Protease-activated CD45-gated CAR

Abstract

A reversibly gated effector polypeptide, such as a chimeric antigen receptor (protease-activated CD45-gated CAR), comprising an extracellular CD45 recruitment domain, a protease-cleavable linker, and a polypeptide comprising an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain. Nucleic acids, including vectors and expression vectors, encoding protease-activated CD45-gated CARs, and cells, including immune cells, such as T cells, that contain and express the nucleic acids. Methods of treating various conditions, including various forms of cancer, comprising administering the cells, including CAR T cell therapy. In some embodiments, the CD45 gate at least partially inhibits activation of the protease-activated CD45-gated CAR when the protease-activated CD45-gated CAR binds to an antigen. The inhibition is at least partially reduced, mitigated, and / or eliminated when the protease-activated CD45-gated CAR is exposed to a protease capable of cleaving the linker.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 128,667, filed December 21, 2020, and U.S. Provisional Patent Application No. 63 / 289,984, filed December 15, 2021, the contents of which are incorporated by reference in their entireties herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on December 16, 2021, is named AT-043_03WO_SL.txt and is 560,820 bytes in size.

[0003] The present disclosure relates generally to the use of immune cells (e.g., T cells) engineered to express a chimeric antigen receptor (CAR) to treat disease. [Background technology]

[0004] Tumor specificity is one of the major obstacles for the development of cancer therapies, such as CAR T cell therapy, in solid tumor indications. This distinguishes solid tumor targets from blood targets in that the expression of many blood targets (e.g., CD19, CD20, BCMA) is largely restricted to the blood lineage. Their expression can be found on healthy blood cells, which can lead to the destruction of both healthy and cancer cells, but repopulation of normal blood cells from their progenitor stem cells makes these targets tolerable. However, many solid tumor targets show significant levels of expression in some healthy organs or tissues, and the concomitant destruction of these healthy tissues by CAR T cells can lead to severe adverse side effects and even death in patients (on-target off-tumor toxicity). Therefore, it would be useful to develop effective strategies to improve the anti-tumor specificity of CAR T cells to address potential on-target off-tumor toxicity. There is a need for cell-based cancer therapies, such as CAR T cells, with reduced on-target off-tumor toxicity. Summary of the Invention

[0005] The present disclosure provides, inter alia, chimeric antigen receptors (CARs) modified to improve target specificity in CAR T cell therapy, as well as related nucleic acids, expression vectors, cells, compositions, and methods of treatment of cancer and other conditions amenable to CAR T cell therapy. The present disclosure provides modifications to the CAR, CD45 gated, that at least partially inhibit activation of the modified CAR in the presence of the CAR's antigen, thereby reducing the activity of the CAR compared to less modified CARs or compared to unmodified CARs. This inhibition and reduction of activity is partially or completely mitigated when the modified CAR is in the presence of certain proteases, as occurs at the site of a tumor that is a CAR T cell therapy target. At least the following aspects and embodiments of the present disclosure are disclosed herein. Also provided are anti-MUC16 CARs, including anti-MUC16 CARs for use as components of the protease-activated CD45-gated CARs disclosed herein.

[0006] CD45 is a phosphatase that plays a dynamic role in regulating T cell activation (1). It has been shown to directly activate TCR signaling by dephosphorylating other kinases, such as LCK, which activates these kinases and downstream TCR signaling (2). CD45 can also directly dephosphorylate CD3 zeta, which leads to inactivation of TCR signaling (3).

[0007] Thus, in one aspect, provided herein is a protease-activated CD45-gated effector polypeptide, comprising a CD45 recruitment domain, optionally connected to an effector entity by a linker. In some embodiments, the effector entity can be reversibly regulated by CD45. In some embodiments, the effector entity is activated when the CD45 recruitment domain is released from the protease-activated CD45-gated effector by protease cleavage. In some embodiments, the CD45 recruiter is released by protease cleavage at the linker. In some embodiments, the effector entity comprises a chimeric antigen receptor (CAR). In some embodiments, the effector entity comprises a T cell receptor (TCR). In some embodiments, a CD45 recruitment domain (e.g., an anti-CD45 scFv) is prepared from a CD45 antibody disclosed, for example, in WO2005 / 026210, WO2013 / 1048804, WO2017 / 009473, WO2020 / 092654, EP1664122B1, and U.S. Pat. No. 6,106,834, using techniques common to those skilled in the art.

[0008] In one aspect, provided herein is a protease-activated CD45-gated chimeric antigen receptor (CD45-gated CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising a CD45 recruitment domain, a linker comprising one or more protease cleavage sites that can be cleaved by at least one protease, and an antigen-binding domain. In some embodiments of the protease-activated CD45-gated CAR, the intracellular domain comprises at least one signaling domain that can be reversibly inactivated by CD45. In one embodiment, the linker is between the CD45 recruitment domain and the antigen-binding domain. In one embodiment, the CD45 recruitment domain comprises one or more linkers. In one embodiment, the CD45 recruitment domain comprises an anti-CD45 antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the linker is between the VH and VL of the anti-CD45 antibody. In some embodiments, the protease-activated CD45-gated CAR comprises an additional flexible region, for example, located between the domains.

[0009] In a further aspect, provided herein is a protease-activated CD45-gated chimeric antigen receptor (CD45-gated CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises: a CD45 recruitment domain; An antigen-binding domain; a linker connecting the carboxy terminus of the CD45 recruitment domain to the amino terminus of the antigen binding domain, the linker comprising at least one protease cleavage site recognized by at least one protease; Additionally, the intracellular domain comprises at least one (eg, one or more) signaling domains that can be reversibly inactivated by CD45.

[0010] In some embodiments, the CD45 recruitment domain comprises one or more of an anti-CD45 antibody antigen binding domain, an anti-CD45 nanobody, an anti-CD45 scFv, a viral protein binder of CD45, a truncated viral protein binder of CD45, an anti-CD45 Fab, an anti-CD45 camelid VHH, a CD45 binding protein, an endogenous CD45 binder, or a truncated endogenous CD45 binder. In some embodiments, the CD45 recruitment domain comprises one or more of a peptide or protein that interacts with CD45, such as full-length or truncated UL11, full-length or truncated sec49K, and full-length or truncated BTN3A1. In some embodiments, the CD45 recruitment domain comprises a truncated BTN3A1, such as a truncated BTN3A1 having the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0011] In some embodiments, the CD45 recruitment domain comprises an anti-CD45 scFv. In some embodiments, the CD45 recruitment domain comprises an anti-CD45 antibody antigen binding domain. In some embodiments, the CD45 recruitment domain comprises one or more of a truncated UL11, a truncated sec49K, and a truncated BTN3A1. In some embodiments, the CD45 recruitment domain comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0012] In some embodiments, the antigen binding domain is selected from the group consisting of BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUC1, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, MHC-NY-ESO1, HER2 (ERBB2), CAIX (carbonic anhydrase IX), LIV1, ADAM10, CHRNA2, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, claudin-18.2 (claudin It specifically binds to claudin-18A2, or claudin 18 isoform 2), PSCA, DLL3 (delta-like protein 3, Drosophila delta homolog 3, delta 3), Mud7 (mucin 17, Muc3, Muc3), FAP alpha (fibroblast activation protein alpha), Ly6G6D (lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGT1, NG25), PSMA, MSLN, or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43).

[0013] In some embodiments, the antigen binding domain specifically binds to MUC16. In some embodiments, the antigen binding domain specifically binds to MUC16 and the protease-activated CD45-gated CAR comprises any of the amino acid sequences of SEQ ID NOs: 11, 56, 75-79, and 121-170, each of which may or may not have the signal sequence of SEQ ID NO: 1. In some embodiments, the antigen binding domain specifically binds to MUC16 and the protease-activated CD45-gated CAR comprises the amino acid sequence of SEQ ID NO: 75, with or without the signal sequence of SEQ ID NO: 1. In some embodiments, the antigen binding domain specifically binds to MUC16 and the protease-activated CD45-gated CAR comprises the amino acid sequence of SEQ ID NO: 76, with or without the signal sequence of SEQ ID NO: 1. In some embodiments, the extracellular domain comprises any one of the amino acid sequences of SEQ ID NOs: 3, 4, 5, 6, 12, 13, 14, 15, 56, and 56, without the signal sequence of SEQ ID NO: 1.

[0014] In some embodiments, the antigen binding domain specifically binds to an antigen expressed by a particular type of tumor, and the protease cleavage site can be recognized and cleaved by a protease expressed by the same type of tumor. In some embodiments, a protease-activated CD45-gated CAR comprises a CD45 recruitment domain-CAR linker that (1) can specifically recognize and bind to an antigen expressed by a tumor, and (2) can be cleaved by a protease secreted by the tumor and / or a protease present or active in the tumor microenvironment.

[0015] In some embodiments, the antigen binding domain specifically binds to a breast cancer tumor antigen or a colorectal cancer tumor antigen, and one or more of the at least one protease cleavage sites can be recognized and cleaved by uPA. In some embodiments, the antigen binding domain of a protease-activated CD45-gated CAR specifically recognizes and binds to an antigen characteristic of any of cervical cancer, breast cancer, ovarian cancer, and colorectal cancer, and at least one of its protease cleavage sites can be recognized and cleaved by MMP-2 and / or MMP-9. In some embodiments, the antigen binding domain of a protease-activated CD45-gated CAR specifically recognizes and binds to an antigen characteristic of any of breast cancer and ovarian cancer, and at least one of its protease cleavage sites can be recognized and cleaved by matriptase.

[0016] In some embodiments, the linker comprises a GS sequence at either or both ends of each of the one or more protease cleavage sites. In some embodiments, the GS sequence is a polypeptide consisting of only glycine and serine residues. In some embodiments, each GS sequence comprises a series of linked GS peptides, e.g., peptides having an amino acid sequence that includes only glycine and serine residues, e.g., peptides having an amino acid sequence that includes one or more glycine residues followed by one or more serine residues, e.g., peptides having an amino acid sequence of GS, GGS, GGGS (SEQ ID NO: 184), or GGGGS (SEQ ID NO: 178), and the linked GS peptides are linked in any order and in any combination. In some embodiments, the GS sequence comprises any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GS peptides, or comprises 1 to 15 GS peptides in any order and combination.

[0017] In some embodiments, the linker of the protease-activated CD45-gated CAR disclosed herein has a length of 15-100 amino acids, 30-100 amino acids, 15-75 amino acids, 20-50 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids, or about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids, or about 50 amino acids.

[0018] In some embodiments of the protease-activated CD45-gated CAR disclosed herein, the linker comprises the amino acid sequence of SEQ ID NO: 32, 91-98, 103-105, or SEQ ID NO: 106. In some embodiments, the linker comprises one or more amino acid sequences of SEQ ID NO: 53, 89, or 90. In some embodiments, the linker comprises one or more amino acid sequences of SEQ ID NO: 8-10, 99-102, 107-120, 172-176, or SEQ ID NO: 177. In some embodiments, the linker comprises two or more protease cleavage sites, each cleavage site being the same or different from any of the other cleavage sites. In some embodiments, the number of protease cleavage sites is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1-15. In some embodiments, when the linker comprises two or more protease cleavage sites, each protease cleavage site is independently the same or different from one or more of the other protease cleavage sites.

[0019] In some embodiments, the linker comprises one protease cleavage site (e.g., any of the protease cleavage sites disclosed herein, e.g., a cleavage site having an amino acid sequence set forth in any of SEQ ID NOs: 32, 89-98, and 103-106) (SEQ ID NOs: 89 and 90 each have two protease cleavage sites, an MMP2 / 9 site and an MTSP site). In some embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 32 [LVPRGS]. In some embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 93. In some embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 94. In some embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 104. In some embodiments, the protease cleavage site comprises the amino acid sequence of any one of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 103, 104, 105, and 106. In some embodiments, the linker comprises multiple protease cleavage sites (e.g., any of the known and / or disclosed protease cleavage sites, e.g., any of the cleavage sites having an amino acid sequence of SEQ ID NOs: 32, 89-98, and 103-104), e.g., 2, 3, 4, 5, 6, or more protease cleavage sites, e.g., up to 10 or up to 15 protease cleavage sites, each of which is the same as or different from the other cleavage sites.In some embodiments, one or more of the multiple protease cleavage sites comprises the amino acid sequence of SEQ ID NO: 32 (amino acid sequence LVPRGS). In some embodiments, the CD45 recruitment domain comprises one or more protease cleavage sites. In some embodiments, the linker amino acid sequence is or comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, or more) of the amino acid sequence of SEQ ID NOs: 99-102, 172-177, and 108-120. In some embodiments, the linker comprises one copy or more (e.g., 1, 2, 3, 4, 5, 6, or more) of the amino acid sequence of any of SEQ ID NOs: 89-98 and 103-106.

[0020] In some embodiments, the protease cleavage site, or one or more of the multiple protease cleavage sites, is recognized by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15, or any range between any two of these values) of thrombin, trypsin, plasmin, prostate specific antigen (PSA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase (MMP), matriptase (MT-SP1), legumain, a disintegrin and metalloproteinase (ADAM), and transmembrane serine proteases (TMPRSS). Stated differently, in some embodiments, the protease that recognizes the protease cleavage site or recognizes one or more of a plurality of protease cleavage sites is selected from the group consisting of thrombin, trypsin, plasmin, prostate specific antigen (PSA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase (MMP), matriptase (MT-SP1), legumain, a disintegrin and metalloproteinase (ADAM), transmembrane serine protease (TMPRSS), granzyme B, activator protein C, caspases ... The protease cleavage site is one or more of a protease, a cathepsin, a chymase, an elastase, a guanidinobenzoatase, HtrAl, a human neutrophil elastase, a lactoferrin, a marapsin, an NS3 / 4A, a PACE4, a tissue plasminogen activator (tPA), a DESC1, a DPP-4, a FAP, a hepsin, a matriptase-2, a secretase, a kallikrein-related peptidase (KLK), and a tryptase, or a serine protease, a cysteine-type lysosomal protease, a metalloproteinase, a coagulation factor protease, or an aspartyl-type lysosomal protease. In some embodiments, the protease cleavage site comprises an amino acid sequence that is cleaved by a protease or type of protease listed herein.In some embodiments, the protease cleavage site, or one or more of the plurality of protease cleavage sites, is recognized by an endogenous protease or by a protease present in the tumor microenvironment, In some embodiments, the protease that recognizes the protease cleavage site, or one or more of the plurality of protease cleavage sites, is a protease present in the tumor microenvironment.

[0021] In some embodiments, the linker comprises any of the following combinations of cleavage sites, e.g., uPA and MMP, uPA and matriptase, MMP and matriptase, matriptase and MMP and uPA, in any order, including, e.g., from amino terminus to carboxy terminus and from carboxy terminus to amino terminus. In some embodiments, the linker further comprises one or more spacer sequences at either or both ends of one or more of each protease cleavage sites to provide flexibility. In some embodiments, the linker further comprises a GS sequence, e.g., one or more of the GS sequences disclosed herein (e.g., any one or more of SEQ ID NOs: 52, 54, 57), at either or both ends of one or more of each protease cleavage sites. In some embodiments, the CD45-gated CAR comprises one or more of the linkers disclosed herein (e.g., SEQ ID NOs: 7-10, 99-102, 172-177, 108-120), e.g., 1, 2, 3, 4, 5, 15, or any intervening integer value or any range between any two such values. The linker can be disposed, for example, between the CD45 recruitment domain, e.g., an anti-CD45 scFv, and the antigen binding domain, e.g., an antigen binding domain, of the CAR. In some embodiments, the CD45 recruitment domain comprises an anti-CD45 antibody, such as an scFv, and the linker can be disposed between the VL and VH, or between the VH and VL of the CD45 binding domain. In some embodiments, the antigen binding domain comprises 53B6, disclosed herein. In some embodiments, the GS sequence provides a spacer sequence that creates flexibility between the functional domains. In some embodiments, one or both of the regions flanking the protease cleavage site, or the spacer sequence that provides flexibility, are not GS sequences, as many flexible sequences that can function equally well are known in the art.See, e.g., Whitlow M. et al., An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability. Protein Eng. 1993 Nov;6(8):989-95. doi:10.1093 / protein / 6.8.989. PMID:8309948. In some embodiments, the spacer sequence creates flexibility and does not interfere with the activity of each functional domain. In some embodiments, the linker can be of any suitable length, e.g., 15-100 amino acids in length, 30-100 amino acids in length, 20-80 amino acids in length, 20-50 amino acids in length, or 25, 30, 35, 40, 45, 50, or 55 amino acids in length, or about 25, about 30, about 35, about 40, about 45, about 50, or about 55 amino acids in length.

[0022] In some embodiments, the protease cleavage site, or one or more of the plurality of protease cleavage sites, is recognized by one or more of a serine protease, a cysteine-type lysosomal protease, an aspartyl-type lysosomal protease, and a metalloproteinase. In some embodiments, the protease that recognizes the protease cleavage site, or recognizes one or more of the plurality of protease cleavage sites, is one or more of a serine protease, a cysteine-type lysosomal protease, an aspartyl-type lysosomal protease, and a metalloproteinase.

[0023] In some embodiments, the linker comprises 1-15, or 1-10, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cleavage sites, each of which may be the same as or different from one or more of the other cleavage sites, and / or the number of proteases recognizing the protease cleavage sites is 1-15, or 1-10, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0024] In some embodiments, the linker comprises an amino GS peptide, a peptide comprising a protease cleavage site, and a carboxy GS peptide. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:9, which comprises an amino GS peptide (SEQ ID NO:52), a cleavage peptide comprising a protease cleavage site (e.g., SEQ ID NO:53), and a carboxy GS peptide (SEQ ID NO:54). In some embodiments, the linker comprises the same amino and carboxy GS peptides, and different cleavage peptides comprising the same or different protease cleavage sites.

[0025] In some embodiments, the linker of a protease-activated CD45-gated CAR disclosed herein comprises any one or more of the amino acid sequences of SEQ ID NOs: 9, 10, 102, 172-177, and 108-120. In some embodiments, the linker comprises one or more protease cleavage sites having an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 89-98, and 103-104, and optionally, the number of protease cleavage sites is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1-15, and when the linker comprises more than one protease cleavage site, each protease cleavage site is independently the same as or different from one or more of the other protease cleavage sites.

[0026] In some embodiments, the linker of a protease-activated CD45-gated CAR disclosed herein comprises one or more sets of contiguous GS sequences at either or both ends of, and adjacent to, one or more of the protease cleavage sites, each GS sequence having an amino acid sequence of GS, GGS, GGGS (SEQ ID NO: 184), or GGGGS (SEQ ID NO: 178), and the set of contiguous GS sequences comprises GS sequences in any combination, optionally the set of contiguous GS sequences comprises any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GS sequences, or comprises 1-15 GS sequences in any combination. Exemplary embodiments of linkers comprising such sets of contiguous GS sequences include SEQ ID NOs: 9, 10, 102, 172-177, and 107-120.

[0027] In some embodiments, a protease-activated CD45-gated CAR comprises two or more linkers comprising a protease cleavage site, each linker having an amino acid sequence independently selected from SEQ ID NOs: 9, 10, 102, 172-177, and 107-120, and optionally, the number of linkers is an integer value between 2, 3, 4, 5, or 2-10. Exemplary embodiments of protease-activated CD45-gated CARs comprising two or more linkers include SEQ ID NOs: 125-128 and 142-145.

[0028] In some embodiments, the extracellular domain comprises a stalk domain between the antigen binding domain and the transmembrane domain.

[0029] In some embodiments, the intracellular signaling domain comprises an activation domain. In some embodiments, the intracellular signaling domain comprises a costimulatory domain. In some embodiments, the intracellular signaling domain comprises an activation domain and a costimulatory domain. In some embodiments, the intracellular signaling domain comprises an activation domain, such as an ITAM-containing domain. In some embodiments, the intracellular signaling domain comprises a CD3 zeta intracellular domain. In some embodiments, the intracellular signaling domain comprises a CD3 zeta domain comprising the amino acid sequence of SEQ ID NO: 34, or a fragment thereof.

[0030] In some embodiments, intracellular signaling domains for use in protease-activated CD45-gated CARs may be, for example, but not limited to, the cytoplasmic sequences of the T cell receptor and co-receptors that act in concert to initiate signaling following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same function.

[0031] In some embodiments, the intracellular domain comprises or further comprises at least one costimulatory domain. In some embodiments, the at least one costimulatory domain comprises at least one of the following: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function associated antigen-1 (LFA-1 (CD1 la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor Body, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 Ia, LFA-1, ITGAM, CD1 Ib, ITGAX, CD1 Ic, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT A cytoplasmic signaling region or domain of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

[0032] In some embodiments, the protease-activated CD45-gated CAR comprises a signal sequence. In some embodiments, the signal sequence is a CD8 signal sequence. In some embodiments, the signal sequence comprises the amino acid sequence of SEQ ID NO: 1 or 44.

[0033] In some embodiments, the amino acid sequence of the protease-activated CD45-gated CAR is or comprises the amino acid sequence of any one of SEQ ID NOs: 16-31 and 121-170, or is or comprises the amino acid sequence of any one of SEQ ID NOs: 16-31 and 121-170, but lacks any one or more of the signal sequence, the hemagglutinin (HA) tag, and / or the V5 tag.

[0034] In alternative aspects of the protease-activated CD45-gated CARs disclosed herein, the CD45 recruitment domain itself comprises one or more protease cleavage sites (e.g., protease cleavage sites disclosed herein), and the linker connecting the CD45 recruitment domain to the antigen binding domain does not comprise a protease cleavage site or comprises one or more protease cleavage sites (e.g., protease cleavage sites disclosed herein). In one embodiment, protease cleavage produces a CAR attached to a fragment of the CD45 protein, and protease cleavage partially or completely relieves CD45-induced inhibition of the activity of the CAR.

[0035] Also provided herein is a CAR that specifically recognizes and binds to MUC16. In one embodiment, the anti-MUC16 CAR comprises the amino acid sequence of any of SEQ ID NOs: 11, 75, 76, 77, 78, and 79.

[0036] The disclosure further provides a protease-activated CD45-gated T cell receptor (TCR). In one embodiment, the protease-activated CD45-gated TCR comprises a modified TCR alpha, beta, gamma, or delta polypeptide, the modification comprising a CD45 recruitment domain linked to the amino terminus of the TCR alpha, beta, gamma, or delta polypeptide by a protease-cleavable linker peptide. In one embodiment, the protease-activated CD45-gated TCR comprises a modified single chain TCR, the modification comprising a CD45 recruitment domain linked to the amino terminus of the single chain TCR by a protease-cleavable linker peptide. In one embodiment, the polypeptide is a CD45-TCR alpha, beta, gamma, or delta polypeptide. In one embodiment, the polypeptide is a CD45 single chain TCR. In another aspect, provided herein is a nucleic acid encoding a CD45-gated TCR disclosed herein.

[0037] In another aspect, provided herein is a nucleic acid encoding the extracellular domain of a protease-activated CD45-gated CAR disclosed herein. In some embodiments, the encoded extracellular domain comprises a CD45 recruitment domain, such as, but not limited to, a CD45 recruitment domain disclosed herein, an extracellular domain of a CAR, such as, but not limited to, a CAR disclosed herein, and a linker connecting the carboxy terminus of the CD45 recruitment domain to the amino terminus of the CAR extracellular domain, such as, but not limited to, a linker disclosed herein.

[0038] In another aspect, provided herein is a nucleic acid encoding a CD45-gated CAR disclosed herein.

[0039] Also provided herein are nucleic acids encoding a polypeptide comprising any of the amino acid sequences of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170. In some embodiments, the polypeptide is an anti-MUC16 CAR disclosed herein. In some embodiments, the polypeptide is a protease-activated CD45-gated CAR disclosed herein.

[0040] In another aspect, provided herein is a vector comprising the nucleic acid disclosed herein.In one embodiment, the vector is an expression vector.In one embodiment, the vector is a viral vector, a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0041] In a further aspect, provided herein is an engineered cell, e.g., an engineered immune cell, comprising a protease-activated CD45-gated CAR as disclosed herein.

[0042] In a further aspect, provided herein is an engineered immune cell comprising a nucleic acid disclosed herein.

[0043] In another aspect, provided herein is an engineered immune cell comprising a vector disclosed herein.

[0044] In various embodiments, any of the engineered cells, e.g., any of the engineered immune cells disclosed herein, functionally express a protease-activated CD45-gated CAR disclosed herein. In various embodiments, any of the engineered cells, e.g., any of the engineered immune cells disclosed herein, functionally express a protease-activated CD45-gated CAR comprising any of the amino acid sequences of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170. Also provided herein are engineered cells, e.g., engineered immune cells, that functionally express a MUC16-specific CAR comprising any of the amino acid sequences of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170. In various embodiments, any of the engineered cells, e.g., any of the engineered immune cells disclosed herein, are isolated cells.

[0045] In various embodiments, the engineered cells disclosed herein, e.g., engineered immune cells, functionally express a protease-activated CD45-gated CAR disclosed herein from a nucleic acid encoding the protease-activated CD45-gated CAR. In various embodiments, the engineered cells disclosed herein, e.g., engineered immune cells, functionally express CD45 from an exogenous nucleic acid encoding CD45 and introduced into the cell, e.g., functionally express the cell's endogenous CD45 gene, or functionally express CD45 or a fragment or variant of CD45. In various embodiments, the engineered cells disclosed herein, e.g., engineered immune cells, functionally express an anti-MUC16 CAR disclosed herein from a nucleic acid encoding the anti-MUC16 CAR. In some embodiments, the anti-MUC16 CAR is a protease-activated CD45-gated MUC16 CAR.

[0046] In various embodiments, the engineered immune cells disclosed herein are engineered B cells, mast cells, myeloid derived phagocytes, T cells, e.g., alpha / beta and / or gamma / delta T cells, tumor infiltrating lymphocytes (TILs), NK cells, TCR expressing cells, dendritic cells, or NK-T cells. In some embodiments, the cells are and / or are derived from autologous T cells. In some embodiments, the cells are and / or are derived from allogeneic T cells or iPSCs (induced pluripotent stem cells). In some embodiments, the cells are and / or are derived from iPSC-derived T cells.

[0047] In one embodiment, the engineered immune cells disclosed herein comprise or further comprise one or more genomic modifications of an endogenous CD52 gene and one or more of an endogenous TCR gene, e.g., a TCR alpha gene. In one embodiment, the engineered immune cells disclosed herein are T cells.

[0048] In another aspect, provided herein is an immune cell population comprising one or more of the engineered immune cells disclosed herein. In one embodiment, the population comprises about or at least about 1×10 4 , about or at least about 1×10 5 , about or at least about 1×10 6 , about or at least about 1×10 7 or about or at least about 1×10 8 10 of the engineered cells, e.g., engineered immune cells, disclosed herein, optionally comprising a population of about 1×10 10 More than 1 x 10 9 More than 5 x 10 9 In one embodiment, the immune cell population disclosed herein is enriched for engineered immune cells disclosed herein. In various embodiments, the immune cell population is at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% engineered cells, and the engineered cells are T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and / or myeloid-derived phagocytes. In various embodiments, the immune cell population is at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% engineered T cells.

[0049] In another aspect, provided herein is a composition comprising an engineered cell disclosed herein, e.g., an engineered immune cell, or a cell population disclosed herein, and a pharma- ceutically acceptable carrier.

[0050] Also provided herein is the use of any of the engineered immune cells, e.g., engineered T cells, provided herein in the manufacture of a medicament for treating cancer or inhibiting tumor growth or progression in a subject in need thereof. Also provided herein is any of the engineered immune cells, e.g., engineered T cells, provided herein for use as a medicament and for use in treating any disease or condition suitable for treatment by CAR T cell therapy. In some embodiments, the disease or condition is cancer, an autoimmune disease, or an infectious disease. In some embodiments, the disease or condition is a form of cancer.

[0051] In another aspect, provided herein is a method of treating a condition in a patient comprising administering to the patient an engineered immune cell as disclosed herein. In one embodiment of the method, the engineered immune cell is an allogeneic engineered immune cell derived from a donor other than the patient.

[0052] In another aspect, provided herein is a method of treating a condition in a patient comprising administering to the patient a population of engineered immune cells disclosed herein. In one embodiment of the method, the engineered immune cells of the population are derived from one or more allogeneic immune cells from a donor other than the patient.

[0053] In another aspect, provided herein is a method of treating a condition in a patient, comprising administering to the patient a pharmaceutical composition disclosed herein. In one embodiment of the method, the composition comprises one or more engineered allogeneic immune cells derived from a donor other than the patient.

[0054] In embodiments of the methods of treating a condition in a patient disclosed herein, the condition may be any disease or condition that is amenable to treatment with CAR T cell therapy. In some embodiments, the disease or condition is cancer, an autoimmune disease, or an infectious disease. In some embodiments, the disease or condition is a form of cancer. In some embodiments, the cancer is a hematological malignancy or a non-solid tumor. In some embodiments, the cancer is a solid cancer or a solid tumor. In some embodiments, the cancer is a hematological malignancy that is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM). In some embodiments, the cancer is a solid cancer that is biliary tract cancer, bladder cancer, bone or soft tissue cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal carcinoma, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumors, head and neck squamous cell carcinoma, liver cancer, lung cancer, melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, renal cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, or uterine cancer.

[0055] In another aspect, provided herein is a method of reducing on-target off-tumor toxicity of CAR T cells, the method comprising administering to a patient a cell disclosed herein, a cell population disclosed herein, or a composition disclosed herein, wherein the on-target, off-tumor toxicity is lower than the on-target, off-tumor toxicity of a control cell, a control cell population, or a composition of control cells, each administered under the same conditions, wherein the control cell, control cell population, or control cell of the composition comprises a CAR that lacks at least one of a CD45 recruitment domain and a linker comprising a protease cleavage site.

[0056] In another aspect, provided herein is a method of increasing the efficiency of a CAR T cell or CAR T cell therapy, the method comprising administering to a patient a cell as disclosed herein, a cell population as disclosed herein, or a composition as disclosed herein, wherein the efficiency is greater than the efficiency of a control cell, a control cell population, or a composition of control cells, each administered under the same conditions, wherein the control cell of the control cell, control cell population, or composition comprises a CAR that lacks at least one of a CD45 recruitment domain and a linker comprising a protease cleavage site.

[0057] In another aspect, provided herein is a method of increasing the efficacy of CAR T cell therapy, the method comprising administering to a patient a cell disclosed herein, a cell population disclosed herein, or a composition disclosed herein, wherein the efficacy is greater than the efficacy of a control cell, a control cell population, or a composition of control cells, each administered under the same conditions, wherein the control cell, control cell population, or control cell of the composition comprises a CAR that lacks at least one of a CD45 recruitment domain and a linker that comprises a protease cleavage site.

[0058] In another aspect, provided herein is a method of increasing the efficacy of CAR T cell therapy against a solid tumor, the method comprising administering a cell disclosed herein, a cell population disclosed herein, or a composition disclosed herein to a patient having a solid tumor, wherein the efficacy against the solid tumor is greater than the efficacy against the solid tumor of a control cell, a control cell population, or a composition of control cells, each administered under the same conditions, wherein the control cell, control cell population, or control cell of the composition comprises a CAR that lacks at least one of a CD45 recruitment domain and a linker that comprises a protease cleavage site.

[0059] In another aspect, provided herein is a method of reducing the incidence of side effects in CAR T cell therapy, the method comprising administering to a patient a cell disclosed herein, a cell population disclosed herein, or a composition disclosed herein, wherein the incidence of side effects is lower than the incidence of side effects when a control cell, a control cell population, or a composition of control cells, respectively, is administered under the same conditions, wherein the control cell of the control cell, control cell population, or composition comprises a CAR that lacks at least one of a CD45 recruitment domain and a linker that comprises a protease cleavage site.

[0060] In another aspect, provided herein is a method for resulting in reversible co-localization of CD45 and a CD45-gated CAR in a CAR T cell, the reversible co-localization comprising a CD45-gated CAR recruitment domain that binds to CD45 protein on the CAR T cell surface and, as a result, co-localizes, and the binding and localization can be disrupted by a protease cleaving a protease cleavage site in the linker of the CD45-gated CAR, thereby reversing the co-localization, the method comprising: (a) providing T cells that express CD45; (b) introducing into a T cell a nucleic acid encoding a protease-activated CD45-gated CAR as disclosed herein; (c) maintaining the T cells under conditions in which both CD45 and CAR are functionally expressed on the cell surface; as a result, the CD45-gated CAR recruitment domain binds to the CD45 protein on the CAR T cell surface, the CAR and the CD45 protein co-localize, and the co-localization can be reversed by a protease that cleaves a protease cleavage site in the linker of the CD45-gated CAR, which cleavage produces a functional CAR that is no longer connected to the CD45-gated CAR recruitment domain by the linker.

[0061] In another aspect, provided herein is a method that results in a reversible reduction of CAR activity by CD45 protein in a CAR T cell, the reversible reduction of CAR activity comprising a CD45-gated CAR that binds to CD45 protein on the surface of the CAR T cell, resulting in inactivation of the chimeric antigen receptor by CD45, which inactivation can be disrupted by a protease cleaving a protease cleavage site in the linker of the CD45-gated CAR, resulting in the chimeric antigen receptor and the CD45 protein dissociating, terminating the inactivation of the chimeric antigen receptor by CD45, the method comprising: (a) providing T cells that express CD45; (b) introducing a nucleic acid encoding a CD45-gated CAR according to any one of claims 1 to 14 into a T cell; (c) maintaining the T cells under conditions in which both CD45 and CAR are functionally expressed on the cell surface; as a result, the CD45-gated CAR recruitment domain binds to the CD45 protein on the surface of the CAR T cell, the CAR and the CD45 protein associate, and the activity of the CAR is reduced by the CD45 protein, which activity reduction can be reversed by a protease cleaving a protease cleavage site in the linker of the CD45-gated CAR, thereby terminating the association of the CAR and the CD45 protein and terminating the reduction or inhibition of the CAR activity of the CD45 protein, and the cleavage produces a functional CAR that is no longer connected to the CD45-gated CAR recruitment domain by the linker.

[0062] In another aspect, provided herein is a method of treating a patient having a tumor characterized by a protease-rich tumor microenvironment with CAR T cell therapy, the method comprising administering to the patient a cell disclosed herein, a cell population disclosed herein, or a composition disclosed herein, wherein CAR T cell activity is lower outside the protease-rich tumor microenvironment than in the protease-rich tumor microenvironment.

[0063] In another aspect, provided herein is a method of modulating the cytotoxic activity of a CAR T cell comprising a CAR disclosed herein, the method comprising inhibiting the cytotoxic activity of the CAR T cell by associating the CAR with CD45 of the CAR T cell, and activating the cytotoxic activity of the CAR T cell by subjecting and / or exposing the CAR T cell to a protease that recognizes and cleaves the protease cleavage site.

[0064] In embodiments of the methods of treating a condition in a patient disclosed herein, the cells, cell populations, or compositions may be administered to the subject once, or may be administered to the subject two or more times spaced at least about 1, 2, 3, 4, 5, 6, 7 days apart, in some embodiments, the disorder may be a viral disease, a bacterial disease, cancer, an inflammatory disease, an immune disease, or an age-related disease.

[0065] Also provided herein are chimeric antigen receptors (CARs) that bind MUC16 ("MUC16-specific CARs," or "anti-MUC16 CARs," or "MUC16 CARs"). Expression of MUC16-specific CARs in T cells has been demonstrated to be effective in activating T cells upon contact with MUC16. The MUC16-specific CARs provided herein bind human MUC16 and exhibit cytotoxic activity upon contact with MUC16-expressing cells.

[0066] Thus, in another aspect, provided herein is a MUC16-specific chimeric antigen receptor (CAR) comprising an extracellular ligand-binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises a single chain variable fragment (scFv) that binds to the extracellular domain of MUC16.

[0067] The extracellular domain of the MUC16-specific CAR comprises an scFv, which comprises a heavy chain variable (VH) region and a light chain variable (VL) region, each of which contains three complementarity determining regions (CDRs) specific for MUC16.

[0068] In some embodiments, the VH region comprises (i) a VH complementarity determining region 1 (CDR1) having the amino acid sequence set forth in SEQ ID NO: 60 or 63, (ii) a VH complementarity determining region 2 (CDR2) having the amino acid sequence set forth in SEQ ID NO: 61 or 64, and (iii) a VH complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO: 62 or 65, and / or the VL region comprises (i) a VL complementarity determining region 1 (CDR1) having the amino acid sequence set forth in SEQ ID NO: 66 or 69, (ii) a VL complementarity determining region 2 (CDR2) having the amino acid sequence set forth in SEQ ID NO: 67 or 70, and (iii) a VL complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO: 68 or 71.

[0069] In some embodiments, a MUC16-specific CAR is provided that comprises an scFv, wherein the scFv comprises a VH region having the sequence shown in SEQ ID NO:58.

[0070] In some embodiments, a MUC16-specific CAR is provided that comprises an scFv, wherein the scFv comprises a VL region having the sequence shown in SEQ ID NO:59.

[0071] In some embodiments, a MUC16-specific CAR is provided comprising an scFV comprising a VH region and a VL region, wherein the VH region has the sequence set forth in SEQ ID NO:58 and the VL region has the sequence set forth in SEQ ID NO:59.

[0072] In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO: 58 or a variant thereof with one or several conservative amino acid substitutions in residues that are not within the CDRs, and / or the VL region comprises the amino acid sequence set forth in SEQ ID NO: 59 or a variant thereof with one or several amino acid substitutions in amino acids that are not within the CDRs.

[0073] In some embodiments, the VH region comprises the amino acid sequence set forth in SEQ ID NO:58, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:59.

[0074] In some embodiments, each CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat and Chothia definitions, the AbM definition, or the contact definition of a CDR.

[0075] In some embodiments, the MUC16-specific CAR intracellular signaling domain comprises a CD3ζ signaling domain. In some embodiments, the intracellular signaling domain comprises a 4-1BB domain. In some embodiments, the MUC16-specific CAR comprises a second intracellular signaling domain. In some embodiments, the second intracellular signaling domain comprises a 4-1BB domain.

[0076] In some embodiments, the MUC16-specific CAR disclosed herein may comprise a stalk domain between the extracellular ligand binding domain and the first transmembrane domain. In some embodiments, the stalk domain is selected from the group consisting of a CD8α hinge, an IgG1 hinge, and an FcγRIIIα hinge. In some embodiments, the stalk domain is a human CD8α hinge, a human IgG1 hinge, or a human FcγRIIIα hinge.

[0077] In some embodiments, the MUC16-specific CAR disclosed herein may comprise one or more epitopes or mimotopes specific for one or more monoclonal antibodies. In some embodiments, the one or more epitopes or mimotopes specific for the monoclonal antibodies comprise a CD20 epitope or mimotope. In some embodiments, the CD20 epitope or mimotope comprises the amino acid sequence set forth in SEQ ID NO:50 or SEQ ID NO:51.

[0078] In some embodiments, a MUC16-specific CAR comprises the amino acid sequence set forth in any of SEQ ID NOs: 11, 75, 76, 77, 78, and 79, with or without a signal sequence. In some embodiments, a MUC16-specific CAR comprises the amino acid sequence set forth in SEQ ID NO: 75, with or without a signal sequence. In some embodiments, a MUC16-specific CAR comprises the amino acid sequence set forth in SEQ ID NO: 76, with or without a signal sequence.

[0079] In some embodiments, the first transmembrane domain comprises a CD8 α chain transmembrane domain.

[0080] In some embodiments, a MUC16-specific CAR can include another extracellular ligand-binding domain that is not specific for MUC16.

[0081] In some embodiments, the extracellular ligand binding domain, the first transmembrane domain, and the intracellular signaling domain are on a single polypeptide.

[0082] In some embodiments, the CAR can comprise a second transmembrane domain, where the first transmembrane domain and the extracellular ligand binding domain are on a first polypeptide and the second transmembrane domain and the intracellular signaling domain are on a second polypeptide. In an exemplary embodiment, the first transmembrane domain comprises a transmembrane domain from the α chain of the high affinity IgE receptor (FcεRI) and the second transmembrane domain comprises a transmembrane domain from the γ or β chain of the FcεRI.

[0083] In some embodiments, the CAR can comprise a third polypeptide comprising a third transmembrane domain fused to an intracellular signaling domain from a costimulatory molecule, wherein the third transmembrane domain comprises a transmembrane domain from the gamma or beta chain of FcεRI.

[0084] In another aspect, provided herein is an isolated polynucleotide comprising a nucleic acid sequence encoding a MUC16-specific CAR described herein.

[0085] In another aspect, provided herein is an expression vector comprising a polynucleotide encoding a MUC16-specific CAR described herein.

[0086] In another aspect, the present invention provides an engineered immune cell that expresses the MUC16-specific CAR described herein on its cell surface membrane.In some embodiments, the engineered immune cell can include another CAR that is not specific for MUC16.In some embodiments, the engineered immune cell can include a polynucleotide that encodes a suicide polypeptide.In some embodiments, the suicide polypeptide is RQR8.

[0087] In some embodiments, the engineered immune cells are derived from inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In some embodiments, the engineered immune cells are inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes.

[0088] In some embodiments, the engineered immune cells can include a disruption of one or more endogenous genes encoding TCR alpha, TCR beta, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), or immune checkpoint proteins such as, for example, programmed death-1 (PD-1).

[0089] In some embodiments, the engineered immune cells are obtained from a healthy donor, hi some embodiments, the engineered immune cells are obtained from an individual suffering from a disease or disorder.

[0090] In another aspect, provided herein is an engineered immune cell expressing a MUC16-specific CAR as described herein on its cell surface membrane for use as a medicament. In another aspect, provided herein is a MUC16-specific antibody for use as a medicament. In some embodiments, the medicament comprising a MUC16-specific CAR-expressing immune cell or a MUC16-specific antibody is for use in treating a MUC16-related disease or disorder.

[0091] In one aspect, provided herein is a cell population comprising engineered immune cells expressing a MUC16-specific CAR as described herein, (ii) the cell population comprises a percentage of stem cell memory cells and central memory cells that is greater than 20%, 30%, or 40%, and / or (iii) the cell population achieves a percentage of lysis of MUC16-expressing cells that is greater than 10%, 20%, 30%, or 40%.

[0092] In another aspect, provided herein is a method of engineering an immune cell to express any one of the MUC16-specific CARs described herein, the method comprising providing an immune cell and introducing into the cell at least one polynucleotide encoding the MUC16-specific CAR, whereby the immune cell expresses the MUC16-specific CAR.

[0093] In some embodiments, the method includes providing an immune cell, introducing into the cell at least one polynucleotide encoding the MUC16-specific CAR, and introducing at least one polynucleotide encoding a CAR that is not specific for MUC16.

[0094] In another aspect, provided herein is a method of treating a subject suffering from a MUC16-associated disease or disorder, the method comprising providing an immune cell expressing a MUC16-specific CAR described herein on its surface and administering the immune cell to the subject. The present disclosure also provides a method of treating a subject suffering from a MUC16-associated disease or disorder, the method comprising providing a MUC16-specific antibody described herein and administering the antibody to the subject.

[0095] In some embodiments, provided herein is a pharmaceutical composition comprising an engineered immune cell expressing a MUC16-specific CAR described herein. In other embodiments, the present disclosure provides a pharmaceutical composition comprising any of the MUC16-specific antibodies described herein.

[0096] In another aspect, provided herein is a method of treating a condition associated with malignant cells expressing MUC16 in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an engineered immune cell as described herein. In some embodiments, the condition is cancer.

[0097] In another aspect, provided herein is a method of inhibiting tumor growth or progression in a subject having malignant cells that express MUC16, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an engineered immune cell as described herein or a MUC16-specific antibody as described herein.

[0098] In another aspect, provided herein is a method of inhibiting metastasis of malignant cells expressing MUC16 in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an engineered immune cell described herein or a MUC16-specific antibody described herein.

[0099] In another aspect, provided herein is a method of inducing tumor regression in a subject having malignant cells that express MUC16, the method comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition comprising an engineered immune cell as described herein or a MUC16-specific antibody as described herein.

[0100] In some embodiments, any of the above methods further comprise administering one or more additional therapies, such as, for example, a monoclonal antibody and / or chemotherapy. In some embodiments, the monoclonal antibody can be an antibody that binds to a checkpoint inhibitor, such as, for example, an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, any of the above methods further comprise administering to the subject a receptor tyrosine kinase inhibitor, an mTOR inhibitor, an epigenetic modulator, a proteasome inhibitor, an immunomodulator such as lenalidomide, a hedgehog inhibitor, or an isocitrate dehydrogenase (IDH) inhibitor. [Brief description of the drawings]

[0101] [Figure 1A-1B]FIG. 1A (left side) shows a potential mechanism of action for an exemplary protease-activated CD45-gated CAR. In the cell membrane of a T cell, for example, a linker peptide tethers (i.e., binds or connects) the CD45 recruiter domain to the CAR. The linker peptide contains at least one protease site. The recruiter recognizes and binds to the CD45 protein, thus holding the CAR and the CD45 protein in relatively close proximity to each other. This proximity allows CD45 to inactivate the CD3 zeta domain. Thus, CAR activity is reduced even when it binds to its target antigen, as long as the linker peptide is intact. When a protease cleaves the linker peptide (FIG. 1A, right side) (e.g., when the CAR T cell is in the environment of a tumor expressing a protease that recognizes the protease site in the linker), the CAR is "released" from CD45 and is no longer inactivated by CD45. Thus, CAR T cells become selectively active primarily in the vicinity of their target tumor, thereby reducing on-target off-tumor toxicity. Figure IB shows a schematic diagram of the domains of an exemplary CAR / CAR construct. Top: An exemplary CAR preprotein comprises a CD8 signal sequence and a CAR polypeptide. Bottom: An exemplary protease-activated CD45-gated CAR preprotein comprises a CD8 signal sequence, an optional hemagglutinin (HA) tag, a CD45 recruiter domain, a linker comprising a glycine-serine (GS)-rich sequence adjacent to a thrombin protease cleavage site, and a CAR. [Figure 2A-2B]CAR T cells containing vectors encoding protease-activated CD45-gated CARs proliferate and express transgenic proteins. T cells were transduced with vectors encoding a CAR that is not a protease-activated CD45-gated CAR (53B6, anti-MUC16 CAR), a protease-activated CD45-gated CAR (CD45 gate1-TMB-53B6, CD45 gate2-TMB-53B6), or an analog of a protease-activated CD45-gated CAR that contains a non-cleavable linker (CD45 gate1-GS-53B6, CD45 gate2-GS-53B6). The linker of the cleavable protein comprised GS region-thrombin cleavage site-V5 peptide-thrombin cleavage site-GS region. The linker of the non-cleavable protein comprised GS region-V5 peptide-GS region. Figure 2A. Percentage of CAR+ cells at days 8 and 15 of production. Figure 2B. Cell counts at day 15 of production. NTD, non-transduced. [Figure 3A] Exposure of the CAR T cells in Figures 2A-2B to thrombin resulted in reduced detection of the V5 tag in the cleavable CAR but not in the non-cleavable CAR. [Figure 3B] The ability of CAR T cells in Figures 2A-2B to bind soluble MUC16 was not significantly affected by the presence or absence of CD45 gating. [Figure 4A-4B] Results of cytotoxicity assays used to evaluate CD45-gated inhibitory activity. Assays were performed in R10 medium without the presence of thrombin or other exogenous proteins. Figure 4A. Ovcar3(MUC16 high), 1st-48h cytotoxicity. Figure 4B. Ovcar3(MUC16 high), 2nd-2x48h cytotoxicity. NTD: non-transduced; Ovcar3(MUC16 high): cell line expressing high density of target antigen MUC16. [Figure 5A-5B]Results of cytotoxicity assays used to assess CD45-gated inhibitory activity. Assays were performed in R10 medium in the absence of thrombin or other exogenous proteins. Figure 5A. Cov644(MUC16 low), 1st-48 hr cytotoxicity. Figure 5B. Cov644(MUC16 low), 2nd-2x48 hr cytotoxicity. NTD: non-transduced; Cov644(MUC16 low): cell line expressing low density of target antigen MUC16. [Figure 6A-6B] Results of a cytotoxicity assay used to assess the reversibility of CD45-gated inhibitory activity. Assays were performed in serum-free medium. Black bars: absence of protease. White bars: presence of protease (thrombin). E:T: effector to target cell number ratio, NTD: non-transduced. [Figure 7A] We successfully generated 53B6 CAR T cells from two different T cell donors using a lentiviral transfer vector containing the 53B6 scFv targeting the MUC16 protein. Flow cytometry was used to detect the CAR+ T cell population with recombinant MUC16 protein (SEA1-4). [Figure 7B] Phenotypes were assigned according to CD62L and CD45RO expression within the CAR+ T cell population as follows: stem cell memory (Tscm; CD45RO- / CD62L+), central memory (Tcm; CD45RO+ / CD62L+), effector memory (Tem; CD45RO+ / CD62L-), effector cell (Teff; CD45RO- / CD62L-). SEA = sperm proteins, enterokinase, agrin, EV: empty vector. [Figure 8A-8C] 1x107 luciferase-expressing ovarian target cells (OVCAR3, COV644, FUOV1) with different MUC16 surface protein expression levels (indicated in brackets) were co-cultured with 53B6 CAR+T cells at various effector:target (E:T) ratios for 72 hours. Empty vector (EV) control T cells were also included in the study. Target cell viability after exposure to T cells was determined by comparing with target cells alone. [Figure 9A-9B]1 x 107 luciferase-expressing OVCAR3 target cells were co-cultured with 53B6 CAR+ T cells at a 1:1 effector:target ratio in a sequential killing assay, with T cells exposed to fresh target cells every 2-3 days. Empty vector (EV) control T cells were also included in the study. Target cell viability after exposure to T cells was determined by comparing with target cells alone. Data presented is the activity of 53B6 CAR T cells from two different donors. [Figure 10A-10B] 53B6 anti-MUC16 CAR T cells show anti-tumor activity against two different orthotopic ovarian tumor models. NSG mice (n=5) injected intraperitoneally (IP) with 3x106 luciferase-labeled OVCAR3 (Figure 10A) or COV644 (Figure 10B) cells received IP injections of 1x106 or 3x106 53B6 CAR+T cells, respectively, 14 days later. Tumor burden was monitored by bioluminescence and results demonstrated anti-tumor activity in both models. Empty vector (EV) control T cells were also included in the study. LOD=limit of detection based on bioluminescence imaging of non-tumor-bearing mice. [Figure 11] Schematic diagram of an exemplary CD45-gated CAR construct. TPS3, TPS4, etc. refer to linkers of various lengths (e.g., TPS6 20aa, TPS6 25aa, etc.) as shown in Table 2, MMP = matrix metalloproteinase, uPA = urokinase plasminogen activator (uPA),

number

[0102] The present disclosure provides protease-activated CD45-gated CARs, e.g., chimeric antigen receptors modified to include a removable CD45 recruitment domain that specifically binds to CD45. The present disclosure further provides related nucleic acids, engineered cells, compositions, and methods. The CD45-binding domain is linked, bound, or tethered to the antigen-binding domain of the CAR by a peptide linker that includes at least one protease cleavage site. The antigen-binding domain of the CAR can specifically bind to an antigen expressed by a target cell, such as a tumor cell. Without wishing to be bound by theory, it is hypothesized that when the protease-activated CD45-gated CAR is functionally expressed in a cell that also functionally expresses CD45, the protease-activated CD45-gated CAR binds to the CD45 protein. This binding is believed to hold CD45 in close proximity to the protease-activated CD45-gated CAR. This is believed to allow CD45, a phosphatase, to dephosphorylate the intracellular signaling domain of the CAR, thereby at least partially suppressing the activity of the CAR. If the CAR binds to its antigen under this condition, it will not be activated to the same extent as it would be in the absence of the CD45 gate. Thus, the CAR is in a "suppressed" or "reduced activity" state. The extracellular linker protease cleavage site of the protease-activated CD45-gated CAR can be recognized and cleaved by a protease present in the microenvironment of one or more tumor cells that the CAR targets. In this situation, the protease detaches the CAR from the CD45 recruitment domain. This allows the CAR to dissociate from CD45, thus terminating the CD45-mediated suppression of the activation of the CAR. Under this condition, if the CAR binds to its antigen, it can be activated to the same extent as it would be if it had never contained the CD45 gate. Thus, when a protease-activated CD45-gated CAR-expressing cell is in the presence of its target cell, the partial suppression of the activity of the CAR is at least partially relieved, in other words, the protease-activated CD45-gated CAR is at least partially activated.Thus, protease-activated CD45-gated CARs are expected to provide more selective CAR activation than CARs lacking a gate.

[0103] general technology The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press, Animal Cell Culture (RIFreshney, ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds., 1993-1998) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DMWeir and CCBlackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (JEColigan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JDCapra, eds., Harwood Academic Publishers, 1995). For example, gene editing techniques using TALEN, CRISPR / Cas9, and megaTAL nucleases are within the skill of the art and are fully described in T. Gaj et al., Genome-Editing Technologies: Principles and Applications, Cold Spring Harb Perspect Biol 2016;8:a023754 and citations therein, C. Sommer et al., Mol. Ther. 27:1126-38(2019)(PMID:31005597), C. Sommer et al., Mol. Ther. 28:2237-51(PMID:32592688).

[0104] definition As used herein, "autologous" means that the cells, cell lines, or cell populations used to treat a subject are derived from that subject.

[0105] As used herein, "allogeneic" means that the cells or cell population used to treat a subject do not originate from the subject, but rather from a donor.

[0106] As used herein, the term "endogenous" refers to any material that is produced from or within an organism, cell, tissue, or system.

[0107] As used herein, the term "exogenous" refers to any material that is introduced from or produced outside an organism, cell, tissue, or system.

[0108] As used herein, "immune cells" refer to cells of hematopoietic origin that are functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. Examples of immune cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0109] As used herein, the term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0110] As used herein, "expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0111] As used herein, "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence if it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).

[0112] As used herein, "expression control sequence" refers to a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0113] "Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. It will be understood by those skilled in the art that different promoters may induce expression of a gene in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions.

[0114] In any of the vectors of the present disclosure, the vector optionally comprises a promoter as disclosed herein.

[0115] A "host cell" includes an individual cell or cell culture that can be or has been a recipient of a vector for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide of the disclosure.

[0116] As used herein, the term "extracellular ligand-binding domain" refers to an oligopeptide or polypeptide capable of binding to a ligand. Preferably, the domain is capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. The term "stalk domain" is used herein to refer to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular ligand-binding domain. In particular, the stalk domain is used to provide additional flexibility and accessibility to the extracellular ligand-binding domain.

[0117] The term "intracellular signaling domain" refers to the portion of a protein that transduces effector functional signals and induces the cell to carry out a specialized function.

[0118] As used herein, a "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include ligands that specifically bind to CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, etc.

[0119] "Costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory signal molecule on a T cell, thereby providing a signal that mediates T cell responses, including, but not limited to, proliferation activation, differentiation, etc., in addition to the primary signal provided, for example, by engagement of the TCR / CD3 complex with a peptide-loaded MHC molecule. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1 These may include, but are not limited to, agonists or antibodies that bind to CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, Toll ligand receptor, and ligands that specifically bind to B7-H3. Costimulatory ligands also encompass antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, CD83, among others.

[0120] An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal or monoclonal antibodies, as well as antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, and Fv), and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site, including, for example, but not limited to, single chain (scFv) and domain antibodies (including, for example, shark antibodies and camelid antibodies), and fusion proteins that contain antibodies. Antibodies include antibodies of any class (or subclass thereof), such as IgG, IgA, or IgM, and an antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which may be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, lgA1, and lgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0121] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. The antigen-binding function of an antibody can be performed by a fragment of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab, Fab', F(ab')2, Fd fragment consisting of the VH and CH1 domains, Fv fragment consisting of the VL and VH domains of a single arm of an antibody, single-domain antibody (dAb) fragments (Ward et al., Nature 341:544-546, 1989), and isolated complementarity-determining regions (CDRs).

[0122] An antibody, antibody conjugate, or polypeptide that "specifically binds" to a target is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates more frequently, more rapidly, with longer duration, and / or with higher affinity with a particular cell or substance than with another cell or substance. An antibody "specifically binds" to a target if it binds with higher affinity, avidity, more readily, and / or with longer duration than it binds to other substances. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope or mimotope) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (although it can include) exclusive binding.

[0123] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs, and the CDRs from the other chain contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948). As used herein, CDRs may refer to CDRs defined by either approach, or by a combination of both approaches.

[0124] The "CDRs" of a variable domain are the amino acid residues within the variable regions identified according to Kabat, Chothia, the combined Kabat and Chothia, AbM, contact and / or structural definitions, or any method of CDR determination known in the art. Antibody CDRs may be identified as hypervariable regions as originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC. The locations of CDRs may also be identified as structural loop structures as originally described by Chothia et al. See, e.g., Chothia et al., Nature 342:877-883, 1989. Other approaches to CDR identification include the "AbM definition," a compromise between Kabat and Chothia, derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the "contact definition" of CDRs based on observed antigen contacts as described in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another approach, referred to herein as the "structural definition" of CDRs, the positions of CDRs can be identified as residues that contribute enthalpic wise to antigen binding. See, e.g., Makabe et al., Journal of Biological Chemistry, 283:1 156-1 166, 2008. Still other CDR boundary definitions may not strictly follow one of the above approaches, but they may nevertheless be shortened or extended, taking into account predictions or experimental findings that certain residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding, but overlap with at least a portion of the Kabat CDR. As used herein, CDR may refer to a CDR defined by any approach known in the art, including a combination of approaches. The methods used herein may utilize CDRs defined according to any of these approaches.In any given embodiment containing two or more CDRs, the CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, contact, and / or structural definitions.

[0125] Antibodies of the disclosure may be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or a combination of such techniques or other techniques readily known in the art (see, e.g., Jayasena, SD, Clin. Chem., 45:1628-50, 1999, and Fellouse, FA, et al, J. Mol. Biol., 373(4):924-40, 2007).

[0126] As known in the art, "polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a chain of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "cap" substitutions with one or more analogs of naturally occurring nucleotides, internucleotide modifications, such as those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides. Additionally, any of the hydroxyl groups normally present in the sugar can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to provide for additional linkages to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides can also contain ribose or deoxyribose sugars in analogous forms commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, alpha or beta anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0127] As used herein, "transfection" refers to the uptake of exogenous or heterologous RNA or DNA by a cell. A cell is "transfected" by exogenous or heterologous RNA or DNA when such RNA or DNA is introduced into the cell. A cell is "transformed" by exogenous or heterologous RNA or DNA when the transfected RNA or DNA affects a phenotypic change. The transforming RNA or DNA may be integrated (covalently linked) into chromosomal DNA constituting the genome of the cell.

[0128] As used herein, "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

[0129] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, and most preferably at least 99% pure. The term "competes" as used herein with respect to antibodies means that a first antibody, or an antigen-binding fragment (or portion) thereof, binds to an epitope or mimotope in a manner sufficiently similar to the binding of a second antibody or antigen-binding portion thereof, such that the result of binding of the first antibody to its cognate epitope or mimotope is detectably reduced in the presence of the second antibody, compared to binding of the first antibody in the absence of the second antibody. The alternative possibility that binding of the second antibody to the epitope or mimotope is also detectably reduced in the presence of the first antibody may, but need not, be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope or mimotope without that second antibody inhibiting the binding of the first antibody to its respective epitope or mimotope. However, if each antibody detectably inhibits the binding of the other antibody to its cognate epitope or mimotope or ligand, whether to the same extent, a greater extent, or a lesser extent, the antibodies are said to "cross-compete" with each other for binding of their respective epitopes or mimotopes. Both competing and cross-competing antibodies are encompassed by the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, or binding to a common epitope or mimotope or a portion thereof), one of skill in the art will understand, based on the teachings provided herein, that such competing and / or cross-competing antibodies are encompassed and may be useful in the methods disclosed herein.

[0130] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, one or more of the following: reduction (or destruction) of tumor or cancer cell proliferation, inhibition of metastasis of tumor cells, reduction or decrease in tumor size, amelioration of a disease (e.g., cancer), reduction of symptoms caused by a disease (e.g., cancer), improvement in the quality of life of a person suffering from a disease (e.g., cancer), reduction in the dosage of other drugs required to treat a disease (e.g., cancer), delay in progression of a disease (e.g., cancer), cure of a disease (e.g., cancer), and / or prolongation of survival of a subject with a disease (e.g., cancer).

[0131] "Ameliorating" refers to the alleviation or improvement of one or more symptoms compared to no treatment. "Ameliorating" also includes shortening or reducing the duration of symptoms. As used herein, an "effective dosage" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to produce any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of a disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes manifested during the development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing or improving the incidence of one or more symptoms of various diseases or conditions (e.g., cancer, etc.), reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug, and / or delaying the progression of the disease. An effective dosage may be administered in one or more administrations. For purposes of this disclosure, an effective dosage of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, either directly or indirectly. As understood in a clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be or is achieved.

[0132] As used herein, a "subject" is any mammal, e.g., a human or a monkey. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats. In an exemplary embodiment, the subject is a human. In an exemplary embodiment, the subject is a monkey, e.g., a cynomolgus monkey.

[0133] As used herein, "vector" refers to a construct that can deliver and preferably express one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0134] As used herein, "pharmaceutical acceptable carrier" or "pharmaceutical acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions such as oil / water emulsions, and various types of wetting agents. A preferred diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or normal (0.9%) saline. The compositions of the present disclosure that include such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, and Remington, The Science and Practice of Pharmacy 21 st Ed. Mack Publishing, 2005).

[0135] As used herein, "allo-reactivity" refers to the ability of T cells to recognize MHC complexes not present during thymic development. Alloreactivity is manifested clinically as host-versus-graft rejection and graft-versus-host disease.

[0136] Reference to "about" a value or parameter herein includes (and describes) an embodiment directed to that value or parameter per se. For example, a reference to "about X" includes the description of "X." Numeric ranges include the numbers defining the range.

[0137] Wherever embodiments are described herein with the term "comprising," it is understood that otherwise similar embodiments are also provided that are described in terms of "consisting of" and / or "consisting essentially of."

[0138] When aspects or embodiments of the disclosure are described in terms of a Markush group or other alternative grouping, the disclosure encompasses not only the entire group recited as a whole, but each individual member of the group and all possible subgroups of the main group, but the main group also is free of one or more of the group members. The disclosure also envisions the explicit exclusion of one or more of the group members in the claimed disclosure.

[0139] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification, including definitions, shall prevail. Throughout this specification and claims, "comprise" or variations such as "comprises" or "comprising" shall be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Unless otherwise required by context, the singular shall include the plural, and the plural shall include the singular.

[0140] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0141] An "antigen binding protein" comprises one or more antigen binding domains. As used herein, "antigen binding domain" refers to any polypeptide that binds to a specified target antigen. In some embodiments, the antigen binding domain binds to an antigen on a tumor cell. In some embodiments, the antigen binding domain binds to an antigen on a cell involved in a hyperproliferative disease, or to a viral or bacterial antigen.

[0142] Antigen-binding domains include, but are not limited to, antibody binding regions that are immunologically functional fragments. The term "immunologically functional fragment" (or "fragment") of an antigen-binding domain is a species of antigen-binding domain that lacks at least a portion of the amino acids present in the full-length chain, but contains a portion of an antibody (regardless of how that portion is obtained or synthesized) that is capable of specifically binding to a target antigen. Such fragments are biologically active in that they bind to the target antigen and can compete with other antigen-binding domains, including intact antibodies, for binding to a given epitope or mimotope.

[0143] Immunologically functional immunoglobulin fragments include, but are not limited to, scFv fragments, Fab fragments (Fab', F(ab')2, etc.), one or more complementarity determining regions ("CDRs"), diabodies (a heavy chain variable domain on the same polypeptide with a light chain variable domain linked via a short peptide linker that is too short to allow pairing between the two domains on the same chain), domain antibodies, bivalent antigen-binding domains (comprising two antigen-binding sites), multispecific antigen-binding domains, and single-chain antibodies. These fragments may be derived from any mammalian source, including, but not limited to, human, mouse, rat, camelid, or rabbit. As will be appreciated by those skilled in the art, the antigen-binding domain may comprise non-proteinaceous components.

[0144] Variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions (CDRs). The CDRs from the two chains of each pair are typically aligned by the framework regions, which allows binding to a specific epitope or mimotope. From the N-terminus to the C-terminus, both light chain variable regions and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. By convention, the CDR regions in the heavy chain are typically referred to as HC CDR1, CDR2, and CDR3. The CDR regions in the light chain are typically referred to as LC CDR1, CDR2, and CDR3.

[0145] In some embodiments, an antigen-binding domain comprises one or more complementary binding regions (CDRs) present in a full-length light or heavy chain of an antibody, and in some embodiments comprises a single heavy and / or light chain or a portion thereof. These fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the antigen-binding domain comprising an intact antibody.

[0146] In some embodiments, the antigen-binding domain is an antibody or fragment thereof and comprises one or more of its complementarity determining regions (CDRs). In some embodiments, the antigen-binding domain is a single chain variable fragment (scFv) comprising light chain CDRs CDR1, CDR2, and CDR3, and heavy chain CDRs CDR1, CDR2, and CDR3.

[0147] The assignment of amino acids to each of the framework, CDR, and variable domains is typically based on the Kabat numbering (see, e.g., Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda Md. 1991), Chothia numbering (see, e.g., Chothia & Lesk, (1987), J Mol Biol 196:901-917; Al-Lazikani et al., (1997) J Mol Biol 273:927-948; Chothia et al., (1992) J Mol Biol 227:799-817; Tramontano et al., (1990) J Mol Biol 215(1):175-82, and U.S. Pat. No. 7,709,226), contact numbering, or the AbM scheme (Antibody Modeling program, Oxford Molecular.

[0148] In some embodiments, the antigen-binding domain is a recombinant antigen receptor. As used herein, the term "recombinant antigen receptor" refers broadly to a non-naturally occurring surface receptor that includes an extracellular antigen-binding domain or an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the recombinant antigen receptor is a chimeric antigen receptor (CAR). Chimeric antigen receptors (CARs) are well known in the art. A CAR is a fusion protein that includes an extracellular domain that includes an antigen-recognizing portion (also referred to herein as an antigen-binding domain), a transmembrane domain, and an intracellular domain that includes one or more T cell activation domains (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724 (1993), and Sadelain et al., Curr. Opin. Immunol, 21(2):215-223 (2009)). The hinge region or domain is typically located between the antigen recognition domain and the transmembrane domain of the CAR.

[0149] In some embodiments, the intracellular domain of the recombinant antigen receptor comprises a costimulatory domain and / or an ITAM-containing domain, hi some embodiments, the intracellular domain of the recombinant antigen receptor comprises an intracellular protein or a functional variant thereof (e.g., truncation, insertion, deletion, or substitution).

[0150] As used herein, the term "extracellular ligand-binding domain" or "extracellular antigen-binding domain" refers to a polypeptide capable of binding to a ligand or antigen or interacting with a cell surface molecule such as a ligand or surface antigen. For example, the extracellular ligand-binding domain or antigen-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state, e.g., a tumor-specific antigen. In some embodiments, the antigen-binding domain comprises an antibody, or an antigen-binding fragment or portion of an antibody. In some embodiments, the antigen-binding domain comprises an Fv or scFv, a Fab or scFab, a F(ab')2 or scF(ab')2, an Fd, a monobody, an affibody, a camelid antibody, a VHH antibody, a single domain antibody, or a DARPin. In some embodiments, the ligand-binding domain comprises a ligand that binds to a surface receptor, or a binding pair partner such as an ectodomain of a surface receptor that binds to the ligand.

[0151] The terms "stalk domain" and "hinge domain" are used interchangeably herein to refer to any polypeptide that functions to link a transmembrane domain to an extracellular ligand-binding domain. In particular, the stalk domain is used to provide additional flexibility and accessibility to the extracellular ligand-binding domain.

[0152] The term "intracellular signaling domain" refers to the portion of a protein that transduces effector functional signals and induces the cell to carry out a specialized function.

[0153] vector Expression vectors and administration of the polynucleotide compositions are further described herein.

[0154] In another aspect, the disclosure provides a method of making any of the polynucleotides described herein.

[0155] Polynucleotides complementary to any such sequences are also encompassed by the present disclosure. Polynucleotides may be single-stranded (coding or antisense) or double-stranded and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond one-to-one to DNA molecules, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of the present disclosure, and polynucleotides may, but need not be, linked to other molecules and / or supporting materials.

[0156] The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or a portion thereof) or may comprise a variant of such a sequence. A polynucleotide variant contains one or more substitutions, additions, deletions, and / or insertions such that the immunoreactivity of the encoded polypeptide is not reduced compared to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be assessed as described herein. A variant preferably exhibits at least about 70% identity, more preferably at least about 80% identity, even more preferably at least about 90% identity, and most preferably at least about 95% identity to a polynucleotide sequence encoding a native antibody or a portion thereof. Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences are aligned for maximum correspondence as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20, usually 30 to about 75, or 40 to about 50 contiguous positions, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0157] Optimal alignment of sequences for comparison can be performed using the Megalign program of the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wl) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins-Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5: 151-153; Myers, EW and Muller W., 1988, CABIOS 4:11-17, Robinson, ED, 1971, Comb. Theor. 11:105, Santou, N., Nes, M., 1987, Mol. Biol. Francisco, CA, Wilbur, W Jand Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

[0158] Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain up to 20 percent, typically 5-15 percent, or 10-12 percent additions or deletions (i.e., gaps) compared to the reference sequence (not including additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0159] Variants may also, or alternatively, be substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[0160] Preferred "moderately stringent conditions" include a prewash in a solution of 5x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization overnight in 5x SSC at 50°C to 65°C, followed by two washes for 20 minutes each in 2x, 0.5x, and 0.2x SSC containing 0.1% SDS at 65°C.

[0161] As used herein, "highly stringent conditions" or "high stringency conditions" refers to (1) conditions employing low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50° C., (2) conditions employing no denaturing agent such as formamide during hybridization, e.g., 50% (v / v) formamide in 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) with 750 mM sodium chloride, 75 mM sodium citrate, and (3) conditions employing no denaturing agent such as formamide during hybridization, e.g., 50% (v / v) formamide in 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) with 750 mM sodium chloride, 75 mM sodium citrate, and (4) conditions employing no denaturing agent such as formamide during hybridization, e.g., 50% (v / v) formamide in 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) with 750 mM sodium chloride, 75 mM sodium citrate, and (5) conditions employing no denaturing agent such as formamide during hybridization. or (3) conditions employing 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg / m), 0.1% SDS, and 10% dextran sulfate at 42° C. with washes in 0.2×SSC (sodium chloride / sodium citrate) at 42° C. and 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1×SSC containing EDTA at 55° C. One of skill in the art will know how to adjust temperature, ionic strength, etc. as necessary to accommodate factors such as probe length.

[0162] It will be understood by those skilled in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that code for the polypeptides described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that differ due to differences in codon usage are specifically contemplated by this disclosure. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of this disclosure. An allele is an endogenous gene that is altered as a result of one or more mutations, such as deletion, addition, and / or substitution of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles can be identified using standard techniques, such as hybridization, amplification, and / or database sequence comparison.

[0163] The polynucleotide of the present disclosure can be obtained by chemical synthesis, recombinant methods, or PCR.Methods of chemical polynucleotide synthesis are well known in the art and do not need to be described in detail herein.Those skilled in the art can use the sequences provided herein and commercially available DNA synthesis equipment to produce desired DNA sequences.

[0164] To prepare a polynucleotide using recombinant methods, as further discussed herein, a polynucleotide containing a desired sequence can be inserted into a suitable vector, and the vector can be introduced into a suitable host cell for replication and amplification. The polynucleotide can be inserted into the host cell by any means known in the art. The cell is transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating, or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or can be integrated into the host cell genome. The polynucleotide thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

[0165] Alternatively, PCR allows the reproduction of DNA sequences. PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and in PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

[0166] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can be isolated using methods well known to those skilled in the art, for example, as described in Sambrook et al., 1989 (see above).

[0167] A suitable cloning vector may be constructed according to standard techniques or may be selected from a large number of cloning vectors available in the art. The cloning vector selected may vary depending on the host cell intended to be used, but useful cloning vectors generally have the ability to replicate autonomously, may have a single target for a specific restriction endonuclease, and / or may carry a marker gene that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

[0168] An expression vector is generally a replicable polynucleotide construct containing a polynucleotide according to the present disclosure. It is implied that an expression vector must be replicable in a host cell, either as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vectors disclosed in PCT Publication No. WO87 / 04462. Vector components generally may include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, suitable transcription control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements are also usually required, such as a ribosome binding site, a translation initiation site, and a stop codon.

[0169] A vector containing a polynucleotide of interest can be introduced into a host cell by any of a number of suitable means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other agents, microprojectile bombardment, lipofection, and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of introduction vector or polynucleotide often depends on the characteristics of the host cell.

[0170] A polynucleotide encoding a protease-activated CD45-gated CAR, its extracellular domain, or another fragment of a protease-activated CD45-gated CAR disclosed herein can be present in an expression cassette or expression vector (e.g., a plasmid for introduction into a bacterial host cell, or a viral vector such as a baculovirus vector for transfection of an insect host cell, or a plasmid or viral vector such as a lentivirus for transfection of a mammalian host cell). In some embodiments, the polynucleotide or vector can include a nucleic acid sequence encoding a ribosomal skipping sequence, such as, but not limited to, a sequence encoding a 2A peptide. The 2A peptide, identified in the aphthovirus subgroup of picornaviruses, causes a ribosomal "skip" from one codon to the next without the formation of a peptide bond between the two amino acids encoded by the codons (see (Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008)). "Codon" refers to three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated into one amino acid residue by a ribosome. Thus, two polypeptides can be synthesized from a single adjacent open reading frame in an mRNA if the polypeptides are separated by a 2A oligopeptide sequence that is in frame. Such ribosome skipping mechanisms are well known in the art and are known to be used by some vectors for the expression of several proteins encoded by a single messenger RNA.

[0171] To direct the transmembrane polypeptide into the secretory pathway of the host cell, in some embodiments, a secretory signal sequence (also known as a leader sequence, prepro sequence, or pre sequence) is provided in the polynucleotide sequence or vector sequence. The secretory signal sequence is operably linked to the transmembrane nucleic acid sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are generally positioned 5' to the nucleic acid sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleic acid sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830). Those skilled in the art will recognize that considerable sequence variation is possible among these polynucleotide molecules, given the degeneracy of the genetic code. In some embodiments, the nucleic acid sequences of the present disclosure are codon-optimized for expression in mammalian cells, preferably for expression in human cells. Codon optimization refers to the replacement of a target sequence of codons that are generally rare in highly expressed genes of a given species with codons that are generally frequent in highly expressed genes of such species, such codons encoding the same amino acids as the codons being replaced.

[0172] Provided herein is a method for preparing immune cells for use in immunotherapy. In some embodiments, the method comprises introducing a protease-activated CD45-gated CAR into an immune cell and expanding the cell. In some embodiments, the present disclosure relates to a method for engineering an immune cell, the method comprises providing an immune cell and expressing at least one protease-activated CD45-gated CAR on the surface of the cell. In some embodiments, the method comprises transfecting the cell with at least one polynucleotide encoding a protease-activated CD45-gated CAR and expressing the at least one polynucleotide in the cell.

[0173] In some embodiments, the polynucleotide encoding the protease-activated CD45-gated CAR is present in an expression vector for stable expression in a cell. In some embodiments, the polynucleotide is present in a viral vector for stable expression in a cell. In some embodiments, the viral vector may be, for example, a lentiviral vector or an adenoviral vector.

[0174] In some embodiments, the polynucleotide encoding the polypeptide according to the present disclosure can be, for example, mRNA that is directly introduced into cells by electroporation. In some embodiments, cytoPulse technology can be used to transiently permeabilize live cells to deliver substances into cells. Parameters can be modified to determine conditions for high transfection efficiency with minimal mortality.

[0175] Also provided herein are methods of transfecting immune cells, e.g., T cells. In some embodiments, the methods include contacting T cells with RNA and applying to the T cells an agile pulse sequence consisting of: (a) an electric pulse having a voltage range of about 2250-3000 V / centimeter, (b) a pulse width of 0.1 ms, (c) a pulse interval of about 0.2-10 ms between the electric pulses of steps (a) and (b), (d) an electric pulse having a voltage range of about 2250-3000 V / centimeter, with a pulse width of about 100 ms and a pulse interval of about 100 ms between the electric pulse of step (b) and the first electric pulse of step (c), and (e) four electric pulses having a voltage of about 325 V, with a pulse width of about 0.2 ms and a pulse interval of 2 ms between each of the four electric pulses. In some embodiments, a method of transfecting a T cell includes contacting the T cell with RNA and administering to the T cell (a) an electrical pulse having a voltage of about 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900, or 3000 V / centimeter, (b) a pulse width of 0.1 ms, (c) and a pulse interval of about 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 between the electrical pulses of steps (a) and (b), (d) a pulse width of about 100 ms, and the electrical pulse of step (b) and applying an agile pulse sequence consisting of an electric pulse having a voltage range of about 2250-3000 V / centimeter, for example, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900, or 3000 V / centimeter, with a pulse interval of about 100 ms between the first electric pulse of step (c) and the second electric pulse of step (d), and (e) four electric pulses having a voltage of about 325 V, with a pulse width of about 0.2 ms, and a pulse interval of about 2 ms between each of the four electric pulses. Any value falling within the above value range is disclosed in the present application. The electroporation medium can be any suitable medium known in the art.In some embodiments, the electroporation medium has a conductivity ranging from about 0.01 to about 1.0 millisiemens.

[0176] In some embodiments, the method may further comprise a step of genetically modifying the cell by inactivating or reducing the expression level of at least one gene expressing, for example, but not limited to, a component of the TCR, a target of an immunosuppressant, an HLA gene, and / or an immune checkpoint protein, such as, for example, PDCD1 or CTLA-4. By inactivating a gene, it is intended that the gene of interest is not expressed in the form of a functional protein. In some embodiments, the inactivated gene is selected from the group consisting of, for example, but not limited to, TCRα, TCRβ, β2-microglobulin ("β2m"), CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4. In some embodiments, the method comprises inactivating or reducing the expression level of one or more genes by introducing into the cell a rare-cutting endonuclease capable of selectively inactivating the gene by selective DNA cleavage. In some embodiments, the infrequently-cutting endonuclease can be, for example, a transcription activator-like effector nuclease (TALE-nuclease or TALEN), or a MegaTAL nuclease, or a Cas9 endonuclease.

[0177] In another aspect, the step of genetically modifying immune cells, e.g., T cells, can include modifying immune cells, e.g., T cells, by inactivating at least one gene expressing a target of an immunosuppressant, and optionally growing the cells in the presence of an immunosuppressant. Immunosuppressants are drugs that suppress immune function by one of several mechanisms of action. Immunosuppressants can reduce the extent and / or voracity of an immune response. Non-limiting examples of immunosuppressants include calcineurin inhibitors, target of rapamycin, interleukin-2 alpha chain blockers, inhibitors of inosine monophosphate dehydrogenase, inhibitors of dihydrofolate reductase, corticosteroids, and immunosuppressant antimetabolites. Some cytotoxic immunosuppressants act by inhibiting DNA synthesis. Other cytotoxic immunosuppressants may act via activation of T cells or by inhibiting activation of helper cells. The method according to the present disclosure allows for conferring immunosuppression resistance to T cells for immunotherapy, e.g., by inactivating the target of the immunosuppressant in the T cells. As non-limiting examples, the target of an immunosuppressant may be a receptor for the immunosuppressant, such as, but not limited to, CD52, glucocorticoid receptor (GR), FKBP family gene members, and cyclophilin family gene members.

[0178] Provided herein are compositions and methods for expressing protease-activated CD45-gated CARs. Also provided are uses of such compositions and methods for improving the functional activity of immune cells, e.g., T cells, such as CAR-T cells. The methods and compositions provided herein are useful for improving the activation specificity and therapeutic efficacy of immune cells, e.g., T cells, such as CAR-T cells.

[0179] The immune cells provided herein, e.g., T cells, express a protease-activated CD45-gated CAR disclosed herein. Advantageously, the immune cells provided herein exhibit improved in vivo activation specificity, e.g., compared to cells expressing a non-gated CAR that expresses the same or equivalent CAR, except that it lacks a functional protease cleavage site.

[0180] Protease-activated CD45-gated CAR In one aspect, provided herein is a protease-activated CD45-gated chimeric antigen receptor (CD45-gated CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises: a CD45 recruitment domain; An antigen-binding domain; a linker connecting the carboxy terminus of the CD45 recruitment domain to the amino terminus of the antigen binding domain, the linker comprising at least one protease cleavage site recognized by at least one protease; In addition, the intracellular domain contains at least one signaling domain that can be reversibly inactivated by CD45.

[0181] In some embodiments, the extracellular domain comprises a stalk domain that connects the extracellular domain to the transmembrane domain.

[0182] In some embodiments, the intracellular signaling domain comprises an activation domain. In some embodiments, the intracellular signaling domain comprises a costimulatory domain. In some embodiments, the intracellular signaling domain comprises an activation domain and a costimulatory domain. In some embodiments, the intracellular signaling domain comprises an activation domain, such as an ITAM-containing domain. In some embodiments, the intracellular signaling domain comprises a CD3 zeta intracellular domain. In some embodiments, the intracellular signaling domain comprises a CD3 zeta domain comprising the amino acid sequence of SEQ ID NO: 34, or a fragment thereof.

[0183] In some embodiments, the intracellular signaling domain comprises one, two, or three ITAM domains selected from the group consisting of wild-type or variants of CD3γ ITAM, CD3δ ITAM, CD3ε ITAM, CD3ζ1 ITAM, CD3ζ2 ITAM, and CD3ζ3 ITAM, with the proviso that when three ITAM domains are selected that are wild-type ITAM domains, the three ITAM domains are not CD3ζ1, CD3ζ2, and CD3ζ3. See, e.g., U.S. Provisional Patent Application No. 63 / 054701, which is incorporated herein by reference in its entirety.

[0184] In some embodiments, the intracellular domain comprises or further comprises at least one costimulatory domain. In some embodiments, the at least one costimulatory domain comprises at least one of the following: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function associated antigen-1 (LFA-1 (CD1 la / CD 18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, I CAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 Ia, LFA-1, ITGAM, CD1 Ib, ITGAX, CD1 Ic, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The signal transduction domains are ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0185] In some embodiments, the CD45 recruitment domain comprises one or more of an anti-CD45 antibody antigen binding domain, an anti-CD45 nanobody, an anti-CD45 scFv, a truncated viral protein binder of CD45, an anti-CD45 Fab, an anti-CD45 camelid VHH, a CD45 binding protein, or a truncated endogenous CD45 binder. In some embodiments, the CD45 recruitment domain comprises one or more of a truncated UL11, a truncated sec49K, and a truncated BTN3A1.

[0186] In some embodiments, the linker comprises one protease cleavage site. In some embodiments, the protease cleavage site comprises any of the amino acid sequences of SEQ ID NO: 32, as well as SEQ ID NO: 89-102 (Table 2) (which when incorporated into the linker comprise (e.g., SEQ ID NO: 99-102) or can be modified to comprise (e.g., any of SEQ ID NO: 89-98) a GS linker at the amino and / or carboxy terminus). In some embodiments, the linker comprises multiple protease cleavage sites, e.g., 2, 3, 4, 5, 6, or more protease cleavage sites, e.g., up to 10 or up to 15 protease cleavage sites, each cleavage site being the same or different from the other cleavage sites. In some embodiments, the linker is or comprises any one or more of the amino acid sequences of SEQ ID NO: 99-102, 172-177, and 108-120 (e.g., 2, 3, 4, 5 copies thereof). In some embodiments, the linker comprises one copy or two or more copies (e.g., 2, 3, 4, 5, 6, 10, or 15) of the amino acid sequence of any of SEQ ID NOs: 89-98, 103, and 104.

[0187] In some embodiments, the protease cleavage site, or one or more of the multiple protease cleavage sites, is recognized by one or more of thrombin, trypsin, plasmin, prostate specific antigen (PSA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase (MMP), matriptase (MT-SP1), legumain, a disintegrin and metalloproteinase (ADAM), and transmembrane serine protease (TMPRSS). Stated differently, in some embodiments, the protease that recognizes the protease cleavage site or recognizes one or more of a plurality of protease cleavage sites is selected from the group consisting of thrombin, trypsin, plasmin, prostate specific antigen (PSA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase (MMP), matriptase (MT-SP1), legumain, a disintegrin and metalloproteinase (ADAM), transmembrane serine protease (TMPRSS), granzyme B, activator protein C, caspases ... The protease cleavage site is one or more of a protease, a cathepsin, a chymase, an elastase, a guanidinobenzoatase, HtrAl, a human neutrophil elastase, a lactoferrin, a marapsin, an NS3 / 4A, a PACE4, a tissue plasminogen activator (tPA), a DESC1, a DPP-4, a FAP, a hepsin, a matriptase-2, a secretase, a kallikrein-related peptidase (KLK), and a tryptase, or a serine protease, a cysteine-type lysosomal protease, a metalloproteinase, a coagulation factor protease, or an aspartyl-type lysosomal protease. In some embodiments, the protease cleavage site comprises an amino acid sequence that is cleaved by a protease or type of protease listed herein. In some embodiments, the protease cleavage site, or one or more of the multiple protease cleavage sites, is recognized by an endogenous protease.In some embodiments, the protease that recognizes the protease cleavage site or that recognizes one or more of the multiple protease cleavage sites is an endogenous protease.

[0188] In some embodiments, the protease cleavage site, or one or more of the plurality of protease cleavage sites, is recognized by one or more of a serine protease, a cysteine-type lysosomal protease, an aspartyl-type lysosomal protease, and a metalloproteinase. In some embodiments, the protease that recognizes the protease cleavage site, or recognizes one or more of the plurality of protease cleavage sites, is one or more of a serine protease, a cysteine-type lysosomal protease, an aspartyl-type lysosomal protease, and a metalloproteinase.

[0189] In some embodiments, the linker comprises an amino GS peptide, a peptide comprising a protease cleavage site, and a carboxy GS peptide. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 9, which comprises an amino GS peptide (SEQ ID NO: 52), a TPS cleavage peptide comprising a protease cleavage site (SEQ ID NO: 53), and a carboxy GS peptide (SEQ ID NO: 54). In some embodiments, the linker comprises the same amino and carboxy GS peptides, and different cleavage peptides comprising the same or (LHF) different protease cleavage sites. Information regarding additional exemplary linkers is set forth in the table below, which shows the linker name, SEQ ID NO:, the protease cleavage site each linker comprises, and their corresponding SEQ ID NO:, as well as the length (in amino acids ("aa")) of each linker. For example, the linker TPS4 45aa (SEQ ID NO: 107) comprises an MMP cleavage site having the amino acid sequence of SEQ ID NO: 93, and a matriptase cleavage site having the amino acid sequence of SEQ ID NO: 91. As shown in Table 2, TPS4 45aa (SEQ ID NO: 107) further contains GS peptides at the amino and carboxy termini. Additional linker information can be found, for example, in M. Geiger et al., Protease-activation using anti-idiotypic masks enables tumor specificity of a folate receptor 1-T cell bispecific antibody, Nature Commun 11:3196 (2020), EJ Kwon et al., Ultrasensitive tumour-penetrating nanosensors of protease activity, Nature Biomed Eng 1:0054 (2017), WO2016 / 118629Al, US20170204139A1, WO2013 / 130683A2, US20130165389A1. [Table 1] [Table 2]

[0190] In some embodiments, the antigen binding domain is selected from the group consisting of BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUC1, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, MHC-NY-ESO1, HER2 (ERBB2), CAIX (carbonic anhydrase IX), LIV1, ADAM10, CHRNA2, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, claudin-18.2 (claudin It specifically binds to claudin-18A2, or claudin 18 isoform 2), PSCA, DLL3 (delta-like protein 3, Drosophila delta homolog 3, delta 3), Mud7 (mucin 17, Muc3, Muc3), FAP alpha (fibroblast activation protein alpha), Ly6G6D (lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGT1, NG25), PSMA, MSLN, or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43).CARs and / or antibodies targeting antigens are disclosed, for example, in: BCMA - WO2016 / 16630, WO2020 / 150339, WO2019 / 196713, WO2016 / 014565, WO2017 / 025038; MUC16: US9,169,328, WO2016 / 149368, WO2020 / 023888; EGFRvIII: WO2017 / 125830, WO2016 / 016341; Flt3: WO2 018 / 222935, WO2020 / 010284, WO2017 / 173410;CD20:WO2018 / 145649, WO2020 / 010235, WO2020 / 123691;CD38:WO2017 / 02 5323;CD70:WO2019 / 152742, WO2018 / 152181;CD33:WO2016 / 014576;CD133:WO2018 / 072025;CS1:WO2019 / 030240;ROR1:W O2016 / 115559;CD19:WO2002 / 077029, US11,077,144;Claudin:WO2018 / 006882, WO2021 / 008463;DLL3:WO2020 / 180591;W T1:US2016 / 0152725A1, US7622119B2;CD23:US6011138A,CN1568198A;CD30:US10815301B2,US10808035B2;PRAME:US20 18 / 0148503A1, WO2020 / 186204A1; LIV1:US2020 / 0231699A1; NKG2D:WO2021 / 179353A1, US2021 / 0269501A1; FAP alpha:US2020 / 0246383A1, US2021 / 0115102A1; PSMA:US2021 / 0277141A1, WO2020 / 108646A1; MSLN:CN109680002A, CN109628492A.

[0191] In some embodiments, the antigen binding domain specifically binds to MUC16. In some embodiments, the antigen binding domain specifically binds to MUC16 and the protease-activated CD45-gated CAR comprises any of the amino acid sequences of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170, with or without a signal sequence. In some embodiments, the antigen binding domain specifically binds to MUC16 and comprises the amino acid sequence of SEQ ID NO: 75 or 76, with or without a signal sequence. In some embodiments, the antigen binding domain specifically binds to an antigen expressed by a particular type of tumor, and the protease cleavage site can be recognized and cleaved by a protease expressed by the same type of tumor. In some embodiments, the protease-activated CD45-gated CAR comprises a CD45-CAR linker that (1) can specifically recognize and bind to an antigen expressed by the tumor, and (2) can be cleaved by a protease secreted by the tumor, and / or a protease present or active in the tumor microenvironment.

[0192] In some embodiments, the antigen binding domain specifically binds to a breast cancer tumor antigen or a colorectal cancer tumor antigen, and one or more of the at least one protease cleavage sites can be recognized and cleaved by uPA. In some embodiments, the antigen binding domain of a protease-activated CD45-gated CAR specifically recognizes and binds to an antigen characteristic of any of cervical cancer, breast cancer, ovarian cancer, and colorectal cancer, and at least one of its protease cleavage sites can be recognized and cleaved by MMP-2 and / or MMP-9. In some embodiments, the antigen binding domain of a protease-activated CD45-gated CAR specifically recognizes and binds to an antigen characteristic of any of breast cancer and ovarian cancer, and at least one of its protease cleavage sites can be recognized and cleaved by matriptase.

[0193] In some embodiments, the protease-activated CD45-gated CAR comprises a signal sequence. In some embodiments, the signal sequence is a CD8 signal sequence. In some embodiments, the signal sequence comprises the amino acid sequence of SEQ ID NO: 1 or 44.

[0194] Also provided herein is a CAR that specifically recognizes and binds to MUC16 (also known as CA125). In one embodiment, the anti-MUC16 CAR comprises the amino acid sequence of any of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170, or a variant thereof that does not contain the signal sequence, or a conservative variant thereof.

[0195] MUC16 has been found to be an overexpressed antigen in several cancers, including ovarian, breast, pancreatic, non-small cell lung cancer, mass-forming intrahepatic cholangiocarcinoma, cervical adenocarcinoma, and gastrointestinal adenocarcinoma. See, e.g., Haridas et al., FASEB J. 28:4183-99 (2014). Thus, the disclosure provides, in some embodiments, methods of treating any of ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, mass-forming intrahepatic cholangiocarcinoma, cervical adenocarcinoma, and gastrointestinal adenocarcinoma, the methods comprising administering to a patient having the condition one or more engineered immune cells that functionally express a MUC16-specific CAR disclosed herein, e.g., comprising any of the amino acid sequences of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170, and variants thereof as described herein, or that functionally express a protease-activated CD45-gated MUC16-specific CAR disclosed herein (e.g., comprising the amino acid sequence of SEQ ID NOs: 11, 16, 18, 20-31, 56, 75, 76, 77, 78, 79, or 121-170, or variants thereof).

[0196] In various embodiments, the protease-activated CD45-gated CAR proteins disclosed herein comprise, for example, any one or more of the amino acid sequences of SEQ ID NOs: 1-178, and conservative variants thereof. In certain embodiments, the protease-activated CD45-gated CAR comprises an amino acid sequence that comprises at least 70%, such as at least 80%, or at least 90%, 95%, 97%, or 99% sequence identity to any one or more of SEQ ID NOs: 1-178.

[0197] In some embodiments, the protease-activated CD45-gated CARs disclosed herein comprise any one of the amino acid sequences of SEQ ID NOs: 11, 16, 17 (control sequence containing no protease cleavage site), 18, 19 (control sequence containing no protease cleavage site), 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 56, 75, 76, 77, 78, 79, and 121-170. Some of these sequences contain one or both of the HA (tag) (SEQ ID NO: 2) and the V5 peptide motif (SEQ ID NO: 55). In some embodiments of the protease-activated CD45-gated CARs of the present disclosure, either or both of the HA (tag) and the V5 peptide motif, and / or the signal peptide are excluded from the protease-activated CD45-gated CAR. Thus, the present disclosure provides protease-activated CD45-gated CARs comprising variants of any one of the amino acid sequences of SEQ ID NOs: 11, 16-31, 56, 75-79, and 121-170, the variants excluding (not including) the amino acid sequences of the HA tag of SEQ ID NO: 2, and / or the amino acid sequence of the V5 peptide motif of SEQ ID NO: 55, and / or the amino acid sequence of the CD8ss SEQ ID NO: 1, or at least 70%, e.g., at least 80%, or at least 90%, 95%, 97%, 99%, or 100% sequence identity thereto. Some embodiments of the protease-activated CD45-gated CARs disclosed herein contain one, two, or all three of the HA(tag) (SEQ ID NO: 2), the V5 peptide motif (SEQ ID NO: 55), and the CD8 signal sequence (SEQ ID NO: 1).

[0198] In some embodiments, the protease-activated CD45-gated CAR comprises a CD45 recruitment domain, a CAR, and a linker connecting the carboxy terminus of the CD45 recruitment domain to the amino terminus of the antigen-binding domain of the CAR. In some embodiments, the protease-activated CD45-gated CAR comprises a CD45 recruitment domain, a CAR, and a linker connecting the carboxy terminus of the CD45 recruitment domain to the amino terminus of the antigen-binding domain of the CAR, wherein the CAR comprises the amino acid sequence of SEQ ID NO:11.

[0199] In some embodiments, the protease-activated CD45-gated CAR extracellular CD45 recruitment domain comprises the amino acid sequence of SEQ ID NO: 3, 4, 12, 13, 14, or 15.

[0200] In some embodiments, the protease-activated CD45-gated CAR extracellular CD45 recruitment domain comprises the amino acid sequence of SEQ ID NO: 5 or 6 together with a protease-cleavable linker.

[0201] In some embodiments, the protease-activated CD45-gated CAR extracellular protease-cleavable linker comprises a protease cleavage site having the amino acid sequence of SEQ ID NO: 8, 9, or 10, and / or the amino acid sequence of SEQ ID NO: 32 (thrombin cleavage site) or 53 (TPS cleavage site), and / or either or both of SEQ ID NO: 52 or 54 (sequences adjacent to the cleavage site). In some embodiments, the protease-activated CD45-gated CAR comprises a non-cleavable linker, and in some embodiments the non-cleavable linker comprises the amino acid sequence of SEQ ID NO: 7.

[0202] In some embodiments, the protease-activated CD45-gated CAR extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO:56.

[0203] In some embodiments, the protease-activated CD45-gated CAR transmembrane domain comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the transmembrane and intracellular domains of the CAR comprise the amino acid sequence of SEQ ID NO: 41.

[0204] In some embodiments, the protease-activated CD45-gated CAR intracellular domain comprises one or more of the amino acid sequences of SEQ ID NOs: 33, 40, and 43.

[0205] In some embodiments, the protease-activated CD45 gated CAR hinge domain comprises the amino acid sequence of SEQ ID NO: 36, 37, or 38.

[0206] In some embodiments, the protease-activated CD45-gated CAR disclosed herein comprises an N-terminal signal sequence. In some embodiments, the signal sequence has the amino acid sequence of SEQ ID NO: 1 or 44. In some embodiments, the nucleic acid encoding the protease-activated CD45-gated CAR encodes a signal sequence, which is removed during processing in the cell, leaving the remaining portion of the encoded amino acid sequence in the mature form of the protein. This mature form of the protease-activated CD45-gated CAR is within the scope of the present disclosure. In some embodiments, the protease-activated CD45-gated CAR disclosed herein does not comprise an N-terminal signal sequence.

[0207] In another aspect, provided herein is a nucleic acid encoding the extracellular domain of a protease-activated CD45-gated CAR disclosed herein. In another aspect, provided herein is a nucleic acid encoding a protease-activated CD45-gated CAR disclosed herein. In certain embodiments, the nucleic acid of the present disclosure encodes a protease-activated CD45-gated CAR comprising an amino acid sequence comprising at least 70%, such as at least 80%, or at least 90%, 95%, 97%, or 99% sequence identity with one or more of the amino acid sequences of SEQ ID NOs: 1-56. In certain embodiments, the nucleic acids of the disclosure encode a protease-activated CD45-gated CAR comprising an amino acid sequence that comprises at least 70%, e.g., at least 80%, or at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequences of SEQ ID NOs: 11, 16, 17 (control sequence that does not contain a protease cleavage site), 18, 19 (control sequence that does not contain a protease cleavage site), 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 56, 75, 76, 77, 78, 79, and 121-170, or a variant thereof that does not include an HA tag and / or a V5 sequence.

[0208] Also provided herein are nucleic acids encoding any of the amino acid sequences of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170. Also provided herein are nucleic acids encoding the amino acid sequence of SEQ ID NO: 56. In certain embodiments, the nucleic acids of the present disclosure encode an anti-MUC16 CAR comprising an amino acid sequence that comprises at least 70%, e.g., at least 80%, or at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170.

[0209] In another aspect, provided herein is a vector comprising the nucleic acid disclosed herein.In one embodiment, the vector is an expression vector.In one embodiment, the vector is a viral vector, a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0210] In a further aspect, provided herein is an engineered cell, e.g., an engineered immune cell, comprising a protease-activated CD45-gated CAR as disclosed herein. In a further aspect, provided herein is an engineered immune cell comprising a nucleic acid as disclosed herein. In another aspect, provided herein is an engineered immune cell comprising a vector as disclosed herein.

[0211] In another aspect, an immune cell, e.g., a T cell, of the disclosure expresses a polypeptide that consists of or includes one or more amino acid sequences, e.g., listed in Table 2. In some embodiments, an immune cell, e.g., a T cell, of the disclosure expresses a nucleic acid encoding one or more amino acid sequences, e.g., listed in Table 2.

[0212] In various embodiments, any of the engineered cells, e.g., any of the engineered immune cells disclosed herein, functionally express a protease-activated CD45-gated CAR disclosed herein. In various embodiments, any of the engineered cells, e.g., any of the engineered immune cells disclosed herein, functionally express a protease-activated CD45-gated CAR comprising the amino acid sequence of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170, or a variant thereof lacking an N-terminal signal sequence. Also provided herein are engineered cells, e.g., engineered immune cells, that functionally express a CAR comprising the amino acid sequence of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170, or a variant thereof lacking an N-terminal signal sequence. In various embodiments, any of the engineered cells, e.g., any of the engineered immune cells disclosed herein, are isolated cells.

[0213] In various embodiments, the engineered cells disclosed herein, e.g., engineered immune cells, functionally express a protease-activated CD45-gated CAR disclosed herein from a nucleic acid encoding the protease-activated CD45-gated CAR. In various embodiments, the engineered cells disclosed herein, e.g., engineered immune cells, functionally express CD45, e.g., functionally express the cell's endogenous CD45 gene, or functionally express CD45 from an exogenous nucleic acid encoding CD45 and introduced into the cell.

[0214] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

[0215] MUC16-specific CAR In another aspect, provided herein is a CAR that specifically binds to MUC16, wherein the CAR comprises an extracellular ligand binding domain comprising a VH region having the sequence set forth in SEQ ID NO: 58, and / or a VL region having the sequence set forth in SEQ ID NO: 59. In some embodiments, the VH and VL are linked to each other by a flexible linker. In some embodiments, the flexible linker comprises the amino acid sequence set forth in SEQ ID NO: 57 ((GGGGS)3).

[0216] In another aspect, a MUC16-specific chimeric antigen receptor (CAR) is provided comprising an extracellular ligand-binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises a single chain Fv fragment (scFv) comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises (i) a VH complementarity determining region 1 (CDR1) having the amino acid sequence set forth in SEQ ID NO: 60 or 63, (ii) a VH complementarity determining region 2 (CDR2) having the amino acid sequence set forth in SEQ ID NO: 61 or 64, and (iii) a VH complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO: 62 or 65.

[0217] In another aspect, a MUC16-specific chimeric antigen receptor (CAR) is provided comprising an extracellular ligand-binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises a single chain Fv fragment (scFv) comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VL region comprises (i) a VL complementarity determining region 1 (CDR1) having the amino acid sequence set forth in SEQ ID NO: 66 or 69, (ii) a VL complementarity determining region 2 (CDR2) having the amino acid sequence set forth in SEQ ID NO: 67 or 70, and (iii) a VL complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO: 68 or 71.

[0218] In another aspect, a MUC16-specific chimeric antigen receptor (CAR) is provided, comprising an extracellular ligand-binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises a single chain Fv fragment (scFv) comprising a heavy chain variable (VH) region and a light chain variable (VL) region, (a) the VH region has (i) a VH complementarity determining region 1 (CDR1) having the amino acid sequence set forth in SEQ ID NO: 60 or 63, (ii) a VH complementarity determining region 2 (CDR3) having the amino acid sequence set forth in SEQ ID NO: 61 or 64. and (iii) a VH complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO: 62 or 65; and (b) the VL region comprises (i) a VL complementarity determining region 1 (CDR1) having the amino acid sequence set forth in SEQ ID NO: 66, (ii) a VL complementarity determining region 2 (CDR2) having the amino acid sequence set forth in SEQ ID NO: 67, and (iii) a VL complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO: 68.

[0219] Also provided herein are CDR portions of the antigen binding domain of an antibody against MUC16 or the CDR portions of the extracellular ligand binding domain of a CAR against MUC16, including Chothia, Kabat CDRs, and CDR contact regions. Determination of the CDR regions is well within the skill of the art. It is understood that in some embodiments, the CDRs may be a combination of Kabat and Chothia CDRs (also referred to as combined CRs or extended CDRs). In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In other words, in embodiments having two or more CDRs, the CDRs may be either Kabat, Chothia, combined CDRs, or a combination thereof. [Table 4] [Table 5-1] [Table 5-2]

[0220] The present disclosure encompasses modifications to the CARs and polypeptides shown in Table 4, including functionally equivalent CARs with modifications that do not significantly affect their properties, as well as variants with enhanced or decreased activity and / or affinity. For example, amino acid sequences can be mutated to obtain antibodies with desired binding affinity to MUC16. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, polypeptides with one or more deletions or additions of amino acids that do not significantly deleteriously alter the functional activity or mature (enhance) the affinity of the polypeptide for its ligand, or polypeptides using chemical analogs.

[0221] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to an epitope or mimotope tag. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the half-life of the antibody in the blood circulation.

[0222] Substitutional variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. The most interesting sites for substitutional mutagenesis include hypervariable regions, but FR changes are also contemplated. Conservative substitutions are shown under the heading of "conservative substitutions" in Table 6. If such substitutions result in a change in biological activity, more substantial changes, indicated as "exemplary substitutions" in Table 6 or further described below for amino acid classes, can be introduced and the products screened.

[0223] In some embodiments, provided herein is a CAR that specifically binds to MUC16, wherein the CAR comprises a VH region comprising the sequence set forth in SEQ ID NO:58, and / or a VL region comprising the sequence set forth in SEQ ID NO:59.

[0224] In some embodiments, a CAR is provided herein that comprises a CDR portion of an antibody against a MUC16 antibody based on CDR contact regions. A CDR contact region is a region of an antibody that confers specificity to an antigen to the antibody. In general, a CDR contact region includes residue positions in the CDR and Vernier zone that are constrained to maintain the appropriate loop structure for the antibody to bind to a specific antigen. See, for example, Makabe et al., J.Biol.Chem., 283:1156-1166, 2007. Determining CDR contact regions is well within the skill of the art.

[0225] The binding affinity (K) of the ligand-binding domain of the MUC16-specific CAR described herein to MUC16 (such as human MUC16 (e.g., Uniprot accession number: Q8WXI7)). D ) can be, for example, about 0.1 to about 1000 nM, for example, about 0.5 nM to about 500 nM, or for example, about 1 nM to about 250 nM. In some embodiments, the binding affinity is about any of 1000 nm, 750 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 19 nm, 18 nm, 17 nm, 16 nm, 15 nM, 10 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, or 0.1 nM.

[0226] In some embodiments, the binding affinity is less than about any of 1000 nm, 900 nm, 800 nm, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, or 5 nM.

[0227] Monoclonal antibody specific epitopes and mimotopes In some embodiments, the extracellular domain of any one of the MUC16-specific CARs disclosed herein may comprise one or more epitopes or mimotopes specific for (i.e., specifically recognized by) a monoclonal antibody. These epitopes or mimotopes are also referred to herein as mAb-specific epitopes or mimotopes. In these embodiments, the extracellular domain comprises VH and VL polypeptides that specifically bind MUC16 and one or more epitopes or mimotopes that bind one or more monoclonal antibodies (mAbs). CARs that comprise mAb-specific epitopes or mimotopes can be single-chain or multi-chain.

[0228] Inclusion of monoclonal antibody specific epitopes or mimotopes in the extracellular domain of a CAR described herein allows for the selection and depletion of engineered immune cells expressing a CAR. In some embodiments, this function also facilitates the restoration of endogenous MUC16 expressing cells that are depleted by administration of engineered immune cells expressing a CAR.

[0229] Thus, in some embodiments, the present disclosure relates to methods for selecting and / or depleting engineered immune cells that have been gifted with CARs that contain mAb-specific epitopes or mimotopes, as well as methods for promoting the recovery of endogenous MUC16-expressing cells, such as bone marrow progenitor cells.

[0230] Some epitope- or mimotope-monoclonal antibody conjugates can be used to generate CARs that contain monoclonal antibody specific epitopes or mimotopes, particularly CARs that are already approved for medical use, such as, as non-limiting examples, CD20 epitopes or mimotopes / rituximab.

[0231] In some embodiments, monoclonal antibodies specific for the epitope or mimotope may be conjugated with a cytotoxic drug. It is also possible to promote CDC cytotoxicity by using engineered antibodies that are grafted components of the complement system. In some embodiments, the activation of CAR-T cells may be regulated by depleting the cells using antibodies that recognize the epitope or mimotope.

[0232] The disclosure also encompasses methods for selecting engineered immune cells loaded with MUC16-specific CARs expressing mAb-specific epitopes or mimotopes, and therapeutic methods in which activation of engineered immune cells loaded with these CARs is modulated by depleting the cells using antibodies targeting the external ligand-binding domain of the CAR.

[0233] CARs comprising one or more epitopes or mimotopes specifically recognized by monoclonal antibodies are disclosed in WO2016 / 120216, which is incorporated herein by reference in its entirety. The one or more epitopes or mimotopes may be selected from any number of epitopes and mimotopes known in the art. In some embodiments, the one or more epitopes or mimotopes may be targets of monoclonal antibodies approved for medical use, such as, but not limited to, CD20 epitopes or mimotopes recognized by rituximab. In some embodiments, the one or more epitopes or mimotopes comprise any one or more of the amino acid sequences set forth in SEQ ID NOs: 50, 51, and 74.

[0234] In some embodiments, the epitope or mimotope can be located between the scFv and the hinge of the CAR. In some embodiments, two instances of the same epitope or mimotope separated by a linker can be used in the CAR. For example, [Sequence Table 1] As shown in TIFF2024500189000034.tif6106 (SEQ ID NO:51), a polypeptide comprising two copies of the mimotope shown in SEQ ID NO:50 separated by a linker may be used within the CAR located between the light chain variable region and the hinge.

[0235] In some embodiments, the VH and VL polypeptides and the extracellular binding domain of a CAR comprising a mAb-specific epitope or mimotope may have different structures depending on the insertion position of the epitope or mimotope and the use of a linker. For example, the extracellular binding domain of a MUC16-specific CAR comprising a mAb-specific epitope or mimotope may have one of the following structures (the mimotope may replace the epitope in the following structures): V 1- L 1- V 2- (L) x -Epitope 1-(L) x -; V 1- L1- V 2- (L) x -Epitope 1-(L) x -Epitope 2-(L) x -; V 1- L 1- V 2- (L) x -Epitope 1-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -; (L) x -Epitope 1-(L) x -V 1- L 1- V2; (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V 1- L 1- V2; Epitope 1(L) x -Epitope 2-(L) x -Epitope 3-(L) x -V 1- L 1- V2; (L) x -Epitope 1-(L) x -V 1- L 1- V 2- (L) x -Epitope 2-(L) x ; (L) x -Epitope 1-(L) x -V 1- L 1- V 2- (L) x -Epitope 2-(L) x -Epitope 3-(L) x -; (L) x -Epitope 1-(L) x -V 1- L 1- V 2- (L) x -Epitope 2-(L) x -Epitope 3-(L)x -Epitope 4-(L) x -; (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V 1- L 1- V 2- (L) x -Epitope 3-(L) x -; (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V 1- L 1- V 2- (L) x -Epitope 3-(L) x -Epitope 4-(L) x -; V 1- (L) x -Epitope 1-(L) x -V2; V 1- (L) x -Epitope 1-(L) x -V 2- (L) x -Epitope 2-(L) x ; V 1- (L) x -Epitope 1-(L) x -V 2- (L) x -Epitope 2-(L) x -Epitope 3-(L) x ; V 1- (L) x -Epitope 1-(L) x -V 2- (L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x ; (L) x -Epitope 1-(L) x -V 1- (L) x -Epitope 2-(L)x -V2; or (L) x -Epitope 1-(L) x -V 1- (L) x -Epitope 2-(L) x -V 2- (L) x -Epitope 3-(L) x . During the ceremony, V1 is V L And V2 is V H or V1 is V H And V2 is V L and L1 is V H Chain V L is a linker suitable for linking to a chain, L is a linker comprising glycine and serine residues, and each occurrence of L in the extracellular binding domain can be identical to or different from other occurrences of L in the same extracellular binding domain, e.g., SGGGG (SEQ ID NO: 185), GGGGS (SEQ ID NO: 178), or SGGGGS (SEQ ID NO: 186) (all of which occur, e.g., in SEQ ID NO: 52); x is 0 or 1, and each occurrence of x is selected independently of the other; Epitope 1, epitope 2, epitope 3, and epitope 4 are mAb-specific epitopes and can be the same or different.

[0236] In some embodiments, the extracellular binding domain of the CAR comprises the following sequence: V 1- L 1- V 2- (L) x -Epitope 1-(L) x -Epitope 2-(L) x -or (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L)x - in which V1, V2, L1, L, x, and epitope 1, epitope 2, epitope 3, and epitope 4 are as defined above.

[0237] In some embodiments, any one of the MUC16-specific CARs disclosed herein is an epitope or mimotope of a CD52 epitope or mimotope, a CD20 epitope or mimotope, a CD3 epitope or mimotope, a CD41 epitope or mimotope, a CD25 epitope or mimotope, a CD30 epitope or mimotope, an EGFR epitope or mimotope, a TNFα epitope or mimotope, a VEGF epitope or mimotope, a complement protein C5 epitope or mimotope, a CD11a epitope or mimotope, a CD33 epitope or mimotope, an alpha-4 integrin epitope or mimotope, an IgE The epitope or mimotope may comprise one or more mAb-specific epitopes or mimotopes selected from an Fc region epitope or mimotope, an RSV protein F epitope or mimotope, an IL-6 receptor epitope or mimotope, a HER2 receptor epitope or mimotope, an integrin α4β7 epitope or mimotope, a BAFF (B cell activatin factor) epitope or mimotope, an IL-1β epitope or mimotope, a RANKL epitope or mimotope, a CTLA4 epitope or mimotope, a CD34 epitope or mimotope, an IL-12 epitope or mimotope, and / or an IL-23 epitope or mimotope.

[0238] In some embodiments, the MUC16-specific CARs disclosed herein are selected from the group consisting of alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, oncolytic stimulants, and serotonin-dependent stimulants. The antibody may comprise one or more mAb-specific epitopes or mimotopes selected from epitopes or mimotopes specifically recognized by marizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, admitamine, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10, and / or ustekinumab.

[0239] In some embodiments, the MUC16-specific CAR comprises one or more mAb-specific epitopes or mimotopes selected from the epitopes and mimotopes disclosed in Table 5. [Table 6]

[0240] The intracellular signaling domain of the CAR disclosed herein is involved in intracellular signaling after the binding of the extracellular ligand binding domain to the target, resulting in the activation of immune cells and immune responses. The intracellular signaling domain has the ability to activate at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell may be a cytolytic activity or a helper activity, including cytokine secretion.

[0241] Expression of Proteins in Cells and Related Embodiments In some embodiments, an immune cell, e.g., a T cell, of the disclosure comprises and / or expresses a polypeptide comprising any one or more of the sequences (SEQ ID NOs: 1-79) listed in Tables 1-3. In some embodiments, an immune cell, e.g., a T cell, of the disclosure comprises and / or expresses a nucleic acid, e.g., a vector, encoding such a polypeptide.

[0242] In some embodiments, an immune cell, e.g., a T cell, of the disclosure comprises and / or expresses a polypeptide comprising the amino acid sequence of MUC16 CAR1 (53B6) (SEQ ID NO: 11). In some embodiments, an immune cell, e.g., a T cell, of the disclosure comprises and / or expresses a polypeptide comprising the amino acid sequence of Sec49k (SEQ ID NO: 12). In some embodiments, an immune cell, e.g., a T cell, of the disclosure comprises and / or expresses a polypeptide comprising the amino acid sequence of UL11 (SEQ ID NO: 13). In some embodiments, an immune cell, e.g., a T cell, of the disclosure comprises and / or expresses a polypeptide comprising the amino acid sequence of BTN3A1-IgV (SEQ ID NO: 14). In some embodiments, an immune cell, e.g., a T cell, of the disclosure comprises and / or expresses a polypeptide comprising the amino acid sequence of BTN3A1-IgC (SEQ ID NO: 15).

[0243] In some embodiments, an immune cell, e.g., a T cell, of the disclosure comprises and / or expresses a nucleic acid, e.g., a vector, encoding a protease-activated CD45-gated CAR disclosed herein. In some embodiments, an immune cell, e.g., a T cell, of the disclosure comprises and / or expresses a nucleic acid, e.g., a vector, comprising a nucleic acid sequence encoding the amino acid sequence of MUC16 CAR (53B6) (any of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170). In some embodiments, an immune cell, e.g., a T cell, of the disclosure comprises and / or expresses a nucleic acid, e.g., a vector, comprising a nucleic acid sequence encoding the amino acid sequence of Sec49k (SEQ ID NO: 12). In some embodiments, an immune cell, e.g., a T cell, of the disclosure comprises and / or expresses a nucleic acid, e.g., a vector, comprising a nucleic acid sequence encoding the amino acid sequence of UL11 (SEQ ID NO: 13). In some embodiments, an immune cell, e.g., T cell, of the disclosure comprises and / or expresses a nucleic acid, e.g., vector, that includes a nucleic acid sequence that encodes the amino acid sequence of BTN3A1-IgV (SEQ ID NO: 14). In some embodiments, an immune cell, e.g., T cell, of the disclosure comprises and / or expresses a nucleic acid, e.g., vector, that includes a nucleic acid sequence that encodes the amino acid sequence of BTN3A1-IgC (SEQ ID NO: 15).

[0244] The present disclosure encompasses modifications to the proteins of the present disclosure shown in Table 2, including functionally equivalent proteins with modifications that do not significantly affect their properties, as well as variants with enhanced or decreased activity and / or affinity. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, polypeptides with one or more deletions or additions of amino acids that do not significantly deleteriously alter the functional activity or that mature (enhance) the affinity of the polypeptide for its ligand, or polypeptides using chemical analogs.

[0245] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope or mimotope tag.

[0246] Conservative substitutions are shown in Table 6 under the heading of "conservative substitutions". If such substitutions result in a change in biological activity, more substantial changes, designated "exemplary substitutions" in Table 6 or described further below for amino acid classes, can be introduced and the products screened. [Table 7]

[0247] Protease-activated CD45-gated CAR proteins and protease-activated CD45-gated CAR protein derivatives may be synthesized in situ after introducing a polynucleotide encoding the protein into a cell. Alternatively, protease-activated CD45-gated CAR proteins and protease-activated CD45-gated CAR protein derivative proteins may be produced extracellularly and then introduced into a cell. Methods for introducing polynucleotide constructs into a cell are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods can be used to transiently express the polynucleotide construct and polynucleotide constructs that are not integrated into the genome of the cell. In other embodiments, viral-mediated methods can be used. The polynucleotide can be introduced into the cell by any suitable means, such as, for example, a recombinant viral vector (e.g., a retrovirus, e.g., a lentivirus, an adenovirus), a liposome, etc. Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or particle bombardment. The polynucleotide can be included in a vector, such as, for example, a plasmid vector or a viral vector.

[0248] In some embodiments, an immune cell of the present disclosure, e.g., a T cell, can comprise at least one protease-activated CD45-gated CAR protein or a protease-activated CD45-gated CAR protein derivative. In some embodiments, an immune cell, e.g., a T cell, can comprise at least one protease-activated CD45-gated CAR protein or a protease-activated CD45-gated CAR protein derivative and one or more additional CARs (gated or non-gated), each of which may comprise a different extracellular ligand binding domain.

[0249] In some embodiments of immune cells, e.g., T cells, provided herein, the CAR expressed by the T cell can include an extracellular ligand binding domain (e.g., a single chain variable fragment (scFv)), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular ligand binding domain, the transmembrane domain, and the intracellular signaling domain are in one polypeptide, i.e., in a single chain. Multi-chain CARs and polypeptides are also provided herein. In some embodiments, the multi-chain CAR contains a first polypeptide that includes a transmembrane domain and at least one extracellular ligand binding domain, and a second polypeptide that includes a transmembrane domain and at least one intracellular signaling domain, where the polypeptides are assembled together to form the multi-chain CAR. The CAR can be modified as described herein to include a CD45 recruitment domain linked to the amino terminus of the extracellular ligand binding domain of the CAR.

[0250] The extracellular ligand binding domain specifically binds to a target of interest. In some embodiments, the target of interest may be any of a variety of proteins, including, but not limited to, BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUC1, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, MHC-NY-ESO1, HER2 (ERBB2), CAIX (carbonic anhydrase IX), LIV1, ADAM10, CHRNA2, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, claudin-18.2 (claudin-18A2, or claudin 18 isoform 2), PSCA, DLL3 (delta-like protein 3, Drosophila delta homolog 3, delta 3), Mud 7 (mucin 17, Muc3, Muc3), FAP alpha (fibroblast activation protein alpha), Ly6G6D (lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGT1, NG25), PSMA, MSLN, or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43).

[0251] In some embodiments, the extracellular ligand-binding domain comprises the light chain variable (V) of a target antigen-specific monoclonal antibody linked by a flexible linker. L ) region and the heavy chain variable (V H) region. Single chain variable region fragments are made by linking light and / or heavy chain variable regions by using a short linking peptide (Bird et al., Science 242:423-426, 1988). One example of a linking peptide is the GS linker, which has the amino acid sequence (GGGGS)3 (SEQ ID NO:57), which bridges approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences have been designed and used (Bird et al., 1988, supra). In general, linkers can be short flexible polypeptides, preferably consisting of about 20 or fewer amino acid residues. Linkers can also be modified for additional functions, such as, for example, attachment of drugs or attachment to solid supports. Single chain variants can be produced recombinantly or synthetically. For synthetic production of scFvs, an automated synthesizer can be used. For recombinant production of scFvs, a suitable plasmid or other vector containing a polynucleotide encoding the scFv can be introduced into a suitable host cell, either a eukaryotic cell such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, or a prokaryotic cell such as E. coli. Polynucleotides encoding the desired scFv can be generated by routine manipulations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0252] In some embodiments, a flexible linker described herein is modified to include a protease cleavage site, and the modified linker connects the CD45 recruitment domain of a protease-activated CD45-gated CAR described herein to the antigen-binding domain of the protease-activated CD45-gated CAR. One example of such a modified linker comprises the amino acid sequence of SEQ ID NO:9.

[0253] The intracellular signaling domain of the protease-activated CD45-gated CAR according to the present disclosure is involved in intracellular signaling following binding of the extracellular ligand-binding domain to a target, resulting in activation of immune cells and immune responses. The intracellular signaling domain has the ability to activate at least one of the normal effector functions of the immune cell in which the protease-activated CD45-gated CAR is expressed. For example, the effector function of a T cell may be a cytolytic activity or a helper activity, including cytokine secretion.

[0254] In some embodiments, the intracellular signaling domain for use in protease-activated CD45-gated CARs may be, for example, but not limited to, the cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signaling following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same function. The intracellular signaling domain contains two distinct classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation, and sequences that act in an antigen-dependent manner to generate secondary or costimulatory signals. Primary cytoplasmic signaling sequences can include signaling motifs known as ITAM immunoreceptor tyrosine-based activation motifs. ITAMs are well-defined signaling motifs present in the intracytoplasmic tails of various receptors that serve as binding sites for the syk / zap70 class of tyrosine kinases. Examples of ITAMs used in the protease-activated CD45-gated CARs of the present disclosure can include, by way of non-limiting example, ITAMs derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain of the CAR can comprise a CD3ζ signaling domain (e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 34).

[0255] In some embodiments, the intracellular signaling domain of a protease-activated CD45-gated CAR of the present disclosure comprises a domain of a costimulatory molecule. In some embodiments, the intracellular signaling domain of a protease-activated CD45-gated CAR of the present disclosure comprises a portion of a costimulatory molecule selected from the group consisting of a fragment of 41BB (GenBank: AAA53133, e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 33), and CD28 (NP_006130.1, e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 35). In some embodiments, the intracellular signaling domain of the CAR comprises an amino acid sequence that comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 33-35.

[0256] CARs are generally expressed on the surface membrane of cells. Thus, the protease-activated CD45-gated CARs disclosed herein can include a transmembrane domain. Suitable transmembrane domains for the protease-activated CD45-gated CARs disclosed herein have (a) the ability to be expressed on the surface of a cell, for example, an immune cell, for example, but not limited to, a lymphocytic cell (e.g., T cell) or a natural killer (NK) cell, and (b) the ability to interact with a ligand-binding domain and an intracellular signaling domain to induce a cellular response of the immune cell against a given target cell. The transmembrane domain can be derived from either natural or synthetic sources. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. As non-limiting examples, the transmembrane polypeptide can be a domain of a T cell receptor, such as the α, β, γ, or δ polypeptides that make up the CD3 complex, the IL-2 receptor, for example, p55 (α chain), p75 (β chain or γ chain), an Fc receptor, particularly a subunit chain of the Fcγ receptor III, or a CD protein. Alternatively, the transmembrane domain may be synthetic and may comprise primarily hydrophobic residues such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8 alpha chain (e.g., NP_001139345.1). The protease-activated CD45-gated CAR may further comprise a stalk domain between the extracellular ligand binding domain and the transmembrane domain. The stalk domain may comprise up to 300 amino acids, e.g., 10-100 amino acids or 25-50 amino acids. The stalk region may be derived from all or a portion of a naturally occurring molecule, such as all or a portion of the extracellular region of CD8, CD4, or CD28, or all or a portion of an antibody constant region. Alternatively, the stalk domain may be a synthetic sequence that corresponds to a natural stalk sequence, or may be an entirely synthetic stalk sequence. In some embodiments, the stalk domain is part of the human CD8 alpha chain (e.g., NP_001139345.1). In another particular embodiment, the transmembrane and hinge domain comprises a portion of the human CD8 alpha chain.In some embodiments, a protease-activated CD45-gated CAR disclosed herein can include a CD45 recruitment domain, a protease-cleavable linker, an extracellular ligand binding domain, a CD8α human stalk and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain. In some embodiments, a nucleic acid encoding a protease-activated CD45-gated CAR disclosed herein can be introduced into an immune cell as a transgene via a vector, e.g., a plasmid vector. In some embodiments, the vector, e.g., a plasmid vector, can also contain a selection marker, e.g., that provides for identification and / or selection of cells that have received the vector.

[0257] The protease-activated CD45-gated CAR polypeptide may be synthesized in situ in the cell after introduction of a polynucleotide encoding the protease-activated CD45-gated CAR polypeptide into the cell. Alternatively, the protease-activated CD45-gated CAR polypeptide may be produced extracellularly and then introduced into the cell. Methods for introducing a polynucleotide construct into a cell are known in the art. In some embodiments, stable transformation methods may be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods may be used to transiently express the polynucleotide construct and the polynucleotide construct that is not integrated into the genome of the cell. In other embodiments, viral-mediated methods may be used. The polynucleotide may be introduced into the cell by any suitable means, such as, for example, a recombinant viral vector (e.g., a retrovirus (e.g., a lentivirus), an adenovirus), a liposome, etc. Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or particle bombardment. The polynucleotide may be included in a vector, such as, for example, a plasmid vector or a viral vector.

[0258] Methods for generating engineered immune cells expressing any of the protease-activated CD45-gated CARs provided herein are described in WO / 2016 / 166630, which is incorporated by reference in its entirety.

[0259] Also provided herein are immune cells, such as isolated T cells, obtained according to any one of the methods described herein, modified, e.g., engineered to contain and express the nucleic acids, vectors, and polypeptides disclosed herein. Any immune cell capable of expressing heterologous DNA can be used to express the protease-activated CD45-gated CAR protein or protease-activated CD45-gated CAR protein derivative, and optionally any additional CAR of interest. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell can be derived from, for example, but not limited to, stem cells. The stem cell can be an adult stem cell, a non-human embryonic stem cell, more particularly, a non-human stem cell, an umbilical cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell, or a hematopoietic stem cell. A representative human cell is a CD34+ cell. The isolated cells may also be dendritic cells, killer dendritic cells, mast cells, NK cells, B cells, or T cells selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In some embodiments, the cells may be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes.

[0260] In some embodiments, the engineered immune cells disclosed herein are engineered B cells, mast cells, myeloid-derived phagocytes, T cells, e.g., alpha / beta and / or gamma / delta T cells, tumor infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, or NK-T cells. In some embodiments, the cells are and / or are derived from autologous T cells. In some embodiments, the cells are and / or are derived from allogeneic T cells.

[0261] In some embodiments, immune cells, e.g., T cells, such as isolated T cells, are further modified, e.g., genetically modified, e.g., by methods described herein (e.g., known gene editing techniques employing TALEN, CRISPR / Cas9, or MegaTAL nucleases).

[0262] Prior to expansion and genetic modification, the cell source can be obtained from a subject through a variety of non-limiting methods. Cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art can be used. In some embodiments, the cells can be derived from a healthy donor, a subject diagnosed with cancer, or a subject diagnosed with an infectious disease. In some embodiments, the cells can be part of a mixed population of cells that exhibit different phenotypic characteristics.

[0263] In some embodiments, the isolated or engineered immune cells according to the present disclosure comprise one inactivated gene selected from the group consisting of CD52, GR, PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRα, and TCRβ, and / or express a CAR, a multi-chain CAR, and / or a pTα transgene. In some embodiments, the isolated cells comprise a polynucleotide encoding a polypeptide comprising a multi-chain CAR. In some embodiments, the isolated cells according to the present disclosure have two inactivated genes selected from the group consisting of: CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ, and / or expressing a CAR (e.g., a protease-activated CD45-gated CAR of the disclosure or an anti-MUC16 CAR of the disclosure), a multi-chain CAR, and a pTα transgene.

[0264] Gene inactivation is performed by methods practiced by those skilled in the art, including but not limited to gene inactivation by use of zinc finger, TALEN®, and CRISPR / Cas-based systems.

[0265] In some embodiments, the protease-activated CD45-gated CAR-containing immune cells have an inactivated CD52 gene. In some embodiments, only one copy of the CD52 gene is inactivated.

[0266] In some embodiments, the protease-activated CD45-gated CAR-containing immune cells have an inactivated TCR alpha gene.

[0267] In some embodiments, the protease-activated CD45-gated CAR-containing immune cells have an inactivated TCR β gene.

[0268] In some embodiments, TALENs are used for gene inactivation. In such embodiments, the efficiency of gene inactivation by TALENs is not 100% and the resulting TCRαβ-negative T cells are enriched by depleting residual TCRαβ-positive T cells before cryopreservation. However, CD52-negative cells are not purified, resulting in a cell product with a different frequency of CD52-negative cells, typically 60-80%. Thus, in some embodiments, the genotype of the protease-activated CD45-gated CAR T cells of the present disclosure is protease-activated CD45-gated CAR+_TCRαβ-_CD52+ / - T cells.

[0269] In some embodiments, the TCR is rendered non-functional in a cell according to the present disclosure by inactivating the TCR alpha and / or TCR beta genes. In some embodiments, a method of obtaining modified cells from an individual is provided, wherein the cells are capable of growing independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells capable of growing independently of the MHC signaling pathway are amenable to being obtained by the present method and are therefore within the scope of the present disclosure. The modified cells disclosed herein may be used to treat host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) in a patient in need thereof, and thus, within the scope of the present disclosure is a method of treating a patient in need thereof, comprising administering to the subject an effective amount of modified cells comprising an inactivated TCR alpha and / or TCR beta gene.

[0270] In some embodiments, the immune cells are engineered to be resistant to one or more chemotherapeutic agents. The chemotherapeutic agents may be, for example, purine nucleotide analogs (PNAs), thus making the immune cells suitable for cancer treatment combining adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) to mono-, di-, and triphosphate PNAs. Their triphosphate forms compete with ATP for DNA synthesis, act as proapoptotic agents, and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in trinucleotide production. Provided herein are protease-activated CD45-gated CAR-T cells that contain an inactivated dCK gene. In some embodiments, dCK knockout cells are generated by transfection of T cells with a polynucleotide encoding a specific TAL nuclease for the dCK gene, for example, by electroporation of mRNA. dCK knockout protease-activated CD45-gated CAR-T cells are resistant to PNAs, including, for example, chlorofarabine and / or fludarabine, and maintain the cytotoxic activity of T cells against cells expressing the ligand of the protease-activated CD45-gated CAR.

[0271] In some embodiments, the isolated cells or cell lines of the present disclosure may comprise pTα or a functional variant thereof, hi some embodiments, the isolated cells or cell lines may be further genetically modified by inactivating the TCRα gene.

[0272] In some embodiments, the CAR-T cell (e.g., a protease-activated CD45-gated CAR-expressing T cell) comprises a polynucleotide encoding a safety switch, such as, for example, RQR8. See, e.g., WO2013153391A, which is incorporated by reference in its entirety. In the CAR-T cell comprising the polynucleotide, the safety switch polypeptide is expressed on the surface of the CAR-T cell. In some embodiments, the safety switch polypeptide comprises the amino acid sequence set forth in SEQ ID NO:48. (SEQ ID NO:48) CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCS GGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY IWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV

[0273] The safety switch polypeptide may also comprise a signal peptide at the amino terminus. In some embodiments, the safety switch polypeptide comprises the amino acid sequence set forth in SEQ ID NO:49. (SEQ ID NO:49) MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVST NVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEA CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRR RVCKCPRPVV

[0274] When the safety switch polypeptide is expressed on the surface of the CAR-T cell, binding of rituximab to the R epitope or mimotope of the polypeptide causes lysis of the cell. Two or more molecules of rituximab can be bound per polypeptide expressed on the cell surface. Each R epitope or mimotope of the polypeptide can bind to a separate molecule of rituximab. Deletion of protease-activated CD45-gated CAR-T cells can occur in vivo, for example, by administering rituximab to the subject. The decision to delete the introduced cells can result from undesirable effects detected in the subject due to the transferred cells, for example, when an unacceptable level of toxicity is detected.

[0275] In some embodiments, the CAR-T cells comprise a selected epitope or mimotope within the scFv domain or extracellular domain (e.g., stalk domain) of a protease-activated CD45-gated CAR, the selected epitope or mimotope having a specificity recognized by a specific antibody. See, e.g., PCT Application No. PCT / EP2016 / 051467, filed Jan. 25, 2016, WO2016 / 120216, "mAb-DRIVEN CHIMERIC ANTIGEN RECEPTOR SYSTEMS FOR SORTING / DEPLETING ENGINEERED IMMUNE CELLS," which is incorporated herein by reference in its entirety. Such epitopes or mimotopes facilitate sorting and / or depletion of CAR-T cells. The epitope or mimotope may be selected from any number of epitopes and mimotopes known in the art. In some embodiments, the epitope or mimotope may be the target of a monoclonal antibody approved for medical use, such as, for example, but not limited to, the CD20 epitope or mimotope recognized by rituximab. In some embodiments, the epitope or mimotope comprises the amino acid sequence set forth in SEQ ID NO:50. CPYSNPSLC (SEQ ID NO:50)

[0276] In some embodiments, the epitope or mimotope is located within the CAR. For example, but not limited to, the epitope or mimotope can be located between the scFv and the hinge of the CAR. In some embodiments, two instances of the same epitope or mimotope separated by a linker can be used in the CAR. For example, a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 51 can be used in the CAR located between the light chain variable region and the hinge. (SEQ ID NO:51) GSGGGGSCPYSNPSLCSGGGGSCPYSNPSLCSGGGGS

[0277] In some embodiments, epitope- or mimotope-specific antibodies may be conjugated with cytotoxic drugs. It is also possible to promote CDC cytotoxicity by using engineered antibodies that are grafted components of the complement system. In some embodiments, the activation of CAR-T cells may be regulated by depleting the cells using antibodies that recognize the epitope or mimotope.

[0278] Adverse events may also be minimized by transducing immune cells (containing one or more CARs) with a suicide gene. It may also be desirable to incorporate an inducible "on" or "facilitator" switch into the immune cells. Suitable techniques include the use of inducible caspase-9 (US Application No. 2011 / 0286980) or thymidine kinase before, after, or simultaneously with the cells being transduced with the CAR constructs of the present disclosure. Additional methods for introducing suicide genes and / or "on" switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques known in the art.

[0279] According to the present disclosure, additional on-off or other types of control switch technologies can be incorporated herein. These technologies can employ the use of dimerization domains and optional activators of such domain dimerization. These technologies include, for example, those described by Wu et al., Science 2014 350(6258), which utilizes the FKBP / rapalog dimerization system in certain cells, the contents of which are incorporated herein by reference in their entirety. Additional dimerization technologies are described, for example, in Fegan et al. Chem. Rev. 2010,110,3315-3336, and U.S. Patent Nos. 5,830,462, 5,834,266, 5,869,337, and 6,165,787, the contents of which are also incorporated herein by reference in their entirety. Additional dimerization pairs may include cyclosporin-A / cyclophilin, receptor, estrogen / estrogen receptor (optionally using tamoxifen), glucocorticoid / glucocorticoid receptor, tetracycline / tetracycline receptor, vitamin D / vitamin D receptor. Further examples of dimerization techniques are described, for example, in WO2014 / 127261, WO2015 / 090229, US2014 / 0286987, US2015 / 0266973, US2016 / 0046700, U.S. Patent No. 8,486,693, US2014 / 0171649, and US2012 / 0130076, the contents of which are incorporated herein by reference in their entireties.

[0280] Also provided herein are cell lines derived from immune cells, e.g., T cells, modified, e.g., transformed, according to any of the methods described herein. In some embodiments, the immune cells, e.g., T cells, according to the present disclosure, comprise a polynucleotide encoding a protease-activated CD45-gated CAR protein or a protease-activated CD45-gated CAR protein derivative. In some embodiments, the immune cells, e.g., T cells, according to the present disclosure, comprise a polynucleotide encoding a protease-activated CD45-gated CAR protein or a protease-activated CD45-gated CAR protein derivative, and a polynucleotide encoding an additional CAR.

[0281] Immune cells, e.g., T cells, of the present disclosure can be derived from, for example, any of the immune cells described in, but not limited to, 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, The cells can be inactivated and expanded either before or after modification using methods generally described in US Patent Application Publication No. 2006 / 0121005, US Patent No. 869, US Patent No. 7,232,566, US Patent No. 7,175,843, US Patent No. 5,883,223, US Patent No. 6,905,874, US Patent No. 6,797,514, US Patent No. 6,867,041, and US Patent Application Publication No. 2006 / 0121005. Immune cells, such as T cells, can be expanded in vitro or in vivo. Generally, the immune cells of the present disclosure can be expanded by contacting with an agent that stimulates the CD3 TCR complex and costimulatory molecules on the surface of the immune cells to create an activation signal for the cells, for example. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to generate activation signals for immune cells, e.g., T cells.

[0282] In some embodiments, a population of T cells can be stimulated in vitro, for example, by contact with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. Co-stimulation of an accessory molecule on the surface of the T cells uses a ligand that binds to the accessory molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640, or X-vivo5, (Lonza)), which can contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFβ, and TNF, or any other additives for cell proliferation known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The medium can include RPMI1640, AIM V, DMEM, MEM, α-MEM, F-12, X-Vivo10, and X-Vivo20, Optimizer with additional amino acids, sodium pyruvate, and vitamins, and is serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in sufficient amounts for T cell growth and proliferation. Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures and not in the cultures of cells injected into the subject. The target cells are maintained under conditions necessary to support growth, such as, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2). Immune cells, such as T cells, exposed to various stimulation times may exhibit different characteristics.

[0283] In some embodiments, the cells of the present disclosure can be expanded by co-culturing with tissue or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after administration of the cells to the subject.

[0284] In another aspect, the disclosure provides compositions (such as pharmaceutical compositions) comprising any of the cells of the disclosure or cell populations of the disclosure. In some embodiments, the compositions comprise T cells comprising a polynucleotide encoding a protease-activated CD45-gated CAR protein or a protease-activated CD45-gated CAR protein derivative, which may further comprise a polynucleotide encoding a second CAR. The compositions include, for example, immune cells of the disclosure, e.g., T cells, e.g., immune cells expressing a protease-activated CD45-gated CAR and / or a protease-activated CD45-gated CAR protein derivative, or comprise a cell population comprising immune cells of the disclosure, e.g., T cells, and one or more pharma- ceutically acceptable carriers or excipients.

[0285] In some embodiments, primary cells isolated from a donor are manipulated as described herein to provide a cell population in which a subpopulation of the resulting cells (e.g., less than 100%, such as 10%, 20%, 30%) contains all of the desired modifications. Such a resulting population, including a mixture of cells that contain and do not contain all of the modifications, can be used in the therapeutic methods of the present disclosure and to prepare the compositions of the present disclosure. Alternatively, this cell population (hereinafter, the "starting population") can be manipulated by known methods, such as cell sorting and / or cell expansion, to provide a cell population enriched for cells that contain one or more of the desired modifications (e.g., enriched for cells expressing a protease-activated CD45-gated CAR protein and / or a protease-activated CD45-gated CAR protein derivative, and / or enriched for cells expressing a desired antigen binding protein), i.e., containing a higher percentage of such modified cells than the starting population. The population enriched for modified cells can then be used in the therapeutic methods of the present disclosure and, for example, to prepare the compositions of the present disclosure. In some embodiments, the enriched cell population contains, or at least contains, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of cells that have one or more of the modifications, hi other embodiments, the percentage of cells in the enriched cell population that contain one or more of the modifications is at least 30% higher than the percentage of cells in the starting population of cells that contain the desired modifications.

[0286] In some embodiments, the immune cell population comprises one or more of the engineered immune cells disclosed herein. In one embodiment, the population comprises about or at least about 1 x 10 4 , about or at least about 1×10 5 , about or at least about 1×10 6 , about or at least about 1×10 7 or about or at least about 1×10 8 10 of the engineered cells, e.g., engineered immune cells, disclosed herein, optionally comprising a population of about 1×10 10 More than 1 x 10 9More than 5 x 10 9 In one embodiment, the immune cell population disclosed herein is enriched for engineered immune cells disclosed herein. In various embodiments, the immune cell population is at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% engineered cells, and the engineered cells are T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and / or myeloid-derived phagocytes. In various embodiments, the immune cell population is at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% engineered T cells.

[0287] Treatment method Immune cells, e.g., T cells, or cell lines derived from such immune cells or T cells obtained by the above method may be used as a medicament. In some embodiments, such a medicament may be used to treat disorders such as, for example, viral diseases, bacterial diseases, cancer, inflammatory diseases, immune diseases, autoimmune diseases, infectious diseases, or age-related diseases. In some embodiments, the cancer is a hematological malignancy or a non-solid tumor. In some embodiments, the cancer is a solid cancer or a solid tumor. In some embodiments, the cancer is a solid cancer or a solid tumor. In some embodiments, the cancer is a hematological malignancy that is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM). In some embodiments, the cancer is a solid cancer that is biliary tract cancer, bladder cancer, bone or soft tissue cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal carcinoma, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumors, head and neck squamous cell carcinoma, liver cancer, lung cancer, melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, renal cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, or uterine cancer. In some embodiments, the cancer may be selected from the group consisting of gastric cancer, sarcoma, osteosarcoma, rhabdomyosarcoma, tissue sarcoma, uterine sarcoma, lymphoma, leukemia, head and neck cancer, thymic cancer, epithelial cancer, salivary gland cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer, and melanoma. In some embodiments, the subject is a previously treated adult subject with locally advanced or metastatic melanoma, squamous cell head and neck cancer (SCHNC), ovarian cancer, sarcoma, or relapsed or refractory classical Hodgkin lymphoma (cHL).In one embodiment, the condition is ovarian cancer and the protease-activated CD45-gated CAR comprises the amino acid sequence of any of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170.

[0288] In some embodiments, an immune cell, e.g., a T cell, or a cell line derived from an immune cell, e.g., a T cell, according to the present disclosure may be used in the manufacture of a medicament for treating a disorder in a subject in need thereof. In some embodiments, the disorder may be, for example, a cancer, an autoimmune disorder, or an infectious disease.

[0289] Also provided herein are methods for treating a subject. In some embodiments, the methods include administering or providing immune cells, e.g., T cells, of the present disclosure to a subject in need thereof. In some embodiments, the methods include administering immune cells, e.g., T cells, of the present disclosure to a subject in need thereof.

[0290] In another aspect, provided herein is a method of treating a condition in a patient comprising administering to the patient an engineered immune cell as disclosed herein, In one embodiment of the method, the engineered immune cell is an allogeneic engineered immune cell derived from a donor other than the patient.

[0291] In another aspect, provided herein is a method of treating a condition in a patient comprising administering to the patient a population of immune cells disclosed herein. In one embodiment of the method, the immune cells of the population are derived from one or more allogeneic immune cells from a donor other than the patient.

[0292] In another aspect, provided herein is a method of treating a condition in a patient, comprising administering to the patient a pharmaceutical composition disclosed herein. In one embodiment of the method, the composition comprises one or more engineered allogeneic immune cells derived from a donor other than the patient.

[0293] In some embodiments, the immune cells, e.g., T cells, of the present disclosure can undergo robust in vivo cell proliferation and can persist for extended periods of time. The therapeutic methods of the present disclosure can be ameliorative, curative, or preventative. The methods of the present disclosure can be part of either an autoimmunotherapy or an allogeneic immunotherapeutic treatment. The present disclosure is particularly suitable for allogeneic immunotherapy. Immune cells, e.g., T cells, from a donor can be transformed into non-allo-reactive cells using standard protocols and regenerated as needed, thereby producing CAR-T cells, which can be administered, for example, to one or more subjects. Such CAR-T cell therapy can be available as an "off the shelf" treatment.

[0294] In another aspect, the disclosure provides a method of inhibiting tumor growth or progression in a subject having a tumor, the method comprising administering to the subject an effective amount of an immune cell, e.g., a T cell as described herein. In another aspect, the disclosure provides a method of inhibiting or preventing metastasis of a cancer cell in a subject, the method comprising administering to a subject in need thereof an effective amount of an immune cell, e.g., a T cell as described herein. In another aspect, the disclosure provides a method of inducing tumor regression in a subject having a tumor, the method comprising administering to the subject an effective amount of an immune cell, e.g., a T cell as described herein.

[0295] In some embodiments, the immune cells provided herein, such as T cells, can be administered parenterally in a subject. In some embodiments, the subject is a human.

[0296] In some embodiments, the method can further include administering an effective amount of a second therapeutic agent, e.g., crizotinib, palbociclib, an anti-CTLA4 antibody, an anti-4-1BB antibody, a PD-1 antibody, or a PD-L1 antibody.

[0297] Also provided herein is the use of any of the immune cells, e.g., T cells, provided herein in the manufacture of a medicament for the treatment of cancer or inhibiting tumor growth or progression in a subject in need thereof.

[0298] Also provided herein are uses and methods described herein, where the condition is ovarian cancer and the protease-activated CD45-gated CAR comprises the amino acid sequence of any of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170. Also provided herein are uses and methods described herein, where the condition is ovarian cancer and a CAR is used that comprises the amino acid sequence of any of SEQ ID NOs: 11, 56, 75, 76, 77, 78, 79, and 121-170 in addition to, or in place of, the protease-activated CD45-gated CAR.

[0299] The degree to which the gate regulates the activity of a protease-activated CD45-gated CAR can be assessed in a variety of ways. One method is to measure the cytotoxicity of T cells expressing a protease-activated CD45-gated CAR in comparison with various controls, such as T cells expressing a protein identical to the protease-activated CD45-gated CAR but lacking a protease cleavage site in the linker connecting the CD45 recruitment domain to the ligand binding domain (non-cleavable gate), T cells expressing a protein containing the same CAR but without the CD45 recruitment domain (non-gated CAR), and untransduced T cells. The cytotoxicity of these cells can be measured by incubating them with target cells expressing a ligand recognized by the CAR antigen-binding domain. The assay can be adjusted by varying the ratio of effector CAR T cells to target ligand-expressing cells. When effector and target cells are incubated together under appropriate conditions, without wishing to be bound by theory, it is expected that CAR T cells will exhibit high cytotoxicity, and that both cleavable and non-cleavable CD45-gated CAR T cells will exhibit lower cytotoxicity, since the gate at least partially inactivates the CAR. Non-transduced cells are expected to exhibit no cytotoxicity. This form of assay helps to confirm that under the conditions of the assay, the gate is able to at least partially inactivate the CAR when the ligand of the CAR is present but the protease is absent. Thus, mimicking the in vivo situation where a protease-activated CD45-gated CAR T cell encounters its antigen-off tumor. In such an assay, in some embodiments, the cytotoxicity of a protease-activated CD45-gated CAR is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a non-gated CAR. The degree of reduction observed depends on the conditions of the assay.

[0300] The same assay can be repeated in the presence of a protease capable of cleaving the linker connecting the CD45 recruitment domain to the ligand binding domain to mimic the in vivo condition where protease-activated CD45-gated CAR T cells encounter antigen at the tumor site. It is expected that the protease will not affect the cytotoxicity of non-gated CAR T cells. It is also expected that the cleavable protease-activated CD45-gated CAR will show reduced cytotoxicity in the absence of protease and will show higher cytotoxicity in the presence of protease compared to the absence of protease, since the gate that inhibited CAR activation has been removed by cleavage of the linker in the presence of protease. It is expected that the cytotoxicity of non-cleavable gated CAR will not be affected by the presence of protease. Without wishing to be bound by theory, it is expected that its cytotoxicity will be reduced in both the absence and presence of protease, since the gate inhibits the activation of the CAR under both conditions. In such assays, in some embodiments, in the absence of proteases, the cytotoxicity of protease-activated CD45-gated CARs is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to non-gated CARs, and in the presence of proteases, the cytotoxicity of the cells is significantly higher. The extent of the observed reduction and increase depends on the conditions of the assay.

[0301] In another aspect, provided herein is a method of reducing on-target off-tumor toxicity of CAR T cells, the method comprising administering to a patient a cell disclosed herein, a cell population disclosed herein, or a composition disclosed herein, wherein the on-target, off-tumor toxicity is lower than the on-target, off-tumor toxicity of a control cell, a control cell population, or a composition of control cells, each administered under the same conditions, wherein the control cell, control cell population, or control cell of the composition comprises a CAR that lacks at least one of a CD45 recruitment domain and a linker comprising a protease cleavage site.

[0302] In another aspect, provided herein is a method of increasing the efficiency of a CAR T cell or CAR T cell therapy, the method comprising administering to a patient a cell as disclosed herein, a cell population as disclosed herein, or a composition as disclosed herein, wherein the efficiency is greater than the efficiency of a control cell, a control cell population, or a composition of control cells, each administered under the same conditions, wherein the control cell of the control cell, control cell population, or composition comprises a CAR that lacks at least one of a CD45 recruitment domain and a linker comprising a protease cleavage site.

[0303] In another aspect, provided herein is a method of increasing the efficacy of CAR T cell therapy, the method comprising administering to a patient a cell disclosed herein, a cell population disclosed herein, or a composition disclosed herein, wherein the efficacy is greater than the efficacy of a control cell, a control cell population, or a composition of control cells, each administered under the same conditions, wherein the control cell, control cell population, or control cell of the composition comprises a CAR that lacks at least one of a CD45 recruitment domain and a linker that comprises a protease cleavage site.

[0304] In another aspect, provided herein is a method of increasing the efficacy of CAR T cell therapy against a solid tumor, the method comprising administering a cell disclosed herein, a cell population disclosed herein, or a composition disclosed herein to a patient having a solid tumor, wherein the efficacy against the solid tumor is greater than the efficacy against the solid tumor of a control cell, a control cell population, or a composition of control cells, each administered under the same conditions, wherein the control cell, control cell population, or control cell of the composition comprises a CAR that lacks at least one of a CD45 recruitment domain and a linker that comprises a protease cleavage site.

[0305] In another aspect, provided herein is a method of reducing the incidence of side effects in CAR T cell therapy, the method comprising administering to a patient a cell disclosed herein, a cell population disclosed herein, or a composition disclosed herein, wherein the incidence of side effects is lower than the incidence of side effects when a control cell, a control cell population, or a composition of control cells, respectively, is administered under the same conditions, wherein the control cell of the control cell, control cell population, or composition comprises a CAR that lacks at least one of a CD45 recruitment domain and a linker that comprises a protease cleavage site.

[0306] In another aspect, provided herein is a method for resulting in reversible co-localization of CD45 and a CD45-gated CAR in a CAR T cell, the reversible co-localization comprising a CD45-gated CAR recruitment domain that binds to CD45 protein on the CAR T cell surface and, as a result, co-localizes, and the binding and localization can be disrupted by a protease cleaving a protease cleavage site in the linker of the CD45-gated CAR, thereby reversing the co-localization, the method comprising: (a) providing T cells that express CD45; (b) introducing into a T cell a nucleic acid encoding a protease-activated CD45-gated CAR as disclosed herein; (c) maintaining the T cells under conditions in which both CD45 and CAR are functionally expressed on the cell surface; as a result, the CD45-gated CAR recruitment domain binds to the CD45 protein on the CAR T cell surface, the CAR and the CD45 protein co-localize, and the co-localization can be reversed by a protease that cleaves a protease cleavage site in the linker of the CD45-gated CAR, which cleavage produces a functional CAR that is no longer connected to the CD45-gated CAR recruitment domain by the linker.

[0307] In another aspect, provided herein is a method that results in a reversible reduction of CAR activity by CD45 protein in a CAR T cell, the reversible reduction of CAR activity comprising a CD45-gated CAR that binds to CD45 protein on the surface of the CAR T cell, resulting in inactivation of the chimeric antigen receptor by CD45, which inactivation can be disrupted by a protease cleaving a protease cleavage site in the linker of the CD45-gated CAR, resulting in the chimeric antigen receptor and the CD45 protein dissociating, terminating the inactivation of the chimeric antigen receptor by CD45, the method comprising: (a) providing T cells that express CD45; (b) introducing a nucleic acid encoding a CD45-gated CAR according to any one of claims 1 to 14 into a T cell; (c) maintaining the T cells under conditions in which both CD45 and CAR are functionally expressed on the cell surface; as a result, the CD45-gated CAR recruitment domain binds to the CD45 protein on the surface of the CAR T cell, the CAR and the CD45 protein associate, and the activity of the CAR is reduced by the CD45 protein, which can be reversed by a protease cleaving a protease cleavage site in the linker of the CD45-gated CAR, thereby terminating the association of the CAR and the CD45 protein and terminating the reduction in CAR activity of the CD45 protein, and the cleavage produces a functional CAR that is no longer connected to the CD45-gated CAR recruitment domain by the linker.

[0308] In another aspect, provided herein is a method of treating a patient having a tumor characterized by a protease-rich tumor microenvironment with CAR T cell therapy, the method comprising administering to the patient a cell disclosed herein, a cell population disclosed herein, or a composition disclosed herein, wherein CAR T cell activity is lower outside the protease-rich tumor microenvironment than in the protease-rich tumor microenvironment.

[0309] In another aspect, provided herein is a method of modulating the cytotoxic activity of a CAR T cell comprising a CAR disclosed herein, the method comprising inhibiting the cytotoxic activity of the CAR T cell by associating the CAR with CD45 of the CAR T cell, and activating the cytotoxic activity of the CAR T cell by subjecting and / or exposing the CAR T cell to a protease that recognizes and cleaves the protease cleavage site.

[0310] In embodiments of the methods of treating a condition in a patient disclosed herein, the cells, cell populations, or compositions may be administered to the subject once, or may be administered to the subject two or more times spaced at least about 1, 2, 3, 4, 5, 6, 7 days apart, in some embodiments, the disorder may be a viral disease, a bacterial disease, cancer, an inflammatory disease, an immune disease, or an age-related disease.

[0311] In some embodiments, the treatment may be in combination with one or more therapies for cancer selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiation therapy.

[0312] In some embodiments, the treatment may be administered or administered to a subject undergoing immunosuppressive therapy. Indeed, the present disclosure may rely on a cell or cell population that has become resistant to at least one immunosuppressive agent due to inactivation of a gene encoding a receptor for such an immunosuppressive agent. In this aspect, the immunosuppressive therapy may assist in the selection and expansion of T cells according to the present disclosure in the subject.

[0313] Administration of cells or cell populations according to the present disclosure may be by any convenient method, including aerosol inhalation, injection, ingestion, infusion, injection, implantation, or transplantation. The compositions described herein may be administered to a subject subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, intravenously or intralymphatically, or intraperitoneally. In one embodiment, the cell composition of the present disclosure is administered by intravenous injection.

[0314] In some embodiments, administration of the cells or cell populations is, for example, about 10 4 ~about 10 9 In some embodiments, the administration of a cell or cell population is about 10 / kg body weight or more, including all integer values ​​of the number of cells within those ranges. 5 ~about 10 6 The ranges may include administration of a single dose of cells, including all integer values ​​of the number of cells within those ranges. The cells or cell populations may be administered in one or more doses. In some embodiments, an effective amount of cells may be administered as a single dose. In some embodiments, an effective amount of cells may be administered as two or more doses over a period of time. The timing of administration is within the judgment of the attending physician and depends on the clinical condition of the subject. The cells or cell populations may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determining the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of one of ordinary skill in the art. An effective amount means an amount that provides a therapeutic or prophylactic benefit. The dosage administered depends on the age, health, and weight of the recipient, the type of concomitant treatment, if any, frequency of treatment, and the nature of the desired effect. In some embodiments, an effective amount of the cells or a composition comprising those cells is administered parenterally. In some embodiments, administration may be intravenous. In some embodiments, administration may be by injection directly into the tumor.

[0315] In some embodiments of the present disclosure, the cells are administered in conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant therapeutic modalities, including, but not limited to, treatment with agents such as monoclonal antibody therapy, CCR2 antagonists (e.g., INC-8761), antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or nataliziimab treatment for MS subjects, or efaliztimab treatment for psoriasis subjects, or other treatments for PML subjects. In some embodiments, the protease-activated CD45-gated CAR-T cells are administered to the subject in conjunction with one or more of the following: an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or PF-06801591), an anti-PD-L1 antibody (e.g., avelumab, atezolizumab, or durvalumab), an anti-OX40 antibody (e.g., PF-04518600), an anti-4-1BB antibody (e.g., PF-05082566), an anti-MCSF antibody (e.g., PD-0360324), an anti-GITR antibody, and / or an anti-TIGIT antibody. In further embodiments, the immune cells, e.g., T cells, of the present disclosure may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, or other antibody therapies, cytoxin, fludarivine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and / or irradiation. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase, which is important in growth factor-induced signal transduction (rapamycin) (Henderson, Naya et al. Immunology. 1991 Jul;73(3):316-321; Liu, Albers et al. Biochemistry 1992 Apr 28;31(16):3896-901; Bierer, Hollander et al. Curr Opin Immunol. 1993 Oct;5(5):763-73).

[0316] In further embodiments, the cell composition of the present disclosure is administered to the subject in conjunction with (e.g., before, simultaneously with, or after) a bone marrow transplant, a T cell ablative therapy using either a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAMPATH. In some embodiments, the cell composition of the present disclosure is administered after a B cell ablative therapy, such as an agent that reacts with CD20, e.g., Rituxan. For example, in one embodiment, the subject may receive standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, after transplant, the subject receives an infusion of the expanded immune cells of the present disclosure. In some embodiments, the expanded cells are administered before or after surgery.

[0317] kit The present disclosure also provides kits for use in the methods. The kits of the present disclosure include one or more containers that include the compositions of the present disclosure, or immune cells, e.g., T cells, or cell populations that include immune cells, e.g., T cells, of the present disclosure. In various embodiments, the immune cells, e.g., T cells, include one or more polynucleotides that encode the protease-activated CD45-gated CAR protein or protease-activated CD45-gated CAR protein derivatives described herein. The kits further include instructions for use with any of the methods of the present disclosure described herein. Generally, these instructions include instructions for administration of the compositions, immune cells, e.g., T cells, or cell populations for the therapeutic treatment described above. In some embodiments, the engineered immune cells are formulated in a solution that includes about 5% DMSO. Additionally, the engineered immune cells can be provided in a frozen state.

[0318] The instructions for use of the kit components generally include information regarding dosages, dosing schedules, and routes of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. The instructions supplied with kits of the present disclosure are typically written instructions on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical memory disk) are also acceptable.

[0319] The kit of the present disclosure is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., atomizers), or injection devices, such as mini-pumps. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an immune cell, e.g., T cell, according to the present disclosure. The container may further comprise a second pharmacologic active agent.

[0320] The kit may optionally provide additional components, such as buffers and interpretive information. Typically, the kit comprises a container and a label or package insert on or associated with the container. EXAMPLES

[0321] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the disclosure in any manner. Indeed, various modifications of the disclosure, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0322] Example 1: Synthesis and demonstration of CD45-gated CAR CD45-gated CAR structure FIG. 1A shows a schematic diagram of a protease-activated CD45-gated CAR. To investigate how the protease-activated CD45 gate can regulate the function of CAR T cells, a second generation CAR ("53B6") that specifically targets MUC16-expressing cells was used. A generic CAR protein is composed of an extracellular target binding domain, a transmembrane domain, and an intracellular signaling domain, which includes, for example, an activation domain (e.g., CD3 zeta cytoplasmic domain), and optionally also a costimulatory domain (e.g., 4-1BB signaling domain). In general, the extracellular domain of the CD45-gated CAR disclosed herein further includes a CD45 recruitment domain (hereinafter "recruiter") fused to the N-terminus of the generic CAR via a flexible linker (which in certain embodiments includes one or more protease cleavable sites), as shown in FIG. 1A and 1B (lower construct). As illustrated in FIG. 1B (lower construct), an additional N-HA tag (sequence YPYDVPDYA (SEQ ID NO: 187), a variant of SEQ ID NO: 2 without the initial G, was also introduced for detection of CD45-gated CAR, and a CD8 signal peptide was fused to the N-terminus of each protein to enable trafficking and surface expression. Non-gated, non-N-HA tagged 53B6 (FIG. 1B, upper construct) was used as a control in some of the experiments.

[0323] CD45-gated CAR expression in T cells To evaluate CAR T cells functionally expressing a protease-activated CD45-gated CAR, two variants of the CD45 gate were prepared, one containing a first anti-CD45 scFv as a CD45 recruiter and the other containing a second, different anti-CD45 scFv as a CD45 recruiter. A flexible protease-cleavable linker (SEQ ID NO: 8, GGGGSGGGGSLVPRGSKPIPNPLLGLDSTLVPRGSGGGGSGGGGS) that contained two thrombin protease cleavage sites flanking a V5 peptide motif that can be used to detect the linker joined the CD45 recruiter to the N-terminus of the CAR. These two protease-activated CD45-gated CAR clones were designated "CD45 gate1-TMB-53B6" (comprising the amino acid sequence of SEQ ID NO: 16) and "CD45 gate2-TMB-53B6" ("TMB" for thrombin-cleavable linker) (comprising the amino acid sequence of SEQ ID NO: 18), and the exemplary anti-MUC16 CAR clone 53B6 was used to demonstrate the proof of concept of such a protease-activated CD45-gated CAR. In parallel, additional control CARs were generated, designated "CD45 gate1-GS-53B6" (comprising the amino acid sequence of SEQ ID NO: 17) and "CD45 gate2-GS-53B6" (comprising the amino acid sequence of SEQ ID NO: 19), using a non-cleavable linker of the same length (SEQ ID NO: 7, GGGGSGGGGSGGGSSKPIPNPLLGLDSTGGGSGSGGGGSGGGGS), both of which are not expected to be cleaved by proteases. CAR T cells expressing either non-gated 53B6 CAR or CD45-gated 53B6 CAR were generated using lentiviral vectors as previously described (14, 15). Briefly (and using the methods described in Example 2, CAR-T production below), primary T cells from healthy donors were transduced with lentiviruses expressing different CAR molecules (MOI of 5) and the percentage of CAR+ cells was recorded on days 8 and 15. As the data in Figure 2A show, CARs with CD45 gate 1 ("CD45 gate 1-TMB-53B6" and "CD45 gate 1-GS-53B6") show a comparable percentage of CAR+ as the 53B6 CAR throughout production.CD45 gated 2 CARs ("CD45 gated 2-TMB-53B6" and "CD45 gated 2-GS-53B6") initially show lower transduction rates, but can match the CAR+ percentage at the end of production (data for Figure 2A was obtained as described in Example 2 below for Figure 7A, flow cytometry analysis). Overall proliferation of T cells remains similar between all clones (see Figure 2B). These data together show that although CD45 is expressed on the surface of CAR T cells, surprisingly, CD45-gated CARs can be successfully produced and expressed in T cells without causing significant fratricide.

[0324] CD45-gated CAR protease cleavage and ligand binding To assess whether the CD45 gate could be released by the corresponding protease, the detection of the V5 tag embedded in the cleavable or non-cleavable linker was evaluated. Protease cleavage was performed by incubating CAR T cells in PBS with 2 micrograms / mL thrombin for 30 minutes at 37°. Successful cleavage of the protease linker was verified by the removal of the V5 tag after cleavage. As the data in Figure 3A show, removal of the V5 tag was only observed in the CD45 gate1-TMB-53B6 and CD45 gate2-TMB-53B6 clones after cleavage, indicating that the CD45 gate could be released from the CAR by specific cleavage by the protease.

[0325] Next, we evaluated whether the CD45 gate directly blocks ligand binding by evaluating the binding of soluble MUC16 ligand to each CAR clone before or after protease cleavage. We observed that 53B6, CD45 gate1-TMB-53B6, CD45 gate1-GS-53B6 showed very similar ligand binding strengths regardless of protease cleavage (see data in Figure 3B). CD45 gate2-TMB-53B6 and CD45 gate2-GS-53B6 showed reduced ligand binding strengths (see data in Figure 3B), likely due to reduced expression of the modified CAR molecule. Nevertheless, binding was not affected by protease cleavage and release of the CD45 gate. Overall, these data demonstrate that the CD45 gate does not block binding to soluble targets.

[0326] CD45 gated CAR T cytotoxicity To understand whether CD45 gating can regulate CAR function, two target cell lines, Ovcar3 (MUC16 high) and Cov644 (MUC16 low), were used to evaluate the cytotoxicity of each clone. Both target cells are integrated with a luciferase gene for easy quantification of viable cells. Briefly, CAR T cells were normalized to the same CAR+ percentage and incubated with 10,000 target cells at an E:T (effector:target) ratio of 3:1, 1:1, or 1:3 in 200 μL of RPMI medium with 10% FBS. After 48 hours of co-culture, 100 μL of supernatant containing CAR T cells was transferred to a new well with 10,000 fresh target cells, while the remaining sample was used to quantify viable target cells by luciferase assay. After another 48 hours of co-culture, a second cytotoxicity evaluation was performed similarly. As Figures 4A-4B show, CD45 gate 1 moderately inhibited CAR function to kill Ovcar3 (MUC16 high) target cells in the first cytotoxicity assay, and the inhibition became more pronounced in the second round. However, CD45 gate 2 showed very limited inhibition of CAR activity, and reduced cytotoxic activity was observed only at low E:T ratios (Figures 4A and 4B). In contrast, both CD45 gate 1 and CD45 gate 2 substantially inhibited CAR cytotoxicity against Cov644 (MUC16 low) target cells (Figures 5A-5B). These data together indicate that the CD45 gate can effectively control CAR function, and the level or extent of inhibition can be influenced by and vary with antigen density.

[0327] Modulation of CD45-gated CAR T cytotoxicity We next assessed whether protease cleavage and removal of the CD45 gate from the CAR could restore its cytotoxicity. To test this, a cytotoxicity assay was performed with MUC16 high Ovcar3 target cells as above, except that serum-free medium was used in this assay to allow thrombin activity. The cytotoxicity of each clone was evaluated against target cells in 1:1 E:T with or without thrombin protease (final concentration of 2 μg / mL). In this condition, regular 53B6 CAR T cells are still able to efficiently kill target cells, even though the activity was weaker than their previous performance in serum-rich medium. There is no change in 53B6 CAR activity when thrombin is introduced into the culture (Figure 6A). Importantly, data from CD45 gate1-TMB-53B6 and CD45 gate2-TMB-53B6 clones demonstrate that the CD45 gate effectively inhibited CAR activity, and cytotoxicity was nearly restored when the protease was present. In contrast, the CD45 gate in the CD45 gate1-GS-53B6 or CD45 gate2-GS-53B6 clones could not be efficiently released, and thus CAR activity remained inhibited despite the presence of the protease.

[0328] Example 2. Expression and activity of anti-MUC16 CAR CAR T production T cells were isolated from human peripheral blood mononuclear cells (PBMCs) obtained by density gradient centrifugation (Ficoll Paque, GE Healthcare, Pittsburgh, PA) using a Pan T cell isolation kit (Miltenyi Biotec, Auburn, CA) and cryopreserved.

[0329] To generate lentivirus encoding 53B6 CAR, HEK-293T cells were seeded at 450,000 cells / mL in 2 mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone or JR Scientific) per well of a 6-well plate on day 0. Lentivirus was prepared by mixing together the lentiviral packaging vector, 1.5 μg of psPAX2, 0.5 μg of pMD2G, and 1 μg of the appropriate transport CAR vector in 250 μL of Opti-MEM (Gibco) per well of a 6-well plate on day 1 ("DNA mix"). 10 μL of Lipofectamine 2000 (Invitrogen) in 250 μL of Opti-MEM was incubated at room temperature for 5 minutes and then added to the DNA mix. The mixture was incubated at room temperature for 20 minutes and a total volume of 500 μL was slowly added to the side of the well containing HEK-293T. Purified T cells were activated in T cell transduction medium (X-Vivo-15 medium [Lonza] with 10% FBS) supplemented with 20 ng / mL human IL-2 (Miltenyi Biotec) and human T cell TransAct reagent (Miltenyi Biotec) as recommended by the manufacturer. On day 2, the medium from each well of the 6-well plate was replaced with 2 mL per well of T cell transduction medium. On day 3, T cells were resuspended in 1 mL of T cell transduction medium at 330,000 cells per mL. Lentiviral supernatant from HEK293T cells was harvested and passed through a 0.45 micron filter (EMD Millipore) to remove cellular debris, then added to 500,000 activated T cells with 20 ng / mL human IL-2. Alternatively, to increase transduction efficiency, lentiviruses can be prepared at a scaled-up ratio as the previous protocol and concentrated with a Lenti-X™ Concentrator (Takara) according to the manufacturer's protocol. Activated T cells were transduced at an MOI of approximately 5 to generate CAR T cells.

[0330] On day 5, 4.5 mL of T cell expansion medium (X-Vivo-15 with 5% human AB serum [Gemini Bio]) was added to each well of a Grex-24 (Wilson Wolf) plate. IL-2 (20 ng / mL) was replenished every 2-3 days. On days 9 and 13, transduction efficiency was determined by detecting the percentage of T cells that recognized recombinant MUC16 protein (in-house) using flow cytometry. On day 14, 53B6 CAR-T cells were cryopreserved.

[0331] Flow cytometry analysis (Figure 7A-7B) To determine the percentage of T cells successfully transduced with 53B6 CAR, T cells were first incubated with 3 μg / mL biotinylated recombinant MUC16 protein (in-house) in FACS buffer (PBS + 0.5% BSA, 2 mM EDTA) for 30 min at 4 °C. Cells were then washed with FACS buffer, stained with streptavidin-PE labeled protein (Thermo Fisher, Cat. No. 21627) at a ratio of 1:250, and analyzed using flow cytometry. As an example, Figure 7A shows that 53B6 CAR T cells were successfully generated from multiple productions using different T cell donors. Flow cytometry plots of 53B6 CAR T cells were from one of the productions. Empty vector (EV) control T cells were included in the study.

[0332] Alternatively, another recombinant MUC16 conjugated with Alexa Fluor® 647 was used to directly stain CAR T cells at a final concentration of approximately 3 μg / mL to assess binding of the 53B6 CAR in Figures 2A and 3B.

[0333] CAR T cell phenotype (Figure 7B) was also determined on day 13 according to expression of CD62L (Biolegend) and CD45RO (Biolegend) within the CAR+ T cell population as follows: stem cell memory (Tscm; CD45RO- / CD62L+), central memory (Tcm; CD45RO+ / CD62L+), effector memory (Tem; CD45RO+ / CD62L-), effector cell (Teff; CD45RO- / CD62L-). Staining also included lineage markers using antibodies against CD3 (BD ​​Biosciences), CD4 (Biolegend), and CD8 (Biolegend).

[0334] Cytotoxicity assay (Figures 8A-8C and Figures 9A-9B) Short-term killing assay This example describes experiments used to determine the specificity and in vitro activity of 53B6 CAR.

[0335] OVCAR3, COV644, and FUOV1 ovarian cell lines were purchased from ATCC or Sigma, and MUC16 protein surface expression was determined using a quantitative analysis kit (Agilent catalog number K007811-8) according to the manufacturer's recommendations. To determine the activity of 53B6 CAR T cells, 1 × 10 4 Luciferase-expressing ovarian target cells were co-cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS with 53B6 CAR+ T cells at defined effector:target (E:T) ratios in 96-well assay plates (Corning). Empty vector (EV) control T cells were also included in the study. Cell viability was measured after 72 hours using One-Glo reagent (Promega). Each condition was assayed in duplicate wells. Target cell viability after exposure to T cells was determined by comparing with target cells alone. The mean percentage of live cells and standard deviation are shown (Figures 8A-8C).

[0336] Long-term sequential killing assay Serial killing assays involve repeatedly exposing CAR T cells to their target, thereby expanding and, in certain cases, causing differentiation and exhaustion of CAR T cells.

[0337] To determine the sustained killing of 53B6 CAR T cells, 1 × 10 4 Luciferase-expressing OVCAR3 target cells were co-cultured with 53B6 CAR+T cells in RPMI1640 medium (Gibco) supplemented with 10% FBS at an effector:target ratio of 1:1 in a sequential killing assay using 96-well assay plates. Every 2–3 days, 100 μL of medium from each well was then transferred to freshly seeded target cells (1 × 10 per well). 4 The viability of previously seeded target cells after transfer into 53B6 CAR T cells (1000 cells) and exposure to CAR T cells was determined by comparing to target cells alone using One-Glo reagent. Each condition was assayed in 3-6 replicates. Empty vector (EV) control T cells were also included in the study. The mean percentage of viable cells and standard deviation are shown. Data presented is the activity of 53B6 CAR T cells from two different donors (Figures 9A-9B).

[0338] In vivo activity (Figures 10A-10B) Two different orthotopic ovarian tumor models were used to test the antitumor activity of 53B6 CAR T cells. Luciferase-labeled OVCAR3 and COV644 ovarian cells were cultured in complete growth medium (RPMI 1640 supplemented with 10% FBS). Cells were harvested and cultured at 15 × 10 6 The cells were diluted to 10 viable cells / mL and the cell suspension was kept on ice until inoculation. 200 μL (3 × 10 per mouse) was added to 7-8 week-old NSG mice (Jackson Laboratory). 6Animals were injected intraperitoneally (IP) with 100 μL of OVCAR3 or COV644 (10 target cells). Tumor burden was monitored using IVIS Spectrum (Perkin Elmer), and animals were injected IP with 200 μL of luciferin (15 mg / mL, Pierce) and bioluminescence intensity was measured. Mice were randomized into five groups based on whole body bioluminescence approximately 2 weeks after tumor implantation. 53B6 CAR T cells were then thawed and resuspended in PBS. OVCAR3 tumor-bearing mice were injected with 1 × 10 per animal. 6 53B6 CAR+T cells per animal, while COV644 tumor-bearing mice received 3 × 10 6 Each animal received 53B6 CAR+ T cells. Empty vector control T cells were also included in the study. All animals injected with T cells were administered via ip injection in a volume of 200 μL / mouse. The in vivo activity of 53B6 CAR T cells against tumors was monitored by bioluminescence, and the results demonstrated antitumor activity in both models (Figures 10A-10B).

[0339] Example 3. Testing CD45-gated CAR T cell activity 3.1. Generation of new CD45-gated CAR T (effector) cells (Figures 12A-12D and Figures 13A-13D) CAR T cells expressing a CD45-gated MUC16 CAR (similar to the CD45-gated CAR illustrated in FIG. 11) containing a TPS linker selected from a panel of TPS linkers were successfully generated and characterized using flow cytometry according to the methods described in Example 2 and below. In addition to cells expressing a CAR containing one of the cleavable TPS linkers, cells expressing a CAR containing a non-cleavable GS linker were prepared as a negative control that is expected to not express CAR activity because the CD45 gate cannot be released by proteases. Cells expressing a non-gated MUC16 CAR (53B6) were included as a second positive control that is expected to be active in the presence or absence of proteases.

[0340] Each CAR+ T cell population was detected with recombinant MUC16 protein (SEA1-4) conjugated to AlexaFluor 647 using flow cytometry (Figure 12A). The overall proliferation of T cells remained similar among all clones, comparable to the non-transduced control (NTD), and showed no obvious fratricide (Figure 12B). The autoactivation and memory phenotype of CAR T cells was also determined using flow cytometry by analyzing the expression of CD25 / 41BB and CD45RO / CD62L on days 9 and 14, respectively. Overall, CD45-gated CAR T cells contained a higher proportion of stem cell memory subsets (CD45RO- / CD62L+, Figure 12C) and showed less sustained signaling (CD25+ / 41BB+, Figure 12D) than naked CAR T cells, suggesting a potential benefit of CD45-gated CARs in controlling T cell exhaustion. At the end of production (days 14-16), all T cells were cryopreserved in 90% FBS / 10% DMSO using a rate-controlled freezing chamber and stored in liquid nitrogen.

[0341] A second set of CAR T cell populations, each expressing a CD45-gated MUC16 CAR comprising a TPS linker selected from a second panel of TPS linkers, were successfully generated and characterized according to the methods described in Example 2 and in this section (results shown in Figures 13A-13D). The successful generation of CD45-gated CAR T cells was surprising since the T cells express endogenous CD45, yet no significant fratricide was observed that would have prevented the production of CD45-gated CAR T cells.

[0342] 3.2. Generation and Analysis of Reagent Cell Lines (Figures 14A-14E) Target cell lines were developed in-house based on three cell lines positive for tumor-associated proteases (i.e., expressing active tumor-associated proteases), including ovarian cancer cell line OVCAR3, breast cancer cell line MDA-MB-231, and lung cancer cell line NCI-H292. Surface expression of matriptase (3 μg / mL, R&D, BAF3946) was confirmed using flow cytometry (Figure 14A), and secretion of urokinase (uPA) was detected in the conditioned medium by ELISA kit (R&D, DUPA00) (Figure 14D). In addition, as already described in Example 1, the activity of surface-expressed matriptase was evaluated through detection of the V5 tag attached to CAR via the matriptase-cleavable linker TPS6 (Figure 14B). Compared to recombinant matriptase, which effectively mediated linker cleavage and V5 tag removal, endogenous matriptase showed limited activity, which, without wishing to be bound to a particular mechanism, is believed to be due to a lack of activation of matriptase in the in vitro assay setting. For example, Figure 14B shows that in this experiment, in vitro matriptase was not active, consistent with a CD45-gated CAR, and TPS6 was also not active.

[0343] To study target-mediated CAR activation, MDA-MB-231 and H292 cell lines engineered to overexpress human MUC16 SEA1-4 and OVCAR3 cell lines expressing endogenous MUC16 were used as target cells. Expression of the target protein MUC16 was verified by flow cytometry (Figure 14C). All three cell lines were then engineered to co-express firefly luciferase and nuclear GFP using the 2A peptide to allow sensitive measurement of tumor killing using luminescence readout or Incucyte imaging.

[0344] 3.3 In vitro analysis of CD45-gated CAR T cell activity, protease dependency, and sensitivity (Figures 15A-15F and Figures 16A-16F) The activity of CD45-gated CAR was evaluated using the Jurkat-NFAT reporter cell line. The Jurkat-NFAT reporter cell line contains the nuclear factor of activated T cells (NFAT) promoter upstream of the luciferase gene, which allows for quantitative measurement of signaling activation after stimulation. To generate CAR reporter cells, Jurkat-NFAT cells were transduced with lentivirus encoding CD45-gated CAR and expression was analyzed after 6 days using recombinant MUC16 protein (SEA1-4). The transduced cells were then mixed with target cells (1 × 10 ) at an E:T ratio of 1:1 in 96-well white flat-bottom plates. 4 Cells were co-cultured with Jurkat cells (0.2 μg / mL / well). Wells containing only Jurkat cells were also included as negative controls. All co-cultures were performed in CTS OpTmizer T cell proliferation SFM (Gibco) with or without human recombinant matriptase (0.2 μg / mL, R&D, 3946-SEB-010). After overnight incubation at 37° C., reporter gene activation was detected by luminescence and normalized to the untreated control signal when no target cells were added.

[0345] As shown in Figures 15A-15F, exposure to MUC16-positive target cells resulted in activation of the 53B6 CAR, whereas no activation was detected for the CD45-gated CAR with a non-cleavable linker. However, activation could be restored via the cleavable linker after exposure to endogenous ("-Matriptase" in Figures 15A-15C) or exogenous ("+Matriptase" in Figures 15A-15C) proteases. This indicates that the CD45 gate inhibits CAR activity, and that inhibition can be protease sensitive if the linker contains one or more protease sites. The data includes clones 1241 (2xTPS6), 1466 (2xTPS6), 1467 (2xTPS6), 1468 (2xTPS6), and 1469 (2xTPS6), each of which utilizes linkers with various lengths (45, 35, 30, 25, 20mer), yet they allow very similar functionality. The following constructs were used in Figures 15D-15F: 0975 (TMB) refers to N-HA-4131scFv-V5-2G4STMB-53B6, which contains the linker GSTMB (45 aa) having the amino acid sequence of SEQ ID NO: 16 (SEQ ID NO: 8). 0976(NC) refers to SEQ ID NO:17 (non-cleavable) 1102 (2xTPS3) is a nucleotide sequence of SEQ ID NO: 24 (SEQ ID NO: 9) containing the linker GSTPS1 (45 aa) and the linker TPS truncation having the amino acid sequence of SEQ ID NO: 53 flanked at both ends by GS sequences, N-HA-4131-VL-RS-VH-TPS1-53B6 1399 (TPS4) refers to SEQ ID NO: 137, which is a construct containing the element V5-GS20-4131scFv-TPS4-53B6, including the linker TPS4 45aa (SEQ ID NO: 107) and the GS sequence 1401 (TPS6) refers to SEQ ID NO: 138, which is a construct containing the element V5-GS20-4131scFv-TPS6-53B6, including the linker TPS6 45aa with the amino acid sequence of SEQ ID NO: 113 1241 (2xTPS6) refers to SEQ ID NO: 144, which is a construct containing the element N-HA-4131scFv-2xTPS6-53B6, which contains the linker TPS6 35aa with the amino acid sequence of SEQ ID NO: 112 and a second linker containing the cleavage site MTSP(3) (SEQ ID NO: 95) flanked at both ends by GS sequences 1466 (2xTPS6) refers to SEQ ID NO: 153, which is a construct containing the element V5-GS20-4131scFv-2XTPS6-35-53B6, which contains two copies of the linker TPS6 35aa, each copy having the amino acid sequence of SEQ ID NO: 112 1467 (2xTPS6) refers to SEQ ID NO: 154, which is a construct containing the element V5-GS20-4131scFv-2XTPS6-30-53B6, including the linker TPS6 35aa (SEQ ID NO: 112) and the linker TPS6 30aa (SEQ ID NO: 111). 1468 (2xTPS6) refers to SEQ ID NO: 155, which is a construct containing the element V5-GS20-4131scFv-2XTPS6-25-53B6, including the linker TPS6 35aa (SEQ ID NO: 112) and the linker TPS6 25aa (SEQ ID NO: 110). 1469 (2xTPS6) refers to SEQ ID NO: 156, which is a construct containing the element V5-GS20-4131scFv-2XTPS6-20-53B6, including the linker TPS6 35aa (SEQ ID NO: 112) and the linker TPS6 20aa (SEQ ID NO: 109).

[0346] 3.4. Cytotoxicity of CD45-gated CAR T cells The short-term cytotoxicity of CD45-gated CAR T cells was characterized using Incucyte. For kinetic analysis of tumor cell killing, target cells MDA-MB-231-MUC16, H292-MUC16, and OVCAR3 cells engineered to express nuclear GFP were plated at 1 × 10 per well in 96-well black flat-bottom plates. 4The following day, T cells were thawed and added at an effector:target (E:T) ratio of 3:1. All co-cultures were performed in CTS OpTmizer T cell proliferation SFM (serum-free medium, Gibco) with or without human recombinant matriptase (0.2 μg / mL, R&D, 3946-SEB-010). Viable cell numbers were monitored by fluorescent imaging every 6 h for 60 h using an IncuCyte Live Cell Analysis System (Essen BioScience). Viable cell numbers were quantified by IncuCyte S3 software (Essen BioScience) and normalized to the initial count at time 0 (time zero). The mean fold change and standard deviation of target cell numbers are shown in Figures 16A-16F. CD45-gated CAR with a non-cleavable GS linker effectively inhibited CAR function. In the presence of endogenous proteases ("w / o [without] exogenous matriptase") (top panels of Figures 16A-16F), CD45-gated CARs with various TPS linkers showed a spectrum of activity, with some of the linkers (e.g., TPS4) showing activity comparable to the non-gated 53B6 CAR. In the presence of exogenous matriptase ("w / [with] exogenous matriptase") (bottom panels of Figures 16A-16F), all CD45-gated CARs with TPS linkers showed cytotoxic activity. Figures 16A-16C show results using linkers from the first linker set (TPS3, TPS4, TPS6) plus controls. Figures 16D-16F show results using linkers from the first and second linker sets (TPS3, TPS4, TPS6, TPS8-TPS13) plus controls.

[0347] 3.5 Long-term killing assay (Figures 17A-17B) To determine the sustained killing of CD45-gated CAR T cells after repeated exposure to targets, luciferase-expressing OVCAR3 cells were plated at 1 × 10 per well in 96-well white flat-bottom plates. 4CAR T cells were seeded in CTS OpTmizer T cell expansion SFM (Gibco) at a concentration of 1 × 10 cells. The next day, T cells were thawed and added at an E:T ratio of 3:1. All co-cultures were performed with or without human recombinant matriptase (0.2 μg / mL, R&D, 3946-SEB-010). Every 2–3 days, half of the supernatant containing CAR T cells from each well was aliquoted onto freshly seeded target cells (1 × 10 4 Cells were transferred to a 5x10 well plate (10 cells / well) and target cell viability was measured by luminescence using the One-Glo assay (Promega) as previously described. Data presented show that CD45-gated CAR T cells with cleavable linkers maintained long-term cytotoxicity in the presence of endogenous (Figure 17A) or exogenous (Figure 17B) proteases.

[0348] In vivo assay (Figures 18A-18C and Figures 19A-19C) The in vivo antitumor efficacy of CD45-gated CAR T cells was evaluated in a breast cancer xenograft model (Figure 18A shows a schematic of the experiment). Seven to eight-week-old NSG mice (Jackson Laboratory) were inoculated with 200 μL of MDA-MB-231-MUC16 cells (5 × 10 cells mixed with Matrigel at a 1:1 ratio). 6 The mean tumor size was 290 mm on day 17. 3 Mice were randomized into six groups (n=7-8) when tumor size reached 1.5 mg / kg / day. Each animal received 5 million CAR+ cells via tail vein injection. The total number of T cells was kept constant across all groups by normalizing with non-transduced T cells. Tumors were measured twice weekly and tumor volume was calculated as length (mm) × width (mm) × height (mm) / 2. Statistical analysis of observed differences was performed by one-way ANOVA followed by Dunnett's test for multiple comparisons.

[0349] As shown in Figure 18B, CD45 gated CARs that only contain non-cleavable GS linkers in MDA-MB-231 models cannot activate anti-tumor CAR activity. In contrast, anti-tumor CAR activity can be activated with CD45 gated CARs that have cleavable linkers (e.g., TPS4). Tumor growth was reduced in mice treated with CD45 gated CAR αCD45-TPS4-53B6.

[0350] Treatment with CD45-gated CAR T cells did not affect mouse body weight (Figure 18C).

[0351] The in vivo antitumor efficacy of CD45-gated CAR T cells was also evaluated in a non-small cell lung cancer xenograft model (Figure 19A shows a schematic of the experiment). NSG mice (Jackson Laboratory) were injected with 200 μL of NCI-H292-MUC16 cells (6 × 10 6 The average tumor size was 150 mm on day 14. 3 When the mice reached 100 mg / kg, they were randomized into six groups (n = 6–7). CAR T cells were then thawed and immediately infused via tail vein injection (5 × 10 per animal). 6 CAR+ cells). The total number of T cells was kept constant across all groups by normalizing with non-transduced T cells. Tumors were measured twice weekly and tumor volume was calculated as length (mm) x width (mm) x height (mm) / 2. Statistical analysis of observed differences was performed by one-way ANOVA followed by Dunnett's test for multiple comparisons. Without wishing to be limited to a particular mechanism, the results likely reflect varying levels of protease activity at the tumor site in this in vivo tumor model.

[0352] As shown in Figure 19B, CD45-gated CARs with non-cleavable GS linkers lacked any CAR anti-tumor activity in the H292 model. In contrast, CD45-gated CARs with cleavable linkers (TPS4) showed anti-tumor activity.

[0353] Treatment with CD45-gated CAR T cells did not affect the body weight of mice (Figure 19C), indicating that CD45-gated CAR T cells did not result in general toxicity in vivo.

[0354] References 1.Ledbetter JA, Tonks NK, Fischer EH, Clark EA.CD45 regulates signal transduction and lymphocyte activation by specific association with receptor molecules on T or B cells.Proc Natl Acad Sci US A.1988.doi:10.1073 / pnas.85.22.8628 2.McNeill L, Salmond RJ, Cooper JC, et al.The Differential Regulation of Lck Kinase Phosphorylation Sites by CD45 Is Critical for T Cell Receptor Signaling Responses.Immunity.2007.doi:10.1016 / j.immuni.2007.07.015 3.Felberg J, Johnson P.Characterization of Recombinant CD45 Cytoplasmic Domain Proteins.J Biol Chem.1998.doi:10.1074 / jbc.273.28.17839 4.Davis SJ, van der Merwe PA.The kinetic-segregation model:TCR triggering and beyond.Nat Immunol.2006.doi:10.1038 / ni1369 5.Chang VT,Fernandes RA,Ganzinger KA,et al.Initiation of T cell signaling by CD45 segregation at“close contacts.”Nat Immunol.2016.doi:10.1038 / ni.3392 6.Razvag Y,Neve-Oz Y,Sajman J,Reches M,Sherman E.Nanoscale kinetic segregation of TCR and CD45 in engaged microvilli facilitates early T cell activation.Nat Commun.2018.doi:10.1038 / s41467-018-03127-w 7.Thiel N,Zischke J,Elbasani E,Kay-Fedorov P,Messerle M.Viral interference with functions of the cellular receptor tyrosine phosphatase CD45.Viruses.2015.doi:10.3390 / v7031540 8.Payne KK,Mine JA,Biswas S,et al.BTN3A1 governs antitumor responses by coordinating ab and gd T cells.Science(80-).2020.doi:10.1126 / science.aay2767 9.Bakalar MH,Joffe AM,Schmid EM,Son S,Podolski M,Fletcher DA.Size-Dependent Segregation Controls Macrophage Phagocytosis of Antibody-Opsonized Targets.Cell.2018.doi:10.1016 / j.cell.2018.05.059 10.Weidle UH,Tiefenthaler G,Georges G.Proteases as activators for cytotoxic prodrugs in antitumor therapy.Cancer Genomics and Proteomics.2014. 11.Gialeli C,Theocharis AD,Karamanos NK.Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting.FEBS J.2011.doi:10.1111 / j.1742-4658.2010.07919.x 12.Geiger M,Stubenrauch KG,Sam J,et al.Protease-activation using anti-idiotypic masks enables tumor specificity of a folate receptor 1-T cell bispecific antibody.Nat Commun.2020.doi:10.1038 / s41467-020-16838-w 13.Desnoyers LR,Vasiljeva O,Richardson JH,et al.Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index.Sci Transl Med.2013.doi:10.1126 / scitranslmed.3006682 14.Sommer C,Cheng HY,Nguyen D,et al.Allogeneic FLT3 CAR T Cells with an Off-Switch Exhibit Potent Activity against AML and Can Be Depleted to Expedite Bone Marrow Recovery.Mol Ther.2020.doi:10.1016 / j.ymthe.2020.06.022 15.Sommer C,Boldajipour B,Kuo TC,et al.Preclinical Evaluation of Allogeneic CAR T Cells Targeting BCMA for the Treatment of Multiple Myeloma.Mol Ther.2019.doi:10.1016 / j.ymthe.2019.04.001

Claims

1. A protease-activated CD45-gated chimeric antigen receptor (CD45-gated CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain is a CD45 recruitment domain; and An antigen-binding domain; a linker connecting the carboxy terminus of the CD45 recruitment domain to the amino terminus of the antigen binding domain, the linker comprising one or more protease cleavage sites recognized by at least one protease; Optionally, the intracellular domain comprises at least one signaling domain that can be reversibly inactivated by CD45.

2. 2. The protease-activated CD45-gated CAR of claim 1, wherein the CD45 recruitment domain comprises one or more of an anti-CD45 scFv, an anti-CD45 antibody antigen-binding domain, an anti-CD45 nanobody, a viral protein binder of CD45, a truncated viral protein binder of CD45, an anti-CD45 Fab, an anti-CD45 camelid VHH, a CD45 binding protein, full length or truncated UL11, full length or truncated sec49K, and full length or full length or truncated BTN3A1.

3. 3. The protease-activated CD45-gated CAR of claim 1 or claim 2, wherein the CD45 recruitment domain comprises an anti-CD45 antibody antigen-binding domain, optionally wherein the anti-CD45 antibody antigen-binding domain comprises an anti-CD45 scFv.

4. The protease-activated CD45-gated CAR according to any one of claims 1 to 3, wherein the CD45 recruitment domain comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:56, or SEQ ID NO:56 without the signal sequence of SEQ ID NO:

1.

5. The antigen binding domain is selected from the group consisting of BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUC1, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, MHC-NY-ESO1, HER2 (ERBB2), CAIX (carbonic anhydrase IX), LIV1, ADAM10, CHRNA2, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, claudin-18.2 (claudin-18A2, or claudin-18 isoform). The protease-activated CD45-gated CAR according to any one of claims 1 to 4, which specifically binds to DLL3 (delta-like protein 3, Drosophila delta homolog 3, delta 3), Mud7 (mucin 17, Muc3, Muc3), FAP alpha (fibroblast activation protein alpha), Ly6G6D (lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGT1, NG25), PSMA, MSLN, or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43).

6. The protease-activated CD45-gated CAR according to any one of claims 1 to 5, wherein the linker comprises a GS sequence at either or both ends of each of the one or more protease cleavage sites.

7. The protease-activated CD45-gated CAR according to any one of claims 1 to 6, wherein the linker comprising at least one protease cleavage site has a length of 15 to 100 amino acids, 30 to 100 amino acids, 15 to 75 amino acids, 20 to 50 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids, or about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids, or about 50 amino acids.

8. The protease-activated CD45-gated CAR of any one of claims 1 to 7, wherein the one or more protease cleavage sites can be cleaved by at least one protease present in a tumor microenvironment.

9. The linker is selected from the group consisting of serine proteases, cysteine-type lysosomal proteases, metalloproteinases, coagulation factor proteases, aspartyl-type lysosomal proteases, matrix metalloproteinases (MMPs), matriptase (MT-SP1), trypsin, plasmin, prostate specific antigen (PSA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR), legumain, a disintegrin and metalloproteinase (ADAM), transmembrane serine proteases (TMPRSS), granzyme B, activating proteins The protease-activated CD45-gated CAR according to any one of claims 1 to 8, comprising at least one protease cleavage site that can be cleaved by C, caspase, cathepsin, chymase, elastase, guanidinobenzoatase, HtrAl, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, tissue plasminogen activator (tPA), thrombin, DESC1, DPP-4, FAP, hepsin, matriptase-2, secretase, kallikrein-related peptidase (KLK), or tryptase.

10. The protease-activated CD45-gated CAR according to any one of claims 1 to 9, wherein the linker comprises one or more protease cleavage sites comprising the amino acid sequence of SEQ ID NO: 32, 91-98, 103-105, or SEQ ID NO:

106.

11. The protease-activated CD45-gated CAR according to any one of claims 1 to 10, wherein the linker comprises one or more amino acid sequences of SEQ ID NOs: 8 to 10, 53, 89, 90, 99 to 102, 107 to 120, 172 to 176, or SEQ ID NO:

177.

12. The protease-activated CD45-gated CAR according to any one of claims 1 to 11, wherein the linker comprises two or more protease cleavage sites, each cleavage site being the same as or different from any of the other cleavage sites.

13. The protease-activated CD45-gated CAR of any one of claims 1 to 12, wherein the extracellular domain further comprises a stalk domain between the antigen-binding domain and the transmembrane domain.

14. The protease-activated CD45-gated CAR according to any one of claims 1 to 13, wherein the intracellular domain comprises one or more cytoplasmic signaling domains of CD3 zeta, CD28, PD1, and CD2, and optionally, the intracellular domain may comprise a CD3 zeta domain comprising the amino acid sequence of SEQ ID NO: 34 or a fragment thereof.

15. The intracellular domain comprises at least one costimulatory domain, and at least one costimulatory domain is selected from the group consisting of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function associated antigen-1 (LFA-1 (CD1 la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor Body, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 Ia, LFA-1, ITGAM, CD1 Ib, ITGAX, CD1 Ic, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL , DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT The protease-activated CD45-gated CAR according to any one of claims 1 to 14, wherein the signal transduction region is a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

16. The protease-activated CD45-gated CAR according to any one of claims 1 to 15, wherein the protease-activated CD45-gated CAR comprises an amino acid sequence of any one of SEQ ID NOs: 11, 16-31, 56, 58, 59, 75, 76, 77, 78, 79, and 121-170, or variants thereof.

17. A protease-activated CD45-gated CAR as described in claim 16, wherein the variant does not contain the amino acid sequence of the HA tag of SEQ ID NO: 2, and / or does not contain the amino acid sequence of the V5 peptide motif of SEQ ID NO: 55, and / or does not contain the signaling sequence of SEQ ID NO:

1.

18. A nucleic acid encoding the protease-activated CD45-gated CAR of any one of claims 1 to 17.

19. A vector comprising the nucleic acid of claim 18.

20. 20. The vector of claim 19, wherein the vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof, optionally wherein the vector is an expression vector.

21. An engineered immune cell comprising the nucleic acid of claim 18 or the vector of claim 19 or 20, or comprising or functionally expressing a protease-activated CD45-gated CAR of any one of claims 1 to 17.

22. 22. The engineered immune cell of claim 21, wherein the immune cell is a T cell, a tumor infiltrating lymphocyte (TIL), an iPSC-derived T cell, an NK cell, a TCR-expressing cell, a dendritic cell, or an NK-T cell.

23. 23. The engineered immune cell of claim 22, wherein the cell is an autologous or allogeneic T cell.

24. At least about 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , or 1 × 10 8 A cell population containing cells.

25. A pharmaceutical composition comprising a cell according to any one of claims 21 to 23 or a cell population according to claim 24, optionally comprising a pharma- ceutically acceptable carrier.

26. 26. The pharmaceutical composition of claim 25 for treating a disease or condition in a patient.

27. ​​The pharmaceutical composition of claim 26, wherein the disease or condition is cancer, and optionally the cancer may be a solid tumor cancer or a liquid tumor cancer.

28. A pharmaceutical composition comprising a cell according to any one of claims 21 to 23 or a cell population according to claim 24 for use in a method for reducing on-target off-tumor toxicity of CAR T cells or reducing the occurrence of side effects in CAR T cell therapy, comprising 11. The pharmaceutical composition of claim 10, wherein the method comprises administering the cell or the cell population to a patient, wherein the on-target, off-tumor toxicity is lower than the incidence of on-target, off-tumor toxicity or side effects of a control cell, a control cell population, or a composition of control cells, each administered under the same conditions, and wherein the control cell, the control cell population, or the control cell of the composition comprises a CAR that lacks at least one of a CD45 recruitment domain and a linker comprising a protease cleavage site.

29. A method for regulating a cytotoxic activity of a CAR T cell comprising the CAR of any one of claims 1 to 17, the method being an in vitro method comprising: inhibiting the cytotoxic activity of the CAR T cell by associating the CAR with CD45 of the CAR T cell; and activating the cytotoxic activity of the CAR T cell by subjecting the CAR T cell to a protease that recognizes the protease cleavage site.

30. 1. A MUC16-specific chimeric antigen receptor (CAR) comprising an extracellular ligand-binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular ligand-binding domain comprises a single chain variable fragment (scFv) having binding specificity for the extracellular domain of MUC16.

31. the extracellular ligand-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; The heavy chain variable (VH) region comprises: (i) a VH complementarity determining region 1 (VHCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 60 or 63, (ii) a VH complementarity determining region 2 (VHCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 61 or 64, and (iii) a VH complementarity determining region 3 (VHCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 62 or 65; and / or The light chain variable (VL) region comprises: The MUC16-specific chimeric antigen receptor (CAR) of claim 30, comprising: (i) a VL complementarity determining region 1 (VLCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 66; (ii) a VL complementarity determining region 2 (VLCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 67; and (iii) a VL complementarity determining region 3 (VLCDR3) comprising the amino acid sequence set forth in SEQ ID NO:

68.

32. 32. The MUC16-specific chimeric antigen receptor (CAR) of claim 30 or 31, wherein the extracellular ligand-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the heavy chain variable (VH) region comprises the amino acid sequence set forth in SEQ ID NO: 58 and / or the light chain variable (VL) region comprises the amino acid sequence set forth in SEQ ID NO:

59.

33. A MUC16-specific chimeric antigen receptor (CAR) described in any one of claims 30 to 32, further comprising a rituximab mimotope sequence of sequence number 50.

34. The MUC16-specific chimeric antigen receptor (CAR) of any one of claims 30 to 33, wherein the MUC16-specific CAR comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 11, 75, 76, 77, 78, and 79, with or without a signal sequence.

35. An isolated polynucleotide comprising a nucleic acid sequence encoding the MUC16-specific CAR of any one of claims 30 to 34.

36. 36. An expression vector comprising the polynucleotide of claim 35.

37. An engineered immune cell comprising the polynucleotide of claim 35 or the vector of claim 36.

38. An engineered immune cell comprising or functionally expressing the MUC16-specific CAR of any one of claims 30 to 34.

39. 39. The method of claim 37 or 38, 3 ~Approx. 1×10 10 , or about 1×10 4 ~Approx. 1×10 9 A cell population comprising engineered immune cells.

40. 40. A pharmaceutical composition comprising the engineered immune cell of claim 37 or 38 or the cell population of claim 39 and a pharma- ceutically acceptable carrier.

41. 41. The pharmaceutical composition of claim 40, for treating a condition in a patient, optionally wherein the condition is cancer, an autoimmune disease, or an infectious disease.

42. The condition is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndromes (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM), biliary tract cancer, bladder cancer, bone or soft tissue cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal carcinoma, endometrial cancer, 42. The pharmaceutical composition of claim 41, wherein the cancer is selected from the group consisting of cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumors, head and neck squamous cell carcinoma, liver cancer, lung cancer, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, renal cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, and uterine cancer.

43. 43. The pharmaceutical composition of claim 41 or 42, wherein the engineered immune cell, the population of engineered immune cells, or the engineered immune cells of the pharmaceutical composition are derived from one or more immune cells from the patient, or from one or more allogeneic immune cells from a donor other than the patient.

44. 35. A method of in vitro engineering an immune cell that expresses the MUC16-specific CAR of any one of claims 30 to 34, said method comprising providing an immune cell and introducing into said cell at least one polynucleotide encoding said MUC16-specific CAR, whereby said immune cell expresses said MUC16-specific CAR.

45. An isolated MUC16-specific antibody comprising a heavy chain variable (VH) region and a light chain variable (VL) region, The heavy chain variable (VH) region comprises: (i) a VH complementarity determining region 1 (VHCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 60 or 63, (ii) a VH complementarity determining region 2 (VHCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 61 or 64, and (iii) a VH complementarity determining region 3 (VHCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 62 or 65; and / or The light chain variable (VL) region comprises: An antibody comprising: (i) a VL complementarity determining region 1 (VLCDR1) having the amino acid sequence set forth in SEQ ID NO:66; (ii) a VL complementarity determining region 2 (VLCDR2) having the amino acid sequence set forth in SEQ ID NO:67; and (iii) a VL complementarity determining region 3 (VLCDR3) having the amino acid sequence set forth in SEQ ID NO:

68.

46. ​​The antibody of claim 45, wherein the heavy chain variable (VH) region comprises the amino acid sequence of SEQ ID NO:58 and / or the light chain variable (VL) region comprises the amino acid sequence of SEQ ID NO:

59.

47. An isolated polynucleotide encoding a MUC16-specific antibody according to claim 45 or 46.

48. A vector comprising the polynucleotide described in claim 47.

49. A host cell comprising the polynucleotide of claim 47 or the vector of claim 48.

50. A host cell expressing the antibody described in claim 45 or 46.

51. A pharmaceutical composition for treating a MUC16-related disease or condition, comprising an antibody described in claim 45 or 46.

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