a collective term for proteins released by one cell group and acting as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factors; fibroblast growth factors; prolactin; placental lactogen; tumor necrosis factors alpha and beta; Mullerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors, such as NGF-beta; platelet growth factors; transforming growth factors (TGFs), such as TGF-alpha and and the like; TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); bone morphogenetic factor; interferons, such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, IL-21, tumor necrosis factors, such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and Kit Ligand (KL). In one embodiment, the term "cytokine" includes "masked" cytokines, including, but not limited to, masked IL-2. As used herein, the term "cytokine" includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0413] In some embodiments, the compositions described herein are administered in combination with an immune checkpoint inhibitor.

[0414] As used herein, "immune checkpoint" refers to costimulatory and inhibitory signals that regulate the amplitude and quality of T cell receptor recognition of antigen. In some embodiments, the immune checkpoint is an inhibitory signal. In some embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1. In some embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86 to displace CD28 binding. In some embodiments, the inhibitory signal is the interaction between a LAG3 molecule and an MHC class II molecule. In some embodiments, the inhibitory signal is the interaction between TIM3 and galectin 9. In some embodiments, the inhibitory signal is the interaction between OX40 and OX40L.

[0415] T cell activation and effector function are balanced by costimulatory and inhibitory signals, referred to as "immune checkpoints". Inhibitory ligands and receptors that regulate T ...

Claims

1. A chimeric antigen receptor (CAR) comprising: an extracellular domain comprising an anti-MUC16 antibody or antigen-binding fragment thereof that binds to one or more epitopes of a human MUC16 polypeptide; a transmembrane domain; one or more intracellular costimulatory signaling domains; and a primary signaling domain.

2. the MUC16 antibody or antigen-binding fragment thereof binds to the membrane-proximal fragment of MUC16 that remains on the cell surface after proteolytic cleavage of the full-length MUC16 polypeptide; the fragment of MUC16 remaining on the cell surface after proteolytic cleavage comprises SEQ ID NO: 151; and / or the full-length MUC16 polypeptide comprises 16 sea urchin sperm, enterokinase, and agrin (SEA) domains numbered 1 to 16 from N-terminus to C-terminus, and the fragment of MUC16 comprises SEA domains 12 to 16; The CAR according to claim 1.

3. the anti-MUC16 antibody or antigen-binding fragment thereof, (a) SEQ ID NO: 7, or (b) any one of SEQ ID NOs: 47, 59, 71, 83, 95, 107, 119, 133, or 145 and comprising a CDRL1, CDRL2, and CDRL3 region within the variable light chain amino acid sequence shown in the anti-MUC16 antibody or antigen-binding fragment thereof, (a) SEQ ID NO: 8, or (b) any one of SEQ ID NOs: 48, 60, 72, 84, 96, 108, 120, 134, or 146 comprising a CDRH1, CDRH2, and CDRH3 region within the variable heavy chain amino acid sequence shown in The CAR according to claim 1 or claim 2.

4. the anti-MUC16 antibody or antigen-binding fragment thereof, (a) any one of SEQ ID NOs: 1 to 3; (b) any one of (i) SEQ ID NOs: 41-43, (ii) SEQ ID NOs: 53-55, (iii) SEQ ID NOs: 65-67, (iv) SEQ ID NOs: 77-79, (v) SEQ ID NOs: 89-91, (vi) SEQ ID NOs: 101-103, (vii) SEQ ID NOs: 113-115, (viii) SEQ ID NOs: 127-129, or (ix) SEQ ID NOs: 139-141; (c) any one of SEQ ID NOs: 4 to 6; or (d) any one of (i) SEQ ID NOs: 44-46, (ii) SEQ ID NOs: 56-58, (iii) SEQ ID NOs: 68-70, (iv) SEQ ID NOs: 80-82, (v) SEQ ID NOs: 92-94, (vi) SEQ ID NOs: 104-106, (vii) SEQ ID NOs: 116-118, (viii) SEQ ID NOs: 130-132, or (ix) SEQ ID NOs: 142-144. The CAR of claim 1 or claim 2, comprising one or more CDRs shown in (a) the anti-MUC16 antibody or antigen-binding fragment thereof is (i) SEQ ID NO: 7, or (ii) any one of SEQ ID NOs: 47, 59, 71, 83, 95, 107, 119, 133, or 145 or comprises a variable light chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to (b) the anti-MUC16 antibody or antigen-binding fragment thereof is (i) SEQ ID NO: 8, or (ii) any one of SEQ ID NOs: 48, 60, 72, 84, 96, 108, 120, 134, or 146 or comprising a variable heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to The CAR according to claim 1 or claim 2.

6. the transmembrane domain is (a) a polypeptide selected from the group consisting of the alpha, beta, or zeta chain of the T-cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1; (b) a polypeptide selected from the group consisting of CD8α, CD4, CD45, PD1, and CD154; (c) from CD8α, or (d) comprising the sequence of SEQ ID NO: 154; The CAR according to claim 1 or claim 2. (a) the one or more costimulatory signaling domains are from a costimulatory molecule selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70; (b) the one or more costimulatory signaling domains are from a costimulatory molecule selected from the group consisting of CD28, CD134, and CD137 (4-1BB); (c) the one or more costimulatory signaling domains are from CD137 (4-1BB), or (d) the costimulatory signaling domain comprises the sequence of SEQ ID NO:

155. The CAR according to claim 1 or claim 2.

8. 3. The CAR of claim 1 or claim 2, wherein the primary signaling domain is from CD3ζ, or wherein the primary signaling domain comprises the sequence of SEQ ID NO:

156.

9. further comprising a hinge region polypeptide, (a) the hinge region polypeptide comprises the hinge region of CD8α; or (b) the hinge region polypeptide comprises the sequence of SEQ ID NO: 153; The CAR according to claim 1 or claim 2.

10. further comprising a signal polypeptide, wherein the signal polypeptide (a) an IgG1 heavy chain signal polypeptide, a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal polypeptide, an Igκ signal polypeptide, or a CD8α signal polypeptide; (b) comprises a CD8α signal polypeptide; or (c) comprising the sequence of SEQ ID NO: 152; The CAR according to claim 1 or claim 2. (a) a polypeptide linker between the variable heavy chain domain and the variable light chain domain, comprising: (i) the polypeptide linker between the variable heavy chain domain and the variable light chain domain comprises the amino acid sequence of any one of SEQ ID NOs: 14-25; or (ii) the polypeptide linker between the variable heavy chain domain and the variable light chain domain comprises a 3xG4S amino acid linker as set forth in SEQ ID NO: 24; a polypeptide linker, and / or (b) a polypeptide linker between the transmembrane domain and one or more intracellular costimulatory signaling domains, wherein the polypeptide linker between the transmembrane domain and one or more intracellular costimulatory signaling domains comprises a sequence of LYC. The CAR according to claim 1 or claim 2, further comprising:

12. The CAR of claim 1 or claim 2, wherein the anti-MUC16 antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 regions within the variable light chain amino acid sequence shown in SEQ ID NO: 7 and CDRH1, CDRH2, and CDRH3 regions within the variable heavy chain amino acid sequence shown in SEQ ID NO: 8; the transmembrane domain is from CD8α; the one or more costimulatory signaling domains are from CD137 (4-1BB); and the primary signaling domain is from CD3ζ. (a) SEQ ID NO: 9 or SEQ ID NO: 11, or (b) any one of SEQ ID NOs: 49, 50, 51, 52, 61, 62, 63, 64, 73, 74, 75, 76, 85, 86, 87, 88, 97, 98, 99, 100, 109, 110, 111, 112, 121, 122, 123, 124, 125, 126, 135, 136, 137, 138, 147, 148, 149, or 150. The CAR of claim 1 or claim 2, comprising an amino acid sequence having or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to:

14. A CAR that competes with the CAR of claim 1 or claim 2 for binding to one or more epitopes of a human MUC16 polypeptide.

15. A polynucleotide encoding the CAR of claim 1 or claim 2.

16. A polynucleotide encoding a CAR, wherein the polynucleotide sequence is: (a) SEQ ID NO: 10 or SEQ ID NO: 12, or (b) any one of SEQ ID NOs: 9, 11, 49, 50, 51, 52, 61, 62, 63, 64, 73, 74, 75, 76, 85, 86, 87, 88, 97, 98, 99, 100, 109, 110, 111, 112, 121, 122, 123, 124, 125, 126, 135, 136, 137, 138, 147, 148, 149, or 150. A polynucleotide encoding a polypeptide having or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to

17. A vector comprising the polynucleotide of claim 15.

18. The vector of claim 17 , wherein the vector is a lentiviral vector.

19. A cell comprising the vector of claim 17.

20. A cell comprising the polynucleotide described in claim 15.

21. A cell comprising the polynucleotide described in claim 16.

22. A cell comprising the CAR of claim 1.

23. The cells (a) an immune effector cell, (i) the immune effector cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell; (b) a T cell, or (c) αβ T cells, γδ T cells, natural killer (NK) cells, or natural killer T (NKT) cells The cell according to any one of claims 19 to 22,

24. A composition comprising the cells of any one of claims 19 to 22.

25. A pharmaceutical composition comprising the cells of any one of claims 19 to 22 and a physiologically acceptable excipient.

26. A method for generating an immune effector cell comprising the CAR of claim 1 or claim 2, the method comprising introducing a polynucleotide encoding the CAR of claim 1 or claim 2, or a vector comprising the polynucleotide, into an immune effector cell.

27. The pharmaceutical composition of claim 25 for use in treating cancer in a subject in need thereof.

28. the cancer is a solid tumor, optionally wherein the solid tumor expresses MUC16; The solid tumor is (a) is selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, cervical cancer, mesothelioma, esophageal cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, fallopian tube cancer, colorectal cancer, bile duct cancer, breast cancer, and bladder cancer; (b) is selected from the group consisting of ovarian cancer, endometrial cancer, cervical cancer, mesothelioma, NSCLC, and SCLC; or (c) ovarian cancer.

28. The pharmaceutical composition of claim 27.

29. 28. The pharmaceutical composition of claim 27, wherein the use of the pharmaceutical composition is in combination with an additional therapeutic agent.

30. 26. The pharmaceutical composition of claim 25 for use in treating cancer in a subject who has received or is receiving a therapeutic agent.

31. 31. The pharmaceutical composition of claim 29 or 30, wherein the use of the composition and the therapeutic agent is sequential or simultaneous.

32. The therapeutic agent is (a) an immune checkpoint inhibitor, an oncolytic virus, or a costimulatory antibody; (b) a vascular endothelial growth factor (VEGF) inhibitor, or (c) cytokines 31. The pharmaceutical composition of claim 29 or 30, wherein

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