Tn-MUC1 chimeric antigen receptor (CAR) T cell therapy

Abstract

Various TnMUC1-specific chimeric antigen receptors (CARs), nucleic acids encoding same, and methods of using same are provided. Compositions and methods comprising TnMUC1-specific CARs for treating MUC1-associated cancers in subjects in need thereof are provided. TIFF2022527297000038.tif122166

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 824,532, filed March 27, 2019, and U.S. Provisional Patent Application No. 62 / 881,269, filed July 31, 2019, which applications are incorporated by reference herein in their entireties. [Background technology]

[0002] Background of the Invention Chimeric antigen receptor (CAR) T cells are effector immune cells that have been genetically modified to recognize specific tumor-associated antigens and subsequently kill tumor cells. Although the success of CAR T therapy has led to its approval for use in hematological malignancies, the efficacy of CAR T therapy in treating solid tumors such as breast cancer remains uncertain. CAR T therapy in solid tumors faces several obstacles. First, most of the identified and most studied cell-expressed antigens expressed by tumors are also expressed by normal tissues, resulting in nonspecific targeting by CAR T cells (off-tumor activity). Second, solid tumors generally possess an immunosuppressive tumor microenvironment, which may inhibit CAR T cell activity after they reach the tumor and recognize antigens. Third, the durability of antitumor responses is highly correlated with the persistence of adoptively transferred cells, and even the highest persistence of CAR T cells in solid tumors still falls short of that observed in hematopoietic malignancies.

[0003] Identifying tumor-specific antigens is essential for the continued application of CAR T cell therapy to solid tumors. There is a need for novel compositions and methods for treating solid tumors, such as breast cancer. The present invention fulfills this need. Summary of the Invention

[0004] overview Mucin 1 (MUC1) is a cell surface mucin that typically undergoes sequential glycan addition to form highly glycosylated proteins (Figure 1). The O-glycosylation process begins with the addition of GalNAc to serine and threonine residues. Elongation begins with the addition of galactose by core 1 synthase (composed of C1GalT1 and its chaperone C1GalT1C1 (Cosmc)) or GlcNAc by core 3 synthase (B3GNT6). Aberrations in this sequential glycosylation, such as epigenetic silencing of Cosmc, can result in an underglycosylated product, Tn-MUC1, which can then be sialic acid-added by ST6GALNAC-1 to form STn-MUC1.

[0005] The present disclosure is based on the discovery that CAR T cells against Tn-MUC1 exhibited potent cytolytic activity against various cancer cell lines in vitro and significant tumor eradication in vivo. In one aspect, modified immune cells or their precursors are provided, comprising a chimeric antigen receptor (CAR) that specifically binds to MUC1, wherein the CAR comprises a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a transmembrane domain; a costimulatory signaling domain; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0006] In certain exemplary embodiments, the MUC1-specific antigen-binding domain is specific for a glycoepitope of MUC1. In certain exemplary embodiments, the MUC1-specific antigen-binding domain is specific for a truncated glycoepitope of MUC1.

[0007] In certain exemplary embodiments, the VH domain comprises the amino acid sequence set forth in SEQ ID NO:5. In certain exemplary embodiments, the VL domain comprises the amino acid sequence set forth in SEQ ID NO:6. In certain exemplary embodiments, the VH domain comprises the amino acid sequence set forth in SEQ ID NO:5 and the VL domain comprises the amino acid sequence set forth in SEQ ID NO:6. In certain exemplary embodiments, the MUC1-specific antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:4.

[0008] In certain exemplary embodiments, the transmembrane domain comprises a transmembrane region of a protein selected from the group consisting of a type I transmembrane protein, T-cell receptor alpha, beta, or zeta chain, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In certain exemplary embodiments, the transmembrane domain comprises a CD8 transmembrane region. In certain exemplary embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:7.

[0009] In certain exemplary embodiments, the costimulatory signaling domain comprises a costimulatory domain of a protein selected from the group consisting of a member of the TNFR superfamily, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD5, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, DAP10, DAP12, Lck, Fas, and any derivative or variant thereof. In certain exemplary embodiments, the costimulatory signaling domain is a CD2 costimulatory signaling domain. In certain exemplary embodiments, the costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO:28.

[0010] In certain exemplary embodiments, the intracellular signaling domain comprises a signaling domain of a protein selected from the group consisting of CD3 zeta, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing cytoplasmic receptor, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In certain exemplary embodiments, the intracellular signaling domain comprises the signaling domain of CD3 zeta. In certain exemplary embodiments, the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:30.

[0011] In certain exemplary embodiments, the CAR further comprises a leader sequence. In certain exemplary embodiments, the leader sequence is a CD8 leader sequence. In certain exemplary embodiments, the leader sequence comprises the amino acid sequence set forth in SEQ ID NO:48.

[0012] In certain exemplary embodiments, the CAR further comprises a hinge domain. In certain exemplary embodiments, the hinge domain is derived from a protein selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, a hinge comprising the amino acid sequence of CD8, and any combination thereof. In certain exemplary embodiments, the hinge domain is a CD8 hinge domain. In certain exemplary embodiments, the hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0013] In certain embodiments, the engineered immune cells further comprise dominant negative receptors and / or switch receptors.

[0014] In certain embodiments, the dominant negative receptor is a truncated variant of the wild-type protein associated with a negative signal. In one embodiment, the truncated variant of the wild-type protein associated with a negative signal comprises the amino acid sequence set forth in SEQ ID NO:76.

[0015] In certain embodiments, the switch receptor comprises a first domain derived from a first polypeptide associated with negative signaling; and a second domain derived from a second polypeptide associated with positive signaling. In one embodiment, the first domain comprises at least a portion of the extracellular domain of the first polypeptide associated with negative signaling, and the second domain comprises at least a portion of the intracellular domain of the second polypeptide associated with positive signaling. In one embodiment, the switch receptor further comprises a transmembrane domain of the switch receptor. In one embodiment, the transmembrane domain of the switch receptor comprises the transmembrane domain of the first polypeptide associated with negative signaling; or the transmembrane domain of the second polypeptide associated with positive signaling. In one embodiment, the first polypeptide associated with negative signaling is selected from the group consisting of CTLA4, PD-1, BTLA, TIM-3, and TGFβR. In one embodiment, the second polypeptide associated with positive signaling is selected from the group consisting of CD28, ICOS, 4-1BB, and IL-12R.

[0016] In one embodiment, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1; a transmembrane domain of the switch receptor comprising at least a portion of the transmembrane domain of CD28; and a second domain comprising at least a portion of the intracellular domain of CD28. In one embodiment, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:78. In one embodiment, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1; a transmembrane domain of the switch receptor comprising at least a portion of the transmembrane domain of PD1; and a second domain comprising at least a portion of the intracellular domain of CD28.

[0017] In one embodiment, the switch receptor comprises the amino acid sequence shown in SEQ ID NO:80.

[0018] In one embodiment, the first domain comprises at least a portion of the extracellular domain of PD1 comprising an alanine (A) to leucine (L) substitution at amino acid position 132.

[0019] In one embodiment, the switch receptor comprises the amino acid sequence shown in SEQ ID NO:82.

[0020] In one embodiment, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1 comprising an alanine (A) to leucine (L) substitution at amino acid position 132; and a second domain comprising at least a portion of the intracellular domain of CD28.

[0021] In one embodiment, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1 comprising an alanine (A) to leucine (L) substitution at amino acid position 132; and a second domain comprising at least a portion of the intracellular domain of 4-1BB.

[0022] In one embodiment, the switch receptor comprises the amino acid sequence shown in SEQ ID NO:86.

[0023] In one embodiment, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of TIM-3; and a second domain comprising at least a portion of the intracellular domain of CD28.

[0024] In one embodiment, the switch receptor comprises the amino acid sequence shown in SEQ ID NO:92.

[0025] In one embodiment, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of TGFβR; and a second domain comprising at least a portion of the intracellular domain of IL12Rα1.

[0026] In one embodiment, the switch receptor comprises the amino acid sequence shown in SEQ ID NO:88.

[0027] In one embodiment, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of TGFβR; and a second domain comprising at least a portion of the intracellular domain of IL12Rβ1.

[0028] In one embodiment, the switch receptor comprises the amino acid sequence shown in SEQ ID NO:90.

[0029] In certain exemplary embodiments, the modified cells are modified natural killer (NK) cells, modified natural killer T (NKT) cells, or modified T cells. In certain exemplary embodiments, the modified immune cells are modified T cells. In certain exemplary embodiments, the modified immune cells are autologous.

[0030] In another aspect, modified T cells are provided that comprise a chimeric antigen receptor (CAR) that specifically binds to MUC1, wherein the CAR comprises a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a hinge domain; a transmembrane domain; a CD2 costimulatory signaling domain; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0031] In another aspect, modified T cells are provided that comprise a chimeric antigen receptor (CAR) that specifically binds to MUC1, wherein the CAR comprises a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a hinge domain; a transmembrane domain; a CD2 costimulatory signaling domain comprising the amino acid sequence set forth in SEQ ID NO:28; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs:22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs:19, 20, and 21.

[0032] In another aspect, modified T cells are provided that comprise a chimeric antigen receptor (CAR) that specifically binds to MUC1, wherein the CAR comprises a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a CD8 hinge domain; a CD8 transmembrane domain; a CD2 costimulatory signaling domain comprising the amino acid sequence set forth in SEQ ID NO:28; and a CD3 zeta intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs:22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs:19, 20, and 21.

[0033] In another aspect, modified T cells are provided that comprise a chimeric antigen receptor (CAR) that specifically binds to MUC1, comprising the amino acid sequence set forth in SEQ ID NO:2, 39, 41, 43, 45, or 47.

[0034] In another aspect, an isolated nucleic acid sequence is provided encoding a chimeric antigen receptor comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a transmembrane domain; a costimulatory signaling domain; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0035] In certain exemplary embodiments, the MUC1-specific antigen-binding domain is specific for a glycoepitope of MUC1. In certain exemplary embodiments, the MUC1-specific antigen-binding domain is specific for a truncated glycoepitope of MUC1.

[0036] In certain exemplary embodiments, the VH domain comprises the amino acid sequence set forth in SEQ ID NO:5. In certain exemplary embodiments, the VL domain comprises the amino acid sequence set forth in SEQ ID NO:6. In certain exemplary embodiments, the VH domain comprises the amino acid sequence set forth in SEQ ID NO:5 and the VL domain comprises the amino acid sequence set forth in SEQ ID NO:6. In certain exemplary embodiments, the MUC1-specific antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:4.

[0037] In certain exemplary embodiments, the MUC1-specific antigen-binding domain is encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:3.

[0038] In certain exemplary embodiments, the transmembrane domain comprises a transmembrane region of a protein selected from the group consisting of a type I transmembrane protein, T-cell receptor alpha, beta, or zeta chain, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In certain exemplary embodiments, the transmembrane domain comprises a CD8 transmembrane region. In certain exemplary embodiments, the transmembrane domain is encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:8.

[0039] In certain exemplary embodiments, the costimulatory signaling domain comprises a costimulatory domain of a protein selected from the group consisting of a member of the TNFR superfamily, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD5, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, DAP10, DAP12, Lck, Fas, and any derivative or variant thereof. In certain exemplary embodiments, the costimulatory signaling domain is a CD2 costimulatory signaling domain. In certain exemplary embodiments, the costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO:28. In certain exemplary embodiments, the costimulatory signaling domain is encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:29.

[0040] In certain exemplary embodiments, the intracellular signaling domain comprises the signaling domain of CD3 zeta. In certain exemplary embodiments, the intracellular signaling domain is encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:31.

[0041] In certain exemplary embodiments, the CAR further comprises a CD8 leader sequence. In certain exemplary embodiments, the leader sequence comprises the amino acid sequence set forth in SEQ ID NO:48.

[0042] In certain exemplary embodiments, the CAR further comprises a CD8 hinge domain. In certain exemplary embodiments, the hinge domain is encoded by a nucleic acid sequence comprising a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:14.

[0043] In another aspect, an isolated nucleic acid sequence is provided encoding a chimeric antigen receptor comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a hinge domain; a transmembrane domain; a CD2 costimulatory signaling domain; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0044] In another aspect, an isolated nucleic acid sequence is provided encoding a chimeric antigen receptor comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a hinge domain; a transmembrane domain; a CD2 costimulatory signaling domain comprising a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:29; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs:22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs:19, 20, and 21.

[0045] In another aspect, an isolated nucleic acid sequence is provided encoding a chimeric antigen receptor comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a CD8 hinge domain; a CD8 transmembrane domain; a CD2 costimulatory signaling domain comprising a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:29; and a CD3 zeta intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs:22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs:19, 20, and 21.

[0046] In another aspect, an isolated nucleic acid sequence encoding a chimeric antigen receptor is provided, comprising a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO: 1, 38, 40, 42, 44, or 46.

[0047] In another aspect, there is provided an isolated nucleic acid sequence encoding an ICOS costimulatory signaling domain comprising the nucleotide sequence set forth in SEQ ID NO:27.

[0048] In another aspect, there is provided a chimeric antigen receptor (CAR) that specifically binds to MUC1, encoded by a nucleic acid of any one of the preceding embodiments.

[0049] In another aspect, a chimeric antigen receptor (CAR) that specifically binds to MUC1 is provided, comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a hinge domain; a transmembrane domain; a costimulatory signaling domain; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0050] In another aspect, a chimeric antigen receptor (CAR) that specifically binds to MUC1 is provided, comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a hinge domain; a transmembrane domain; a CD2 costimulatory signaling domain comprising the amino acid sequence set forth in SEQ ID NO:28; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs:22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs:19, 20, and 21.

[0051] In another aspect, a chimeric antigen receptor (CAR) that specifically binds to MUC1 is provided, comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a CD8 hinge domain; a CD8 transmembrane domain; a CD2 costimulatory signaling domain comprising the amino acid sequence set forth in SEQ ID NO:28; and a CD3 zeta intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs:22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs:19, 20, and 21.

[0052] In another aspect, a chimeric antigen receptor (CAR) that specifically binds to MUC1 is provided, comprising the amino acid sequence set forth in SEQ ID NO:47.

[0053] In another aspect, there is provided an expression construct comprising the isolated nucleic acid of any one of the preceding embodiments. In certain exemplary embodiments, the expression construct further comprises an EF-1α promoter. In certain exemplary embodiments, the expression construct further comprises a rev response element (RRE). In certain exemplary embodiments, the expression construct further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In certain exemplary embodiments, the expression construct further comprises a cPPT sequence. In certain exemplary embodiments, the expression construct further comprises an EF-1α promoter, a rev response element (RRE), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a cPPT sequence.

[0054] In certain exemplary embodiments, the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. In certain exemplary embodiments, the expression construct is a lentiviral vector. In certain exemplary embodiments, the expression construct is a self-inactivating lentiviral vector.

[0055] In another aspect, there is provided a method for generating an modified immune cell, or a progenitor thereof, according to any one of the preceding aspects, comprising introducing into an immune cell, or a progenitor thereof, an isolated nucleic acid according to any one of the preceding aspects or an expression construct according to any one of the preceding aspects.

[0056] In another aspect, there is provided a method of treating a MUC1-associated cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective composition comprising the modified immune cells of any one of the preceding embodiments.

[0057] In certain exemplary embodiments, the MUC1-associated cancer is selected from the group consisting of multiple myeloma, non-small cell lung cancer, breast cancer, pancreatic adenocarcinoma, and ovarian and fallopian tube cancer.

[0058] In certain exemplary embodiments, the MUC1-associated cancer is breast cancer. In certain exemplary embodiments, the breast cancer is characterized by aberrant glycosylation of MUC1. In certain exemplary embodiments, the breast cancer is selected from the group consisting of hormone receptor-positive breast cancer, hormone receptor-negative breast cancer, estrogen receptor-negative breast cancer, progesterone receptor-negative breast cancer, and Her2 receptor-negative breast cancer. In certain exemplary embodiments, the breast cancer is metastatic breast cancer. In certain exemplary embodiments, the breast cancer is triple-negative breast cancer.

[0059] In another aspect, a method of treating a MUC1-associated cancer in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective composition comprising engineered T cells comprising a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; optionally a hinge domain; a transmembrane domain; a costimulatory signaling domain; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0060] In another aspect, a method of treating MUC1-associated multiple myeloma in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective composition comprising engineered T cells comprising a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; optionally a hinge domain; a transmembrane domain; a costimulatory signaling domain; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0061] In another aspect, a method of treating MUC1-associated non-small cell lung cancer in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective composition comprising engineered T cells comprising a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; optionally a hinge domain; a transmembrane domain; a costimulatory signaling domain; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0062] In another aspect, a method of treating MUC1-associated triple-negative breast cancer in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective composition comprising engineered T cells comprising a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; optionally a hinge domain; a transmembrane domain; a costimulatory signaling domain; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0063] In another aspect, a method of treating MUC1-associated pancreatic adenocarcinoma in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective composition comprising engineered T cells comprising a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; optionally a hinge domain; a transmembrane domain; a costimulatory signaling domain; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0064] In another aspect, a method of treating MUC1-associated ovarian and fallopian tube cancer in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective composition comprising engineered T cells comprising a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; optionally a hinge domain; a transmembrane domain; a costimulatory signaling domain; and an intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0065] In certain exemplary embodiments, the method according to any one of the above embodiments further comprises administering lymphodepleting chemotherapy to the subject.In certain exemplary embodiments, the lymphodepleting chemotherapy comprises administering a therapeutically effective amount of cyclophosphamide to the subject.In certain exemplary embodiments, the lymphodepleting chemotherapy comprises administering a therapeutically effective amount of fludarabine to the subject.In certain exemplary embodiments, the lymphodepleting chemotherapy comprises administering a therapeutically effective amount of cyclophosphamide and a therapeutically effective amount of fludarabine to the subject.

[0066] In certain exemplary embodiments, the method of any one of the preceding embodiments further comprises administering to the subject a cytokine release syndrome (CRS) management regimen. In certain exemplary embodiments, the CRS management regimen comprises a therapeutically effective amount of tocilizumab. In certain exemplary embodiments, the CRS management regimen comprises a therapeutically effective amount of tocilizumab and / or a corticosteroid.

[0067] In certain exemplary embodiments, the modified immune cells or modified T cells are autologous.

[0068] In certain exemplary embodiments, the administration of the modified immune cells or modified T cells is via intratumoral delivery. In certain exemplary embodiments, the administration of the modified immune cells or modified T cells is via intravenous delivery. In certain exemplary embodiments, the administration of the modified immune cells or modified T cells is via intraperitoneal delivery.

[0069] The modified immune cell or progenitor thereof of any one of the preceding aspects is for use in a method according to any one of the preceding aspects. The isolated nucleic acid sequence of any one of the preceding aspects is for use in a method according to any one of the preceding aspects. The chimeric antigen receptor of any one of the preceding aspects is for use in a method according to any one of the preceding aspects. The expression vector of any one of the preceding aspects is for use in a method according to any one of the preceding aspects. [The present invention 1001] 1. A modified immune cell or a precursor thereof comprising a chimeric antigen receptor (CAR) that specifically binds to MUC1, CAR, a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; with a transmembrane domain; with costimulatory signaling domains; Intracellular signaling domains and Including, Modified immune cells or their precursor cells. [The present invention 1002] 1001. The modified immune cell of the present invention, wherein the MUC1-specific antigen-binding domain is specific for a glycoepitope of MUC1. [The present invention 1003] The modified immune cell of invention 1001 or 1002, wherein the MUC1-specific antigen-binding domain is specific for a truncated glycoepitope of MUC1. [The present invention 1004] Any of the aforementioned modified immune cells of the present invention, wherein the VH domain comprises the amino acid sequence shown in SEQ ID NO:5. [The present invention 1005] Any of the aforementioned modified immune cells of the present invention, wherein the VL domain comprises the amino acid sequence shown in SEQ ID NO:6. [The present invention 1006] Any of the modified immune cells of the present invention, wherein the VH domain comprises the amino acid sequence shown in SEQ ID NO:5 and the VL domain comprises the amino acid sequence shown in SEQ ID NO:6. [The present invention 1007] Any of the aforementioned modified immune cells of the present invention, wherein the MUC1-specific antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:4. [The present invention 1008] any of the aforementioned modified immune cells of the present invention, wherein the transmembrane domain comprises a transmembrane region of a protein selected from the group consisting of a type I transmembrane protein, T cell receptor alpha chain, T cell receptor beta chain, T cell receptor zeta chain, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. [The present invention 1009] Any of the aforementioned modified immune cells of the present invention, wherein the transmembrane domain comprises a CD8 transmembrane region. [The present invention 1010] Any of the aforementioned modified immune cells of the present invention, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:7. [The present invention 1011]

[0023] Any of the aforementioned modified immune cells of the present invention, wherein the costimulatory signaling domain comprises a costimulatory domain of a protein selected from the group consisting of a member of the TNFR superfamily, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD5, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, DAP10, DAP12, Lck, Fas, and any derivative or variant thereof. [The present invention 1012] Any of the aforementioned modified immune cells of the present invention, wherein the costimulatory signaling domain is a CD2 costimulatory signaling domain. [The present invention 1013] Any of the aforementioned modified immune cells of the present invention, wherein the costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO:28. [The present invention 1014] Any of the aforementioned modified immune cells of the present invention, wherein the intracellular signaling domain comprises a signaling domain of a protein selected from the group consisting of CD3 zeta, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-containing cytoplasmic receptor, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. [The present invention 1015] Any of the modified immune cells of the present invention, wherein the intracellular signaling domain comprises the signaling domain of CD3 zeta. [The present invention 1016] Any of the aforementioned modified immune cells of the present invention, wherein the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:30. [The present invention 1017] Any of the aforementioned modified immune cells of the present invention, wherein the CAR further comprises a leader sequence. [The present invention 1018] 1017. The modified immune cell of the present invention, wherein the leader sequence is a CD8 leader sequence. [The present invention 1019] The modified immune cell of the present invention 1017 or 1018, wherein the leader sequence comprises the amino acid sequence shown in SEQ ID NO:48. [The present invention 1020] Any of the aforementioned modified immune cells of the present invention, wherein the CAR further comprises a hinge domain. [The present invention 1021] The modified immune cell of the present invention 1020, wherein the hinge domain is derived from a protein selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, an amino acid hinge sequence of CD8, and any combination thereof. [The present invention 1022] The modified immune cell of the present invention 1020 or 1021, wherein the hinge domain is a CD8 hinge domain. [The present invention 1023] The modified immune cell of any of claims 1020 to 1022, wherein the hinge domain comprises the amino acid sequence shown in SEQ ID NO:13. [The present invention 1024] Any of the modified immune cells of the present invention, which are modified natural killer (NK) cells, modified natural killer T (NKT) cells, or modified T cells. [The present invention 1025] Any of the modified immune cells of the present invention, which are modified T cells. [The present invention 1026] Any of the modified immune cells of the present invention, which are autologous. [The present invention 1027] 1. A modified T cell comprising a chimeric antigen receptor (CAR) that specifically binds to MUC1, CAR, a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; with the hinge domain; with a transmembrane domain; with the CD2 costimulatory signaling domain; Intracellular signaling domains and Including, Engineered T cells. [The present invention 1028] 1. A modified T cell comprising a chimeric antigen receptor (CAR) that specifically binds to MUC1, CAR, a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; with the hinge domain; with a transmembrane domain; a CD2 costimulatory signaling domain comprising the amino acid sequence set forth in SEQ ID NO:28; Intracellular signaling domains and Including, Engineered T cells. [The present invention 1029] 1. A modified T cell comprising a chimeric antigen receptor (CAR) that specifically binds to MUC1, CAR, a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; CD8 hinge domain and; with the CD8 transmembrane domain; a CD2 costimulatory signaling domain comprising the amino acid sequence set forth in SEQ ID NO:28; CD3 zeta intracellular signaling domain Including, Engineered T cells. [The present invention 1030] A chimeric antigen receptor (CAR) that specifically binds to MUC1, comprising the amino acid sequence set forth in SEQ ID NO: 2, 39, 41, 43, 45, or 47. 1. A modified T cell comprising: [The present invention 1031] a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; with a transmembrane domain; with costimulatory signaling domains; Intracellular signaling domains and 1. An isolated nucleic acid sequence encoding a chimeric antigen receptor comprising: [The present invention 1032] 1031. The isolated nucleic acid sequence of the present invention, wherein the MUC1-specific antigen-binding domain is specific for a glycoepitope of MUC1. [The present invention 1033] 1032. The isolated nucleic acid sequence of claim 1031, wherein the MUC1-specific antigen-binding domain is specific for a truncated glycoepitope of MUC1. [The present invention 1034] 4. The isolated nucleic acid sequence of any of claims 1031 to 1033, wherein the VH domain comprises the amino acid sequence shown in SEQ ID NO:5. [This invention 1035] 1035. The isolated nucleic acid sequence of any of claims 1031 to 1034, wherein the VL domain comprises the amino acid sequence shown in SEQ ID NO:6. [The present invention 1036] 6. The isolated nucleic acid sequence of any of claims 1031 to 1035, wherein the VH domain comprises the amino acid sequence shown in SEQ ID NO:5 and the VL domain comprises the amino acid sequence shown in SEQ ID NO:6. [This invention 1037] 1037. The isolated nucleic acid sequence of any of claims 1031 to 1036, wherein the MUC1-specific antigen-binding domain is encoded by a nucleic acid sequence comprising the nucleotide sequence shown in SEQ ID NO:3. [The present invention 1038] 8. The isolated nucleic acid sequence of any of claims 1031 to 1037, wherein the transmembrane domain comprises a transmembrane region of a protein selected from the group consisting of a type I transmembrane protein, T cell receptor alpha chain, T cell receptor beta chain, T cell receptor zeta chain, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. [This invention 1039] 1039. The isolated nucleic acid sequence of any of claims 1031 to 1038, wherein the transmembrane domain comprises the CD8 transmembrane region. [The present invention 1040] 1039. The isolated nucleic acid sequence of any of claims 1031 to 1039, wherein the transmembrane domain is encoded by a nucleic acid sequence comprising the nucleotide sequence shown in SEQ ID NO:8. [This invention 1041] 1040. The isolated nucleic acid sequence of any of claims 1031 to 1040, wherein the costimulatory signaling domain comprises a costimulatory domain of a protein selected from the group consisting of a member of the TNFR superfamily, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD5, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, DAP10, DAP12, Lck, Fas, and any derivative or variant thereof. [The present invention 1042] 1042. The isolated nucleic acid sequence of any of claims 1031 to 1041, wherein the costimulatory signaling domain is a CD2 costimulatory signaling domain. [This invention 1043] 1043. The isolated nucleic acid sequence of any of claims 1031 to 1042, wherein the costimulatory signaling domain is encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:29. [This invention 1044] 1044. The isolated nucleic acid sequence of any of claims 1031 to 1043, wherein the intracellular signaling domain comprises the signaling domain of CD3 zeta. [This invention 1045] 104. The isolated nucleic acid sequence of any of claims 1031 to 1044, wherein the intracellular signaling domain is encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:31. [The present invention 1046] 1046. The isolated nucleic acid sequence of any of claims 1031 to 1045, wherein the CAR further comprises a CD8 leader sequence. [This invention 1047] 1046. An isolated nucleic acid sequence of the present invention, wherein the leader sequence comprises the amino acid sequence shown in SEQ ID NO:48. [This invention 1048] The isolated nucleic acid sequence of any of claims 1031 to 1047, wherein the CAR further comprises a CD8 hinge domain. [This invention 1049] 1048. The isolated nucleic acid sequence of the present invention, wherein the hinge domain is encoded by a nucleic acid sequence comprising the nucleotide sequence shown in SEQ ID NO:14. [The present invention 1050] a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; with the hinge domain; with a transmembrane domain; with the CD2 costimulatory signaling domain; Intracellular signaling domains and 1. An isolated nucleic acid sequence encoding a chimeric antigen receptor comprising: [This invention 1051] a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; with the hinge domain; with a transmembrane domain; a CD2 costimulatory signaling domain comprising a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:29; Intracellular signaling domains and 1. An isolated nucleic acid sequence encoding a chimeric antigen receptor comprising: [This invention 1052] a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; CD8 hinge domain and; with the CD8 transmembrane domain; a CD2 costimulatory signaling domain comprising a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:29; CD3 zeta intracellular signaling domain 1. An isolated nucleic acid sequence encoding a chimeric antigen receptor comprising: [This invention 1053] An isolated nucleic acid sequence encoding a chimeric antigen receptor, comprising a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO: 1, 38, 40, 42, 44, or 46. [This invention 1054] An isolated nucleic acid sequence encoding an ICOS costimulatory signaling domain, comprising a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:27. [This invention 1055] A chimeric antigen receptor (CAR) that specifically binds to MUC1, which is encoded by any of the nucleic acids of the present inventions 1031 to 1053. [This invention 1056] a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; the hinge domain; with a transmembrane domain; with costimulatory signaling domains; Intracellular signaling domains and A chimeric antigen receptor (CAR) that specifically binds to MUC1, comprising: [This invention 1057] a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; the hinge domain; with a transmembrane domain; a CD2 costimulatory signaling domain comprising the amino acid sequence set forth in SEQ ID NO:28; Intracellular signaling domains and A chimeric antigen receptor (CAR) that specifically binds to MUC1, comprising: [This invention 1058] a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; CD8 hinge domain and; with the CD8 transmembrane domain; a CD2 costimulatory signaling domain comprising the amino acid sequence set forth in SEQ ID NO:28; CD3 zeta intracellular signaling domain A chimeric antigen receptor (CAR) that specifically binds to MUC1, comprising: [This invention 1059] A chimeric antigen receptor (CAR) that specifically binds to MUC1, comprising the amino acid sequence set forth in SEQ ID NO: 2, 39, 41, 43, 45, or 47. [The present invention 1060] An expression construct comprising any one of the isolated nucleic acids of the present inventions 1031 to 1053. [This invention 1061] 1060. An expression construct of the present invention further comprising an EF-1α promoter. [This invention 1062] The expression construct of invention 1060 or 1061, further comprising a rev response element (RRE). [This invention 1063] 1062. The expression construct of any of claims 1060 to 1062, further comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). [This invention 1064] An expression construct according to any one of claims 1060 to 1063, further comprising a cPPT sequence. [This invention 1065] The expression construct of any of claims 1060 to 1064, further comprising an EF-1α promoter, a rev response element (RRE), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a cPPT sequence. [The present invention 1066] The expression construct of any one of claims 1060 to 1065, which is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. [This invention 1067] An expression construct according to any one of claims 1060 to 1066, which is a lentiviral vector. [The present invention 1068] An expression construct according to any one of claims 1060 to 1067, which is a self-inactivating lentiviral vector. [The present invention 1069] A method for generating a modified immune cell or progenitor cell thereof according to any of claims 1001 to 1030, comprising the step of introducing an isolated nucleic acid according to any of claims 1031 to 1053, or an expression construct according to any of claims 1060 to 1067, into an immune cell or progenitor cell thereof. [The present invention 1070] A method of treating MUC1-associated cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective composition comprising the modified immune cells of any of claims 1001 to 1030. [This invention 1071] The method of claim 1070, wherein the MUC1-associated cancer is selected from the group consisting of multiple myeloma, non-small cell lung cancer, breast cancer, pancreatic adenocarcinoma, and ovarian and fallopian tube cancer. [This invention 1072] The method of claim 1071, wherein the MUC1-associated cancer is breast cancer. [This invention 1073] The method of claim 1072, wherein the breast cancer is characterized by aberrant glycosylation of MUC1. [This invention 1074] 1073. The method of claim 1071 or 1072, wherein the breast cancer is selected from the group consisting of hormone receptor-positive breast cancer, hormone receptor-negative breast cancer, estrogen receptor-negative breast cancer, progesterone receptor-negative breast cancer, and Her2 receptor-negative breast cancer. [This invention 1075] The method of any one of claims 1071 to 1074, wherein the breast cancer is metastatic breast cancer. [This invention 1076] The method of any one of claims 1071 to 1075, wherein the breast cancer is triple-negative breast cancer. [This invention 1077] 1. A method of treating a MUC1-associated cancer in a subject in need thereof, comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; optionally with a hinge domain; with a transmembrane domain; with costimulatory signaling domains; Intracellular signaling domains and administering to a subject a therapeutically effective composition comprising modified T cells comprising A method comprising: [This invention 1078] 1. A method of treating MUC1-associated multiple myeloma in a subject in need thereof, comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; optionally with a hinge domain; with a transmembrane domain; with costimulatory signaling domains; Intracellular signaling domains and administering to a subject a therapeutically effective composition comprising modified T cells comprising A method comprising: [This invention 1079] 1. A method of treating MUC1-associated non-small cell lung cancer in a subject in need thereof, comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; optionally with a hinge domain; with a transmembrane domain; with costimulatory signaling domains; Intracellular signaling domains and administering to a subject a therapeutically effective composition comprising modified T cells comprising A method comprising: [The present invention 1080] 1. A method of treating MUC1-associated triple-negative breast cancer in a subject in need thereof, comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; optionally with a hinge domain; with a transmembrane domain; with costimulatory signaling domains; Intracellular signaling domains and administering to a subject a therapeutically effective composition comprising modified T cells comprising A method comprising: [This invention 1081] 1. A method of treating MUC1-associated pancreatic adenocarcinoma in a subject in need thereof, comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; optionally with a hinge domain; with a transmembrane domain; with costimulatory signaling domains; Intracellular signaling domains and administering to a subject a therapeutically effective composition comprising modified T cells comprising A method comprising: [This invention 1082] 1. A method of treating MUC1-associated ovarian and fallopian tube cancer in a subject in need thereof, comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; optionally with a hinge domain; with a transmembrane domain; with costimulatory signaling domains; Intracellular signaling domains and administering to a subject a therapeutically effective composition comprising modified T cells comprising A method comprising: [This invention 1083] 1083. The method of any one of claims 1070 to 1082, further comprising administering lymphodepleting chemotherapy to the subject. [This invention 1084] The method of claim 1083, wherein the lymphodepleting chemotherapy comprises administering to the subject a therapeutically effective amount of cyclophosphamide. [This invention 1085] 1085. The method of claim 1083 or 1084, wherein the lymphodepleting chemotherapy comprises administering to the subject a therapeutically effective amount of fludarabine. [The present invention 1086] 6. The method of any of claims 1083 to 1085, wherein the lymphodepleting chemotherapy comprises administering to the subject a therapeutically effective amount of cyclophosphamide and a therapeutically effective amount of fludarabine. [This invention 1087] The method of any of claims 1070 to 1086, further comprising administering to the subject a cytokine release syndrome (CRS) management regimen. [This invention 1088] The method of claim 1087, wherein the management regimen for CRS comprises a therapeutically effective amount of tocilizumab. [This invention 1089] The method of any one of claims 1087 to 1088, wherein the management regimen for CRS comprises a therapeutically effective amount of tocilizumab and / or a corticosteroid. [The present invention 1090] 1089. The method of any of claims 1070 to 1089, wherein the modified immune cells or modified T cells are autologous. [This invention 1091] The method of any of claims 1070 to 1090, wherein the administration of the modified immune cells or modified T cells is via intratumoral delivery. [This invention 1092] The method of any of claims 1070 to 1090, wherein the administration of the modified immune cells or modified T cells is via intravenous delivery. [This invention 1093] The method of any of claims 1070 to 1090, wherein the administration of the modified immune cells or modified T cells is via intraperitoneal delivery. [Brief explanation of the drawings]

[0070] The following detailed description of certain embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0071] [Figure 1] FIG. 1 shows the initiation of O-glycan biosynthesis highlighting the core glycan and associated glycotransferases. [Figure 2A] A set of plots showing gene expression analysis of MUC1 and glycosylation enzymes by qPCR. Gene expression was measured in four breast cancer cell lines (BT-20, MCF7, MDA-MB-231, and MDA-MB-453) and compared to the MCF10A non-tumorigenic breast epithelial cell line. [Figure 2B] See legend to Figure 2A. [Figure 3]

[0023] Figure 3 is a set of images showing Tn-MUC1 expression in breast cancer tissues assessed by immunohistochemistry using anti-5E5 antibody. Figure 3A shows 3+ staining in breast cancer tissues with no staining in the surrounding stroma. Figure 3B shows 2+ staining in breast cancer tissues with no staining in the surrounding stroma. [Figure 4]

[0023] Figure 1 is a series of plots illustrating results from a cytotoxicity assay using anti-Tn-MUC1 CAR T cells and four breast cancer cell lines. 5E5-CAR, CD19-specific CAR, or NTD T cells were co-cultured with breast cancer cell lines at an effector:target ratio of 10:1. After addition of the T cells, cytolysis was measured by measuring impedance in real time every 15 minutes for 100 hours. [Figure 5A]1 is a series of plots and images showing the finding that intraperitoneal and intratumoral delivery of 5E5-CAR T cells enhances antitumor efficacy. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6] 1 is a series of plots and images showing the finding that intraperitoneal delivery of murine HMFG1-CAR T cells in human MUC1 transgenic mice causes extratumoral on-target toxicity that is not observed from murine 5E5-CAR T cells. [Figure 7A] 1 is a series of plots showing gene expression of MUC1, ST6GALNAC1, B3GNT6, C1GALT1, and C1GALT1C1 in 50 patient-derived breast cancer samples compared to the average gene expression in 10 matched patient-derived normal breast tissue samples. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 8] 1 is a set of flow cytometry plots showing the expression of various TnMUC1 CAR transgenes as indicated. [Figure 9] 1 shows the results of a CFSE assay demonstrating that the indicated various TnMUC1 CAR-T cells proliferate in response to MCF7 cells. [Figure 10] From left to right is a set of three charts showing the secretion levels of IL-2, TNFa, and IFNg of the various TnMUC1 CAR-T cells indicated. [Figure 11] Graph showing total flux (photons / second) measured over time in mice following intravenous administration of the various indicated TnMUC1 CAR-T cells. [Figure 12] 1 is a plot showing the levels of various TnMUC1 CAR-T cells measured in the peripheral blood of infused mice 42 days post-infusion. [Figure 13]1 is a graph showing the cytotoxicity of CART-TnMUC1, CART-TnMUC1-BBz, and negative control cells (CART-19 and NTD) against the Hs766T pancreatic cancer cell line. [Figure 14] 1 is a series of graphs showing targeted cell killing by CART-TnMUC1 cells of various cell lines as indicated. [Figure 15] 1 is a series of graphs showing targeted cell killing by CART-TnMUC1 cells in response to Tn antigens of various cell lines as indicated. [Figure 16] 1 shows a series of graphs plotting bioluminescence imaging of tumor burden in a mouse model of pancreatic cancer. [Figure 17] 1 is a series of graphs and images showing the proliferation of CART-TnMUC1 cells in response to antigen-expressing target cells. [Figure 18] 1 is a series of graphs showing cytokine and chemokine production in various indicated cell lines. [Figure 19] 1 is a graph showing data obtained from an IFNγ ELISA experiment. [Figure 20] 1 is a series of graphs showing quantification of various indicated T cells in the peripheral blood of mice 21 and 42 days after T cell infusion. [Figure 21] Micrographs of Jurkat CBG / GFP CD19-P2A-Cosmc cells (left) and MCF-7 cells (right) stained with anti-TnMUC1 antibody. [Figure 22] FIG. 1 is a schematic diagram showing the experimental setup for testing the reproducibility of the TnMUC1 CTA assay. [Figure 23] FIG. 1 is a schematic representation of the plasmid map of pTRPE_5E5(H2L)_CD2z. [Figure 24] FIG. 1 is a schematic representation of the plasmid map of pGEM-SS1-CD2z. [Figure 25] FIG. 1 is a schematic representation of the plasmid map of pTRPE_5E5-BBz. [Figure 26] FIG. 1 is a schematic diagram showing the backbone of the pTRPE_5E5(H2L) vector. [Figure 27] FIG. 1 is a schematic diagram showing the study design for the Phase 1 and Phase 1a portions of the clinical trial. [Figure 28] FIG. 1 is a schematic diagram showing the overall patient pathway for a clinical trial. [Figure 29] FIG. 1 is a schematic diagram showing the dose escalation scheme of the clinical trial. [Figure 30] FIG. 1 is a schematic diagram showing the dose escalation cohorts of the clinical trial. [Figure 31] 1 is a series of graphs showing total cell numbers of T cells from five different healthy donors transduced with the indicated CARs. [Figure 32] 1 is a series of graphs showing population doublings of T cells from five different healthy donors transduced with the indicated CARs. [Figure 33] 1 is a series of graphs showing the mean cell volume of T cells from four different healthy donors transduced with the indicated CARs. [Figure 34] 1 is a set of flow cytometry plots showing expression of the indicated CARs in T cells from five different healthy donors. DETAILED DESCRIPTION OF THE INVENTION

[0072] Detailed Description definition Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0073] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0074] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0075] As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is intended to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are reasonable in practicing the disclosed methods.

[0076] As used herein, "activation" refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" particularly refers to T cells undergoing cell division.

[0077] As used herein, "alleviating" a disease means reducing the severity of one or more symptoms of the disease.

[0078] "Allogeneic" refers to any material derived from different animals of the same species.

[0079] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or can be an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies in the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single-chain antibodies (scFv) and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0080] The term "antibody fragment" refers to a portion of an intact antibody, and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0081] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0082] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. The α and β light chains refer to the two major antibody light chain isotypes.

[0083] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA techniques, such as, for example, the bacteriophage-expressed antibodies described herein. The term should also be taken to mean an antibody produced by synthesis of an antibody-encoding DNA molecule that expresses an antibody protein or an amino acid sequence defining that antibody, where the DNA or amino acid sequence is available and well known in the art, and obtained using DNA or amino acid sequence synthesis techniques.

[0084] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may include either or both antibody production and activation of specific immunocompetent cells. Those skilled in the art will understand that virtually any macromolecule, including all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be generated, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0085] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is later reintroduced into that individual.

[0086] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell receptor that is engineered to be expressed on immune effector cells and specifically bind to an antigen. CARs can be used as a therapy involving adoptive cell transfer. T cells are removed from a patient and modified to express a receptor specific to a particular form of antigen. In some embodiments, the CAR has specificity for a selected target, such as MUC1. CARs can also include an extracellular domain containing an intracellular activation domain, a transmembrane domain, and an antigen-binding region.

[0087] The term "cleavage" refers to the breaking of covalent bonds or the hydrolysis of peptide bonds in the backbone of a nucleic acid molecule. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand cleavage are possible. Double-strand cleavage can occur as a result of two separate single-strand cleavage events. DNA cleavage can result in the production of either blunt ends or cohesive ends. In certain embodiments, fusion polypeptides can be used to target cleaved double-stranded DNA.

[0088] As used herein, the term "conservative sequence modification" is intended to refer to an amino acid modification that does not significantly affect or alter the binding properties of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be tested for its ability to bind to an antigen using the functional assays described herein.

[0089] "Costimulatory ligand," as that term is used herein, includes a molecule on an antigen-presenting cell (e.g., an aAPC, a dendritic cell, a B cell, etc.) that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing signals that mediate T cell responses, including but not limited to proliferation, activation, differentiation, etc., in addition to the primary signal provided by engagement of the TCR / CD3 complex with, for example, a peptide-loaded MHC molecule. Costimulatory ligands can 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, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, among others, and ligands that specifically bind to CD83.

[0090] 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 T 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.

[0091] A "costimulatory signal," as used herein, refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up- or down-regulation of key molecules.

[0092] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and in which the animal's health will continue to deteriorate unless the disease is ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than it would be without the disorder. If left untreated, a disorder does not necessarily cause a further decline in the animal's health.

[0093] "Donor antigen" means an antigen expressed by donor cells that are transplanted into a recipient.

[0094] "Recipient antigen" means the target of an immune response against a donor antigen.

[0095] The term "downregulation," as used herein, refers to a decrease or elimination of gene expression of one or more genes.

[0096] "Effective amount" or "therapeutically effective amount," as used interchangeably herein, refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, an amount that, when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. Immune responses can be readily assessed by numerous art-recognized methods. Those skilled in the art will understand that the amount of a composition administered herein will vary and can be readily determined based on numerous factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, and the particular compound being administered.

[0097] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0098] As used herein, "endogenous" refers to any material that originates or is produced within an organism, cell, tissue, or system.

[0099] The term "epitope," as used herein, is defined as a small chemical molecule on an antigen that can elicit an immune response and induce a B-cell response and / or a T-cell response. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. Generally, epitopes are approximately 10 amino acids and / or sugars in size. In some exemplary embodiments, epitopes are about 4-18 amino acids, about 5-16 amino acids, about 6-14 amino acids, about 7-12 amino acids, or about 8-10 amino acids. Those skilled in the art will understand that, in general, the overall three-dimensional structure of a molecule, rather than its specific linear sequence, is the primary criterion for antigen specificity, and therefore, this distinguishes one epitope from another. Based on the present disclosure, a peptide of the present invention can be an epitope.

[0100] As used herein, the term "exogenous" refers to any material that is introduced from or produced outside an organism, cell, tissue or system.

[0101] As used herein, the term "expand" refers to an increase in number, such as an increase in the number of T cells. In one embodiment, T cells expanded ex vivo are increased in number relative to the number originally present in the culture. In another embodiment, T cells expanded ex vivo are increased in number relative to other cell types in the culture. As used herein, the term "ex vivo" refers to cells removed from an organism (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0102] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0103] "Expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) incorporating a recombinant polynucleotide.

[0104] As used herein, "homologous" refers to the identity of subunit sequences between two polymer molecules, for example, between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. If the subunit position in both molecules is occupied by the same monomer subunit; for example, if a position in each of two DNA molecules is occupied by adenine, they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in two sequences (for example, 5 positions in a polymer 10 subunits long) are homologous, the two sequences are 50% homologous; if 90% of the positions (for example, 9 out of 10) are matching or homologous, the two sequences are 90% homologous.

[0105] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are from a human immunoglobulin sequence. A humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0106] "Fully human" refers to an immunoglobulin, such as an antibody, where the entire molecule is of human origin or consists of an amino acid sequence identical to that of a human form of the antibody.

[0107] As used herein, "identity" refers to the identity of the subunit sequence between two amino acid molecules, such as between two polymer molecules, particularly between two polypeptide molecules. If two amino acid sequences have the same residue at the same position; for example, if a position in each of two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that match or are identical; for example, if half of the positions in the two sequences (e.g., 5 positions in a 10-amino acid long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are identical or matched, the two amino acid sequences are 90% identical.

[0108] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called BCRs (B cell receptors) or antigen receptors. Five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and respiratory and genitourinary mucus. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important for defense against bacteria and viruses. IgD is an immunoglobulin with unknown antibody function but may serve as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity reactions by triggering the release of mediators from mast cells and basophils upon allergen exposure.

[0109] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify the antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0110] The term "immunostimulatory" is used herein to refer to increasing the overall immune response.

[0111] The term "immunosuppressive" is used herein to refer to reducing the overall immune response.

[0112] As used herein, "instructional material" includes publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present invention. The instructional material of the kits of the present invention may, for example, be affixed to a container containing the nucleic acids, peptides, and / or compositions of the present invention, or may be shipped together with the container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound will be used cooperatively by the recipient.

[0113] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0114] As used herein, the term "knockdown" refers to a decrease in gene expression of one or more genes.

[0115] As used herein, the term "knockout" refers to the elimination of gene expression of one or more genes.

[0116] " Lentivirus " used herein refers to the genus of Retroviridae.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; it can deliver a significant amount of genetic information into the DNA of host cell, so it is one of the most efficient methods of gene delivery vector.HIV, SIV and FIV are all examples of lentivirus.Lentivirus-derived vectors provide a means to achieve a significant level of in vivo gene transfer.

[0117] The term "limited toxicity" as used herein refers to the peptides, polynucleotides, cells and / or antibodies of the invention that demonstrate a lack of substantial negative biological effects, anti-tumor effects, or substantial negative physiological symptoms on healthy cells, non-tumor cells, non-diseased cells, non-target cells, or populations of such cells, either in vitro or in vivo.

[0118] As used herein, the term "modified" refers to an altered state or structure of a molecule or cell of the present invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0119] As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of a treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. This term encompasses perturbing and / or affecting a natural signal or response in a subject, such as a human, thereby mediating a beneficial therapeutic response.

[0120] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0121] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" can also include introns, to the extent that a nucleotide sequence encoding a protein, depending on its form, may contain introns.

[0122] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.

[0123] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Therefore, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™, as well as synthetic means.

[0124] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined together by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide can be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0125] The term "self-antigen" as used herein is defined as an antigen expressed by host cells or tissues. Self-antigens can be tumor antigens, but in certain embodiments, they are expressed by both normal cells and tumor cells. Those skilled in the art will readily understand that self-antigens can be overexpressed in cells.

[0126] As used herein, the term "specifically binds" with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not, in itself, change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not, in itself, change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than the entire protein. If an antibody is specific for epitope "A," then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0127] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta and / or rearrangement of cytoskeletal structures.

[0128] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0129] As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., an aAPC, a dendritic cell, a B cell, etc.), is capable of specifically binding to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, inter alia, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0130] The term "subject" is intended to include organisms (e.g., mammals) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. In an exemplary embodiment, the subject is a human.

[0131] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types with which it is normally associated in its natural state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, the term simply refers to cells that have been separated from the cells with which they are originally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0132] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0133] As used herein, the term "T cell receptor" or "TCR" refers to a membrane protein complex involved in the activation of T cells in response to antigen presentation. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. Although TCRs are composed of a heterodimer of alpha (a) and beta (β) chains, in some cells, TCRs consist of gamma and delta (γ / δ) chains. TCRs can exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains: a variable and a constant domain. In some embodiments, TCRs can be engineered in any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0134] As used herein, the term "therapeutic" means treatment and / or prophylaxis. A therapeutic effect is achieved by suppression, amelioration, or eradication of a disease state.

[0135] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0136] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder from which a subject suffers.

[0137] A "vector" is a material composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0138] "Xenogenic" refers to any material derived from an animal of a different species.

[0139] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges and individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0140] explanation The present invention provides MUC1-specific chimeric antigen receptors (CARs; e.g., Tn-MUC1 CARs) and modified cells comprising the same. Compositions and methods for utilizing MUC1-specific CARs to treat cancer are also provided. In particular, the Tn-MUC1 CARs of the present invention may be suitable for treating both liquid tumors (e.g., multiple myeloma, etc.) and solid tumors (e.g., breast cancer, non-small cell lung cancer, ovarian and fallopian tube cancer, pancreatic adenocarcinoma, etc.).

[0141] We demonstrate herein that Tn-MUC1 is an attractive tumor-specific antigen for antibody-directed adoptive immunotherapy in various cancers. CAR T cells against Tn-MUC1 exhibited potent cytolytic activity against cancer cell lines in vitro and significant tumor eradication in vivo. While strategies for targeting MUC1 outside the context of tumor-specific glycosylation can exhibit on-target off-tumor toxicity, CAR T cells targeting Tn-MUC1 overcome this potential toxicity and broaden the therapeutic window for solid tumors such as breast cancer.

[0142] Chimeric antigen receptor (CAR) The present invention provides compositions and methods for engineered immune cells or their precursor cells, e.g., engineered T cells, comprising a chimeric antigen receptor (CAR) with affinity for MUC1 or a glycosylated form of MUC1 (e.g., Tn-MUC1). A subject CAR of the present invention comprises an antigen-binding domain (e.g., a Tn-MUC1-binding domain), a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain. A subject CAR of the present invention may optionally comprise a hinge domain. Thus, a subject CAR of the present invention comprises an antigen-binding domain (e.g., a Tn-MUC1-binding domain), a hinge domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain. In some embodiments, each domain of a subject CAR is separated by a linker.

[0143] The antigen-binding domain may be operably linked to another domain of the CAR for expression in a cell, such as a transmembrane domain, a costimulatory signaling domain, or an intracellular signaling domain, each of which is described elsewhere herein. In one embodiment, a first nucleic acid sequence encoding the antigen-binding domain is operably linked to a second nucleic acid encoding the transmembrane domain, and is further operably linked to a third nucleic acid sequence encoding the costimulatory signaling domain.

[0144] The antigen binding domains described herein can be combined with any transmembrane domain, any costimulatory signaling domain, any intracellular signaling domain, or any other domain described herein that can be included in a CAR of the present invention.

[0145] In one aspect, the invention includes a chimeric antigen receptor (CAR) that specifically binds to MUC1, comprising a MUC1-specific antigen binding domain, and optionally a hinge domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.

[0146] In one aspect, the invention includes a chimeric antigen receptor (CAR) that specifically binds to MUC1, comprising a TnMUC1-specific antigen binding domain, and optionally a hinge domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.

[0147] In one exemplary embodiment, the invention includes a chimeric antigen receptor (CAR) that specifically binds to MUC1, comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a CD8 hinge domain; a CD8 transmembrane domain; a CD2 costimulatory signaling domain; and a CD3 zeta intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0148] In one exemplary embodiment, the invention includes a chimeric antigen receptor (CAR) that specifically binds to MUC1, comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a CD8 hinge domain; a CD8 transmembrane domain; a 4-1BB costimulatory signaling domain; and a CD3 zeta intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0149] In one exemplary embodiment, the invention includes a chimeric antigen receptor (CAR) that specifically binds to MUC1, comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a CD8 hinge domain; a CD8 transmembrane domain; an ICOS costimulatory signaling domain; and a CD3 zeta intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0150] In some embodiments, the genetically modified immune cells (e.g., T cells) or progenitor cells thereof of the present invention comprise a chimeric antigen receptor (CAR) with affinity for MUC1. In some embodiments, the genetically modified immune cells (e.g., T cells) or progenitor cells thereof of the present invention comprise a chimeric antigen receptor (CAR) with affinity for Tn-MUC1.

[0151] In certain embodiments, the genetically modified cells are T cells. In certain embodiments, the genetically modified cells are natural killer (NK) cells. In certain embodiments, the genetically modified cells are NKT cells.

[0152] Thus, in one exemplary aspect, provided herein are genetically modified T cells comprising a chimeric antigen receptor (CAR) that specifically binds to MUC1, the chimeric antigen receptor comprising a MUC1-specific antigen-binding domain, an optional hinge domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.

[0153] Thus, in one exemplary aspect, provided herein is a genetically modified T cell comprising a chimeric antigen receptor (CAR) that specifically binds to MUC1, the chimeric antigen receptor comprising a TnMUC1-specific antigen-binding domain, an optional hinge domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.

[0154] Thus, in one exemplary embodiment, provided herein is a genetically modified T cell comprising a chimeric antigen receptor (CAR) that specifically binds to MUC1, the chimeric antigen receptor (CAR) comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a CD8 hinge domain; a CD8 transmembrane domain; a CD2 costimulatory signaling domain; and a CD3 zeta intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0155] Thus, in one exemplary embodiment, provided herein is a genetically modified T cell comprising a chimeric antigen receptor (CAR) that specifically binds to MUC1, the chimeric antigen receptor (CAR) comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a CD8 hinge domain; a CD8 transmembrane domain; a 4-1BB costimulatory signaling domain; and a CD3 zeta intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0156] Thus, in one exemplary embodiment, provided herein is a genetically modified T cell comprising a chimeric antigen receptor (CAR) that specifically binds to MUC1, the chimeric antigen receptor (CAR) comprising: a MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; a CD8 hinge domain; a CD8 transmembrane domain; an ICOS costimulatory signaling domain; and a CD3 zeta intracellular signaling domain, wherein the VH domain comprises the heavy chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and the VL domain comprises the light chain complementarity-determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21.

[0157] In certain embodiments of the invention, the CAR is encoded by a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 1, 38, 40, 42, 44, or 46. In certain embodiments of the invention, the CAR comprises the amino acid sequence of SEQ ID NO: 2, 39, 41, 43, 45, or 47.

[0158] The sequences of the individual domains and CARs are found in Table 1.

[0159] (Table 1) TIFF0007654557000001.tif157153TIFF0007654557000002.tif233153TIFF0007654557000003.tif255153TIFF000 7654557000004.tif201153TIFF0007654557000005.tif228153TIFF0007654557000006.tif230153TIFF0007654557 000007.tif231153TIFF0007654557000008.tif231153TIFF0007654557000009.tif230153TIFF0007654557000010. tif229153TIFF0007654557000011.tif229153TIFF0007654557000012.tif229153TIFF0007654557000013.tif81153

[0160] Thus, a subject CAR may be a CAR with affinity for Tn-MUC1, comprising a Tn-MUC1-binding domain comprising the amino acid sequence set forth in SEQ ID NO:4, 5, 6, and / or 19-24. A subject Tn-MUC1 CAR may further comprise a leader sequence comprising the amino acid sequence set forth in SEQ ID NO:48. A subject Tn-MUC1 CAR may further comprise a hinge domain comprising the amino acid sequence set forth in SEQ ID NO:13. A subject Tn-MUC1 CAR may further comprise a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:7 and / or 15. A subject Tn-MUC1 CAR may further comprise a costimulatory signaling domain comprising the amino acid sequence set forth in SEQ ID NO:9, 17, 25, 28, 32, 34, and / or 36. A subject Tn-MUC1 CAR may further comprise an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:11 and / or 30. A subject Tn-MUC1 CAR can comprise the amino acid sequence set forth in SEQ ID NOs: 2, 39, 41, 43, 45, and / or 47.

[0161] Thus, a subject CAR may be a CAR with affinity for Tn-MUC1, comprising a Tn-MUC1-binding domain comprising the amino acid sequence set forth in SEQ ID NO:4, 5, 6, and / or 19-24. A subject Tn-MUC1 CAR may further comprise a leader sequence comprising the amino acid sequence set forth in SEQ ID NO:48. A subject Tn-MUC1 CAR may further comprise a hinge domain comprising the amino acid sequence set forth in SEQ ID NO:13. A subject Tn-MUC1 CAR may further comprise a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:7 or 15. A subject Tn-MUC1 CAR may further comprise a costimulatory signaling domain comprising the amino acid sequence set forth in SEQ ID NO:9, 17, 25, 28, 32, 34, or 36. A subject Tn-MUC1 CAR may further comprise an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:11 or 30. A subject Tn-MUC1 CAR can comprise the amino acid sequence set forth in SEQ ID NO:2, 39, 41, 43, 45, or 47.

[0162] antigen-binding domain The antigen-binding domain of a CAR is the extracellular region of the CAR that binds to a specific target antigen, including proteins, carbohydrates, and glycolipids. In some embodiments, a CAR comprises affinity for a target antigen (e.g., a tumor-associated antigen) on a target cell (e.g., a cancer cell). The target antigen can include any type of protein associated with the target cell or its epitope. For example, a CAR can comprise affinity for a target antigen on a target cell that indicates a specific state of the target cell.

[0163] In certain embodiments, the CAR of the present invention comprises an antigen-binding domain that binds to MUC1. In certain embodiments, the antigen-binding domain binds to a glycosylated form or glycoepitope of MUC1. In certain embodiments, the antigen-binding domain is specific for a truncated glycoepitope of MUC1. In certain embodiments, the antigen-binding domain is specific for Tn-MUC1. In certain embodiments, the antigen-binding domain of the present invention comprises an antibody or fragment thereof that binds to the MUC1 molecule or a glycoepitope of MUC1 (Tn-MUC1). In certain exemplary embodiments, the antigen-binding domain is an scFv antibody that binds to Tn-MUC1. The selection of the antigen-binding domain depends on the type and number of antigens present on the surface of the target cell. For example, the antigen-binding domain can be selected to recognize an antigen that serves as a cell surface marker on the target cell associated with a particular state of the target cell.

[0164] As described herein, a CAR of the present disclosure having affinity for a specific target antigen on a target cell can comprise a target-specific binding domain. In some embodiments, the target-specific binding domain is a mouse target-specific binding domain, e.g., the target-specific binding domain is of mouse origin. In some embodiments, the target-specific binding domain is a human target-specific binding domain, e.g., the target-specific binding domain is of human origin. In exemplary embodiments, a CAR of the present disclosure having affinity for Tn-MUC1 on a target cell can comprise a Tn-MUC1 binding domain. In some embodiments, the Tn-MUC1 binding domain is a mouse Tn-MUC1 binding domain, e.g., the Tn-MUC1 binding domain is of mouse origin. In some embodiments, the Tn-MUC1 binding domain is a humanized Tn-MUC1 binding domain. In some embodiments, the Tn-MUC1 binding domain is a human Tn-MUC1 binding domain, e.g., the Tn-MUC1 binding domain is of human origin.

[0165] In some embodiments, the Tn-MUC1 binding domain is derived from the 5E5 antibody disclosed in PCT Publication No. WO2008 / 040362, the disclosure of which is incorporated herein by reference in its entirety. Thus, a CAR of the present disclosure comprises a Tn-MUC1 binding domain derived from the 5E5 antibody disclosed in PCT Publication No. WO2008 / 040362. In some embodiments, the Tn-MUC1 binding domain is a humanized Tn-MUC1 binding domain. In some embodiments, the humanized Tn-MUC1 binding domain is derived from any one of the humanized 5E5 heavy chain and light chain sequences disclosed in PCT Publication No. WO2015 / 159076, the disclosure of which is incorporated herein by reference in its entirety. Thus, a CAR of the present disclosure comprises a humanized Tn-MUC1 binding domain derived from any one of the humanized 5E5 heavy and light chain sequences disclosed in PCT Publication No. WO2015 / 159076. A CAR of the present disclosure may comprise a humanized Tn-MUC1 binding domain, an optional transmembrane domain, an optional hinge domain, an optional costimulatory domain, and an optional intracellular signaling domain disclosed herein.

[0166] In some embodiments, the CAR of the present disclosure may have affinity for one or more target antigens on one or more target cells. In some embodiments, the CAR may have affinity for one or more target antigens on a single target cell. In such embodiments, the CAR is a bispecific or multispecific CAR. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for the same target antigen. For example, a CAR comprising one or more target-specific binding domains with affinity for the same target antigen can also bind to distinct epitopes of the target antigen. When multiple target-specific binding domains are present in a CAR, the binding domains may be arranged in tandem and separated by a linker peptide. For example, in a CAR comprising two target-specific binding domains, the binding domains are covalently linked to each other on a single polypeptide chain via a polypeptide linker, an Fc hinge region, or a membrane hinge region.

[0167] The antigen-binding domain can comprise any domain that binds to an antigen, and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, and any fragment thereof. Thus, in one embodiment, the antigen-binding domain portion comprises a mammalian antibody or a fragment thereof. In another embodiment, the antigen-binding domain of the CAR is selected from the group consisting of an anti-Tn-MUC1 antibody or a fragment thereof. In some embodiments, the antigen-binding domain is selected from the group consisting of an antibody, an antigen-binding fragment (Fab), and a single-chain variable fragment (scFv). In some embodiments, the Tn-MUC1-binding domain of the present invention is selected from the group consisting of a Tn-MUC1-specific antibody, a Tn-MUC1-specific Fab, and a Tn-MUC1-specific scFv. In one embodiment, the Tn-MUC1-binding domain is a Tn-MUC1-specific antibody. In one embodiment, the Tn-MUC1-binding domain is a Tn-MUC1-specific Fab. In one embodiment, the Tn-MUC1 binding domain is a Tn-MUC1-specific scFv.

[0168] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein in which the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin (e.g., murine or human) are covalently linked to form a VH::VL heterodimer. The heavy (VH) and light (VL) chains are either directly linked or linked by a linker or spacer encoding a peptide connecting the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. The terms "linker" and "spacer" are used interchangeably herein. In some embodiments, the antigen-binding domain (e.g., the Tn-MUC1 binding domain) comprises an scFv having, from the N-terminus to the C-terminus, the following configuration: VH - linker - VL. In some embodiments, the antigen-binding domain (e.g., the Tn-MUC1 binding domain) comprises an scFv having, from the N-terminus to the C-terminus, the following configuration: VL - linker - VH. Those skilled in the art will be able to select suitable configurations for use in the present invention.

[0169] The linker is typically rich in serine or threonine for solubility, in addition to glycine for flexibility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. Glycine-serine (GS) linkers, such as (GS) n , (GSGGS) n (SEQ ID NO: 52), (GGGS) n (SEQ ID NO: 53), and (GGGGS) n A variety of linker sequences are known in the art, including, but not limited to, (SEQ ID NO:54), where n represents an integer of at least 1. Exemplary linker sequences can comprise amino acid sequences, including, but not limited to, GGSG (SEQ ID NO:55), GGSGG (SEQ ID NO:56), GSGSG (SEQ ID NO:57), GSGGG (SEQ ID NO:58), GGGSG (SEQ ID NO:59), GSSSG (SEQ ID NO:60), GGGGS (SEQ ID NO:61), GGGSGGGGSGGGGS (SEQ ID NO:62), and the like. One of skill in the art will be able to select a suitable linker sequence for use in the present invention. In one embodiment, the antigen-binding domain of the present invention (e.g., the Tn-MUC1 binding domain) comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are represented by the nucleotide sequences The fragments are separated by a linker sequence having the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO:62), which can be encoded by a nucleic acid sequence comprising TIFF0007654557000014.tif11128.

[0170] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids containing VH and VL coding sequences as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. Antagonist scFvs with inhibitory activity have been described (e.g., Zhao et al., Hybridoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40). Agonist scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0171] As used herein, "Fab" refers to the fragment of an antibody structure that binds to an antigen, but is monovalent and does not have the Fc portion; for example, digestion of an antibody with the enzyme papain yields two Fab fragments and an Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind to antigen).

[0172] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, wherein the fragment has two antigen-binding (ab') (bivalent) regions, each of which contains two separate amino acid chains, a portion of an H chain and a light (L) chain linked by an S-linkage for antigen binding, with the remaining H chain portions linked together. The "F(ab')2" fragment can be divided into two separate Fab' fragments.

[0173] In some instances, the antigen-binding domain may be derived from the same species that the CAR will ultimately be used in. For example, for human use, the antigen-binding domain of the CAR may comprise a human antibody or fragment thereof, as described elsewhere herein.

[0174] In exemplary embodiments, a Tn-MUC1 CAR of the invention comprises a Tn-MUC1 binding domain, e.g., a Tn-MUC1-specific scFv. In one embodiment, the Tn-MUC1 binding domain comprises the amino acid sequence set forth in SEQ ID NO:4. In one embodiment, the Tn-MUC1 binding domain is encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:3.

[0175] In one embodiment, the Tn-MUC1-binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6. The light chain variable region of the Tn-MUC1-binding domain comprises light chain complementarity-determining regions (CDRs). As used herein, "complementarity-determining region" or "CDR" refers to the region of the variable chain of an antigen-binding molecule that binds to a specific antigen. Thus, the Tn-MUC1-binding domain may comprise a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19; CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20; and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21.

[0176] In one embodiment, the Tn-MUC1 binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5. The Tn-MUC1 binding domain may comprise a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0177] Those of skill in the art will know acceptable variations of the Tn-MUC1 binding domain that retain specific binding to Tn-MUC1. For example, in some embodiments, the Tn-MUC1 binding domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs:4-6 and 19-24.

[0178] In some embodiments, the Tn-MUC1 binding domain is encoded by a nucleic acid sequence comprising a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:3.

[0179] The antigen binding domain can be operably linked to another domain of the CAR, such as a transmembrane domain or a costimulatory signaling domain, both of which are described elsewhere herein. In one embodiment, the nucleic acid encoding the antigen binding domain is operably linked to a nucleic acid encoding the transmembrane domain and a nucleic acid encoding the costimulatory signaling domain.

[0180] An antigen binding domain described herein, such as an antibody or fragment thereof that binds to Tn-MUC1, can be combined with any of the transmembrane domains described herein, any of the intracellular or cytoplasmic domains described herein, or any of the other domains described herein that can be included in a CAR.

[0181] Transmembrane domain Regarding the transmembrane domain, the CAR of the present invention (e.g., Tn-MUC1-CAR) can be designed to include a transmembrane domain that connects the antigen-binding domain of the CAR with the intracellular domain. The transmembrane domain of the subject CAR is a region that can penetrate the plasma membrane of a cell (e.g., an immune cell or its precursor cell). The transmembrane domain is for insertion into a cell membrane, for example, a eukaryotic cell membrane. In some embodiments, the transmembrane domain is sandwiched between the antigen-binding domain and the intracellular domain of the CAR.

[0182] In one embodiment, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some examples, the transmembrane domain can be modified by selection or amino acid substitution to avoid binding of such domain with the transmembrane domain of the same or different surface membrane protein, thereby minimizing interaction with other members of the receptor complex.

[0183] Transmembrane domain can be derived from either natural or synthetic source.When source is natural, domain can be derived from any membrane-binding protein or transmembrane protein, for example, type I transmembrane protein.When source is synthetic, transmembrane domain can be any artificial sequence, for example, artificial hydrophobic sequence, that facilitates the insertion of CAR into cell membrane. Examples of transmembrane regions of particular use in the present invention include, but are not limited to, transmembrane domains derived from (i.e., comprising at least the transmembrane regions of) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some embodiments, the transmembrane domain may be synthetic, in which case it will primarily comprise hydrophobic residues such as leucine and valine. In some exemplary embodiments, a phenylalanine, tryptophan, and valine triplet will be found at each end of the synthetic transmembrane domain.

[0184] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the costimulatory signaling domains described herein, any of the intracellular signaling domains described herein, or any of the other domains described herein that may be included in a subject CAR.

[0185] In some embodiments, the transmembrane domain further comprises a hinge region. The CAR of the present invention may also comprise a hinge region. The hinge region of a CAR is a hydrophilic region located between the antigen-binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component of a CAR. The hinge region may comprise a domain selected from an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, the CD8a hinge, an artificial hinge composed of a polypeptide that can be as small as three glycines (Gly), and the CH1 and CH3 domains of IgG (such as human IgG4).

[0186] In some embodiments, the CAR of the present disclosure comprises a hinge region that connects the antigen-binding domain to the transmembrane domain, which in turn connects it to the intracellular domain. The hinge region preferably can assist the antigen-binding domain in recognizing and binding to the target antigen on the target cell (see, for example, Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, allowing the antigen-binding domain to have a structure that optimally recognizes the specific structure and density of the target antigen on a cell, such as a tumor cell. The flexibility of the hinge region allows the hinge region to adopt many different conformations.

[0187] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD8-derived hinge region).

[0188] The hinge region can have a length of about 4 amino acids to about 50 amino acids, for example, about 4 amino acids to about 10 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 40 amino acids, or about 40 amino acids to about 50 amino acids.

[0189] A suitable hinge region can be readily selected and can be any of several suitable lengths, for example, 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0190] For example, the hinge region may be a glycine polymer (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 52) and (GGGS) n(SEQ ID NO:53), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively conformationally undefined and can therefore serve as neutral tethers between components. Glycine polymers can be used; glycine has significantly more access to the φ-ψ space than alanine and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2:73-142). Exemplary hinge regions can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:55), GGSGG (SEQ ID NO:56), GSGSG (SEQ ID NO:57), GSGGG (SEQ ID NO:58), GGGSG (SEQ ID NO:59), GSSSG (SEQ ID NO:60), and the like.

[0191] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. The amino acid sequences of immunoglobulin hinge regions are known in the art; see, for example, Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1):162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. As a non-limiting example, an immunoglobulin hinge region may have the following amino acid sequence: TIFF0007654557000015.tif11138 (see, e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); It can contain one of the following: TIFF0007654557000016.tif32140.

[0192] The hinge region can comprise the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge region. In one embodiment, the hinge region can comprise one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge can be replaced by Tyr, thereby causing the hinge region to have the sequence see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one embodiment, the hinge region can comprise an amino acid sequence derived from human CD8 or a variant thereof.

[0193] In one embodiment, the transmembrane domain comprises a CD8α transmembrane domain. In some embodiments, a subject CAR comprises a CD8α transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:7, which can be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:8.

[0194] In another embodiment, a subject CAR comprises a CD8α hinge domain and a CD8α transmembrane domain. In one embodiment, the CD8α hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 13, which can be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO: 14.

[0195] In one embodiment, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, a subject CAR comprises a CD28 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 15, which can be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO: 16.

[0196] Acceptable variations of the transmembrane and / or hinge domain while retaining its intended function will be known to those of skill in the art. For example, in some embodiments, the transmembrane or hinge domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs:7, 13, and 15. For example, in some embodiments, the transmembrane domain or hinge domain is encoded by a nucleic acid sequence comprising a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the nucleotide sequences set forth in SEQ ID NOs:8, 14, and 16.

[0197] The transmembrane domain may be combined with any hinge domain and / or may comprise one or more transmembrane domains described herein.

[0198] A transmembrane domain described herein, such as the transmembrane regions of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9, can be combined with any of the antigen binding domains described herein, any of the costimulatory signaling domains or intracellular or cytoplasmic domains described herein, or any of the other domains described herein that may be included in a CAR.

[0199] In one aspect, the transmembrane domain may be synthetic, in which case it will comprise primarily hydrophobic residues such as leucine and valine. In an exemplary aspect, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0200] In some embodiments, a subject CAR may further comprise a spacer domain between the extracellular domain and transmembrane domain of the CAR, or between the intracellular domain and transmembrane domain of the CAR. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to either the extracellular domain or the intracellular domain within a polypeptide chain. A spacer domain may comprise up to 300 amino acids, e.g., 10 to 100 amino acids, or 25 to 50 amino acids. In some embodiments, a spacer domain may be a short oligopeptide or polypeptide linker, e.g., 2 to 10 amino acids in length. For example, a glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain of a subject CAR.

[0201] Thus, a subject CAR of the present disclosure can comprise any of the transmembrane domains, hinge domains, or spacer domains described herein.

[0202] Intracellular domain The subject CAR of the present invention also comprises an intracellular domain. The intracellular domain of the CAR is responsible for activating at least one effector function of the cell (e.g., immune cell) in which the CAR is expressed. The intracellular domain transmits an effector function signal, directing the cell (e.g., immune cell) to perform its specialized function, such as damaging and / or destroying target cells.

[0203] The intracellular or otherwise cytoplasmic domain of a CAR is responsible for the activation of the cell in which the CAR is expressed. Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portions of surface receptors, costimulatory molecules, and any molecules that act in concert to initiate signaling in T cells, as well as any derivatives or variants of these elements and any synthetic sequences that have the same functional properties.

[0204] In certain embodiments, the intracellular domain comprises a costimulatory signaling domain. In certain embodiments, the intracellular domain comprises an intracellular signaling domain. In certain embodiments, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain. In certain embodiments, the intracellular domain comprises 4-1BB and CD3 zeta. In certain embodiments, the costimulatory signaling domain comprises 4-1BB. In certain embodiments, the intracellular signaling domain comprises CD3 zeta.

[0205] In one embodiment, the intracellular domain of the CAR comprises at least one signaling domain from a costimulatory signaling domain comprising any portion of one or more costimulatory molecules, e.g., CD2, CD3, CD8, CD27, CD28, OX40, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof having the same functional properties, and any combination thereof.

[0206] Examples of intracellular signaling domains include, but are not limited to, the ζ chain of the T cell receptor complex or any of its homologs, such as the η chain, FcsRI γ and β chains, MB1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction such as CD2, CD5, and CD28. In one aspect, the intracellular signaling domain can be human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing cytoplasmic receptor, and combinations thereof.

[0207] Other examples of intracellular domains include TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma RiIa, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD lib, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT These include fragments or domains derived from one or more molecules or receptors, including, but not limited to, AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0208] Additional examples of intracellular domains include the intracellular signaling domains of several different immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins, including, but not limited to, CD3, B7 family costimulatory receptors, and tumor necrosis factor receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, the intracellular signaling domain can include signaling domains used by NK cells and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195), such as the signaling domains of NKp30(B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0209] Intracellular signaling domains suitable for use in the subject CARs of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; altered transcription of a target gene; altered activity of a protein; altered cellular behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; modulation of a cell signaling response, etc.) in response to activation of the CAR (i.e., activated by an antigen and a dimerizing agent). In some embodiments, the intracellular signaling domain comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described below. In some embodiments, the intracellular signaling domain comprises a DAP10 / CD28-type signaling chain. In some embodiments, the intracellular signaling domain is not covalently linked to the membrane-bound CAR, but instead is diffused in the cytoplasm.

[0210] Intracellular signaling domains suitable for use in the subject CARs of the present invention comprise immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, the ITAM motif is repeated twice within the intracellular signaling domain, wherein the first and second instances of the ITAM motif are separated from each other by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain of a subject CAR comprises three ITAM motifs. In some embodiments, the intracellular signaling domain comprises a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, or Fc RL5 (see, e.g., Gillis et al., Front. (2014) Immunol. 5:254).

[0211] A suitable intracellular signaling domain can be an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain does not need to contain the entire sequence of the protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0212] In one aspect, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase-binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer-activating receptor-associated protein; killer-activating receptor-associated protein, etc.). In one aspect, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcR gamma; fceRl gamma; high-affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high-affinity gamma chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one aspect, the intracellular signaling domain is derived from CD79A (B cell antigen receptor complex-associated protein alpha chain; also known as CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; Ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.).In one embodiment, the intracellular signaling domain suitable for use in the subject CARs of the present disclosure comprises a DAP10 / CD28-type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the subject CARs of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR comprises the cytoplasmic signaling domain of human CD3 zeta.

[0213] Typically, the entire intracellular signaling domain can be used, although in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain so long as it transmits the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0214] The intracellular signaling domains described herein can be combined with any of the costimulatory signaling domains described herein, any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that can be included in a CAR.

[0215] Furthermore, variant intracellular signaling domains suitable for use in the subject CAR are known in the art.YMFM motif is an SH2 binding motif found in ICOS, which recruits both p85 and p50 alpha subunits of PI3K, resulting in enhanced AKT signal transduction.See, for example, Simpson et al. (2010) Curr. Opin. Immunol., 22:326-332.In one embodiment, CD28 intracellular domain variants can be generated to contain YMFM motif.

[0216] In one embodiment, the intracellular domain of a subject CAR comprises a 4-1BB costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:9, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:10. In one embodiment, the intracellular domain of a subject CAR comprises a CD28 costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:17, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:18. In one embodiment, the intracellular domain of a subject CAR comprises an ICOS costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:25, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:26 or 27. In one embodiment, the intracellular domain of a subject CAR comprises a CD2 costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:28, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:29. In one embodiment, the intracellular domain of a subject CAR comprises a CD28 YMFM variant costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 32, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO: 33. In one embodiment, the intracellular domain of a subject CAR comprises a CD27 costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 34, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO: 35. In one embodiment, the intracellular domain of a subject CAR comprises an OX40 costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 36, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO: 37.

[0217] In one embodiment, the intracellular domain of a subject CAR comprises a CD3 zeta intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:11 or 30, which can be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:12 or 31.

[0218] Permissible variations in the intracellular domain while retaining specific activity will be known to those of skill in the art. For example, in some embodiments, the intracellular domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs:9, 11, 17, 25, 28, 32, 34, or 36. For example, in some embodiments, the intracellular domain is encoded by a nucleic acid sequence comprising a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the nucleotide sequences set forth in SEQ ID NOs: 10, 12, 18, 26, 27, 29, 31, 33, 35, or 37.

[0219] In one embodiment, the intracellular domain of the subject CAR comprises an ICOS costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, the intracellular domain of the subject CAR comprises a CD28 costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, the intracellular domain of the subject CAR comprises a CD28 YMFM variant costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, the intracellular domain of the subject CAR comprises a CD27 costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, the intracellular domain of the subject CAR comprises an OX40 costimulatory domain and a CD3 zeta intracellular signaling domain. In one exemplary embodiment, the intracellular domain of the subject CAR comprises a 4-1BB costimulatory domain and a CD3 zeta intracellular signaling domain. In one exemplary embodiment, the intracellular domain of the subject CAR comprises a CD2 costimulatory domain and a CD3 zeta intracellular signaling domain.

[0220] CAR sequence A subject CAR of the present invention may be a CAR with affinity for MUC1 (e.g., MUC1). In one embodiment, a Tn-MUC1 CAR of the present invention comprising the amino acid sequence set forth in SEQ ID NO:2, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:1, comprises a 4-1BB costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, a Tn-MUC1 CAR of the present invention comprising the amino acid sequence set forth in SEQ ID NO:39, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:38, comprises a CD28 costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, a Tn-MUC1 CAR of the present invention comprising the amino acid sequence set forth in SEQ ID NO:41, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:40, comprises a CD28 YMFM variant costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, a Tn-MUC1 CAR of the present invention comprising the amino acid sequence set forth in SEQ ID NO:43, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:42, comprises a CD27 costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, a Tn-MUC1 CAR of the present invention comprising the amino acid sequence set forth in SEQ ID NO:45, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:44, comprises an OX40 costimulatory domain and a CD3 zeta intracellular signaling domain. In one embodiment, a Tn-MUC1 CAR of the present invention comprising the amino acid sequence set forth in SEQ ID NO:47, which may be encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:46, comprises a CD2 costimulatory domain and a CD3 zeta intracellular signaling domain.

[0221] Acceptable variations of a CAR while retaining specific activity will be known to those of skill in the art. For example, in some embodiments, a CAR comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 39, 41, 43, 45, or 47. For example, in some embodiments, the CAR is encoded by a nucleic acid sequence comprising a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, 38, 40, 42, 44, or 46.

[0222] In some embodiments, the subject CARs of the present invention comprise a MUC1 binding domain and a transmembrane domain. In one embodiment, the CAR comprises a MUC1 binding domain and a transmembrane domain, wherein the transmembrane domain comprises a CD8 hinge region. In one embodiment, the CAR comprises a MUC1 binding domain and a transmembrane domain, wherein the transmembrane domain comprises a CD8α transmembrane domain. In one embodiment, the CAR comprises a MUC1 binding domain and a transmembrane domain, wherein the transmembrane domain comprises a CD8 hinge region and a CD8α transmembrane domain.

[0223] In some embodiments, a subject CAR of the present invention comprises a MUC1 binding domain, a transmembrane domain, and an intracellular domain. In one embodiment, a CAR comprises a Tn-MUC1 binding domain, a transmembrane domain, and an intracellular domain. In one embodiment, a CAR comprises a Tn-MUC1 binding domain, a transmembrane domain, and an intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta domain. In one embodiment, a CAR comprises a Tn-MUC1 binding domain, a transmembrane domain, and an intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta domain. In one embodiment, a CAR comprises a Tn-MUC1 binding domain, a transmembrane domain, and an intracellular domain comprising a CD28 YMFM variant costimulatory domain and a CD3 zeta domain. In one embodiment, a CAR comprises a Tn-MUC1 binding domain, a transmembrane domain, and an intracellular domain comprising a CD27 domain and a CD3 zeta domain. In one embodiment, the CAR comprises a Tn-MUC1 binding domain, a transmembrane domain, and an intracellular domain comprising an OX40 domain and a CD3 zeta domain. In one embodiment, the CAR comprises a Tn-MUC1 binding domain, a transmembrane domain, and an intracellular domain comprising a CD2 domain and a CD3 zeta domain.

[0224] Accordingly, the present invention provides modified immune cells or precursor cells thereof, e.g., modified T cells, modified NK cells, modified NKT cells, comprising a chimeric antigen receptor (CAR) with affinity for MUC1, as described herein.

[0225] Human antibodies It may be preferable that the antigen-binding domain of CAR comprises a human antibody or its fragment.Fully human antibodies are particularly desirable for the therapeutic treatment of human subjects.Human antibodies can be produced by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences (including improvements to these techniques).See also U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735 and WO91 / 10741; each of which is incorporated herein by reference in its entirety.

[0226] Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered nonfunctional by homologous recombination, either separately or simultaneously with the introduction of human immunoglobulin loci. For example, homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice has been described to result in complete inhibition of endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the usual manner with a selected antigen, such as all or a portion of a polypeptide of the present invention. Antibodies against the selected target can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such technology, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies, including, but not limited to, IgG1 (gamma 1) and IgG3. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (Int. Rev. Immunol., 13:65-93 (1995)).For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT Publication Nos. WO98 / 24893, WO96 / 34096, and WO96 / 33735; and U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, each of which is incorporated by reference in its entirety. Additionally, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be contracted to provide human antibodies against a selected antigen using technology similar to that described above. For a specific discussion of the transfer of human germ-line immunoglobulin gene arrays in germ-line mutant mice that will result in the production of human antibodies upon antigen challenge, see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993); and Duchosal et al., Nature, 355:258 (1992).

[0227] Human antibodies can also be derived from phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309 (1996)). Phage display technology (McCafferty et al., Nature, 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires derived from unimmunized donors. According to this technique, antibody V domain genes are cloned into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of an antibody also results in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in a variety of formats; for reviews, see, e.g., Johnson, Kevin S., and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). V gene segments from several sources can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of unimmunized mice.V gene repertoires from unimmunized human donors can be constructed, and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially according to the techniques described by Marks et al., J. Mol. Biol., 222:581-597 (1991), or Griffith et al., EMBO J., 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905, each of which is incorporated herein by reference in its entirety.

[0228] Human antibodies can also be generated by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275, each of which is incorporated by reference in its entirety.) Human antibodies may also be generated in vitro using hybridoma techniques, such as, but not limited to, those described by Roder et al. (Methods Enzymol., 121:140-167 (1986)).

[0229] Dominant-negative receptors and switch receptors The present invention provides compositions and methods for engineered immune cells or their precursor cells, e.g., engineered T cells, comprising dominant-negative receptors and / or switch receptors. Thus, in some embodiments, immune cells are genetically engineered to express dominant-negative receptors and / or switch receptors. The sequences of dominant-negative receptors and switch receptors are found in Table 1. As used herein, the term "dominant-negative receptor" refers to a molecule designed to reduce the effect of a negative signaling molecule, e.g., on engineered immune cells of the present invention. Dominant-negative receptors of the present invention may bind to negative signaling molecules, e.g., TGF-β or PD-1, via their extracellular domains associated with negative signals, thereby reducing the effect of the negative signaling molecule. Such dominant-negative receptors are described herein. For example, engineered immune cells comprising dominant-negative receptors may bind to negative signaling molecules in the microenvironment of the engineered immune cells, thereby reducing the effect the negative signaling molecule may have on the engineered immune cells.

[0230] In addition to reducing the effect of negative signaling molecules, the switch receptors of the present invention may be designed to convert negative signals into positive signals by including an intracellular domain associated with a positive signal. Switch receptors designed to convert negative signals into positive signals are described herein. Thus, the switch receptors include an extracellular domain associated with a negative signal and / or an intracellular domain associated with a positive signal.

[0231] Tumor cells generate an immunosuppressive microenvironment that serves to protect them from immune recognition and elimination. This immunosuppressive microenvironment can limit the effectiveness of immunosuppressive therapies, such as CAR-T cell therapy. The secreted cytokine transforming growth factor-β (TGFβ) directly inhibits the function of cytotoxic T cells and additionally induces the formation of regulatory T cells, further suppressing the immune response. T cell immunosuppression by TGFβ in the context of prostate cancer has previously been demonstrated (Donkor et al., 2011; Shalapour et al., 2015). To reduce the immunosuppressive effects of TGFβ, immune cells can be modified to express a dominant-negative receptor for TGF-β.

[0232] In some embodiments, the dominant-negative receptor is a truncated variant of a wild-type protein associated with negative signaling. In some embodiments, the dominant-negative receptor is a dominant-negative receptor for TGF-β. Thus, in some embodiments, the dominant-negative receptor for TGF-β is a truncated variant of a wild-type TGF-β receptor. In some embodiments, the dominant-negative receptor is a truncated dominant-negative variant of TGF-β receptor type II (TGFβRII-DN). In one embodiment, TGFβRII-DN comprises the amino acid sequence of SEQ ID NO:76, which can be encoded by the nucleic acid sequence of SEQ ID NO:77.

[0233] Acceptable variations in the sequence of a TGFβRII-DN while retaining its intended function will be known to those of skill in the art. For example, in some embodiments, a dominant-negative receptor of the present invention is a TGFβRII-DN comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:76. In one embodiment, the dominant-negative receptor is a TGFβRII-DN comprising the amino acid sequence set forth in SEQ ID NO:76.

[0234] In some embodiments, the dominant negative receptor of the present invention is a TGFβRII-DN encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 77. In one embodiment, the dominant negative receptor is a TGFβRII-DN encoded by the nucleic acid sequence set forth in SEQ ID NO: 77.

[0235] In one embodiment, a switch receptor suitable for use in the present invention is a PD1-CTM-CD28 receptor. The PD1-CTM-CD28 receptor converts a negative PD1 signal into a positive CD28 signal when expressed in a cell. The PD1-CTM-CD28 receptor comprises a variant of the PD1 extracellular domain, the CD28 transmembrane domain, and the CD28 cytoplasmic domain. In one embodiment, the PD1-CTM-CD28 receptor comprises the amino acid sequence of SEQ ID NO:79, which can be encoded by the nucleic acid sequence of SEQ ID NO:78.

[0236] Acceptable variations of the PD1-CTM-CD28 receptor while retaining its intended biological activity (e.g., converting a negative PD1 signal to a positive CD28 signal when expressed in a cell) will be known to those of skill in the art. Thus, the PD1-CTM-CD28 receptor of the present invention can comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the PD1-CTM-CD28 receptor set forth in SEQ ID NO:78. Thus, the PD1-CTM-CD28 receptor of the present invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the PD1-CTM-CD28 receptor set forth in SEQ ID NO:79.

[0237] In one embodiment, a switch receptor suitable for use in the present invention is a PD1-PTM-CD28 receptor. The PD1-PTM-CD28 receptor converts a negative PD1 signal into a positive CD28 signal when expressed in a cell. The PD1-PTM-CD28 receptor comprises a variant of the PD1 extracellular domain, the PD1 transmembrane domain, and the CD28 cytoplasmic domain. In one embodiment, the PD1-PTM-CD28 receptor comprises the amino acid sequence of SEQ ID NO:81, which can be encoded by the nucleic acid sequence of SEQ ID NO:80.

[0238] Acceptable variations of the PD1-PTM-CD28 receptor while retaining its intended biological activity (e.g., converting a negative PD1 signal to a positive CD28 signal when expressed in a cell) will be known to those of skill in the art. Thus, the PD1-PTM-CD28 receptor of the present invention can comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the PD1-PTM-CD28 receptor set forth in SEQ ID NO:80. Thus, the PD1-PTM-CD28 receptor of the present invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the PD1-PTM-CD28 receptor set forth in SEQ ID NO:81.

[0239] In one aspect, a switch receptor suitable for use in the present invention is PD1 A132L -PTM-CD28 receptor. PD1 A132LThe -PTM-CD28 receptor converts negative PD1 signals into positive CD28 signals when expressed in cells. A point mutation (A132L) substituting alanine with leucine at amino acid position 132 of PD1 was found to increase its affinity for PD-L1 by two-fold (see, e.g., Zhang et al., Immunity (2004) 20(3), 337-347). PD1 A132L -PTM-CD28 receptor comprises a variant of the PD1 extracellular domain with an amino acid substitution at position 132 (A132L), the PD1 transmembrane domain, and the CD28 cytoplasmic domain. A132L -PTM-CD28 receptor comprises the amino acid sequence of SEQ ID NO:83, which can be encoded by the nucleic acid sequence of SEQ ID NO:82.

[0240] PD1 while preserving its intended biological activity (e.g., converting a negative PD1 signal to a positive CD28 signal when expressed in cells). A132L Acceptable variations of the -PTM-CD28 receptor will be known to those of skill in the art. A132L -PTM-CD28 receptor is PD1 as shown in SEQ ID NO:82 A132L -PTM-A CD28 receptor. A132L -PTM-CD28 receptor is PD1 as shown in SEQ ID NO:83 A132L-PTM- It can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a nucleic acid sequence of a CD28 receptor.

[0241] In one embodiment, a switch receptor suitable for use in the present invention is a PD1-4-1BB receptor. The PD1-4-1BB receptor (also referred to herein as PD1-BB) converts a negative PD1 signal into a positive 4-1BB signal when expressed in a cell. In one embodiment, the PD1-4-1BB receptor comprises the amino acid sequence of SEQ ID NO:85, which can be encoded by the nucleic acid sequence of SEQ ID NO:84.

[0242] Acceptable variations of the PD1-4-1BB receptor while retaining its intended biological activity (e.g., converting a negative PD1 signal to a positive 4-1BB signal when expressed in a cell) will be known to those of skill in the art. Thus, the PD1-4-1BB receptor of the present invention can include an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the PD1-4-1BB receptor set forth in SEQ ID NO:84. Thus, the PD1-4-1BB receptor of the present invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the PD1-4-1BB receptor set forth in SEQ ID NO:85.

[0243] In one aspect, a switch receptor suitable for use in the present invention is PD1 A132L -4-1BB receptor. PD1 A132L The PD1-4-1BB receptor (also referred to herein as PD1*BB) converts a negative PD1 signal into a positive 4-1BB signal when expressed in a cell. A132L The -4-1BB receptor comprises the amino acid sequence of SEQ ID NO:87, which can be encoded by the nucleic acid sequence of SEQ ID NO:86.

[0244] PD1 while preserving its intended biological activity (e.g., converting a negative PD1 signal to a positive 4-1BB signal when expressed in cells). A132L Acceptable variations of the PD1-4-1BB receptor will be known to those skilled in the art. A132L The PD1 -4-1BB receptor is shown in SEQ ID NO:86 A132L The PD1 receptor of the present invention may comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of the PD1 receptor. A132L The PD1 -4-1BB receptor is shown in SEQ ID NO:87 A132L The nucleic acid may be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the -4-1BB receptor.

[0245] In one embodiment, a switch receptor suitable for use in the present invention is a TGFβR-IL12Rβ1 receptor, which converts a negative TGF-β signal into a positive IL-12 signal when expressed in a cell. In one embodiment, the TGFβR-IL12Rβ1 receptor comprises the amino acid sequence of SEQ ID NO:89, which can be encoded by the nucleic acid sequence of SEQ ID NO:88.

[0246] Acceptable variations of the TGFβR-IL12Rβ1 receptor while retaining its intended biological activity (e.g., converting a negative TGF-β signal into a positive IL-12 signal when expressed in a cell) will be known to those of skill in the art. Thus, the TGFβR-IL12Rβ1 receptor of the present invention can comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the TGFβR-IL12Rβ1 receptor set forth in SEQ ID NO:88. Thus, the TGFβR-IL12Rβ1 receptor of the present invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the TGFβR-IL12Rβ1 receptor set forth in SEQ ID NO:89.

[0247] In one embodiment, a switch receptor suitable for use in the present invention is a TGFβR-IL12Rβ2 receptor, which converts a negative TGF-β signal into a positive IL-12 signal when expressed in a cell. In one embodiment, the TGFβR-IL12Rβ2 receptor comprises the amino acid sequence of SEQ ID NO:91, which can be encoded by the nucleic acid sequence of SEQ ID NO:90.

[0248] Acceptable variations of the TGFβR-IL12Rβ2 receptor while retaining its intended biological activity (e.g., converting a negative TGF-β signal into a positive IL-12 signal when expressed in a cell) will be known to those of skill in the art. Thus, the TGFβR-IL12Rβ2 receptor of the present invention can comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the TGFβR-IL12Rβ2 receptor set forth in SEQ ID NO:90. Thus, the TGFβR-IL12Rβ2 receptor of the present invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the TGFβR-IL12Rβ2 receptor set forth in SEQ ID NO:91.

[0249] In one embodiment, a switch receptor suitable for use in the present invention is a TIM3-CD28 receptor, which converts a negative TIM-3 signal into a positive CD28 signal when expressed in a cell. In one embodiment, the TIM3-CD28 receptor comprises the amino acid sequence of SEQ ID NO:93, which can be encoded by the nucleic acid sequence of SEQ ID NO:92.

[0250] Those skilled in the art will know permissible variations of the TIM3-CD28 receptor while retaining its intended biological activity (e.g., converting a negative TIM-3 signal to a positive CD28 signal when expressed in a cell). Thus, the TIM3-CD28 receptor of the present invention can comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the TIM3-CD28 receptor set forth in SEQ ID NO:92. Thus, the TIM3-CD28 receptor of the present invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the TIM3-CD28 receptor shown in SEQ ID NO:93.

[0251] Other dominant-negative receptors and switch receptors suitable for use in the present invention are described in PCT Publication No. WO2013019615A2, the disclosure of which is incorporated herein by reference.

[0252] Engineered immune cells The present invention provides modified immune cells or their precursor cells (e.g., modified T cells, modified NK cells, modified NKT cells) containing the target CAR. Thus, such modified cells have the specificity directed by the CAR expressed therein. For example, modified cells of the present invention containing TnMUC1 CAR have specificity for MUC1 on target cells.

[0253] Any modified cell comprising a CAR comprising any antigen binding domain, any hinge, any transmembrane domain, any intracellular costimulatory domain, and any intracellular signaling domain described herein is contemplated and can be readily understood and produced by one of skill in the art in light of the disclosure herein.

[0254] In some embodiments, the modified cells are immune cells or precursor cells thereof. In exemplary embodiments, the modified cells are T cells. In exemplary embodiments, the modified cells are autologous cells. In exemplary embodiments, the modified cells are autologous immune cells or precursor cells thereof. In exemplary embodiments, the modified cells are autologous T cells.

[0255] The present invention provides modified immune cells or their precursor cells (e.g., T cells) comprising a CAR and / or a dominant-negative receptor and / or a switch receptor. Thus, such modified cells retain the specificity dictated by the CAR expressed therein. For example, modified cells of the present invention comprising a TnMUC1-CAR retain specificity for TnMUC1 on target cells.

[0256] In some embodiments, the modified cells of the present invention comprise a CAR. In one embodiment, the modified cells of the present invention comprise a CAR with affinity for TnMUC1 on target cells. In some embodiments, the modified cells of the present invention comprise a dominant-negative receptor and / or a switch receptor. In one embodiment, the modified cells of the present invention comprise a dominant-negative receptor capable of reducing the effect of negative signaling molecules in the microenvironment. In one embodiment, the modified cells of the present invention comprise a switch receptor capable of reducing the effect of negative signaling molecules in the microenvironment and converting a negative signal to a positive signal within the modified cell. In some embodiments, the modified cells of the present invention comprise a CAR and a dominant-negative receptor and / or a switch receptor. In one embodiment, the modified cells of the present invention comprise a CAR with affinity for TnMUC1 on target cells and a dominant-negative receptor and / or a switch receptor. The modified cells comprising the dominant-negative receptor and / or switch receptor of the present invention can associate with negative signaling molecules (e.g., inhibitory ligands) in the microenvironment via their respective extracellular domains. In some embodiments, modified cells of the invention comprising a dominant-negative receptor can reduce the effect of a negative signaling molecule in the microenvironment, wherein the dominant-negative receptor comprises an extracellular domain associated with a negative signal. In some embodiments, modified cells of the invention comprising a switch receptor can convert the effect of a negative signaling molecule in the microenvironment into a positive signal, wherein the switch receptor comprises an extracellular domain associated with a negative signal and an intracellular domain associated with a positive signal.

[0257] In exemplary embodiments, the modified cells of the invention comprise a dominant-negative receptor capable of reducing the effect of a negative signaling molecule. In one embodiment, the modified cells of the invention comprise TGFβRII-DN.

[0258] In exemplary embodiments, the modified cells of the invention comprise a switch receptor capable of converting the effect of a negative signaling molecule into a positive (e.g., activating) signal within the modified cell. In one embodiment, the modified cells of the invention comprise PD1-CTM-CD28. In one embodiment, the modified cells of the invention comprise PD1 A132L -PTM-CD28. In one embodiment, the modified cells of the invention comprise TIM3-CD28.

[0259] In an exemplary embodiment, the modified cells of the present invention comprise a TnMUC1-CAR and a dominant-negative receptor capable of reducing the effects of negative signaling molecules. In one embodiment, the modified cells of the present invention comprise a TnMUC1-CAR and a TGFβRII-DN. In addition to having affinity for TnMUC1 on target cells, such modified cells (e.g., modified T cells) are capable of reducing inhibitory TGF-β signals from the microenvironment in which they reside.

[0260] In exemplary embodiments, the modified cells of the invention comprise a MUC1-CAR and a switch receptor capable of converting the inhibitory effect of a negative signaling molecule into a positive signal within the modified cell. In one embodiment, the modified cells of the invention comprise a MUC1-CAR and a PD1-CTM-CD28. In one embodiment, the modified cells of the invention comprise a MUC1-CAR and a PD1 A132L In one embodiment, the modified cells of the present invention comprise MUC1-CAR and TIM3-CD28. In one embodiment, the modified cells of the present invention comprise MUC1 CAR and PD1-4-1BB. In one embodiment, the modified cells of the present invention comprise MUC1-CAR and PD1 A132LIn one embodiment, the modified cells of the present invention comprise MUC1-CAR and TGFβR-IL12Rβ1. In addition to having affinity for MUC1 on target cells, such modified cells (e.g., modified T cells) are capable of converting inhibitory PD-1, TIMI1, or TGFβ signals from the microenvironment into positive (e.g., activating) signals within the modified cells. In addition to having affinity for MUC1 on target cells, such modified cells (e.g., modified T cells) are capable of converting inhibitory PD-1 or TIM-3 signals from the microenvironment into positive (e.g., activating) CD28 signals within the modified cells.

[0261] In an exemplary embodiment, the modified cells of the invention comprise MUC1-CAR, TGFβRII-DN, and PD1-CTM-CD28.

[0262] Nucleic acids and expression vectors The present invention provides nucleic acids encoding CARs having affinity for MUC1 (e.g., Tn-MUC1). The subject CARs described herein comprise an antigen-binding domain (e.g., a MUC1-binding domain), a transmembrane domain, and an intracellular domain. Thus, the present invention provides nucleic acids encoding the antigen-binding domain (e.g., a MUC1-binding domain), a transmembrane domain, and an intracellular domain of a subject CAR.

[0263] In exemplary embodiments, the nucleic acid encoding the MUC1 CAR of the present invention is encoded by a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:1, 38, 40, 42, 44, or 46.

[0264] The present invention provides nucleic acids encoding CARs and / or dominant-negative receptors and / or switch receptors. In one embodiment, the nucleic acids of the present disclosure comprise a nucleic acid sequence encoding a subject CAR of the present invention (e.g., TnMUC1-CAR). In one embodiment, the nucleic acids of the present disclosure comprise a nucleic acid sequence encoding a dominant-negative receptor and / or a switch receptor (e.g., PD1-PTM-CD28 receptor).

[0265] In some embodiments, the nucleic acids of the present disclosure provide for the production of a CAR and / or a dominant negative receptor and / or a switch receptor, e.g., as described herein, in a mammalian cell. In some embodiments, the nucleic acids of the present disclosure provide for the amplification of nucleic acids encoding a CAR and / or a dominant negative receptor and / or a switch receptor.

[0266] As described herein, the target CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.Therefore, the present disclosure provides a nucleic acid that encodes the antigen binding domain, the transmembrane domain, and the intracellular domain of the target CAR.As described herein, various dominant negative receptors and switch receptors are provided.Therefore, the present invention provides a nucleic acid that encodes a dominant negative receptor and / or a switch receptor.

[0267] In some embodiments, the nucleic acid encoding the CAR is separate from the nucleic acid encoding the dominant negative receptor and / or the switch receptor. In exemplary embodiments, the nucleic acid encoding the CAR and the nucleic acid encoding the dominant negative receptor and / or the switch receptor are present within the same nucleic acid.

[0268] In some embodiments, nucleic acids of the present invention include nucleic acids comprising a coding sequence for a CAR and a coding sequence for a dominant-negative receptor and / or a switch receptor. In some embodiments, nucleic acids of the present invention include nucleic acids comprising a coding sequence for a CAR and a coding sequence for a dominant-negative receptor and / or a switch receptor, separated by a linker. Linkers for use in the present invention (e.g., in the context of linking a coding sequence for a CAR and a coding sequence for a dominant-negative receptor and / or a switch receptor) allow multiple proteins to be encoded by the same nucleic acid sequence (e.g., a polycistronic or bicistronic sequence), and the proteins to be translated as a polyprotein that is dissociated into separate protein components. For example, linkers for use in nucleic acids of the present disclosure comprising a coding sequence for a CAR and a coding sequence for a dominant-negative receptor and / or a switch receptor allow the CAR and the dominant-negative receptor and / or switch receptor to be translated as a polyprotein that is dissociated into separate CAR and dominant-negative receptor and / or switch receptor components.

[0269] In some embodiments, the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into a start codon, such as ATG, of a protein-coding region, thereby resulting in cap-independent translation of the gene. A variety of internal ribosome entry sites are known to those skilled in the art, including, but not limited to, IRESs available from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRESs available from, for example, cardioviruses, rhinoviruses, aphthoviruses, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select a suitable IRES for use in the present invention.

[0270] In some embodiments, the linker comprises a nucleic acid sequence encoding a self-cleaving peptide. As used herein, a "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows encoding of multiple proteins as a polyprotein that dissociates into component proteins upon translation. The use of the term "self-cleaving" is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those of skill in the art, including, but not limited to, those found in members of the Picornaviridae virus family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), zosea siagnavirus (TaV), and porcine teschovirus-1 (PTV-1); and cardioviruses such as tylovirus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. Those of skill in the art will be able to select a suitable self-cleaving peptide for use in the present invention.

[0271] In some embodiments, the nucleic acid of the present disclosure comprises a nucleic acid sequence comprising a coding sequence for a CAR and a coding sequence for a dominant negative receptor and / or a switch receptor, separated by a linker comprising a T2A peptide sequence. In some embodiments, the T2A peptide sequence is An amino acid sequence that can be encoded by TIFF0007654557000018.tif11139 TIFF0007654557000019.tif4128. In some embodiments, a linker comprising a T2A peptide sequence can further comprise a spacer sequence as described herein. For example, a linker comprising a T2A peptide sequence can further comprise a spacer sequence as described herein. It may further comprise a spacer sequence comprising the amino acid sequence SGRSGGG (SEQ ID NO:96), which may be encoded by TIFF0007654557000020.tif4128.

[0272] In some embodiments, the nucleic acids of the present disclosure comprise a nucleic acid sequence comprising a coding sequence for a CAR and a coding sequence for a dominant negative receptor and / or a switch receptor, separated by a linker comprising an F2A peptide sequence. In some embodiments, the F2A peptide sequence is An amino acid sequence that can be encoded by TIFF0007654557000021.tif11139 Contains TIFF0007654557000022.tif4128.

[0273] In some embodiments, the linker further comprises a nucleic acid sequence encoding a furin cleavage site. Furin is a ubiquitously expressed protease that resides in the trans-Golgi and processes protein precursors prior to their secretion. Furin cleaves at the COOH-terminus of its consensus recognition sequence. Various furin consensus recognition sequences (or "furin cleavage sites") are known to those skilled in the art, including, but not limited to, Arg-X-Lys-Arg (SEQ ID NO:100) or Arg-X-Arg-Arg (SEQ ID NO:101), such as Arg-Gln-Lys-Arg (SEQ ID NO:103), and Arg-XX-Arg (SEQ ID NO:102), where X is any naturally occurring amino acid. Another example of a furin cleavage site is X1-Arg-X2-X3-Arg (SEQ ID NO:104), where X1 is Lys or Arg, X2 is any naturally occurring amino acid, and X3 is Lys or Arg. Those skilled in the art will be able to select a suitable furin cleavage site for use in the present invention.

[0274] In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a furin cleavage site and a 2A peptide. Examples include, but are not limited to, a linker comprising a nucleic acid sequence encoding furin and F2A, a linker comprising a nucleic acid sequence encoding furin and E2A, a linker comprising a nucleic acid sequence encoding furin and P2A, or a linker comprising a nucleic acid sequence encoding furin and T2A. Those skilled in the art will be able to select a suitable combination for use in the present invention. In such embodiments, the linker may further comprise a spacer sequence between the furin and the 2A peptide. Various spacer sequences are known in the art, including, but not limited to, glycine serine (GS) spacers such as (GS)n, (GSGGS)n (SEQ ID NO:52), and (GGGS)n (SEQ ID NO:53), where n represents an integer of at least 1. Exemplary spacer sequences can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO:55), GGSGG (SEQ ID NO:56), GSGSG (SEQ ID NO:57), GSGGG (SEQ ID NO:58), GGGSG (SEQ ID NO:59), and GSSSG (SEQ ID NO:60), etc. One of skill in the art will be able to select a suitable spacer sequence for use in the present invention.

[0275] In some embodiments, the nucleic acids of the present disclosure comprise a nucleic acid sequence comprising a coding sequence for a CAR and a coding sequence for a dominant-negative receptor and / or a switch receptor, separated by a furin-(G4S)2-T2A (F-GS2-T2A) linker. The F-GS2-T2A linker is a nucleic acid sequence TIFF0007654557000023.tif18141, and the amino acid sequence TIFF0007654557000024.tif4128. One of skill in the art will recognize that the linkers of the present invention may include sequence variations that are tolerated.

[0276] In some embodiments, the present invention provides a nucleic acid comprising a nucleic acid sequence encoding a dominant-negative receptor and / or a switch receptor as described herein. In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding the dominant-negative receptor and / or a switch receptor and a nucleic acid sequence encoding a CAR (e.g., TnMUC1-CAR) as described herein. In one embodiment, the nucleic acid sequence encoding the dominant-negative receptor and / or a switch receptor and the nucleic acid sequence encoding the CAR are present on separate nucleic acids. In one embodiment, the nucleic acid sequence encoding the dominant-negative receptor and / or a switch receptor and the nucleic acid sequence encoding the CAR are present in the same nucleic acid. In such embodiments, the nucleic acid sequence encoding the dominant-negative receptor and / or a switch receptor and the nucleic acid sequence encoding the CAR are separated by a linker as described herein.

[0277] For example, a nucleic acid of the present disclosure may comprise a nucleic acid sequence encoding a dominant receptor, a linker, and a nucleic acid sequence encoding a CAR. In one embodiment, the linker comprises a nucleic acid sequence encoding a 2A peptide (e.g., T2A). In an exemplary embodiment, a nucleic acid of the present disclosure may comprise a nucleic acid sequence encoding a dominant negative receptor and / or a switch receptor and a nucleic acid sequence encoding a CAR, separated by a linker sequence comprising a nucleic acid sequence encoding T2A.

[0278] Thus, in one embodiment, a nucleic acid of the disclosure comprises, in the 5' to 3' direction: a nucleic acid sequence encoding a dominant negative receptor and / or a switch receptor, a nucleic acid sequence encoding a linker, and a nucleic acid sequence encoding a CAR. In one embodiment, a nucleic acid of the disclosure comprises, in the 5' to 3' direction: a nucleic acid sequence encoding a CAR, a nucleic acid sequence encoding a linker, and a nucleic acid sequence encoding a dominant negative receptor and / or a switch receptor.

[0279] Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5' to 3': a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising a 2A peptide (e.g., T2A), and a nucleic acid sequence encoding MUC1-CAR (e.g., SEQ ID NOs: 1, 38, 40, 42, 44, or 46). In one embodiment, a nucleic acid of the disclosure comprises, from 5' to 3': a nucleic acid encoding MUC1-CAR, a nucleic acid encoding a linker comprising a 2A peptide (e.g., T2A), and a nucleic acid encoding a dominant-negative receptor and / or a switch receptor.

[0280] In some aspects, the nucleic acids of the present disclosure may be operably linked to transcriptional control elements, such as promoters, enhancers, etc. Suitable promoter and enhancer elements are known to those of skill in the art.

[0281] Suitable promoters for expression in bacterial cells include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. Suitable promoters for expression in eukaryotic cells include, but are not limited to, the light and / or heavy chain immunoglobulin gene promoter and enhancer element; the cytomegalovirus immediate early promoter; the herpes simplex virus thymidine kinase promoter; the early and late SV40 promoter; the promoter present in the long terminal repeat of retrovirus; the mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. Suitable reversible promoters, including reversibly inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from many organisms, for example, eukaryotes and prokaryotes. The modification of a reversible promoter from a first organism for use in a second organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.) is well known in the art.Such reversible promoters, and systems based on such reversible promoters but also including additional regulatory proteins, include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, alcohol transactivator protein (A1cR) responsive promoter, etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivator, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid-regulated promoter, ethylene-regulated promoter, benzothiadiazole-regulated promoter, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0282] In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK cell-specific promoter.For example, the CD4 gene promoter can be used; see, for example, Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416.As another example, the CD8 gene promoter can be used.NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, for example, Eckelhart et al. Blood (2011) 117:1565.

[0283] Suitable promoters for expression in yeast cells are constitutive promoters such as the ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter, etc.; or regulatable promoters such as the GAL1 promoter, GAL10 promoter, ADH2 promoter, PHOS promoter, CUP1 promoter, GALT promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (e.g., for use in Pichia). Selection of an appropriate vector and promoter is well within the level of one skilled in the art. Suitable promoters for use in prokaryotic host cells include the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, e.g., the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, etc.; the araBAD promoter; in vivo regulated promoters such as the ssaG promoter or related promoters (see, e.g., U.S. Patent Application Publication No. 20040131637), the pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83), the nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813) (e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol.(1992) 10:888-892); sigma70 promoters, e.g., consensus sigma70 promoters (see, e.g., GenBank accession numbers AX798980, AX798961, and AX798183); stationary phase promoters, e.g., dps promoters, spv promoters, and the like; promoters from pathogenicity island SPI-2 (see, e.g., WO96 / 17951); actA promoters (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087-1096); rpsM promoters (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); tet promoters (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein--Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res.(1984) 12:7035). Strong promoters suitable for use in prokaryotes, such as Escherichia coli, include, but are not limited to, Trc, Tac, T5, T7, and Plambda. Non-limiting examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80:21-25).

[0284] Other examples of suitable promoters include the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, EF-1 alpha promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence operably linked to it when such expression is desired, or turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0285] In some embodiments, a locus or construct or transgene containing a suitable promoter is irreversibly switched via induction of an inducible system. Suitable systems for inducing irreversible switches are well known in the art; for example, induction of irreversible switches may utilize Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, ligases, recombination sites, and the like known in the art may be used to generate irreversibly switchable promoters. The methods, mechanisms, and requirements for performing site-specific recombination described elsewhere herein are used to generate irreversibly switched promoters and are well known in the art. See, for example, Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, MA), the disclosures of which are incorporated herein by reference.

[0286] In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CAR-inducing expression cassette. In one embodiment, the CAR-inducing expression cassette is for producing a transgenic polypeptide product that is released upon CAR signaling. See, for example, Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015) 7(5): 535-544.

[0287] The nucleic acids of the present disclosure can be present in an expression vector and / or a cloning vector. Expression vectors can include a selectable marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. Numerous suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating recombinant constructs of interest. The following vectors are provided by way of example and should not be construed as limiting in any way: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Prokaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0288] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769, WO 93 / 19191, WO 94 / 28938, WO 95 / 11984, and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86; Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4: 683-690; Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci.USA (1993) 90: 10613-10617); SV40; herpes simplex virus; viral vectors based on human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); retroviral vectors (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and the like.

[0289] Additional expression vectors suitable for use include, but are not limited to, lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors, engineered hybrid virus vectors, transposon-mediated vectors, etc. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0290] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0291] In some embodiments, an expression vector (e.g., a lentiviral vector) may be used to introduce a CAR into immune cells or their precursor cells (e.g., T cells). Thus, an expression vector (e.g., a lentiviral vector) of the present invention may comprise a nucleic acid encoding a CAR. In some embodiments, an expression vector (e.g., a lentiviral vector) will comprise additional elements that aid in the functional expression of a CAR encoded therein. In some embodiments, an expression vector comprising a nucleic acid encoding a CAR further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation factor-1-alpha promoter (EF-1α promoter). Use of the EF-1α promoter may increase the efficiency of expression of a downstream transgene (e.g., a nucleic acid sequence encoding a CAR). A physiological promoter (e.g., an EF-1α promoter) may be less likely to induce integration-mediated genotoxicity and may negate the ability of a retroviral vector to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those skilled in the art and may be incorporated into the vectors of the present invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises a non-essential cis-acting sequence that may improve titer and gene expression. A non-limiting example of a non-essential cis-acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear import. Other non-essential cis-acting sequences are known to those skilled in the art and may be incorporated into the vectors (e.g., lentiviral vectors) of the present invention. In some embodiments, the vector further comprises a post-transcriptional regulatory element. Post-transcriptional regulatory elements may improve RNA translation, improve transgene expression, and stabilize RNA transcripts. An example of a post-transcriptional regulatory element is the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Thus, in some embodiments, the vector for the present invention further comprises a WPRE sequence. Various post-transcriptional regulatory elements are known to those skilled in the art and may be incorporated into the vectors (e.g., lentiviral vectors) of the present invention.The vectors of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, a packaging sequence, and 5' and 3' long terminal repeats (LTRs). The term "long terminal repeat" or "LTR" refers to a base-pair domain located at the end of retroviral DNA, including the U3, R, and U5 regions. LTRs generally provide functions necessary for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. In one embodiment, the vectors of the present invention (e.g., lentiviral vectors) comprise a 3' U3-deleted LTR. Thus, the vectors of the present invention (e.g., lentiviral vectors) may comprise any combination of the elements described herein to increase the efficiency of functional expression of the transgene. For example, the vectors of the present invention (e.g., lentiviral vectors) may comprise a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, and a 3' U3-deleted LTR' in addition to the nucleic acid encoding a CAR.

[0292] The vector of the present invention may be a self-inactivating vector. As used herein, the term "self-inactivating vector" refers to a vector in which the 3' LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). A self-inactivating vector can prevent viral transcription beyond the first round of viral replication. As a result, a self-inactivating vector can infect and then be integrated into the host genome (e.g., a mammalian genome) only once, but cannot be further passaged. Therefore, a self-inactivating vector can greatly reduce the risk of generating a replication-competent virus.

[0293] In some embodiments, the nucleic acid of the present invention can be RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those skilled in the art; any known method can be used to synthesize RNA comprising a sequence encoding a CAR of the present disclosure. Methods for introducing RNA into host cells are known in the art. See, for example, Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a CAR of the present disclosure into host cells can be performed in vitro, ex vivo, or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated with RNA comprising a nucleotide sequence encoding a CAR of the present disclosure in vitro or ex vivo.

[0294] To evaluate the expression of a polypeptide or a portion thereof, the expression vector to be introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a cell population to be transfected or infected with a viral vector. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be adjacent to appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, but are not limited to, antibiotic resistance genes.

[0295] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences.Generally, reporter genes are genes that are not present in or expressed by recipient organisms or tissues, and encode polypeptides whose expression is revealed by some easily detectable characteristics, such as enzymatic activity.The expression of reporter genes is evaluated at an appropriate time after DNA is introduced into recipient cells.Suitable reporter genes may include, but are not limited to, luciferase, beta-galactosidase, chloramphenicol acetyltransferase, genes encoding secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

[0296] Methods for generating modified immune cells The present invention provides methods for producing / generating modified immune cells or their precursors (e.g., T cells / NK cells / NKT cells). The cells are generally engineered by introducing a nucleic acid encoding a CAR of interest (e.g., a MUC1 CAR).

[0297] Methods for introducing nucleic acids into cells include physical, biological, and chemical methods. Physical methods for introducing polynucleotides such as RNA into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. RNA can be introduced into target cells using commercially available methods, including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, MA) or Gene Pulser II (BioRad, Denver, CO), Multiporator (Eppendorf, Hamburg, Germany). RNA can also be introduced into cells using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems such as "gene guns" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0298] The biological method for introducing the polynucleotide of interest into host cell includes the use of DNA and RNA vector.Virus vector, especially retrovirus vector, has become the most widely used method for inserting genes into mammalian, for example, human cell.Other virus vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus, etc.See, for example, U.S. Patent No. 5,350,674 and U.S. Patent No. 5,585,362.

[0299] In some embodiments, the nucleic acid encoding the CAR of the present invention is introduced into cells by an expression vector.Provided herein is an expression vector comprising a nucleic acid encoding the CAR of interest (e.g., MUC1-CAR).Suitable expression vectors include lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA including but not limited to transposon-mediated vectors, such as Sleeping Beauty, Piggyback, and Phi31 integrase.Some other suitable expression vectors include herpes simplex virus (HSV) and retrovirus expression vectors.

[0300] Adenovirus expression vectors are based on adenovirus and have a low capacity for integration into genomic DNA, but high efficiency for transfection into host cells. Adenovirus expression vectors contain sufficient adenovirus sequences to (a) support the packaging of expression vectors and (b) ultimately express the target CAR in host cells. In some embodiments, the adenovirus genome is a 36 kb linear double-stranded DNA, into which a foreign DNA sequence (e.g., a nucleic acid encoding the target CAR) can be inserted to replace a large piece of adenovirus DNA to create the expression vector of the present invention (see, for example, Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714).

[0301] Another expression vector is based on adeno-associated virus and utilizes an adenovirus coupled system. This AAV expression vector has a high integration rate into the host genome. This vector can infect non-dividing cells, and is therefore useful for delivering genes into mammalian cells, for example, in tissue culture or in vivo. AAV vectors have a wide host range for infectivity. Details regarding the generation and use of AAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0302] Retroviral expression vectors can be integrated into the host genome, deliver large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in special cell lines. Retroviral vectors are constructed by inserting nucleic acid (e.g., nucleic acid encoding the target CAR) into a specific location in the viral genome to produce a replication-deficient virus. Retroviral vectors can infect a wide variety of cell types, but the integration and stable expression of the target CAR requires the division of host cells.

[0303] Lentiviral vectors are composite retroviruses derived from lentiviruses and contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions (see, e.g., U.S. Patent Nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency viruses (HIV-1, HIV-2) and simian immunodeficiency viruses (SIV). Lentiviral vectors have been generated by multiple attenuation of HIV pathogenic genes, for example, deletion of the genes env, vif, vpr, vpu, and nef, making the vector biologically safe. Lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of, for example, a nucleic acid encoding a CAR of interest (see, e.g., U.S. Patent No. 5,994,136).

[0304] An expression vector containing a nucleic acid of the present disclosure can be introduced into a host cell by any means known to those skilled in the art. The expression vector may contain viral sequences for transfection, if desired. Alternatively, the expression vector may be introduced by fusion, electroporation, biolistics, transfection, lipofection, etc. The host cell may be grown and expanded in culture before introducing the expression vector, followed by treatment appropriate for the introduction and integration of the vector. The host cell may then be expanded and screened for a marker present in the vector. Various markers that can be used are known in the art and may include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. In some embodiments, the host cell is an immune cell or its precursor cell, such as a T cell, NK cell, or NKT cell.

[0305] The present invention also provides genetically engineered cells that contain and stably express the subject CAR of the present disclosure. In some embodiments, the genetically engineered cells are genetically engineered T lymphocytes (T cells), regulatory T cells (Treg), naive T cells (TN), memory T cells (e.g., central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), natural killer T cells (NKT cells), and macrophages that can give rise to therapeutically relevant offspring. In one embodiment, the genetically engineered cells are autologous cells.

[0306] Modified cells (e.g., expressing and secreting the subject CAR) can be produced by stably transfecting host cells with an expression vector containing the nucleic acid of the present disclosure. Additional methods for generating modified cells of the present disclosure include, but are not limited to, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes, and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, phototransformation, gene electrotransfer and / or hydrodynamic delivery), and / or particle-based methods (e.g., impalefection and / or magnetofection using a gene gun). Transfected cells expressing the subject CAR of the present disclosure can be expanded ex vivo.

[0307] Physical methods for introducing an expression vector into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0308] Chemical means for introducing polynucleotides into host cells include macromolecule complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0309] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. Before forming a closed structure, the lipid components undergo self-reorganization, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions with structures different from the usual vesicular structure in solution are also encompassed. For example, lipids may exhibit a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0310] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose the cells to the inhibitors of the present invention, various assays may be performed to confirm the presence of the nucleic acid in the host cells. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern blotting, Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, for example, by immunological means (ELISA and Western blot), or by the assays described herein to identify agents within the scope of the present invention.

[0311] Moreover, the nucleic acids may be introduced by any means, such as transducing expanded T cells, transfecting expanded T cells, and electroporating expanded T cells. One nucleic acid may be introduced by one method and another nucleic acid may be introduced into the T cells by another method.

[0312] RNA In one embodiment, the nucleic acid introduced into host cell is RNA.In another embodiment, RNA is mRNA, including in vitro transcribed RNA or synthetic RNA.RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR).The DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase.The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other suitable DNA source.

[0313] PCR may be used to generate a template for in vitro transcription of mRNA, which is then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have a region substantially complementary to the DNA region to be used as a template for PCR. As used herein, "substantially complementary" refers to a nucleotide sequence in which most or all of the bases in the primer sequence are complementary, or in which one or more bases are non-complementary or mismatched. A substantially complementary sequence is capable of annealing or hybridizing with the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify a gene portion (open reading frame) that is normally transcribed in cells, including the 5' UTR and 3' UTR. Primers may also be designed to amplify a portion of a gene encoding a specific domain of interest. In one embodiment, primers are designed to amplify the coding region of a human cDNA, including all or part of the 5' UTR and 3' UTR. Primers useful for PCR are generated by synthetic methods well known in the art. A "forward primer" is a primer that contains a nucleotide region that is substantially complementary to a nucleotide on a DNA template upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to the 5' position of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a nucleotide region that is substantially complementary to a double-stranded DNA template downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to the 3' position of the DNA sequence to be amplified relative to the coding strand.

[0314] Chemical structures capable of promoting RNA stability and / or translation efficiency may also be used. The RNA preferably has a 5' UTR and a 3' UTR. In one embodiment, the 5' UTR is 0 to 3,000 nucleotides long. The lengths of the 5' UTR and 3' UTR sequences to be added to the coding region can be varied by various methods, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, those skilled in the art can modify the lengths of the 5' UTR and 3' UTR required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0315] 5'UTR and 3'UTR can be the natural endogenous 5'UTR and 3'UTR of the gene of interest.Alternatively, UTR sequence that is not endogenous to the gene of interest can be added by incorporating UTR sequence into forward primer and reverse primer, or by any other modification of template.The use of UTR sequence that is not endogenous to the gene of interest can be useful for modifying RNA stability and / or translation efficiency.For example, it is known that AU-rich elements in 3'UTR sequence can reduce mRNA stability.Therefore, 3'UTR can be selected or designed to increase the stability of transcribed RNA based on the properties of UTR that are well known in the art.

[0316] In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, if a 5' UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding a 5' UTR sequence. Although the Kozak sequence can increase the translation efficiency of some RNA transcripts, it is unlikely that all RNAs are necessary to enable efficient translation. The necessity of a Kozak sequence for many mRNAs is known in the art. In another embodiment, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In another embodiment, various nucleotide analogs can be used in the 3' UTR or 5' UTR to prevent exonuclease degradation of mRNA.

[0317] To enable RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter should be attached to the DNA template upstream of the sequence to be transcribed.If a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter will be incorporated upstream of the open reading frame to be transcribed in the PCR product.In one embodiment, the promoter is the T7 polymerase promoter as described elsewhere herein.Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters.Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0318] In one embodiment, mRNA has both a 5'-end cap and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in cells. On circular DNA templates, such as plasmid DNA, RNA polymerase produces long concatemeric products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR produces mRNA of normal size, which is ineffective for eukaryotic transfection, even when polyadenylated after transcription.

[0319] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the final base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).

[0320] The traditional method for incorporating a polyA / T stretch into a DNA template is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other abnormalities. This not only makes the cloning procedure laborious and time-consuming, but also often reduces its reliability. Therefore, a method that allows the construction of DNA templates with a 3' stretch of polyA / T without cloning would be highly ideal.

[0321] The poly(A) / T segment of the transcription DNA template can be generated during PCR by using a reverse primer containing a poly(T) tail (which can range in size from 50 to 5000 T), such as a 100T tail, or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. The poly(A) tail also provides stability to RNAs and reduces their degradation. Generally, the length of the poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100 to 5000 adenosines.

[0322] The poly(A) tail of RNA can be further extended after in vitro transcription using a poly(A) polymerase, such as Escherichia coli (E. coli) poly(A) polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides results in an approximately two-fold increase in RNA translation efficiency. Additionally, the addition of different chemical groups to the 3' end can increase mRNA stability. Such additions can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. The ATP analogs can further increase RNA stability.

[0323] The 5' cap also provides stability to the RNA molecule. In certain exemplary embodiments, the RNA produced by the methods disclosed herein includes a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0324] The RNA produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and promotes translation initiation. Any solute suitable for cell electroporation can be included, including factors such as sugars, peptides, lipids, proteins, antioxidants, and surfactants that promote cell permeability and viability.

[0325] In some embodiments, RNA, such as in vitro transcribed RNA, is electroporated into cells.

[0326] The disclosed methods can be applied to modulating T cell activity in basic research and therapy in the areas of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including assessing the ability of genetically modified T cells to kill target cancer cells.

[0327] These methods also provide the ability to control expression levels over a wide range, for example, by varying the amount of promoter or input RNA, allowing expression levels to be individually regulated. Furthermore, PCR-based mRNA production techniques greatly facilitate the design of mRNAs with different structures and combinations of their domains.

[0328] One advantage of the RNA transfection method of the present invention is that RNA transfection is essentially transient and does not use vectors.RNA transgenes can be delivered to lymphocytes and expressed therein after a short period of in vitro cell activation as a minimal expression cassette without the need for any additional viral sequences.Under these conditions, the integration of transgenes into the host cell genome is unlikely to occur.Due to the transfection efficiency of RNA and the ability of RNA to uniformly modify the entire lymphocyte population, cell cloning is unnecessary.

[0329] Genetic modification of host cells with in vitro transcribed RNA (IVT-RNA) utilizes two different strategies, both of which have been successfully tested in various animal models. In vitro transcribed RNA is transfected into cells by lipofection or electroporation. To achieve long-term expression of the transfected IVT-RNA, it is desirable to stabilize the IVT-RNA using various modifications.

[0330] Several IVT vectors, which are typically used as templates for in vitro transcription, are known in the literature and have been genetically modified to produce stabilized RNA transcripts. Currently, protocols used in the art are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by a gene of interest flanked by untranslated regions (UTRs) on either the 3' and / or 5' ends, and a 3' polyadenylation cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylation cassette using a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). Thus, the polyadenylation cassette corresponds to the subsequent poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. It is unclear whether this non-physiological overhang affects the amount of protein produced in cells from such constructs.

[0331] RNA has several advantages over more traditional plasmid or viral approaches. Gene expression from RNA sources does not require transcription, and protein products are rapidly produced after transfection. Furthermore, because RNA only needs access to the cytoplasm rather than the nucleus, typical transfection methods result in extremely high transfection rates. Additionally, plasmid-based approaches require that the promoter driving expression of the gene of interest be active in the cells under test.

[0332] In another aspect, RNA constructs are delivered into cells by electroporation.For example, refer to the formulation and methodology of electroporation of nucleic acid constructs into mammalian cells as taught in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, US 2004 / 0092907A1.Various parameters, including the strength of the electric field required for electroporation of any known cell type, are generally known from relevant research literature and numerous patents and applications in this field.For example, refer to US Patent No. 6,678,556, US Patent No. 7,171,264 and US Patent No. 7,173,116. Devices for therapeutic applications of electroporation are commercially available, such as the MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, CA), and are described in patents such as U.S. Patent Nos. 6,567,694; 6,516,223; 5,993,434; 6,181,964; 6,241,701; and 6,233,482; electroporation may also be used for in vitro transfection of cells, as described, for example, in US20070128708A1. Electroporation may also be used to deliver nucleic acids into cells in vitro. Thus, electroporation-mediated administration of nucleic acids, including expression constructs, into cells using any of the many available devices and electroporation systems known to those skilled in the art represents an exciting new means for delivering RNA of interest to target cells.

[0333] Accordingly, the present invention provides methods for generating modified immune cells or precursors thereof, comprising introducing into a cell an isolated nucleic acid (e.g., an expression construct) encoding a subject CAR described herein using any of the delivery methods described herein or known to those of skill in the art.

[0334] Source of immune cells Prior to expansion, a source of immune cells for ex vivo manipulation is obtained from a subject. The source of target cells for ex vivo manipulation may also include, for example, autologous or heterologous donor blood, umbilical cord blood, or bone marrow. For example, the source of immune cells may be derived from the subject to be treated with the modified immune cells of the present invention, such as the subject's blood, the subject's umbilical cord blood, or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. In certain exemplary embodiments, the subject is a human.

[0335] Immune cells can be obtained from several sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of innate or adaptive immunity, for example, lymphocytes, typically myeloid or lymphoid cells, including T cells and / or NK cells and / or NKT cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In certain aspects, the cells are human cells. With respect to the subject to be treated, the cells can be allogeneic and / or autologous. The cells are typically primary cells, for example, primary cells isolated directly from the subject and / or isolated and frozen from the subject.

[0336] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cell), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), a natural killer T cell (NKT cell), or a dendritic cell. In some embodiments, the cell is a monocyte or granulocyte, e.g., a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil. In certain embodiments, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell that has been generated from a subject and engineered to alter (e.g., induce mutations in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

[0337] In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., the entire T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturation, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation. T cells and / or CD4+ and / or CD8+ T cell subtypes and subpopulations include, among others, naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, innate and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In certain embodiments, any number of T cell lines available in the art may be used.

[0338] In some embodiments, the method includes isolating immune cells from a subject, preparing, treating, culturing, and / or manipulating them. In some embodiments, preparing the engineered cells includes one or more culturing and / or preparation steps. Cells for manipulation as described may be isolated from a sample, such as a biological sample, e.g., a sample obtained or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject with a disease or condition, or a subject in need of or to whom cell therapy will be administered. The subject, in some embodiments, is a human in need of a specific therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, treated, and / or manipulated. Thus, the cells, in some embodiments, are primary cells, e.g., primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from a biological source or processed samples. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0339] In certain aspects, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in connection with cell therapy, e.g., adoptive cell therapy.

[0340] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are obtained from heterologous sources, e.g., mice, rats, non-human primates, and pigs. In some embodiments, the isolation of cells includes one or more preparative steps and / or non-affinity-based cell separation steps. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove undesired components, enrich for desired components, or lyse or remove cells sensitive to a particular reagent. In some examples, the cells are separated based on one or more properties, such as density, adhesive properties, size, sensitivity and / or resistance to a particular component.

[0341] In some examples, cells from a subject's circulating blood are obtained, for example, by apheresis or leukapheresis. In certain aspects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, and B cells, other nucleated leukocytes, red blood cells, and / or platelets, and in certain aspects, cells other than red blood cells and platelets. In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and place the cells in a buffer or medium suitable for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In certain aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In certain embodiments, the cells are resuspended in various biocompatible buffers after washing. In certain embodiments, components of the blood cell sample are removed, and the cells are resuspended directly in culture medium. In some embodiments, the methods involve density-based cell separation methods, such as preparation of white blood cells from peripheral blood by lysing red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0342] In one embodiment, immune cells are obtained from an individual's circulating blood by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and place the cells in a suitable buffer or medium, such as phosphate-buffered saline (PBS), or the wash solution may lack calcium and magnesium, or may lack many, if not all, divalent cations, for subsequent processing steps. As those skilled in the art will readily appreciate, the washing step can be accomplished by methods known to those skilled in the art, such as using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processing device, a Baxter CytoMate, or a Harmonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells are immersed in a variety of biocompatible buffers, such as Ca2+, HCl, or HCl. 2+ Contains no Mg 2+ The cells may be resuspended in a medium such as free PBS, PlasmaLyte A, or saline solutions with or without buffers. In some embodiments, undesirable components of the apheresis sample may be removed and the cells resuspended directly in medium.

[0343] In some embodiments, the isolation method involves separating different cell types based on the expression or presence in cells of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method can be used for such marker-based separation. In some embodiments, the separation is affinity- or immunoaffinity-based. For example, in certain aspects, separation involves separating cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, by, for example, incubation with an antibody or binding partner that specifically binds to such marker, typically followed by a washing step, and separating cells bound to the antibody or binding partner from cells that are not bound to the antibody or binding partner. Such separation steps can be based on positive selection, in which cells bound to the reagent are retained for further use, and / or negative selection, in which cells not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In certain aspects, negative selection can be particularly useful when antibodies that specifically identify cell types in heterogeneous populations are unavailable, so that separation is best performed based on markers expressed by cells other than the desired population.Separation does not need to result in 100% enrichment or removal of a specific cell population or cells that express a specific marker.For example, positive selection or enrichment of a specific type of cell, such as cells that express a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express a marker.Similarly, negative selection, removal, or depletion of a specific type of cell, such as cells that express a marker, refers to reducing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0344] In certain exemplary embodiments, multiple rounds of separation steps are performed, in which the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In certain exemplary embodiments, cells that simultaneously express multiple markers can be depleted in a single separation step, such as by incubating cells with multiple antibodies or binding partners, each specific to a marker targeted for negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed on various cell types.

[0345] In some embodiments, one or more of the T cell populations are positive for or express high levels of one or more particular markers, e.g., surface markers (marker+). high ) cells, or cells that are negative for one or more markers ( - ) or express it at a relatively low level (marker low) cells are enriched or depleted. For example, in certain aspects, specific subpopulations of T cells, e.g., cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but present or expressed at relatively high levels on certain other populations of T cells (e.g., memory cells). In one embodiment, cells (CD8+ cells, or T cells, such as CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive for or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, and / or are depleted for (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched for or depleted for cells that are positive for or express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In certain exemplary embodiments, CD8+ T cells are enriched for cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0346] In some embodiments, T cells are separated from PBMC samples by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, e.g., CD14. In certain aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed at a relatively high level on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, e.g., by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is performed to increase efficacy, e.g., to improve long-term survival, expansion, and / or engraftment after administration, which in certain aspects is particularly robust in such subpopulations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0347] In some embodiments, memory T cells exist in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions using, for example, anti-CD8 and anti-CD62L antibodies. In some embodiments, the CD4+ T cell population and / or CD8+ T cell population is a subpopulation enriched for central memory (TCM) cells. In some embodiments, enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in certain aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In certain aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is then subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such selections are carried out simultaneously in certain aspects, and sequentially in other aspects in either order. In some specific aspects, the same selection step based on CD4 expression used to prepare a CD8+ cell population or subpopulation is also used to generate a CD4+ cell population or subpopulation, whereby both the positive and negative fractions from CD4-based separation are retained and used in subsequent steps of the method, optionally following one or more additional positive or negative selection steps.

[0348] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations bearing cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or matrix, such as magnetic or paramagnetic beads, to enable separation of cells for positive and / or negative selection.

[0349] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic manipulation. Incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of a stimulatory substance. Such conditions include conditions designed to induce proliferation, expansion, activation, and / or survival of cells in a population, to mimic antigen exposure, and / or to prime cells for genetic manipulation, e.g., introduction of a recombinant antigen receptor. Conditions can include one or more of a specific medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells. In some embodiments, the stimulatory conditions or stimulatory substances include one or more agents, e.g., ligands, capable of activating the intracellular signaling domain of a TCR complex. In certain aspects, the agent triggers or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such an agent can include, for example, an antibody bound to a solid support such as a bead, e.g., an antibody specific for a TCR component and / or a costimulatory receptor, e.g., anti-CD3, anti-CD28, and / or one or more cytokines. Optionally, the expansion method can further include adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agent includes IL-2 and / or IL-15, e.g., an IL-2 concentration of at least about 10 units / mL.

[0350] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from umbilical cords. In any event, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0351] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using isolated antibodies, antibody-containing biological samples such as ascites fluid, antibodies bound to a physical support, or antibodies bound to cells.

[0352] Enrichment of a T cell population by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. Exemplary methods include cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can enrich for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0353] To isolate a desired population of cells by positive or negative selection, cell concentration and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which beads and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in one embodiment, a cell concentration of 2 billion cells / ml is used. In one embodiment, a cell concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml are used. In a further embodiment, a cell concentration of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml is used. In further embodiments, cell concentrations of 125 or 150 million / ml can be used. Using higher concentrations can result in increased cell yield, cell activation, and cell expansion.

[0354] T cells can also be frozen after a washing step that does not require a monocyte removal step. Without wishing to be bound by theory, the freezing and subsequent thawing step provides a more uniform product by removing granulocytes and some monocytes from the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While numerous freezing solutions and parameters are known in the art and would be useful in this context, in a non-limiting example, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or other suitable cell freezing medium. The cells are then frozen to -80°C at a rate of 1°C / min and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen, may also be used.

[0355] In one embodiment, the T cell population is comprised in cells such as peripheral blood mononuclear cells, umbilical cord blood cells, purified populations of T cells, and T cell lines. In another embodiment, peripheral blood mononuclear cells comprise the T cell population. In yet another embodiment, purified T cells comprise the T cell population.

[0356] Increased immune cells Whether before or after modification of the cells to express the CAR of interest, e.g., U.S. Pat. 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 The cells can be activated and their numbers increased using methods such as those described in U.S. Patent Nos. 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. For example, the immune cells of the present invention can be increased by contact with a surface to which is attached an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of immune cells. In particular, the immune cell population can be stimulated by contact with a surface-immobilized anti-CD3 antibody, or an antigen-binding fragment thereof, or anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. For costimulation of accessory molecules on the surface of immune cells, the ligand that binds to accessory molecules is used.For example, immune cells can be contacted with anti-CD3 antibody and anti-CD28 antibody under the conditions suitable for stimulating the proliferation of immune cells.Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which can be used in the present invention, but other methods and reagents known in the art can also be used (see, for example, ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).

[0357] Immune cells can be expanded by the methods disclosed herein by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more, and any and all whole integers and partial integers therebetween. In one embodiment, immune cells are expanded by about 20-fold to about 50-fold.

[0358] Following culturing, the immune cells can be incubated in the cell culture medium in the culture device for a period of time, or until the cells reach confluence or a high cell density for optimal passage before passaging the cells to another culture device. The culture device can be any culture device commonly used for in vitro cell culture. In certain exemplary embodiments, the level of confluence is 70% or greater before passaging the cells to another culture device. In certain exemplary embodiments, the level of confluence is 90% or greater. The period can be any time suitable for in vitro cell culture. The immune cell medium may be changed during the culture of the immune cells at any time. In certain exemplary embodiments, the immune cell medium is changed approximately every 2-3 days. The immune cells are then harvested from the culture device, from which they can be used immediately or cryopreserved and stored for later use. In one embodiment, the present invention includes cryopreserving the expanded immune cells. The cryopreserved immune cells are thawed before introducing a nucleic acid into the immune cells.

[0359] In another embodiment, the method comprises isolating immune cells and expanding the immune cells. In another embodiment, the invention further comprises cryopreserving the immune cells prior to expansion. In yet another embodiment, the cryopreserved immune cells are thawed for electroporation with RNA encoding the chimeric membrane protein.

[0360] Another procedure for ex vivo expansion of cells is described in U.S. Patent No. 5,199,942 (incorporated herein by reference). Expansion as described in U.S. Patent No. 5,199,942 can be an alternative to or in addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of immune cells includes the addition of cell growth factors or other factors, such as those described in U.S. Patent No. 5,199,942, such as flt3-L, IL-1, IL-3, and c-kit ligand. In one embodiment, expanding immune cells includes culturing immune cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0361] The culturing step as described herein (following contact with an agent as described herein or electroporation) can be very short, e.g., less than 24 hours, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as further described herein (contact with an agent as described herein) can be longer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0362] Various terms are used to describe cultured cells. Cell culture generally refers to cells obtained from a living organism and grown under controlled conditions. Primary cell culture is a culture of cells, tissues, or organs obtained directly from an organism before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger cell population. When cells are expanded in culture, the growth rate of the cells is typically measured by the time required for the cells to double in number, otherwise known as the doubling time.

[0363] Each round of subculture is called a passage. When cells are subcultured, they are said to be passaged. A particular cell population or cell line is sometimes referred to or characterized by the number of times they have been subcultured. For example, a cultured cell population that has been subcultured 10 times may be called a P10 culture. The primary culture, i.e., the first culture after isolating cells from tissue, is called P0. After the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those skilled in the art that there may be multiple population doublings during the subculture period; therefore, the population doubling number of a culture is greater than the passage number. The expansion of cells during the period between subcultures (i.e., the number of population doublings) depends on many factors, including but not limited to the seeding density, substrate, medium, and subculture interval.

[0364] In one embodiment, cells may be cultured for a few hours (about 3 hours) to about 14 days, or any integer value therebetween. Conditions suitable for immune cell culture include an appropriate medium (e.g., Minimal Essential Medium or RPMI Medium 1640 or X-vivo 15, (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-alpha, or any other additives for cell growth 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. Culture media can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and serum-free or supplemented with an appropriate amount of serum (or plasma) or a predetermined set of hormones and / or cytokines in amounts sufficient for the growth and expansion of immune cells. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and are not included in cultures of cells to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0365] The medium used to culture immune cells may contain an agent that can costimulate immune cells. For example, an agent that can stimulate CD3 is an antibody against CD3, and an agent that can stimulate CD28 is an antibody against CD28. For, as shown by the data disclosed herein, cells isolated by the methods disclosed herein can be expanded by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more. In one embodiment, immune cells are expanded by about 2-fold to about 50-fold or more by culturing the electroporated population. In one embodiment, human regulatory T cells are expanded via KT64.86 artificial antigen-presenting cells (aAPCs) coated with anti-CD3 antibodies. Methods for expanding and activating immune cells can be found in U.S. Patent Nos. 7,754,482, 8,722,400, and 9,555,105, the contents of which are incorporated herein in their entireties.

[0366] In one aspect, the method of expanding immune cells can further include isolating the expanded immune cells for further application. In another aspect, the method of expanding can further include subsequent electroporation of the expanded immune cells followed by culturing. The subsequent electroporation can include introducing a nucleic acid encoding an agent, e.g., transducing the expanded immune cells, transfecting the expanded immune cells, or electroporating the expanded immune cells with a nucleic acid to create an expanded immune cell population, where the agent further stimulates the immune cells. The agent can stimulate the immune cells, e.g., by stimulating further expansion, effector function, or another immune cell function.

[0367] Treatment method Mucins are high-molecular-weight glycosylated proteins that, when overexpressed in epithelial cells, function as physicochemical defenses against toxins and mutagens in healthy cells. Expression in other healthy cell types has been shown to allow mucins to function as adhesion modulators or to play a role in regulating signal transduction and cell proliferation (Winterford et al. (1999) J Histochem Cytochem, 47(8):1063-1074). Tumorigenesis and metastasis have been shown to increase with changes in cell surface glycosylation (protein modification following the addition of sugar moieties to specific amino acids) of various mucin proteins (Ren et al. (2014) Tumour Biol, 35(10):9603-9612; Tarp et al. (2008) Glycobiology, 17(2):197-209; Taylor-Papadimitriou et al. (1999) 1455(2-3):301-313). At least nine of the 20 amino acids can be modified with various carbohydrates (Stowell et al. (2015) Annu Rev Pathol, 10:473-510). Tn (GalNAca1-O-Ser / Thr) and sialyl-Tn (STn) (NeuAca2-6-GalNAca1-O-Ser / Thr) are among the most common aberrant glycoforms found in cancer (Springer (1984) Science, 224(4654):1198-1206).This aberrant glycosylation also results in a tumor-specific form of the full-length mucin 1 glycoprotein, called TnMUC1, which is thought to play a key role in carcinogenesis (Ju et al. (2005) Nature, 437(7063):1252; Ju et al. (2008) Cancer Res, 68(6):1636-1646; Ju et al. (2014) Cancer Biomark, 14(1):63-81; Varki et al. (2017) Essentials of Glycobiology [Internet]. 3rd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017). Aberrant expression of Tn / sTn glycoforms has been observed, particularly in membrane-associated mucin 1 (MUC1), a large protein with tandem repeats bearing O-glycans that is overexpressed in most adenocarcinomas (Cascio et al. (2017) Oncotarget, 8(62):105284-98; Finn et al. (2011) Immunol Research, 50(2-3):261-268). Several healthy tissues of epithelial origin express MUC1 on the cell surface (Winterford et al. (1999) J Histochem Cytochem, 47(8):1063-1074); the aberrantly glycosylated version (TnMUC1) is expressed in the Golgi apparatus and is the precursor to the full-length MUC1 observed on the cell surface (Posey et al. (2016) Immunity, 44(6):1444-1454).Tumor-associated TnMUC1 is overexpressed in some multiple myeloma cases (Andrulis et al. (2014) Histopathology, 64:799-806; Cloosen et al. (2006) British Journal of Haematology, 135:513-516) and in a variety of solid tumors, including those of the breast, colon, lung, stomach, ovary, and pancreas, where loss of membrane polarity and aberrant O-glycosylation result in the expression of Tn and STn glycoforms on the tumor cell surface (Lavrsen et al. (2013) Glycoconjugates, 30(3):227-236; Pinto et al. (2012) J Cellular Mol Medicine, 16:1474-1484; Sorensen et al. (2006) Glycobiology, 16:96-107).

[0368] In one aspect, the present invention includes a method of treating a MUC1-associated cancer in a subject in need thereof. In another aspect, the present invention includes a method of treating a MUC1-associated cancer in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective population of modified immune cells of the present invention. In some embodiments, the MUC1-associated cancer is selected from the group consisting of multiple myeloma, breast cancer, colon cancer, lung cancer, gastric cancer, ovarian cancer, and pancreatic cancer. In some embodiments, the MUC1-associated cancer is selected from the group consisting of MUC1-associated breast cancer, MUC1-associated multiple myeloma, MUC1-associated non-small cell lung cancer, MUC1-associated pancreatic adenocarcinoma, and MUC1-associated ovarian and fallopian tube cancer.

[0369] The method includes administering to the subject an engineered immune cell of the invention (e.g., a MUC1 CAR T cell).

[0370] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods of the present invention. The terms "subject" and "patient" may be used interchangeably herein. Such organisms include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and in exemplary embodiments, humans. As used herein, the terms "treat," "treatment," and "treating" include any effect that results in improvement of a condition, disease, disorder, etc., such as lowering, reducing, modulating, ameliorating, or eliminating, or ameliorating the symptoms thereof, such as reducing the number of cancer cells, reducing tumor size, reducing tumor burden, reducing the rate of cancer cell invasion into peripheral organs, or reducing the rate of tumor metastasis or tumor growth.

[0371] A positive therapeutic effect in cancer can be measured in several ways (see W. A. ​​Weber, J. Null. Med. 50:1S-10S (2009); Eisenhauer et al., Eur. J. Cancer 45:228-247 (2009)). In some embodiments, the response to a subject CAR T cell therapy (e.g., TN-MUC1 CAR T cell therapy) is assessed using RECIST 1.1 criteria (Eisenhauer et al., supra). In some embodiments, the treatment achieved by a therapeutically effective amount (e.g., of TN-MUC1 CAR T cell therapy) is either a partial response (PR), a complete response (CR), progression-free survival (PFS), disease-free survival (DFS), an objective response (OR), a change in duration of response (e.g., an increase in duration of response), a change in time to response (e.g., a decrease in time to response), or overall survival (OS). The therapeutically effective amount described herein effective to treat breast cancer in a patient may vary depending on various factors, such as the patient's condition, age, and weight, and the ability of the treatment to elicit an anti-cancer response in the subject.

[0372] As used herein, "RECIST 1.1 Response Criteria" means the definitions set forth in Eisenhauer et al. (2009) Eur J Cancer, 45(2):228-247 for target lesions or non-target lesions, as appropriate, based on the context in which response is being measured.

[0373] "Tumor" as applied to a subject diagnosed with or suspected of having cancer (e.g., MUC1-associated breast cancer, MUC1-associated multiple myeloma, MUC1-associated non-small cell lung cancer, MUC1-associated pancreatic adenocarcinoma, MUC1-associated ovarian and fallopian tube cancer) refers to a malignant or potentially malignant neoplasm or mass of tissue of any size.

[0374] "Tumor burden," also known as "tumor volume," refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells throughout the body, including lymph nodes and bone marrow, or the total size of the tumor. Tumor burden can be determined by a variety of methods known in the art, such as by measuring the size of a tumor removed from a subject, for example, using a caliper, or by measuring internally using imaging techniques, such as ultrasound, bone scan, computed tomography (CT) or magnetic resonance imaging (MRI) scan.

[0375] The term "tumor size" refers to the total size of a tumor, which can be measured as the length and width of the tumor.Tumor size can be determined by a variety of methods known in the art, for example, by measuring the dimensions of the tumor when removed from a subject, for example, using a vernier caliper, or by measuring in vivo using imaging techniques, for example, ultrasound, bone scan, CT or MRI scan.

[0376] In one aspect, the present invention includes a method of treating MUC1-associated breast cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective population of modified immune cells, wherein the modified immune cells comprise a chimeric antigen receptor (CAR). In certain embodiments, the CAR comprises a MUC1-specific antigen-binding domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.

[0377] In one aspect, the present invention includes a method of treating MUC1-associated multiple myeloma in a subject in need thereof, comprising administering to the subject a therapeutically effective population of modified immune cells, wherein the modified immune cells comprise a chimeric antigen receptor (CAR). In certain embodiments, the CAR comprises a MUC1-specific antigen-binding domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain. In other embodiments, the modified cells further comprise a dominant-negative receptor and / or a switch receptor.

[0378] In one aspect, the present invention includes a method of treating MUC1-associated non-small cell lung cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective population of modified immune cells, wherein the modified immune cells comprise a chimeric antigen receptor (CAR). In certain embodiments, the CAR comprises a MUC1-specific antigen-binding domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain. In other embodiments, the modified cells further comprise a dominant-negative receptor and / or a switch receptor.

[0379] In one aspect, the present invention includes a method of treating MUC1-associated pancreatic adenocarcinoma in a subject in need thereof, comprising administering to the subject a therapeutically effective population of modified immune cells, wherein the modified immune cells comprise a chimeric antigen receptor (CAR). In certain embodiments, the CAR comprises a MUC1-specific antigen-binding domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain. In other embodiments, the modified cells further comprise a dominant-negative receptor and / or a switch receptor.

[0380] In one aspect, the present invention includes a method of treating MUC1-associated ovarian and / or fallopian tube cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective population of modified immune cells, wherein the modified immune cells comprise a chimeric antigen receptor (CAR). In certain embodiments, the CAR comprises a MUC1-specific antigen-binding domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain. In other embodiments, the modified cells further comprise a dominant-negative receptor and / or a switch receptor.

[0381] In certain embodiments, a MUC1-specific antigen-binding domain binds to a glycosylated form of MUC1, i.e., is specific for a glycoepitope of MUC1. In certain embodiments, a MUC1-specific antigen-binding domain is specific for a truncated glycoepitope of MUC1. In certain embodiments, a MUC1-specific antigen-binding domain is specific for TnMUC1. In certain embodiments, a MUC1-specific antigen-binding domain may comprise the heavy chain complementarity-determining region (CDR) sequences of SEQ ID NOs:22, 23, and 24 and / or the light chain complementarity-determining region (CDR) sequences of SEQ ID NOs:19, 20, and 21. In certain embodiments, a MUC1-specific antigen-binding domain may comprise all six complementarity-determining region (CDR) sequences of SEQ ID NOs:19-24. In certain embodiments, the MUC1-specific antigen-binding domain may comprise the heavy chain variable domain (VH) sequence of SEQ ID NO:5 and / or the light chain variable domain (VL) sequence of SEQ ID NO:6. In certain embodiments, the MUC1-specific antigen-binding domain comprises the amino acid sequence of SEQ ID NO:2.

[0382] The CAR used in the methods of the present invention may comprise a transmembrane domain selected from the group consisting of an artificial hydrophobic sequence, a type I transmembrane protein, a T cell receptor alpha, beta, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154 transmembrane domain. In certain exemplary embodiments, the transmembrane domain comprises a CD8a transmembrane domain.

[0383] The CAR may comprise a costimulatory signaling domain comprising a costimulatory domain of a protein selected from the group consisting of a member of the TNFR superfamily, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD5, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, DAP10, DAP12, Lck, Fas, and any combination thereof. In certain exemplary embodiments, the costimulatory signaling domain comprises a 4-1BB costimulatory domain.

[0384] The intracellular signaling domain may comprise a signaling domain of a protein selected from the group consisting of CD3 zeta, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing cytoplasmic receptor, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In certain exemplary embodiments, the intracellular signaling domain comprises a CD3 zeta signaling domain.

[0385] The CAR may further comprise a CD8a leader sequence and / or an extracellular hinge domain selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, a hinge comprising the amino acid sequence of CD8, and any combination thereof. In certain exemplary embodiments, the extracellular hinge domain comprises a CD8a extracellular hinge domain.

[0386] In certain embodiments, the CAR is encoded by a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 1, 38, 40, 42, 44, or 46. In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 2, 39, 41, 43, 45, or 47.

[0387] In certain exemplary embodiments, the CAR is encoded by a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 46. In certain exemplary embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 47.

[0388] The CARs of the present disclosure, when present in T lymphocytes or NK cells, can mediate cytotoxicity toward target cells. The CARs of the present disclosure bind to an antigen present on the target cell, thereby mediating the killing of the target cell by T lymphocytes or NK cells genetically modified to produce the CAR. The antigen-binding domain of the CAR (e.g., anti-TN-MUC1 scFv) binds to an antigen (e.g., TN-MUC1 antigen) present on the surface of the target cell. Target cells include, but are not limited to, cancer cells, such as breast cancer cells. Thus, the present disclosure provides a method for killing or inhibiting the growth of target cancer cells, comprising contacting cytotoxic immune effector cells (e.g., cytotoxic T cells or NK cells) genetically modified to produce the subject CAR, whereby the T lymphocytes or NK cells recognize the antigen present on the surface of the target cancer cell and mediate the killing of the target cell.

[0389] The present disclosure provides methods for treating cancer in a subject having cancer, the methods comprising: i) introducing a chimeric antigen receptor of the present disclosure or an expression vector of the present disclosure into cells to produce modified cells; and ii) administering the modified cells to the subject. In some embodiments, the cells are obtained from the subject (i.e., the cells are autologous), manipulated ex vivo, and administered to the same subject. In some embodiments, the cells are obtained from one subject, manipulated ex vivo, and administered to a second suitable subject (i.e., the cells are allogeneic).

[0390] In some embodiments, a method is provided, comprising: collecting cytotoxic cells from a subject; genetically modifying the cytotoxic cells by introducing the CAR gene of the present invention into the cytotoxic cells; and administering the modified cytotoxic cells to the subject. In some embodiments, the cytotoxic cells are selected from T cells, naive T cells, memory T cells, effector T cells, natural killer cells, and macrophages. In one embodiment, the cytotoxic cells are T cells.

[0391] In one embodiment, T cells are obtained from a subject.T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumor.In some embodiments of the present invention, any number of T cell lines available in the art may be used.In some embodiments of the present invention, T cells can be obtained from the blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation.

[0392] For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a period of time sufficient to positively select the desired T cells. In one embodiment, the period is approximately 30 minutes. In one embodiment, the period ranges from 30 minutes to 36 hours or more, and integer values ​​therebetween. In one embodiment, the period is at least 1, 2, 3, 4, 5, or 6 hours. In one embodiment, the period is 10 to 24 hours. In one embodiment, the incubation period is 24 hours. For isolating T cells from patients with leukemia, the use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Furthermore, the use of longer incubation times can increase the efficiency of CD8+ T cel...

Claims

1. 1. A modified immune cell or a precursor thereof comprising a chimeric antigen receptor (CAR) that specifically binds to Tn-MUC1, CAR, A Tn-MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH domain comprises the amino acid sequence shown in SEQ ID NO:5; and The VL domain comprises the amino acid sequence shown in SEQ ID NO:

6. a Tn-MUC1 specific antigen-binding domain; a transmembrane domain; a CD2 costimulatory signaling domain; CD3 zeta intracellular signaling domain and Including, Modified immune cells or their precursors.

2. The modified immune cell of claim 1, wherein the CD2 costimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:

28.

3. The transmembrane domain, (i) a transmembrane region of a protein selected from the group consisting of type I transmembrane proteins, T cell receptor alpha chain, T cell receptor beta chain, T cell receptor zeta chain, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9; or (ii) the CD8 transmembrane domain; or (iii) the amino acid sequence shown in SEQ ID NO: 7 The modified immune cell of claim 1 , comprising:

4. The modified immune cell of any one of claims 1 to 3, wherein the CAR further comprises a hinge domain, the hinge domain being derived from a protein selected from the group consisting of an Fc fragment of an antibody, an antibody hinge region, an antibody CH2 region, an antibody CH3 region, an artificial spacer sequence, a CD8 hinge domain, and any combination thereof.

5. The modified immune cell described in claim 4, wherein the hinge domain is a CD8 hinge domain or the hinge domain comprises the amino acid sequence shown in SEQ ID NO:

13.

6. The modified immune cell of claim 1, wherein the CAR further comprises a leader sequence.

7. The modified immune cell described in claim 6, wherein the leader sequence is a CD8 leader sequence or the leader sequence comprises the amino acid sequence shown in SEQ ID NO:

48.

8. The modified immune cell of claim 4, wherein the hinge domain is a CD8 hinge domain, the transmembrane domain is a CD8 transmembrane domain, the CD2 costimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:28, the CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:30, and the modified immune cell is a T cell.

9. The modified immune cell of any one of claims 1 to 4, wherein the modified immune cell comprises an amino acid sequence shown in SEQ ID NO:47, and the modified immune cell is a T cell.

10. (a) a switch receptor comprising an extracellular domain of a signaling protein associated with a negative signal, a transmembrane domain, and an intracellular domain of a signaling protein associated with a positive signal, wherein the signaling protein associated with a negative signal is selected from the group consisting of CTLA4, PD-1, BTLA, TIM-3, and TGFβR, and the signaling protein associated with a positive signal is selected from the group consisting of CD28, ICOS, 4-1BB, and IL-12R; or (b) A dominant-negative receptor that contains the extracellular domain and the transmembrane domain of a signal transduction protein associated with a negative signal and lacks an intracellular signal transduction domain, wherein the signal transduction protein associated with a negative signal is selected from the group consisting of CTLA4, PD-1, BTLA, TIM-3, and TGFβR. The modified immune cell of claims 1 to 9, further comprising:

11. (a) The extracellular and intracellular domains of the switch receptors are PD1 and CD28, respectively, and PD1 A132L and CD28, PD1 and 4-1BB, PD1 A132L and 4-1BB, PD1 and IL12Rβ1, PD1 A132L and IL12Rβ1, PD1 and IL12Rβ2, PD1 A132L and IL12Rβ2, TGFβRII and IL12Rβ1, TGFβRII and IL12Rβ2, TGFβRII and CD28, or TIM-3 and CD28; or (b) the switch receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, and 92; or (c) the dominant negative receptor comprises the amino acid sequence set forth in SEQ ID NO: 76; or (d) the dominant negative receptor is a TGFβR or a TGFβRII dominant negative receptor; The modified immune cell of claim 10.

12. The modified immune cell of any one of claims 1 to 4, wherein the modified immune cell is a modified natural killer (NK) cell, a modified natural killer T (NKT) cell, or a modified T cell.

13. A Tn-MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH domain comprises the amino acid sequence shown in SEQ ID NO:5; and The VL domain comprises the amino acid sequence shown in SEQ ID NO:

6. a Tn-MUC1 specific antigen-binding domain; a hinge domain; a transmembrane domain; a CD2 costimulatory signaling domain; CD3 zeta intracellular signaling domain and Including, A chimeric antigen receptor (CAR) that specifically binds to Tn-MUC1.

14. (a) the CD2 costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO:28; or (b) the CAR comprises the amino acid sequence set forth in SEQ ID NO: 47; The CAR of claim 13.

15. A Tn-MUC1-specific antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH domain comprises the amino acid sequence shown in SEQ ID NO:5; and The VL domain comprises the amino acid sequence shown in SEQ ID NO:

6. a Tn-MUC1 specific antigen-binding domain; a transmembrane domain; a CD2 costimulatory signaling domain; CD3 zeta intracellular signaling domain and 1. An isolated nucleic acid encoding a chimeric antigen receptor comprising: (a) a nucleotide sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:48, or a combination thereof; or (b) The nucleotide sequence of SEQ ID NO:46 An isolated nucleic acid comprising:

16. The method of claim 1, further comprising a second nucleic acid sequence, the second nucleic acid sequence comprising: (a) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, and 93; or (b) the nucleic acid sequence shown in SEQ ID NO: 77 16. The isolated nucleic acid of claim 15, wherein the nucleic acid encoding the CAR and the second nucleic acid sequence are separated by a nucleic acid sequence encoding a self-cleaving peptide.

17. An expression construct comprising the isolated nucleic acid of claim 15 or 16.

18. (a) a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector; or (b) a self-inactivating lentiviral vector; 18. The expression construct of claim 17. (a) the EF-1α promoter; and / or (b) a rev response element (RRE); and / or (c) the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and / or (d) a cPPT sequence; or (e) EF-1α promoter, rev response element (RRE), woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and cPPT sequence 19. The expression construct of claim 17 or 18, further comprising:

20. 20. A method for generating an modified immune cell according to any one of claims 1 to 12, comprising introducing into an immune cell an isolated nucleic acid according to claim 15 or 16, or an expression construct according to any one of claims 17 to 19.

21. A pharmaceutical composition for treating Tn-MUC1 associated cancer in a subject in need thereof, comprising a therapeutically effective composition comprising the modified immune cell of any one of claims 1 to 12.

22. Tn-MUC1-associated cancers (a) selected from the group consisting of multiple myeloma, non-small cell lung cancer, breast cancer, pancreatic adenocarcinoma, and ovarian and fallopian tube cancer; or (b) breast cancer; or (c) multiple myeloma; or (d) non-small cell lung cancer; or (e) pancreatic adenocarcinoma; or (f) ovarian and fallopian tube cancer, 22. The pharmaceutical composition of claim 21.

23. Breast cancer, (a) is characterized by aberrant glycosylation of MUC1; or (b) selected from the group consisting of hormone receptor positive breast cancer, hormone receptor negative breast cancer, estrogen receptor negative breast cancer, progesterone receptor negative breast cancer, and Her2 receptor negative breast cancer; or (c) metastatic breast cancer; or (d) triple-negative breast cancer; 23. The pharmaceutical composition of claim 22.

24. (a) in combination with lymphodepleting chemotherapy; or (b) used in combination with a cytokine release syndrome (CRS) management regimen; or (c) the modified cells are autologous; or (d) administration of the pharmaceutical composition is via intratumoral delivery; or (e) administration of the pharmaceutical composition is via intravenous delivery; or (f) administration of the pharmaceutical composition is via intraperitoneal delivery; 24. The pharmaceutical composition of any one of claims 21 to 23.

25. (a) the lymphodepleting chemotherapy is a therapeutically effective amount of cyclophosphamide; or (b) the lymphodepleting chemotherapy is a therapeutically effective amount of fludarabine; or (c) the lymphodepleting chemotherapy is a therapeutically effective amount of cyclophosphamide and a therapeutically effective amount of fludarabine; or (d) the management regimen for CRS includes a therapeutically effective amount of tocilizumab; or (e) the management regimen for CRS comprises a therapeutically effective amount of tocilizumab and / or a corticosteroid; 25. The pharmaceutical composition of claim 24.

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