Engineered cancer antigens with modified domains and related methods and uses

Abstract

Provided are engineered cancer antigens and uses of such engineered cancer antigens for treating cancer, such as in combination with a chimeric antigen receptor therapy that recognizes the engineered cancer antigen on tumor cells.

Description

ENGINEERED CANCER ANTIGENS WITH MODIFIED DOMAINS AND RELATED METHODS AND USESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 552,039 filed on February 9, 2024, entitled “ENGINEERED CANCER ANTIGENS AND RELATED METHODS AND USES” and U.S. Provisional Patent Application No. 63 / 726,226 filed on November 27, 2024, entitled “ENGINEERED CANCER ANTIGENS WITH MODIFIED DOMAINS AND RELATED METHODS AND USES”, the contents of which are incorporated by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 307612001940SeqList.XML created February 10, 2025, which is 323,917 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.FIELD

[0003] The present disclosure generally relates to engineered cancer antigens and uses of such engineered cancer antigens for treating cancer, such as in combination with a chimeric antigen receptor therapy that recognizes the engineered cancer antigen on tumor cells.BACKGROUND

[0004] Adoptive transfer of modified chimeric-antigen-receptor (CAR)-bearing T lymphocytes against tumor-associated antigens (TAAs) has shown promising results against several hematological malignancies (Rossig et al., Mol Ther. (2004) 10(l):5- 18). Despite their potential, these therapies have not been successfully adapted to the treatment of solid tumors due to several factors. The primary factors include, for instance, CAR-T cell immunosuppression in the tumor microenvironment, limited migration of T cells to the tumor site, and “on-target, off- tumor” toxicity (Wang et al., Front Med. (2020) 14(6):726-45; Tahmasebi et al., Stem Cell Rev Rep. (2019) 15(5):619— 36).SUMMARY

[0005] Provided herein is an engineered cancer antigen, comprising a target domain and a tumor targeting molecule, wherein the target domain is the extracellular domain or a truncated portion thereof of a target antigen. In some of any of the provided embodiments, the engineered cancer antigen is secretable. In some of any of the provided embodiments, the tumor targeting molecule binds to a tumor-associated antigen expressed on the surface of a tumor cell. In some of any of the provided embodiments, the tumor-associated antigen is selected from the group consisting of EpCAM, CEA (Carcinoembryonic antigen), gpA33 (Glycoprotein A33 (Transmembrane)), mucins, TAG-72 (Tumor-associated glycoprotein 72), CAIX (Carbonic anhydrase IX), PSMA (Pro state- specific membrane antigen), and FBP (Folate-binding protein), EGFR / ERBB1 / HER1 (epidermal growth factor receptor 1), ERBB3 (epidermal growth factor receptor 3), MET (Tyrosine-Protein Kinase IGF1R (insulin-like growth factor 1 receptor), EPHA3 (EPH Receptor A3), TRAILR1 (Death receptor 4), and RANK-L (Receptor activator of nuclear factor kappa-B ligand). In some of any of the provided embodiments, the tumor cell is a tumor cell of a solid tumor. In some of any of the provided embodiments, the tumor cell is a tumor cell of a cancer selected from the group consisting of multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, medulloblastoma, liver cancer, stomach cancer, thyroid cancer, bile duct cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, endometrial cancer, and cervical cancer. In some of any of the provided embodiments, the tumor cell is a tumor cell of a cancer selected from the group consisting of bladder cancer, breast cancer, skin cancer, head and neck cancer, colorectal cancer, endometrial cancer, liver cancer, kidney cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, thyroid cancer, and ovarian cancer.

[0006] In some of any of the provided embodiments, the tumor targeting molecule comprises an antibody or an antigen-binding fragment thereof. In some of any of the provided embodiments, the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv), DARPin, variable heavy (VH) domain, or VHH, more optionally is a singlechain variable fragment (scFv).

[0007] In some of any of the provided embodiments, the tumor-associated antigen is EpCAM. In some of any of the provided embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region,wherein the VH region comprises a heavy chain complementarity determining region 1 (CDR- Hl), a CDR-H2, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NOS: 15, 16, and 17, respectively, and the VL region comprises a light chain complementarity determining region 1 (CDR-L1), a CDR-L2, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NOS: 18, 19, and 20, respectively. In some of any of the provided embodiments, the antibody or an antigen-binding fragment thereof comprises a VH region comprising an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10; and a VL region comprising an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some of any of the provided embodiments, the antibody or an antigen-binding fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 10, and a VL region comprising the amino acid sequence of SEQ ID NO: 8.

[0008] In some of any of the provided embodiments, the tumor domain and the tumor targeting molecule are directly linked. In some of any of the provided embodiments, the tumor domain and the tumor targeting molecule are linked by a linker. In some of any of the provided embodiments, the linker has a length of between 1 and 100 amino acids, between 1 and 75 amino acids, between 1 and 50 amino acids, between 1 and 25 amino acids, between 5 and 100 amino acids, between 5 and 75 amino acids, between 5 and 50 amino acids, between 5 and 25 amino acids, between 10 and 100 amino acids, between 10 and 75 amino acids, between 10 and 50 amino acids, or between 10 and 25 amino acids. In some of any of the provided embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOS: 9 and 138-144.

[0009] In some of any of the provided embodiments, the target antigen is a membrane- expressed antigen. In some of any of the provided embodiments, the target antigen is a cell surface receptor. In some of any of the provided embodiments, the target domain is recognizable by a cognate binder. In some of any of the provided embodiments, the target domain is a truncated portion of an extracellular domain of the target antigen that retains an epitope recognizable by a cognate binder. In some of any of the provided embodiments, the target antigen is selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104,CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyro sine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein-coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), R0R1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

[0010] In some of any of the provided embodiments, the target antigen is BCMA. In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 3. In some of any of the provided embodiments, the target domain is recognizable by a cognate binder comprising: a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 32; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 35.

[0011] In some of any of the provided embodiments, the target antigen is CD70. In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 106. In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 107 or 124.

[0012] In some of any of the provided embodiments, the target antigen is CD 19. In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 71. In some of any of the provided embodiments, the target domain is recognizable by a cognate binder comprising: (a) a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable (VL) region comprising the amino acidsequence of SEQ ID NO: 47; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 48 or 49; or (b) a VH region comprising the amino acid sequence of SEQ ID NO: 50 and a VL region comprising the amino acid sequence of SEQ ID NO: 51; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 52.

[0013] In some of any of the provided embodiments, the target antigen is CD20 (e.g., CD20 extracellular domain). In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 72. In some of any of the provided embodiments, the target domain is recognizable by a cognate binder comprising: a VH region comprising the amino acid sequence of SEQ ID NO: 53 and a VL region comprising the amino acid sequence of SEQ ID NO: 54; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 55.

[0014] In some of any of the provided embodiments, the target antigen is CD22. In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 73. In some of any of the provided embodiments, the target domain is recognizable by a cognate binder comprising: (a) a VH region comprising the amino acid sequence of SEQ ID NO: 56 and a VL region comprising the amino acid sequence of SEQ ID NO: 57; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 58; or (b) a VH region comprising the amino acid sequence of SEQ ID NO: 59 and a VL region comprising the amino acid sequence of SEQ ID NO: 60; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 61.

[0015] In some of any of the provided embodiments, the target antigen is CD70. In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 124. In some of any of the provided embodiments, the target domain is recognizable by a cognate binder comprising: a VH region comprising the amino acid sequence of SEQ ID NO: 125 and a VL region comprising the amino acid sequence of SEQ ID NO: 126; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 127.

[0016] In some of any of the provided embodiments, the cognate binder is a chimeric antigen receptor (CAR) that binds to the target domain. In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of a cell surface protein antigen selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyro sine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein- coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancertestis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostatespecific membrane antigen (PSMA), R0R1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

[0017] In some of any of the provided embodiments, the engineered cancer antigen does not comprise an intracellular domain.

[0018] Provided herein is an engineered cancer antigen comprising a target domain and a heterologous membrane targeting domain, wherein the target domain is an extracellular domain or a truncated portion thereof of a target antigen. In some of any of the provided embodiments, the engineered cancer antigen is membrane-bound when expressed from a cell. In some of any of the provided embodiments, the heterologous membrane targeting domain is a heterologous transmembrane domain. In some of any of the provided embodiments, the heterologous transmembrane domain is derived from the transmembrane domain of the alpha, beta, or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, CD1 la, CD18, ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49d, ITGA6, VLA-6, CD49f, ITGAD, CD1D, ITGAE, CD103, ITGAL, ITGAM, CD1B, ITGAX, CD1C, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, CD229, CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44,NKp30, NKp46, NKG2D, or NKG2C. In some of any of the provided embodiments, the transmembrane domain is a CD8 transmembrane domain. In some of any of the provided embodiments, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 4.

[0019] In some of any of the provided embodiments, the target domain and the heterologous membrane targeting domain are each derived from a different cell surface receptor. In some of any of the provided embodiments, the target antigen is a membrane-expressed antigen. In some of any of the provided embodiments, the target antigen is a cell surface receptor. In some of any of the provided embodiments, the target domain is recognizable by a cognate binder. In some of any of the provided embodiments, the target domain is a truncated portion of an extracellular domain of the target antigen that retains an epitope recognizable by a cognate binder. In some of any of the provided embodiments, the target antigen is selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyro sine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein-coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

[0020] In some of any of the provided embodiments, the target antigen is BCMA. In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQID NO: 3. In some of any of the provided embodiments, wherein the target domain is recognizable by a cognate binder comprising: a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 32; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 35.

[0021] In some of any of the provided embodiments, the target antigen is CD70. In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 106. In some of any of the provided embodiments, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 107 or 124. In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 124. In some of any of the provided embodiments, the target domain is recognizable by a cognate binder comprising: a VH region comprising the amino acid sequence of SEQ ID NO: 125 and a VL region comprising the amino acid sequence of SEQ ID NO: 126; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 127.

[0022] In some of any of the provided embodiments, the target antigen is CD 19. In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 71. In some of any of the provided embodiments, the target domain is recognizable by a cognate binder comprising: (a) a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 47; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 48 or 49; or (b) a VH region comprising the amino acid sequence of SEQ ID NO: 50 and a VL region comprising the amino acid sequence of SEQ ID NO: 51; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 52.

[0023] In some of any of the provided embodiments, the target antigen is CD20. In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 72. In some of any of the provided embodiments, the target domain is recognizable by a cognate binder comprising: a VH region comprising the amino acid sequence of SEQ ID NO: 53 and a VL region comprising the amino acid sequence of SEQ ID NO: 54; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 55.

[0024] In some of any of the provided embodiments, the target antigen is CD22. In some of any of the provided embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 73. In some of any of the provided embodiments, the target domain is recognizable by acognate binder comprising: (a) a VH region comprising the amino acid sequence of SEQ ID NO: 56 and a VL region comprising the amino acid sequence of SEQ ID NO: 57; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 58; or (b) a VH region comprising the amino acid sequence of SEQ ID NO: 59 and a VL region comprising the amino acid sequence of SEQ ID NO: 60; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 61.

[0025] In some of any of the provided embodiments, the cognate binder is a chimeric antigen receptor (CAR) that binds to the target domain. In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of a cell surface protein antigen selected from the group consisting of carbonic anhydrase DC (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P- selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyro sine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein- coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancertestis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostatespecific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

[0026] In some of any of the provided embodiments, the engineered cancer antigen does not comprise an intracellular domain. In some of any of the provided embodiments, the engineered cancer antigen further comprises an intracellular domain. In some of any of the provided embodiments, the intracellular domain is a heterologous intracellular domain from the target antigen or is a truncated portion thereof. In some of any of the provided embodiments, theintracellular domain is a truncated and / or is a non-functional intracellular domain. In some of any of the provided embodiments, the intracellular domain comprises a death domain. In some of any of the provided embodiments, the death domain is derived from a death receptor selected from the group consisting of FAS (CD95), tumor necrosis factor receptor superfamily member 10A (TNFRSF10A; also known as death receptor 4 (DR4), DR5, TNFR1, DR3, Leucine-rich repeat and death domain-containing protein 1, Ankyrin repeat and death domain-containing protein IB, Interleukin- 1 receptor-associated kinase- like 2, Tumor necrosis factor receptor superfamily member 21, Netrin receptor UNC5A, Netrin receptor UNC5B, Netrin receptor UNC5C, Netrin receptor UNC5D, UNC5C-like protein, Tumor necrosis factor receptor superfamily member 16, Ankyrin-1, Ankyrin-2, Ankyrin-3, Nuclear factor NF-kappa-B pl05 subunit, Tumor necrosis factor receptor superfamily member 6, Interleukin- 1 receptor-associated kinase 1, Death-associated protein kinase 1, Death domain-containing protein CRADD, Nuclear factor NF-kappa-B plOO subunit, FAS-associated death domain protein (FADD), Receptorinteracting serine / threonine-protein kinase 1, Ankyrin repeat and death domain-containing protein 1A, Death domain-containing protein 1, Ectodysplasin-A receptor-associated adapter protein, Tumor necrosis factor receptor superfamily member 25, THO complex subunit 1, Myeloid differentiation primary response protein MyD88, p53-induced death domain-containing protein 1, and interleukin-1 receptor-associated kinase 3. In some of any of the provided embodiments, the death domain is derived from FAS, FADD, or TNFRSF10A. In some of any of the provided embodiments, the death domain is derived from FAS. In some of any of the provided embodiments, the death domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 93, 97, and 99. In some of any of the provided embodiments, the death domain is derived from FADD. In some of any of the provided embodiments, the death domain comprises the amino acid sequence of SEQ ID NO: 101. In some of any of the provided embodiments, the death domain is derived from TNFRSF10A. In some of any of the provided embodiments, the death domain comprises the amino acid sequence of SEQ ID NO: 103 or 105.

[0027] Provided herein is a cell expressing any of the provided engineered cancer antigens. Also provided herein is a cell expressing any of the provided engineered cancer antigens and comprising any of the providing engineered cancer antigens.

[0028] In some of any of the provided embodiments, the cell is a tumor cell. In some of any of the provided embodiments, the tumor cell is a tumor cell of a cancer selected from the group consisting of non-Hodgkin’s lymphoma (NHL), diffuse large cell lymphoma (DLCL), diffuse large B cell lymphoma (DLBCL), Hodgkin’s lymphoma, multiple myeloma, renal cellcarcinoma (RCC), neuroblastoma, skin cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, medulloblastoma, liver cancer, stomach cancer, thyroid cancer, bile duct cancer, liver cancer, bone cancer, colon cancer, rectal cancer, endometrial cancer, and cervical cancer. In some of any of the provided embodiments, the tumor cell is a tumor cell of a cancer selected from the group consisting of bladder cancer, breast cancer, skin cancer, head and neck cancer, colorectal cancer, endometrial cancer, liver cancer, kidney cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, thyroid cancer, and ovarian cancer.

[0029] Provided herein is a polynucleotide encoding any of the provided engineered cancer antigens. Also provided herein is a polynucleotide comprising a nucleic acid sequence encoding any of the engineered cancer antigens provided herein and a nucleic acid sequence encoding any of the provided engineered cancer antigens.

[0030] In some of any of the provided embodiments, the nucleic acid sequence encoding the engineered cancer antigen are separated by a cleavable linker. In some of any of the provided embodiments, the cleavable linker is a self-cleaving linker. In some of any of the provided embodiments, the self-cleaving linker is or comprises a 2A peptide.Provided herein is a vector comprising any of the provided polynucleotides. In some of any of the provided embodiments, the vector is for delivery of the polynucleotide to a cancer cell. In some of any of the provided embodiments, the vector is for delivery specifically to a blood cancer cell. In some of any of the provided embodiments, the vector is for delivery specifically to a solid tumor cancer cell. In some of any of the provided embodiments, the vector is a viral vector.

[0031] In some of any of the provided embodiments, the viral vector is an oncolytic virus. In some of any of the provided embodiments, the oncolytic virus is selected from the group consisting of an adenovirus, a herpes simplex virus, a vaccinia virus, a mumps virus, a newcastle disease virus, a poliovirus, a seneca valley virus, a measles virus, a sindbis virus, a parvovirus, a coxsackie virus, a vesicular stomatitis virus, a reovirus, and a maraba and rhabdovirus.

[0032] In some of any of the provided embodiments, the viral vector exhibits tropism to a tumor cell. In some of any of the provided embodiments, the viral vector enters a cell by binding to a cell surface receptor expressed by a tumor cell. In some of any of the provided embodiments, the viral vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, or a herpes simplex viral (HSV) vector. In some of any ofthe provided embodiments, the viral vector is an adenoviral vector. In some of any of the provided embodiments, the viral vector is a lentiviral vector.

[0033] Provided herein is a pharmaceutical composition comprising any of the provided polynucleotides or any of the provided vectors. In some of any of the provided embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0034] Provided herein is a combination comprising any of the provided vectors and a population of lymphocytes expressing a chimeric antigen receptor (CAR) that recognizes the target domain of the engineered cancer antigen.

[0035] Provided herein is a combination comprising a vector comprising a polynucleotide encoding an engineered cancer antigen comprising a target domain and a membrane targeting domain, wherein the target domain is an extracellular domain or a truncated portion thereof of a target antigen and a population of lymphocytes expressing a chimeric antigen receptor (CAR) that recognizes the target domain of the engineered cancer antigen.

[0036] In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of a cell surface protein antigen selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand- 1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein-coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappalight chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelialgrowth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

[0037] In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of BCMA. In some of any of the provided embodiments, the CAR comprises: a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 32; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 35.

[0038] In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of CD19. In some of any of the provided embodiments, the CAR comprises: (a) a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 47; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 48 or 49; or (b) a VH region comprising the amino acid sequence of SEQ ID NO: 50 and a VL region comprising the amino acid sequence of SEQ ID NO: 51; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 52.

[0039] In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of CD20. In some of any of the provided embodiments, the CAR comprises: a VH region comprising the amino acid sequence of SEQ ID NO: 53 and a VL region comprising the amino acid sequence of SEQ ID NO: 54; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 55.

[0040] In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of CD22. In some of any of the provided embodiments, (a) a VH region comprising the amino acid sequence of SEQ ID NO: 56 and a VL region comprising the amino acid sequence of SEQ ID NO: 57; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 58; or (b) a VH region comprising the amino acid sequence of SEQ ID NO: 59 and a VL region comprising the amino acid sequence of SEQ ID NO: 60; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 61.

[0041] In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of CD70. In some of any of the provided embodiments, the CAR comprises: a VH region comprising the amino acid sequence of SEQ ID NO: 125 and a VL region comprising the amino acid sequence of SEQ ID NO: 126; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 127.

[0042] Provided herein is a kit comprising any of the provided pharmaceutical compositions and instructions for using the polynucleotide or the vector. In some of any of the provided embodiments, the provided kit further comprises instructions for using the vector and the population of lymphocytes.

[0043] Provided herein is a method of treatment comprising administering a therapeutically effective amount of any of the provided polynucleotides, any of the provided vectors, or any of the provided pharmaceutical compositions, to a subject having a cancer, and administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) that bind to the target domain of the engineered cancer antigen.

[0044] Provided herein is a method of treatment comprising administering a therapeutically effective amount of a vector comprising a polynucleotide encoding an engineered cancer antigen comprising a target domain and a membrane targeting domain, wherein the target domain is an extracellular domain or a truncated portion thereof of a target antigen, to a subject having a cancer, and administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) that bind to the target domain of the engineered cancer antigen.

[0045] Provided herein is a method of treatment comprising administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) to a subject having a cancer who was previously administered any of the provided polynucleotides, any of the provided vectors, or any of the provided pharmaceutical compositions, wherein the CAR binds to the target domain of the engineered cancer antigen.

[0046] Provided herein is a method of treatment comprising administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) to a subject having a cancer who was previously administered a vector comprising a polynucleotide encoding an engineered cancer antigen comprising a target domain and a membrane targeting domain, wherein the target domain is an extracellular domain or a truncated portion thereof of a target antigen, wherein the CAR binds to the target domain of the engineered cancer antigen.

[0047] In some of any of the provided embodiments, the lymphocytes are T cells. In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of a cell surface protein antigen selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74,CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand- 1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyro sine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein-coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), R0R1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

[0048] In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of BCMA. In some of any of the provided embodiments, the CAR comprises: a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 32; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 35.

[0049] In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of CD19. In some of any of the provided embodiments, the CAR comprises: (a) a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 47; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 48 or 49; or (b) a VH region comprising the amino acid sequence of SEQ ID NO: 50 and a VL region comprising the amino acid sequence of SEQ ID NO: 51; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 52.

[0050] In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of CD20. In some of any of the provided embodiments, theCAR comprises: a VH region comprising the amino acid sequence of SEQ ID NO: 53 and a VL region comprising the amino acid sequence of SEQ ID NO: 54; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 55.

[0051] In some of any of the provided embodiments, the CAR binds to an extracellular domain or truncated portion thereof of CD22. In some of any of the provided embodiments, the CAR comprises: (a) a VH region comprising the amino acid sequence of SEQ ID NO: 56 and a VL region comprising the amino acid sequence of SEQ ID NO: 57; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 58; or (b) a VH region comprising the amino acid sequence of SEQ ID NO: 59 and a VL region comprising the amino acid sequence of SEQ ID NO: 60; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 61.

[0052] In some of any of the provided embodiments, the target antigen is CD70. In some of any of the provided embodiments, the target domain is recognizable by a cognate binder comprising: a VH region comprising the amino acid sequence of SEQ ID NO: 125 and a VL region comprising the amino acid sequence of SEQ ID NO: 126; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 127.

[0053] Provided herein is a method of tagging a tumor cell in vivo, comprising contacting the tumor cell with any of the provided polynucleotides, any of the provided vectors, or any of the provided the pharmaceutical compositions.

[0054] Provided herein is a method of tagging a tumor cell of a subject having a cancer, comprising administering a therapeutically effective amount of any of the provided polynucleotides, any of the provided vectors, or any of the provided pharmaceutical compositions, to the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG. 1 shows the design of an exemplary truncated membrane-bound BCMA-ECD target construct.

[0056] FIG. 2A shows the expression level of mCherry and BCMA in cells that were transduced with lentiviral vectors expressing the truncated membrane-bound BCMA-ECD construct, or in untransduced (“UTD”) cells. FIG. 2B shows the expression level of FSC and BCMA in cells that were transduced with the truncated membrane-bound BCMA-ECD construct, or were untransduced. A representative flow cytometry dot plot is shown with mCherry or FSC staining on the y-axis and anti-BCMA APC staining on the X-axis (FIGS. 2A and 2B).

[0057] FIG. 3A shows the killing of membrane -bound BCMA-ECD expressing A549 tumor cells by different anti-BCMA CAR-T cells. FIG. 3B shows the killing of membrane -bound BCMA-ECD expressing PC3 cells with anti-BCMA CAR-T cells.

[0058] FIG. 4 shows expression of a transgene polynucleotide encoding truncated membrane-bound BCMA-ECD in A549 tumor cells cultured with different concentrations of the AdV-truncated BCMA-ECD. Expression is shown on the y-axis and days post infection are shown on the x-axis.

[0059] FIG. 5 shows the killing of Ad3 / 11 truncated BCMA-ECD-expressing cancer cells by anti-BCMA CAR-T cells.

[0060] FIG. 6A and FIG. 6B show the killing of tumor cells infected with different concentrations (particles per cell; PPC) of truncated BCMA-ECD by anti-BCMA CAR T cells. Concentrations: 10 PPC (FIG. 6A) and 100 PPC (FIG. 6B).

[0061] FIG. 7A shows the expression of AdV-BCMA in PC3 tumor cells that were cultured with Ad3 / 11 BCMA-ECD. FIG. 7B shows the killing of PC3 tumor cells infected with Ad3 / 11 BCM A viral vectors.

[0062] FIG. 8 shows the design of an exemplary modified membrane-bound BCMA- intracellular domain with a death receptor signaling domain.

[0063] FIG. 9 shows the design of an exemplary secretable BCMA target construct.

[0064] FIG. 10A shows flow cytometry histograms depicting the expression of an exemplary secretable BCMA target construct at different dilutions on the surface of A549, HCT- 116, HT-29, and SK-OV-3 cells.

[0065] FIG. 10B shows the expression of an exemplary secretable BCMA target construct at different dilutions on the surface of HT29, HCT-116, and SK-OV-3 cells. PE-molecules / cell are shown on the y-axis and the secretable BCMA dilutions are shown on the x-axis.DETAILED DESCRIPTION

[0066] Provided herein are engineered cancer antigens for use as targets for the treatment of cancer, e.g., solid cancers, such as in combination with a CAR therapy, such as a CART cell therapy, that recognizes an extracellular target domain of the engineered cancer antigen on the tagged cancer cells. Thus, provided embodiments, relate to a CAR therapy including a cell, e.g., a population of immune effector cells, including (e.g., expressing) a chimeric antigen receptor (CAR) for use in combination with engineered cancer antigens. Also provided herein are polynucleotides encoding the engineered cancer antigens and vectors such as viral vectorscomprising the same. In some embodiments, the viral vector is a tumor-tropic vector that is able to specifically target and express the engineered cancer antigen in tumor cells. The provided embodiments permit tumor specific gene delivery in which the engineered cancer antigen is able to selectively tag tumor cells of various types with a targetable antigen. In some embodiments, the engineered cancer antigen is expressed as a membrane protein, such as containing a transmembrane domain. In some embodiments, the engineered cancer antigen is expressed as a soluble protein that is secretable from cells in which it is expressed. In some embodiments, a secretable chimeric cancer antigen tag is linked to a tumor-targeting molecule able to bind to a tumor antigen to tag cancer cells. In some embodiments, polynucleotides and / or viral vectors encode both an engineered cancer antigen membrane protein and a secretable engineered cancer antigen.

[0067] In some embodiments, upon delivery of the polynucleotide or viral vector to tumor cells with the engineered cancer antigen, the engineered cancer antigen can be recognized by a cognate binder, such as a CAR that contains an extracellular antigen binding domain for recognizing or binding the engineered cancer antigen. CAR activation directed against (i.e. contains an extracellular domain that recognizes or binds) the engineered cancer antigen initiates cytotoxic killing by cells in which it is expressed of the tumor cells. In some embodiments, the killing activates viral release so that the virus is able to infect neighboring tumor cells and spread expression of the engineered cancer antigen to other tumor cells for recognition by the CAR cell therapy. Moreover, embodiments herein also include those in which an infected cell can both express a membrane form of the engineered cancer antigen as well as secrete a soluble version of the engineered cancer antigen to tag adjacent or neighboring tumors. Such aspects further allow for improved targeting of tumor cells for recognition by CAR cell therapy. In combination with an autologous CAR cell therapy (e.g., CAR-T cells) directed against the engineered cancer antigen, provided embodiments allow for directed killing of tumor cells, including solid tumors.

[0068] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0069] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. DEFINITIONS

[0070] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0071] The terms “a”, “an”, and “the”, as used herein, include plural references unless the context clearly dictates otherwise.

[0072] The terms “or” and “and / or”, as used herein, include any, and all, combinations of one or more of the associated listed items.

[0073] The term “about”, as used herein, in reference to a number or range of numbers, is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0074] The terms “including”, “includes”, “included”, and other forms, as used herein, are not limiting.

[0075] The terms “comprise” and its grammatical equivalents, as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0076] The term “administer”, “administration”, or “administering”, as used herein refers to the act of injecting or otherwise physically delivering a substance (e.g., a pharmaceutical composition provided herein) to a subject (e.g., human), such as by oral, mucosal, topical, intradermal, parenteral, intravenous, intravitreal, intraarticular, subretinal, intramuscular, intrathecal delivery and / or any other method of physical delivery described herein or known in the art. The delivery can be systemic or to a specific tissue.

[0077] The term “antibody,” “immunoglobulin,” or “Ig” is used interchangeably herein, and is used in the broadest sense and specifically covers, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, and multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity). An antibody can be human, humanized, chimeric and / or affinity matured, as well as an antibody from other species, for example, mouse and rabbit, etc. The term “antibody” is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck, ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies or their humanized variants, and intrabodies. A “molecule derived from an antibody” refers to a functional antigen-binding fragment of any of the above. It is a portion of an antibody heavy and / or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of functional fragments include single-chain Fvs (scFv), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody. Such functional antigen-binding fragment can be found in, for example, Harlow and Fane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, ed., 1995); Huston, et al, 1993, Cell Biophysics 22:189-224; Pliickthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistrv (2d ed. 1990). The antibodies and molecules derived from antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.

[0078] The term “chimeric antigen receptor” or “CAR”, as used herein, refers to a genetically engineered receptor, which can be used to graft one or more antigen specificity onto immune effector cells, such as T cells and NK cells. CARs are also known as “artificial T-cell receptors,” “chimeric T cell receptors,” or “chimeric immune receptors.” In some embodiments, the CAR comprises an extracellular antigen binding domain specific for one or more engineered cancer antigens disclosed herein, a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptors. “CAR-T cell” refers to a T cell that expresses a CAR. “CAR- NK cell” refers to an NK cell that expresses a CAR.

[0079] The term “coding sequence” or a polynucleotide which “encodes” a polypeptide, as used herein, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A transcription termination sequence may be located 3’ to the coding sequence.

[0080] The term “constant region” or “constant domain”, as used herein, refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. This portion has a more conserved amino acid sequence relative to the variable region. The constant region may contain the CHI, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0081] The term “effective amount” or “therapeutically effective amount”, as used herein, refers to an amount of a therapeutic (e.g., a pharmaceutical composition provided herein) which is sufficient to treat, diagnose, prevent, delay the onset of, reduce and / or ameliorate the severity and / or duration of a given condition, disorder or disease and / or a symptom related thereto. The term also encompasses an amount necessary for the reduction, slowing, or amelioration of the advancement or progression of a given disease, reduction, slowing, or amelioration of the recurrence, development or onset of a given disease, and / or to improve or enhance the prophylactic or therapeutic effect (s) of another therapy or to serve as a bridge to another therapy.

[0082] The term “engineered”, as applied to a nucleic acid or a polypeptide, refers to a nucleic acid or polypeptide created using genetic engineering techniques. The term “engineered”, as applied to a cell or an organism, refers to a cell or organism that cannot be directly isolated from a source in nature. In some embodiments, the engineered nucleic acid, polypeptide, cell, or organism is altered or changed as compared to the corresponding naturally-occurring one. In some embodiments, the engineered nucleic acid, polypeptide, cell, or organism is produced by functionally linking or combining different fragments of sourcing nucleic acids, polypeptides, cells, or organisms together. For instance, an engineered polypeptide can comprise different sourcing polypeptides functionally linked together. In such examples, an engineered polypeptide may be a chimeric protein.

[0083] The term “Fab” or “Fab region”, as used herein, refers to an antibody region that binds to antigens. A conventional IgG usually comprises two Fab regions, each residing on oneof the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of each of the heavy and the light chain. More specifically, the variable region and the constant region of the heavy chain in a Fab region are VH and CHI regions, and the variable region and the constant region of the light chain in a Fab region are VE and CE regions. The VH, CHI, VE, and CL in a Fab region can be arranged in various ways to confer an antigen binding capability. For example, VH and CHI regions can be on one polypeptide, and VL and CL regions can be on a separate polypeptide, similarly to a Fab region of a conventional IgG. Alternatively, VH, CHI, VL and CL regions can all be on the same polypeptide and oriented in different orders.

[0084] The term “fragment”, as used herein, refers to a portion of a polypeptide or polynucleotide molecule containing less than the entire polypeptide or polynucleotide sequence. In some embodiments, a fragment of a polypeptide or polynucleotide comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire length of the reference polypeptide or polynucleotide. In some embodiments, a fragment of a polypeptide or polynucleotide comprises about 10%-99%, 20%-99%, 30%-99%, 40%-99%, 50%-99%, 60%-99%, 70%-99%, 80%-99%, 90%-99%, 95%-99%, 96%-99%, 97%-99%, or 98%-99%, of the entire length of the reference polypeptide or polynucleotide. In some embodiments, a polypeptide or polynucleotide fragment may contain 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more nucleotides or amino acids.

[0085] The term “heavy chain”, when used in reference to an antibody, refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy -terminal portion includes a constant region. The constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha, delta, epsilon, gamma, and mu, based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: alpha, delta, and gamma contain approximately 450 amino acids, while epsilon and mu contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3, and IgG4.

[0086] The term “light chain”, when used in reference to an antibody, refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy -terminal portion includes a constantregion. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa or lambda based on the amino acid sequence of the constant domains.

[0087] The term “leucine zipper domain”, as used herein, refers to an amphipathic a helix containing heptad repeats of Leu residues on one face of the helix and serves as a dimerization module. In some embodiments, the leucine zipper domain is an active leucine zipper domain. On dimerization with another leucine zipper domain, the leucine-zipper a helices form a parallel- coiled coil based on hydrophobic interfacial side-chain packing. In some embodiments, the leucine zipper domain is an inactive leucine zipper domain that cannot form a dimer with another leucine zipper domain.

[0088] The term “naturally-occurring”, as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is naturally occurring.

[0089] The term “oncolytic virus”, as used herein, refers to a type of virus that preferentially infects cancer cells but not non-cancer cells. Oncolytic viruses can occur naturally or can be made in the laboratory by altering and modifying existing viruses.

[0090] The term “operatively linked” and similar phrases (e.g., genetically fused), as used herein, refer to the operational linkage of nucleic acid sequences or amino acid sequences placed in functional relationships with each other. For example, a promoter operatively linked to a polynucleotide encoding a polypeptide result in the transcription of the polynucleotide and ultimately the expression of the polypeptide. As another example, an operatively linked peptide is one in which the functional domains are placed with appropriate distance from each other to impart the intended function of each domain.

[0091] The term “pharmaceutically acceptable excipient, carrier or diluent”, as used herein, refers to any substance formulated alongside the active ingredient of a pharmaceutical composition that allows the active ingredient to retain biological activity and is non-reactive with the subject's immune system. Such a substance can be included for the purpose of long-term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating absorption, reducing viscosity, or enhancing solubility. The selection of appropriate substance can depend upon the route of administration and the dosage form, as well as the active ingredient and other factors. Compositions having such substances can beformulated by well-known conventional methods (see, e.g., Remington, The Science and Practice of Pharmacy, 23rd edition, A. Adejare, ed., Academic Press, 2020).

[0092] The term “pharmaceutical composition” or “therapeutic composition”, as used here, refers to a composition capable of being administered to a subject for the treatment of a particular disease or disorder.

[0093] The term “polynucleotide” or “nucleic acid”, as used herein, refers to polymers of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. Unless specified otherwise, the left-hand end of any singlestranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double- stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction.

[0094] As used herein, the terms “nucleotide” or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA / RNA hybrids.

[0095] The terms “polypeptide” and “peptide” and “protein”, as used herein, refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art.

[0096] The term “population” of cells, as used herein, refers to any number of cells greater than 1, but is preferably at least IxlO3cells, at least IxlO4cells, at least IxlO5cells, at least IxlO6cells, at least IxlO7cells, at least IxlO8cells, at least IxlO9cells, at least IxlO10cells, at least IxlO11or more cells. A population of cells may refer to an in vitro population (e.g., a population of cells in culture) or an in vivo population (e.g., a population of cells residing in a particular tissue).

[0097] The term “sequence identity”, as used herein, refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certainpercentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. The term “reference sequence” refers to a molecule to which a test sequence is compared. Methods of sequence alignment for comparison and determination of percent sequence identity and percent complementarity are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. NatT. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology), by use of algorithms know in the art including the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0098] The term “subject”, as used herein, refers to a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkey and human). In some embodiments, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder provided herein. In some embodiment, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder provided herein. In some embodiment, the subject is human.

[0099] The term “target domain”, as applied to an engineered cancer antigen disclosed herein, refers to the extracellular domain of the engineered cancer antigen. It can be expressed on the surface of a cancer cell and be recognized by a cognate binder. It can be bound by a chimeric antigen receptor comprising the cognate binder and expressing on the surface of a cancer cell.

[0100] The term “transduced”, as used herein, refers to a process by which a transgene is introduced into a host cell from a virus particle.

[0101] The term “transgene”, as used herein, refers to any heterologous polynucleotide incorporated in a viral vector, e.g., for transcription or expression in a target cell. An example of a transgene is a polynucleotide encoding a therapeutic polypeptide or a detectable marker.

[0102] The term “transmembrane domain”, as used herein, refers to any protein structure that is thermodynamically stable in a cell membrane (e.g., a eukaryotic cell membrane). The transmembrane domain may be derived either from a natural or from a synthetic source.

[0103] The terms “treatment” and “treating”, as used herein, refer to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0104] The term “variable region”, “variable domain”, “V region”, or “V domain”, as used herein, refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as “VH.” The variable region of the light chain may be referred to as “VL.” The term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” or “complementarity determining regions” that are each about 9-12 amino acids long. The variable regions of heavy and light chains each comprise four FRs, largely adopting a P sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the P sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see, e.g., Rabat et al, Sequences of Proteins of Immunological Interest (5th ed. 1991)).

[0105] The term, “viral vector”, as used herein, refers to a nucleic acid to be delivered into a host cell via a recombinantly produced virus or viral particle. The nucleic acid may be singlestranded or double stranded, linear or circular, segmented or non-segmented. The nucleic acid may comprise DNA, RNA, or a combination thereof. Non-limiting examples of viruses or viral particles that can deliver a viral vector include retroviruses (e.g., lentiviruses and y-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses. A viral vector delivered by such viruses or viral particles may be referred to by the type of virus to deliver the viral vector (e.g., a lentiviral vector is a viral vector that is to be delivered by a lentivirus). A viral vector can contain viral elements (e.g., nucleotide sequences) necessary for packaging of the viral vector into the virus or viral particle, replicating the virus, or other desired viral activities. A virus containing a viral vector may be replication competent, replication deficient or replication defective. In some embodiments, the viral vector is delivered via an oncolytic virus.

[0106] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.II. ENGINEERED CANCER ANTIGENS AND COGNATE BINDERS

[0107] Provided herein are engineered cancer antigens that can be used to tag cancer cells for recognition by a cognate binder. In some embodiments, the engineered cancer antigen is a chimeric cancer antigen comprising heterologous domains.

[0108] In some embodiments, the engineered cancer antigens are chimeric proteins that contain a target domain that is an extracellular domain (hereinafter also called “ectodomain”) or a truncated portion thereof of a target antigen that can be recognized by a cognate binder, and one or more heterologous transmembrane domain sequence that permits display or expression of the engineered cancer antigen on the surface of a cancer cell. As such, the engineered cancerantigen with a heterologous transmembrane protein is a membrane-bound engineered cancer antigen that is capable of being membrane-associated when expressed from a cell. In provided embodiments, the target antigen is an extracellular domain or a truncated portion thereof of a cell surface membrane protein. In some embodiments, provided herein is an engineered cancer antigen comprising a target domain and a heterologous membrane targeting domain, wherein the target domain is an extracellular domain or a truncated portion thereof of a target antigen. In one embodiment, the engineered cancer antigen is a membrane-associated protein that contains the extracellular domain or a truncated portion thereof of a target antigen and a heterologous transmembrane domain. In provided embodiments, expression of the engineered cancer antigen on the surface of a cancer cell allows for exposure of the extracellular domain or truncated portion of the cancer antigen tag on the surface of the cell for recognition by a cognate binder.

[0109] In another embodiment, the engineered cancer antigen is a secretable protein that contains a target domain that is an extracellular domain or a truncated portion thereof of a target antigen that can be recognized by a cognate binder, and a tumor-targeting domain in which the extracellular domain or a truncated portion thereof is linked to the tumor targeting molecule. In such embodiments, the engineered cancer antigen is without a transmembrane protein so that it is able to be secreted from a cell in which it is expressed. As such, the engineered cancer antigen with a tumor-targeting domain is a secretable engineered cancer antigen that is capable of being targeted via the tumor-targeting domain for display on the surface of a tumor cell when it is expressed and secreted from a cell. In some embodiments, provided herein is an engineered cancer antigen comprising a target domain and a tumor targeting molecule, wherein the target domain is the extracellular domain or a truncated portion thereof of a target antigen. In such embodiments, binding of the tumor targeting molecule to a protein on the surface of a cancer cell directs the extracellular domain or truncated portion (i.e., the target domain) to the cancer cell for display of the extracellular domain or truncated portion of the cancer antigen tag on the surface of the cell for recognition by a cognate binder.

[0110] In such embodiments, the cognate binder may be any known binding molecule that is known or able to bind to the extracellular domain or truncated region of the engineered cancer antigen, such as of the membrane protein or the secretable protein. For instance, the extracellular domain or the truncated region thereof of the target antigen that is the target domain of the engineered cancer antigen contains an epitope recognized by a cognate binder. In some embodiments, the cognate binder may be contained as part of the extracellular domain of a chimeric antigen receptor (CAR).A. Target Domains

[0111] In some embodiments, the provided engineered cancer antigens contain a target domain that is or comprises an extracellular domain or a truncated portion thereof of a target antigen, e.g., a cell surface protein. In some embodiments, the target antigen is a cell surface protein. In some embodiments, the extracellular domain or a truncated portion thereof is from a native cell surface protein. Among such cell surface proteins are those that are normally expressed as membrane proteins on cells and can be targetable by cognate binders, such as antibodies (including antigen-binding fragments thereof), including those as contained as part of an antibody therapy, such as contained as part of a bispecific (e.g., BiTe), ADC or CAR T cell therapy. In some embodiments, the extracellular domain or truncated portion thereof is from a target antigen that is able to be targeted by an antibody or antigen-binding fragment (e.g., scFv) contained in the extracellular domain of a CAR.

[0112] Non-limiting examples of cell surface protein antigens include carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand- 1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyro sine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein-coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

[0113] In some embodiments, the cell surface protein target antigen is one recognized by the CAR present in any of a variety of known engineered cell products. The CAR may include, but is not limited to a CAR engineered into cells of idecabtagene vicleucel (ABECMA®), Orvacabtagene Autoleucel (JCARH125), ciltacabtagene autoleucel (CARVYKTI™, also called NJ-68284528; Janssen / Legend), P-BCMA-101 (Poseida), PBCAR269A (Poseida), P-BCMA- Allol (Poseida), Allo-715 (Pfizer / Allogene), CT053 (Carsgen), Descartes-08 (Cartesian), PHE885 (Novartis), CTX120 (CRISPR Therapeutics); axicabtagene ciloleucel (YESCARTA®), tisagenlecleucel (KYMRIAH®), brexucabtagene autoleucel (TECARTUS®), or lisocabtagene maraleucel (BREYANZI®).

[0114] In particular embodiments, the target domain is not cleavable by a gamma secretase. Gamma secretase is a multi- subunit protease complex that cleaves single -pass transmembrane proteins at residues within the transmembrane domain. In provided embodiments, the target domain contains a target domain that is not cleavable by a gamma secretase. In some embodiments, the target domain is part of a membrane engineered cancer antigen, such as described in Section II.C, that contains a heterologous transmembrane domain that is not cleavable by a gamma secretase inhibitor. In some embodiments, the target domain is part of any engineered cancer antigen that is secretable, such as described in Section II.B, that lacks a transmembrane domain so that there is no membrane association of the tag molecule.

[0115] In some embodiments, the target antigen is BCMA, such as human BCMA. B-cell maturation antigen, also known as BCMA, CD269, TNFRSF17 (e.g., wildtype BCMA, UniProt Q02223; SEQ ID NO: 129), is a member of the tumor necrosis receptor superfamily that is preferentially expressed in differentiated plasma cells (Laabi et al. (1992) EMBO J 11(11):3897- 3904; Madry et al. (1998) Int Immunol 10(11): 1693-1702). BCMA is a non-glycosylated type I transmembrane protein, which is involved in B cell maturation, growth and survival. BCMA is a receptor for two ligands of the TNF superfamily: APRIL (a proliferation-inducing ligand, CD256, TNFSF13), the high- affinity ligand to BCMA and the B cell activation factor BAFF (THANK, BlyS, B lymphocyte stimulator, TALL-1 and zTNF4), the low-affinity ligand to BCMA.

[0116] In some embodiments, the extracellular domain of BCMA or the truncated portion thereof comprises an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 3. In some embodiments, the extracellular domain of BCMA or the truncated portion thereof is the sequence of SEQ ID NO: 3that is able to be recognized or bound by a cognate binder. In some embodiments, the extracellular domain of BCMA or the truncated portion thereof of SEQ ID NO: 3 is encoded by the nucleotide sequence set forth in SEQ ID NOs: 1, 175, 176, 180 or 181.

[0117] In some embodiments, the target antigen is CD19, such as human CD19. Cluster of Differentiation 19 (CD 19) is an antigenic determinant detectable on leukemia precursor cells. The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found as UniProt / Swiss-Prot Accession No. P15391 and the nucleotide sequence encoding of the human CD19 can be found at Accession No. NM_001178098. In some embodiments, the extracellular domain of CD 19 or the truncated portion thereof is the sequence of SEQ ID NO: 71 or a truncated portion that contains an epitope that is able to be recognized or bound by a cognate binder.

[0118] In some embodiments, the target antigen is CD20. Human CD20 is also called membrane-spanning 4-domains, subfamily A, member 1 (MS4A1). The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD20 can be found at Accession Nos. NP_690605.1 (SEQ ID NO: 162) and NP_068769.2, and the nucleotide sequence encoding transcript variants 1 and 3 of the human CD20 can be found at Accession No. NM_152866.2 and NM_021950.3, respectively. CD20 is a 33-kD, Type III, tetraspan- transmembrane phosphoprotein with a 44-amino acid extracellular domain. In some embodiments, the extracellular domain of CD20 or the truncated portion thereof is the sequence of SEQ ID NO: 72 or a truncated portion that contains an epitope that is able to be recognized or bound by a cognate binder. In some embodiments, the extracellular domain of CD20 or the truncated portion thereof is the sequence of SEQ ID NO: 158 that contains an epitope that is able to be recognized or bound by a cognate binder.

[0119] In some embodiments, the target antigen is CD22. Human CD22 is also called SIGLEC2 or BL-CAM. The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD22 can be found at UniProt Accession No. P20273. CD22 has seven extracellular IgG-like domains, which are commonly identified as Ig domain 1 to Ig domain 7, with Ig domain 7 being most proximal to the B cell membrane and Ig domain 1 being the most distal from the Ig cell membrane. In some embodiments, the extracellular domain of CD22 includes amino acids Asp20 - Arg687 (Accession: P20273) or a truncated portion thereof thatcontains an epitope that is able to be recognized or bound by a cognate binder. In some embodiments, the extracellular domain of CD22 or the truncated portion thereof is the sequence of SEQ ID NO: 73 or a truncated portion that contains an epitope that is able to be recognized or bound by a cognate binder.

[0120] In some embodiments, the target antigen is Cluster of Differentiation 70 (CD70). In some embodiments, the target antigen is wild-type CD70. CD70 (also known as CD27LG or TNFSF7) is a member of the tumor necrosis factor (TNF) superfamily and the ligand for CD27, a TNF superfamily receptor. The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD70 (e.g., wildtype) can be found at UniProt Accession No. P32970 (SEQ ID NO: 106). In some embodiments, the extracellular domain of CD70 or the truncated portion thereof is the sequence of SEQ ID NO: 130 or a truncated portion that contains an epitope that is able to be recognized or bound by a cognate binder. In some embodiments, the truncated portion of the CD70 target antigen is the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 124.B. Secretable Engineered cancer antigens

[0121] In some embodiments, provided herein is an engineered cancer antigen comprising a target domain comprising at least one target domain described in Section II. A and a tumor targeting molecule (e.g., antibody or antigen binding fragment specific to a tumor-associated antigen). In some embodiments, provided herein is an engineered cancer antigen, comprising a target domain and a tumor targeting molecule, wherein the target domain is the extracellular domain or a truncated portion thereof of a target antigen. In such embodiments, it is understood that the engineered cancer antigen is thus a chimeric cancer antigen composed of sequences that are not normally present in a protein in nature. In some embodiments, the amino acid sequence of the target domain and the tumor targeting molecule are directly linked. In some embodiments, the amino acid sequence of the target domain and the tumor targeting molecule are indirectly linked via a linker, such as a peptide linker. In particular embodiments, such an engineered cancer antigen is secretable and is not membrane associated with a cell. It is thus understood that an engineered cancer antigen provided herein that contains a tumor-targeting molecule is one in which a transmembrane domain is not present, such that the engineered cancer antigen is not able to be embedded in the lipid bilayer of a cell membrane from which is expressed. In certain embodiments in which the engineered cancer antigen is expressed from a cell it can be secretedfrom the cell. In some embodiments, the engineered cancer antigen is soluble. Thus, it is understood that an engineered cancer antigen that is secretable is soluble.

[0122] In some embodiments, the tumor targeting molecule of the engineered cancer antigen is able to bind to an antigen expressed on a target cell, e.g., a tumor cell, so that the coupled target domain is exposed on the outside surface of the cell. In some embodiments, the tumor targeting molecule is an antibody or antibody fragment. In some embodiments, tumor targeting molecule is a tumor-associated antigen (TAA) binding molecule. In some embodiments, the tumor targeting molecule is an antibody or antigen binding fragment targeting a tumor- associated antigen. In some embodiments, the tumor targeting molecule is an antibody or antigen binding fragment targeting a tumor-associated antigen expressed on the surface of a tumor cell. In some embodiments, the tumor-targeting molecule is an antibody comprising means for binding to an tumor-associated antigen, such as a tumor-associated antigen expressed on the surface of a tumor cell. In some embodiments, the antigen is associated with a cancer. In some embodiments, the antigen is associated with a solid tumor.

[0123] For example, tumor-associated antigens include antigens expressed on tumors including, but are not limited to, adenoma, carcinoma, or sarcoma. In some embodiments, the tumor-associated antigen is expressed on a cancer cell, including but not limited to multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, medulloblastoma, liver cancer, stomach cancer, thyroid cancer, bile duct cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, endometrial cancer, or cervical cancer. In some embodiments, the cancer is bladder cancer, breast cancer, skin cancer, head and neck cancer, colorectal cancer, endometrial cancer, liver cancer, kidney cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, thyroid cancer, or ovarian cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is kidney cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is insensitive or resistant.

[0124] In some embodiments, the tumor-associated antigen is selected from the group consisting of EpCAM, CEA (Carcinoembryonic antigen), gpA33 (Glycoprotein A33 (Transmembrane)), mucins, TAG-72 (Tumor-associated glycoprotein 72), CAIX (Carbonic anhydrase IX), PSMA (Pro state- specific membrane antigen), and FBP (Folate-binding protein), EGFR / ERBB1 / HER1 (epidermal growth factor receptor 1), ERBB2 / HER2 (epidermal growth factor receptor 2), ERBB3 (epidermal growth factor receptor 3), MET (Tyrosine-Protein Kinase IGF1R (insulin-like growth factor 1 receptor), EPHA3 (EPH Receptor A3), TRAILR1 (Death receptor 4), and RANK-L (Receptor activator of nuclear factor kappa-B ligand).

[0125] In some embodiments, the tumor targeting molecule is a molecule derived from an antibody is one or more functional fragments of the antibody. Non-limiting examples of functional fragments include heavy chain, light chain, heavy chain variable domain, light chain variable domain, single-chain Fvs (scFv), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody. In some embodiments, the tumor targeting molecule is an antibody without the heavy chain CH3 domain. In some embodiments, the tumor targeting molecule is an antibody without the heavy chain CH2 domain. In some embodiments, the tumor targeting molecule is a Fab. In some embodiments, the tumor targeting molecule is an antibody without the heavy chain CH2 and CH3 domain. In some embodiments, the tumor targeting molecule is a single-chain variable fragment (scFv). In some embodiments, the tumor targeting molecule is the heavy chain of an antibody. In some embodiments, the tumor targeting molecule is the light chain of an antibody. In some embodiments, the tumor targeting molecule is the variable domain of the heavy chain of an antibody. In some embodiments, the tumor targeting molecule is the variable domain of the light chain of an antibody.

[0126] In some embodiments, the tumor targeting molecule is a single-chain variable fragment (scFv). As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VE) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH::VE heterodimer. The heavy (VH) and light chains (VE) are either joined directly or joined by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids), which connects the N-terminus of the VH with the C--iterminus of the VE, or the C-terminus of the VH with the N-terminus of the VE. Exemplary linkers that can be used herein are disclosed in the Examples section of the present application. Additional linkers are known to a skilled artisan. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 9.

[0127] In some embodiments, the tumor targeting molecule is a tumor targeting antibody. In some embodiments, the tumor targeting antibody binds to an antigen expressed on the surface of a tumor cell. In some embodiments, a tumor targeting antibody is an antibody or antibody fragment with means for binding to an antigen expressed on the surface of a tumor, such as any described in Table 1. In some embodiments, the tumor targeting antibody or the molecule derived from an antibody provided herein comprises VL, VH or CDRs having amino acid sequences of the VL, VH or CDR contained in certain known antibodies. In some embodiments, the tumor targeting antibody or the molecule derived from an antibody provided herein comprises VL, VH or CDRs having amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the VL, VH or CDR contained in certain known antibodies.

[0128] Exemplary tumor-associated antigens and their exemplary known antibodies include but are not limited to those described in Table 1 below.

[0129] Table 1. Exemplary tumor-associated antigens and their known antibodies.

[0130] In some embodiments, the tumor-associated antigen targeted by the tumor targeting molecule is expressed on a solid tumor.

[0131] In some embodiments, the tumor-associated antigen is expressed on the solid tumor. Exemplary solid tumor associated antigens include but are not limited to EPC AM (Epithelial cell adhesion molecule), CEA (Carcinoembryonic antigen), gpA33 (Glycoprotein A33 (Transmembrane)), mucins, TAG-72 (Tumor-associated glycoprotein 72), CAIX (Carbonic anhydrase IX), PSMA (Pro state- specific membrane antigen), and FBP (Folate-binding protein), EGFR / ERBB1 / HER1 (epidermal growth factor receptor 1), ERBB2 / HER2 (epidermal growth factor receptor 2), ERBB3 (epidermal growth factor receptor 3), MET (Tyrosine-Protein Kinase IGF1R (insulin-like growth factor 1 receptor), EPHA3 (EPH Receptor A3), TRAILR1 (Death receptor 4), MMP (matrix metalloproteinase, e.g., MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, MMP28), GPC2, GPC3 (glypican-2 and glypican-3), mesothelin, PSCA (Prostate stem cell antigen), claudin 6 and claudinl8.2, and RANK-L (Receptor activator of nuclear factor kappa-B ligand).

[0132] In some embodiments, the tumor-associated antigen targeted by the tumor targeting molecule is epithelial cell adhesion / activating molecule (EpCAM), epidermal growth factor receptor (EGFR), or human epidermal growth factor receptor 2 (HER2). In some embodiments, the tumor-associated antigen targeted by the tumor targeting molecule is EpCAM.

[0133] Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein mediating Ca2+-independent homotypic cell-cell adhesion in epithelia. EpCAM is also involved in cell signaling, migration, proliferation, and differentiation. Additionally, EpCAM has oncogenic potential via its capacity to upregulate c-myc, e-fabp, and cyclins A and E. Since EpCAM is expressed exclusively in epithelia and epithelial-derived neoplasms, EpCAM can be used as therapeutic marker for various cancers, such as head and neck cancer, ovarian cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, esophageal cancer, and lung cancer.

[0134] In some embodiments, the tumor targeting molecule is adecatumumab or an antigenbinding fragment of adecatumumab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of adecatumumab.

[0135] In some embodiments, the tumor targeting molecule is edrecolomab or an antigenbinding fragment of edrecolomab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of edrecolomab.

[0136] In some embodiments, the tumor targeting molecule is tucotuzumab or an antigenbinding fragment of tucotuzumab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of tucotuzumab.

[0137] In some embodiments, the tumor targeting molecule binds to a tumor-associated antigen that is EpCAM. In some embodiments, the tumor targeting molecule is an antibody or antigen-binding fragment thereof that binds to a tumor-associated antigen that is EpCAM.

[0138] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3 as contained in the VH set forth in SEQ ID NO: 10, and the VL region comprises a light chain complementarity determining region 1 (CDR-L1), a CDR-L2, and a CDR-L3 as contained in the CL set forth in SEQ ID NO: 8. In some embodiments, the antibody or antigenbinding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NOS: 15, 16, and 17, respectively, and the VL region comprises a light chain complementarity determining region 1 (CDR-L1), a CDR-L2, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NOS: 18, 19, and 20, respectively.

[0139] In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence of SEQ ID NO: 8. In some embodiments, the tumor targeting molecule comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10.

[0140] In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence of SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 10. In some embodiments, thetumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 10. In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 10.

[0141] In some embodiments, the VL region of SEQ ID NO: 8 is encoded by the nucleotide sequence of SEQ ID NO: 5, and the VH region of SEQ ID NO: 10 is encoded by the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the VL of SEQ ID NO: 8 and the VH of SEQ ID NO: 10 are linked via a linker comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the linker of SEQ ID NO: 9 is encoded by the nucleotide sequence set forth in SEQ ID NO: 6.

[0142] In some embodiments, the tumor-associated antigen targeted by the tumor targeting molecule is GPC2.

[0143] In some embodiments, the tumor-associated antigen targeted by the tumor targeting molecule is mesothelin.

[0144] In some embodiments, the tumor targeting molecule is Anetumab or an antigenbinding fragment of Anetumab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of Anetumab.

[0145] In some embodiments, the tumor targeting molecule is Amatuximab or an antigenbinding fragment of Amatuximab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of Amatuximab.

[0146] In some embodiments, the tumor-associated antigen targeted by the tumor targeting molecule is prostate stem cell antigen (PSCA).

[0147] In some embodiments, the tumor targeting molecule is AGS -PSCA or an antigenbinding fragment of AGS- PSCA. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of AGS- PSCA.

[0148] In some embodiments, the tumor-associated antigen targeted by the tumor targeting molecule is Claudin6.

[0149] In some embodiments, the tumor-associated antigen targeted by the tumor targeting molecule is GPC3.

[0150] In some embodiments, the tumor targeting molecule is codrituzumab or an antigenbinding fragment of codrituzumab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of codrituzumab.

[0151] In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence of SEQ ID NO: 135 (e.g., Anti-GPC3 VL). In some embodiments, the tumor targeting molecule comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 134 (e.g., Anti-GPC3 VH). In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 135. In some embodiments, the tumor targeting molecule comprises a heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 134.

[0152] In some embodiments, the tumor-associated antigen targeted by the tumor targeting molecule is Claudinl8.2.

[0153] In some embodiments, the tumor targeting molecule is Zolbetuximab or an antigenbinding fragment of Zolbetuximab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of Zolbetuximab.

[0154] In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence of SEQ ID NO: 137 (e.g., Anti-Claudin 18.2 VL). In some embodiments, the tumor targeting molecule comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 136 (e.g., Anti-Claudin 18.2 VH). In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 137. In some embodiments, the tumor targeting molecule comprises a heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 136.

[0155] In some embodiments, the tumor-associated antigen targeted by the tumor targeting molecule is MMP. In some embodiments, the tumor-associated antigen targeted by the tumor targeting molecule is selected from the groups consisting of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, MMP28. In some embodiments, the tumor-associated antigen targeted by the tumor targeting molecule is MMP9.

[0156] In some embodiments, the tumor targeting molecule is Andecaliximab or an antigenbinding fragment of Andecaliximab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of Andecaliximab.

[0157] In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence of SEQ ID NO: 133 (e.g., Anti-MMP9 VL). In some embodiments, the tumor targeting molecule comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 132 (e.g., Anti-MMP9 VH). In some embodiments, the tumor targeting molecule comprises a light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 133. In some embodiments, the tumor targeting molecule comprises a heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 132.

[0158] In some embodiments, the different elements of the secretable engineered cancer antigen are operatively linked. In some embodiments, the different elements are directly linked to each other. In some embodiments, the different elements are linked via linkers. Accordingly, in some embodiments, the extracellular domain of the target antigen and the tumor targeting molecule are directly linked. In some embodiments, the extracellular domain of the target antigen and the tumor targeting molecule are linked by a linker. Exemplary linkers can be used herein are disclosed in the Example section of the present application. Additional linkers are known to a skilled artisan.

[0159] In some embodiments, the linkers may be flexible linkers. Linkers include peptides, polymers, nucleotides, nucleic acids, polysaccharides, and lipid organic species (such as polyethylene glycol). In some embodiments, the linker is a peptide linker. Peptide linkers may be from about 2-100, 10-50, or 15-30 amino acids long. In some embodiments, peptide linkers may be at least 10, at least 15, or at least 20 amino acids long and no more than 80, no more than 90, or no more than 100 amino acids long. In some embodiments, the linker has a length of between 1 and 100 amino acids, between 1 and 75 amino acids, between 1 and 50 amino acids, between 1 and 25 amino acids, between 5 and 100 amino acids, between 5 and 75 amino acids, between 5 and 50 amino acids, between 5 and 25 amino acids, between 10 and 100 amino acids, between 10 and 75 amino acids, between 10 and 50 amino acids, or between 10 and 25 amino acids. In some embodiments, the linker has a length of between 5 and 50 amino acids.

[0160] The linkers can be naturally-occurring, synthetic or a combination of both. Particularly suitable linker polypeptides predominantly include amino acid residues selectedfrom Glycine (Gly), Serine (Ser), Alanine (Ala), and Threonine (Thr). For example, the linker may contain at least 75% (calculated on the basis of the total number of residues present in the peptide linker), such as at least 80%, at least 85%, or at least 90% of amino acid residues selected from Gly, Ser, Ala, and Thr. The linker may also consist of Gly, Ser, Ala and / or Thr residues only. In some embodiments, the linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues, or 8-12 glycine residues. In some aspects, suitable peptide linkers typically contain at least 50% glycine residues, such as at least 75% glycine residues. In some embodiments, a peptide linker comprises glycine residues only. In some embodiments, a peptide linker comprises glycine and serine residues only.

[0161] In some embodiments, these linkers are composed predominately of the amino acids Glycine and Serine, denoted as GS-linkers herein. In some embodiments, the linker contains (GGS)n, wherein n is 1 to 10, such as 1 to 5, for example 1 to 3, such as GGS(GGS)n (SEQ ID NO: 145), wherein n is 1 to 10. In particular embodiments, the linker contains the sequence (GGGGS)n (SEQ ID NO: 146), wherein n is 1 to 10 or n is 1 to 5, such as 1 to 3. In further embodiments, the linker contains (GGGGGS)n (SEQ ID NO: 147), wherein n is 1 to 4, such as 1 to 3. The linker can include combinations of any of the above, such as repeats of 2, 3, 4, or 5 GS, GGS, GGGGS, and / or GGGGGS linkers may be combined. In some embodiments, such a linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids in length.

[0162] In some embodiments, the linker is or comprises (in one-letter amino acid code): GGS, GGGGS (SEQ ID NO: 148), or GGGGGS (SEQ ID NO: 149). In some embodiments, the GS-linker is or comprises an amino acid sequence of GGSGGS, i.e., (GGS)2 (SEQ ID NO: 150); GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 151); GGSGGSGGSGGS, i.e., (GGS)4(SEQ ID NO: 152); GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 153);GGGGGSGGGGGSGGGGGS, i.e., (G5S)3(SEQ ID NO: 154), GGSGGGGSGGGGSGGGGS (SEQ ID NO: 155) and GGGGSGGGGSGGGGS (SEQ ID NO: 156). In some embodiments, the linker is or comprises GGGG (SEQ ID NO: 157). In some embodiments, the linker is or comprises GGGGG (SEQ ID NO: 142). In some embodiments, the linker is or comprises GGGGGGGG (SEQ ID NO: 143). In some of any of the above examples, serine can be replaced with alanine (e.g., (Gly4Ala) or (Gly3Ala)).

[0163] In some embodiments, the linker is or comprises GSAGSAAGSGEF (SEQ ID NO: 144). In some embodiments, the linker is or comprises GSGSGS (SEQ ID NO: 139).

[0164] In some cases, it may be desirable to provide some rigidity into the peptide linker. This may be accomplished by including proline residues in the amino acid sequence of thepeptide linker. Thus, in some embodiments, a linker comprises at least one proline residue in the amino acid sequence of the peptide linker. For example, a peptide linker can have an amino acid sequence wherein at least 25% (e.g., at least 50% or at least 75%) of the amino acid residues are proline residues. In one particular embodiment, the peptide linker comprises proline residues only. In In some embodiments, the linker is or comprises PAPAP (SEQ ID NO: 141).

[0165] In some embodiments, the linker is or comprises EAAAK (SEQ ID NO: 140).

[0166] In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 9 or 138-157, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 9 or 138-157. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 9 or 138-157. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 9. In some embodiments, the linker comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9.

[0167] In some embodiments, the linker is or comprises GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 110). In some embodiments, the linker is or comprises GGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 128).

[0168] In some embodiments, the secretable engineered cancer antigen provided herein can comprise any of the target domains described herein and any of the tumor targeting molecules described herein.

[0169] In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 164. In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 164. In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 164. In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 164. In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 164. In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 164. In some embodiments, the secretable engineered cancer antigencomprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 164. In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 164. In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 164. In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 164. In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 164. In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 164. In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence set forth in SEQ ID NO: 164. In some embodiments, the amino acid sequence of SEQ ID NO: 164 is encoded by the nucleotide sequence of SEQ ID NOs: 187-191.

[0170] In some embodiments, the secretable engineered cancer antigen comprises an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 166. In some embodiments, the secretable engineered cancer antigen comprises the amino acid sequence set forth in SEQ ID NO: 166. In some embodiments, the amino acid sequence of SEQ ID NO: 166 is encoded by the nucleotide sequence of SEQ ID NOs: 182-186.

[0171] In some embodiments, the secretable engineered cancer antigen is encoded by the nucleotide sequence of SEQ ID NOs: 163, 165, or 182, 187.C. Membrane Engineered cancer antigens

[0172] In some embodiments, provided herein is an engineered cancer antigen comprising at least one target domain described in Section II.A and a heterologous membrane targeting domain. In some embodiments, the amino acid sequence of the target domain and the heterologous membrane targeting domain are directly linked. In some embodiments, the amino acid sequence of the target domain and the heterologous membrane targeting domain are indirectly linked via a linker, such as a peptide linker. In provided embodiments in which an engineered cancer antigen contains a heterologous membrane-targeting domain, the engineered cancer antigen is one in which the engineered cancer antigen is able to be embedded in the lipid bilayer of a cell membrane from which is expressed. In some embodiments, the heterologous membrane target domain directs display of the target domain on the surface of a cell from which it has been expressed. In some embodiments, the membrane engineered cancer antigen isexpressed on the surface of a cell in which the target domain is exposed on the outside surface of the cell. In some embodiments, the heterologous membrane target domain is a transmembrane domain. In some embodiments, the target domain is any as described in Section II.A.

[0173] The heterologous membrane targeting domain of engineered cancer antigens provided herein can be the transmembrane domain of any known transmembrane protein or from a synthetic source. The transmembrane domains disclosed herein for engineered cancer antigens can also be used as the transmembrane domain of CAR molecules disclosed below.

[0174] Transmembrane domains are classified based on the three-dimensional structure of the transmembrane domain. For example, transmembrane domains may form an alpha helix, a complex of more than one alpha helix, a beta-barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell. Furthermore, transmembrane domains may also or alternatively be classified based on the transmembrane domain topology, including the number of passes that the transmembrane domain makes across the membrane and the orientation of the protein. For example, single-pass membrane proteins cross the cell membrane once, and multipass membrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 or more times). Membrane proteins may be defined as Type I, Type II or Type III depending upon the topology of their termini and membrane-passing segment(s) relative to the inside and outside of the cell. Type I membrane proteins have a single membrane-spanning region and are oriented such that the N-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is present on the cytoplasmic side. Type II membrane proteins also have a single membrane- spanning region but are oriented such that the C-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the N- terminus of the protein is present on the cytoplasmic side. Type III membrane proteins have multiple membrane- spanning segments and may be further sub-classified based on the number of transmembrane segments and the location of N- and C-termini.

[0175] In some embodiments, the transmembrane domain is derived from a Type I singlepass membrane protein. In some embodiments, transmembrane domains from multi-pass membrane proteins may also be compatible. Multi-pass membrane proteins may comprise a complex (at least 2, 3, 4, 5, 6, 7 or more) alpha helices or a beta sheet structure. In some embodiments, the N-terminus and the C-terminus of a multi-pass membrane protein are present on opposing sides of the lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side.

[0176] Transmembrane domains can also comprise at least a portion of a synthetic, non- naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is about 15-100 amino acids. In some embodiments, the protein segment is at least approximately 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in U.S. Pat. No. 7,052,906 and PCT Publication No. WO 2000 / 032776, the relevant disclosures of which are incorporated by reference herein.

[0177] In some embodiments, the size of the transmembrane domain is at least about 15 amino acids. In some embodiments, the size of the transmembrane domain is about 15 amino acids to about 45 amino acids. In some embodiments, the size of the transmembrane domain is at most about 45 amino acids. In some embodiments, the size of the transmembrane domain is about 15 amino acids to about 20 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 30 amino acids, about 15 amino acids to about 35 amino acids, about 15 amino acids to about 40 amino acids, about 15 amino acids to about 45 amino acids, about 20 amino acids to about 25 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 45 amino acids, about 25 amino acids to about 30 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 45 amino acids, about 30 amino acids to about 35 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 35 amino acids to about 45 amino acids, or about 40 amino acids to about 45 amino acids. In some embodiments, the size of the transmembrane domain is about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, or about 45 amino acids.

[0178] In some embodiments, the transmembrane domain is about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, about 25 amino acids, about 26 amino acids, about 27 amino acids, about 28 amino acids, about 29 amino acids, about 30 amino acids, about 31 amino acids, about 32 amino acids or about 33 amino acids in length. In some embodiments, the transmembrane domain is 21 amino acids in length. In some embodiments, the transmembrane domain is 22 amino acids in length. In some embodiments, the transmembrane domain is 23 amino acids in length. In some embodiments, the transmembrane domain is 24 amino acids in length. In some embodiments, the transmembrane domain is 25 amino acids inlength. In some embodiments, the transmembrane domain is 26 amino acids in length. In some embodiments, the transmembrane domain is 27 amino acids in length. In some embodiments, the transmembrane domain is 28 amino acids in length. In some embodiments, the transmembrane domain is 29 amino acids in length. In some embodiments, the transmembrane domain is 30 amino acids in length. In some embodiments, the transmembrane domain is 31 amino acids in length. In some embodiments, the transmembrane domain is 32 amino acids in length. In some embodiments, the transmembrane domain is 33 amino acids in length.

[0179] The transmembrane domain provided herein may comprise a transmembrane region and a cytoplasmic region located at the C-terminal side of the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids and, in some embodiments, helps to orient the transmembrane domain in the lipid bilayer. In some embodiments, one or more cysteine residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

[0180] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain provided herein comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan and valine may be present at the C terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence. The hydropathy, or hydrophobic or hydrophilic characteristics of a protein or protein segment, can be assessed by any method known in the art, for example the Kyte and Doolittle hydropathy analysis.

[0181] In some embodiments, the transmembrane domain comprises hydrophobic amino acid residues and allows the engineered cancer antigen to be anchored into the cell membrane of the cell. In some embodiments, the transmembrane domain comprises an amino acid sequence derived from a transmembrane protein.

[0182] In some embodiments, the transmembrane domain is heterologous to the extracellular target domain, such that the transmembrane domain is from a different natural protein as theextracellular domain of the target domain. In such embodiments, the provided membrane engineered cancer antigen is a chimeric antigen comprising the target domain and a heterologous transmembrane domain. In such embodiments, it is understood that the chimeric cancer antigen is composed of sequences that are not normally present in a protein in nature.

[0183] In some embodiments, the transmembrane domain, such as the heterologous transmembrane domain, is from a transmembrane glycoprotein. In some embodiments, the transmembrane domain, such as the heterologous transmembrane domain, comprises a transmembrane domain chosen from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, CD1 la, CD18, ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49d, ITGA6, VLA-6, CD49f, ITGAD, CD ID, ITGAE, CD 103, ITGAL, ITGAM, CD1B, ITGAX, CD1C, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, CD229, CD 160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain is a CD8 transmembrane domain.

[0184] In some embodiments, the transmembrane domain comprises an amino acid sequence derived from the transmembrane domain of the alpha, beta, or zeta chain of the T-cell receptor, CD27, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, and CD154.

[0185] In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the transmembrane has an amino acid sequence derived from the transmembrane domain of CD8. In some embodiments, the transmembrane domain has an amino acid sequence derived from the transmembrane domain of human CD8 alpha. In some embodiments, the transmembrane domain of CD8 alpha has the sequence of amino acids set forth in SEQ ID NO: 68 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 68. In some embodiments, the transmembrane domain is set forth in SEQ ID NO: 68.

[0186] In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acidsequence that is at least 70% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 4.

[0187] In some embodiments, the CD8 transmembrane domain of SEQ ID NO: 4 is encoded by the nucleotide sequence set forth in SEQ ID NO: 2.

[0188] In some embodiments, the transmembrane has an amino acid sequence derived from the transmembrane domain of CD28. In some embodiments, the transmembrane domain has an amino acid sequence derived from the transmembrane domain of human CD28. In some embodiments, the transmembrane domain of CD28 has the sequence of amino acids set forth in SEQ ID NO: 62 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 62. In some embodiments, the transmembrane domain is set forth in SEQ ID NO: 62.

[0189] In some embodiments, the size of the transmembrane domain is about 40 amino acids to about 90 amino acids. In some embodiments, the size of the transmembrane domain is at least about 40 amino acids. In some embodiments, the size of the transmembrane domain is at most about 90 amino acids. In some embodiments, the size of the transmembrane domain is about 40 amino acids to about 50 amino acids, about 40 amino acids to about 60 amino acids, about 40amino acids to about 70 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 90 amino acids, about 50 amino acids to about 60 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 90 amino acids, about 60 amino acids to about 70 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 90 amino acids, about 70 amino acids to about 80 amino acids, about 70 amino acids to about 90 amino acids, or about 80 amino acids to about 90 amino acids. In some embodiments, the size of the transmembrane domain is about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, or about 90 amino acids.

[0190] The engineered cancer antigens disclosed herein can further comprise additional elements. In some embodiments, the engineered cancer antigen comprises a detection tag. In some embodiments, the detection tag is at the N-terminus of the engineered cancer antigen. In some embodiments, the detection tag is at the C-terminus of the engineered cancer antigen. In some embodiments, the detection tag is a flag tag. In some embodiments, the detection tag is a c- Myc tag. In some embodiments, the detection tag is a His tag. In some embodiments, the detection tag comprises an amino acid sequence of SEQ ID NO: 111. In some embodiments, the detection tag is a HA tag. In some embodiments, the detection tag comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the detection tag comprises an amino acid sequence of SEQ ID NO: 30.

[0191] In some embodiments, the different elements of the engineered cancer antigen are operatively linked. In some embodiments, the different elements are directly linked to each other. In some embodiments, the different elements are linked via linkers. Exemplary linkers can be used herein are disclosed in the Example section of the present application. Additional linkers are known to a skilled artisan.

[0192] In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 110. In some embodiments, the linker comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 110. In some embodiments, the linker of SEQ ID NO: 110 is encoded by the nucleotide sequence set forth in SEQ ID NO: 114. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 128. In some embodiments, the linker comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 128.

[0193] In some embodiments, the membrane-bound engineered cancer antigen provided herein can comprise any of the target domains described herein and any of the heterologous membrane targeting domains described herein.

[0194] In some embodiments, the membrane-bound engineered cancer antigen comprises an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 70. In some embodiments, the membranebound engineered cancer antigen comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 70. In some embodiments, the membrane -bound engineered cancer antigen comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 70. In some embodiments, the membrane-bound engineered cancer antigen comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 70. In some embodiments, the membranebound engineered cancer antigen comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 70. In some embodiments, the membrane -bound engineered cancer antigen comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 70. In some embodiments, the membrane-bound engineered cancer antigen comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 70. In some embodiments, the membranebound engineered cancer antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 70. In some embodiments, the membrane -bound engineered cancer antigen comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 70. In some embodiments, the membrane-bound engineered cancer antigen comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 70. In some embodiments, the membranebound engineered cancer antigen comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 70. In some embodiments, the membrane -bound engineered cancer antigen comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 70. In some embodiments, the membrane-bound engineered cancer antigen comprises an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 113.

[0195] In some embodiments, the membrane-bound engineered cancer antigen of SEQ ID NO: 70 is encoded by the nucleotide sequence set forth in SEQ ID NOs: 69, 179 or 187-191. In some embodiments, the membrane-bound engineered cancer antigen comprises a nucleotide sequence having 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%, or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NOs: 69, 179 or 187-191.J. Intracellular Domains

[0196] In some embodiments, the engineered cancer antigen comprising a target domain comprising at least one target domain described in Section II.A and a heterologous membrane targeting domain such as described above further comprises an intracellular domain. For instance, in some embodiments, the engineered cancer antigen further comprises an intracellular domain, such as a death domain, that promotes apoptosis of the cancer, e.g., tumor, cell whose membrane it is expressed on. In provided aspects, this may be advantageous because, inter alia, such an engineered cancer antigen promotes cell death of the tumor cell by at least two mechanisms: (1) including a target domain that is recognized by a therapy, e.g., a CAR-T cell therapy, and (2) eliciting a cell death signaling cascade in response to the target domain being bound by the therapy, e.g., CAR-T cell therapy. In some embodiments, the intracellular domain comprises a death domain.

[0197] In other embodiments, the engineered cancer antigen does not comprise an intracellular domain, or does not comprise a native intracellular domain of the target antigen. For instance, in some embodiments, the engineered cancer antigen does not comprise an intracellular domain in an effort to avoid or eliminate intracellular signaling that has the potential to promote tumor growth. For instance, the native intracellular domain associated with the target antigen, e.g., BCMA, has the potential to promote tumor growth and / or survival through activation of certain signaling pathways, e.g., NFKB signaling. Accordingly, in some aspects, it would be advantageous to eliminate such an intracellular domain and / or replacing it with an intracellular domain comprising a pro-apoptotic death domain.

[0198] In some embodiments, the engineered cancer antigen comprises an intracellular domain, in which the intracellular domain comprises a signaling domain that is a death domain.

[0199] In some embodiments, the death domain is derived from a death receptor. In general, a death receptor is a membrane-bound receptor characterized by the ability to induce cellular apoptosis in response to ligand binding through its intracellular death domain. For instance, death receptors of the tumor necrosis factor (TNF) receptor superfamily (TNF-SF) include a death domain that is an intracellular sequence of around 80 amino acids. See, e.g., Ashkenzai et al., Science. 1998 Aug. 28; 281(5381): 1305-8; Locksley et al., Cell. 2001 Feb. 23; 104(4):487- 501; Wajant Cell Death Differ. 2015 November; 22(11):1727-41; and Nagata et al., Cell. 1997Feb. 7; 88(3):355-65. Ligand-mediated activation of a death domain is known to trigger apoptosis, such as by activating a caspase cascade that leads to cell death.

[0200] In provided embodiments in which the intracellular domain comprises a signaling domain that is a death domain, the engineered cancer antigen is able to mediate apoptotic signaling upon binding to the target domain. In some embodiments, the apoptotic signaling induces killing of the cancer or tumor cell in which the membrane engineered cancer antigen is expressed. Accordingly, in some embodiments, the engineered cancer antigen further comprises an intracellular domain. In some embodiments, the intracellular domain comprises a death domain.

[0201] In some embodiments, the death domain is a death domain of a TNF-SF member. In some embodiments, the death domain is, or is derived from, a death receptor selected from the group consisting of FAS (CD95), DR3, tumor necrosis factor receptor superfamily member 10A (TNFRSF10A; also known as death receptor 4 (DR4), DR5, DR6, TNFR1, Leucine-rich repeat and death domain-containing protein 1, Ankyrin repeat and death domain-containing protein IB, Interleukin- 1 receptor-associated kinase-like 2, Tumor necrosis factor receptor superfamily member 21, Netrin receptor UNC5A, Netrin receptor UNC5B, Netrin receptor UNC5C, Netrin receptor UNC5D, UNC5C-like protein, Tumor necrosis factor receptor superfamily member 16, Ankyrin- 1, Ankyrin-2, Ankyrin-3, Nuclear factor NF-kappa-B p 105 subunit, Tumor necrosis factor receptor superfamily member 6, Interleukin- 1 receptor-associated kinase 1, Death- associated protein kinase 1, Death domain-containing protein CRADD, Nuclear factor NF- kappa-B p 100 subunit, FAS-associated death domain protein (FADD), Receptor-interacting serine / threonine-protein kinase 1, Ankyrin repeat and death domain -containing protein 1A, Death domain-containing protein 1, Ectodysplasin-A receptor, Ectodysplasin-A receptor- associated adapter protein, Tumor necrosis factor receptor superfamily member 25, THO complex subunit 1, Myeloid differentiation primary response protein MyD88, p53- induced death domain-containing protein 1, interleukin- 1 receptor-associated kinase 3, and nerve growth factor receptor (NGFR). In some embodiments, the death domain is derived from FAS, FADD, or TNFRSF10A.

[0202] In some embodiments, the death domain is derived from FAS. In some embodiments, the death domain is derived from FAS and comprises an amino acid sequence that is at least 85%, 90%, 95% or 97% identical to the sequence set forth in any of SEQ ID NOS: 93, 97, and 99. In some embodiments, the death domain is derived from FAS and comprises an amino acid sequence set forth in any of SEQ ID NOS: 93, 97, or 99. In some embodiments, the FAS aminoacid sequence set forth in SEQ ID NOS: 93, 97 and 99 are encoded by the sequence set forth in SEQ ID NOS: 92, 96 and 98, respectively.

[0203] In some embodiments, the death domain derived from FAS also has an amino acid sequence derived from the transmembrane domain of FAS. In some embodiments, the transmembrane domain has an amino acid sequence derived from the transmembrane domain of human FAS. In some embodiments, the transmembrane domain of FAS has the sequence of amino acids set forth in SEQ ID NO: 95 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 95. In some embodiments, the transmembrane domain is set forth in SEQ ID NO: 95. In some embodiments, the amino acid sequence of SEQ ID NO: 95 is encoded by the nucleotide sequence of SEQ ID NO: 94.

[0204] In some embodiments, the death domain is derived from FADD. In some embodiments, the death domain is derived from FADD and comprises an amino acid sequence that is at least 85%, 90%, 95% or 97% identical to the sequence set forth in SEQ ID NO: 101. In some embodiments, the death domain is derived from FADD and comprises the amino acid sequence of SEQ ID NO: 101. In some embodiments, the death domain is derived from FADD and is encoded by a nucleotide sequence that is at least 85%, 90%, 95% or 97% identical to the sequence set forth in SEQ ID NO: 100. In some embodiments, the death domain is derived from FADD and is encoded by a nucleotide sequence of SEQ ID NO: 100.

[0205] In some embodiments, the death domain is derived from TNFRSF10A. In some embodiments, the death domain is derived from TNFRSF10A and comprises an amino acid sequence that is at least 85%, 90%, 95% or 97% identical to the sequence set forth in SEQ ID NO: 103 or 105. In some embodiments, the death domain is derived from TNFRSF10A and comprises the amino acid sequence of SEQ ID NO: 103 or 105. In some embodiments, the death domain is derived from TNFRSF10A and is encoded by a nucleotide sequence that is at least 85%, 90%, 95% or 97% identical to the sequence set forth in SEQ ID NO: 102 or 104. In some embodiments, the death domain is derived from TNFRSF10A and is encoded by a nucleotide sequence of SEQ ID NO: 102 or 104.

[0206] In some embodiments, the membrane-bound engineered cancer antigen provided herein can comprise any of the target domains, transmembrane domains and intracellular domains provided herein.

[0207] In some embodiments, the membrane-bound engineered cancer antigen comprises a sequence of amino acids that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, or about 99% identical to a sequence selected from the group consisting of SEQ ID NOS: 75, 77, 79, 81, 83, 85, 87, 89, 91, and 115-123. In some embodiments, the membrane-bound engineered cancer antigen comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 75, 77, 79, 81, 83, 85, 87, 89, 91, and 115-123.

[0208] In some embodiments, the membrane-bound engineered cancer antigen comprises a sequence of nucleotides that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to a sequence selected from the group consisting of SEQ ID NOS: 74, 76, 78, 80, 82, 84, 86, 88, and 90. In some embodiments, the membrane-bound cancer antigen comprises the nucleotide sequence selected from the group consisting of SEQ ID NOS: 74, 76, 78, 80, 82, 84, 86, 88, and 90.D. Cognate Binders

[0209] In provided aspects, embodiments herein also relate to cognate binders that specifically bind to the engineered cancer antigens disclosed herein. The target domain containing the extracellular domain or truncated portion thereof of the target antigen is recognizable by the cognate binder. Specifically, in provided embodiments, the cognate binder specifically binds to the extracellular domain or the truncated portion thereof of the engineered cancer antigens disclosed herein.

[0210] In some embodiments, the cognate binder binds to an extracellular domain or truncated portion thereof of a cell surface protein antigen from carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyro sine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein-coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2,MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), R0R1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

[0211] Binding of a cognate binder (for example, an scFv) to a target domain, such as an extracellular domain or a truncated portion thereof of a target antigen, can be confirmed by, for example, enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detect the presence of binding complexes (e.g., protein- antibody complexes) of particular interest by employing a labeled reagent (e.g., an antibody, or an scFv) specific for the complex of interest.

[0212] In some embodiments, the size of the cognate binder is about 20 amino acids to about 700 amino acids. In some embodiments, the size of the cognate binder is at least about 20 amino acids. In some embodiments, the size of the cognate binder is at most about 700 amino acids. In some embodiments, the size of the cognate binder is about 20 amino acids to about 40 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 250 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 700 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 250 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 700 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 250 amino acids, about 60 amino acids to about 300 amino acids, about 60 amino acids to about 400 amino acids, about 60 amino acids to about 500 amino acids, about 60 amino acids to about 600 amino acids, about 60 amino acids to about 700 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 250amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 700 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 250 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 700 amino acids, about 200 amino acids to about 250 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 700 amino acids, about 250 amino acids to about 300 amino acids, about 250 amino acids to about 400 amino acids, about 250 amino acids to about 500 amino acids, about 250 amino acids to about 600 amino acids, about 250 amino acids to about 700 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 700 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 700 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 700 amino acids, or about 600 amino acids to about 700 amino acids. In some embodiments, the size of the cognate binder is about 20 amino acids, about 40 amino acids, about 60 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, or about 700 amino acids.

[0213] In some embodiments, the cognate binder is an antibody. In some embodiments, the cognate binder is a molecule derived from an antibody. In some embodiments, a molecule derived from an antibody is one or more functional fragments of the antibody. Non-limiting examples of functional fragments include heavy chain, light chain, heavy chain variable domain, light chain variable domain, single-chain Fvs (scFv), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody. In some embodiments, the cognate binder is an antibody or antibody fragment with means for binding the target domain.

[0214] Cognate binders based on antibodies or molecules derived from antibodies may be made using the hybridoma method first described by Kohler, et al., Nature, 1975, 256:495-7, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

[0215] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice 59-103 (1986)).

[0216] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, which, in some embodiments, contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0217] Exemplary fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Exemplary myeloma cell lines are murine myeloma lines, such as SP-2 and derivatives, for example, X63-Ag8-653 cells available from the American Type Culture Collection (Manassas, VA), and those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA). Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, Immunol. 1984, 133:3001-05; and Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, 1987, 51-63).

[0218] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as RIA or ELISA. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 1980, 107:220-39.

[0219] Once hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, forexample, DMEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal, for example, by i.p. injection of the cells into mice.

[0220] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ionexchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

[0221] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells can serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells, such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra, et al, Curr. Opinion in Immunol., 1993, 5:256-62 and Pluckthun, Immunol. Revs., 1992, 130:151- 88.

[0222] Cognate binders based on antibodies or molecules derived from antibodies may be made from antibody phage libraries generated using the techniques described in, for example, Antibody Phage Display: Methods and Protocols (O’Brien and Aitken, eds., 2002). In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. Examples of phage display methods that can be used to make the antibodies described herein include those disclosed in Brinkman, et al, J. Immunol. Methods, 1995, 182:41-50; Ames, et al, Immunol. Methods, 1995, 184:177-86; Kettleborough, et al., Eur. J. Immunol., 1994, 24:952-8; Persic, et al, Gene, 1997, 187:9-18; Burton et a / ., Advances in Immunology, 1994, 57:191-280; PCT Application No. PCT / GB91 / 01 134; International Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 1 1236, WO 95 / 15982, WO 95 / 20401, and W097 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.

[0223] In principle, synthetic antibody clones are selected by screening phage libraries containing phages that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are screened against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thusseparated from the non-binding clones in the library. The binding clones are then eluted from the antigen and can be further enriched by additional cycles of antigen absorption / elution.

[0224] Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described, for example, in Winter et al., 1994, Ann. Rev. Immunol. 12:433-55.

[0225] Repertoires of VH and VL genes can be separately cloned by PCR and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al, supra. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of nonself and also self antigens without any immunization as described by Griffiths et al, EMBO J, 1993, 12:725-34. Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described, for example, by Hoogenboom and Winter, J. Mol. Biol., 1992, 227:381-88.

[0226] Screening of the libraries can be accomplished by various techniques known in the art. For example, the engineered cancer antigens disclosed herein can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning display libraries. The selection of antibodies with slow dissociation kinetics (e.g., good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass, et al, Proteins, 1990, 8:309-14 and WO 92 / 09690, and by use of a low coating density of antigen as described in Marks et al, BiotechnoL, 1992, 10:779-83.

[0227] In some embodiments, the cognate binder is an antibody or antigen-binding fragment thereof directed against BCMA, CD19, CD20, CD22, ROR1 or GPRC5D. In some embodiments, the cognate binding is an antibody or antigen-binding fragment with means for binding BCMA, CD19, CD20, CD22, ROR1 or GPRC5D.

[0228] In some embodiments, the cognate binder can be part of an antibody therapy for targeting to the engineered cancer antigen displayed on the cancer cell. In some embodiments, the cognate binder is part of an antibody drug conjugate containing the cognate binder and a cytotoxic pay load. In some embodiments, the cognate binder is part of a bispecific antibody. In some embodiments, the bispecific antibody is a bispecific T cell engager (BiTe) containing thecognate binder and an antibody targeting a T cells, such as an anti-CD3 antibody. In some embodiments, the cognate binder is part of the extracellular domain of a chimeric antigen receptor. Exemplary CAR comprising cognate binders are further described below in Section II.D.2. / . Exemplary cognate Under antibody or antigen binding fragments

[0229] In some embodiments, the cognate binder is an antibody or antigen-binding fragment (e.g., scFv) thereof directed against BCMA, such as human BCMA. In some embodiments, the cognate binder is a part of an extracellular binding domain of any known CAR, such as described in Section II.D.l. In some embodiments, the cognate binder is derived from an antibody specific for BCMA, including, for example, belantamab, erlanatamab, teclistamab, LCAR-B38M, C11D5.3, C12A3.2, BB2121 or FHVH33. In any of these embodiments, the cognate binder can comprise or consist of the variable heavy chain (VH), the variable light chain (VL), and / or one or more CDRs of any known anti-BCMA antibodies.

[0230] In some embodiments, the cognate binder contains a variable heavy chain (VH) and a variable light chain (VL). In some embodiments, the cognate binder is a single chain antibody. In some embodiments, the cognate binder is an scFv antibody containing a VH and a VL, linked in any order (e.g., VH-VL or VL-VH) by a linker. In some embodiments, the linker separating the VH and VE in the scFv is the GS linker set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34. Exemplary linkers separating VH and VL chains of an scFv are set forth in Table 2.Table 2: Exemplary linkers separating VH and VL chains of an scFv

[0231] In some embodiments, the cognate binder is a variable heavy chain fragment only (VH antibody), such as a single variable fragments of two heavy chains (VHH) or a fully human heavy-chain variable domain (FHVH).

[0232] In some embodiments, the cognate binder comprises an scFv derived from Cl 1D5.3 (e.g., BCMA), a murine monoclonal antibody as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013). See also PCT Application Publication No. WO2010 / 104949. The Cl lD5.3-derived scFv may comprise the heavy chain variable region (VH) and the light chain variable region (VL) of Cl 1D5.3. In some embodiments, the VH has the sequence of amino acidsset forth in SEQ ID NO: 31 and the VL has the sequence of amino acids set forth in SEQ ID NO: 32. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 35. In some embodiments, it is understood the cognate binder may include any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to BCMA. Exemplary sequences of a cognate binder are set forth in Table 3.TABLE 3: Exemplary sequences of a cognate binder

[0233] In some embodiments, the cognate binder comprises an scFv derived from another murine monoclonal antibody, C12A3.2, as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013) and PCT Application Publication No. WO2010 / 104949. In some embodiments, the VH has the sequence of amino acids set forth in SEQ ID NO: 36 and the VL has the sequence of amino acids set forth in SEQ ID NO: 37. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34. In some embodiments, the scFv comprises the sequence of amino acids setforth in SEQ ID NO: 38. In some embodiments, it is understood the cognate binder may include any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to BCMA. Exemplary sequences of a cognate binder are set forth in Table 4.Table 4: Exemplary sequences of a cognate binder

[0234] In some embodiments, the cognate binder comprises a murine monoclonal antibody with high specificity to human BCMA, referred to as BB2121 in Friedman et al., Hum. Gene Ther. 29(5):585-601 (2018)). See also, PCT Application Publication No. WO2012163805. BB2121 is also known as anti-BCMA02 CAR. In some embodiments, the VH has the sequence of amino acids set forth in SEQ ID NO: 39 and the VL has the sequence of amino acids set forth in SEQ ID NO: 40. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 41. In some embodiments, it is understood the cognate binder may include any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to BCMA. Exemplary sequences of a cognate binder are set forth in Table 5.Table 5: Exemplary sequences of a cognate binder

[0235] In some embodiments, the cognate binder comprises single variable fragments of two heavy chains (VHH) that can bind to two epitopes of BCMA as described in Zhao et al., J. Hematol. Oncol. 11(1): 141 (2018), also referred to as LCAR-B38M. See also, PCT Application Publication No. WO2018 / 028647.

[0236] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy-chain variable domain (FHVH) as described in Lam et al., Nat. Commun. 11(1):283 (2020), also referred to as FHVH33. In some embodiments, the extracellular binding domain comprises an amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, it is understood the cognate binder may include any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to BCMA. Exemplary sequences of a cognate binder are set forth in Table 6.Table 6: Exemplary sequences of a cognate binder

[0237] In some embodiments, the cognate binder comprises an scFv derived from CT 103 A (or CAR0085) as described in U.S. Patent No. 11,026,975 B2. In some embodiments, the VH has the sequence of amino acids set forth in SEQ ID NO: 43 and the VL has the sequence of amino acids set forth in SEQ ID NO: 44. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 45. In some embodiments, it is understood the cognate binder may include any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to BCMA. Exemplary sequences of a cognate binder are set forth in Table 7.Table 7: Exemplary sequences of a cognate binder

[0238] In some embodiments, the cognate binder comprises an scFv derived from CT053 (or Zevorcabtagene autoleucel) as described in Yang et al., Haematologica, 107(8): 1960-1965 (2022). In some embodiments, the VH has the sequence of amino acids set forth in SEQ ID NO: 168 and the VL has the sequence of amino acids set forth in SEQ ID NO: 169. In someembodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 170. In some embodiments, it is understood the cognate binder may include any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to BCMA. Exemplary sequences of a cognate binder are set forth in Table 8.Table 8: Exemplary sequences of a cognate binder

[0239] In some embodiments, the cognate binder is an antibody or antigen-binding fragment (e.g., scFv) thereof directed against CD19, such as human CD19. In some embodiments, the cognate binder is a part of an extracellular binding domain of any known CAR, such as described in Section II.D.l. In some embodiments, the cognate binder is derived from an antibody specific to CD19, including, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol. 138(9):2793-2799 (1987)), 4G7 (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418- 427 (1987)), B4 HB12b (Kansas & Tedder, J. Immunol. 147:4094-4102 (1991); Yazawa et al., Proc. Natl. Acad. Sci. USA 102:15178-15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther.335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10): 2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol. 118:368-381(1989)), each of which is incorporated by reference in their entirety. Other cognate binders include fully human or humanized antibodies, such as described in WO2014153270, WO2018068766, WO2019159193, or U.S. Patent Publication No. US 2016 / 0152723, each of which is incorporated by reference in their entirety. In any of these embodiments, the cognate binder can comprise or consist of the variable heavy chain (VH), the variable light chain (VL), and / or one or more CDRs of any known anti-CD19 antibodies.

[0240] In some embodiments, the cognate binder is from a mouse derived antibody FMC63. In some embodiments the cognate binder includes a VH and / or VL derived from FMC63. In some embodiments, the cognate binder is an scFv antibody containing a VH and a VL, linked in any order (e.g, VH-VL or VL-VH) by a linker. In some embodiments, the linker separating the VH and VL in the scFv is the GS linker set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34. In some embodiments, the FMC63 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:46 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 48. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 49. In some embodiments, it is understood the cognate binder may include any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD19. Exemplary sequences of a cognate binder are set forth in Table 9.Table 9: Exemplary sequences of a cognate binder

[0241] In some embodiments, the cognate binder is from a mouse derived antibody SJ25C1 that binds CD19. In some embodiments the cognate binder includes a VH and / or VL derived from SJ25C1. In some embodiments, the cognate binder is an scFv antibody containing a VH and a VL, linked in any order (e.g, VH-VL or VL-VH) by a linker. In some embodiments, the linker separating the VH and VL in the scFv is the GS linker set forth in SEQ ID NO:33. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34. In some embodiments, the SJ25C1 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 50 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 52. In some embodiments, it is understood the cognate binder may include any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD 19. Exemplary sequences of a cognate binder are set forth in Table 10.Table 10: Exemplary sequences of a cognate binder

[0242] In some embodiments, the cognate binder is an antibody or antigen-binding fragment (e.g., scFv) thereof directed against CD20, such as human CD20. In some embodiments, the cognate binder is a part of an extracellular binding domain of any known CAR, such as described in Section II.D.l. In some embodiments, the cognate binder is derived from an antibody specific to CD20, including, for example, Leul6 as described in Rufener et al., Cancer Immunol. Res. 2016 4:509-519. See also, GenBank accession # KX055828), IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab. In any of these embodiments, the cognate binder can comprise or consist of the variable heavy chain (VH), the variable light chain (VL), and / or one or more CDRs of any known anti-CD20 antibodies.

[0243] In some embodiments the cognate binder includes a VH and / or VL derived from Leul6. In some embodiments, the cognate binder is an scFv antibody containing a VH and a VL, linked in any order (e.g, VH-VL or VL-VH) by a linker. In some embodiments, the linker separating the VH and VL in the scFv is the GS linker set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34. In some embodiments, the antibody comprises the heavy chain variable region(VH) comprising the amino acid sequence of SEQ ID NO: 53 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 55. In some embodiments, it is understood the cognate binder may include any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD20. Exemplary sequences of a cognate binder are set forth in Table 11.Table 11: Exemplary sequences of a cognate binder

[0244] In some embodiments, the cognate binder is an antibody or antigen-binding fragment (e.g., scFv) thereof directed against CD22, such as human CD22. In some embodiments, the cognate binder is a part of an extracellular binding domain of any known CAR, such as described in Section II.D.l. In some embodiments, the cognate binder is derived from an antibody specific to CD22, including, for example, for example, SM03, inotuzumab, epratuzumab, moxetumomab, pinatuzumab, and m971 monoclonal antibody (m971) or m971-L7. Exemplary anti-CD22 antibodies include those described in US2023 / 0174654, W02009124109, WO2014065961 and W02020014482, incorporated by reference in its entirety. In any of these embodiments, the cognate binder can comprise or consist of the variable heavy chain (VH), the variable light chain(VL), and / or one or more CDRs of any known anti-CD22 antibodies. In some embodiments, the cognate binder is an scFv antibody containing a VH and a VL, linked in any order (e.g, VH-VL or VL-VH) by a linker. In some embodiments, the linker separating the VH and VL in the scFv is the GS linker set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34.

[0245] In some embodiments, the antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 56 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 58. In some embodiments, the antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 59 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 61. In some embodiments, it is understood the cognate binder may include any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD22. Exemplary sequences of a cognate binder are set forth in Table 12.Table 12: Exemplary sequences of a cognate binder

[0246] In some embodiments, the cognate binder is an antibody or antigen-binding fragment (e.g., scFv) thereof directed against CD70, such as human CD70. In some embodiments, the cognate binder is a part of an extracellular binding domain of any known CAR, such as described in Section II.D.2. In some embodiments, the cognate binder is derived from an antibody specific to CD70, including, for example, any as described in WO2022 / 078344, WO2022 / 150831 (e.g., Table 3 therein), WO2022 / 238962, W02021 / 047208, WO2021 / 055437, WO2019 / 152742 or US2023 / 0399412. In any of these embodiments, the cognate binder can comprise or consist of the variable heavy chain (VH), the variable light chain (VL), and / or one or more CDRs of any known anti-CD70 antibodies. In some embodiments, the cognate binder is an scFv antibody containing a VH and a VL, linked in any order (e.g, VH-VL or VL-VH) by a linker. In some embodiments, the linker separating the VH and VL in the scFv is the GS linker set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34. In some embodiments, the antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 160 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 159. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 161. In some embodiments, it is understood the cognate binder may include any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD70. Exemplary sequences of a cognate binder are set forth in Table 13.Table 13: Exemplary sequences of a cognate binder2. Chimeric A niigen Jieceptors

[0247] In some embodiments, the cognate binder of the engineered cancer antigen is or is present as part of the extracellular domain of a chimeric antigen receptor (CAR). In some embodiments, the CAR is expressed by genetically engineered cells and is able to bind via its extracellular domain to the target domain (comprising the extracellular domain or truncated portion thereof of a target antigen) of the engineered cancer antigen. In some embodiments, the CAR further comprises a transmembrane domain and an intracellular signaling domain. In some embodiments, the CAR is expressed on the surface of an immune effector cell. In some embodiments, the immune effector cell is a cytotoxic T cell. In some embodiments, the immune effector cell is a natural killer cell. In provided embodiments, the engineered cells (e.g., T cells) that comprise the CAR may be used for immunotherapy to target and destroy cancer cells that have been tagged to express the engineered cancer antigen recognized by the CAR.

[0248] In some embodiments, the CAR is a fusion protein that includes the extracellular domain comprising the cognate binder as an extracellular antigen-binding domain for recognizing the target domain; a transmembrane domain; and an intracellular signaling domain. The ectodomain and transmembrane domains may be linked by a flexible linker (also called aspacer). In some embodiments, the extracellular domain of the CAR is any of the cognate binders as described above. In some embodiments, the intracellular signaling domain includes an immunoreceptor tyrosine-based activation motif (IT AM). Activation of the CAR fusion protein results in cellular activation in response to recognition by the cognate binder of the target domain of the engineered cancer antigen. When a cell expresses such a CAR, it can recognize and kill target cells that express, or that are tagged to display on their surface, the engineered cancer antigen.

[0249] In some embodiments, the CAR is typically encoded by a nucleic acid sequence (polynucleotide) that includes a leader sequence for directing expression of the CAR at the membrane of a cell. In some embodiments, the antigen receptor (e.g. CAR) is encoded by a polynucleotide that encodes a CAR with an Nth-terminal leader sequence. The leader sequence (also known as the signal peptide) allows the expressed CAR construct to enter the endoplasmic reticulum (ER) and target the cell surface. The leader sequence is cleaved in the ER and the mature cell surface CAR does not possess a leader sequence. In general, the leader sequence length will be in the range of 5 to 30 amino acids, and comprise a stretch of hydrophobic amino acids. In some embodiments, the leader sequence is 5, 10, 15, 20, or 25 amino acids in length, or any value between any of the foregoing. Suitably, the leader sequence comprises a sequence derived from any secretory protein. In some embodiments, the leader sequence can be any of the signal peptide sequences described herein. An exemplary CD8a signal peptide is set forth in SEQ ID NO: 64. In some embodiments, the leader sequence or signal peptide sequence comprising the amino acid sequence of SEQ ID NO: 64. An exemplary GM-CSFRa signal peptide is set forth in SEQ ID NO: 65. An exemplary IgK signal peptide is set forth in SEQ ID NO: 66. An exemplary IgK signal peptide is set forth in SEQ ID NO: 67. In some embodiments, the signal peptide sequences described herein can be from any mammal species. In some embodiments, the signal peptide sequences can be a murine signal peptide sequence. In some embodiments, the murine signal peptide can be a murine heavy chain signal peptide. An exemplary murine heavy chain signal peptide is set forth in SEQ ID NO: 109 (corresponding nucleotide sequence set forth in SEQ ID NO: 14).

[0250] The CARs disclosed herein comprise a transmembrane domain that can be directly or indirectly fused to the extracellular domain. The transmembrane domains disclosed above for the engineered cancer antigens can be used as the transmembrane domain of CARs disclosed herein.

[0251] In some embodiments, the transmembrane domain of the CAR comprises hydrophobic amino acid residues and allows the CAR to be anchored into the cell membrane ofthe engineered cell. Suitably, the transmembrane domain comprises an amino acid sequence derived from a transmembrane protein. Suitably, the transmembrane domain comprises an amino acid sequence derived from the transmembrane domain of the alpha, beta, or zeta chain of the T- cell receptor, CD27, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, and CD154.

[0252] In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the CAR comprises a transmembrane with an amino acid sequence derived from the transmembrane domain of CD8. In some embodiments, the CAR comprises a transmembrane domain with an amino acid sequence derived from the transmembrane domain of human CD8 alpha. In some embodiments, the CAR contains a transmembrane domain of CD8 alpha that has the sequence of amino acids set forth in SEQ ID NO: 68 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 68. In some embodiments, the transmembrane domain is set forth in SEQ ID NO: 68.

[0253] In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequencethat is at least 98% identical to SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 4.

[0254] In some embodiments, suitably, the CAR comprises a transmembrane with an amino acid sequence derived from the transmembrane domain of CD28. Suitably, the CAR comprises a transmembrane domain with an amino acid sequence derived from the transmembrane domain of human CD28. In some embodiments, the CAR contains a transmembrane domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO: 62 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 62. In some embodiments, the transmembrane domain is set forth in SEQ ID NO: 62.

[0255] In some embodiments, the CARs can also comprise a spacer region located between the cognate binder of the extracellular antigen binding domain and the transmembrane domain. In some embodiments, the spacer region comprises hydrophilic amino acids and allows flexibility of the targeting domain with respect to the cell surface. In some embodiments, the spacer region comprises greater than 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the spacer region comprises less than 10, 15, 20, 25, 30, or 35 amino acids. In some embodiments, the spacer region is a hinge domain.

[0256] The CARs disclosed herein may comprise a hinge domain that is located between the extracellular domain and the transmembrane domain. A hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular domain relative to the transmembrane domain of the effector molecule can be used.

[0257] In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8a. In some embodiments, the hinge domain is a portion of the hinge domain of CD8a, e.g., a fragment containing about 15-100 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8a. In some embodiments, the CAR contains a CD8 hinge spacer sequence that has the sequence of amino acids set forth in SEQ ID NO: 21 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 21. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO: 21.

[0258] In some embodiments, the hinge domain is the hinge domain of an antibody, such as an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the hinge domain is the hinge domain that joins the constant domains CHI and CH2 of an antibody. In some embodiments, the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgGl, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgGl antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgGl antibody.

[0259] In some embodiments, the spacer region includes all or a portion containing the hinge domain of an IgGl Fc or an IgG4 Fc. In some embodiments, the spacer is an IgG4 Fc spacer. In some embodiments, the CAR contains an IgG4 Fc spacer that has the sequence of amino acids set forth in SEQ ID NO: 22 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 22. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO: 22. In some embodiments, the sequence of the spacer is the hinge portion of the IgGl Fc or IgG4 Fc. In some embodiments, the CAR contains an IgG4 hinge spacer. In some embodiments, the IgG4 hinge spacer has the sequence of amino acids set forth in SEQ ID NO: 23 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 23. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO: 23.

[0260] Non-naturally occurring peptides may also be used as hinge domains. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain of an Fc receptor and the N- terminus of the transmembrane domain is a peptide linker, such as a (GxS)n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0261] The hinge domain may contain about 10-100 amino acids, e.g., about any one of 15- 75 amino acids, 20-50 amino acids, or 30-60 amino acids. In some embodiments, the hinge domain may be at least about any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0262] The intracellular signaling domain in the CARs provided herein is responsible for activation of at least one of the normal effector functions of the immune effector cell expressing the CARs. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term “cytoplasmic signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire cytoplasmic signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term cytoplasmic signaling domain is thus meant to include any truncated portion of the cytoplasmic signaling domain sufficient to transduce the effector function signal.

[0263] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. “Primary intracellular signaling domain” refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as immunoreceptor tyrosine -based activation motif, or IT AM. An “IT AM,” as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells. The motif may comprises two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxL / Ix(6-8)YxxL / I. IT AMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the IT AM following activation of the signaling molecule. ITAMs may also function as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3z, FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0264] In some embodiments, the intracellular signaling domain is derived from CD3 zeta chain (CD3z). In some embodiments, the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3z. In some embodiments, the intracellular signaling domain is a cytoplasmic signaling domain of wild-type CD3z.

[0265] In some embodiments, the CAR contains an intracellular signaling domain that contains a signaling domain of CD3zeta that has the sequence of amino acids set forth in SEQ ID NO: 24 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 24. In some embodiments, the CAR contains an intracellular signaling domain that contains the signaling domain of CD3zeta that has the sequence of amino acids set forth in SEQ ID NO: 24. In some embodiments, the CAR contains an intracellular signaling domain that contains a signaling domain of CD3zeta that has the sequence of amino acids set forth in SEQ ID NO: 25 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 25. In some embodiments, the CAR contains an intracellular signaling domain that contains a signaling domain of CD3zeta that has the sequence of amino acids set forth in SEQ ID NO: 25.

[0266] The CARs disclosed herein may further comprise at least one co-stimulatory signaling domain. Thus, in some embodiments, the chimeric antigen receptor (CAR) comprises: 1) the cognate binder; 2) a spacer, such as a hinge sequence; 3) a transmembrane domain; and 4) an intracellular signaling region comprising a first primary intracellular signaling domain, such as a CD3 zeta intracellular signaling domain and second co-stimulatory intracellular signaling domain. The term “co-stimulatory signaling domain,” as used herein, refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function. Many immune effector cells require co- stimulation, in addition to stimulation of an antigen- specific signal, to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell.

[0267] The co-stimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. “Co-stimulatory signaling domain” can be the cytoplasmic portion of a co-stimulatory molecule.

[0268] In some embodiments, the intracellular signaling domain comprises a single co- stimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (such as about any of 2, 3, 4, or more) co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same co- stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein. In some embodiments, theintracellular signaling domain comprises a primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3z) and one or more co- stimulatory signaling domains (such as 4- IBB signal). In some embodiments, the one or more co- stimulatory signaling domains (such as 4- IBB signal) and the primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3z) are fused to each other via optional peptide linkers. The primary intracellular signaling domain, and the one or more co- stimulatory signaling domains may be arranged in any suitable order. Multiple co-stimulatory signaling domains may provide additive or synergistic stimulatory effects.

[0269] In some embodiments, a costimulatory domain can be CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, lymphocyte function- associated antigen-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, and / or B7-H3 costimulatory domains.

[0270] In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO: 26 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 26. In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO: 26. In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO: 27 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 27. In some embodiments, the CAR contains an intracellular signaling domain that contains the costimulatory signaling domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO: 27.

[0271] In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of 4- IBB that has the sequence of amino acids set forth in SEQ ID NO: 28 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 28. In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of 4- IBB that has the sequence of amino acids set forth in SEQ ID NO: 28.

[0272] In some embodiments, an intracellular signaling domain can be a domain of CD3zeta, CD28 and / or 4- IBB. In some embodiments, the CAR comprises at least two intracellular signaling domains derived from CD3 zeta and 4-1BB. In other embodiments, the CAR comprises at least two intracellular signaling domains derived from CD3 zeta and CD28.

[0273] In some embodiments, the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 63. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 63.

[0274] In some embodiments, the CAR is an anti-BCMA CAR. Chimeric antigen receptors containing anti-BCMA antibodies, and cells expressing such chimeric antigen receptors have been previously described. See Carpenter et al., Clin Cancer Res., 2013, 19(8):2048-2060, W02010104949, WO 2016090320, W02016090327, W02016094304, WO2016014789, WO2017173256, WO2017130223, WO2018028647, WO2019 / 089968, WO2019089969, W02019085102, WO2019241358, W02019108900, W02020014333, WO2020243546, W02020018825, W02020038147, WO2021091978, WO2021256724, WO2021057866, WO2021146147, WO2021121228, WO2021162394, WO2021231213, WO2022089353, W02022046730, W02022046730, WO2022143870, WO2022119923, WO2023226921, WO2023288185, WO2023193662, WO2023109257, WO2023016576, W02023020474,WO2023019398, WO2023068382, each of which is incorporated by reference in their entirety. Any anti-BCMA CAR previously described or known or based on any of such CARs can be used in the provided embodiments.

[0275] In some embodiments, the anti-BCMA CAR includes any of the cognate binders as described in Section II.D.l. In some embodiments, the anti-BCMA CAR includes an extracellular binding domain containing any of the anti-BCMA cognate binders described above, a hinge domain, a transmembrane domain and an intracellular domain containing an intracellular costimulatory domain and a CD3zeta intracellular primary signaling domain. In some embodiments, the CAR also may include a signal peptide.

[0276] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from Cl 1D5.3, a murine monoclonal antibody as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013). See also PCT Application Publication No. WO2010 / 104949. The Cl lD5.3-derived scFv may comprise the heavy chain variable region (VH) and the light chain variable region (VL) of Cl 1D5.3. In some embodiments, the VH has the sequence of amino acids set forth in SEQ ID NO: 31 and the VL has the sequence of amino acids set forth in SEQ ID NO: 32. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:34. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO:35. In some embodiments, the spacer is a hinge sequence, such as a CD8a hinge or an immunoglobulin hinge. In some embodiments, the transmembrane domain is any as described such as a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain contains a 4- IBB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to BCMA and intracellular signaling and cytotoxic activity.

[0277] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from another murine monoclonal antibody, C12A3.2, as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013) and PCT Application Publication No. WO2010 / 104949. In some embodiments, the VH has the sequence of amino acids set forth inSEQ ID NO: 36 and the VL has the sequence of amino acids set forth in SEQ ID NO: 37. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO: 38. In some embodiments, the spacer is a hinge sequence such as any as described, for example a CD8a hinge or an immunoglobulin hinge. In some embodiments, the transmembrane domain is any as described such as a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain contains a 4- IBB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to BCMA and intracellular signaling and cytotoxic activity.

[0278] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a murine monoclonal antibody with high specificity to human BCMA, referred to as BB2121 in Friedman et al., Hum. Gene Ther. 29(5):585-601 (2018)). See also, PCT Application Publication No. WO2012163805. BB2121 is also known as anti-BCMA02 CAR. In some embodiments, the VH has the sequence of amino acids set forth in SEQ ID NO: 39 and the VL has the sequence of amino acids set forth in SEQ ID NO: 40. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:34. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO:41. In some embodiments, the spacer is a hinge sequence such as any as described, for example a CD 8 a hinge or an immunoglobulin hinge. In some embodiments, the transmembrane domain is any as described such as a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain contains a 4- 1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or moresequence identity thereto, and retain binding to BCMA and intracellular signaling and cytotoxic activity.

[0279] In some embodiments, the extracellular binding domain of the BCMA CAR comprises single variable fragments of two heavy chains (VHH) that can bind to two epitopes of BCMA as described in Zhao et al., J. Hematol. Oncol. 11(1): 141 (2018), also referred to as LCAR-B38M. See also, PCT Application Publication No. WO2018 / 028647. In some embodiments, the spacer is a hinge sequence such as any as described, for example a CD8a hinge or an immunoglobulin hinge. In some embodiments, the transmembrane domain is any as described such as a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain contains a 4- IBB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to BCMA and intracellular signaling and cytotoxic activity.

[0280] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy-chain variable domain (FHVH) as described in Lam et al., Nat. Commun. 11(1):283 (2020), also referred to as FHVH33. In some embodiments, the extracellular binding domain comprises an amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the spacer is a hinge sequence such as any as described, for example a CD8a hinge or an immunoglobulin hinge. In some embodiments, the transmembrane domain is any as described such as a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain contains a 4- IBB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to BCMA and intracellular signaling and cytotoxic activity.

[0281] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from CT103A (or CAR0085) as described in U.S. Patent No. 11,026,975 B2. In some embodiments, the VH has the sequence of amino acids set forth in SEQID NO: 39 and the VL has the sequence of amino acids set forth in SEQ ID NO: 40. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:34. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO:41. In some embodiments, the spacer is a hinge sequence such as any as described, for example a CD8a hinge or an immunoglobulin hinge. In some embodiments, the transmembrane domain is any as described such as a CD 8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain contains a 4- IBB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to BCMA and intracellular signaling and cytotoxic activity.

[0282] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from CT053 (or Zevor-cel) as described in Yang et al., Haematologica, 107(8):1960-1965 (2022). In some embodiments, the VH has the sequence of amino acids set forth in SEQ ID NO: 168 and the VL has the sequence of amino acids set forth in SEQ ID NO: 169. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:34. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO: 170. In some embodiments, the spacer is a hinge sequence such as any as described, for example a CD8a hinge or an immunoglobulin hinge. In some embodiments, the transmembrane domain is any as described such as a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain contains a 4- IBB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to BCMA and intracellular signaling and cytotoxic activity. In someembodiments, the anti-BCMA CAR comprises an amino acid sequence that is at least 85%, at least 90% or at least 95% identical to the anti-BCMA CAR set forth in SEQ ID NO: 167.

[0283] In some embodiments, the CAR comprises an anti-BCMA CAR of a commercial CAR cell therapy. Non-limiting examples of an anti-BCMA CAR in commercial cell-based therapies include the anti-BCMA CAR engineered in cells of idecabtagene vicleucel (ABECMA®) or ciltacabtagene autoleucel (CARVYKTI™). In some embodiments, the commercial cell-based therapy includes the anti-BCMA CAR engineered in cells of Zevorcabtagene autoleucel (Zevor-cel).

[0284] In some embodiments, the anti-BCMA CAR has features of the CAR in idecabtagene vicleucel. In some embodiments, the CAR has the sequence of amino acids set forth as FDA unique ingredient identifier (UNII) code No. H4ON8SV8LK (drugs.ncats.io / substance / H4ON8SV8LK). In some embodiments, the anti-BCMA CAR is set forth by the sequence of amino acids set forth in SEQ ID NO: 192. In some embodiments, the anti-BCMA CAR is set forth by a sequence of amino acids that has at least 85%, at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 192.

[0285] In some embodiments, the anti-BCMA CAR has features of the CAR in ciltacabtagene autoleucel. In some embodiments, the CAR has the sequence of amino acids set forth as FDA unique ingredient identifier (UNII) code No. 7S3Z5YP744 (drugs.ncats.io / substance / 7S3Z5YP744). The CAR is also described, for example, in International PCT publication No. WO2023164695. In some embodiments, the anti-BCMA CAR is set forth by the sequence of amino acids set forth in SEQ ID NO: 193. In some embodiments, the anti-BCMA CAR is set forth by a sequence of amino acids that has at least 85%, at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 193.

[0286] In some embodiments, the anti-BCMA CAR has features of the CAR in Zevorcabtagene autoleucel. In some embodiments, the anti-BCMA CAR is set forth by the sequence of amino acids set forth in SEQ ID NO: 167. In some embodiments, the anti-BCMA CAR is set forth by a sequence of amino acids that has at least 85%, at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 167.

[0287] In some embodiments, the CAR is an anti-CD19 CAR. Chimeric antigen receptors containing anti-CD19 antibodies, and cells expressing such chimeric antigen receptors have been previously described. In some embodiments, the anti-CD19 CAR includes any of the cognate binders as described in Section II.D.l. In some embodiments, the anti-CD19 CAR includes an extracellular binding domain containing any of the anti-CD19 cognate binders described above, ahinge domain, a transmembrane domain and an intracellular domain containing an intracellular costimulatory domain and a CD3zeta intracellular primary signaling domain. In some embodiments, the CAR also may include a signal peptide.

[0288] In some embodiments, the extracellular binding domain of the CD 19 CAR is from a mouse derived antibody FMC63. In some embodiments, the extracellular binding domain of the CD19 CAR may comprise the heavy chain variable region (VH) set forth in SEQ ID NO:46 and the light chain variable region (VL) set forth in SEQ ID NO:47. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:34. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO:48. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO:49. In some embodiments, the spacer is a hinge sequence such as any as described, for example a CD 8 a hinge or an immunoglobulin hinge. In some embodiments, the transmembrane domain is any as described such as a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain contains a 4- 1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD 19 and intracellular signaling and cytotoxic activity.

[0289] In some embodiments, the extracellular binding domain of the CD 19 CAR is from a mouse derived antibody SJ25C1. In some embodiments, the extracellular binding domain of the CD19 CAR may comprise the heavy chain variable region (VH) set forth in SEQ ID NO:50 and the light chain variable region (VL) set forth in SEQ ID NO:51. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO: 34. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO: 52. In some embodiments, the spacer is a hinge sequence such as any as described, for example a CD 8 a hinge or an immunoglobulin hinge. In some embodiments, the transmembrane domain is any as described such as a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain contains a 4-IBB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD 19 and intracellular signaling and cytotoxic activity.

[0290] In some embodiments, the CAR comprises an anti-CD19 CAR of a commercial CAR cell therapy. Non-limiting examples of an anti-CD19 CAR in commercial cell-based therapies include the anti-CD19 CAR engineered in cells of YESCARTA®, KYMRIAH®, TECARTUS®, or BREYANZI®.

[0291] In some embodiments, the CAR is an anti-CD20 CAR. Chimeric antigen receptors containing anti-CD20 antibodies, and cells expressing such chimeric antigen receptors have been previously described. In some embodiments, the anti-CD20 CAR includes any of the cognate binders as described in Section II.D.l. In some embodiments, the anti-CD20 CAR includes an extracellular binding domain containing any of the anti-CD20 cognate binders described above, a hinge domain, a transmembrane domain and an intracellular domain containing an intracellular costimulatory domain and a CD3zeta intracellular primary signaling domain. In some embodiments, the CAR also may include a signal peptide.

[0292] In some embodiments, the extracellular binding domain of the CD20 CAR may comprise the heavy chain variable region (VH) set forth in SEQ ID NO:53 and the light chain variable region (VL) set forth in SEQ ID NO:54. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:34. In some embodiments, the anti-CD20 scFv is set forth in SEQ ID NO: 55. In some embodiments, the spacer is a hinge sequence such as any as described, for example a CD8a hinge or an immunoglobulin hinge. In some embodiments, the transmembrane domain is any as described such as a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain contains a 4- IBB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above ordescribed SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD20 and intracellular signaling and cytotoxic activity.

[0293] In some embodiments, the CAR is an anti-CD22 CAR. Chimeric antigen receptors containing anti-CD22 antibodies, and cells expressing such chimeric antigen receptors have been previously described. In some embodiments, the anti-CD22 CAR includes any of the cognate binders as described in Section II.D.l. In some embodiments, the anti-CD22 CAR includes an extracellular binding domain containing any of the anti-CD22 cognate binders described above, a hinge domain, a transmembrane domain and an intracellular domain containing an intracellular costimulatory domain and a CD3zeta intracellular primary signaling domain. In some embodiments, the CAR also may include a signal peptide.

[0294] In some embodiments, the extracellular binding domain of the CD22 CAR may comprise the heavy chain variable region (VH) set forth in SEQ ID NO:56 and the light chain variable region (VL) set forth in SEQ ID NO:57. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:34. In some embodiments, the anti-CD22 scFv is set forth in SEQ ID NO: 58. In some embodiments, the extracellular binding domain of the CD22 CAR may comprise the heavy chain variable region (VH) set forth in SEQ ID NO:59 and the light chain variable region (VL) set forth in SEQ ID NO:60. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:34. In some embodiments, the anti-CD22 scFv is set forth in SEQ ID NO: 61. In some embodiments, the spacer is a hinge sequence such as any as described, for example a CD8a hinge or an immunoglobulin hinge. In some embodiments, the transmembrane domain is any as described such as a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain contains a 4- IBB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD22 and intracellular signaling and cytotoxic activity.

[0295] In some embodiments, the CAR is an anti-CD70 CAR. Chimeric antigen receptors containing anti-CD70 antibodies, and cells expressing such chimeric antigen receptors have been previously described. In some embodiments, the anti-CD70 CAR includes any of the cognate binders as described in Section II.D.l. In some embodiments, the anti-CD70 CAR includes an extracellular binding domain containing any of the anti-CD70 cognate binders described above, a hinge domain, a transmembrane domain and an intracellular domain containing an intracellular costimulatory domain and a CD3zeta intracellular primary signaling domain. In some embodiments, the CAR also may include a signal peptide. In some embodiments, the CAR may be a CAR with a CD70 binding domain as described in WO2022 / 150831 (see e.g., Table 3 for exemplary CD70 binding domains and CARs). In some embodiments, the extracellular binding domain of the CD70 CAR may comprise the heavy chain variable region (VH) set forth in SEQ ID NO: 160 and the light chain variable region (VL) set forth in SEQ ID NO: 159. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 9. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:34. In some embodiments, the anti-CD70 scFv is set forth in SEQ ID NO: 161. In some embodiments, the spacer is a hinge sequence such as any as described, for example a CD8a hinge or an immunoglobulin hinge. In some embodiments, the transmembrane domain is any as described such as a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain contains a 4- 1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD70 and intracellular signaling and cytotoxic activity. In some embodiments, the CAR has a sequence that has at least 85%, 90%, 95% or 97% sequence identity to SEQ ID NO: 108. In some embodiments, the CAR has a sequence set forth in SEQ ID NO: 108.

[0296] In some embodiments, the extracellular binding domain of the CD70 CAR may comprise the heavy chain variable region (VH) set forth in SEQ ID NO: 125 and the light chain variable region (VL) set forth in SEQ ID NO: 126. In some embodiments, the anti-CD70 scFv is set forth in SEQ ID NO: 127.III. POLYNUCLEOTIDES

[0297] Also provided herein are polynucleotides encoding one or more of any of the engineered cancer antigens disclosed herein.

[0298] In some embodiments, provided herein is a polynucleotide encoding any of the engineered cancer antigens that comprises a target domain and a tumor targeting molecule, e.g., as disclosed in Section II.B. In some embodiments, provided herein is a polynucleotide encoding any of the engineered cancer antigens that comprises a target domain and a heterologous membrane targeting domain, e.g., as disclosed in Section II.C.

[0299] In some embodiments, provided herein is a polynucleotide encoding an engineered cancer antigen that comprises a target domain and a tumor targeting molecule, e.g., any of those disclosed in Section II.B., and an engineered cancer antigen that comprises a target domain and a heterologous membrane targeting domain, e.g., any of those disclosed in Section II.C. In some embodiments, the engineered cancer antigen that comprises a target domain and a tumor targeting molecule and the engineered cancer antigen that comprises a target domain and a heterologous membrane targeting domain are separated by a cleavable linker. In some embodiments, the cleavable linker comprises a 2A self-cleaving peptide.

[0300] In some embodiments, provided herein is a polynucleotide comprising a first nucleic acid sequence encoding any of the engineered cancer antigens that comprise a target domain and a tumor targeting molecule, e.g., any of those disclosed in Section II.B., and a second nucleic acid sequence encoding any of the engineered cancer antigens that comprise a target domain and a heterologous membrane targeting domain, e.g., any of those disclosed in Section II.C. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a cleavable linker. In some embodiments, the cleavable linker is a self-cleaving linker. In some embodiments, the self-cleaving linker is or comprises a 2A peptide.

[0301] In some embodiments, the polynucleotide is a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA) or a DNA / RNA hybrid. Polynucleotides may be single- stranded or double- stranded and either recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotides refer to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides or a modified formof either type of nucleotide, as well as all intermediate lengths. It will be readily understood that “intermediate lengths, ” in this context, means any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.-, 151, 152, 153, etc.-, 201, 202, 203, etc.

[0302] In some embodiments, polynucleotides are codon-optimized. As used herein, the term “codon-optimized” refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, (x) systematic variation of codon sets for each amino acid, (xi) isolated removal of spurious translation initiation sites and / or (xii) elimination of fortuitous polyadenylation sites otherwise leading to truncated RNA transcripts.

[0303] It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide, or fragment of variant thereof, as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and / or primate codon selection. Further, alleles of the genes comprising the polynucleotide sequences provided herein may also be used. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides.

[0304] In some embodiments, the polynucleotides provided herein, regardless of the length of the coding sequence itself, further comprise other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides,epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may be employed in particular embodiments, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0305] Polynucleotides can be prepared, manipulated and / or expressed using any of a variety of well-established techniques known and available in the art.IV. VECTORS FOR DELIVERY OF ENGINEERED CANCER ANTIGENS

[0306] Provided herein are vectors comprising any of the polynucleotides encoding any of the engineered cancer antigens disclosed herein.

[0307] Also provided herein are vectors comprising polynucleotides encoding two or more of the engineered cancer antigens disclosed herein, such as an engineered cancer antigen comprising a tumor targeting molecule, and an engineered cancer antigen comprising a heterologous membrane targeting domain.

[0308] Also provided herein are vectors comprising a first polynucleotide encoding an engineered cancer antigen that comprises a target domain and a tumor targeting molecule, e.g., any of those disclosed in Section II.B., and a second polynucleotide encoding an engineered cancer antigen that comprises a target domain and a heterologous membrane targeting domain, e.g., any of those disclosed in Section II.C. In some embodiments, the first polynucleotide and the second polynucleotide are separated by a cleavable linker. In some embodiments, the cleavable linker comprises a 2A self-cleaving peptide.

[0309] In some embodiments, the engineered cancer antigen is delivered by a viral vector comprising a polynucleotide encoding the engineered cancer antigen, such as any of the polynucleotides disclosed in Section II.

[0310] In order to express the engineered cancer antigens and / or cognate binders described herein in a cell, e.g., a tumor cell, an expression cassette encoding the engineered cancer antigen can be inserted into a nucleic acid vector. The “expression cassette” contains the gene of interest, which is the engineered cancer antigen. The cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergo appropriate post-translational modifications required for activity in the transformed cell, and be translocated to the appropriate compartment for biological activity by targeting to appropriate intracellular compartments or secretion into extracellular compartments. Preferably, the cassette has its 3' and 5' ends adaptedfor ready insertion into a vector, e.g., it has restriction endonuclease sites at each end. The cassette can be removed and inserted into a plasmid or viral vector as a single unit.

[0311] The term “nucleic acid vector” is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A nucleic acid vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA.

[0312] In some embodiments, vectors include, without limitation, plasmids, phagemids, cosmids, transposons, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. In some embodiments, the coding sequences of the engineered cancer antigen disclosed herein can be ligated into such vectors for expression in mammalian cells.

[0313] In some embodiments, the vector is a viral vector or a non-viral vector.

[0314] In some embodiments, the vector is a non-viral vector. In some embodiments, non- viral vectors are used to deliver one or more polynucleotides contemplated herein. In some embodiments, the recombinant vector comprising a polynucleotide encoding the engineered cancer antigen described herein is a plasmid. Numerous suitable plasmid expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for eukaryotic host cells: pXTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other plasmid vector may be used so long as it is compatible with the host cell.

[0315] In some embodiments, the vector is a viral vector. In some embodiments, viral vectors are used to deliver one or more polynucleotides contemplated herein. Suitable viral vectors include, but are not limited to, viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191 ; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., U.S. Patent No. 7,078,387; Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al„ PNAS 94:6916 6921 , 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al„Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:78127816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. Examples of vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.

[0316] In some embodiments, the vector is a non-integrating vector, including but not limited to, an episomal vector or a vector that is maintained extrachromosomally. As used herein, the term “episomal” refers to a vector that is able to replicate without integration into host’s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally. The vector is engineered to harbor the sequence coding for the origin of DNA replication or “ori” from a lymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast, specifically a replication origin of a lymphotrophic herpes virus or a gamma herpesvirus corresponding to oriP of EBV. In some embodiments, the lymphotrophic herpes virus may be Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV), Herpes virus saimiri (HS), or Marek's disease virus (MDV). Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus.

[0317] In some embodiments, the vector is an integrating vector. In some embodiments, a polynucleotide is introduced into a target or host cell using a transposon vector system. In some embodiments, the transposon vector system comprises a vector comprising transposable elements and a polynucleotide contemplated herein; and a transposase. In one embodiment, the transposon vector system is a single transposase vector system, see, e.g., WO 2008 / 027384. Exemplary transposases include, but are not limited to: piggyBac, Sleeping Beauty, Mosl, Tcl / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and derivatives thereof. The piggyBac transposon and transposase are described, for example, in U.S. Patent 6,962,810, which is incorporated herein by reference in its entirety. The Sleeping Beauty transposon and transposase are described, for example, in Izsvak et al., J. Mol. Biol. 302: 93-102 (2000), which is incorporated herein by reference in its entirety. The Tol2 transposon which was first isolated from the medaka fish Oryzias latipes and belongs to the hAT family of transposons is describedin Kawakami et al. (2000). Mini-Tol2 is a variant of Tol2 and is described in Balciunas et al. (2006). The Tol2 and Mini-Tol2 transposons facilitate integration of a transgene into the genome of an organism when co-acting with the Tol2 transposase. The Frog Prince transposon and transposase are described, for example, in Miskey et al., Nucleic Acids Res. 31:6873-6881 (2003).

[0318] In some embodiments, a polynucleotide sequence encoding the engineered cancer antigen disclosed herein is operably linked to one or more control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells. “Control elements” refer those non-translated regions of the vector which interact with host cellular proteins to carry out transcription and translation. Non-limiting examples of control elements include origin of replication, selection cassettes, constitutive and inducible promoters, enhancers, translation initiation signals (Shine Dalgamo sequence or Kozak sequence) introns, transcription terminators, 5' and 3' untranslated regions. See e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544) Such elements may vary in their strength and specificity. The transcriptional control element may be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., bacterial or archaeal cell).

[0319] In some embodiments, polynucleotides encoding the engineered cancer antigen described herein is operably linked to a promoter and / or an enhancer. The term “promoter” as used herein refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter. In some embodiments, promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and / or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide. The term “enhancer” refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and / or other enhancer elements.

[0320] Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, a viral simian virus 40 (SV40) (e.g., early and late SV40), a spleen focus forming virus (SFFV) promoter, long terminal repeats (LTRs) from retrovirus (e.g., a Moloney murine leukemia virus (MoMLV) LTR promoter or a Rous sarcoma virus (RSV)LTR), a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pl l promoters from vaccinia virus, an elongation factor 1-alpha (EFla) promoter, early growth response 1 (EGR1) promoter, a ferritin H (FerH) promoter, a ferritin L (FerL) promoter, a Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a eukaryotic translation initiation factor 4A1 (EIF4A1) promoter, a heat shock 70kDa protein 5 (HSPA5) promoter, a heat shock protein 90kDa beta, member 1 (HSP90B1) promoter, a heat shock protein 70kDa (HSP70) promoter, a P-kinesin (P-KIN) promoter, the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C (UBC) promoter, a phosphoglycerate kinase- 1 (PGK) promoter, a cytomegalovirus enhancer / chicken P-actin (CAG) promoter, a P-actin promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primerbinding site substituted (MND) promoter, and mouse metallothionein-1. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

[0321] In some embodiments, a polynucleotide sequence encoding the engineered cancer antigen described herein is operably linked to a constitutive promoter. In such embodiments, the polynucleotides encoding the engineered cancer antigen described herein are constitutively and / or ubiquitously expressed in a cell.

[0322] In some embodiments, a polynucleotide sequence encoding the engineered cancer antigen described herein is operably linked to an inducible promoter. In such embodiments, polynucleotides encoding the engineered cancer antigen described herein are conditionally expressed. As used herein, “conditional expression” may refer to any type of conditional expression including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological, or disease state (e.g., cell type or tissue specific expression) etc. Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the “GeneSwitch” mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), the cumate inducible gene switch (WO 2002 / 088346), tetracycline-dependent regulatory systems, etc.

[0323] In some embodiments, the vectors described herein further comprise a transcription termination signal. Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increases heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. In someembodiments, vectors comprise a poly adenylation sequence 3' of a polynucleotide encoding a polypeptide to be expressed. The term “polyA site” or “polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Cleavage and poly adenylation are directed by a poly(A) sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage -polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5' cleavage product. In some embodiments, the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA). In some embodiments, the poly (A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit P-globin polyA sequence (rPgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.

[0324] In some embodiments, a vector may also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, mitochondrial localization), fused to the polynucleotide encoding the engineered cancer antigens or the cognate binders. For example, a vector may comprise a nuclear localization sequence (e.g., from SV40) fused to the polynucleotide encoding the engineered cancer antigens or the cognate binders. In some embodiments, the signal peptide is an Igk signal peptide. In some embodiments, the signal peptide is a CD8 signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 64. In some embodiments, the signal peptide comprises the nucleic acid sequence of SEQ ID NO: 12.

[0325] In some embodiments, the expression vector further comprises nucleotide sequences encoding one or more protein tags (e.g., 6xHis tag (SEQ ID NO: 111), hemagglutinin tag, green fluorescent protein, etc.) that are fused to polynucleotide encoding the engineered cancer antigen, thereby resulting in an engineered cancer antigen that further comprises a protein tag.

[0326] Methods of introducing polynucleotides and recombinant vectors into a host cell are known in the art. Suitable methods include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al., Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9), microfluidics delivery methods (See e.g., International PCT Publication No. WO 2013 / 059343), and the like.

[0327] In some embodiments, delivery via electroporation comprises mixing the cells with the polynucleotides encoding the engineered cancer antigens or the cognate binders in a cartridge, chamber, or cuvette and applying one or more electrical impulses of defined duration and amplitude. In some embodiments, cells are mixed with polynucleotides encoding the engineered cancer antigens or the cognate binders in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber, or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel. Illustrative examples of polynucleotide delivery systems suitable for use in particular embodiments contemplated include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, NeonTM Transfection Systems, and Copernicus Therapeutics Inc. Lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient lipofection of polynucleotides have been described in the literature. See e.g., Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12.

[0328] In some embodiments, polynucleotides encoding the engineered cancer antigen described herein are introduced to a cell in a non- viral delivery vehicle, such as a transposon, a nanoparticle (e.g., a lipid nanoparticle), a liposome, an exosome, an attenuated bacterium, or a virus-like particle. In some embodiments, the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis including Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli), bacteria having nutritional and tissue-specific tropism to target specific cells, and bacteria having modified surface proteins to alter target cell specificity. In some embodiments, the vehicle is a genetically modified bacteriophage (e.g., engineered phages having large packaging capacity, less immunogenicity, containing mammalian plasmid maintenance sequences and having incorporated targeting ligands). In some embodiments, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject and wherein tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), secretory exosomes, or subject-derived membrane-bound nanovescicles (30 -100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need for targeting ligands).

[0329] In some embodiments, vectors comprising polynucleotides encoding the engineered cancer antigen described herein are introduced to cells by viral delivery methods, e.g., by viral transduction. A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the engineered mammalian cell in vitro or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In some embodiments, lentivirus vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying the immunomodulator (such as immune checkpoint inhibitor) coding sequence and / or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged with protocols known in the art. The resulting lentiviral vectors can be used to transduce a mammalian cell (such as primary human T cells) using methods known in the art. Vectors derived from retroviruses such as lentivirus are suitable tools to achieve long-term gene transfer, because they allow long-term, stable integration of a transgene and its propagation in progeny cells. Lentiviral vectors also have low immunogenicity, and can transduce nonproliferating cells.

[0330] In some embodiments, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the “empty” particles followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be engineered to incorporate targeting ligands to alter target tissue specificity.A. Tumor Tropic Viral Vectors

[0331] In some embodiments, the engineered cancer antigen is delivered by a vector that is a viral vector exhibiting tumor tropism.

[0332] Accordingly, in some embodiments, the vector comprising the polynucleotide encoding the engineered cancer antigen is a viral vector having tropism to a tumor cell, i.e., is a tumor tropic viral vector. By having tropism for a tumor cell, the viral vector is able to deliver the polynucleotide encoding the engineered cancer antigen to the tumor cell being targeted, e.g., a tumor cell of a subject’s cancer.

[0333] In some embodiments, the viral vector is a non-replicating viral vector, e.g., is replication defective. In some embodiments, the viral vector is a replication defective adenoviral vector that lacks the E1A gene that is essential for adenovirus replication.

[0334] In some embodiments, the viral vector is a replicating viral vector.

[0335] In some embodiments, the viral vector is a viral vector or modified form thereof of a viral vector as disclosed in, e.g., W02005 / 118825, WO2015 / 097220, WO2015 / 059303, WO2015 / 155370, WO2016 / 174200, WO2017 / 103291, WO2018 / 041838, WO2018 / 041827, W02017 / 103290, WO2018 / 220207, WO2019 / 043020, WO2022 / 171853, the contents of which are hereby incorporated by reference in their entity.

[0336] In some embodiments, the viral vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, or a herpes simplex viral (HSV) vector.

[0337] Exemplary adenoviral vectors, AAV vectors, and lentiviral vectors, among other viral vectors, are described in, e.g., Bezeljak, Radiol Oncol., 2022, 56(1): 1-13. Exemplary adenoviral vectors include those disclosed, e.g., in Engelhardt et al., Hum Gene Ther 5.1217-1229, 1994; US 5,756,283; US 5,707,618; Tessarollo et al., Cancers (Basel), 2021, 13(8): 1863; Wold et al., Curr. Gene Ther., 2013, 13(6): 421-433; and WO2014198852 A2. There are greater than 50 human adenovirus serotypes that are known, including Ad2, Ad3, Ad5, Ad7, Ad9, Adi l, Adl2, Ad 17, and Ad40.

[0338] In some embodiments, the viral vector is an adenoviral vector. In some embodiments, the viral vector is a non-replicating adenoviral vector, such as any of those as described in Tessarollo et al., supra; and Wold et al., supra. Adenoviruses are non-enveloped viruses with a single linear strand of double- stranded DNA inside of an icoasahedral capsid.

[0339] Adenoviruses of animal original can be obtained from deposited strains, and then amplified in competent cell lines, and, optionally, modified as desired. Complete genome sequences of adenoviruses have been determined for, e.g., human adenovirus type 2 (GenBank Accession No. J01917;), human adenovirus type 5 (GenBank Accession No. M73260,; and GenBank Accession No. NC 001406), human adenovirus type 12 (GenBank Accession No. NC— 001460, X73487); human adenovirus type 17 (GenBank Accession No. NC— 002067, AF108105), and human adenovirus type 40 (GenBank Accession No. L19443). Techniques for producing, isolating, and modifying adenoviruses have been described in the literature. See, e.g., Akli et al., Nature Genetics 3 (1993) 224; Stratford- Perricaudet et al., Human Gene Therapy 1 (1990) 241; EP 185 573; Levrero et al., Gene 101 (1991) 195; Le Gal la Salle et al., Science 259(1993) 988; Roemer and Friedmann, Eur. J. Biochem. 208 (1992) 211; Dobson et al., Neuron 5 (1990) 353; Chiocca et al., New Biol. 2 (1990) 739; Miyanohara et al., New Biol. 4 (1992) 238; WO 91 / 18088, WO 90 / 09441; WO 88 / 10311; and WO 91 / 11525. Such adenoviruses can be modified, for instance, by deletion, addition, and / or substitution.

[0340] In some embodiments, the adenoviral vector is of any one of subgroups A-G. In some embodiments, the adenoviral vector is of subgroup A. In some embodiments, the adenoviral vector is of subgroup B. In some embodiments, the adenoviral vector is of subgroup B, type 1. In some embodiments, the adenoviral vector is of subgroup B, type 2. In some embodiments, the adenoviral vector is of subgroup C. In some embodiments, the adenoviral vector is of subgroup D. In some embodiments, the adenoviral vector is of subgroup E. In some embodiments, the adenoviral vector is of subgroup F. In some embodiments, the adenoviral vector is of subgroup G.

[0341] Subgroup A adenoviruses includes, e.g., serotypes 12, 18, and 31; subgroup B, type 1 adenoviruses includes, e.g., serotypes 3, 7, 16, and 21; subgroup B, type 2 adenoviruses includes, e.g., serotypes 11, 14, 34, and 35; subgroup C adenoviruses includes, e.g., serotypes 1, 2, 5, and 6; subgroup D adenoviruses includes, e.g., serotypes 8-10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36- 39, and 42-49; subgroup E adenoviruses includes, e.g., serotype 4; subgroup F adenoviruses includes, e.g., serotypes 40 and 41; and subgroup G adenoviruses includes, e.g., serotype 52. See, e.g., Ghebremedhin, Eur. J. Microbiol. Immunol., 2014, 4(1): 26-33.

[0342] Most adenoviruses, e.g., those in subgroups A, C, D, E, and F, enter target cells by binding primarily to the coxsackie-adenovirus receptor. However, generally, the subgroup B, type 1 group of adenoviruses bind primarily to CD46, whereas, generally, the subgroup B, type 2 group of adenoviruses bind primarily to desmoglein-2, with some exceptions. Adi l, Adl4, Ad 16, Ad21, Ad35, and Ad50 of subgroup B bind to CD46, as well as Ad 17 and Ad47 of subgroup D. Ad3, Ad7, and Adl4 of subgroup B bind to desmoglein-2. See, e.g., Hensen et al., Int. J. Mol. Sci., 2020, 21(18): 6828.

[0343] The levels of coxsackie-adenovirus receptor vary by tissue and by tumor type, and can be upregulated or downregulated in tumor cells, depending on the type of cancer. See, e.g., Hensen et al., supra.

[0344] All nucleated cells express CD46, thereby making adenoviruses from subgroup B, type 1 desirable for its broad tropism. Moreover, the expression of CD46 is reported as being low or moderate in most normal tissues, but upregulated in many different types of cancer, thereby making adenoviruses that primarily bind CD46, e.g., Adi l, Adl4, Adl6, Adl7, Ad21,Ad35, Ad47, and Ad50, desirable for use in delivering engineered cancer antigens to tumor cells. See, e.g., Do et al., Int. J. Mol. Sci., 2018, 19: 2694; Su et al., JCI Insight, 2018, 3: el21497; and Hensen et al., supra.

[0345] Desmoglein-2 is a transmembrane glycoprotein that is expressed in a variety of tissue types, including bladder, colon, kidney, prostate, and stomach, and, like CD46, has been reported to be upregulated in cancers. See, e.g., Hensen et al., supra; and Brennan et al., Cell Adhes. Migr., 2009, 3: 148-154. As such, like adenoviruses that primarily bind to CD46, adenoviruses that primarily bind to desmoglein-2, such as Ad3, Ad7, and Ad 14, are desirable for use in delivering engineered cancer antigens to tumor cells.

[0346] Accordingly, in some embodiments, the viral vector, e.g., adenoviral vector, binds to CD46 and / or desmoglein-2. In some embodiments, the viral vector that binds to CD46 and / or desmoglein-2 is Ad3, Ad7, Adi l, Adl4, Adl6, Adl7, Ad21, Ad35, Ad47, or Ad50.

[0347] In some embodiments, the adenoviral vector is based on Ad3, Ad7, Adi l, Adl4, Adl6, Adl7, Ad21, Ad35, Ad47, and Ad50, or any combination thereof. In some embodiments, the adenoviral vector is based on Ad2. In some embodiments, the adenoviral vector is based on Ad3. In some embodiments, the adenoviral vector is based on Ad5. In some embodiments, the adenoviral vector is based on Ad7. In some embodiments, the adenoviral vector is based on Ad9. In some embodiments, the adenoviral vector is based on Adi l. In some embodiments, the Adi 1 is an Adi Ip. In some embodiments, the adenoviral vector is based on Adl2. In some embodiments, the adenoviral vector is based on Adl4. In some embodiments, the adenoviral vector is based on Adl7. In some embodiments, the adenoviral vector is based on Ad35. In some embodiments, the adenoviral vector is based on Ad40. In some embodiments, the adenoviral vector is based on Ad50.

[0348] In some embodiments, the adenoviral vector is a chimeric virus based on any two or more serotypes of adenovirus. When an adenovirus is chimeric then the characteristics of the outer capsid will be employed to determine the serotype. Chimeric as employed herein refers to a virus that comprises DNA from at least two different virus serotypes, including different serotypes within the same group. In some embodiments, the adenoviral vector is a chimeric virus based on serotype 3 (Ad3) and / or serotype 5 (Ad5) adenoviral vectors. In some embodiments, the adenoviral vector is a chimeric virus based on serotype 3 (Ad3) and / or serotype 11 (Adi l) adenoviral vectors.

[0349] In some embodiments, the adenoviral vector is Ad5. In some embodiments, the adenoviral vector is Ad5 or a variant or derivative thereof. The Ad5 adenoviral vector generally enters the cell through the coxsackie-adenovirus receptor.

[0350] In some embodiments, the adenovirus is a chimeric adenovirus. In some embodiments, the chimeric adenovirus is Ad3 / l lp or is Ad5 / 3.

[0351] In some embodiments, the adenoviral vector is Ad3 / 1 Ip, which is a chimeric adenovirus composite of Ad3 and Adi Ip. In some embodiments, the Ad3 / 1 Ip is referred to as ColoAdl (also known as “enadenotucirev”). ColoAdl is a chimeric adenovirus in which the major coat proteins are derived from Adi Ip, and, relative to Adi l, includes an almost complete 2444 base pair deletion of the E3 gene, a smaller 25-base pair deletion of the E4 gene region, and a chimeric Ad3 / Adl 1pm E3B gene region. See Kuhn et al., PLoS ONE, 2008, 3: e2409; WO2014198852 A2; US8765463. In particular, ColoAdl has a chimeric E2B region, which features DNA from Adi Ip and Ad3, and deletions in E3 / E4. The structural changes result in a genome that is approximately 3.5 kb smaller than Adi Ip and thereby provides space for insertion of transgenes. Moreover, ColoAdl is a replication competent oncolytic chimeric adenovirus which has enhanced properties compared to wild-type adenoviruses, see e.g. W02005 / 118825. The Adi l adenovirus generally enters a cell through the CD46 receptor, which is expressed at low levels in all nucleated cells, but typically exhibits increased surface expression in tumor cells, thereby preferentially infecting tumor cells. Both Ad3 and Adi Ip are subgroup B adenoviruses (Bl and B2 respectively). Chimeric adenoviruses, such as Ad3 / l lp are typically generated using a method called “directed evolution,” such as described in, e.g., Kuhn et al., supra, in which pools of Ad serotypes, representing the different Ad subgroups, are passaged on human tumor cell lines representative of major solid tumor indications (e.g., breast cancer, colon cancer, pancreatic cancer, or prostate cancer) to invite recombination and selection of potent viral variants or serotypes.

[0352] In some embodiments, the Ad3 / l lp viral vector is ColoAdl. See, e.g., Kuhn et al., supra; US8765463; and W02005 / 118825. ColoAdl has the nucleic acid sequence of SEQ ID NO: 13.

[0353] In some embodiments, the adenoviral vector is Ad5 / 3, such as described in, e.g., Hemminki et al., Oncotarget, 2015, 6: 4467-81; and Safar et al., Cancer Gene Therapy, 2021, 28: 442-454. The Ad5 / 3 chimeric adenoviral vector includes the serotype 3 fiber knob in an Ad5 capsid, thereby using the Ad3 receptor (desmoglein-2), which is highly expressed in advanced tumors. In some embodiments, the vector is an adeno-associated viral (AAV) vector. In someembodiments, the AAV is an AAV of any serotype, such as AAV1, AAV2, AAV3, or AAV4. In some embodiments, the AAV is an AAV1. In some embodiments, the AAV is an AAV2. In some embodiments, the AAV is an AAV3. In some embodiments, the AAV is an AAV4.

[0354] The genomes of AAVs have been determined. For instance, the genomic sequences of AAV2 are provided in GenBank Accession No. J01901 and GenBank No. NC_001401.

[0355] In some embodiments, the viral vector is an oncolytic virus or exhibits oncolytic activity. Oncolytic viruses are capable of infecting and killing cancer cells. The oncolytic virus can be, but is not limited to, adenoviruses, herpes simplex virus (HSV), parvoviruses, and poxviruses such as vaccinia virus (VACV) and myxoma virus (MYXV). In some embodiments, the oncolytic virus is selected from the group consisting of an adenovirus, a herpes simplex virus, a vaccinia virus, a mumps virus, a newcastle disease virus, a poliovirus, a seneca valley virus, a measles virus, a sindbis virus, a parvovirus, a coxsackie virus, a vesicular stomatitis virus, a reovirus, and a maraba and rhabdovirus. In some embodiments, the viral vector exhibits weak oncolytic activity.

[0356] In some embodiments, the viral vector comprises a tumor- specific promoter element that promotes increased expression of the encoded engineered antigen tag when expressed in a tumor cell, or a tumor cell of a specific cancer type, as compared to its expressed in a non-tumor cell, e.g., a normal non-cancerous cell of the same cell or tissue type as the tumor cell. In some embodiments, the tumor- specific promoter element is a STAT3 promoter, a survivin promoter, a cyclooxygenase-2 (COX-2) promoter, a prostate specific antigen (PSA) promoter, a CXCR4 promoter, or any other promoter that promoters increased expression in a tumor cell as compared to a normal cell of the same cell or tissue type. Examples of tumor- specific promoter elements and methods for modifying viral vectors to include the same can be found in, e.g., US20190275093A1.

[0357] It is within the level of a skilled artisan to determine the insertion site of a transgene within an oncolytic vector. Typically, an insert, such as encoding a transgene sequence (e.g., engineered cancer antigen) is inserted into a non-coding region of genomic virus DNA, such as an intron or intergenic sequence. In some embodiments, the transgene is under the control of a promoter.

[0358] For example, with reference to adenovirus, including a chimeric adenovirus, such as ColoAdl, among regions for insertion include the L5 region, such as between L5 and the E4 region.

[0359] In some embodiments, an adenovirus, including a chimeric adenovirus such as ColoAdl, include the structural elements of adenoviruses as described in WO2018 / 041838, which is incorporated by reference, including the formula set forth by formula (I), (la), 1(b), 1(c), or 1(e), but in which the transgene encodes an engineered cancer antigen as described herein.

[0360] In some embodiments, an adenovirus, including a chimeric adenovirus such as ColoAdl, that includes a transgene, has a sequence with the following formula (I): 5’ ITR-Bi- BA-B2-BX-BB-BY-B3-3’ ITR.

[0361] In such an embodiment, the Inverted Terminal Repeat (ITR) sequences are common to all known adenoviruses and are so named because of their symmetry, and are the viral chromosome origins of replication. Another property of these sequences is their ability to form a hairpin. The 5'ITR as employed herein refers to part or all of an ITR from the 5' end of an adenovirus, which retains the function of the ITR when incorporated into an adenovirus in an appropriate location. In one embodiment, the 5'ITR comprises or consists of the sequence from about 1 bp to 138bp of SEQ ID NO: 13 or a sequence 90, 95, 96, 97, 98 or 99% identical thereto along the whole length, in particular the sequence consisting of from about 1 bp to 138bp of SEQ ID NO: 13. In some embodiments, the 3'ITR with reference to an adenovirus refers to part or all of an ITR from 3' end of an adenovirus which retains the function of the ITR when incorporated into an adenovirus in an appropriate location. In one embodiment the 3'ITR comprises or consists of the sequence from about 32189bp to 32326bp of SEQ ID NO: 13 or a sequence 90, 95, 96, 97, 98 or 99% identical thereto along the whole length, in particular the sequence consisting of from about 32189bp to 32326bp of SEQ ID NO: 13.

[0362] In embodiments of the formula (I), Bi is a bond or comprises E1A, E1B or E1A-E1B. In some embodiments, Bi as employed herein refers to the DNA sequence encoding: part or all of an E1A from an adenovirus, part or all of the El 8 region of an adenovirus, and independently part or all of E1A and E1B region of an adenovirus. When Bi is a bond then E1A and E1B sequences will be omitted from the virus. In one embodiment Bi is a bond and thus the virus is a vector.

[0363] In one embodiment Bi further comprises a transgene. It is known in the art that the Bi region can accommodate a transgene which may be inserted in a disruptive way into the El region (i.e. in the "middle" of the sequence) or part or all of the El region may be deleted to provide more room to accommodate genetic material.

[0364] E1A as employed herein refers to the DNA sequence encoding part or all of an adenovirus E1A region. The latter here is referring to the polypeptide / protein E1A It may bemutated such that the protein encoded by the E1A gene has conservative or non-conservative amino acid changes, such that it has: the same function as wild-type (i.e. the corresponding nonmutated protein); increased function in comparison to wild-type protein; decreased function, such as no function in comparison to wild-type protein; or has a new function in comparison to wild-type protein or a combination of the same as appropriate.

[0365] E1B as employed herein refers to the DNA sequence encoding part or all of an adenovirus E1B region (i.e. polypeptide or protein), it may be mutated such that the protein encoded by the El 8 gene / region has conservative or non-conservative amino acid changes, such that it has: the same function as wild-type (i.e. the corresponding non-mutated protein); increased function in comparison to wild-type protein; decreased function, such as no function in comparison to wild-type protein; or has a new function in comparison to wild-type protein or a combination of the same as appropriate.

[0366] Thus Bi can be modified or unmodified relative to a wild- type El region, such as a wild- type El A and / or E1B. The skilled person can easily identify whether El A and / or El 8 are present or (part) deleted or mutated.

[0367] In one embodiment Bi has the sequence from 139bp to 3932bp of SEQ ID NO: 13.

[0368] In embodiments of the formula (I), BA as employed herein refers to the DNA sequence encoding the E2B-L1-L2-L3-E2A-L4 regions including any non-coding sequences, as appropriate. Generally this sequence will not comprise a transgene. In one embodiment the sequence is substantially similar or identical to a contiguous sequence from a known adenovirus, for example a serotype of a group B virus, for example Ad3, Ad7, Adi l, Adl4, Adl6, Ad21, Ad34, Ad35, Ad51 or a combination thereof, such as Ad3, Adi l or a combination thereof. In one embodiment, E2B-L1-L2-L3-E2A-L4 refers to comprising these elements and other structural elements associated with the region, for example BA will generally include the sequence encoding the protein IV2a, for example as follows: IV2A IV2a-E2B-Ll-L2-L3-E2A- L4.

[0369] In one embodiment the E2B region is chimeric. That is, it comprises DNA sequences from two or more different adenoviral serotypes, for example from Ad3 and Adi l, such as Adi Ip. In one embodiment the E2B region has the sequence from 5068bp to 10355bp of SEQ ID NO: 13 or a sequence 95%, 96%, 97%, 98% or 99% identical thereto over the whole length.

[0370] In one embodiment BA has the sequence from 3933bp to 27184bp of SEQ ID NO: 13.

[0371] E3 as employed herein refers to the DNA sequence encoding part or all of an adenovirus E3 region (i.e. pro tein / polypep tide), it may be mutated such that the protein encodedby the E3 gene has conservative or non-conservative amino acid changes, such that it has the same function as wildtype (the corresponding unmutated protein); increased function in comparison to wild-type protein; decreased function, such as no function in comparison to wildtype protein or has a new function in comparison to wild-type protein or a combination of the same, as appropriate.

[0372] In one embodiment the E3 region is from an adenovirus serotype, in particular a group B serotype, for example Ad3, Ad7, Adi 1 (in particular Adi Ip), Adl4, Adl6, Ad21, Ad34, Ad35, Ad51 or a combination thereof, such as Ad3, Adi 1 (in particular Adi Ip) or a combination thereof.

[0373] In one embodiment, the E3 region is partially deleted, for example is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% deleted. In one embodiment B2 with reference to formula (I) is a bond, wherein the DNA encoding the E3 region is absent.

[0374] In one embodiment the DNA encoding the E3 region can be replaced or interrupted by a transgene. As employed herein "E3 region replaced by a transgene” as employed herein include part or all of the E3 region is replaced with a transgene.

[0375] In one embodiment the B2 region comprises the sequence from 27185bp to 28165bp of SEQ ID NO: 13. In one embodiment B2 region consists of the sequence from 27185bp to 28165bp of SEQ ID NO: 13.

[0376] In embodiments of the formula (I), Bx as employed herein refers to the DNA sequence in the vicinity of the 5' end of the L5 gene in BB. In the vicinity of or proximal to the 5' end of the L5 gene as employed herein refers to: adjacent (contiguous) to the 5' end of the L5 gene or a non-coding region inherently associated herewith i.e. abutting or contiguous to the 5' prime end of the L5 gene or a non-coding region inherently associated therewith. Alternatively, in the vicinity of or proximal to may refer to being close the L5 gene, such that there are no coding sequences between the Bx region and the 5' end of L5 gene.

[0377] Thus in one embodiment Bx is joined directly to a base of L5 which represents, for example the start of a coding sequence of the L5 gene.

[0378] Thus in one embodiment Bx is joined directly to a base of L5 which represents, for example the start of a non-coding sequence, or joined directly to a non-coding region naturally associated with L5. A non-coding region naturally associated L5 as employed herein refers to part of all of a non-coding regions which is part of the L5 gene or contiguous therewith but not part of another gene.

[0379] In one embodiment Bx has the sequence from 28166bp to 28366bp of SEQ ID NO: 13. The insert(s) can occur anywhere in this sequence from the 5' end, the 3' end or at any point between bp 1 to 201 of the sequence corresponding to 28166bp to 28366bp of SEQ ID NO: 13. In one embodiment Bx has a DNA sequence inserted between a place corresponding to between positions 28192bp and 28193bp of SEQ ID NO: 13.

[0380] In one embodiment, Bx is a bond.

[0381] This sequence is an artificial non-coding sequence wherein a DNA sequence, for example comprising a transgene (or transgene cassette), a restriction site or a combination thereof may be inserted therein. This sequence is advantageous because it acts as a buffer in that allows some flexibility on the exact location of the transgene while minimizing the disruptive effects on virus stability and viability.

[0382] In embodiments of the formula (I), BB as employed herein refers to the DNA sequence encoding the L5 region. As employed herein the L5 region refers to the DNA sequence containing the gene encoding the fibre polypeptide / protein, as appropriate in the context. The fibre gene / region encodes the fibre protein which is a major capsid component of adenoviruses. The fibre functions in receptor recognition and contributes to the adenovirus' ability to selectively bind and infect cells.

[0383] In viruses of the present disclosure the fibre can be from any adenovirus serotype and adenoviruses which are chimeric as result of changing the fibre for one of a different serotype are also envisaged with the present disclosure. In one embodiment the fibre is from a group B virus, in particular Ad 11, such as Adi Ip.

[0384] In one embodiment BB has the sequence from 28367bp to 29344bp of SEQ ID NO:13.

[0385] DNA sequence in relation to By of formula (I) as employed herein refers to the DNA sequence in the vicinity of the 3' end of the L5 gene of BB. In the vicinity of or proximal to the 3' end of the L5 gene as employed herein refers to: adjacent (contiguous) to the 3’ end of the L5 gene or a non-coding region inherently associated therewith i.e. abutting or contiguous to the 3’ prime end of the L5 gene or a non-coding region inherently associated therewith (i.e. all or part of an non-coding sequence endogenous to L5). Alternatively, in the vicinity of or proximal to may refer to being close the L5 gene, such that there are no coding sequences between the By region and the 3’ end of the L5 gene.

[0386] Thus, in one embodiment By is joined directly to a base of L5 which represents the "end" of a coding sequence.I l l

[0387] Thus, in one embodiment By is joined directly to a base of L5 which represents the "end" of a non-coding sequence, or joined directly to a non-coding region naturally associated with L5.

[0388] In one embodiment By has the sequence from 29345bp to 29379bp of SEQ ID NO: 13. This sequence is a non-coding sequence wherein a DNA sequence, for example comprising a transgene (or transgene cassette), a restriction site or a combination thereof may be inserted. This sequence is advantageous because it acts a buffer in that allows some flexibility on the exact location of the transgene while minimizing the disruptive effects on virus stability and viability. The insert(s) can occur anywhere within the sequence from the 5' end, the 3' end or at any point between bp 1 to 35 of the sequence from 29345bp to 29379bp of SEQ ID NO: 13. In one embodiment By has a DNA sequence inserted between a place corresponding to positions 29356bp and 29357bp in SEQ ID NO: 13. In one embodiment the insert is at about position 29356bp of SEQ ID NO: 13. In one embodiment the insert is a restriction site insert. In one embodiment the insert is a transgene cassette comprising one or more transgenes, such as an engineered cancer antigen as described herein. In some embodiments, the transgene is under the control of a promoter.

[0389] The term “insert” as employed herein refers to a DNA sequence that is incorporated either at the 5' end, the 3' end or within a given DNA sequence reference segment such that it interrupts the reference sequence. A reference sequence employed as a reference point relative to which the insert is located. An insert can be either a restriction site insert, a transgene cassette or both. In the context of the present disclosure inserts generally occur within a sequence corresponding to either at about or after position 29356bp of SEQ ID NO: 13 or between positions 28192bp and 28193bp of SEQ ID NO:13. When the sequence is interrupted the virus will still comprise the original sequence, but generally it will be as two fragments sandwiching the insert.

[0390] In embodiments in relation to formula (I), B3 is the sequence of a partially deleted E4 region. In some embodiments B3 is a bond, i.e. wherein E4 is absent.

[0391] E4 as employed herein refers to the DNA sequence encoding part or all of an adenovirus E4 region (i.e. polypeptide / protein region), which may be mutated such that the protein encoded by the E4 gene has conservative or non-conservative amino acid changes (e.g. 1, 2, 3, 4 or 5 amino acid changes, additions and / or deletions), and has the same function as wildtype (the corresponding non-mutated protein); increased function in comparison to wild-typeprotein; decreased function, such as no function in comparison to wild-type protein or has a new function in comparison to wild-type protein or a combination of the same as appropriate.

[0392] In one embodiment, the E4 region is partially deleted, for example is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% deleted. In one embodiment the E4 region has the sequence from 32188bp to 29380bp of SEQ ID NO: 13.

[0393] In one embodiment E4 is present except for the E4orf4 region which is deleted.

[0394] In one embodiment B3 is a bond, i.e. wherein E4 is absent.

[0395] In one embodiment B3 has the sequence consisting of from 32188bp to 29380bp of SEQ ID NO: 13.

[0396] The sites identified herein, that are associated with the L5 region (for example between L5 and the E4 region), are suitable for accommodating a transgene sequence, such as an engineered cancer antigen as described herein. In some embodiments, the transgene is under the control of a promoter

[0397] In some embodiments, provided herein is a replication deficient or replication capable oncolytic adenovirus, comprising a transgene cassette encoding any of the engineered cancer antigens herein located between the virus fibre gene L5 and the virus E4 gene and wherein the transgene is under the control of a promoter. In some embodiments, the virus is a group B adenovirus, such as Adi l. In some embodiments, the virus is a chimeric virus, for example a virus that comprises a modified E2B region. In some embodiments, the virus is ColAdl 1 (enadenotucirev) .

[0398] In some embodiments, the promoter is endogenous to the virus. Thus, endogenous promoter as employed herein refers to a promoter that naturally occurs in (i.e. is native to) the adenovirus (or construct) into which the transgene, is being inserted. In one or more embodiments, the endogenous promoter employed is the naturally occurring promoter in the virus in its original location in the virus genome, in particular this is the primary or only promoter employed in the expression of the transgene or transgenes. In one embodiment the endogenous promoter used to promote the translation and optionally the transcription of the transgene is one resident, i.e. is one integrated in the genome of the adenovirus and not previously introduced by recombinant techniques.

[0399] Under the control of an endogenous promoter as employed herein refers to where the transgene / transgene cassette is inserted in the appropriate orientation to be under the control ofthe endogenous promoter. That is, where the promoter is generally on the antisense strand, the cassette is inserted, for example in the antisense orientation.

[0400] Having said this, genes can be expressed in one of two orientations. However, generally one orientation provides increased levels of expression over the other orientation, for a given (particular) transgene. In one embodiment, the cassette is in the sense orientation. That is, is transcribed in a 5' to 3' direction. In one embodiment, the cassette is in the antisense orientation. That is, transcribed in the 3' to 5' orientation.

[0401] In one embodiment the transgene, transgenes, or transgene cassette are under the control of an E4 promoter or a major late promoter, such as the major late promoter (ML promoter). Under the control of as employed herein means that the transgene is activated, i.e. transcribed, when a particular promoter dictates.

[0402] In some embodiments, the promoter is an MLP. The Major Late Promoter (ML promoter or MLP) as employed herein refers to the adenovirus promoter that controls expression of the "late expressed" genes, such as the L5 gene. The MLP is a "sense strand" promoter. That is, the promoter influences genes that are downstream of the promoter in the 5'-3' direction. The major late promoter as employed herein refers the original major late promoter located in the virus genome.

[0403] In some embodiments, the promoter is the E4 promoter. The E4 promoter as employed herein refers to the adenovirus promoter of the E4 region. The E4 region is an antisense region; therefore the promoter is an anti-sense promoter. That is, the promoter is upstream of the E4 region in the 3'-5' direction. Therefore any transgene cassette under control of the E4 promoter may need to be oriented appropriately. In one embodiment the cassette under the control of the E4 promoter is in the antisense orientation. In one embodiment the cassette is under the control of the E4 promoter in the sense orientation. The E4 promoter as employed herein refers to the original E4 promoter located in the virus genome.

[0404] Thus in one embodiment there is provided a replication competent oncolytic adenovirus serotype 11 (such as Adi Ip) or virus-derivative thereof wherein the fibre, hexon and capsid are serotype 11 (such as Adi Ip), wherein the virus genome comprises a transgene (DNA sequence) encoding any of the provided engineered cancer antigens, wherein said DNA sequence is under the control of a promoter endogenous to the adenovirus selected from consisting of E4 and the major late promoter (i.e. the E4 promoter or the major late promoter), such that the transgene does not interfere with virus replication, for example is associated with the L5 region(i.e. located before or after said region), such as located after L5 in the virus genome, in particular located between L5 and the E4 region.

[0405] In one embodiment, the transgene cassette comprises an exogenous promoter. Exogenous promoter as employed herein refers to a promoter that is not naturally occurring in the adenovirus into which the transgene is being inserted. Typically, exogenous promoters are from other viruses or are mammalian promoters. Exogenous promoter as employed herein means a DNA element, usually located upstream of the gene of interest, that regulates the transcription of the gene.

[0406] In one embodiment, the regulator of gene expression is an exogenous promoter, for example CMV (cytomegalovirus promoter), CBA (chicken beta actin promoter) or PGK (phosphoglycerate kinase 1 promoter), such as CMV promoter.

[0407] In one embodiment there is provided a replication competent oncolytic adenovirus serotype 11 (such as Adi Ip) or virus-derivative thereof wherein the fibre, hexon and capsid are serotype 11 (such as Adi Ip), wherein the virus genome comprises a DNA sequence encoding any of the provided engineered cancer antigens located in a part of the virus genome which is expressed late in the virus replication cycle and such that the transgene does not interfere with virus replication, wherein said DNA sequence under the control of a promoter exogenous to the adenovirus (for example the CMV promoter). In one embodiment, the DNA sequence encoding the engineered cancer antigen is associated with the L5 region as described elsewhere herein, in particular located between L5 and E4 region.

[0408] Other features of a viral vector, such as an adenovirus, known to a skilled artisan also are present as part of the adenoviral vector. These include regulatory sequences, splice acceptor sequences, poly adenylation sequences and other sequences well known to a skilled artisan. Any of such sequences are known and described, for example, in W02005 / 118825, WO2015 / 097220, W02015 / 059303, WO2015 / 155370, WO2016 / 174200, WO2017 / 103291, WO2018 / 041838, WO20 18 / 041827, W02017 / 103290, WO2018 / 220207, WO2019 / 043020, WO2022 / 171853, the contents of which are hereby incorporated by reference in their entity.V. COMPOSITIONS AND KITS

[0409] Provided herein are compositions, e.g., pharmaceutical compositions, comprising one or more of any of the vectors disclosed herein, e.g., any of the vectors comprising one or more polynucleotides encoding one or more of the engineered cancer antigens disclosed herein.

[0410] In some embodiments, the composition is a pharmaceutical composition comprising one or more of any of the vectors disclosed herein, e.g., any of the vectors comprising one ormore polynucleotides encoding one or more of the engineered cancer antigens disclosed herein, and a pharmaceutically acceptable carrier.

[0411] In some embodiments, the pharmaceutical composition comprises a vector comprising a polynucleotide encoding any of the engineered cancer antigens comprising a tumor targeting molecule.

[0412] In some embodiments, the pharmaceutical composition comprises a vector comprising a polynucleotide encoding any of the engineered cancer antigens comprising a heterologous membrane targeting domain.

[0413] Provided herein are compositions, e.g., pharmaceutical compositions, comprising a population of lymphocytes expressing a CAR, such as any of the CARs disclosed in Section I.D. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells are CD4+ and / or CD8+ T cells.

[0414] Also provided herein are compositions, e.g., pharmaceutical compositions, comprising any of the CAR-T cell therapies disclosed herein, e.g., any of the CAR-T cell therapies comprising CAR-T cells that specifically bind to the target domain of any of the engineered cancer antigens disclosed herein.

[0415] Also provided herein are compositions, e.g., pharmaceutical compositions, comprising: (i) one or more of any of the vectors disclosed herein, e.g., any of the vectors comprising one or more polynucleotides encoding one or more of the engineered cancer antigens disclosed herein; and (ii) any of the CAR-T cell therapies disclosed herein, e.g., any of the CAR- T cell therapies comprising CAR-T cells that specifically bind to the target domain of any of the engineered cancer antigens disclosed herein.

[0416] Also provided herein are kits comprising: (i) a first composition, e.g., a first pharmaceutical composition, comprising one or more of any of the vectors disclosed herein, e.g., any of the vectors comprising one or more polynucleotides encoding one or more of the engineered cancer antigens disclosed herein; and (ii) a second composition, e.g., a second pharmaceutical composition, comprising any of the CAR-T cell therapies disclosed herein, e.g., any of the CAR-T cell therapies comprising CAR-T cells that specifically bind to the target domain of any of the engineered cancer antigens disclosed herein.

[0417] In some embodiments, the kit further comprises instructions for use of the first composition and the second composition, e.g., in the treatment of a cancer in a subject.

[0418] In some embodiments, the first composition, e.g., the first pharmaceutical composition, and the second composition, e.g., the second pharmaceutical composition, areadministered concurrently. In some embodiments, the first composition, e.g., the first pharmaceutical composition is administered before the second composition, e.g., the second pharmaceutical composition, being administered. In some embodiments, the first composition, e.g., the first pharmaceutical composition is administered after the second composition, e.g., the second pharmaceutical composition being administered.

[0419] In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the first composition and the second composition are each a pha...

Claims

WHAT IS CLAIMED IS:

1. An engineered cancer antigen, comprising a target domain and a tumor targeting molecule, wherein the target domain is the extracellular domain or a truncated portion thereof of a target antigen.

2. The engineered cancer antigen of claim 1, that is secretable when expressed from a cell.

3. The engineered cancer antigen of claim 1 or claim 2, wherein the tumor targeting molecule binds to a tumor-associated antigen expressed on the surface of a tumor cell.

4. The engineered cancer antigen of claim 3, wherein the tumor-associated antigen is selected from the group consisting of EpCAM, CEA (Carcinoembryonic antigen), gpA33 (Glycoprotein A33 (Transmembrane)), mucins, TAG-72 (Tumor-associated glycoprotein 72), CAIX (Carbonic anhydrase IX), PSMA (Prostate-specific membrane antigen), and FBP (Folate- binding protein), EGFR / ERBB1 / HER1 (epidermal growth factor receptor 1), ERBB3 (epidermal growth factor receptor 3), MET (Tyrosine-Protein Kinase IGF1R (insulin-like growth factor 1 receptor), EPHA3 (EPH Receptor A3), TRAILR1 (Death receptor 4), and RANK-L (Receptor activator of nuclear factor kappa-B ligand).

5. The engineered cancer antigen of claim 3 or claim 4, wherein the tumor cell is a tumor cell of a solid tumor.

6. The engineered cancer antigen of any one of claims 3-5, wherein the tumor cell is a tumor cell of a cancer selected from the group consisting of multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, medulloblastoma, liver cancer, stomach cancer, thyroid cancer, bile duct cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, endometrial cancer, and cervical cancer.

7. The engineered cancer antigen of any one of claims 3-6, wherein the tumor cell is a tumor cell of a cancer selected from the group consisting of bladder cancer, breast cancer, skincancer, head and neck cancer, colorectal cancer, endometrial cancer, liver cancer, kidney cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, thyroid cancer, and ovarian cancer.

8. The engineered cancer antigen of any one of claims 1-7, wherein the tumor targeting molecule comprises an antibody or an antigen-binding fragment thereof.

9. The engineered cancer antigen of claim 8, wherein the antibody or antigenbinding fragment thereof is a single-chain variable fragment (scFv), DARPin, variable heavy (VH) domain, or VHH, more optionally is a single-chain variable fragment (scFv).

10. The engineered cancer antigen of any one of claims 1-9, wherein the tumor- associated antigen is EpCAM.

11. The engineered cancer antigen of any one of claims 8-10, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region comprises a heavy chain complementarity determining region 1 (CDR- Hl), a CDR-H2, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NOS: 15, 16, and 17, respectively, and the VL region comprises a light chain complementarity determining region 1 (CDR-L1), a CDR-L2, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NOS: 18, 19, and 20, respectively.

12. The engineered cancer antigen of any one of claims 8-11, wherein the antibody or an antigen-binding fragment thereof comprises a VH region comprising an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10; and a VL region comprising an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 8.

13. The engineered cancer antigen of any one of claims 8-12, wherein the antibody or an antigen-binding fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 10, and a VL region comprising the amino acid sequence of SEQ ID NO: 8.

14. The engineered cancer antigen of any one of claims 1-13, wherein the target domain and the tumor targeting molecule are directly linked.

15. The engineered cancer antigen of any one of claims 1-13, wherein the target domain and the tumor targeting molecule are linked by a linker.

16. The engineered cancer antigen of claim 15, wherein the linker has a length of between 1 and 100 amino acids, between 1 and 75 amino acids, between 1 and 50 amino acids, between 1 and 25 amino acids, between 5 and 100 amino acids, between 5 and 75 amino acids, between 5 and 50 amino acids, between 5 and 25 amino acids, between 10 and 100 amino acids, between 10 and 75 amino acids, between 10 and 50 amino acids, or between 10 and 25 amino acids.

17. The engineered cancer antigen of claim 15 or claim 16, wherein the linker comprises the amino acid sequence of any one of SEQ ID NOS: 9 and 138-144.

18. The engineered cancer antigen of any one of claims 1-17, wherein the target antigen is a membrane-expressed antigen.

19. The engineered cancer antigen of any one of claims 1-18, wherein the target antigen is a cell surface receptor.

20. The engineered cancer antigen of any one of claims 1-19, wherein the target domain is recognizable by a cognate binder.

21. The engineered cancer antigen of any one of claims 1-20, wherein the target domain is a truncated portion of an extracellular domain of the target antigen that retains an epitope recognizable by a cognate binder.

22. The engineered cancer antigen of any one of claims 1-21, wherein the target antigen is selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyro sine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein-coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), R0R1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

23. The engineered cancer antigen of any one of claims 1-22, wherein the target antigen is BCMA.

24. The engineered cancer antigen of any one of claims 1-23, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 3.

25. The engineered cancer antigen of claim 23 or claim 24, wherein the target domain is recognizable by a cognate binder comprising: a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 32; and / ora single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 35.

26. The engineered cancer antigen of any one of claims 1-22, wherein the target antigen is CD70.

27. The engineered cancer antigen of any one of claims 1-22 and 26, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 106.

28. The engineered cancer antigen of claim 26 or claim 27, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 107 or 124.

29. The engineered cancer antigen of any one of claims 1-22, wherein the target antigen is CD 19.

30. The engineered cancer antigen of any one of claims 1-22 and 29, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 71.

31. The engineered cancer antigen of claim 29 or claim 30, wherein the target domain is recognizable by a cognate binder comprising:(a) a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 47; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 48 or 49; or(b) a VH region comprising the amino acid sequence of SEQ ID NO: 50 and a VL region comprising the amino acid sequence of SEQ ID NO: 51; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 52.

32. The engineered cancer antigen of any one of claims 1-22, wherein the target antigen is CD20.

33. The engineered cancer antigen of any one of claims 1-22 and 32, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 72.

34. The engineered cancer antigen of claim 32 or claim 33, wherein the target domain is recognizable by a cognate binder comprising: a VH region comprising the amino acid sequence of SEQ ID NO: 53 and a VL region comprising the amino acid sequence of SEQ ID NO: 54; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 55.

35. The engineered cancer antigen of any one of claims 1-22, wherein the target antigen is CD22.

36. The engineered cancer antigen of any one of claims 1-22 and 35, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 73.

37. The engineered cancer antigen of claim 35 or claim 36, wherein the target domain is recognizable by a cognate binder comprising:(a) a VH region comprising the amino acid sequence of SEQ ID NO: 56 and a VL region comprising the amino acid sequence of SEQ ID NO: 57; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 58; or(b) a VH region comprising the amino acid sequence of SEQ ID NO: 59 and a VL region comprising the amino acid sequence of SEQ ID NO: 60; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 61.

38. The engineered cancer antigen of any one of claims 1-22, wherein the target antigen is CD70.

39. The engineered cancer antigen of any one of claims 1-22 and 38, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 124.

40. The engineered cancer antigen of claim 38 or claim 39, wherein the target domain is recognizable by a cognate binder comprising:a VH region comprising the amino acid sequence of SEQ ID NO: 125 and a VL region comprising the amino acid sequence of SEQ ID NO: 126; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 127.

41. The engineered cancer antigen of any one of claims 20-40, wherein the cognate binder is a chimeric antigen receptor (CAR) that binds to the target domain.

42. The engineered cancer antigen of claim 41, wherein the CAR binds to an extracellular domain or truncated portion thereof of a cell surface protein antigen selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand- 1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein-coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappalight chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

43. The engineered cancer antigen of any one of claims 1-42, wherein the engineered cancer antigen does not comprise an intracellular domain.

44. An engineered cancer antigen comprising a target domain and a heterologous membrane targeting domain, wherein the target domain is an extracellular domain or a truncated portion thereof of a target antigen.

45. The engineered cancer antigen of claim 44, that is membrane-bound when expressed from a cell.

46. The engineered cancer antigen of claim 44 or claim 45, wherein the heterologous membrane targeting domain is a heterologous transmembrane domain.

47. The engineered cancer antigen of claim 46, wherein the heterologous transmembrane domain is derived from the transmembrane domain of the alpha, beta, or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, CD1 la, CD18, ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49d, ITGA6, VLA-6, CD49f, ITGAD, CD1D, ITGAE, CD103, ITGAL, ITGAM, CD1B, ITGAX, CD1C, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, CD229, CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or NKG2C.

48. The engineered cancer antigen of claim 46 or claim 47, wherein the transmembrane domain is a CD8 transmembrane domain.

49. The engineered cancer antigen of claim 48, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 4.

50. The engineered cancer antigen of any one of claims 44-49, wherein the target domain and the heterologous membrane targeting domain are each derived from a different cell surface receptor.

51. The engineered cancer antigen of any one of claims 44-50, wherein the target antigen is a membrane-expressed antigen.

52. The engineered cancer antigen of any one of claims 44-51, wherein the target antigen is a cell surface receptor.

53. The engineered cancer antigen of any one of claims 44-52, wherein the target antigen is recognizable by a cognate binder.

54. The engineered cancer antigen of any one of claims 44-53, wherein the target domain is a truncated portion of an extracellular domain of the target antigen that retains an epitope recognizable by a cognate binder.

55. The engineered cancer antigen of any one of claims 44-54, wherein the target antigen is selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyro sine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein-coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2),Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

56. The engineered cancer antigen of any one of claims 44-55, wherein the target antigen is BCMA.

57. The engineered cancer antigen of any one of claims 44-56, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 3.

58. The engineered cancer antigen of claim 56 or claim 57, wherein the target domain is recognizable by a cognate binder comprising: a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 32; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 35.

59. The engineered cancer antigen of any one of claims 44-55, wherein the target antigen is CD70.

60. The engineered cancer antigen of any one of claims 44-55 and 59, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 106.

61. The engineered cancer antigen of claim 59 or claim 60, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 107 or 124.

62. The engineered cancer antigen of any one of claims 59-61, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 124.

63. The engineered cancer antigen of any one of claims 59-62, wherein the target domain is recognizable by a cognate binder comprising: a VH region comprising the amino acid sequence of SEQ ID NO: 125 and a VL region comprising the amino acid sequence of SEQ ID NO: 126; and / oran scFv comprising the amino acid sequence of SEQ ID NO: 127.

64. The engineered cancer antigen of any one of claims 44-55, wherein the target antigen is CD 19.

65. The engineered cancer antigen of any one of claims 44-55 and 64, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 71.

66. The engineered cancer antigen of claim 64 or claim 65, wherein the target domain is recognizable by a cognate binder comprising:(a) a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 47; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 48 or 49; or(b) a VH region comprising the amino acid sequence of SEQ ID NO: 50 and a VL region comprising the amino acid sequence of SEQ ID NO: 51; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 52.

67. The engineered cancer antigen of any one of claims 44-55, wherein the target antigen is CD20.

68. The engineered cancer antigen of any one of claims 44-55 and 67, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 72.

69. The engineered cancer antigen of claim 67 or claim 68, wherein the target domain is recognizable by a cognate binder comprising: a VH region comprising the amino acid sequence of SEQ ID NO: 53 and a VL region comprising the amino acid sequence of SEQ ID NO: 54; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 55.

70. The engineered cancer antigen of any one of claims 44-55, wherein the target antigen is CD22.

71. The engineered cancer antigen of any one of claims 44-55 and 70, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 73.

72. The engineered cancer antigen of claim 70 or claim 71, wherein the target domain is recognizable by a cognate binder comprising:(a) a VH region comprising the amino acid sequence of SEQ ID NO: 56 and a VL region comprising the amino acid sequence of SEQ ID NO: 57; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 58; or(b) a VH region comprising the amino acid sequence of SEQ ID NO: 59 and a VL region comprising the amino acid sequence of SEQ ID NO: 60; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 61.

73. The engineered cancer antigen of any one of claims 53-72, wherein the cognate binder is a chimeric antigen receptor (CAR) that binds to the target domain.

74. The engineered cancer antigen of claim 73, wherein the CAR binds to an extracellular domain or truncated portion thereof of a cell surface protein antigen selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand- 1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein-coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappalight chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1),Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

75. The engineered cancer antigen of any one of claims 44-74, wherein the engineered cancer antigen does not comprise an intracellular domain.

76. The engineered cancer antigen of any one of claims 44-74, further comprising an intracellular domain.

77. The engineered cancer antigen of claim 75 or claim 76, wherein the intracellular domain is a heterologous intracellular domain from the target antigen or is a truncated portion thereof.

78. The engineered cancer antigen of claim 76 or claim 77, wherein the intracellular domain is a truncated and / or is a non-functional intracellular domain.

79. The engineered cancer antigen of any of claims 76-78, wherein the intracellular domain comprises a death domain.

80. The engineered cancer antigen of claim 79, wherein the death domain is derived from a death receptor selected from the group consisting of FAS (CD95), tumor necrosis factor receptor superfamily member 10A (TNFRSF10A; also known as death receptor 4 (DR4), DR5, TNFR1, DR3, Eeucine-rich repeat and death domain-containing protein 1, Ankyrin repeat and death domain-containing protein IB, lnterleukin-1 receptor-associated kinase-like 2, Tumor necrosis factor receptor superfamily member 21, Netrin receptor UNC5A, Netrin receptor UNC5B, Netrin receptor UNC5C, Netrin receptor UNC5D, UNC5C-like protein, Tumor necrosis factor receptor superfamily member 16, Ankyrin-1, Ankyrin-2, Ankyrin-3, Nuclear factor NF-kappa-B pl05 subunit, Tumor necrosis factor receptor superfamily member 6, lnterleukin-1 receptor-associated kinase 1, Death-associated protein kinase 1, Death domain-containing protein CRADD, Nuclear factor NF-kappa-B plOO subunit, FAS- associated death domain protein (FADD), Receptor-interacting serine / threonine-protein kinase 1, Ankyrin repeat and death domain-containing protein 1A, Death domain-containing protein 1, Ectodysplasin-A receptor-associated adapter protein, Tumor necrosis factor receptor superfamily member 25, THO complex subunit 1, Myeloid differentiation primary response protein MyD88, p53-induced death domain-containing protein 1, and interleukin- 1 receptor-associated kinase 3.

81. The engineered cancer antigen of claim 79 or claim 80, wherein the death domain is derived from FAS, FADD, or TNFRSF10A.

82. The engineered cancer antigen of any one of claims 79-81, wherein the death domain is derived from FAS.

83. The engineered cancer antigen of claim 82, wherein the death domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 93, 97, and 99.

84. The engineered cancer antigen of any one of claims 79-81, wherein the death domain is derived from FADD.

85. The engineered cancer antigen of claim 84, wherein the death domain comprises the amino acid sequence of SEQ ID NO: 101.

86. The engineered cancer antigen of any one of claims 79-81, wherein the death domain is derived from TNFRSF10A.

87. The engineered cancer antigen of claim 86, wherein the death domain comprises the amino acid sequence of SEQ ID NO: 103 or 105.

88. A cell expressing the engineered cancer antigen of any one of claims 1-43.

89. A cell comprising the engineered cancer antigen of any one of claims 44-87.

90. A cell expressing the engineered cancer antigen of any one of claims 1-43 and comprising the engineered cancer antigen of any one of claims 44-87.

91. The cell of any one of claims 88-90, wherein the cell is a tumor cell.

92. The cell of claim 91, wherein the tumor cell is a tumor cell of a cancer selected from the group consisting of non-Hodgkin’s lymphoma (NHL), diffuse large cell lymphoma (DLCL), diffuse large B cell lymphoma (DLBCL), Hodgkin’s lymphoma, multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, skin cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, medulloblastoma, liver cancer, stomach cancer, thyroid cancer, bile duct cancer, liver cancer, bone cancer, colon cancer, rectal cancer, endometrial cancer, and cervical cancer.

93. The cell of claim 91 or claim 92, wherein the tumor cell is a tumor cell of a cancer selected from the group consisting of bladder cancer, breast cancer, skin cancer, head and neck cancer, colorectal cancer, endometrial cancer, liver cancer, kidney cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, thyroid cancer, and ovarian cancer.

94. A polynucleotide encoding the engineered cancer antigen of any one of claims 1- 43.

95. A polynucleotide encoding the engineered cancer antigen of any one of claims 44-87.

96. A polynucleotide comprising a nucleic acid sequence encoding the engineered cancer antigen of any one of claims 1-43 and a nucleic acid sequence encoding the engineered cancer antigen of any one of claims 44-87.

97. The polynucleotide of claim 96, wherein the nucleic acid sequence encoding the engineered cancer antigen of any one of claims 1-43 and the nucleic acid sequence encoding the engineered cancer antigen of any one of claims 44-87 are separated by a cleavable linker.

98. The polynucleotide of claim 97, wherein the cleavable linker is a self-cleaving linker.

99. The polynucleotide of claim 98, wherein the self-cleaving linker is or comprises a 2 A peptide.

100. A vector comprising the polynucleotide of claim 94 or claim 95.

101. A vector comprising the polynucleotide of any one of claims 96-99.

102. A vector comprising the polynucleotide of claim 94 and the polynucleotide of claim 95.

103. The vector of any one of claims 100-102, wherein the vector is for delivery of the polynucleotide specifically to a cancer cell.

104. The vector of claim 103, wherein the vector is for delivery specifically to a blood cancer cell.

105. The vector of claim 103, wherein the vector is for delivery specifically to a solid tumor cancer cell.

106. The vector of any one of claims 100-105, wherein the vector is a viral vector.

107. The vector of claim 106, wherein the viral vector is an oncolytic virus.

108. The vector of claim 107, wherein the oncolytic virus is selected from the group consisting of an adenovirus, a herpes simplex virus, a vaccinia virus, a mumps virus, a newcastle disease virus, a poliovirus, a seneca valley virus, a measles virus, a sindbis virus, a parvovirus, a coxsackie virus, a vesicular stomatitis virus, a reovirus, and a maraba and rhabdovirus.

109. The vector of any one of claims 106-108, wherein the viral vector exhibits tropism to a tumor cell.

110. The vector of any one of claims 106-109, wherein the viral vector enters a cell by binding to a cell surface receptor expressed by a tumor cell.

111. The vector of any one of claims 106- 110, wherein the viral vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, or a herpes simplex viral (HSV) vector.

112. The vector of any one of claims 106-111, wherein the viral vector is an adenoviral vector.

113. The vector of any one of claims 106-111, wherein the viral vector is a lentiviral vector.

114. A pharmaceutical composition comprising the polynucleotide of any one of claims 94-99 or the vector of any one of claims 100-113.

115. The pharmaceutical composition of claim 114, further comprising a pharmaceutically acceptable carrier.

116. A combination comprising a vector of any one of claims 100-113 and a population of lymphocytes expressing a chimeric antigen receptor (CAR) that recognizes the target domain of the engineered cancer antigen.

117. A combination comprising, a vector comprising a polynucleotide encoding an engineered cancer antigen comprising a target domain and a membrane targeting domain, wherein the target domain is an extracellular domain or a truncated portion thereof of a target antigen, and a population of lymphocytes expressing a chimeric antigen receptor (CAR) that recognizes the target domain of the engineered cancer antigen.

118. The combination of claim 116 or claim 117, wherein the CAR binds to an extracellular domain or truncated portion thereof of a cell surface protein antigen selected fromthe group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand- 1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein-coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappalight chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

119. The combination of any one of claims 116-118, wherein the CAR binds to an extracellular domain or truncated portion thereof of BCMA.

120. The combination of claim 119, wherein the CAR comprises: a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 32; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 35.

121. The combination of any of claims 116-118, wherein the CAR binds to an extracellular domain or truncated portion thereof of CD 19.

122. The combination of claim 121, wherein the CAR comprises:(a) a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 47; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 48 or 49; or(b) a VH region comprising the amino acid sequence of SEQ ID NO: 50 and a VL region comprising the amino acid sequence of SEQ ID NO: 51; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 52.

123. The combination of any one of claims 116-118, wherein the CAR binds to an extracellular domain or truncated portion thereof of CD20.

124. The combination of claim 123, wherein the CAR comprises: a VH region comprising the amino acid sequence of SEQ ID NO: 53 and a VL region comprising the amino acid sequence of SEQ ID NO: 54; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 55.

125. The combination of any one of claims 116-118, wherein the CAR binds to an extracellular domain or truncated portion thereof of CD22.

126. The combination of claim 125, wherein the CAR comprises:(a) a VH region comprising the amino acid sequence of SEQ ID NO: 56 and a VL region comprising the amino acid sequence of SEQ ID NO: 57; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 58; or(b) a VH region comprising the amino acid sequence of SEQ ID NO: 59 and a VL region comprising the amino acid sequence of SEQ ID NO: 60; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 61.

127. The combination of any one of claims 116-118, wherein the CAR binds to an extracellular domain or truncated portion thereof of CD70.

128. The combination of claim 127, wherein the CAR comprises: a VH region comprising the amino acid sequence of SEQ ID NO: 125 and a VL region comprising the amino acid sequence of SEQ ID NO: 126; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 127.

129. A kit comprising the pharmaceutical composition of claim 114 or claim 115 and instructions for using the polynucleotide or the vector.

130. A kit comprising the combination of any one of claims 116-128.

131. The kit of claim 130, further comprising instructions for using the vector and the population of lymphocytes.

132. A method of treatment, comprising: administering a therapeutically effective amount of the polynucleotide of any one of claims 94-99, the vector of any one of claims 100-113, or the pharmaceutical composition of claim 114 or claim 115 to a subject having a cancer, and administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) that bind to the target domain of the engineered cancer antigen.

133. A method of treatment, comprising: administering a therapeutically effective amount of a vector comprising a polynucleotide encoding an engineered cancer antigen comprising a target domain and a membrane targeting domain, wherein the target domain is an extracellular domain or a truncated portion thereof of a target antigen, to a subject having a cancer, and administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) that bind to the target domain of the engineered cancer antigen.

134. A method of treatment, comprising administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) to a subject having a cancer who was previously administered the polynucleotide of any one ofclaims 94-99, the vector of any one of claims 100-113, or the pharmaceutical composition of claim 114 or claim 115, wherein the CAR binds to the target domain of the engineered cancer antigen.

135. A method of treatment, comprising administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) to a subject having a cancer who was previously administered a vector comprising a polynucleotide encoding an engineered cancer antigen comprising a target domain and a membrane targeting domain, wherein the target domain is an extracellular domain or a truncated portion thereof of a target antigen, wherein the CAR binds to the target domain of the engineered cancer antigen.

136. The method of any one of claims 132-135, wherein the lymphocytes are T cells.

137. The method of any one of claims 132-135, wherein the CAR binds to an extracellular domain or truncated portion thereof of a cell surface protein antigen selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD70, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand- 1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), G protein-coupled receptor, class C, group 5, member D (GPRC5D), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappalight chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), R0R1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelialgrowth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

138. The method of any one of claims 132-137, wherein the CAR binds to an extracellular domain or truncated portion thereof of BCMA.

139. The method of claim 138, wherein the CAR comprises: a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 32; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 35.

140. The method of any one of claims 132-137, wherein the CAR binds to an extracellular domain or truncated portion thereof of CD 19.

141. The method of claim 140, wherein the CAR comprises:(a) a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 47; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 48 or 49; or(b) a VH region comprising the amino acid sequence of SEQ ID NO: 50 and a VL region comprising the amino acid sequence of SEQ ID NO: 51; and / or a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 52.

142. The method of any one of claims 132-137, wherein the CAR binds to an extracellular domain or truncated portion thereof of CD20.

143. The method of claim 142, wherein the CAR comprises: a VH region comprising the amino acid sequence of SEQ ID NO: 53 and a VL region comprising the amino acid sequence of SEQ ID NO: 54; and / oran scFv comprising the amino acid sequence of SEQ ID NO: 55.

144. The method of any one of claims 132-137, wherein the CAR binds to an extracellular domain or truncated portion thereof of CD22.

145. The method of claim 144, wherein the CAR comprises:(a) a VH region comprising the amino acid sequence of SEQ ID NO: 56 and a VL region comprising the amino acid sequence of SEQ ID NO: 57; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 58; or(b) a VH region comprising the amino acid sequence of SEQ ID NO: 59 and a VL region comprising the amino acid sequence of SEQ ID NO: 60; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 61.

146. The method of any one of claims 132-137, wherein the target antigen is CD70.

147. The method of claim 146, wherein the target antigen is recognizable by a cognate binder comprising: a VH region comprising the amino acid sequence of SEQ ID NO: 125 and a VL region comprising the amino acid sequence of SEQ ID NO: 126; and / or an scFv comprising the amino acid sequence of SEQ ID NO: 127.

148. A method of tagging a tumor cell in vivo, comprising contacting the tumor cell with the polynucleotide of any one of claims 94-99, the vector of any one of claims 100-113, or the pharmaceutical composition of claim 114 or claim 115.

149. A method of tagging a tumor cell of a subject having a cancer, comprising administering a therapeutically effective amount of the polynucleotide of any one of claims 94- 99, the vector of any one of claims 100-113, or the pharmaceutical composition of claim 114 or claim 115, to the subject.