CDH17 CAR

Abstract

The present invention relates to chimeric antigen receptors (CARs) that target cadherin-17 (CDH17) and cells comprising the CARs. Methods and uses comprising the CARs of the invention are also provided.

Description

[Technical Field]

[0001] The present invention relates to chimeric antigen receptors (CARs) and cells comprising the CARs. Methods and uses comprising the CARs of the invention are also provided. [Background technology]

[0002] Chimeric antigen receptors (CARs) are synthetic biology molecules commonly constructed by fusing an antigen-binding portion, often derived from a tumor-reactive monoclonal antibody, with an intracellular signaling domain derived from a T lymphocyte. Clinical trials of CAR-T cell therapy have rapidly increased in recent years, leading to the marketing approval of different CAR-T cell products targeting either CD19 or BCMA in relapsed / refractory B-cell lymphoma, pediatric and young adult B-cell acute lymphoblastic leukemia, and multiple myeloma. Despite the success of CAR-T therapy, objective response rates in patients with solid tumors are far less frequent, and improving therapeutic efficacy against these malignancies represents one of the greatest challenges in the field.

[0003] Effective targeting of solid tumors requires overcoming several barriers, such as poor transport to the tumor site and suboptimal invasion of the tumor mass. Further challenges arise due to the highly immunosuppressive tumor microenvironment, which leads to T cell anergy, exhaustion, and senescence.

[0004] In addition to the obstacles associated with targeting solid tumors, antigen selection is an important and difficult consideration.Most antigens expressed by solid malignant tumors are shared with healthy tissues, which increases the risk of harmful on-target off-tumor toxicity.The ideal CAR target is a surface molecule that is highly and uniformly expressed in tumor cells but not in healthy tissues.In addition, advantageously, preferred antigens will be expressed by multiple tumor types and involved in cancer cell survival, reducing antigen loss.

[0005] Thus, there remains an unmet need to identify new antigens for CAR cell therapy. Furthermore, there remains a need for CARs that effectively target relevant antigens and can be used in therapy. Summary of the Invention

[0006] The present inventors have identified the antigen cadherin-17 (CDH-17) and developed a CAR that targets CDH-17. The inventors have demonstrated that the CAR can redirect the specificity and functional activity of human T cells to tumor cells that express the CDH-17 target antigen.

[0007] Using a rational process that integrated data from multiple sources, including analysis of liver metastases from patients with colorectal cancer (CRC), we identified novel antigens suitable for CAR T cell therapy, of which cadherin-17 (CDH-17) or Li-cadherin was the most promising candidate. We then designed and generated a CDH-17-targeting CAR construct that exhibited high reactivity against CDH17+ tumors.

[0008] The CARs of the present invention can be used to generate CAR-containing cells, such as CAR T cells specific for the CDH-17 antigen, which can be applied to the treatment of different gastrointestinal tumors, including colorectal cancer, pancreatic cancer, and gastric cancer. Importantly, the CAR cells of the present invention can target liver metastases (LV MTS) of colorectal cancer and pancreatic ductal adenocarcinoma (PDAC), which represent a major cause of death and a significant unmet clinical need.

[0009] In one aspect, there is provided a chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises: a) a heavy chain variable region (VH) complementarity determining region (CDR) having the following sequences: CDR1-DHTIHWMR (SEQ ID NO: 1), CDR2-YIYPRDGITGYNERFRGK (SEQ ID NO: 2), and CDR3-WGYSYRNYAYYYDYWGQGTL (SEQ ID NO: 3), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) A CAR is provided, comprising light chain variable region (VL) CDRs having the following sequences: CDR1-INCRSSQSLLHSSNQR (SEQ ID NO: 4), CDR2-PPKVLIYWASTRES (SEQ ID NO: 5), and CDR3-QQYYSYPWTFGQ (SEQ ID NO: 6), or variants thereof each having up to three amino acid substitutions, additions, or deletions.

[0010] Preferably, the antigen-binding domain binds to CDH-17.

[0011] In one embodiment, the antigen binding domain comprises: a) a VH domain comprising the sequence of SEQ ID NO: 7, and b) comprises a VL domain comprising the sequence of SEQ ID NO:8.

[0012] In one embodiment, the antigen binding domain comprises: a) a VH domain comprising the sequence of SEQ ID NO: 7, and b) a VL domain comprising the sequence of SEQ ID NO: 8; or variants thereof having at least 75% sequence identity thereto, for example at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0013] In one embodiment, the antigen binding domain comprises: a) a VH domain comprising the sequence of SEQ ID NO: 7, and b) a VL domain comprising the sequence of SEQ ID NO: 8; or variants thereof each having at least 90% sequence identity thereto.

[0014] In one embodiment, the antigen-binding domain comprises a single-chain variable fragment (scFv).

[0015] In one embodiment, the antigen-binding domain consists of a single-chain variable fragment (scFv).

[0016] In one embodiment, the antigen binding domain comprises the sequence of SEQ ID NO:9.

[0017] In one embodiment, the antigen binding domain consists of the sequence of SEQ ID NO:9.

[0018] In one embodiment, the antigen-binding domain comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:9.

[0019] In one embodiment, the antigen-binding domain comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:9, e.g., at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO:9.

[0020] In another aspect, there is provided a chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises: a) a heavy chain variable region (VH) complementarity determining region (CDR) having the following sequences: CDR1-DYYMY (SEQ ID NO: 42), CDR2-SISFDGTYTYYTDRVKG (SEQ ID NO: 43), and CDR3-DRPAWFPY (SEQ ID NO: 44), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) A CAR is provided, comprising light chain variable region (VL) CDRs having the following sequences: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO: 39), CDR2-KVSNRFS (SEQ ID NO: 40), and CDR3-FQGSHVPLT (SEQ ID NO: 41), or variants thereof each having up to three amino acid substitutions, additions, or deletions.

[0021] In one embodiment, the antigen binding domain comprises: a) a VH domain comprising the sequence of SEQ ID NO: 58, and b) comprises a VL domain comprising the sequence of SEQ ID NO: 59.

[0022] In one embodiment, the antigen binding domain comprises: a) a VH domain comprising the sequence of SEQ ID NO: 58, and b) a VL domain comprising the sequence of SEQ ID NO: 59; or variants thereof having at least 75% sequence identity thereto, for example at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0023] In one embodiment, the antigen binding domain comprises: a) a VH domain comprising the sequence of SEQ ID NO: 58, and b) a VL domain comprising the sequence of SEQ ID NO: 59; or variants thereof each having at least 90% sequence identity thereto.

[0024] In one embodiment, the antigen-binding domain comprises a single-chain variable fragment (scFv).

[0025] In one embodiment, the antigen-binding domain consists of a single-chain variable fragment (scFv).

[0026] In one embodiment, the antigen binding domain comprises the sequence of SEQ ID NO:45.

[0027] In one embodiment, the antigen binding domain consists of the sequence of SEQ ID NO:45.

[0028] In one embodiment, the antigen-binding domain comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:45.

[0029] In one embodiment, the antigen-binding domain comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 45, e.g., at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO: 45.

[0030] In one embodiment, the CAR comprises a CD28, CD8, and / or CD4 transmembrane domain.

[0031] In one embodiment, the CAR comprises a CD28 transmembrane domain.

[0032] In one embodiment, the CD28 transmembrane domain comprises the sequence of SEQ ID NO:61.

[0033] In one embodiment, the CD28 transmembrane domain consists of the sequence of SEQ ID NO:61.

[0034] In one embodiment, the CD28 transmembrane domain comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:61.

[0035] In one embodiment, the CD28 transmembrane domain comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:61, e.g., at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO:61.

[0036] In one embodiment, the CAR comprises a CD28 transmembrane and costimulatory domain.

[0037] In one embodiment, the CD28 transmembrane domain and the costimulatory domain are not separated by another sequence.

[0038] In one embodiment, the CD28 transmembrane domain and costimulatory domain comprises the sequence of SEQ ID NO:12.

[0039] In one embodiment, the CD28 transmembrane and costimulatory domain consists of the sequence of SEQ ID NO:12.

[0040] In one embodiment, the CD28 transmembrane and costimulatory domain comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:12.

[0041] In one embodiment, the CD28 transmembrane domain and costimulatory domain comprise or consist of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 12, e.g., at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO: 12.

[0042] In one embodiment, the CAR comprises a CD8 transmembrane domain.

[0043] In one embodiment, the CAR comprises a CD4 transmembrane domain.

[0044] In one embodiment, the CAR comprises an IgG1 hinge, a LNGFR spacer, or a mCH2CH3 spacer.

[0045] In one embodiment, the CAR comprises an IgG1 hinge.

[0046] In one embodiment, the IgG1 hinge comprises the sequence of SEQ ID NO:10.

[0047] In one embodiment, the IgG1 hinge consists of the sequence of SEQ ID NO:10.

[0048] In one embodiment, the IgG1 hinge comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:10.

[0049] In one embodiment, the IgG1 hinge comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 10, such as at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO: 10.

[0050] In one embodiment, the CAR comprises a LNGFR spacer.

[0051] In one embodiment, the LNGFR spacer is a LNGFR mutated short (NMS) spacer. In another embodiment, the LNGFR spacer is a LNGFR wild type long spacer (NWL).

[0052] In one embodiment, the LNGFR spacer comprises the sequence of SEQ ID NO: 11 or 27.

[0053] In one embodiment, the LNGFR spacer consists of the sequence of SEQ ID NO: 11 or 27.

[0054] In one embodiment, the LNGFR spacer comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 11 or 27.

[0055] In one embodiment, the LNGFR spacer comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 11, 27, 32, or 33.

[0056] In one embodiment, the LNGFR spacer comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 11, 27, 32, or 33, for example, at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO: 11, 27, 32, or 33.

[0057] In one embodiment, the CAR comprises a mCH2CH3 spacer.

[0058] In one embodiment, the mCH2CH3 spacer comprises the sequence of SEQ ID NO:28.

[0059] In one embodiment, the mCH2CH3 spacer consists of the sequence of SEQ ID NO:28.

[0060] In one embodiment, the mCH2CH3 spacer comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:28.

[0061] In one embodiment, the mCH2CH3 spacer comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:28, e.g., at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO:28.

[0062] In one embodiment, the CAR comprises a CD28 and / or 4-1BB costimulatory domain.

[0063] In one embodiment, the CAR comprises a CD28 costimulatory domain.

[0064] In one embodiment, the costimulatory domain is a CD28 costimulatory domain.

[0065] In one embodiment, the CD28 costimulatory domain comprises the sequence of SEQ ID NO:60.

[0066] In one embodiment, the CD28 costimulatory domain consists of the sequence of SEQ ID NO:60.

[0067] In one embodiment, the CD28 costimulatory domain comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:60.

[0068] In one embodiment, the CD28 costimulatory domain comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:60, e.g., at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO:60.

[0069] In one embodiment, the CAR comprises a 4-1BB costimulatory domain.

[0070] In one embodiment, the CAR comprises a CD3 zeta signaling domain.

[0071] In one embodiment, the CD3 zeta signaling domain comprises the sequence of SEQ ID NO:13.

[0072] In one embodiment, the CD3 zeta signaling domain consists of the sequence of SEQ ID NO:13.

[0073] In one embodiment, the CD3 zeta signaling domain comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:13.

[0074] In one embodiment, the CD3 zeta signaling domain comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 13, e.g., at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO: 13.

[0075] In one embodiment, the CAR is a) CD28, CD8, and / or CD4 transmembrane domains; b) an IgG1 hinge, an LNGFR spacer, or an mCH2CH3 spacer; c) CD28 and / or 4-1BB costimulatory domains, and / or d) Contains the CD3 zeta signaling domain.

[0076] In one embodiment, the CAR is a) a transmembrane domain derived from the CD28, CD8, and / or CD4 transmembrane domain; b) a spacer domain comprising an IgG1 hinge, an LNGFR spacer, or an mCH2CH3 spacer; c) one or more costimulatory domains selected from the group consisting of CD28 and / or 4-1BB costimulatory domains, and / or d) Contains the CD3 zeta signaling domain.

[0077] In one embodiment, the CAR comprises or consists of a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 9, 14, 15, 23, 24, or 47, for example at least 80%, at least 85%, 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%, or 100% sequence identity to any one of SEQ ID NOs: 9, 14, 15, 23, 24, or 47.

[0078] In one embodiment, the CAR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9, 14, 15, 23, 24, or 47.

[0079] In one embodiment, the CAR consists of a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14, 15, 23, 24, or 47.

[0080] In one embodiment, the CAR comprises the sequence of any one of SEQ ID NOs: 9, 14, 15, 23, 24, or 47.

[0081] In one embodiment, the CAR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9, 14, 15, 23, 24, or 47, and the sequence comprises any one or more of the sequences according to SEQ ID NOs: 1-6.

[0082] In one embodiment, the CAR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9, 14, 15, 23, 24, or 47, including sequences according to SEQ ID NOs: 1-6.

[0083] In one embodiment, the CAR comprises a sequence having at least 90% sequence identity to SEQ ID NO: 45, or any one of SEQ ID NOs: 48-50.

[0084] In one embodiment, the CAR consists of a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 48-50.

[0085] In one embodiment, the CAR comprises the sequence of any one of SEQ ID NOs: 45, or 48-50.

[0086] In one embodiment, the CAR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 45, or 48-50, and the sequence comprises any one or more of the sequences according to SEQ ID NOs: 39-44.

[0087] In one embodiment, the CAR comprises a sequence having at least 90% sequence identity to SEQ ID NO: 45, or any one of SEQ ID NOs: 48-50, including a sequence according to SEQ ID NOs: 39-44.

[0088] In one embodiment, a single chain variable fragment (scFv) comprising: a) a heavy chain variable region (VH) complementarity determining region (CDR) having the following sequences: CDR1-DHTIHWMR (SEQ ID NO: 1), CDR2-YIYPRDGITGYNERFRGK (SEQ ID NO: 2), and CDR3-WGYSYRNYAYYYDYWGQGTL (SEQ ID NO: 3), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) An scFv is provided, comprising light chain variable region (VL) CDRs having the following sequences: CDR1-INCRSSQSLLHSSNQR (SEQ ID NO: 4), CDR2-PPKVLIYWASTRES (SEQ ID NO: 5), and CDR3-QQYYSYPWTFGQ (SEQ ID NO: 6), or variants thereof each having up to three amino acid substitutions, additions, or deletions.

[0089] In one embodiment, the scFv comprises the sequence of SEQ ID NO:9.

[0090] In one embodiment, the scFv consists of the sequence of SEQ ID NO:9.

[0091] In one embodiment, the scFv comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:9.

[0092] In one embodiment, the scFv comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:9, for example at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO:9.

[0093] In one embodiment, a single chain variable fragment (scFv) comprising: a) a heavy chain variable region (VH) complementarity determining region (CDR) having the following sequences: CDR1-DYYMY (SEQ ID NO: 42), CDR2-SISFDGTYTYYTDRVKG (SEQ ID NO: 43), and CDR3-DRPAWFPY (SEQ ID NO: 44), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) An scFv is provided, comprising light chain variable region (VL) CDRs having the following sequences: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO: 39), CDR2-KVSNRFS (SEQ ID NO: 40), and CDR3-FQGSHVPLT (SEQ ID NO: 41), or variants thereof each having up to three amino acid substitutions, additions, or deletions.

[0094] In one embodiment, the scFv comprises the sequence of SEQ ID NO:45.

[0095] In one embodiment, the scFv consists of the sequence of SEQ ID NO:45.

[0096] In one embodiment, the scFv comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:45.

[0097] In one embodiment, the scFv comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:45, for example at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO:45.

[0098] In one aspect, a polynucleotide is provided comprising one or more nucleotide sequences encoding a CAR or scFv of the invention.

[0099] In one embodiment, the polynucleotide comprises a sequence encoding an scFv comprising the sequence of SEQ ID NO: 16, or a variant thereof having at least 75% sequence identity thereto, for example at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0100] In one embodiment, the polynucleotide comprises a sequence encoding an scFv comprising the sequence of SEQ ID NO:16.

[0101] In one embodiment, the polynucleotide comprises a sequence encoding a CAR comprising the sequence of any one of SEQ ID NOs: 16-18, 25-26, or 51, or a variant thereof having at least 75% sequence identity thereto, for example, at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0102] In one embodiment, the polynucleotide comprises a sequence encoding a CAR comprising the sequence of any one of SEQ ID NOs: 16-18, 25-26, or 51.

[0103] In one embodiment, the polynucleotide is a) a sequence encoding an scFv comprising the sequence of SEQ ID NO: 16, and / or b) a sequence encoding a CAR comprising any one of SEQ ID NOs: 16 to 18, 25 to 26, or 51; or variants thereof each having at least 75% sequence identity thereto.

[0104] In one embodiment, the polynucleotide comprises a sequence encoding a CAR comprising the sequence of any one of SEQ ID NOs: 16-18, 25-26, 51, or 85, or a variant thereof having at least 75% sequence identity thereto, for example at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0105] In one embodiment, the polynucleotide comprises a sequence encoding a CAR comprising the sequence of any one of SEQ ID NOs: 16-18, 25-26, 51, or 85.

[0106] In one embodiment, the polynucleotide is a) a sequence encoding an scFv comprising the sequence of SEQ ID NO: 16, and / or b) a sequence encoding a CAR comprising any one of SEQ ID NOs: 16 to 18, 25 to 26, 51, or 85; or variants thereof each having at least 75% sequence identity thereto.

[0107] In one embodiment, the polynucleotide comprises a sequence encoding an scFv comprising the sequence of SEQ ID NO: 46, or a variant thereof having at least 75% sequence identity thereto, for example at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0108] In one embodiment, the polynucleotide comprises a sequence encoding an scFv comprising the sequence of SEQ ID NO:46.

[0109] In one embodiment, the polynucleotide comprises a sequence encoding a CAR comprising the sequence of any one of SEQ ID NOs: 46, or 52-54, or a variant thereof having at least 75% sequence identity thereto, for example, at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0110] In one embodiment, the polynucleotide comprises a sequence encoding a CAR comprising the sequence of any one of SEQ ID NOs: 46, or 52-54.

[0111] In one embodiment, the polynucleotide is a) a sequence encoding an scFv comprising the sequence of SEQ ID NO: 46, and / or b) a sequence encoding a CAR comprising any one of SEQ ID NOs: 46, or 52 to 54; or variants thereof each having at least 75% sequence identity thereto.

[0112] In one embodiment, the polynucleotide comprises a sequence encoding an IgG1 hinge comprising or consisting of the sequence of SEQ ID NO: 19, or a variant thereof having at least 75% sequence identity thereto, for example at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0113] In one embodiment, the polynucleotide comprises a sequence encoding an LNGFR spacer comprising or consisting of the sequence of SEQ ID NO: 20 or 29, or a variant thereof having at least 75% sequence identity thereto, for example, at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0114] In one embodiment, the polynucleotide comprises a sequence encoding an mCH2CH3 spacer that comprises or consists of the sequence of SEQ ID NO: 30, or a variant thereof having at least 75% sequence identity thereto, for example at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0115] In one embodiment, the polynucleotide comprises a sequence encoding a CD28 costimulatory domain and transmembrane domain that comprises or consists of the sequence of SEQ ID NO: 21, or a variant thereof having at least 75% sequence identity thereto, for example at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0116] In one embodiment, the polynucleotide comprises a sequence encoding a CD3 zeta signaling domain comprising or consisting of the sequence of SEQ ID NO: 22, or a variant thereof having at least 75% sequence identity thereto, for example, at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0117] In one embodiment, the polynucleotide further encodes one or more nucleotides of interest.

[0118] In one embodiment, the polynucleotide further encodes a cytokine.

[0119] In one embodiment, the polynucleotide further encodes a selectable marker.

[0120] In one embodiment, the polynucleotide further encodes a suicide gene.

[0121] In one embodiment, the polynucleotide further encodes CD20, or a modified version thereof.

[0122] In one embodiment, the polynucleotide further encodes thymidine kinase, or a modified version thereof.

[0123] In one embodiment, the polynucleotide further encodes human epidermal growth factor, or a modified version thereof.

[0124] In one embodiment, the polynucleotide further encodes a functionally inactive truncated version of human epidermal growth factor receptor (EGFRt) or an enhanced version of EGFRt (eEGFRt).

[0125] In one embodiment, the polynucleotide further encodes a caspase.

[0126] In another embodiment, the polynucleotide further encodes a glycosidase.

[0127] In one embodiment, the glycosidase comprises any one or more of SEQ ID NOs: 66-69, or a fragment or variant thereof.

[0128] In one embodiment, the glycosidase consists of the sequence of any one of SEQ ID NOs: 66-69, or a fragment or variant thereof.

[0129] In one embodiment, the glycosidase comprises or consists of any one of SEQ ID NOs: 66-69, or a variant thereof having at least 75% sequence identity thereto, for example at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0130] In one aspect, there is provided a vector comprising a polynucleotide according to the invention.

[0131] In one embodiment, the vector is a viral vector.

[0132] In one embodiment, the vector is a lentiviral vector.

[0133] In one embodiment, the vector is a bidirectional vector, such as a bidirectional lentiviral vector.

[0134] In one embodiment, the vector is an adeno-associated viral (AAV) vector.

[0135] In one embodiment, the vector is in the form of a nanoparticle.

[0136] In one embodiment, the polynucleotide or vector comprises one or more promoters operably linked to the nucleotide sequence encoding the CAR or scFv.

[0137] In one embodiment, the polynucleotide or vector comprises the human phosphoglycerate kinase (PGK) promoter.

[0138] In one embodiment, the polynucleotide or vector comprises a human phosphoglycerate kinase (PGK) promoter operably linked to a nucleotide sequence encoding a CAR.

[0139] In one embodiment, the polynucleotide or vector comprises one or more nucleotide sequences encoding a suicide gene.

[0140] In one embodiment, the suicide gene is CD20. In one embodiment, the suicide gene is thymidine kinase (TK) or a modified version thereof. Preferably, the TK or modified version thereof is TK mut2.

[0141] In one embodiment, the polynucleotide or vector comprises one or more nucleotide sequences encoding a selectable marker.

[0142] In one embodiment, the selectable marker is CD20. In one embodiment, the selectable marker is thymidine kinase (TK), or a modified version thereof.

[0143] In one embodiment, the polynucleotide or vector comprises one or more promoters operably linked to a nucleotide sequence encoding CD20. In one embodiment, the polynucleotide or vector comprises one or more promoters operably linked to a nucleotide sequence encoding thymidine kinase (TK), or a modified version thereof.

[0144] In one embodiment, the polynucleotide or vector comprises a human phosphoglycerate kinase (PGK) promoter. In one embodiment, the polynucleotide or vector comprises a human phosphoglycerate kinase (PGK) promoter operably linked to a nucleotide sequence encoding a suicide gene or a selectable marker.

[0145] In one embodiment, the polynucleotide or vector comprises a human phosphoglycerate kinase (PGK) promoter operably linked to a nucleotide sequence encoding CD20. In one embodiment, the polynucleotide or vector comprises a human phosphoglycerate kinase (PGK) promoter operably linked to a nucleotide sequence encoding thymidine kinase (TK), or a modified version thereof.

[0146] In one embodiment, the polynucleotide or vector comprises a minimal core promoter derived from cytomegalovirus (minCMV). In one embodiment, the polynucleotide or vector comprises a minimal core promoter derived from cytomegalovirus (minCMV) operably linked to a nucleotide sequence encoding a suicide gene or a selectable marker.

[0147] In one embodiment, the polynucleotide or vector comprises a minimal core promoter from cytomegalovirus (minCMV) operably linked to a nucleotide sequence encoding CD20. In one embodiment, the polynucleotide or vector comprises a minimal core promoter from cytomegalovirus (minCMV), or a modified version thereof, operably linked to a nucleotide sequence encoding thymidine kinase (TK).

[0148] In one embodiment, the nucleotide sequence encoding the suicide gene or selectable marker is encoded in antisense orientation relative to the nucleotide sequence encoding the CAR, hi one embodiment, a polynucleotide or vector comprises a nucleotide sequence encoding a CAR and a nucleotide sequence encoding a suicide gene or selectable marker.

[0149] In one embodiment, the polynucleotide or vector comprises a human phosphoglycerate kinase (PGK) promoter operably linked to a nucleotide sequence encoding a CAR, and a minimal core promoter derived from cytomegalovirus (minCMV) operably linked to a nucleotide sequence encoding a suicide gene or a selectable marker.

[0150] In one embodiment, the polynucleotide or vector comprises a nucleotide sequence encoding a CAR and a nucleotide sequence encoding CD20. In one embodiment, the polynucleotide or vector comprises a nucleotide sequence encoding a CAR and a nucleotide sequence encoding thymidine kinase (TK), or a modified version thereof.

[0151] In one embodiment, the polynucleotide or vector comprises a human phosphoglycerate kinase (PGK) promoter operably linked to a nucleotide sequence encoding a CAR, and a minimal core promoter from cytomegalovirus (minCMV) operably linked to a nucleotide sequence encoding CD20. In one embodiment, the polynucleotide or vector comprises a human phosphoglycerate kinase (PGK) promoter operably linked to a nucleotide sequence encoding a CAR, and a minimal core promoter from cytomegalovirus (minCMV) operably linked to a nucleotide sequence encoding a thymidine kinase (TK), or a modified version thereof. In a preferred embodiment, the nucleotide sequence encoding thymidine kinase (TK), or a modified version thereof, is encoded in an antisense orientation relative to the nucleotide sequence encoding the CAR.

[0152] In one embodiment, the nucleotide sequence encoding the suicide gene or selectable marker is encoded in the antisense orientation and the nucleotide sequence encoding the CAR is in the sense orientation. In one embodiment, the nucleotide sequence encoding the suicide gene or selectable marker is encoded in the sense orientation and the nucleotide sequence encoding the CAR is in the antisense orientation.

[0153] In one aspect, a cell comprising a CAR, scFv, polynucleotide, or vector according to the invention is provided.

[0154] In another aspect, there is provided a cell comprising a polynucleotide or vector according to the invention.

[0155] In a further aspect, a cell comprising a CAR, or scFv according to the invention is provided.

[0156] In one embodiment, the cell is a eukaryotic cell, such as a mammalian cell, hi one embodiment, the cell is selected from the group consisting of a rodent cell, such as a mouse or rat cell, a feline cell, a canine cell, and a human cell.

[0157] In a preferred embodiment, the cells are human cells.

[0158] In one embodiment, the cell is an immune cell.

[0159] In one embodiment, the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, an Invariant NK T cell, a Cytokine-Induced Killer (CIK) cell, and a macrophage, and optionally the cell is an autologous or allogeneic cell.

[0160] In one embodiment, the cell is a T cell.

[0161] In one embodiment, the cell is an invariant NK T cell.

[0162] In one embodiment, the cells are cytokine-induced killer (CIK) cells.

[0163] In one embodiment, the cell is a macrophage.

[0164] In one embodiment, the cells are autologous cells.

[0165] In one embodiment, the cells are allogeneic cells.

[0166] In one aspect, a composition comprising a CAR, scFv, polynucleotide, vector, or cell according to the invention is provided.

[0167] In one aspect, a pharmaceutical composition comprising a CAR, scFv, polynucleotide, vector, or cell according to the invention is provided.

[0168] In one aspect, there is provided a CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition according to the invention for use in therapy.

[0169] In one aspect, there is provided a CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition according to the invention for use in treating cancer.

[0170] In one embodiment, there is provided a CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition according to the invention for use in treating cancer, comprising: a) the cancer is a primary cancer, optionally gastrointestinal cancer, colorectal cancer, pancreatic cancer, and / or gastric cancer; b) the cancer is a secondary cancer, optionally liver metastasis, optionally liver metastasis of colorectal cancer and / or liver metastasis of pancreatic ductal adenocarcinoma (PDAC), and / or c) The cancer is a neuroendocrine tumor.

[0171] In one embodiment, the cancer is a primary cancer.

[0172] In one embodiment, the cancer is gastrointestinal cancer, colorectal cancer, pancreatic cancer, and / or gastric cancer.

[0173] In one embodiment, the cancer is gastrointestinal cancer.

[0174] In one embodiment, the cancer is colorectal cancer.

[0175] In one embodiment, the cancer is pancreatic cancer. In one embodiment, the cancer is pancreatic ductal adenocarcinoma (PDAC). In one embodiment, the cancer is pancreatic neuroendocrine tumor.

[0176] In one embodiment, the cancer is gastric cancer.

[0177] In one embodiment, the cancer is a secondary cancer.

[0178] In one embodiment, the cancer is liver metastasis.

[0179] In one embodiment, the cancer is liver metastasis of colorectal cancer.

[0180] In one embodiment, the cancer is liver metastasis of pancreatic ductal adenocarcinoma (PDAC).

[0181] In one embodiment, the cancer is a neuroendocrine tumor.

[0182] In one aspect there is provided the use of an scFv of the invention for determining the level of cadherin-17 (CDH-17) in a sample.

[0183] In one embodiment, there is provided use of an scFv of the invention for determining the level of cadherin-17 (CDH-17) in a sample, wherein the sample is derived from a subject.

[0184] In one embodiment, the subject is a human subject.

[0185] In another embodiment, the subject is a human subject with cancer.

[0186] In one aspect, a method is provided for identifying a subject suitable for treatment with an anti-CDH-17 therapy, the method comprising determining the CDH-17 expression level in a sample isolated from the subject, wherein the CDH-17 expression level is determined using an scFv of the invention.

[0187] In one embodiment, the subject is a human subject. [Brief explanation of the drawings]

[0188] [Figure 1] Target discovery strategy. Schematic representation of the steps taken in the rational search for candidate CAR targets. [Figure 2-1] Identification of target antigens. Heatmap showing the expression of known (A) and unknown (B) target antigens in liver metastasis samples recovered from patients with colorectal cancer. Data are obtained from RNA sequencing analysis of samples containing more than 70% tumor cells. After obtaining the expression matrix with TPM (Transcripts Per Kilobase Million), values ​​were converted to deciles, and highly expressed genes were defined as those belonging to the first three deciles (10, 9, and 8). (C) Expression of the 30 top antigens in healthy tissues. Starting from a global matrix containing mRNA expression data for all genes in all available tissues, the mean and standard deviation of all genes in each tissue were calculated to identify four expression classes: high, medium, low, and not detected. (D) Expression of CDH-17 (transcripts per million) in tumors (red dots) and adjacent normal tissues (green dots) from different patients with cancer, plotted using data from The Cancer Genome Atlas (TCGA). Highlighting tumor types in red indicates higher expression in tumors relative to normal tissue. Right: Focus on expression data in CRC at different stages. [Figure 2-2]Identification of target antigens. Heatmap showing the expression of known (A) and unknown (B) target antigens in liver metastasis samples recovered from patients with colorectal cancer. Data are obtained from RNA sequencing analysis of samples containing more than 70% tumor cells. After obtaining the expression matrix with TPM (Transcripts Per Kilobase Million), values ​​were converted to deciles, and highly expressed genes were defined as those belonging to the first three deciles (10, 9, and 8). (C) Expression of the 30 top antigens in healthy tissues. Starting from a global matrix containing mRNA expression data for all genes in all available tissues, the mean and standard deviation of all genes in each tissue were calculated to identify four expression classes: high, medium, low, and not detected. (D) Expression of CDH-17 (transcripts per million) in tumors (red dots) and adjacent normal tissues (green dots) from different patients with cancer, plotted using data from The Cancer Genome Atlas (TCGA). Highlighting tumor types in red indicates higher expression in tumors relative to normal tissue. Right: Focus on expression data in CRC at different stages. [Figure 2-3]Identification of target antigens. Heatmap showing the expression of known (A) and unknown (B) target antigens in liver metastasis samples recovered from patients with colorectal cancer. Data are obtained from RNA sequencing analysis of samples containing more than 70% tumor cells. After obtaining the expression matrix with TPM (Transcripts Per Kilobase Million), values ​​were converted to deciles, and highly expressed genes were defined as those belonging to the first three deciles (10, 9, and 8). (C) Expression of the 30 top antigens in healthy tissues. Starting from a global matrix containing mRNA expression data for all genes in all available tissues, the mean and standard deviation of all genes in each tissue were calculated to identify four expression classes: high, medium, low, and not detected. (D) Expression of CDH-17 (transcripts per million) in tumors (red dots) and adjacent normal tissues (green dots) from different patients with cancer, plotted using data from The Cancer Genome Atlas (TCGA). Highlighting tumor types in red indicates higher expression in tumors relative to normal tissue. Right: Focus on expression data in CRC at different stages. [Figure 2-4]Identification of target antigens. Heatmap showing the expression of known (A) and unknown (B) target antigens in liver metastasis samples recovered from patients with colorectal cancer. Data are obtained from RNA sequencing analysis of samples containing more than 70% tumor cells. After obtaining the expression matrix with TPM (Transcripts Per Kilobase Million), values ​​were converted to deciles, and highly expressed genes were defined as those belonging to the first three deciles (10, 9, and 8). (C) Expression of the 30 top antigens in healthy tissues. Starting from a global matrix containing mRNA expression data for all genes in all available tissues, the mean and standard deviation of all genes in each tissue were calculated to identify four expression classes: high, medium, low, and not detected. (D) Expression of CDH-17 (transcripts per million) in tumors (red dots) and adjacent normal tissues (green dots) from different patients with cancer, plotted using data from The Cancer Genome Atlas (TCGA). Highlighting tumor types in red indicates higher expression in tumors relative to normal tissue. Right: Focus on expression data in CRC at different stages. [Figure 3]

[0033] Figure 1 shows a schematic of an exemplary CAR construct. Schematic of a bidirectional lentiviral construct encoding a CDH-17-specific CAR and a CD20 marker gene. From left to right, each construct contains boxes representing the following domains / regions: selectable marker / suicide gene (CD20), mCMV promoter or "minCMV," human PGK promoter, single-chain fragment variable region (e.g., Lic3 or A4_4R), extracellular spacer or linker (LNGFR spacer (NMS) or "H" [IgG1-derived]), costimulatory domain (e.g., CD28), and signaling domain (e.g., CD3 zeta). The transmembrane domain is not explicitly shown, but may include sequences from the costimulatory domain or, alternatively, linker / spacer sequences. [Figure 4]Evaluation of CAR T cell production. Peripheral blood-derived human T cells were bead-activated, transduced with lentiviral vectors, and expanded in IL-7 and IL-15. CAR T cell products were analyzed at the end of manufacturing. (A) Fold expansion of T cells (n=5 donors). (B) Frequency of CD4 and CD8 T cells. (C) Frequency of memory subsets by CD45RA and CD62L expression. (D) Frequency of HLA-DR-expressing T cells. (E) Frequency of PD-1-expressing T cells. TSCM: stem memory T cells. TCM: central memory T cells, TEM: effector memory T cells, TEMRA: effector memory RA+ T cells. [Figure 5] In vitro CAR T cell-mediated tumor cell killing. (A) Killing of LoVo and BxPC3 tumor cells was quantified after coculture with CDH17.28z CAR T cells at different E:T ratios (n=4 donors). Killing is expressed as a depletion index, calculated relative to untransduced T cells. (B) Tumor cell killing comparing constructs with different spacer regions (LNGFR spacer (NMS) vs. hinge) or scFvs (Lic3 vs. A4_4R). P values ​​(**P<0.01, ***P<0.001, ****P<0.0001) were determined by two-way ANOVA (A) or paired t-test (B). Data are presented as mean + / - SEM. [Figure 6] In vitro CAR T cell-mediated tumor cell killing. (A) Immunodeficient NSG mice were intraperitoneally injected with LoVo cells labeled with secreted luciferase and treated 10 days later with A4_4R-NMS CAR T cells, A4_4R.hinge CAR T cells, or untransduced T cells (6 × 10 cells / mouse). Tumor growth was tracked by measuring bioluminescence signals in peripheral blood. (B) Left: Tumor growth kinetics. Center: Tumor level 6 days after T cell injection. Right: Tumor level 25 days after T cell injection. P values ​​(*P<0.05, **P<0.01) were determined by two-way ANOVA. Data are presented as mean + / - SEM. [Figure 7]Evaluation of CAR T cell production. A. Schematic of the bidirectional lentiviral construct containing a CDH17-specific CAR and CD20 marker gene. Red box: single-chain variable fragment. Orange box: extracellular spacer. Purple box: costimulatory endodomain. Green box: activation endodomain. Peripheral blood-derived human T cells were bead-activated, transduced with the lentiviral vector, and expanded in IL-7 and IL-15. CAR T cell products were analyzed at the end of production. Fold expansion of B cells (n=10 donors for Ut, IgG1h, and NWL conditions, n=3 donors for NMS condition). C. Frequency of NGFR-spaced CAR-expressing T cells. D. Frequency of CD20-expressing T cells. E. Frequency of CD4 and CD8 T cells. F. Frequency of memory subsets by CD45RA and CD62L expression. TSCM: stem memory T cells. TCM: central memory T cells, TEM: effector memory T cells, TEMRA: effector memory RA+ T cells. P values ​​(****P<0.0001) were determined by one-way analysis of variance (B). Data are presented as mean + / - SEM. [Figure 8] CDH17 immunohistochemical staining of human tissues. Representative staining of primary colorectal cancer (A), liver metastasis from colorectal cancer (B), primary pancreatic ductal adenocarcinoma (C), and pancreatic neuroendocrine tumor (D) with a commercially available anti-CDH17 antibody (Abcam, clone EPR 3996). Experiments were performed on multiple primary samples with similar results. [Figure 9]Generation of CDH17 truncation mutants and identification of extracellular cadherins containing the A4_4R CAR epitope. Schematic diagram of CDH17 truncation mutants. SP, signal peptide; EC, extracellular cadherin; TM, transmembrane; IC, intracellular domain. B. CDH17negSW620 colorectal cancer cells were transduced with a bidirectional lentiviral vector expressing WT CDH17 or truncation mutants and NGFR as a marker gene, followed by flow cytometry analysis with NGFR (left) and a commercially available anti-CDH17 antibody (Santa Cruz, clone H1-1) that binds EC7 (right). C. SW620 cell killing quantified after coculture with CDH17.28z cells bearing IgG1 hinge (left), NMS (center), and NWL (right) as extracellular spacers at an effector-to-target ratio of 1:5 (n = 3 donors). Killing is expressed as a depletion index, calculated relative to untransduced T cells. P values ​​(**P<0.01, ***P<0.001, ****P<0.0001) were determined by one-way analysis of variance (A) or paired t-test (B). Data are presented as mean + / - SEM. [Figure 10]In vivo efficacy of CDH17 CAR T cells in a subcutaneous model of CRC. Antitumor efficacy of CDH17.28z cells in a xenograft mouse model of colorectal cancer. Immunodeficient NSG mice were subcutaneously injected with secreted luciferase-labeled LoVo cells and treated with CDH17.28z_H CAR T cells (n=8), CDH17.28z_NMS CAR T cells (n=8), CDH17.28z_NWL CAR T cells (n=7), or untransduced T cells (n=4) (10x106 cells / mouse). Tumor growth was tracked by measuring bioluminescence signals in peripheral blood. A. Kinetics of tumor growth. RLU, relative light units. B. Tumor-associated Kaplan-Meier survival. C, D. Post-infusion CAR T cell proliferation in peripheral blood (C) and HLA-DR expression (RFI, relative fluorescence intensity, D) at day 8. E, F. CD20 expression (percentage of positive cells, E) and NGFR expression (percentage of positive cells, F) in tumor-infiltrating lymphocytes at the time of sacrifice. P values ​​(*P<0.05, ***P<0.001, ****P<0.0001) were determined by the log-rank Mantel-Cox test (B) or by one-way ANOVA (D) or by two-tailed t-test (F). Data are presented as mean + / - SEM. [Figure 11]In vivo efficacy of CDH17 CAR T cells in a subcutaneous model of PDAC. Antitumor efficacy of CDH17.28z cells in a xenograft mouse model of pancreatic adenocarcinoma. Immunocompromised NSG mice were subcutaneously injected with secreted luciferase-labeled AsPC-1 cells and treated with CDH17.28z_H CAR T cells (n=8), CDH17.28z_NMS CAR T cells (n=8), CDH17.28z_NWL CAR T cells (n=7), or untransduced T cells (n=3) (10x106 cells / mouse). Tumor growth was tracked by measuring bioluminescence signals in peripheral blood. A. Kinetics of tumor growth. RLU, relative light units. B. Tumor-associated Kaplan-Meier survival. C, D. Post-infusion CAR T cell proliferation (C) and HLA-DR expression (D) in peripheral blood at day 9 (RFI, relative fluorescence intensity). P values ​​(*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001) were determined by the log-rank Mantel-Cox test (B) or by one-way analysis of variance (D). Data are presented as mean + / - SEM. [Figure 12] Cytokine production of CDH17 CAR T cells in vitro. IFN-γ (A) and TNF-α (B) production after 24 h coculture of CDH17.28z_H or CDH17.28z_NWL cells with LoVo colorectal cells at an effector-to-target ratio of 1:10 (n=3 donors in technical duplicates). UT, untransduced control T cells. P values ​​(*P<0.05) were determined by one-way ANOVA. Data are presented as mean + / - SEM. [Figure 13]A. In vitro efficacy of CEA and CDH17 CAR T cells. Schematic of bidirectional lentiviral constructs containing CEA or CDH17 CAR and CD20 marker genes. The CEA CAR was generated as the CDH17.28z_H construct and contains a specific scFv derived from the BW431-26 mAb. Peripheral blood-derived human T cells were bead-activated, transduced with the lentiviral vector, and expanded in IL-7 and IL-15. CAR T cell products were analyzed at the end of production. Ut indicates untransduced control T cells. Fold expansion of BT cells (n = 3 donors). C. Frequency of NGFR-expressing T cells. D. Frequency of CD4 and CD8 T cells. E. Killing of LoVo cells quantified after coculture with CDH17.28z or CEA.28z cells at multiple effector-to-target ratios (n = 3-6 donors). Killing is expressed as a depletion index, calculated relative to untransduced T cells. F. IFN-γ and TNF-α production after 24 h coculture of CDH17.28z or CEA.28z cells with LoVo colorectal cells at an E:T ratio of 1:10 (n=3 donors in technical duplicates). UT, untransduced control T cells. (**P<0.01, ***P<0.001) determined by paired t-test (C) and two-way ANOVA (E). Data are presented as mean + / - SEM. [Figure 14] In vitro efficacy of CDH17 CAR T cells against patient-derived organoids from CRC-LM. Representative brightfield microscopy images (left) and quantification (right) of killing of organoids derived from a patient with metastatic colorectal cancer to the liver after co-culture with CDH17.28z CAR T cells derived from a healthy donor at an E:T ratio of 1:1 (n=1 in technical triplicates). P values ​​(*P<0.05) were determined by paired t-test. Data are presented as mean + / - SEM. [Figure 15]CAR T cell production from a patient with liver metastases from colorectal cancer. Peripheral blood-derived human T cells were bead-activated, transduced with a lentiviral vector, and expanded in IL-7 and IL-15. CAR T cell products were analyzed at the end of production. Fold expansion of AT cells (n = 3-7 donors). Frequency (left) and intensity (right) of CD20 expression by BT cells. RFI, relative fluorescence intensity. C. Frequency of NGFR-expressing T cells. D. Frequency of CD4 and CD8 T cells. E. Frequency of memory subsets by CD45RA and CD62L expression. TSCM: stem memory T cells. TCM: central memory T cells; TEM: effector memory T cells; TEMRA: effector memory RA+ T cells. P values ​​(*P<0.05, **P<0.01, ***P<0.001) were determined by one-way ANOVA (B, C). Data are presented as mean + / - SEM. [Figure 16] Production of CAR T cells from a patient with liver metastases from pancreatic ductal adenocarcinoma. Peripheral blood-derived human T cells were bead-activated, transduced with lentiviral vectors, and expanded in IL-7 and IL-15. CAR T cell products were analyzed at the end of production. Fold expansion of AT cells (n=3 donors). Frequency (left) and intensity (right) of CD20 expression by BT cells. RFI, relative fluorescence intensity. C. Frequency of NGFR-expressing T cells. D. Frequency of CD4 and CD8 T cells. E. Frequency of memory subsets by CD45RA and CD62L expression. TSCM: stem memory T cells. TCM: central memory T cells, TEM: effector memory T cells, TEMRA: effector memory RA+ T cells. P values ​​(**P<0.01) were determined by one-way ANOVA. Data are presented as mean + / - SEM. [Figure 17]Production of CAR T cells from a patient with primary pancreatic ductal adenocarcinoma. Peripheral blood-derived human T cells were bead-activated, transduced with a lentiviral vector, and expanded in IL-7 and IL-15. CAR T cell products were analyzed at the end of production. Fold expansion of AT cells (n=2 donors). Frequency (left) and intensity (right) of CD20 expression by BT cells. RFI, relative fluorescence intensity. C. Frequency of NGFR-expressing T cells. D. Frequency of CD4 and CD8 T cells. E. Frequency of memory subsets by CD45RA and CD62L expression. TSCM: stem memory T cells. TCM: central memory T cells, TEM: effector memory T cells, TEMRA: effector memory RA+ T cells. P values ​​(*P<0.05) were determined by one-way ANOVA. Data are presented as mean + / - SEM. [Figure 18] In vitro efficacy of patient-derived CDH17 CAR T cells from CRC-LM against patient-derived organoids from CRC-LM. Killing of luciferase-labeled patient-derived organoids (PDO) after coculture with CDH17.28z CAR T cells derived from a patient with liver metastases from colorectal cancer at a 1:1 E:T ratio. PDO was detected by measuring the bioluminescence signal. A, B. Representative brightfield microscopy images (A) and quantification of PDO killing (B) by autologous CDH17.28z_H (left) and CDH17.28z_NWL (right) CAR T cells (n=1 in technical duplicates). C-F. Representative brightfield microscopy images (C, E) and quantification of PDO killing by allogeneic CDH17.28z_H CAR T cells (D left, F left) and allogeneic CDH17.28z_NWL CAR T cells (D right, F right). RLU, relative light units. P values ​​(*P<0.05, **P<0.01) were determined by paired t-test. Data are presented as mean + / - SEM. [Figure 19] Immunofluorescence staining of CDH17 localization in primary healthy colon and tumor tissue. Representative confocal images of healthy (A) and tumor (B) colon and liver metastasis from colorectal cancer (C) stained for CDH17 (green) and occludin (red, tight junctions). Zoomed images at 63x magnification. [Figure 20] Validation of the route of CDH17.28z CAR-T cell administration in a xenograft mouse model of colorectal cancer. Immunodeficient NSG mice were intrahepatically injected with secreted luciferase-labeled LoVo cells and treated either intrahepatically with CDH17.28z_H CAR T cells (n=5) or CDH17.28z_NWL CAR T cells (n=5) or intravenously with CDH17.28z_H CAR T cells (n=5) or CDH17.28z_NWL CAR T cells (n=5) (10x106 cells / mouse). Non-transduced T cells (n=4) were administered as a negative control. Tumor growth was tracked by measuring bioluminescence signals in the peripheral blood. A. Kinetics of tumor growth. RLU, relative light units. B. Proliferation of CDH17.28z_H (left) and CDH17.28z_NWL (right) CAR T cells after infusion in peripheral blood. Data are presented as mean + / - SEM. [Figure 21]Validation of the route of CDH17.28z CAR-T cell administration in the HSPC-humanized SGM3 mouse model of colorectal cancer. NSG mice transgenic for expression of human SCF, GM-CSF, and IL-3 (SGM3) were infused with human cord blood-derived hematopoietic stem and progenitor cells (CB HSPCs), intrahepatic LoVo cells labeled with secreted luciferase, and treated with either CDH17.28z_H CAR T cells (n=7) or CDH17.28z_NWL CAR T cells (n=7) intrahepatically or CDH17.28z_H CAR T cells (n=7) or CDH17.28z_NWL CAR T cells (n=7) intravenously (5x106 cells / mouse). Untransduced T cells (n=4) were administered as a negative control. Tumor growth was tracked by measuring bioluminescence signals in peripheral blood. A. Kinetics of tumor growth. RLU, relative light units. B. Tumor level (left), HLA-DR expression (percentage of positive cells) (center), and T cells in peripheral blood (right) 7 days after CDH17.28z_H CAR T cell infusion. C. Tumor level (left), HLA-DR expression (percentage of positive cells) (center), and T cells in peripheral blood (right) 7 days after CDH17.28z_NWL CAR T cell infusion. P values ​​(*P<0.05, **P<0.01) were determined by two-tailed t-test. Data are presented as mean + / - SEM. [Figure 22]Evaluation of the potential systemic toxicity of CDH17.28z CAR-T cells in the HSPC-humanized SGM3 mouse model of colorectal cancer. A-C. Percentage of body weight loss (A), concentrations of circulating bone marrow-derived cytokines (B), and weight loss-associated Kaplan-Meier survival (C) for mice from Figure 20 treated either intrahepatically or intravenously with CDH17.28z_H CAR T cells. D-F. Percentage of body weight loss (D), concentrations of circulating bone marrow-derived cytokines (E), and weight loss-associated Kaplan-Meier survival (F) for mice from Figure 20 treated either intrahepatically or intravenously with CDH17.28z_NWL CAR T cells. P values ​​(*P<0.05, **P<0.01, ***P<0.001) were determined by two-tailed t-test (B) or log-rank Mantel-Cox test (C, F). Data are presented as mean + / - SEM. [Figure 23] Validation of the route of CDH17.28z CAR-T cell administration in a xenograft mouse model of pancreatic adenocarcinoma. Immunodeficient NSG mice were intrapancreatically injected with AsPC-1 cells labeled with secreted luciferase and treated either intrapancreatically with CDH17.28z_H CAR T cells (n=5) or CDH17.28z_NWL CAR T cells (n=5) or intravenously with CDH17.28z_H CAR T cells (n=3) or CDH17.28z_NWL CAR T cells (n=3) (10x106 cells / mouse). Non-transduced T cells (n=3) were administered as a negative control. Tumor growth was tracked by measuring the bioluminescent signal in the peripheral blood. A. Kinetics of tumor growth. RLU, relative light units. B. Tumor-associated Kaplan-Meier survival of CDH17.28z_H (left) and CDH17.28z_NWL (right) CAR T cell-treated mice. P values ​​were determined by the log-rank Mantel-Cox test (B). [Figure 24-1]Generation and in vitro efficacy of third-generation CDH17 CAR-T cells. A. Schematic of bidirectional lentiviral constructs containing a CDH17-specific CAR and a CD20 marker gene. Red indicates a second-generation CAR with the A4-4R scFv, blue indicates a third-generation CAR with the A4-4R scFv, and green indicates a third-generation CAR with the VHH1 nanobody. Peripheral blood-derived human T cells were bead-activated, transduced with lentiviral vectors, and expanded in IL-7 and IL-15. Fold expansion of BT cells (n=3 donors). C. Frequency of CD20-expressing T cells at day 9 of manufacture. D. Frequency of NGFR-expressing T cells at day 9 of manufacture. E. Frequency of CD4 and CD8 T cells at day 9 of manufacture. F-H. Killing of LoVo cells quantified after co-culture with CAR T cells (n=6 donors). F. Killing at multiple effector-to-target ratios. Killing is expressed as a depletion index, calculated relative to non-transduced T cells. G, I. Killing of LoVo cells at an E:T ratio of 1:25. H. Production of IFN-γ (left) and TNF-α (right) after 24 hours of co-culture of LoVo cells with CAR T cells at an E:T ratio of 1:10. P values ​​(*P<0.05, **P<0.01) were determined by paired t-test (G, I) and one-way ANOVA (H). Data are presented as mean + / - SEM. [Figure 24-2]Generation and in vitro efficacy of third-generation CDH17 CAR-T cells. A. Schematic of bidirectional lentiviral constructs containing a CDH17-specific CAR and a CD20 marker gene. Red indicates a second-generation CAR with the A4-4R scFv, blue indicates a third-generation CAR with the A4-4R scFv, and green indicates a third-generation CAR with the VHH1 nanobody. Peripheral blood-derived human T cells were bead-activated, transduced with lentiviral vectors, and expanded in IL-7 and IL-15. Fold expansion of BT cells (n=3 donors). C. Frequency of CD20-expressing T cells at day 9 of manufacture. D. Frequency of NGFR-expressing T cells at day 9 of manufacture. E. Frequency of CD4 and CD8 T cells at day 9 of manufacture. F-H. Killing of LoVo cells quantified after co-culture with CAR T cells (n=6 donors). F. Killing at multiple effector-to-target ratios. Killing is expressed as a depletion index, calculated relative to non-transduced T cells. G, I. Killing of LoVo cells at an E:T ratio of 1:25. H. Production of IFN-γ (left) and TNF-α (right) after 24 hours of co-culture of LoVo cells with CAR T cells at an E:T ratio of 1:10. P values ​​(*P<0.05, **P<0.01) were determined by paired t-test (G, I) and one-way ANOVA (H). Data are presented as mean + / - SEM. [Figure 25] Production of third-generation anti-CDH17 CAR T cells from a patient with liver metastases from colorectal cancer. Peripheral blood-derived human T cells were bead-activated, transduced with a lentiviral vector, and expanded in IL-7 and IL-15. CAR T cell products were analyzed at the end of production. A. Fold expansion of A-T cells (n=3 donors). B. Frequency of CD20-expressing T cells. C. Frequency of CD4 and CD8 T cells. D. Frequency of memory subsets by CD45RA and CD62L expression. TSCM: stem memory T cells. TCM: central memory T cells, TEM: effector memory T cells, TEMRA: effector memory RA+ T cells. P values ​​(**P<0.01) were determined by unpaired t-test (B). Data are presented as mean + / - SEM. [Figure 26]Generation of CAR-T cells expressing the thymidine kinase (TK) suicide gene. A-C. The TK suicide gene was cloned under the control of the mCMV promoter in CAR backbones expressing the CDH17.28z_H (A), CDH17.28z_NWL (B), and CDH17.28BBz_H (C) CARs. DETAILED DESCRIPTION OF THE INVENTION

[0189] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including" or "includes," or "containing" or "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0190] It will be understood that when a protein or polypeptide is referred to herein, it can equally apply to the polynucleotide encoding it, and vice versa where relevant (i.e., when referring to a coding sequence within a polynucleotide).

[0191] Cadherin-17 (CDH-17) Cadherin-17 (CDH-17) is an intercellular adhesion glycoprotein that is overexpressed in several cancers of the gastrointestinal tract, including pancreatic, colorectal, gastric, liver, and esophageal, as well as in neuroendocrine tumors. Upon interaction with α2β1 integrin, CDH17 promotes tumor cell proliferation, adhesion, and metastatic colonization of the liver.

[0192] Chimeric antigen receptor (CAR) As used herein, "chimeric antigen receptor" or "CAR" refers to an engineered receptor that can confer antigen specificity to a cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors.

[0193] CARs are generally classified into "generations" according to the composition of their intracellular signaling domain. All CARs typically contain an extracellular antigen-binding domain, usually an scFv, connected to a membrane-anchoring transmembrane domain by a linker or spacer sequence. First-generation CARs contain a single intracellular signaling domain, typically a single CD3 zeta chain, while second- and third-generation CARs further contain one or two additional costimulatory domains (respectively), typically CD28, 4-1BB, and / or OX-40. Fourth-generation CARs are structurally similar to second-generation CARs, but are typically provided (e.g., in CAR T cells) with expression cassettes encoding additional transgenes, such as cytokines.

[0194] In one embodiment, a CAR of the invention is a first generation CAR.

[0195] In one embodiment, a CAR of the invention is a second generation CAR.

[0196] In one embodiment, a CAR of the invention is a third generation CAR.

[0197] In one embodiment, a CAR of the invention is a fourth generation CAR.

[0198] Preferably, the CAR of the present invention comprises an antigen-specific targeting region, which may include an extracellular domain such as an scFv, a linker or a hinge, a transmembrane domain, optionally one or more costimulatory domains, and an intracellular signaling domain.

[0199] In some embodiments, the CAR of the present invention is provided with one or more nucleotides or transgenes of interest. In some embodiments, the transgene encodes a cytokine.

[0200] Antigen-specific targeting domains The antigen-specific targeting domain (or antigen-binding domain) provides the CAR with the ability to bind to a target antigen of interest. The antigen-specific targeting domain preferably targets an antigen of clinical interest against which it is desirable to elicit an effector immune response that results in cell killing.

[0201] An antigen-specific targeting domain can be any protein or peptide capable of specifically recognizing and binding to a biological molecule (e.g., a cell surface receptor or tumor protein, or component thereof). Antigen-specific targeting domains include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for the biological molecule of interest.

[0202] Exemplary antigen-specific targeting domains include antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands for cell surface molecules / receptors or their receptor-binding domains, and tumor-binding proteins.

[0203] In a preferred embodiment, the antigen-specific targeting domain is an antibody or is derived from an antibody. An antibody-derived targeting domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of an antibody, which fragments are responsible for binding to the antigen. Examples include a variable region (Fv), a complementarity-determining region (CDR), a Fab, a single-chain variable fragment (scFv), a heavy chain variable region (VH), a light chain variable region (VL), and a camelid antibody (VHH).

[0204] In a preferred embodiment, the binding domain is a single chain variable fragment (scFv), which can be, for example, a murine, human, or humanized scFv.

[0205] "Complementarity-determining region" or "CDR" with respect to an antibody or antigen-binding fragment thereof refers to the hypervariable loops in the variable region of the heavy or light chain of the antibody. CDRs interact with the antigen conformation and can primarily determine antigen binding (although some framework regions are known to be involved in binding). The heavy and light chain variable regions each contain three CDRs.

[0206] "Heavy chain variable region" or "VH" refers to the fragment of an antibody heavy chain containing the three CDRs interposed between adjacent stretches known as framework regions, which are more highly conserved than the CDRs and form the scaffold that supports them.

[0207] "Light chain variable region" or "VL" refers to the fragment of the light chain of an antibody containing the three CDRs interposed between framework regions.

[0208] "Fv" refers to the minimum fragment of an antibody containing a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0209] "Single chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other either directly or via a peptide linker sequence.

[0210] For targeting domains that target cancer antigens, the choice of targeting domain will depend on the type of cancer being treated and the tumor antigen that may be targeted. A tumor sample from a subject may be characterized for the presence of specific biomarkers or cell surface markers.

[0211] A preferred target antigen of the present invention is CDH17.

[0212] A tumor antigen or cell surface molecule is selected that is found on tumor cells of an individual subject. Preferably, the antigen-specific targeting domain targets a cell surface molecule that is found on tumor cells and is substantially absent from normal tissues.

[0213] Preferably, the antigen-binding domain specifically binds to a tumor antigen (e.g., CDH17). In one embodiment, the antigen-binding domain specifically binds to CDH17.

[0214] In a preferred embodiment, the antigen-binding domain is an scFv.

[0215] In a preferred embodiment, the antigen binding domain is an scFv with specificity for CDH17.

[0216] In one embodiment, the antigen binding domain (eg, scFv) specifically binds to the EC1 domain of CDH17.

[0217] In one embodiment, the antigen binding domain comprises: a) a heavy chain variable region (VH) complementarity determining region (CDR) having the following sequences: CDR1-DHTIHWMR (SEQ ID NO: 1), CDR2-YIYPRDGITGYNERFRGK (SEQ ID NO: 2), and CDR3-WGYSYRNYAYYYDYWGQGTL (SEQ ID NO: 3), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) Light chain variable region (VL) CDRs having the following sequences: CDR1-INCRSSQSLLHSSNQR (SEQ ID NO: 4), CDR2-PPKVLIYWASTRES (SEQ ID NO: 5), and CDR3-QQYYSYPWTFGQ (SEQ ID NO: 6), or variants thereof each having up to three amino acid substitutions, additions, or deletions.

[0218] In one embodiment, the antigen binding domain comprises: a) a VH domain comprising the sequence of SEQ ID NO: 7, and b) a VL domain comprising the sequence of SEQ ID NO: 8; or variants thereof each having at least 75% sequence identity thereto.

[0219] Exemplary VH Domain (SEQ ID NO:7): QVQLVQSGAEVKKPGASVKVSCKASGYTLTDHTIHWMRQAPGQRLEWIGYIYPRDGITGYNERFRGKATLTADTSASTAYMELSSLRSEDTAVYYCARWGYSYRNYAYYYDYWGQGTLVTVSS

[0220] Exemplary VL Domain (SEQ ID NO: 8): DIVMTQSPDSLAVSLGERATINCRSSQSLLHSSNQRNYLAWYQQKPGQPPKVLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIK

[0221] In one embodiment, the antigen-binding domain comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 9, e.g., at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0222] Exemplary scFv (SEQ ID NO: 9): MEAPAQLLFLLLLWLPDTTGDIVMTQSPDSLAVSLGERATINCRSSQSLLHSSNQRNYLAWYQQKPGQPPKVLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIKGGGG SGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTLTDHTIHWMRQAPGQRLEWIGYIYPRDGITGYNERFRGKATLTADTSASTAYMELSSLRSEDTAVYYCARWGYSYRNYAYYYDYWGQGTLVTVSSSPV

[0223] In one embodiment, the antigen-binding domain is encoded by SEQ ID NO: 16, or a sequence having at least 75% sequence identity thereto.

[0224] Nucleotide sequence encoding an exemplary scFv (SEQ ID NO: 16): ATGGAGGCCCCCGCCAGCCTGCTGTTCCTGCTGCTGCTGTGGCTGCCCGACACCACCGGCGACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGAGAGCCACCATCAACTGCAGAAGCAGCCAGAGCCTGCTGCACAGCAGCAACCAGAGAAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCA GCCCCCCAAGGTGCTGATCTACTGGGCCAGCACCAGAGAGAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTACTACAGCTACCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGGGCGGCGG CGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCCTGACCGACCACACCATCCACTGGATGAGACAGGCCCCCGGCCAGAGACTGGAGTGGATCGGCTACATCTACCCCAGAGACGG CATCACCGGCTACAACGAGAGATTCAGAGGCAAGGCCACCCTGACCGCCGACACCAGCGCCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGCCAATGGGGCTACAGCTACAGAAACTACGCCTACTACTACGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCTC

[0225] In one aspect, there is provided a chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises: a) a heavy chain variable region (VH) complementarity determining region (CDR) having the following sequences: CDR1-DYYMY (SEQ ID NO: 42), CDR2-SISFDGTYTYYTDRVKG (SEQ ID NO: 43), and CDR3-DRPAWFPY (SEQ ID NO: 44), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) A CAR is provided, comprising light chain variable region (VL) CDRs having the following sequences: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO: 39), CDR2-KVSNRFS (SEQ ID NO: 40), and CDR3-FQGSHVPLT (SEQ ID NO: 41), or variants thereof each having up to three amino acid substitutions, additions, or deletions.

[0226] In one embodiment, the antigen binding domain comprises: a) a VH domain comprising the sequence of SEQ ID NO: 58, and b) a VL domain comprising the sequence of SEQ ID NO: 59; or variants thereof having at least 75% sequence identity thereto, for example at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0227] Exemplary VH Domain (SEQ ID NO:58): QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVASISFDGTYTYYTDRVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRPAWFPYWGQGTLVTVSA

[0228] Exemplary VL Domain (SEQ ID NO:59): DIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGAGTKLELKGAP

[0229] In one embodiment, the antigen-binding domain comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:45.

[0230] In one embodiment, the antigen-binding domain comprises a sequence having at least 75% sequence identity to SEQ ID NO:45, e.g., at least 80%, at least 85%, 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%, or 100% sequence identity to SEQ ID NO:45.

[0231] Exemplary scFv (SEQ ID NO: 45): MEAPAQLLFLLLLWLPDTTGQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVASISFDGTYTYYTDRVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRPAWFPYWGQGTLVT VSAGGGGSGGGGSGGGGSGDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGAGTKLELKGAP

[0232] In one embodiment, the antigen-binding domain is encoded by SEQ ID NO: 46, or a sequence having at least 75% sequence identity thereto.

[0233] Nucleotide sequence encoding an exemplary scFv (SEQ ID NO:46): ATGGAGGCCCCGCCCAGCTGCTGTTCCTGCTGCTGCTGGCTGCCCGACACCACGGCCAGGTGCAGCTGGTGGAGAGCGGCGGCGTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCGACTACTACATGTACTGGGTGAGACAGGCCCCGGCAAGGGCCTGGAGTGGG GTGGCCAGCATCAGCTTCGACGGCACCTACACCTACTACACCGACAGGTGAAGGGCAGATTCACCATCAGCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGACAGACCCGCTGGTTCCCCTACTGGGGCCAGGGCACCCTGGTGACC GTGAGCGCCGGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGCAGCGGCGGACATCGTGATGACCCAGACCCCCCTGAGCCTGAGCGTGACCCCGGCCAGCCCGCCAGCATCAGCTGCAGAAGCAGCCAGAGCATCGTGCACAGCAACGGCAACACCTACCTGGAGTGGTACCTGCAGAAGCCCGGCCAGAGC CCCCAGCTGCTGATCTACAAGGTGAGCAACAGATTCAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCGAGGACGTGGGCGTGTACTACTGCTTCCAGGGCAGCCACGTGCCCCTGACCTTCGGCCGGCACCAAGCTGGAGCTGAAGGGCGCCCCCC

[0234] In one embodiment, the antigen binding domain contains the sequence number 45.

[0235] In one embodiment, the antigen binding domain is composed of the sequence number 45.

[0236] In one embodiment, the antigen-binding domain comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:45.

[0237] In one embodiment, the antigen-binding domain comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 45, e.g., at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO: 45.

[0238] In one embodiment, a single chain variable fragment (scFv) comprising: a) a heavy chain variable region (VH) complementarity determining region (CDR) having the following sequences: CDR1-DYYMY (SEQ ID NO: 42), CDR2-SISFDGTYTYYTDRVKG (SEQ ID NO: 43), and CDR3-DRPAWFPY (SEQ ID NO: 44), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) An scFv is provided, comprising light chain variable region (VL) CDRs having the following sequences: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO: 39), CDR2-KVSNRFS (SEQ ID NO: 40), and CDR3-FQGSHVPLT (SEQ ID NO: 41), or variants thereof each having up to three amino acid substitutions, additions, or deletions.

[0239] In one embodiment, the scFv comprises the sequence of SEQ ID NO:45.

[0240] In one embodiment, the scFv consists of the sequence of SEQ ID NO:45.

[0241] In one embodiment, the scFv comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:45.

[0242] In one embodiment, the scFv comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:45, for example at least 80%, at least 85%, 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%, or 100% sequence identity to the sequence of SEQ ID NO:45.

[0243] In one embodiment, the antigen binding domain comprises: a) a heavy chain variable region (VH) complementarity determining region (CDR) having the following sequences: CDR1-DHTIHWMR (SEQ ID NO: 1), CDR2-YIYPRDGITGYNERFRGK (SEQ ID NO: 2), and CDR3-WGYSYRNYAYYYDYWGQGTL (SEQ ID NO: 3), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and Light chain variable region (VL) CDRs having the following sequences: CDR1-INCRSSQSLLHSSNQR (SEQ ID NO: 4), CDR2-PPKVLIYWASTRES (SEQ ID NO: 5), and CDR3-QQYYSYPWTFGQ (SEQ ID NO: 6), or variants thereof each having up to three amino acid substitutions, additions, or deletions; or b) heavy chain variable region (VH) CDRs having the following sequences: CDR1-DYYMY (SEQ ID NO: 42), CDR2-SISFDGTYTYYTDRVKG (SEQ ID NO: 43), and CDR3-DRPAWFPY (SEQ ID NO: 44), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and The light chain variable region (VL) CDRs have the following sequences: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO: 39), CDR2-KVSNRFS (SEQ ID NO: 40), and CDR3-FQGSHVPLT (SEQ ID NO: 41), or variants thereof each having up to three amino acid substitutions, additions, or deletions.

[0244] Costimulatory domain The CARs of the invention can also include one or more costimulatory domains, which can enhance cell proliferation, cell survival, and the generation of memory cells.

[0245] In some embodiments, the costimulatory domains are fused together.

[0246] In some embodiments, the costimulatory domains are separated by a linker sequence.

[0247] In some embodiments, the costimulatory domains are not separated by a linker sequence.

[0248] Each costimulatory domain can include, for example, any one or more costimulatory domains from members of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-1, TNFR-II, Fas, CD30, CD40, or combinations thereof. Costimulatory domains from other proteins can also be used with the CARs of the present invention. Additional costimulatory domains will be apparent to those skilled in the art.

[0249] In some embodiments, the costimulatory domain is a 4-1BB costimulatory domain.

[0250] In some embodiments, the costimulatory domain is a CD28 costimulatory domain.

[0251] In some embodiments, the CAR comprises two costimulatory domains.

[0252] In some embodiments, the one or more costimulatory domains comprise a CD28 costimulatory domain and a 4-1BB costimulatory domain.

[0253] In one embodiment, the CD28 costimulatory domain and the 4-1BB costimulatory domain are fused together.

[0254] In one embodiment, the costimulatory domain comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:60.

[0255] Exemplary CD28 costimulatory domain (SEQ ID NO: 60): RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0256] In some embodiments, the costimulatory domain may comprise a composite costimulatory domain comprising one or more costimulatory domains according to the present invention, or fragments thereof. As such, the domains may be fused in their entirety or smaller portions of the domains may be fused.

[0257] In one embodiment, the costimulatory domain is a composite costimulatory domain comprising fragments or entire costimulatory domains selected from any two or more of the following: CD28, CD137 (4-1BB), CD134 (OX40), DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-1, TNFR-II, Fas, CD30, and CD40.

[0258] Intracellular signaling domains The CARs of the present invention can also include an intracellular signaling domain, which can be cytoplasmic and transduce effector function signals to direct the cell to carry out its specialized function.

[0259] Examples of intracellular signaling domains include, but are not limited to, the zeta chain of the T cell receptor or any of its homologs (e.g., eta chain, FcεR1 gamma and beta chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (e.g., Syk, ZAP 70, etc.), src family tyrosine kinases (e.g., Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, e.g., CD2, CD5, and CD28. The intracellular signaling domain can be, for example, human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an immunoreceptor tyrosine-based activation motif (ITAM), or a combination thereof.

[0260] Additional intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternative embodiments of the invention.

[0261] In a preferred embodiment, the intracellular signaling domain is a CD3 zeta signaling domain.

[0262] In one embodiment, the intracellular signaling domain comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:13.

[0263] Exemplary CD3 zeta signaling domain (SEQ ID NO: 13): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0264] In one embodiment, the intracellular signaling domain is encoded by SEQ ID NO: 22, or a sequence having at least 75% sequence identity thereto.

[0265] Nucleotide sequence encoding an exemplary CD3 zeta signaling domain (SEQ ID NO:22): AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAG GCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCTAA

[0266] Transmembrane domain The CAR of the present invention can also comprise a transmembrane domain. The transmembrane domain can comprise a transmembrane sequence derived from any protein having a transmembrane domain, including type I, type II, or type III transmembrane proteins. The transmembrane domain of the CAR of the present invention can also comprise an artificial hydrophobic sequence. The transmembrane domain of the CAR of the present invention can be selected so as not to dimerize. Additional transmembrane domains will be apparent to those skilled in the art.

[0267] Examples of transmembrane (TM) regions used in CAR constructs include: 1) the CD28 TM region (Pule et al., Mol Ther, 2005, Nov;12(5):933-41; Brentjens et al., CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Casucci et al., Blood, 2013, Nov 14;122(20):3461-72.), 2) the OX40 TM region (Pule et al., Mol Ther, 2005, Nov;12(5):933-41), 3) the 41BB TM region (Brentjens et al., CCR, 2007, Sep 15;13(18 Pt 1):5426-35), and 4) the CD3 zeta TM region (Pule et al., Mol Ther, 2005, Nov;12(5):933-41). Ther,2005,Nov;12(5):933-41;Savoldo B,Blood,2009,Jun 18;113(25):6392-402.), 5)CD8a TM region(Maher et al,Nat Biotechnol,2002,Jan;20(1):70-5.,Imai C, Leukemia,2004,Apr;18(4):676-84, Brentjens et al,CCR,2007,Sep 15;13(18 Pt 1):5426-35, Milone et al,Mol Ther,2009,Aug;17(8):1453-64.).

[0268] In some embodiments, the transmembrane domain is a CD28 transmembrane domain.

[0269] In some embodiments, the transmembrane domain and the intracellular costimulatory domain can be derived from the same molecule.

[0270] In one embodiment, the transmembrane domain comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:61.

[0271] Exemplary CD28 Transmembrane Domain (SEQ ID NO: 61) FWVLVVVGGVLACYSLLVTVAFIIFWV

[0272] In one embodiment, the transmembrane and costimulatory domains comprise a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:12.

[0273] Exemplary CD28 Transmembrane and Costimulatory Domain (SEQ ID NO: 12) FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0274] In some embodiments, the transmembrane domain may comprise a composite transmembrane domain comprising one or more transmembrane domains according to the invention or fragments thereof. As such, the domains may be fused in their entirety or smaller portions of the domains may be fused.

[0275] In one embodiment, the transmembrane domain is a composite transmembrane domain comprising a transmembrane domain or a fragment thereof derived from any two or more of the transmembrane domains selected from the group consisting of: CD28 transmembrane domain, CD8a transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, and CD3 zeta transmembrane domain.

[0276] spacer domain The CAR of the present invention may comprise an extracellular spacer domain. The extracellular spacer domain may be linked to the antigen-specific targeting region and the transmembrane domain. The spacer domain may also be referred to as a hinge or linker.

[0277] In some embodiments, the spacer is a hinge spacer derived from IgG1.

[0278] In one embodiment, the spacer comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:10.

[0279] Exemplary IgG1 Hinge (SEQ ID NO: 10) EPKSCDKTHTCPPCP

[0280] In one embodiment, the spacer is encoded by SEQ ID NO: 19, or a sequence having at least 75% sequence identity thereto.

[0281] Nucleotide sequence encoding an exemplary IgG1 hinge (SEQ ID NO: 19): GAGCCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCCTGCCCC

[0282] In one embodiment, the spacer comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:57.

[0283] Exemplary IgG4 Hinge (SEQ ID NO: 57) ATSSGESKYGPPCPPCP

[0284] The CAR of the invention may comprise an extracellular spacer comprising at least a portion of the extracellular domain of human low affinity nerve growth factor receptor (LNGFR) or a derivative thereof.

[0285] LNGFR is not expressed in the majority of human hematopoietic cells, thus allowing quantitative analysis of transduced gene expression by immunofluorescence at single-cell resolution. Fluorescence-activated cell sorter analysis of LNGFR expression can therefore be performed in transduced cells to examine gene expression. Further details regarding analyses using LNGFR can be found in Mavilio (1994) Blood 83, 1988-1997.

[0286] In one embodiment, the CAR of the present invention comprises a truncated LNGFR (also known as ΔLNGFR). Preferably, the LNGFR used in the present invention is truncated in its cytoplasmic domain. Such truncations are described in Mavilio (1994) Blood 83, 1988-1997.

[0287] Therefore, preferably, the LNGFR spacer of the present invention comprises at least a portion of the extracellular domain of LNGFR or a derivative thereof, but lacks its intracellular domain. The extracellular domain may comprise amino acids 29 to 250 of LNGFR or a derivative thereof.

[0288] Exemplary human LNGFR [UNIPROT Accession P08138, TNR16_HUMAN] (SEQ ID NO: 31): MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQ EPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWNSCKQNKQGANSRPVNQTPPPEGEKLHSDSGISVDSQSLHDQQPH TQTASGQALKGDGGLYSSLPPAKREEVEKLLNGSAGDTWRHLAGELGYQPEHIDSFTHEACPVRALLASWATQDSATLDALLAALRRIQRADLVESLCSESTATSPV

[0289] An exemplary extracellular domain of human LNGFR [UNIPROT accession P08138, TNR16_HUMAN, positions 29-250] (SEQ ID NO: 27) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQ DKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN

[0290] Preferably, the LNGFR lacks a signal peptide.

[0291] SEQ ID NO: 27 may also be referred to as LNGFR wild-type long spacer (NWL).

[0292] In one embodiment, the spacer comprises at least a portion of a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the extracellular domain of LNGFR (e.g., SEQ ID NO: 27). In one embodiment, the spacer comprises at least a portion of a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 29-250 of LNGFR (e.g., SEQ ID NO: 31).

[0293] In one embodiment, the spacer comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27. In one embodiment, the spacer comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 29-250 of SEQ ID NO: 31.

[0294] In one embodiment, the spacer is encoded by SEQ ID NO: 29, or a sequence having at least 75% sequence identity thereto.

[0295] Nucleotide sequence encoding an exemplary LNGFR spacer (NWL) (SEQ ID NO:29): AAAGAGGCCTGCCCCACCGGCCTGTACACCCACAGCGGAGAGTGCTGCAAGGCCTGCAACCTGGGAGAGGGCGTGGCCCAGCCTTGCGGCGCCAATCAGACCGTGTGCGAGCCCTGCCTGGACAGCGTGACCTTCAGCGACGTGGTGTCCGCCACCGAGCCCTGCA AGCCTTGCACCGAGTGTGTGGGCCTGCAGAGCATGAGCCCCCTGCGTGGAAGCCGACGACGCCGTGTGTAGATGCGCCTACGGCTACTACCAGGACGAGACAACCGGCAGATGCGAGGCCTGTAGAGTGTGCGAGGCCGGCAGCGGCCTGGTGTTCAGTTGTCAA GACAAGCAGAATACCGTGTGTGAAGAGTGCCCCGACGGCACCTACAGCGACGAGGCCAACCACGTGGACCCCTGCCTGCCCTGCACTGTGTGCGAGGACACCGAGCGGCAGCTGCGCGAGTGCACAAGATGGGCCGACGCCGAGTGCGAAGAGATCCCCGGCAGAT GGATCACCAGAAGCACCCCCCCTGAGGGCAGCGACAGCACCGCCCCTAGCACCCAGGAACCTGAGGCCCCTCCCGAGCAGGACCTGATCGCCTCTACAGTGGCCGGCGTGGTGACAACCGTGATGGGCAGCTCTCAGCCCGTGGTGACACGGGGCACCACCGACAAT

[0296] In one embodiment, the spacer comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:11.

[0297] Exemplary LNGFR spacer (SEQ ID NO: 11) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEAARAADAECEE

[0298] SEQ ID NO: 11 may also be referred to as LNGFR Mutant Short Spacer (NMS) In one embodiment, the spacer is encoded by SEQ ID NO: 20, or a sequence having at least 75% sequence identity thereto.

[0299] Nucleotide sequence encoding an exemplary LNGFR spacer (SEQ ID NO:20): AAAGAGGCCTGCCCCACCGGCCTGTACACCCACAGCGGAGAGTGCTGCAAGGCCTGCAACCTGGGAGAGGGCGTGGCCCAGCCTTGCGGCGCCAATCAGACCGTGTGCGAGCCCTGCCTGGACAGCGTGACCTTCAGCGACGTGGTGTCCGCCACCGAGCCCTGCAAGCCTTGCACCGAGTGTGTGGGCCTGCAGAGCATGAGCGCCCCC TGCGTGGAAGCCGACGACGCCGTGTTAGATGCGCCTACGGCTACTACCAGGACGAGACAACCGGCAGATGCGAGGCCTGTAGAGTGTGCGAGGCCGGCAGCGGCCTGGTGTTCAGTTGTCAGGACAAGCAGAACACCGTGTGTGAAGAGTGCCCCGACGGCACCTACAGCGACGAGGGCCGCCCGGGCCGCCGACGCCGAGTGCGAGGAA

[0300] Further exemplary spacers are shown below.

[0301] Exemplary LNGFR Spacer (LNGFR Wild-Type Short (NWS)) (SEQ ID NO: 32) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEE

[0302] Exemplary LNGFR Spacer (LNGFR Mutant Long (NML)) (SEQ ID NO: 33) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEAARAADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN

[0303] In one embodiment, the spacer comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 11, 31, or 32.

[0304] LNGFR contains four TNFR-Cys domains (TNFR-Cys1, TNFR-Cys2, TNFR-Cys3, and TNFR-Cys4). The sequences of the domains are illustrated below: TNFR-Cys1 (SEQ ID NO: 34) ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC TNFR-Cys2 (SEQ ID NO: 35) PCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPPCVEADDAVC TNFR-Cys3 (SEQ ID NO: 36) RCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVC TNFR-Cys4 (SEQ ID NO: 37) ECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAEC

[0305] In one embodiment, the spacer comprises TNFR-Cys 1, 2, and 3 domains or fragments or derivatives thereof. In another embodiment, the spacer comprises TNFR-Cys 1, 2, 3, and 4 domains or fragments or derivatives thereof.

[0306] In one embodiment, the spacer comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TNFR-Cys1 (SEQ ID NO: 34), a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TNFR-Cys2 (SEQ ID NO: 35), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TNFR-Cys3 (SEQ ID NO: 36). The spacer may further comprise a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TNFR-Cys4 (SEQ ID NO: 37).

[0307] Rather than comprising the entire TNFR-Cys4 domain, the spacer may comprise the TNFR-Cys4 domain with the following amino acids deleted from that domain: NHVDPCLPCTVCEDTERQLRECTRW (SEQ ID NO: 38).

[0308] In one embodiment, the NHVDPCLPCTVCEDTERQLRECTRW (SEQ ID NO: 38) amino acids are substituted with the following amino acids: ARA.

[0309] In one embodiment, the spacer lacks the LNGFR serine / threonine-rich stalk, hi another embodiment, the spacer comprises the LNGFR serine / threonine-rich stalk.

[0310] The spacer may comprise or consist of the sequence of SEQ ID NO:34 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:34.

[0311] The spacer may comprise or consist of the sequence of SEQ ID NO:35 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:35.

[0312] The spacer may comprise or consist of the sequence of SEQ ID NO:36 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:36.

[0313] The spacer may comprise or consist of the sequence of SEQ ID NO:37 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:37.

[0314] The spacer may comprise a mutated version of the IgG1 CH2CH3 spacer (mCH2CH3) that is unable to recognize FcγRI (Hombach et al., Gene Ther. 2000).

[0315] In one embodiment, the spacer comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:28.

[0316] Exemplary mCH2CH3 spacer (SEQ ID NO: 28): EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0317] In one embodiment, the spacer is encoded by SEQ ID NO: 30, or a sequence having at least 75% sequence identity thereto.

[0318] Nucleotide sequence encoding an exemplary mCH2CH3 spacer (SEQ ID NO:30): GAGCCCAAGAGCCCCGACAAGACCCACACCTGTCCCCCCTGTCCTGCCCCTCCAGTGGCCGGACCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCGCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAA GTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCTGCCCCCA TCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACACTGCCCCCAGCCGGGACGAGCTGACCAAGAACCAGGTGTCCTGACCTGCCTCGTGAAAGGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAAC AACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG

[0319] The spacer can confer properties to the CAR that allow for immune selection of cells, preferably T cells, that express the CAR.

[0320] The CARs of the invention (e.g., including a spacer as referred to herein) preferably allow T cells expressing the CAR to proliferate in the presence of cells expressing the antigen for which the CAR is designed.

[0321] The CARs of the invention (e.g., comprising a spacer as referred to herein) preferably enable T cells expressing the CAR to mediate therapeutically important anti-cancer effects against the cancer that the CAR is designed to target.

[0322] The CARs of the present invention (e.g., comprising a spacer as referred to herein) are preferably suitable for facilitating immune selection of cells transduced with the CAR.

[0323] Exemplary CARs of the invention comprising an LNGFR-based spacer may avoid the activation of unwanted and potentially toxic off-target immune responses and may allow CAR-expressing T cells to persist in vivo without being prematurely eliminated by the host immune system.

[0324] As described herein, the present invention also encompasses the use of variants, derivatives, homologues and fragments of the spacer elements described herein.

[0325] Exemplary CAR In one embodiment, the CAR is an anti-CDH17 CAR In one embodiment, the CAR is a) CD28, CD8, or CD4 transmembrane domain; b) an IgG1 hinge, an LNGFR spacer, or an mCH2CH3 spacer; c) CD28 and / or 4-1BB costimulatory domains; d) the CD3 zeta signaling domain, and / or e) Contains anti-CDH17 scFv.

[0326] In one embodiment, the CAR comprises an antigen binding domain comprising an anti-CDH 17 scFv, an IgG1 hinge, a CD28 transmembrane and costimulatory domain, and a CD3 zeta signaling domain.

[0327] In one embodiment, the CAR comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:14.

[0328] An exemplary CAR[A4_4R-hinge.28z] (SEQ ID NO: 14): MEAPAQLLFLLLLWLPDTTGDIVMTQSPDSLAVSLGERATINCRSSQSLLHSSNQRNYLAWYQQKPGQPPKVLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYY SYPWTFGQGTKVEIKGGGGSGGGGSGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTLTDHTIHWMRQAPGQRLEWIGYIYPRDGITGYNERFRGKATLTADTSASTAYMELSSLRS EDTAVYYCARWGYSYRNYAYYYDYWGQGTLVTVSSSPVEPKSCDKTHTCPPCPPLIKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0329] In one embodiment, the CAR comprises a protein encoded by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17.

[0330] Nucleotide sequence encoding an exemplary CAR[A4_4R-hinge.28z] (SEQ ID NO: 17):

[0331] In one embodiment, the CAR comprises an antigen-binding domain comprising an anti-CDH17 scFv, a LNGFR mutated short spacer (NMS), a CD28 transmembrane and costimulatory domain, and a CD3 zeta signaling domain.

[0332] In one embodiment, the CAR comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:15.

[0333] An exemplary CAR [A4_4R-NMS.28z] (SEQ ID NO: 15): MEAPAQLLFLLLLWLPDTTGDIVMTQSPDSLAVSLGERATINCRSSQSLLHSSNQRNYLAWYQQKPGQPPKVLIYWASTRESGVPDRFSGSGSGTFTLTISSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGSQ VQLVQSGAEVKKPGASVKVSCKASGYTLTDHTIHWMRQAPGQRLEWIGYIYPRDGITGYNERFRGKATLTADTSASTAYMELSSLRSEDTAVYYCARWGYSYRNYAYYYDYWGQGTLVTVSSSPVKEACPTGLYTHSGECCKACNLGEGV AQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEAARAADAECEEPLIKFWVLVVVGGVLACYSLLVTVAFIIFWVRSK RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0334] In one embodiment, the CAR comprises a protein encoded by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18.

[0335] Nucleotide sequence encoding an exemplary CAR [A4_4R-NMS.28z] (SEQ ID NO: 18):

[0336] In one embodiment, the CAR comprises an antigen-binding domain comprising an anti-CDH17 scFv, an LNGFR wild-type long spacer (NWL), a CD28 transmembrane and costimulatory domain, and a CD3 zeta signaling domain.

[0337] In one embodiment, the CAR comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:23.

[0338] Exemplary CAR[A4_4R-NWL.28z] (SEQ ID NO: 23): MEAPAQLLFLLLLWLPDTTGDIVMTQSPDSLAVSLGERATINCRSSQSLLHSSNQRNYLAWYQQKPGQPPKVLIYWASTRESGVPDRFSGSGSGTFTLTISSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKKKPGASVKVSC KASGYTLTDHTIHWMRQAPGQRLEWIGYIYPRDGITGYNERFRGKATLTADTSASTAYMELSSLRSEDTAVYYCARWGYSYRNYAYYYDYWGQGTLVTVSSSPVKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQS MSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNPLIKFWVLVVVGGV LACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0339] In one embodiment, the CAR comprises a protein encoded by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:25.

[0340] Nucleotide sequence encoding an exemplary CAR [A4_4R-NWL.28z] (SEQ ID NO: 25):

[0341] In one embodiment, the CAR comprises an antigen binding domain comprising an anti-CDH17 scFv, a mCH2CH3 spacer, a CD28 transmembrane and costimulatory domain, and a CD3 zeta signaling domain.

[0342] In one embodiment, the CAR comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:24.

[0343] An exemplary CAR[A4_4R-CH2CH3mut.28z] (SEQ ID NO: 24): MEAPAQLLFLLLLWLPDTTGDIVMTQSPDSLAVSLGERATINCRSSQSLLHSSNQRNYLAWYQQKPGQPPKVLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKA SGYTLTDHTIHWMRQAPGQRLEWIGYIYPRDGITGYNERFRGKATLTADTSASTAYMELSSLRSEDTAVYYCARWGYSYRNYAYYYDYWGQGTLVTVSSSPVEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPLIKFWVLVVV GGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0344] In one embodiment, the CAR comprises a protein encoded by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:26.

[0345] Nucleotide sequence encoding an exemplary CAR [A4_4R-CH2CH3mut.28z] (SEQ ID NO: 26):

[0346] In one embodiment, the CAR comprises an antigen binding domain comprising an anti-CDH17 scFv, an IgG4 hinge, a CD28 transmembrane and costimulatory domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

[0347] In one embodiment, the CAR comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:47.

[0348] Exemplary CAR [A4_4R-IgG4h.28BBz] (SEQ ID NO: 47): MEAPAQLLFLLLLWLPDTTGDIVMTQSPDSLAVSLGERATINCRSSQSLLHSSNQRNYLAWYQQKPGQPPKVLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPWTFGQGTK VEIKGGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTLTDHTIHWMRQAPGQRLEWIGYIYPRDGITGYNERFRGKATLTADTSASTAYMELSSLRSEDTAVYYCARWGYSYRNYAYYY DYWGQGTLVTVSSAAATSSGESKYGPPCPPCPDIFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0349] In one embodiment, the CAR comprises a protein encoded by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:51.

[0350] Nucleotide sequence encoding an exemplary CAR [A4_4R-IgG4h.28BBz] (SEQ ID NO:51):

[0351] In one embodiment, the CAR comprises a protein encoded by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:85.

[0352] Additional nucleotide sequence encoding exemplary CAR [A4_4R-IgG4h.28BBz] (SEQ ID NO:85):

[0353] In one embodiment, the CAR comprises an antigen-binding domain comprising an anti-CDH17 scFv, a LNGFR mutated short (NMS) spacer, a CD28 transmembrane and costimulatory domain, and a CD3 zeta signaling domain.

[0354] In one embodiment, the CAR comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:48.

[0355] An exemplary CAR [Lic3-NMS.28z] (SEQ ID NO: 48): MEAPAQLLFLLLLWLPDTTGQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVASISFDGTYTYYTDRVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRPAWFPYWGQGTLVTVSAGGGGGSGGGGSG GGGSGDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGAGTKLELKGAPSPVKEACPTGLYTHSGECCKACNLGEGVA QPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEAARAADAECEEPLIKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0356] In one embodiment, the CAR comprises a protein encoded by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:52.

[0357] Nucleotide sequence encoding an exemplary CAR [Lic3-NMS.28z] (SEQ ID NO:52):

[0358] In one embodiment, the CAR comprises an antigen-binding domain comprising an anti-CDH17 scFv, a LNGFR wild-type long (NWL) spacer, a CD28 transmembrane and costimulatory domain, and a CD3 zeta signaling domain.

[0359] In one embodiment, the CAR comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:49.

[0360] An exemplary CAR [Lic3-NWL.28z] (SEQ ID NO: 49): MEAPAQLLFLLLLWLPDTTGQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVASISFDGTYTYYTDRVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRPAWFPYWGQGTLVTVSAGGGGSGGGGSGGGGSGDIVMTQTPLSLSVTPG QPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGAGTKLELKGAPSPVKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSM SAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNPLIKFWVLVVVGGV LACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0361] In one embodiment, the CAR comprises a protein encoded by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:53.

[0362] Nucleotide sequence encoding an exemplary CAR [Lic3-NWL.28z] (SEQ ID NO:53):

[0363] In one embodiment, the CAR comprises an antigen binding domain comprising an anti-CDH 17 scFv, an IgG1 hinge, a CD28 transmembrane and costimulatory domain, and a CD3 zeta signaling domain.

[0364] In one embodiment, the CAR comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:50.

[0365] An exemplary CAR[Lic3-hinge.28z] (SEQ ID NO: 50): MEAPAQLLFLLLLWLPDTTGQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVASISFDGTYTYYTDRVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RDRPAWFPYWGQGTLVTVSAGGGGSGGGGSGGGGSGDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR VEAEDVGVYYCFQGSHVPLTFGAGTKLELKGAPSPVEPKSCDKTHTCPPCPPLIKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0366] In one embodiment, the CAR comprises a protein encoded by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:54.

[0367] Nucleotide sequence encoding an exemplary CAR[Lic3-hinge.28z] (SEQ ID NO:54):

[0368] In one embodiment, the CAR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9, 14, 15, 23, 24, 45, or 47-50.

[0369] In one embodiment, the CAR consists of a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14, 15, 23, 24, or 47-50.

[0370] In one embodiment, the CAR comprises the sequence of any one of SEQ ID NOs: 9, 14, 15, 23, 24, 45, or 47-50.

[0371] In one embodiment, the CAR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9, 14, 15, 23, 24, 45, or 47-50, and the sequence comprises any one or more of the sequences according to SEQ ID NOs: 1-6 or 39-44.

[0372] In one embodiment, the CAR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9, 14, 15, 23, 24, 45, or 47-50, including a sequence according to SEQ ID NOs: 1-6 or 39-44.

[0373] In one embodiment, the CAR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 45 or 47-50.

[0374] In one embodiment, the CAR consists of a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 47-50.

[0375] In one embodiment, the CAR comprises the sequence of any one of SEQ ID NOs: 45 or 47-50.

[0376] In one embodiment, the CAR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 45 or 47-50, and the sequence comprises any one or more of the sequences according to SEQ ID NOs: 39-44.

[0377] In one embodiment, the CAR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 45 or 47-50, which sequence comprises a sequence according to SEQ ID NOs: 39-44.

[0378] Tags and Linkers The polynucleotide and polypeptide sequences of the present invention may further comprise tag and / or linker sequences.

[0379] Linker At both the polynucleotide and polypeptide levels, specific functional sequence elements can be separated by linkers, which typically comprise short polynucleotide or polypeptide sequences. Linkers can be used to physically separate functional sequences, for example, to improve the functionality of the sequences.

[0380] In one embodiment, a polynucleotide of the invention comprises one or more linker sequences.

[0381] In one embodiment, a sequence encoding a functional domain of a CAR is separated from another sequence encoding a functional domain by a linker.

[0382] In one embodiment, a CAR of the invention comprises one or more linker sequences.

[0383] In one embodiment, the linker is a GS linker or a GGS linker.

[0384] The GGS linker may be composed of GGS repeats, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 GGS repeats. The GGS linker may comprise more than 10 repeats of the amino acid sequence GGS, for example more than 20, more than 30, or more than 40 repeats.

[0385] The GGS linker may contain variations of the GGS sequence, such as GGGS (SEQ ID NO: 62), GGGGS (SEQ ID NO: 63). Furthermore, the GGS motif and its variants may also contain imperfect motifs, such as GS.

[0386] Exemplary GGS repeat-containing linker sequences are exemplified as SEQ ID NOs: 64 and 65. GGGGSGGGGSGGGGSG (SEQ ID NO: 64) GGGGSGGGGSGGGGS (SEQ ID NO: 65)

[0387] A GGS linker, for example, one comprising or consisting of any one of SEQ ID NOs: 62 to 65, can be placed between other functional sequences, such as VH and VL sequences in an scFV, an antigen-specific binding domain, or a CAR.

[0388] tag Both polynucleotides and polypeptides of the invention can contain tags. Tags are typically sequences that facilitate detection or isolation of the molecule to which they are attached. Tags can be particularly useful in experimental tests that utilize the polynucleotides or polypeptides of the invention.

[0389] In one embodiment, a polynucleotide of the invention comprises one or more tag sequences.

[0390] In one embodiment, the CAR or scFv of the invention comprises one or more tag sequences.

[0391] Further polypeptides or polynucleotides of interest In addition to the CAR of the present invention, additional polypeptides or polynucleotides of interest can be provided, which can be provided together with the CAR or the polynucleotide encoding it, for example, in the same molecule or vector, or in different molecules or vectors.

[0392] In some embodiments, the polynucleotide of interest encodes a polypeptide of interest.

[0393] In one aspect, the invention provides an article of manufacture (e.g., a composition or kit) comprising a CAR of the invention and a polypeptide of interest.

[0394] In some embodiments, a polynucleotide of the present invention further comprises a nucleotide sequence encoding a polypeptide of interest.

[0395] In some embodiments, the cells of the invention further comprise a polypeptide of interest or a polynucleotide encoding same.

[0396] The polypeptide or polynucleotide of interest can be used for a variety of purposes, for example, to select successfully transduced cells, to increase the efficacy of CAR-containing cells, or to provide a mechanism for CAR cell inactivation.

[0397] immunomodulatory molecules The polypeptide of interest can be an immunomodulatory molecule. Such a molecule can, for example, stimulate an immune response and increase the effectiveness of cells comprising a CAR according to the present invention.

[0398] In one aspect, a polynucleotide is provided that comprises: (a) one or more nucleotide sequences encoding a chimeric antigen receptor (CAR) of the present invention; and (b) a nucleotide sequence encoding an immunomodulatory molecule (e.g., a cytokine).

[0399] In one aspect, an article of manufacture is provided comprising: (a) a first polynucleotide comprising one or more nucleotide sequences encoding a chimeric antigen receptor (CAR) of the present invention; and (b) a second polynucleotide comprising a nucleotide sequence encoding an immunomodulatory molecule (e.g., a cytokine). The article of manufacture can be, for example, a kit or a composition. In one aspect, a kit is provided comprising: (a) a first polynucleotide comprising one or more nucleotide sequences encoding a chimeric antigen receptor (CAR) of the present invention; and (b) a second polynucleotide comprising a nucleotide sequence encoding an immunomodulatory molecule (e.g., a cytokine). In another aspect, a composition is provided comprising: (a) a first polynucleotide comprising one or more nucleotide sequences encoding a chimeric antigen receptor (CAR) of the present invention; and (b) a second polynucleotide comprising a nucleotide sequence encoding an immunomodulatory molecule (e.g., a cytokine).

[0400] In one embodiment, the polypeptide of interest is a cytokine.

[0401] In one embodiment, the cytokine is IL-12.

[0402] Selectable markers and suicide genes Additional genes or polynucleotides may be provided together with the CAR and scFv of the invention.

[0403] Genes can also be provided that allow for the selection of engineered cells, such as cells containing a CAR according to the invention, for example, in vitro or in vivo.

[0404] In one aspect, a polynucleotide is provided comprising: (a) one or more nucleotide sequences encoding a chimeric antigen receptor (CAR) of the invention; and (b) a nucleotide sequence encoding a selectable marker or suicide gene.

[0405] In one aspect, an article of manufacture is provided comprising: (a) a first polynucleotide comprising one or more nucleotide sequences encoding a chimeric antigen receptor (CAR) of the present invention; and (b) a second polynucleotide comprising a nucleotide sequence encoding a selectable marker or a suicide gene. The article of manufacture can be, for example, a kit or a composition. In one aspect, a kit is provided comprising: (a) a first polynucleotide comprising one or more nucleotide sequences encoding a chimeric antigen receptor (CAR) of the present invention; and (b) a second polynucleotide comprising a nucleotide sequence encoding a selectable marker or a suicide gene. In another aspect, a composition is provided comprising: (a) a first polynucleotide comprising one or more nucleotide sequences encoding a chimeric antigen receptor (CAR) of the present invention; and (b) a second polynucleotide comprising a nucleotide sequence encoding a selectable marker or a suicide gene.

[0406] In one embodiment, the polypeptide of interest is a selectable marker.

[0407] In one embodiment, the polynucleotide of interest encodes a selectable marker.

[0408] Human cell surface proteins, such as ΔNGFR, CD34, CD19, CD20, and CD4, and CD90, have been used as surrogate markers for the identification of ex vivo genetically modified cells. In one embodiment, the polypeptide of interest is any one or more of ΔNGFR, CD34, CD19, CD20, and CD4, and CD90.

[0409] In one embodiment, the selectable marker is thymidine kinase (TK), or a modified version thereof.

[0410] An example of a TK amino acid sequence is (SEQ ID NO: 80, TK mut2): MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHVGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVL AFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWWEDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN

[0411] An example of a nucleotide sequence encoding TK is (SEQ ID NO:81):

[0412] An example of a nucleotide sequence encoding TK is (SEQ ID NO:82):

[0413] In some embodiments, TK comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 80. In some embodiments, TK consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 80.

[0414] In some embodiments, TK is encoded by a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 81 or 82.

[0415] In one embodiment, the polypeptide of interest is a functionally inactive truncated version of human epidermal growth factor receptor (EGFRt) or an enhanced version of EGFRt (eEGFRt), as described in WO 2021 / 229075.

[0416] An example of an EGFRt amino acid sequence is (SEQ ID NO: 55): RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCK ATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0417] An example of a nucleotide sequence encoding EGFRt is (SEQ ID NO: 56):

[0418] In some embodiments, the EGFRt comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 55. In some embodiments, the EGFRt consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 55. Preferably, the EGFRt comprises an epitope recognizable by an antibody, such as cetuximab. Preferably, the EGFRt lacks signaling or transport activity.

[0419] In one embodiment, the selectable marker is human epidermal growth factor receptor or a modified version thereof.

[0420] In one embodiment, the selectable marker is a truncated human epidermal growth factor receptor (EGFRt), or a modified version thereof.

[0421] In one embodiment, the selectable marker is a truncated human epidermal growth factor receptor (EGFRt) encoded by SEQ ID NO: 56, or a sequence having at least 75% sequence identity thereto.

[0422] In one embodiment, the selectable marker is enhanced truncated human epidermal growth factor receptor (eEGFRt), or a modified version thereof.

[0423] In one embodiment, the selectable marker is CD20.

[0424] In one embodiment, the polynucleotide or vector comprises one or more promoters operably linked to the nucleotide sequence encoding CD20.

[0425] An example of a CD20 sequence is (SEQ ID NO: 70): MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLK MESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP

[0426] In some embodiments, CD20 comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 70. In some embodiments, CD20 consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 70.

[0427] In one embodiment, the selectable marker is NGFR.

[0428] Suicide genes act as a means to selectively induce the elimination of cells that contain them, to provide a safe mechanism to mitigate potential adverse effects of adoptive cell therapy, such as severe autoimmune disease or graft-versus-host disease.

[0429] In one embodiment, the polynucleotide of interest is a suicide gene.

[0430] Advantageously, the polynucleotide of interest can act as both a selectable marker and a suicide gene.

[0431] In a preferred embodiment, the polynucleotide of interest acts as both a suicide gene and a selectable marker.

[0432] In one embodiment, the suicide gene is CD20.

[0433] In one embodiment, the suicide gene is thymidine kinase (TK), or a modified version thereof.

[0434] In one embodiment, the suicide gene is human epidermal growth factor receptor, or a modified version thereof.

[0435] In one embodiment, the suicide gene is a truncated human epidermal growth factor receptor (EGFRt) or an enhanced truncated human epidermal growth factor receptor (EGFRt).

[0436] In one embodiment, the suicide gene is a caspase.

[0437] An example of a caspase amino acid sequence is (SEQ ID NO: 83): MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLE LAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSASRA

[0438] An example of a nucleotide sequence encoding a caspase is (SEQ ID NO: 84):

[0439] In some embodiments, the caspase comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 83. In some embodiments, the caspase consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 83.

[0440] In some embodiments, the caspase is encoded by a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84.

[0441] In one embodiment, the polynucleotide or vector comprises the minimal core promoter from cytomegalovirus (minCMV) operably linked to a nucleotide sequence encoding CD20.

[0442] In one embodiment, the nucleotide sequence encoding CD20 is encoded in an antisense orientation relative to the nucleotide sequence encoding the CAR.

[0443] In one embodiment, the polynucleotide or vector comprises a nucleotide sequence encoding a CAR and a nucleotide sequence encoding CD20.

[0444] In one embodiment, the polynucleotide or vector comprises a human phosphoglycerate kinase (PGK) promoter operably linked to a nucleotide sequence encoding a CAR, and a minimal core promoter from cytomegalovirus (minCMV) operably linked to a nucleotide sequence encoding CD20.

[0445] In one embodiment, CD20 and CAR are encoded in opposite orientations within a polynucleotide or vector.

[0446] It will be understood that, where appropriate, any of the embodiments described for a selectable marker may also be applied when the selectable marker is also functioning as a suicide gene, and vice versa.

[0447] glycosidases Additional polypeptides or polynucleotides may be provided together with the CAR and scFv of the invention. In one embodiment, the polypeptide of interest is a glycosidase.

[0448] In one aspect, a polynucleotide is provided that comprises: (a) one or more nucleotide sequences encoding a chimeric antigen receptor (CAR) of the invention; and (b) a nucleotide sequence encoding a glycosidase.

[0449] In one aspect, an article of manufacture is provided comprising (a) a first polynucleotide comprising one or more nucleotide sequences encoding a chimeric antigen receptor (CAR) of the present invention, and (b) a second polynucleotide comprising a nucleotide sequence encoding a glycosidase. The article of manufacture can be, for example, a kit or composition. In one aspect, a kit is provided comprising (a) a first polynucleotide comprising one or more nucleotide sequences encoding a chimeric antigen receptor (CAR) of the present invention, and (b) a second polynucleotide comprising a nucleotide sequence encoding a glycosidase. In another aspect, a composition is provided comprising (a) a first polynucleotide comprising one or more nucleotide sequences encoding a chimeric antigen receptor (CAR) of the present invention, and (b) a second polynucleotide comprising a nucleotide sequence encoding a glycosidase.

[0450] Glycosidases (or glycoside hydrolases / glycosyl hydrolases) are enzymes that catalyze the hydrolysis of glycosidic bonds in sugars. Glycosidases that act on carbohydrates that are attached to proteins can be referred to herein as deglycosylating enzymes, as they catalyze the removal of the glycan or sugar moiety, for example, as an N- or O-linked glycan.

[0451] Removal of glycans or sugars from extracellular proteins, or degradation of extracellular glycans, can facilitate target recognition by CARs, thereby enhancing their activity.

[0452] Glycosidases that are functional in the extracellular environment can catalyze the removal of glycans (or "act on" glycans) from a variety of substrates. In a preferred embodiment, the glycosidase is a secreted glycosidase. In another embodiment, the glycosidase is active in the extracellular environment.

[0453] In one embodiment, the glycosidase has a substrate that is an N-linked glycan. In one embodiment, the glycosidase deglycosylates the N-linked glycan. In one embodiment, the glycosidase is an N-glycanase.

[0454] In one embodiment, the glycosidase has a substrate that is an O-linked glycan. In one embodiment, the glycosidase deglycosylates the O-linked glycan. In one embodiment, the glycosidase is an O-glycanase.

[0455] In one embodiment, the glycosidase catalyzes the removal of one or more sugar moieties.

[0456] In one embodiment, the glycosidase catalyzes the removal of the entire glycan chain.

[0457] As used herein, "glycan" can refer to the entire glycan structure or to fragments of the structure, such as individual sugars (monosaccharides, disaccharides, polysaccharides).

[0458] An exemplary glycosidase is peptide-N(4)-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase), such as human PNGase and modified versions thereof.

[0459] In one embodiment, the glycosidase is PNGase.

[0460] In one embodiment, the glycosidase is human PNGase.

[0461] Exemplary human PNGase [Uniprot(Q96IV0-1)] (SEQ ID NO: 66): AAAALGSSSGSASPAVAELCQNTPETFLEASKLLLTYADNILRNPNDEKYRSIRIGNTAFSTRLLPVRGAVECLFEMGFEEGETHLIFPKKASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSAST VAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANCFTLCCRAVGFEA RYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPGELGGRISGSVAWRVARGEMGLQRKETLFIPCENEKIS KQLHLCYNIVKDRYVRVSNNNQTISGWENGVWKMESIFRKVETDWHMVYLARKEGSSFAYISWKFECGSVGLKVDSISIRTSSQTFQTGTVEWKLRSDTAQVELTGDNSLHSYADFSGATEVILEAELSRGDGDVAWQHTQLFRQSLNDHEENCLEIIIKFSDL

[0462] In one embodiment, the glycosidase is a variant of human PNGase. In one embodiment, the glycosidase comprises or consists of a sequence having at least 70% sequence identity to SEQ ID NO: 66, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity, or a fragment thereof.

[0463] In one embodiment, the PNGase variant is a truncated PNGase.

[0464] In one embodiment, PNGase lacks the PUB domain. In another embodiment, PNGase lacks the PAW domain. In a further embodiment, PNGase lacks both the PUB and PAW domains.

[0465] An exemplary PUB-cleaving human PNGase (SEQ ID NO: 67): KASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLL ELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANCFTLCCRAVGFEARYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPLLYEIGWGKK LSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPGELGGRISGSVAWRVARGEMGLQRKETLFIPCENEKISKQLHLCYNIVKDRYVRVSNNNNQT ISGWENGVWKMESIFRKVETDWHMVYLARKEGSSFAYISWKFECGSVGLKVDSISIRTSSQTFQTGTVEWKLRSDTAQVELTGDNSLHSYADFSGATEVILEAELSRGDGDVAWQHTQLFRQSLNDHEENCLEIIIKFSDL

[0466] An exemplary PUB and PAW cleaved human PNGase (SEQ ID NO: 68): KASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLPSDDELK WGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANCFTLCCRAVGFEARYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPG

[0467] Exemplary PAW-cleaved human PNGase (SEQ ID NO: 69): AAAALGSSSGSASPAVAELCQNTPETFLEASKLLLTYADNILRNPNDEKYRSIRIGNTAFSTRLLPVRGAVECLFEMGFEEGETHLIFPKKASVEQLQKIRDLIAIERSSRLD GSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDED FLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANCFTLCCRAVGFEARYVWDYTDHVWTE VYSPSQQRWLHCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPG

[0468] In one embodiment, the glycosidase is (a) a sequence having at least 70% sequence identity to SEQ ID NO: 66, or a fragment thereof; (b) a sequence having at least 70% sequence identity to SEQ ID NO: 67, or a fragment thereof; (c) a sequence having at least 70% sequence identity to SEQ ID NO: 68, or a fragment thereof; or (d) comprising or consisting of a sequence having at least 70% sequence identity to SEQ ID NO: 69, or a fragment thereof.

[0469] In one embodiment, the glycosidase comprises or consists of a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 66-69, e.g., at least 75%, at least 80%, at least 85%, 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%, or 100% sequence identity, or a fragment thereof.

[0470] The glycosidase may include a signal peptide to facilitate its secretion. In one embodiment, the glycosidase includes a signal peptide. The signal peptide may be cleaved from the glycosidase during its export from the cell.

[0471] In a preferred embodiment, the nucleotide sequence encoding the glycosidase further encodes a signal peptide operably linked to the glycosidase.

[0472] In one embodiment, the signal peptide is selected from the group consisting of a CD8 signal peptide, an IgG variable region heavy chain signal peptide, an Ig kappa chain V-III region VG signal peptide, a GM-CFS / CSF signal peptide, and a CSFR2A signal peptide.

[0473] In one embodiment, the signal peptide is the CD8 signal peptide.

[0474] In one embodiment, the signal peptide is an IgG variable region heavy chain signal peptide.

[0475] An exemplary CD8 signal peptide [residues 1-12, Uniprot accession P01732] (SEQ ID NO: 71) is: MALPVTALLLPLALLLHAARP

[0476] In one embodiment, the signal peptide comprises or consists of a sequence having at least 70% sequence identity to SEQ ID NO: 71, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or a fragment thereof. In one embodiment, the signal peptide comprises SEQ ID NO: 71. In one embodiment, the signal peptide consists of SEQ ID NO: 71.

[0477] An exemplary IgG variable region heavy chain signal peptide [residues 1-19, Uniprot accession P01768] (SEQ ID NO: 72) is: MEFGLSWVFLVALLRGVQC

[0478] In one embodiment, the signal peptide comprises or consists of a sequence having at least 70% sequence identity to SEQ ID NO: 72, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or a fragment thereof. In one embodiment, the signal peptide comprises SEQ ID NO: 72. In one embodiment, the signal peptide consists of SEQ ID NO: 72.

[0479] An exemplary GM-CFS / CSF signal peptide [residues 1-17, Uniprot accession P04141] (SEQ ID NO: 73) is: MWLQSLLLLGTVACSIS

[0480] In one embodiment, the signal peptide comprises or consists of a sequence having at least 70% sequence identity to SEQ ID NO: 73, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or a fragment thereof. In one embodiment, the signal peptide comprises SEQ ID NO: 73. In one embodiment, the signal peptide consists of SEQ ID NO: 73.

[0481] An exemplary Ig kappa chain V-III region VG signal peptide [residues 1-20, Uniprot accession P04433] (SEQ ID NO: 74) is: MEAPAQLLFLLLLWLPDTTG

[0482] In one embodiment, the signal peptide comprises or consists of a sequence having at least 70% sequence identity to SEQ ID NO: 74, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or a fragment thereof. In one embodiment, the signal peptide comprises SEQ ID NO: 74. In one embodiment, the signal peptide consists of SEQ ID NO: 74.

[0483] An exemplary CSFR2A signal peptide [residues 1-22, NCBI Reference Sequence NP_758452.1] (SEQ ID NO: 75) is: MLLLVTSLLLCELPHPAFLLIP

[0484] In one embodiment, the signal peptide comprises or consists of a sequence having at least 70% sequence identity to SEQ ID NO: 75, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 75, or a fragment thereof. In one embodiment, the signal peptide comprises SEQ ID NO: 75. In one embodiment, the signal peptide consists of SEQ ID NO: 75.

[0485] Further exemplary glycosidase sequences are shown below: An exemplary human PNGase with CD8 signal peptide (SEQ ID NO: 76): MALPVTALLLPLALLLHAARPAAAALGSSSGSASPAVAELCQNTPETFLEASKLLLTYADNILRNPNDEKYRSIRIGNTAFSTRLLPVRGAVECLFEMGEEGETHLIFPKKASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQH TRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANCFT LCCRAVGFEARYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPGELGGRISGSVAWRVARGEMGLQRKETLFIPCE NEW

[0486] An exemplary human PNGase with IgG variable region heavy chain signal peptide (SEQ ID NO: 77): MEFGLSWVFLVALLRGVQCAAAAALGSSSGSASPAVAELCQNTPETFLEASKLLLTYADNILRNPNDEKYRSIRIGNTAFSTRLLPVRGAVECLFEMGEEGETHLIFPKKASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTR NRQGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANCFTL CCRAVGFEARYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPGELGGRISGSVAWRVARGEMGLQRKETLFIPCEN EKISKQLHLCYNIVKDRYVRVSNNNQTISGWENGVWKMESIFRKVETDWHMVYLARKEGSSFAYISWKFECGSVGLKVDSISIRTSSQTFQTGTVEWKLRSDTAQVELTGDNSLHSYADFSGATEVILEAELSRGDGDVAWQHTQLFRQSLNDHEENCLEIIIKFSDL

[0487] An exemplary PUB-cleaving human PNGase with IgG variable region heavy chain signal peptide (SEQ ID NO: 78): MEFGLSWVFLVALLRGVQCKASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKRKSQEKLSRARKL DKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRDRSLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANCFTLCCRAVGFEARYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPL LYEIGWGKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPGELGGRISGSVAWRVARGEMGLQRKETLFIPCENEKISKQLHLCYNIVKDRYVRVS NNNQTISGWENGVWKMESIFRKVETDWHMVYLARKEGSSFAYISWKFECGSVGLKVDSISIRTSSQTFQTGTVEWKLRSDTAQVELTGDNSLHSYADFSGATEVILEAELSRGDGDVAWQHTQLFRQSLNDHEENCLEIIIKFSDL

[0488] An exemplary PUB and PAW cleaved human PNGase with IgG variable region heavy chain signal peptide (SEQ ID NO: 79): MEFGLSWVFLVALLRGVQCKASVEQLQKIRDLIAIERSSRLDGSNKSHKVKSSQQPAASTQLPTTPSSNPSGLNQHTRNRQGQSSDPPSASTVAADSAILEVLQSNIQHVLVYENPALQEKALACIPVQELKRKSQEKLSRARKLDKGINISDEDFLLLELLHWFKEEFFHWVNNVLCSKCGGQTRSRDR SLLPSDDELKWGAKEVEDHYCDACQFSNRFPRYNNPEKLLETRCGRCGEWANCFTLCCRAVGFEARYVWDYTDHVWTEVYSPSQQRWLHCDACEDVCDKPLLYEIGWGKKLSYVIAFSKDEVVDVTWRYSCKHEEVIARRTKVKEALLRDTINGLNKQRQLFLSENRRKELLQRIIVELVEFISPKTPKPG

[0489] In one embodiment, the glycosidase comprises or consists of a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 76-79, for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or a fragment thereof.

[0490] Expression control sequences The polynucleotide of the present invention may comprise one or more expression control sequences. Preferably, the nucleic acid sequence encoding a CAR and / or any other polynucleotide of interest is operably linked to one or more expression control sequences. As used herein, the term "operably linked" means that parts (e.g., a nucleic acid sequence encoding a CAR and one or more expression control sequences) are linked together in a manner that allows both to perform their functions substantially unimpeded.

[0491] As used herein, "expression control sequence" may refer to a nucleotide sequence that controls the expression of a transgene, e.g., to facilitate and / or increase expression.

[0492] The expression control sequence and transgene can be in any suitable configuration in the polynucleotide, provided that the expression control sequence is operably linked to the transgene (e.g., a nucleic acid sequence encoding a CAR or any other nucleotide sequence of interest).

[0493] promoter In some embodiments, the expression control sequence is a promoter.

[0494] Any suitable promoter can be used, and its selection can be easily performed by those skilled in the art. The promoter sequence can be constitutively active (i.e., operable in any host cell background), or alternatively, can be active only in a specific host cell environment, thus allowing targeted expression of the nucleotide of interest (e.g., CAR) in a specific cell type (e.g., tissue-specific promoter). The promoter can also exhibit inducible expression in response to the presence of another factor, for example, a factor present in the host cell. In any case, when the vector is administered for therapy, it is preferable that the promoter should be functional in the target cell background.

[0495] In some embodiments, the polynucleotide further comprises a promoter operably linked to the nucleic acid sequence encoding the CAR, scFv, or other nucleotide of interest.

[0496] In some embodiments, the promoter is a constitutive promoter.

[0497] In one embodiment, the nucleotide sequence encoding the CAR is operably linked to one or more promoters.

[0498] In one embodiment, the nucleotide sequence encoding the CAR and any other polynucleotide of interest is operably linked to one or more promoters.

[0499] In one embodiment, the nucleotide sequences encoding the CAR and any other polynucleotide of interest are operably linked to the same promoter.

[0500] The nucleotide sequences encoding the CAR and any other polynucleotide of interest can share a promoter so that their expression can be regulated by a single regulatory sequence. In one embodiment, the nucleotide sequences encoding the CAR and the polynucleotide of interest are independently operably linked to one or more promoters.

[0501] The nucleotide sequence encoding the polynucleotide of interest and the CAR can each be operably linked to a separate promoter such that their expression can be independently regulated by separate regulatory sequences. In one embodiment, the nucleotide sequence encoding the polynucleotide of interest and the CAR are operably linked to separate promoters.

[0502] In one embodiment, the polynucleotide of interest and the CAR are encoded in opposite orientations.

[0503] In one embodiment, the polynucleotide of interest and the CAR are encoded in opposite orientations and are independently operably linked to separate promoters.

[0504] In one embodiment, the polynucleotide of interest and the CAR are encoded in the same orientation.

[0505] In one embodiment, the promoter is selected from the group consisting of a cytomegalovirus promoter (CMV), a human phosphoglycerate kinase promoter (PGK), an EF-1α promoter, and an inducible NFAT promoter.

[0506] In one embodiment, the promoter is a cytomegalovirus (CMV) promoter.

[0507] In another embodiment, the promoter is a minimal cytomegalovirus (mCMV or minCMV) promoter (mCMV, see, e.g., Amendola (2005) Nat Biotech 23:108-116).

[0508] In one embodiment, the promoter is the human phosphoglycerate kinase (PGK) promoter.

[0509] In one embodiment, the promoter is the EF-1α promoter.

[0510] In one embodiment, the promoter is an inducible NFAT promoter. The inducible module may consist of a synthetic NFAT response element, typically containing repeats of consensus NFAT binding sites, placed upstream of a minimal promoter.

[0511] protein As used herein, the term "protein" includes single polypeptide chain molecules and multi-polypeptide complexes in which individual constituent polypeptides are linked by covalent or non-covalent means. As used herein, the terms "polypeptide" and "peptide" refer to polymers in which the monomers are amino acids and are linked together through peptide or disulfide bonds.

[0512] Proteins of the invention include any of the proteins disclosed herein that have an N-terminal methionine.

[0513] Polynucleotides The polynucleotides of the present invention may comprise, for example, DNA or RNA. They may be single-stranded or double-stranded. Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. In addition, it should be understood that those skilled in the art can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention to reflect the codon usage of any particular host organism in which the polypeptide of the present invention will be expressed.

[0514] Polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of polynucleotides of the invention.

[0515] Polynucleotides, such as DNA polynucleotides, may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.

[0516] Longer polynucleotides are generally produced using recombinant means, for example, polymerase chain reaction (PCR) cloning techniques. This may involve creating a pair of primers (e.g., about 15-30 nucleotides) that flank the target sequence desired to be cloned, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing the polymerase chain reaction under conditions that result in amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. Primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0517] In one embodiment, the polynucleotide comprises a sequence encoding a CAR comprising the sequence of any one of SEQ ID NOs: 16-18, 25-26, 46, or 51-54, or a variant thereof having at least 75% sequence identity thereto, for example at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0518] In one embodiment, the polynucleotide comprises a sequence encoding a CAR comprising the sequence of any one of SEQ ID NOs: 16-18, 25-26, 46, or 51-54.

[0519] In one embodiment, the polynucleotide is a) a sequence encoding an scFv comprising the sequence of SEQ ID NO: 16 or 46, and / or b) a sequence encoding a CAR comprising any one of SEQ ID NOs: 16 to 18, 25 to 26, or 51 to 54; or variants thereof each having at least 75% sequence identity thereto.

[0520] In one embodiment, the polynucleotide comprises a sequence encoding a CAR comprising the sequence of any one of SEQ ID NOs: 16-18, 25-26, 46, 51-54, or 85, or a variant thereof having at least 75% sequence identity thereto, for example at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0521] In one embodiment, the polynucleotide comprises a sequence encoding a CAR comprising the sequence of any one of SEQ ID NOs: 16-18, 25-26, 46, 51-54, or 85.

[0522] In one embodiment, the polynucleotide is a) a sequence encoding an scFv comprising the sequence of SEQ ID NO: 16 or 46, and / or b) a sequence encoding a CAR comprising any one of SEQ ID NOs: 16 to 18, 25 to 26, 51 to 54, or 85; or variants thereof each having at least 75% sequence identity thereto.

[0523] In one embodiment, the polynucleotide comprises a sequence encoding a CAR comprising the sequence of any one of SEQ ID NOs: 46 or 52-54, or a variant thereof having at least 75% sequence identity thereto, for example, at least 80%, at least 85%, 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%, or 100% sequence identity thereto.

[0524] In one embodiment, the polynucleotide comprises a sequence encoding a CAR comprising the sequence of any one of SEQ ID NOs: 46 or 52-54.

[0525] In one embodiment, the polynucleotide is a) a sequence encoding an scFv comprising the sequence of SEQ ID NO: 46, and / or b) a sequence encoding a CAR comprising any one of SEQ ID NOs: 52 to 54; or variants thereof each having at least 75% sequence identity thereto.

[0526] vector A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present invention, for example, some vectors used in recombinant nucleic acid technology allow the transfer of an entity, such as a nucleic acid segment (e.g., a heterologous DNA segment, such as a heterologous cDNA segment), into a target cell. A vector can serve the purpose of maintaining heterologous nucleic acid (DNA or RNA) in a cell, promoting replication of a vector containing a nucleic acid segment, or promoting expression of a protein encoded by the nucleic acid segment. A vector can be non-viral or viral. Examples of vectors used in recombinant nucleic acid technology include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNA), chromosomes, artificial chromosomes, and viruses. A vector can also be, for example, naked nucleic acid (e.g., DNA). In its simplest form, a vector can be the nucleotide of interest itself.

[0527] The vectors used in the present invention may be, for example, plasmid, mRNA or viral vectors and may comprise a promoter for the expression of the polynucleotide and optionally a regulator of the promoter.

[0528] Vectors containing polynucleotides used in the present invention can be introduced into cells using a variety of techniques known in the art, such as transfection, transformation, and transduction. Several such techniques are known in the art, such as infection with recombinant viral vectors such as retroviruses, lentiviruses (e.g., integration-deficient lentiviruses), adenoviruses, adeno-associated viruses, baculoviruses, and herpes simplex virus vectors; direct injection of nucleic acids; and biolistic transformation.

[0529] Non-viral delivery systems include, but are not limited to, DNA transfection methods. Here, transfection refers to the process of delivering genes to target cells using non-viral vectors. Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic drug-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14:556), and combinations thereof.

[0530] Transfection of cells with mRNA vectors can be achieved using nanoparticles, such as liposomes.

[0531] In some embodiments, the vector is contained in a nanoparticle. In some embodiments, the nanoparticle is a polymeric nanoparticle, an inorganic nanoparticle, or a lipid nanoparticle. In some embodiments, the nanoparticle is a liposome.

[0532] Nanoparticles can be targeted to specific cell types using one or more ligands displayed on their surface.

[0533] In one embodiment, polynucleotide delivery is transposon-mediated.

[0534] In one embodiment, the polynucleotide is mRNA. The mRNA may be contained in a nanoparticle.

[0535] viral vectors In a preferred embodiment, the vector is a viral vector. The viral vector may be in the form of a viral vector particle.

[0536] The viral vector can be, for example, a retroviral, lentiviral, adeno-associated viral (AAV), or adenoviral vector.

[0537] In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an AAV vector.

[0538] Retroviral and lentiviral vectors Retroviral vectors may be derived from or be derivable from any suitable retrovirus. A number of different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), and avian erythroblastosis virus (avian erythroblastosis virus). A detailed list of retroviruses can be found in Coffin et al. (1997) Retroviruses, Cold Spring Harbor Laboratory Press, 758-63.

[0539] Retroviruses can be broadly classified into two categories: "simple" and "complex." Retroviruses can also be further divided into seven groups. Five of these groups represent retroviruses with oncogenic properties. The remaining two groups are lentiviruses and spumaviruses. A review of these retroviruses is presented in Coffin et al. (1997) Retroviruses, Cold Spring Harbor Laboratory Press, 758-63.

[0540] The basic structure of retrovirus and lentivirus genomes share many common features, such as a 5'LTR and a 3'LTR. Between or within these are located a packaging signal that enables packaging of the genome, a primer binding site, an integration site that enables integration into the host cell genome, and the gag, pol, and env genes that encode packaging components, which are polypeptides necessary for the assembly of viral particles. Lentiviruses have additional features, such as the rev and RRE sequences found in HIV, which enable efficient transport of integrated proviral RNA transcripts from the nucleus to the cytoplasm of infected target cells.

[0541] In the provirus, these genes are flanked on both sides by regions called long terminal repeats (LTRs). LTRs are responsible for proviral integration and transcription. LTRs can also function as enhancer-promoter sequences and control viral gene expression.

[0542] The LTRs themselves are identical sequences that can be divided into three elements: U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA. U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably between different retroviruses.

[0543] In a defective retroviral vector genome, gag, pol, and env may be absent or non-functional.

[0544] In a typical retroviral vector, at least a portion of one or more protein coding regions essential for replication can be removed from the virus, rendering the viral vector replication-deficient.

[0545] Lentiviral vectors are part of a larger group of retroviral vectors. A detailed list of lentiviruses can be found in Coffin et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. Lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototypic "slow virus" visna / maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0546] The lentivirus family differs from retroviruses in that lentiviruses have the ability to infect both dividing and non-dividing cells (Lewis et al. (1992) EMBO J. 11:3053-8; Lewis et al. (1994) J. Virol. 68:510-6). In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells, such as cells that make up muscle, brain, lung, and liver tissue.

[0547] As used herein, a lentiviral vector is a vector that contains at least one component part derived from a lentivirus, preferably the component part being involved in the biological mechanism by which the vector infects cells, expresses genes, or replicates.

[0548] Lentiviral vectors can be "primate" vectors. Lentiviral vectors can be "non-primate" vectors (i.e., derived from viruses that do not primarily infect primates, especially humans). Examples of non-primate lentiviruses can be any member of the Lentiviridae family that does not naturally infect primates.

[0549] Preferably, the viral vectors used in the present invention have a minimal viral genome.

[0550] By "minimal viral genome" it is to be understood that the viral vector has been engineered to remove non-essential elements and retain essential elements to provide the functionality necessary to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998 / 017815.

[0551] Preferably, the plasmid vector used to produce the viral genome in the host / packaging cell will have sufficient lentiviral genetic information to allow packaging of the RNA genome into viral particles that, in the presence of packaging components, are capable of infecting target cells but are incapable of independently replicating to produce infectious viral particles in the final target cell. Preferably, the vector lacks functional gag-pol and / or env genes and / or other genes essential for replication.

[0552] However, the plasmid vector used to produce the viral genome in the host cell / packaging cell will also contain transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in the host cell / packaging cell. These regulatory sequences may be the native sequences associated with the transcribed viral sequence (i.e., the 5' U3 region), or they may be a heterologous promoter, such as another viral promoter (e.g., the CMV promoter).

[0553] The vector can be a self-inactivating (SIN) vector, in which viral enhancer and promoter sequences are deleted. SIN vectors can generate and transduce non-dividing cells in vivo with efficacy similar to that of wild-type vectors. Transcriptional inactivation of the long terminal repeats (LTRs) in the SIN provirus should prevent recruitment by replication-competent virus. This should also allow regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTRs.

[0554] The vector may be integration-defective. Integration-defective lentiviral vectors (IDLV) can be produced, for example, by packaging the vector with a catalytically inactive integrase (such as HIV integrase with a D64V mutation in the catalytic site), or by modifying or deleting essential att sequences from the vector LTR, or by a combination of the above.

[0555] cell The present invention provides a cell comprising a CAR, scFv, polynucleotide, or vector of the present invention.

[0556] In one embodiment, the cell is a mammalian cell, hi another embodiment, the cell is a human cell.

[0557] In one embodiment, the cell is a cell from a subject, hi one embodiment, the subject is a human subject.

[0558] In one embodiment, the cell is a T cell.

[0559] In one embodiment, the cells are natural killer (NK) cells.

[0560] In one embodiment, the cell is a hematopoietic stem cell (HSC).

[0561] In one embodiment, the cells are hematopoietic stem and / or progenitor cells (HSPCs).

[0562] In one embodiment, the cell is a tumor-infiltrating lymphocyte (TIL).

[0563] In one embodiment, the cell is an invariant NK T cell, a cytokine-induced killer cell (CIK), or a macrophage.

[0564] TILs are T cells that can be isolated from tumors. TILs are enriched in natural T cells that recognize tumor antigens. Isolated TILs can be expanded and modified, for example, transduced ex vivo with a polynucleotide or vector according to the present invention, and reintroduced into the tumor or subject.

[0565] The present invention also contemplates cells expressing the CAR of the present invention that have been engineered to disrupt one or more endogenous MHC genes. Disruption of endogenous MHC genes can reduce or prevent MHC expression on the surface of the engineered cells. Thus, such engineered cells with reduced or no MHC expression will have limited or no ability to present antigens on their cell surface. Such cells are particularly advantageous for adoptive cell transfer because they are non-alloreactive, e.g., they do not present antigens that can be recognized by the immune system of the subject receiving the adoptively transferred cells. As a result, the transferred cells are not recognized as "non-self," avoiding harmful immune reactions against the cells. Such cells are referred to as "universal cells" because they are suitable for adoptive transfer into a variety of different hosts, regardless of HLA type.

[0566] In some embodiments, the cells are non-allo-reactive universal T cells.

[0567] Variants, derivatives, analogs, homologs, and fragments In addition to the specific proteins and polynucleotides mentioned herein, the present invention also encompasses the use of variants, derivatives, analogs, homologs, and fragments thereof.

[0568] In the context of the present invention, a variant of any given sequence is a sequence in which a specific sequence of residues (either amino acid or nucleic acid residues) has been modified in such a way that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement, and / or modification of at least one residue present in the naturally occurring protein.

[0569] The term "derivative" as used herein in reference to a protein or polypeptide of the present invention includes any substitution, variation, modification, exchange, deletion, and / or addition of one (or more) amino acid residues from or to the sequence, provided that the resulting protein or polypeptide substantially retains at least one of its endogenous functions.

[0570] The term "analog" as used herein with respect to a polypeptide or polynucleotide includes any mimetic, i.e., a compound that has at least one of the endogenous functions of the polypeptide or polynucleotide that it mimics.

[0571] Typically, amino acid substitutions can be made, for example, from 1, 2, or 3 to 10 or 20 substitutions, provided the modified sequence substantially retains the required activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogues.

[0572] The proteins used in the present invention may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions may be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues, so long as the intrinsic function is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0573] Conservative substitutions may be made, for example, according to the following table: Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

[0574] [Table 1]

[0575] The term "homologue" as used herein means an entity that has a certain homology with a wild-type amino acid sequence or a wild-type nucleotide sequence. The term "homology" can be equated with "identity."

[0576] A homologous sequence may comprise an amino acid sequence that may be at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the subject sequence, preferably at least 95%, 97%, or 99% identical. Typically, a homolog will contain the same active site, etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of the present invention, it is preferred to express homology in terms of sequence identity.

[0577] A homologous sequence may comprise a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the subject sequence, preferably at least 95%, 97%, or 99% identical. Although homology may also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity.

[0578] Preferably, reference to a sequence having a percent identity to any one of the SEQ ID NOs disclosed herein refers to a sequence having the stated percent identity over the entire length of the referenced SEQ ID NO.

[0579] Homology comparisons can be performed by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage homology or identity between two or more sequences.

[0580] The homology percentage can be calculated for consecutive sequences, i.e., one sequence is aligned with the other sequence, and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0581] While this is a very simple and consistent method, it does not take into account that, for example, in an otherwise identical pair of sequences, a single insertion or deletion in a nucleotide sequence can cause the subsequent codon to be out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. As a result, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to attempt to maximize local homology.

[0582] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, such that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible, reflecting a higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. An "affine gap cost" is typically used, which imposes a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. Higher gap penalties will, of course, result in an optimized alignment with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, it is preferable to use the default values ​​when using such software for sequence comparison. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0583] Therefore, the calculation of the maximum homology percentage first requires the creation of an optimal alignment, taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA, Devereux et al. (1984) Nucleic Acids Res. 12:387). Examples of other software capable of performing sequence comparison include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid-Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), and the GENEWORKS comparison tool suite. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferable to use the GCG Bestfit program. Another tool called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174:247-50, FEMS Microbiol. Lett. (1999) 177:187-8).

[0584] Although the final homology percentage can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​generally used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. One example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the public default values ​​or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferable to use the public default values ​​for the GCG package, or a default matrix, such as BLOSUM62, in the case of other software.

[0585] Once the software has produced an optimal alignment, it is possible to calculate percentage homology, preferably percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0586] "Fragment" is also a variant, and the term typically refers to a selected region of a polypeptide or polynucleotide of interest functionally or, for example, in an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0587] Such variants can be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. When an insertion is made, synthetic DNA can be made that encodes the insert, along with 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion site. The flanking regions contain convenient restriction sites corresponding to sites in the naturally occurring sequence, allowing the sequence to be cleaved with appropriate enzymes and the synthetic DNA to be ligated to the cleavage product. The DNA is then expressed in accordance with the invention to produce the encoded protein. These methods are merely illustrative of the many standard techniques known in the art for manipulating DNA sequences; other known techniques can also be used.

[0588] Codon optimization The polynucleotides used in the present invention can be codon-optimized. Codon optimization has been previously described in International Publication Nos. 1999 / 41397 and 2001 / 79518. Different cells have different frequencies of use of certain codons. This codon bias corresponds to the bias in the relative abundance of certain tRNAs in cell types. Expression can be increased by modifying the codons in the sequence so that they are adjusted to match the relative abundance of the corresponding tRNAs. Expression can also be reduced by deliberately selecting codons whose corresponding tRNAs are known to be rare in certain cell types. Thus, an additional degree of translational control can be utilized.

[0589] composition The polynucleotides, proteins (e.g., CARs), vectors, and cells of the present invention can be formulated with a pharmaceutically acceptable carrier, diluent, or excipient for administration to a subject. Suitable carriers and diluents include isotonic saline, such as phosphate-buffered saline, and may contain human serum albumin.

[0590] Materials used to formulate pharmaceutical compositions should be non-toxic and should not interfere with the effectiveness of the active ingredient. The precise nature of the carrier or other material can be determined by one skilled in the art according to the route of administration.

[0591] Pharmaceutical compositions are typically in liquid form. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. Physiological saline, magnesium chloride, dextrose, or other sugar solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included. In some cases, surfactants such as pluronic acid (PF68) 0.001% may be used. In some cases, serum albumin may be used in the composition.

[0592] For injection, the active ingredient may be in the form of an aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0593] For delayed release, the pharmaceutical agent may be included in a pharmaceutical composition formulated for sustained release, e.g., in microcapsules formed from biocompatible polymers or in liposome carrier systems, according to methods known in the art.

[0594] Handling of cell therapy products is preferably performed in accordance with the FACT-JACIE International Standard for Cell Therapy.

[0595] Treatment method In one aspect, the invention provides a CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition of the invention for use in therapy.

[0596] In one aspect, the invention provides a CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition of the invention for use in treating cancer.

[0597] All references herein to treatment include curative, palliative and prophylactic treatment. Mammalian, particularly human, treatment is preferred. Both human and veterinary treatment are within the scope of the present invention.

[0598] In some embodiments, the treatment method provides a CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition of the present invention to a tumor.

[0599] In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is a CDH17+ cancer.

[0600] In one embodiment, a) the cancer is a primary cancer, optionally gastrointestinal cancer, colorectal cancer, pancreatic cancer, and / or gastric cancer; b) the cancer is a secondary cancer, optionally liver metastasis, optionally liver metastasis of colorectal cancer and / or liver metastasis of pancreatic ductal adenocarcinoma (PDAC), and / or c) The cancer is a neuroendocrine tumor.

[0601] In one embodiment, the cancer is a primary cancer.

[0602] In one embodiment, the cancer is gastrointestinal cancer, colorectal cancer, pancreatic cancer, and / or gastric cancer.

[0603] In one embodiment, the cancer is gastrointestinal cancer.

[0604] In one embodiment, the cancer is colorectal cancer.

[0605] In one embodiment, the cancer is pancreatic cancer. In one embodiment, the cancer is pancreatic ductal adenocarcinoma (PDAC). In one embodiment, the cancer is pancreatic neuroendocrine tumor.

[0606] In one embodiment, the cancer is gastric cancer.

[0607] In one embodiment, the cancer is a secondary cancer.

[0608] In one embodiment, the cancer is liver metastasis.

[0609] In one embodiment, the cancer is liver metastasis of colorectal cancer.

[0610] In one embodiment, the cancer is liver metastasis of pancreatic ductal adenocarcinoma (PDAC).

[0611] In one embodiment, the cancer is a neuroendocrine tumor.

[0612] In one embodiment, the solid tumor is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, and central nervous system cancer. The cancer is selected from the group consisting of a central nervous system (CNS) tumor, a primary central nervous system (CNS) lymphoma, a tumor angiogenesis, a spinal axis tumor, a brain stem glioma, a pituitary adenoma, Kaposi's sarcoma, an epidermoid carcinoma, a squamous cell carcinoma, an environmentally induced cancer, a combination of such cancers, and a metastatic lesion of such cancer.

[0613] In one aspect, there is provided a use of a polynucleotide, vector, or cell according to the invention for improving CAR cell activity.

[0614] In one aspect, a method of producing a CAR cell is provided, comprising introducing a CAR, scFv, polynucleotide, or vector into a cell.

[0615] In one embodiment, the cell is a CAR-T cell.

[0616] In a further embodiment, a method or use is provided, wherein the therapeutic activity of CAR cells or CAR-T cells is improved.

[0617] In one embodiment, the therapeutic activity is target cell killing.

[0618] In one aspect, a method of treatment is provided that includes producing CAR cells according to the methods of the invention and administering the CAR cells to a subject in need thereof.

[0619] Administration In some embodiments, the CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition is administered locally to the subject.

[0620] Local administration may include administration to the tumor of interest.

[0621] In a preferred embodiment, the CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition is administered to a tumor.

[0622] In some embodiments, the CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition is administered to the liver of the subject. In some embodiments, the CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition is administered to the pancreas of the subject.

[0623] In some embodiments, the CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition is administered to the subject systemically, hi some embodiments, the CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition is administered to the subject intravenously.

[0624] As used herein, the term "systemic delivery" or "systemic administration" means that an agent of the invention is administered to the circulatory system, e.g., to achieve widespread distribution of the agent. In contrast, topical or local administration restricts delivery of an agent to a localized area, e.g., a tumor.

[0625] In some embodiments, the CAR, scFv, polynucleotide, vector, cell, composition, or pharmaceutical composition is administered in a nanoparticle that targets T cells in vivo.

[0626] Dosage Those skilled in the art can easily determine the appropriate dosage of the agent of the present invention to be administered to a subject.Typically, a doctor will determine the actual dosage that is most suitable for each individual patient, which will depend on various factors, including the activity of the specific compound used, the metabolic stability and duration of action of the compound, age, body weight, general health, sex, diet, mode and time of administration, excretion rate, drug combination, the severity of the specific condition, and the individual undergoing therapy.Of course, there may be individual cases where higher or lower dosage ranges are advantageous, and such are within the scope of the present invention.

[0627] subject As used herein, the term "subject" refers to either a human or non-human animal.

[0628] Examples of non-human animals include vertebrates, e.g., mammals, such as non-human primates (especially higher primates), dogs, rodents (e.g., mice, rats, or guinea pigs), pigs, and cats. The non-human animals may be companion animals.

[0629] Preferably, the subject is a human.

[0630] Those skilled in the art will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention disclosed.

[0631] Preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0632] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology that are within the capabilities of those skilled in the art, and such techniques are explained in the literature. For example, Sambrook, J., Fritsch, EFand Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Ausubel, FM et al. Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley&Sons, Polak, JMand McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press, Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, and Lilley, DMand. Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part See A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference.

[0633] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the disclosed polypeptides, polynucleotides, vectors, cells, compositions, uses, and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been disclosed in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention that are apparent to those skilled in the art are intended to be within the scope of the following claims. [Example]

[0634] Materials and Methods Expression analysis in liver metastases Colorectal cancer (CRC) liver metastasis samples containing over 70% tumor cells were analyzed by mRNA sequencing. After obtaining an expression matrix using Transcripts Per Kilobase Million (TPM), values ​​were converted to deciles, and highly expressed genes were defined as those belonging to the first three deciles (10, 9, and 8).

[0635] Expression analysis in healthy tissues The approach described by Perna et al., Cancer Cell 2017, was applied to expression analysis in healthy tissues. The starting point was a global matrix containing mRNA expression data for all genes in all available tissues. The mean and standard deviation of all genes in each tissue were calculated to identify four expression classes: high, medium, low, and not detected. The class to which each candidate gene belonged in each tissue was determined, and the criteria described by Perna et al. were applied to identify safe targets, i.e., "no high expression in any normal tissue except the tissue of origin" and "no moderate expression across all healthy tissues."

[0636] Generation of CAR constructs Anti-CDH17 CARs were generated by including scFvs derived from either the Lic3 or A4_4R mAb (WO 2017120557 and WO 2012054084). Both scFvs were synthesized by GeneArt (Thermo Fisher Scientific) and cloned into CARs incorporating the CD28 transmembrane and costimulatory domains and the CD3z endodomain (Casucci, et al., Blood 2013). As CAR spacers, either an IgG1 hinge or NGFR-derived sequence was incorporated as previously described (Casucci et al., Front Immunol 2018). All CARs were cloned into bidirectional lentiviral vectors under the direct control of the human phosphoglycerate kinase promoter (hPGK), while CD20 was cloned in antisense under the control of the cytomegalovirus-derived minimal core promoter (minCMV or mCMV). Viral supernatant was produced in 293T packaging cells.

[0637] Cells and cell culture T cells were derived from peripheral blood of healthy donors after gradient centrifugation. All procedures were approved by the Institutional Review Board of the IRCCS San Raffaele Scientific Institute and complied with all relevant ethical regulations. T cells were activated with CD3 / CD28 beads (Gibco, 40203D) at a 3:1 ratio, transduced on day 2, and cultured in RPMI-1640 containing interleukin (IL)-7 and IL-15 (5 ng / ml, Peprotech, 200-07, 200-15). On day 6, beads were removed, and CAR T cells were expanded in complete medium until day 21. Cell lines (LoVo and BxPC-3) were cultured in RPMI-1640 (Euroclone, ECB90062L). All media for tumor cells were supplemented with penicillin / streptomycin (100 UI / ml, Lonza, DE17-602E), glutamine (2 mM, Lonza, LOBE17-605E), and 10% FBS (fetal bovine serum, Carlo Erba, FA30WS1810500). All cells were routinely tested for mycoplasma contamination by PCR (Mycoplasmacheck, Eurofins Genomics) and proved negative.

[0638] In vitro co-culture assay with CAR T cells CAR T cells were co-cultured with target cells at different effector:target (E:T) ratios in fully supplemented RPMI-1640 in the absence of cytokines. After 4 days, viable cells were counted using Flow-Count Fluorospheres (Beckman Coulter, 7547053) and analyzed by flow cytometry. Untransduced or irrelevant CAR (19.28z)-transduced T cells were used as controls. The depletion index was calculated as follows: 1 - (number of remaining target cells with experimental CAR T cells / number of remaining target cells with control T cells).

[0639] Flow cytometry Samples were washed with phosphate-buffered saline (PBS) containing 1% fetal bovine serum (FBS) and stained for 20 minutes at 4° C. Prior to use, all antibodies were validated and titrated for optimal on-target / off-target activity against human peripheral blood cells or tumor cell lines. The following specific mouse anti-human fluorophore-conjugated antibodies were used: CD3 allophycocyanin (APC)-Cy7 (clone SK7, BioLegend, 344818), CD45 APC-Cy7 (clone HI30, BioLegend, 304014), CD45 PE-Cy7 (clone HI30, BioLegend, 304016), CD45 BV510 (clone 30-F11, BioLegend, 103137), CD45RA fluorescein isothiocyanate (FITC, clone HI100, BioLegend, 983002), and CD62L. APC (clone DREG-56, BioLegend, 304810), major histocompatibility complex II receptor (HLA-DR) APC-Cy7 (clone L243, BioLegend, 307618), programmed cell death protein 1 (PD-1) PE-Cy7 (clone EH12.2H7, BioLegend, 329918), and 4',6-diamidino-2-phenylindole (DAPI). CAR transduction efficiency was determined by staining with anti-CD20 Pe (clone 2H7, BioLegend, 302306). Data were collected using a FACS Canto (BD Biosciences) flow cytometer and analyzed using FlowJo software.

[0640] statistical analysis All data are presented as mean ± sem. Statistical analysis was performed with GraphPad Prism 8 software. Data sets were analyzed by Student's t-test, one-way, or two-way ANOVA, depending on the experimental design, as described in the figure legends. Differences with a P value < 0.05 were considered statistically significant.

[0641] Mouse experiments All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the IRCCS San Raffaele Scientific Institute and the Italian Government Health Agency. Female or male 6- to 9-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl (NSG) mice (Charles River Laboratories) were housed in individually ventilated cages in a specific-pathogen-free (SPF) facility. Mice were inoculated with luciferase-inducible (Luc+) LoVo CRC cells (0.5 × 10 6 ) was injected intraperitoneally (ip), and on day 11, 6 × 10 6 CAR T cells were treated i.p. Tumor growth was monitored by bioluminescence assay using QUANTI-Luc detection reagent (InvivoGen, rep-qlc1) and expressed as relative light units (RLU). Mice were treated i.p. with CAR T cells in the control group and in the control group, with RLU ≥ 5.5 × 10 5 He was euthanized.

[0642] Example 1: Identification of CDH17 as a promising target for CAR T cell therapy of multiple solid tumors. The identification of new targets for CRC liver metastases was carried out in a sequential manner, as broadly depicted in Figure 1. First, we included surface proteome analysis of CRC and PDAC tumors, selecting articles that included at least adjacent tissues as comparisons. In addition, we included a list of CAR targets already in clinical development to provide an internal control. From this analysis, we obtained a list of over 200 genes, which we analyzed for their expression in LV MTS derived from the first cohort of CRC patients enrolled in the LiMeT protocol. This list was then reduced to 133 genes to meet the requirements for surface expression according to HPA and UniProt. Analysis of 47 known CAR antigens confirmed the validity of our process, as we did not detect any unexpected antigens, such as CD19, but did find high expression of antigens, such as CEACAM5, which has previously been reported to be highly expressed in LV MTS from various carcinoma types (Figure 2A). Interestingly, analysis of 86 new candidate antigens revealed 30 genes with high expression in over 70% of patient samples (Figure 2B). The expression of these 30 antigens in healthy tissues was confirmed by applying the approach described by Perna et al. (Cancer Cell 2017) to generate a matrix reporting the expression level of each candidate gene in each available tissue (Figure 2C). By applying the filtering criteria reported by Perna et al., CDH17 or Li-cadherin was identified as an antigen that met all of the criteria and was absent from all healthy tissues except the intestine.

[0643] CDH17 is an intercellular adhesion glycoprotein that is overexpressed in several cancers of the gastrointestinal tract, including pancreatic, colorectal, gastric, liver, and esophageal cancers, as well as in neuroendocrine tumors. Upon interaction with α2β1 integrin, CDH17 promotes tumor cell proliferation, adhesion, and metastatic colonization of the liver.

[0644] Through the use of the online database GEPIA, we confirmed that CDH17 is overexpressed not only in CRC but also in PDAC and gastric adenocarcinoma (Figure 2D). Importantly, CDH17 is overexpressed 10-fold in tumor cells compared to healthy counterparts, and such high expression is maintained across all CRC stages, including metastatic stages.

[0645] Example 2: Successful generation of a CDH17 CAR construct. We designed and engineered novel CAR constructs targeting CDH17. Single-chain fragment variable fragments (scFvs) from two humanized antibody clones, Lic3 and A4_R4, were selected to limit the potential immunogenicity of the new CAR proteins. For each scFv, we generated two constructs containing different extracellular spacers: either an IgG1-derived hinge region or an LNGFR-derived spacer. The LNGFR-derived spacer contains four TNFR cysteine-rich regions, the fourth of which has a deletion that abrogates NGF signaling and lacks the serine / threonine-rich stalk (LNGFR Mutant Short or NMS, Casucci et al., Front Immunol 2018).

[0646] All constructs used CD28 as the starting point for the transmembrane and intracellular costimulatory domains. Four constructs were cloned into a bidirectional lentiviral vector along with the CD20 gene, which acts as both a selectable marker and a suicide gene in conjunction with rituximab (Figure 3A).

[0647] Example 3: CDH17 CAR T cells demonstrate potent and specific recognition of CDH17+ tumor cells. Primary human T lymphocytes were transduced with the described LV vector. To generate CDH17 CAR T cells, a protocol capable of preserving T cell fitness, based on the use of anti-CD3 / CD28 beads and IL-7 / IL-15, was applied. No problems were encountered during production, and a high proliferation rate was observed at the end of the culture (Figure 4A). The CAR-T cell product obtained at the end of the process showed a large representation of both CD4 and CD8 T cells, an extremely high percentage of stem memory T cells (T SCM ), a low activation profile, and low expression of PD-1 exhaustion markers (Figures 4B-E). When challenged in coculture experiments, CDH17 CAR T cells demonstrated potent elimination of CDH17-positive tumor cells, but not CDH17-negative tumor cells, indicating that all CARs were functional and could specifically recognize the CDH17 antigen naturally expressed by malignant cells (Figure 5A). Interestingly, although no difference was observed between the two spacers, the A4_4R scFv outperformed the Lic3 scFv (Figure 5B).

[0648] A4_4R.NMS CAR T cells and A4_4R.hinge CAR T cells were then tested in a xenograft mouse model of CRC with the LoVo cell line (Figure 6A). In this model, LoVo cells were transduced to express secreted luciferase, which allows for easy monitoring of tumor progression by serial peripheral blood analysis. Control untransduced T cells failed to control tumor progression, as expected, but both A4_4R.NMS CAR T cells and A4_4R.hinge CAR T cells proved remarkably effective (Figure 6B).

[0649] Because the A4_4R scFv proved to be the best performing, in addition to the construct with the NMS spacer (SEQ ID NO: 15) and IgG1 hinge (SEQ ID NO: 14), we used this scFv to generate two additional CARs containing other spacer domains (SEQ ID NOs: 23 and 24). The first contains the entire extracellular portion of the LNGFR molecule (LNGFR wild-type long or NWL) and can be recognized by the clinical-grade anti-LNGFR mAb ME20.4. The second contains a mutated version of the IgG1 CH2CH3 spacer (mCH2CH3) that is unable to recognize FcγRI.

[0650] Example 4: Successful generation of a CDH17 CAR construct. After selecting the A4_4R scFv as the best performer, we designed and generated additional CAR constructs to expand manufacturing testing to more donors. To this end, we generated an NGFR-derived mutant short (i.e., NMS) as described in Casucci et al. (2018) Front Immunol., along with an IgG1 hinge and NWL-derived spacer. The NMS-spaced CDH17 CAR was then cloned into a lentiviral backbone carrying the CD20 gene in the antisense orientation (Figure 7A). To generate CDH17 CAR-T cells, primary human T lymphocytes from healthy donors were stimulated with anti-CD3 / CD28 beads, transduced with the described LV vector, and expanded with interleukin-7 (IL-7) and IL-15 according to a protocol that preserves T cell fitness. The CAR-T cell products showed similar proliferation kinetics and transduction efficiency despite lower surface expression of NGFR by NMS space CAR-T cells compared to NWL space CAR-T cells (Figure 7B-D). All T cell products contained significant representation of both CD4 and CD8 (Figure 7E) and a very high proportion of stem memory T cells (TSCM, Figure 7F).

[0651] Example 5: CDH17 expression in human primary tumors Immunohistochemical analysis was performed to evaluate CDH17 protein expression in primary human tissue samples. Tissues from 20 patients with primary colorectal cancer (CRC, Figure 8A) and 10 patients with liver metastases from CRC (Figure 8B) were stained with a commercially available anti-CDH17 antibody (Abcam, clone EPR 3996). CDH17 expression was found to be high and uniform in all tissue samples tested. This result is consistent with previously reported findings showing high levels of CDH17 expression in tumor samples from CRC patients with advanced tumors and distant metastases. Immunohistochemical analysis of CDH17 expression was positive for tissues from primary pancreatic ductal adenocarcinoma (PDAC, Figure 8C), as well as pancreatic neuroendocrine tumors (Figure 8D).

[0652] Example 6: Identification of the extracellular domain containing the epitope recognized by A4_4R CAR To better characterize and understand the interaction between A4_4R CAR T cells and the CDH17 target antigen, we sought to identify the extracellular cadherin (EC) on which the scFv binding site resides. Because CDH17 consists of seven EC domains that mediate calcium-dependent cell adhesion, we designed and generated seven different CDH17 mutants with individual EC domain deletions (Figure 9A). An additional construct encoding the wild-type (WT) form of CDH17 served as a positive control. Full-length CDH17 and CDH17 mutant coding sequences were cloned into a bidirectional lentiviral vector carrying the ΔNGFR selection marker in the antisense orientation under the mCMV promoter, and then expressed as CDH17. negSW620 colorectal cancer cells were transduced with CDH17. The cells were efficiently transduced, as assessed by high levels of surface NGFR expression via flow cytometry, and expressed similar levels of CDH17 truncation mutants, as assessed by staining with a commercially available anti-CDH17 antibody (Santa Cruz anti-CDH17, clone H-1). As expected, because the anti-CDH17 antibody binds to an epitope located within EC7, cleavage of EC7 by the ΔEC7 mutant caused a loss of detectable signal (Figure 9B). The transduced cells were then cocultured with A4_4R CAR T cells bearing three different extracellular spacers (i.e., CDH17.28z_H, CDH17.28z_NMS, and CDH17.28z_NWL). A4_AR CAR T cells mediated effective killing of all CDH17 mutants except the EC1 domain deletion mutant (Figure 9C). ΔEC1 escaped CAR T cell killing, as did WT SW620 cells, which do not endogenously express CDH17, serving as a negative control and demonstrating that the epitope recognized by A4_4R scFv is within EC1.

[0653] Example 7: In vivo evaluation of the efficacy of CDH17.28z CAR T cells with different extracellular spacers in CRC and PDAC xenograft models We evaluated the efficacy of CDH17 CAR T cells with three different extracellular spacers (i.e., CDH17.28z_H, CDH17.28z_NMS, and CDH17.28z_NWL) in vivo. We established tumor xenograft mouse models of colorectal cancer and pancreatic adenocarcinoma using LoVo and AsPC-1 cell lines, respectively. In both mouse models, tumor cells were injected subcutaneously, and in the setting of low tumor burden, CDH17.28z CAR T cells were administered intravenously. In a CRC xenograft model, CDH17 CAR T cells provided a significant survival benefit to mice compared with control untransduced T cells (Figure 10A and B). However, CDH17.28z NMS showed no peripheral proliferation and low T cell activation, whereas CDH17.28z_H and CDH17.28z_NWL CAR-T cells showed high peak proliferation and activation rates (Figures 10C and 10D). Analysis of tumor-infiltrating lymphocytes collected at sacrifice showed that all three CDH17 CAR-T cells had high levels of the transduction marker gene CD20, indicating that the integrated vector expressing the CAR was stably expressed under all conditions. However, CDH17.28z_NMS CAR-T cells had lower levels of surface NGFR compared with CDH17.28z_NWL CAR-T cells, indicating lower surface expression of the CAR by CDH17.28z_NMS CAR-T cells. Similar results were obtained in a PDAC xenograft model, whereby all three CDH17 CAR-T cells provided a significant survival benefit to mice compared with control untransduced T cells, whereas CDH17.28z NMS CAR-T cells were the least effective in doing so (Figures 11A and 11B) and showed a deficit in their ability to proliferate and activate in the periphery compared with CDH17.28z_H and CDH17.28z_NWL CAR-T cells (Figures 11C and 11D). Together, these results demonstrated the efficacy of CDH17.28z CAR T cells in recognizing and killing CDH17+ tumors in vivo and highlighted the lower efficacy of NMS-spaced CDH17 CAR-T cells.

[0654] Example 8: Evaluating cytokine production by CDH17.28z_H and CDH17.28z_NWL CAR-T cells in vitro IFNg and TNFa production by CDH17.28z_H and CDH17.28z_NWL CAR-T cells was measured upon stimulation with the CRC LoVo cell line in vitro. Compared to control untransduced T cells, both CDH17 CAR-T cells produced significant levels of cytokines (Figures 12A and B).

[0655] Example 9: Efficacy of CEA and CDH17 CAR-T cells in vitro CEA CAR-T cells are currently being used in clinical trials for the treatment of liver metastases arising from different solid tumors, including CRC. Therefore, we generated CEA CAR-T cells and compared their efficacy with CDH17 CAR-T cells. CEA CAR-T cells were generated by including an IgG hinge extracellular spacer and CD28 transmembrane and costimulatory domains. The CEA CAR was cloned into a bidirectional lentiviral vector carrying NGFR in the antisense orientation as a marker gene (Figure 13A). Primary human T lymphocytes from healthy donors were stimulated with anti-CD3 / CD28 beads, transduced with the described LV vector, and expanded with interleukin-7 (IL-7) and IL-15 (Figure 13B). At the end of production, the CEA CAR-T cells demonstrated high transduction efficiency and significant expression of both CD4 and CD8 (Figures 13C and D). When cocultured with LoVo tumor cells, both CEA and CDH17 CAR-T cells demonstrated potent tumor recognition. Nevertheless, CDH17 CAR-T cells appeared more potent than CEA CAR-T cells in terms of both tumor killing and cytokine production (Figures 13E and 13F).

[0656] Example 10: Efficacy of CDH17 CAR-T cells in primary human samples To test the efficacy of CAR-T cells in a more relevant setting, healthy donor-derived CDH17 CAR-T cells (see Figure 7) were first tested against patient-derived organoids from CRC-LM. Patient-derived organoids are highly relevant because they retain the histological complexity and genetic heterogeneity of the parental tumor. CDH17.28z_H and CDH17.28z_NWL demonstrated potent recognition and killing of CRC-LM-derived organoids (Figure 14).

[0657] Next, CDH17 CAR-T cells were generated from patients with CRC-LM, PDAC-LM, and PDAC (Figures 15, 16, and 17). CDH17 CAR-T cells generated from CRC-LM patients showed similar proliferation kinetics and transduction efficiency to CEA CAR-T cells, which have already been used in clinical trials (Figures 15A-C). The distribution of CD4 and CD8 T cell compartments and T cell memory subsets was also comparable between the two CAR specificities (Figures 15D and E). Similar results were obtained in T cell generation from PDAC-LM and PDAC patients, whereby CDH17 CAR-T cells showed high proliferation and transduction (Figures 16 and 17, A-C) and good representation of early memory T cell subsets, which are associated with improved antitumor activity in vivo (Figures 16 and 17, D and E).

[0658] Finally, patient-derived CDH17 CAR T cells from CRC-LM were co-cultured with autologous or allogeneic patient-derived organoids from CRC-LM. Compared with control untransduced T cells, CDH17 CAR-T cells demonstrated significantly more potent recognition and killing of CRC-LM-derived organoids (Figure 18).

[0659] Example 11: Expression and localization of CDH17 in normal and tumor human colon According to literature data and expression analysis of healthy tissues (Figure 2C), CDH17 is expressed exclusively by epithelial cells in normal colon and small intestine, specifically in the tissue of origin of colon cancer. In normal cells, CDH17 expression is reportedly localized to the intercellular junctions between epithelial cells. This polarized expression is lost during malignant transformation, and tumor cells express the antigen across the entire cell surface. Murine CDH17 shows a similar expression pattern in normal colon, and animal models have demonstrated that antigen localization at intercellular junctions masks recognition by anti-CDH17 CAR-T cells (Feng et al. (2022) Nat Cancer), encouraging the safety of using CDH17-directed CAR-T cells. To address whether CDH17 is localized to the lateral junctions between epithelial cells, we performed immunofluorescence staining of human healthy colon tissues using CDH17 and occludin, a marker of apical tight junctions in colonic epithelial cells. In normal colon, CDH17 was predominantly expressed on the lateral surfaces of epithelial cells, but not on the basal surfaces, which are accessible to CAR-T cells (Figure 19A). In contrast, tumor tissue showed disruption of tissue architecture, and CDH17 was expressed at high and continuous levels by tumor epithelial cells (Figures 19B and C).

[0660] Example 12: Validation of CDH17.28z cell administration route in a xenograft mouse model of colorectal cancer After evaluating the in vivo efficacy of CDH17.28z_H and NWL CAR-T cells, we first moved on to optimizing their administration route to potentially enhance their therapeutic efficacy. In contrast to conventional systemic administration, locoregional delivery of CAR-T cells has been explored as a means to overcome poor T cell trafficking and inefficient T cell penetration into solid tumors. To test locoregional delivery of CDH17.28z CAR-T cells, we used an immunodeficient NSG xenograft mouse model in which LoVo CRC cells were injected directly into the liver and CDH17.28z_H CAR-T cells and CDH17.28z_NWL CAR-T cells were administered intrahepatically or intravenously. Both IgG1-hinge and NWL-space CAR-T cells demonstrated efficient tumor growth suppression compared to control untransduced T cells, with no significant difference between systemic and locoregional administration (Figure 20A). Consistent with this, CDH17.28z_H and CDH17.28z_NWL CAR-T cells administered locoregionally or systemically expanded in a comparable manner, peaking at day 11 postinfusion (Figure 20B). Locoregional versus systemic delivery of CDH17.28z CAR-T cells was also tested in a humanized mouse model in which the CRC LoVo cell line was injected intrahepatically (Figure 21). Briefly, in this model, NSG mice transgenic for expression of human IL-3, GM-CSF, and CSF were reconstituted with human hematopoietic stem and progenitor cells to reconstitute a functional human immune system. Here, tumor growth and normal hematopoiesis coexist, which offers several advantages. First, the presence of cells and cytokines of human origin maintains human CAR-T cell dynamics over the long term, allowing for more representative efficacy testing, primarily through the production of IL-15 by bone marrow cells. Second, the model allows for the reproduction of human CAR-T cytotoxicity, such as cytokine release syndrome (CRS), due to the presence of circulating monocytes, which are primarily responsible for the systemic release of cytokines that cause the syndrome. In this model, no significant overall differences were observed in the ability to combat tumor growth by systemically or locoregionally infused CDH17 CAR-T cells (Figure 21A).Nevertheless, intrahepatic infusion of CDH17 CAR-T cells achieved faster tumor debulking, T cell activation, and T cell proliferation (Figures 21B and C). However, perhaps the stronger activation observed with intrahepatic delivery of CDH17 CAR-T cells caused severe cytokine release syndrome, manifested as rapid weight loss (Figures 22A and D), higher levels of bone marrow-derived cytokines (Figures 22B and E), and all intrahepatically treated mice ultimately succumbed to toxicity (Figures 22C and F).

[0661] Example 13: Validation of CDH17.28z CAR-T cell administration routes in a xenograft mouse model of pancreatic adenocarcinoma Next, we investigated the effects of locoregional and systemic delivery of CDH17 CAR-T cells in a xenograft mouse model of pancreatic adenocarcinoma. In this model, AsPC-1 PDAC cell lines were intrapancreatically injected into immunodeficient NSG mice, which were then treated with CDH17 CAR-T cells either intrapancreatically or intravenously. Both IgG1-hinge and NWL-space CAR-T cells demonstrated antitumor activity compared with control untransduced T cells, regardless of whether they were administered locally or systemically (Figure 23A). However, intrapancreatically injected CDH17 CAR-T cells achieved the best survival benefit (Figure 23B), indicating that CAR-T cell performance and trafficking can be affected by unique structural features that may differ between distinct tumor types, and therefore the optimal delivery route should be evaluated on a case-by-case basis depending on the CAR specificity and tumor setting.

[0662] Example 14: Generation and in vitro efficacy of third-generation CDH17 CAR-T cells Third-generation CARs are generated by combining two or more costimulatory domains, most frequently CD28 and 4-1BB, in the CAR intracellular portion. Because CD28 and 4-1BB activate distinct signaling pathways with distinct downstream effects on T cells, third-generation CARs exploit the synergistic properties of the two costimulators to enhance CAR-T cell fitness and efficacy. In several reported studies, third-generation CARs have demonstrated stronger cytokine release and tumor killing in vitro, as well as increased proliferation and longer persistence in vivo. Therefore, we decided to generate third-generation CDH17.28BBz CAR-T cells to test whether they confer advantages over second-generation CDH17.28z CAR-T cells. The CDH17.28BBz A4_4R CAR was generated by combining the A4_4R scFv, an IgG4 hinge spacer, a CD28 transmembrane domain, and an intracellular domain consisting of both CD28 and 4-1BB costimulatory domains, as well as CD3ζ. We produced a third-generation CAR construct developed by Feng et al. (Feng et al. (2022) Nat Cancer) containing a llama-derived VHH1 nanobody (CDH17.28BBZ VHH1) with the intention of comparing the effect of our A4_4R scFv versus their antigen recognition domains on CAR efficacy. All CAR constructs were then cloned into a bidirectional lentiviral vector containing the NGFR selection marker in the antisense orientation and the CAR gene downstream of the PGK promoter (Figure 24A). Primary human T lymphocytes from healthy donors were stimulated with anti-CD3 / CD28 beads, transduced with the described LV vectors, and expanded with interleukin-7 (IL-7) and IL-15 (Figure 24B). At the end of production, all generated CAR-T cells showed high and similar transduction efficiency and high expression of both CD4 and CD8 (Figure 24C-E). When cocultured with LoVo tumor cells, second-generation CDH17.28z CAR-T cells showed the best antitumor killing (Figure 24F and G) and the highest production of inflammatory cytokines (Figure 24H).

[0663] Focusing on third-generation CARs with different antigen recognition domains, A4_4R CAR T cells exhibited higher tumor killing than VHH1 CAR T cells at low E:T ratios (Figures 24F and I).

[0664] CDH17.28BBz A4_4R CAR-T cells were successfully produced from patients with CRC-LM, as they showed good proliferation kinetics, high transduction efficiency, and good representation of CD4 and CD8 T cells and memory T cell subsets upon stimulation with anti-CD3 / CD28 beads and interleukin-7 (IL-7) and IL-15 (Figures 25A-D).

[0665] Additional Materials and Methods (Examples 4-14) Cells and culture conditions T cells were derived from peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with colorectal cancer, pancreatic ductal adenocarcinoma, or liver metastases from primary pancreatic ductal adenocarcinoma. All procedures were approved by the Institutional Review Board of the IRCCS San Raffaele Scientific Institute and complied with all relevant ethical regulations. Patient samples were collected at the Clinical Department of Ospedale San Raffaele (Milan, Italy) under written informed consent in accordance with the Declaration of Helsinki (protocol "LiMet", Milan, Italy). Samples obtained for routine diagnostic or monitoring purposes were processed and stored by the Institutional Biobank Biological Resource Center (CRB-OSR) (Num ID CRB in BBMRI-ERIC: bbmri-eric: ID: IT_1383758011993577). T cells were activated with a 3:1 ratio of CD3 / CD28 beads (Gibco, 40203D), transduced on day 2, and cultured in RPMI-1640 (Euroclone, ECB90062L) supplemented with penicillin / streptomycin (100 UI / ml, Lonza, DE17-602E), glutamine (2 mM, Lonza, LOBE17-605E), 10% FBS (fetal bovine serum, Carlo Erba, FA30WS1810500), and interleukins IL-7 and IL-15 (5 ng / ml, Peprotech, 200-07, 200-15). On day 6, beads were removed, and CAR T cells were expanded in complete medium as previously described (Casucci et al. (2013) Blood; Casucci et al. (2018) Front Immunol). Phenotypic analysis and functional testing were performed at the end of production. Human colorectal cancer cell lines (LoVo and SW620) and a human pancreatic cancer cell line (AsPC-1) were cultured in RPMI-1640 (Euroclone) supplemented with penicillin / streptomycin (100 UI / ml, Lonza, DE17-602E), glutamine (2 mM, Lonza, LOBE17-605E), and 10% FBS (fetal bovine serum, Carlo Erba, FA30WS1810500).Human HEK-293T cells were used as a packaging line for lentivirus production and cultured in Iscove's Modified Dulbecco's Medium (IMDM, Euroclone, ECB2072L). For in vivo studies, tumor cells were transduced with secreted luciferase to allow tumor growth monitoring. All cells were routinely tested for mycoplasma contamination by PCR (Mycoplasmacheck, Eurofins Genomics) and proved negative.

[0666] Generation of CAR constructs The second-generation CEA.28z CAR construct contains an scFv derived from the BW431-26 mAb. The CAR incorporates an IgG1-derived hinge spacer, CD28 transmembrane and costimulatory domains, and a CD3ζ endodomain. The third-generation CDH17.28BBz_A4_4R and CDH17.28BBz_VHH1 CAR constructs were generated by cloning the CAR coding sequence into a bidirectional LV with a human PGK strong promoter and mCMV to enhance upstream transcription. For both of these constructs, the ΔLNGFR selection marker was placed in the antisense orientation, while the CAR coding sequence was cloned immediately downstream of the PGK promoter. The CDH17.28BBz_A4_4R CAR construct contains the A4_4R scFv, an IgG4 hinge, a CD28 transmembrane domain, CD28 and 4-1BB costimulatory domains, and a CD3ζ endodomain. The CDH17.28BBz_VHH1 CAR construct contains a VHH1 nanobody (Feng et al. (2022) Nature Cancer), an IgG4 hinge, a CD28 transmembrane domain, CD28 and 4-1BB costimulatory domains, and a CD3ζ endodomain.

[0667] Generation of CDH17 deletion mutants and identification of the A4_4R scFv binding site CDH17 deletion mutants (ΔEC1-ΔEC7) were generated by cloning CDH17 complementary DNA sequences with individual EC domain deletions, synthesized by Twist Bioscience and GeneArt (Thermo Fisher Scientific), into a bidirectional lentiviral vector containing the human PGK strong promoter and mCMV to enhance upstream transcription. In this platform, the ΔNGFR selection marker was placed in the antisense orientation, while the CDH17 mutant coding sequences were cloned immediately downstream of the PGK promoter. To determine the binding site of CDH17 by the A4_4R scFv, full-length CDH17 or CDH17 deletion mutants were transfected with CDH17. neg SW620 colorectal cancer cells were transduced. Transduced cells were stained with anti-human fluorophore-conjugated antibodies specific for CDH17 FITC (Santa Cruz, clone H1-1) and nerve growth factor receptor (NGFR) PE-Cy7 (clone C40-1457, BD Biosciences, 562122). Transduced cells were co-cultured with CDH17.28z cells or control untransduced T cells. The depletion index was calculated as follows: 1 - (number of remaining target cells with experimental CAR T cells / number of remaining target cells with control T cells). SW620 wild-type cells were used as a negative control.

[0668] Sequencing To confirm that the CDH17 deletion mutant cDNA insert was correctly cloned into the lentiviral backbone, DNA sequencing was performed using the forward primer 5′-GACCGAATCACCGACCTCTCT-3′ and the reverse primer 5′-AATCCAGAGGTTGATT GTCGA-3′ (Eurofins Genomics).

[0669] LV vector production and titration To produce LV supernatants, 293T cells were cotransfected with the transfer vector, packaging plasmid (pMDLg / pRRE, encoding the viral gag-pol gene; Addgene, Cambridge, MA), REV plasmid (pRSV-Rev, encoding the viral rev gene; Addgene), and ENV plasmid (pMD2.VSV-G, encoding the G glycoprotein of vesicular stomatitis virus pericapsis-VSV-G; Addgene) using CaCl precipitation. The lentivirus-containing supernatants were collected 48 hours later, ultracentrifuged, and cryopreserved. To titrate the LV supernatants, 293T cells were transduced with different supernatant dilutions. Six days after titration, 293T cells were analyzed by FACS, and the titer of the LV supernatants was calculated.

[0670] Generation of patient-derived organoids (PDO) from CRC liver metastasis samples Tissue sections were obtained from patients undergoing surgery at IRCCS San Raffaele Hospital for the removal of clinically confirmed liver metastases from colorectal cancer. Human samples were obtained after written informed consent and IRB approval (protocol "Limet", Milan, Italy). Tissue samples were kept refrigerated in phosphate-buffered saline (PBS) (Euroclone) until processing and for up to 24 hours after surgical resection. Tissues were cut into small pieces with a scalpel and processed by incubation with PBS + 5 mM EDTA (Invitrogen). Tissue fragments were washed in PBS and then incubated for 1 hour at room temperature with a digestion solution consisting of PBS / EDTA 1 mM + TrypLE 10X (Gibco) + 10X DNAse I buffer + DNAse I (Roche). Dissociated cells were collected in Advanced DMEM / F12 medium (Gibco), pelleted, resuspended in 130 μl of Growth Factor Reduced (GFR) Matrigel matrix (Corning), and seeded onto a single 24-microwell plate. After Matrigel solidification, complete human organoid medium was added to the plate. Basal medium was prepared using the following reagents: Advanced DMEM F / 12 (Gibco) supplemented with 1% penicillin / streptomycin (100 U / ml, 0.1 mg / ml, Euroclone), 1% glutamine (2 mM, Euroclone), 1x B-27 (Gibco), 1x N-2 (Gibco), and 0.1% BSA. Basal medium was supplemented with the following additives: EGF (PrepoTech), Noggin (PrepoTech), R-spondin-1 (PrepoTech), Gastrin (Sigma-Aldrich), FGF-10 (PrepoTech), FGF-basic (PrepoTech), WNT-3A (R&D Systems), Prostaglandin E2 (Tocris), Y-27632 (Stem Cell Therapeutics), Nicotinamide (Sigma-Aldrich), A 83-01 (Tocris), SB202190 (Sigma-Aldrich), and HGF (PrepoTech). Supplementary solutions were prepared at 1000x concentrations and freshly added to the basal medium.To perform in vitro functional assays, PDO cells were transduced with a luciferase-expressing lentiviral vector and selected in puromycin. For in vitro functional assays, PDO cells were harvested by gently pipetting them out of Matrigel using 1 ml of Cell Recovery Solution (Corning) per well and incubated at 4°C for 45 minutes. After incubation, PDO cells were collected in a Falcon tube, diluted 5-fold with HBSS, and centrifuged. The supernatant was discarded, and the pellet was resuspended in a 1:1 ratio of Matrigel and basal medium.

[0671] In vitro functional assays CAR T cells or control non-transduced T cells were co-cultured with target cells at different E:T ratios in fully supplemented RPMI-1640 in the absence of cytokines. After 24 hours, supernatants were collected and analyzed using a LEGENDplex™ bead-based cytokine immunoassay (BioLegend, 740725). After 4 days, viable (tumor) cells were counted using Flow-Count Fluorospheres (Beckman Coulter, 7547053) and analyzed by flow cytometry. The depletion index was calculated as follows: 1 - (number of remaining target cells with experimental CAR T cells / number of remaining target cells with control T cells). In the co-culture assay addressing PDO killing after 3 days, surviving organoids were quantified by bioluminescence assay using a Tristar 3 filter-based multimode plate reader (Berthold), and representative brightfield microscopy images were taken. Residual tumor levels were expressed as relative light units (RLU). The depletion index was calculated as follows: 1-(RLU of remaining target cells with experimental CAR T cells / RLU of remaining target cells with control T cells).

[0672] Mouse experiments All experiments were approved by the Institutional Animal Care and Use Committee of the IRCCS San Raffaele Scientific Institute and the Italian Government Health Agency. Female or male 6- to 9-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl (NSG) mice (Charles River Laboratories) and NSGTgCMV-IL3, CSF2, KITLG1Eav / MloySzJ (SGM3) mice (Charles River Laboratories) were housed in a specific pathogen-free facility in individually ventilated cages. For experiments addressing the antitumor efficacy of CDH17.28z cells, 1.5 × 10 6 LoVo LUCIA+NGFR+ cells and 4 × 10 6 AsPC-1 LUCIA+NGFR+ cells were injected subcutaneously, and 10 × 10 6 CAR T cells were administered intravenously. For experiments validating the route of treatment, 0.1 × 10 6 LoVo LUCIA+NGFR+ cells were injected intrahepatically, with 1.5 × 10 6 10×10 AsPC-1 LUCIA+NGFR+ cells were administered intrapancreatically. 6 For experiments assessing the potential systemic toxicity of CDH17.28z cells, SGM3 mice were sublethally irradiated and delivered 1 × 10 purified CAR T cells from umbilical cord CB samples collected at the Department of Gynecology at IRCCS Ospedale San Raffaele (Milan, Italy) under written informed consent approved by the IRCCS Ospedale San Raffaele Ethics Committee (Protocol 34CB, Milan, Italy). 5 Human cord blood (CB)-derived hematopoietic stem and progenitor cells (HSPCs) CD34+ were intravenously infused. After reconstitution, 0.1 × 10 HSPC-humanized SGM3 mice were injected with 0.1 × 10 65 x 10 LoVo LUCIA+NGFR+ cells were injected intrahepatically and delivered either intrahepatically or intravenously. 6 Each mouse was treated with 100 CAR T cells. To assess the development of cytokine release syndrome, body weight loss was monitored daily, and serum human cytokine concentrations were assessed on day 10 using the LEGENDplex™ bead-based cytokine immunoassay (BioLegend, 740724). In all experiments, tumor growth was monitored by bioluminescence assay using QUANTI-Luc detection reagent (InvivoGen, rep-qlc1) and expressed as relative light units (RLU). Mice were treated with 100 CAR T cells in the control group and 100 CAR T cells in the control group. 5 At the time of euthanasia, subcutaneous tumor masses were collected, dissociated using gentleMACS (Miltenyi Biotec, 130-093-235) and tumor dissociation reagent (Miltenyi Biotec, 130-095-929), and analyzed by flow cytometry.

[0673] Flow cytometry Samples were washed with phosphate-buffered saline (PBS) containing 5% fetal bovine serum (FBS) and stained for 20 minutes at 4° C. Prior to use, all antibodies were validated and titrated for optimal on-target / off-target activity against human peripheral blood cells or tumor cell lines. We used mouse anti-human fluorophore-conjugated antibodies specific for the following: CD3 allophycocyanin (APC)-Cy7 (clone SK7, BioLegend, 344818), CD45 BV510 (clone 30-F11, BioLegend, 103137), CD8 PerCP (clone SK1, BD Biosciences, 345774), CD4 PB (clone OKT4, BioLegend, 317423), CD45RA fluorescein isothiocyanate (FITC, clone HI100, BioLegend, 983002), CD62L APC (clone DREG-56, BioLegend, 304810), nerve growth factor receptor (NGFR) PE (clone C40-1457, BD Biosciences, 557196), and CD20 PE (clone 2H7, BioLegend, 980214), CDH17 FITC (clone H1-1, Santa Cruz, sc-393533), and major histocompatibility complex II receptor (HLA-DR) APC-Cy7 (clone L243, BioLegend, 307618). Cell viability was determined using 4',6-diamidino-2-phenylindole (DAPI). CAR transduction efficiency was determined by staining with an anti-CD20 monoclonal antibody or an anti-NGFR monoclonal antibody reactive against an antisense gene marker. Relative fluorescent intensity (RFI) was calculated as the ratio of the mean fluorescence intensity (MFI) of a specific fluorophore-conjugated antibody to the fluorophore-conjugated control. Either a secondary antibody or a control isotype was used as a control. Data were collected using a FACS Canto II (BD Biosciences) or CytoFLEX (Beckman Coulter) flow cytometer and analyzed with FlowJo software.

[0674] Immunohistochemistry and immunofluorescence analysis For IHC, tumor samples were incubated with a commercially available anti-CDH17 antibody (clone EPR3996, Abcam, ab109190) according to the manufacturer's instructions. Briefly, tumor sections were deparaffinized and heat-mediated antigen retrieval was performed before the IHC staining protocol. For IF, healthy and tumor samples were incubated with the indicated primary antibodies according to the manufacturer's instructions. Confocal images were acquired as z-stacks using a Leica TCS SP8 confocal microscope (Leica Microsystems) equipped with a HC PL APO CS 263X (NA 1.4) oil objective lens with a white light laser (470-670 nm). Digital images were recorded in separately scanned channels with no overlap in the detection of emission from each fluorochrome. Final image processing was performed with ImageJ software with minimum contrast and brightness adjustments.

[0675] statistical analysis Statistical analysis was performed using GraphPad Prism 10.0.2 software and is presented as mean + / - SEM as described in the figure legends. Data sets were analyzed with paired or unpaired Student's t-test, one-way or two-way analysis of variance (ANOVA), and log-rank Mantel-Cox test, depending on the experimental design. Appropriate statistical tests were used as described in the figure legends. Differences with a P value <0.05 were considered statistically significant.

[0676] Embodiment Various preferred features and embodiments of the present invention are described with reference to the following numbered paragraphs.

[0677] 1. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain is a) a heavy chain variable region (VH) complementarity determining region (CDR) having the following sequences: CDR1-DHTIHWMR (SEQ ID NO: 1), CDR2-YIYPRDGITGYNERFRGK (SEQ ID NO: 2), and CDR3-WGYSYRNYAYYYDYWGQGTL (SEQ ID NO: 3), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) A CAR comprising light chain variable region (VL) CDRs with the following sequences: CDR1-INCRSSQSLLHSSNQR (SEQ ID NO: 4), CDR2-PPKVLIYWASTRES (SEQ ID NO: 5), and CDR3-QQYYSYPWTFGQ (SEQ ID NO: 6), or variants thereof each with up to three amino acid substitutions, additions, or deletions. 2. The antigen-binding domain is a) a VH domain comprising the sequence of SEQ ID NO: 7, and b) a VL domain comprising the sequence of SEQ ID NO: 8; or a variant thereof, each having at least 75% sequence identity thereto. 3. The CAR of paragraph 1 or 2, wherein the antigen-binding domain comprises a single-chain variable fragment (scFv). 4. The CAR of any one of paragraphs 1 to 3, wherein the antigen-binding domain comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 9. 5.CAR, a) CD28, CD8, and / or CD4 transmembrane domains; b) an IgG1 hinge, an LNGFR spacer, or an mCH2CH3 spacer; c) CD28 and / or 4-1BB costimulatory domains, and / or d) A CAR described in any one of paragraphs 1 to 4, comprising a CD3 zeta signaling domain. 6. The CAR of any one of paragraphs 1 to 5, wherein the CAR comprises an IgG1 hinge comprising a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 10. 7. A CAR described in any one of paragraphs 1 to 5, wherein the CAR comprises a LNGFR spacer comprising a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 11, 27, 32, or 33. 8. The CAR of any one of paragraphs 1 to 5, wherein the CAR comprises a mCH2CH3 spacer comprising a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 28. 9. The CAR of any one of paragraphs 1 to 8, wherein the CAR comprises a CD3 zeta signaling domain comprising a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 13. 10. The CAR of any one of paragraphs 1 to 9, wherein the CAR comprises a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 9, 14, 15, 23, 24, or 47. 11. Single-chain variable fragments (scFv), a) a heavy chain variable region (VH) complementarity determining region (CDR) having the following sequences: CDR1-DHTIHWMR (SEQ ID NO: 1), CDR2-YIYPRDGITGYNERFRGK (SEQ ID NO: 2), and CDR3-WGYSYRNYAYYYDYWGQGTL (SEQ ID NO: 3), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) An scFv comprising light chain variable region (VL) CDRs having the following sequences: CDR1-INCRSSQSLLHSSNQR (SEQ ID NO: 4), CDR2-PPKVLIYWASTRES (SEQ ID NO: 5), and CDR3-QQYYSYPWTFGQ (SEQ ID NO: 6), or variants thereof each having up to three amino acid substitutions, additions, or deletions. 12. The scFv according to paragraph 11, wherein the scFv comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 9. 13. A polynucleotide comprising one or more nucleotide sequences encoding a CAR according to any one of paragraphs 1 to 10, or an scFv according to paragraph 11 or 12. 14. A polynucleotide comprising: a) a sequence encoding an scFv comprising the sequence of SEQ ID NO: 16, and / or b) a sequence encoding a CAR comprising any one of SEQ ID NOs: 16 to 18, 25 to 26, 51, or 85; or a variant thereof, each having at least 75% sequence identity thereto. 15. A vector comprising a polynucleotide according to paragraph 13 or 14. 16. A cell comprising a CAR according to any one of paragraphs 1 to 10, an scFv according to paragraph 11 or 12, a polynucleotide according to paragraph 13 or 14, or a vector according to paragraph 15. 17. The cell of paragraph 16, wherein the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, an invariant NK T cell, a cytokine-induced killer (CIK) cell, and a macrophage, and optionally the cell is an autologous or allogeneic cell. 18. A pharmaceutical composition comprising a CAR described in any one of paragraphs 1 to 10, an scFv described in paragraph 11 or 12, a polynucleotide described in paragraph 13 or 14, a vector described in paragraph 15, or a cell described in paragraph 16 or 17. 19. A CAR according to any one of paragraphs 1 to 10, an scFv according to paragraph 11 or 12, a polynucleotide according to paragraph 13 or 14, a vector according to paragraph 15, a cell according to paragraph 16 or 17, or a pharmaceutical composition according to paragraph 18, for use in therapy. 20. The CAR, scFv, polynucleotide, vector, cell, or pharmaceutical composition for use according to paragraph 19, wherein the therapy is treatment of cancer. twenty one. a) the cancer is a primary cancer, optionally gastrointestinal cancer, colorectal cancer, pancreatic cancer, and / or gastric cancer; b) the cancer is a second cancer, optionally liver metastasis, optionally liver metastasis of colorectal cancer and / or liver metastasis of pancreatic ductal adenocarcinoma (PDAC), and / or c) The CAR, scFv, polynucleotide, vector, cell, or pharmaceutical composition for use according to paragraph 19 or 20, wherein the cancer is a neuroendocrine tumor. 22. Use of an scFv according to paragraph 11 or 12 for determining the level of cadherin-17 (CDH-17) in a sample, optionally wherein the sample is derived from a subject. 23. A method for identifying a subject suitable for treatment with an anti-CDH-17 therapy, the method comprising determining the CDH-17 expression level in a sample isolated from the subject, wherein the CDH-17 expression level is determined using an scFv described in paragraph 11 or 12. 24. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain is: a) a heavy chain variable region (VH) complementarity determining region (CDR) having the following sequences: CDR1-DYYMY (SEQ ID NO: 42), CDR2-SISFDGTYTYYTDRVKG (SEQ ID NO: 43), and CDR3-DRPAWFPY (SEQ ID NO: 44), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) A CAR comprising light chain variable region (VL) CDRs having the following sequences: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO: 39), CDR2-KVSNRFS (SEQ ID NO: 40), and CDR3-FQGSHVPLT (SEQ ID NO: 41), or variants thereof each having up to three amino acid substitutions, additions, or deletions. 25. The antigen-binding domain is a) a VH domain comprising the sequence of SEQ ID NO: 58, and b) a VL domain comprising the sequence of SEQ ID NO: 59; or a variant thereof, each having at least 75% sequence identity thereto. 26. The CAR of paragraph 24 or 25, wherein the antigen-binding domain comprises a single-chain variable fragment (scFv). 27. The CAR of any one of paragraphs 24 to 26, wherein the antigen-binding domain comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 45. 28.CAR, a) CD28, CD8, and / or CD4 transmembrane domains; b) an IgG1 hinge, an LNGFR spacer, or an mCH2CH3 spacer; c) CD28 and / or 4-1BB costimulatory domains, and / or d) A CAR described in any one of paragraphs 24 to 27, comprising a CD3 zeta signaling domain. 29. The CAR according to any one of paragraphs 24 to 28, wherein the CAR comprises an IgG1 hinge comprising a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 10. 30. A CAR described in any one of paragraphs 24 to 28, wherein the CAR comprises a LNGFR spacer comprising a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 11, 27, 32, or 33. 31. A CAR according to any one of paragraphs 24 to 28, wherein the CAR comprises a mCH2CH3 spacer comprising a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 28. 32. A CAR described in any one of paragraphs 24 to 31, wherein the CD3 zeta signaling domain comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 13. 33. A CAR according to any one of paragraphs 24 to 32, wherein the CAR comprises a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 48 to 50. 34. Single-chain variable fragments (scFv), a) a heavy chain variable region (VH) complementarity determining region (CDR) having the following sequences: CDR1-DYYMY (SEQ ID NO: 42), CDR2-SISFDGTYTYYTDRVKG (SEQ ID NO: 43), and CDR3-DRPAWFPY (SEQ ID NO: 44), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) An scFv comprising light chain variable region (VL) CDRs having the following sequences: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO: 39), CDR2-KVSNRFS (SEQ ID NO: 40), and CDR3-FQGSHVPLT (SEQ ID NO: 41), or variants thereof each having up to three amino acid substitutions, additions, or deletions. 35. The scFv according to paragraph 34, wherein the scFv comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 45. 36. A polynucleotide comprising one or more nucleotide sequences encoding a CAR according to any one of paragraphs 24 to 33, or an scFv according to paragraph 34 or 35. 37. A polynucleotide is a) a sequence encoding an scFv comprising the sequence of SEQ ID NO: 46, and / or b) a sequence encoding a CAR comprising any one of SEQ ID NOs: 46 and / or 52 to 54; or a variant thereof, each having at least 75% sequence identity thereto. 38. A vector comprising a polynucleotide according to paragraph 36 or 37. 39. A cell comprising a CAR according to any one of paragraphs 24 to 33, an scFv according to paragraph 34 or 35, a polynucleotide according to paragraph 36 or 37, or a vector according to paragraph 38. 40. The cell of paragraph 39, wherein the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, an invariant NK T cell, a cytokine-induced killer (CIK) cell, and a macrophage, and optionally the cell is an autologous or allogeneic cell. 41. A pharmaceutical composition comprising a CAR according to any one of paragraphs 24 to 33, an scFv according to paragraph 34 or 35, a polynucleotide according to paragraph 36 or 37, a vector according to paragraph 38, or a cell according to paragraph 39 or 40. 42. A CAR according to any one of paragraphs 24 to 33, an scFv according to paragraph 34 or 35, a polynucleotide according to paragraph 36 or 37, a vector according to paragraph 38, a cell according to paragraph 39 or 40, or a pharmaceutical composition according to paragraph 41, for use in therapy. 43. The CAR, scFv, polynucleotide, vector, cell, or pharmaceutical composition for use according to paragraph 42, wherein the therapy is the treatment of cancer. 44. a) the cancer is a primary cancer, optionally gastrointestinal cancer, colorectal cancer, pancreatic cancer, and / or gastric cancer; b) the cancer is a second cancer, optionally liver metastasis, optionally liver metastasis of colorectal cancer and / or liver metastasis of pancreatic ductal adenocarcinoma (PDAC), and / or c) The CAR, scFv, polynucleotide, vector, cell, or pharmaceutical composition for use according to paragraph 42 or 43, wherein the cancer is a neuroendocrine tumor. 45. Use of an scFv according to paragraph 34 or 35 for determining the level of cadherin-17 (CDH-17) in a sample, optionally wherein the sample is derived from a subject. 46. ​​A method for identifying a subject suitable for treatment with anti-CDH-17 therapy, the method comprising determining the CDH-17 expression level in a sample isolated from the subject, wherein the CDH-17 expression level is determined using an scFv described in paragraph 34 or 35.

Claims

1. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises: (i) a) heavy chain variable region (VH) complementarity determining regions (CDRs) having the following sequences: CDR1-DHTIHWMR (SEQ ID NO: 1), CDR2-YIYPRDGITGYNERFRGK (SEQ ID NO: 2), and CDR3-WGYSYRNYAYYYDYWGQGTL (SEQ ID NO: 3), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) light chain variable region (VL) CDRs having the following sequences: CDR1-INCRSSQSLLHSSNQR (SEQ ID NO: 4), CDR2-PPKVLIYWASTRES (SEQ ID NO: 5), and CDR3-QQYYSYPWTFGQ (SEQ ID NO: 6), or variants thereof each having up to three amino acid substitutions, additions, or deletions; or (ii) a) a heavy chain variable region (VH) complementarity determining regions (CDRs) having the following sequences: CDR1-DYYMY (SEQ ID NO: 42), CDR2-SISFDGTYTYYTDRVKG (SEQ ID NO: 43), and CDR3-DRPAWFPY (SEQ ID NO: 44), or variants thereof having up to three amino acid substitutions, additions, or deletions, each of which has up to three amino acid substitutions, additions, or deletions; and b) a light chain variable region (VL) CDRs having the following sequences: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO: 39), CDR2-KVSNRFS (SEQ ID NO: 40), and CDR3-FQGSHVPLT (SEQ ID NO: 41), or variants thereof having up to three amino acid substitutions, additions, or deletions, each of which has up to three amino acid substitutions, additions, or deletions.

2. the antigen-binding domain a) a VH domain comprising the sequence of SEQ ID NO: 7 and a VL domain comprising the sequence of SEQ ID NO: 8, or a variant thereof, each having at least 75% sequence identity thereto; or b) The CAR of claim 1, comprising a VH domain comprising the sequence of SEQ ID NO: 58 and a VL domain comprising the sequence of SEQ ID NO: 59, or a variant thereof, each having at least 75% sequence identity thereto.

3. 3. The CAR of claim 1 or 2, wherein the antigen-binding domain comprises a single-chain variable fragment (scFv), and optionally, the antigen-binding domain comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 9 or SEQ ID NO:

45.

4. The CAR is a) the CD28, CD8, and / or CD4 transmembrane domains; b) an IgG1 hinge, an LNGFR spacer, or an mCH2CH3 spacer; c) CD28 and / or 4-1BB costimulatory domains, and / or d) comprises a CD3 zeta signaling domain; Optionally, (i) the CAR comprises a CD28 transmembrane domain comprising a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 61; (ii) the CAR comprises an IgG1 hinge comprising a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 10; (iii) the CAR comprises an LNGFR spacer comprising a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 11, 27, 32, or 33; (iv) the CAR comprises a mCH2CH3 spacer comprising a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 28, and / or (v) The CAR of any one of claims 1 to 3, wherein the CAR comprises a CD3 zeta signaling domain comprising a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:

13.

5. The CAR of any one of claims 1 to 4, wherein the CAR comprises a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 9, 14, 15, 23, 24, 47, 48, 49, or 50.

6. A single chain variable fragment (scFv), (i) a) heavy chain variable region (VH) complementarity determining regions (CDRs) having the following sequences: CDR1-DHTIHWMR (SEQ ID NO: 1), CDR2-YIYPRDGITGYNERFRGK (SEQ ID NO: 2), and CDR3-WGYSYRNYAYYYDYWGQGTL (SEQ ID NO: 3), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) light chain variable region (VL) CDRs having the following sequences: CDR1-INCRSSQSLLHSSNQR (SEQ ID NO: 4), CDR2-PPKVLIYWASTRES (SEQ ID NO: 5), and CDR3-QQYYSYPWTFGQ (SEQ ID NO: 6), or variants thereof each having up to three amino acid substitutions, additions, or deletions; or (ii) a) heavy chain variable region (VH) complementarity determining regions (CDRs) having the following sequences: CDR1-DYYMY (SEQ ID NO: 42), CDR2-SISFDGTYTYYTDRVKG (SEQ ID NO: 43), and CDR3-DRPAWFPY (SEQ ID NO: 44), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and b) light chain variable region (VL) CDRs having the following sequences: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO: 39), CDR2-KVSNRFS (SEQ ID NO: 40), and CDR3-FQGSHVPLT (SEQ ID NO: 41), or variants thereof each having up to three amino acid substitutions, additions, or deletions; Optionally, the scFv comprises a sequence having at least 75% sequence identity to the sequence of SEQ ID NO:9 or SEQ ID NO:

45.

7. A polynucleotide comprising one or more nucleotide sequences encoding the CAR according to any one of claims 1 to 5, or the scFv according to claim 6.

8. A vector comprising the polynucleotide of claim 7.

9. A cell comprising the CAR of any one of claims 1 to 5, the scFv of claim 6, the polynucleotide of claim 7, or the vector of claim 8, optionally wherein the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, an invariant NK T cell, a cytokine-induced killer (CIK) cell, and a macrophage, and further optionally wherein the cell is an autologous or allogeneic cell.

10. A pharmaceutical composition comprising the CAR according to any one of claims 1 to 5, the scFv according to claim 6, the polynucleotide according to claim 7, the vector according to claim 8, or the cell according to claim 9.

11. 11. The CAR according to any one of claims 1 to 5, the scFv according to claim 6, the polynucleotide according to claim 7, the vector according to claim 8, the cell according to claim 9, or the pharmaceutical composition according to claim 10, for use in therapy.

12. 12. The CAR, scFv, polynucleotide, vector, cell, or pharmaceutical composition for use according to claim 11, wherein the therapy is the treatment of cancer.

13. a) the cancer is a primary cancer, optionally gastrointestinal cancer, colorectal cancer, pancreatic cancer, and / or gastric cancer; b) the cancer is a secondary cancer, optionally a liver metastasis, optionally a liver metastasis of colorectal cancer and / or a liver metastasis of pancreatic ductal adenocarcinoma (PDAC), and / or c) The CAR, scFv, polynucleotide, vector, cell, or pharmaceutical composition for use according to claim 11 or 12, wherein the cancer is a neuroendocrine tumor.

14. 10. Use of the scFv of claim 6 for determining the level of cadherin-17 (CDH-17) in a sample, optionally wherein the sample is derived from a subject.

15. 10. A method for identifying a subject suitable for treatment with an anti-CDH-17 therapy, said method comprising determining a CDH-17 expression level in a sample isolated from said subject, wherein said CDH-17 expression level is determined using the scFv of claim 6.

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