Systems and methods for expression of synthetic cancer antigens and cytokine receptors

Abstract

The present disclosure generally relates to a system comprising a vector carrying a synthetic cancer antigen and a cell expressing a cognate binder of the synthetic cancer antigen. In some embodiments, the system further comprises enhancements, such as a synthetic cytokine receptor and cytokine. Also provided herein are uses of the system for treating different types of cancers.

Description

307612002240SYSTEMS AND METHODS FOR EXPRESSION OF SYNTHETIC CANCER ANTIGENS AND CYTOKINE RECEPTORSCROSS-REFRENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Patent Application No. 63 / 726,228 filed on November 27, 2024, entitled “SYSTEMS AND METHODS FOR EXPRESSION OF SYNTHETIC CANCER ANTIGENS AND CYTOKINE RECEPTORS,” U. S. Provisional Patent Application No. 63 / 863,358 filed on August 13, 2025, entitled “SYSTEMS AND METHODS FOR EXPRESSION OF SYNTHETIC CANCER ANTIGENS AND CYTOKINE RECEPTORS,” U. S. Provisional Patent Application No. 63 / 864,187 filed on August 14, 2025, entitled “SYSTEMS AND METHODS FOR EXPRESSION OF SYNTHETIC CANCER ANTIGENS AND CYTOKINE RECEPTORS,” U. S. Provisional Patent Application No 63 / 910,750 filed on November 3, 2025, entitled “SYSTEMS AND METHODS FOR EXPRESSION OF SYNTHETIC CANCER ANTIGENS AND CYTOKINE RECEPTORS,” 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 307612002240SeqList. XML created November 26, 2025, which is 680,821 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 a system comprising a vector carrying a synthetic cancer antigen and a cell expressing a cognate binder of the synthetic cancer antigen. In some embodiments, the system further comprises enhancements, such as a synthetic cytokine receptor and cytokine. Also provided herein are uses of the system for treating different types of cancers. 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( 1):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 CAR-T immunosuppression in the tumor microenvironment, limited migration of T cells to the tumor site, and most significantly, “on-target, off-tumor” toxicity (Wang et al., Front Med. (2020) l4t6):726M-5; Tahmasebi et al., Stem Cell Rev Rep. (2019) 15(5):619— 36).1MF-364447969307612002240Improved systems and methods are needed for cell therapies including for treating solid tumors. Provided herein are embodiments that meet such needs.SUMMARY

[0005] Provided herein is a tumor-targeting system, comprising: (a) a first composition comprising a vector encoding (i) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain and (ii) a C-X-C motif chemokine ligand 9 (CXCL9); and (b) a second composition comprising an immune cell expressing (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR), wherein the CAR binds to the target domain of the mtSCA.

[0006] In some of any of the provided embodiments, the vector further encodes (iii) a tumortargeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule. In some of any of the provided embodiments, the vector encodes one or more copies of the mtSCA and / or ttSCA, each comprising the target domain and the membrane-targeting domain or tumor-targeting binding molecule. In some of any of the provided embodiments, the target domain of the mtSCA and the ttSCA comprise the same epitope for a cognate binder. In some of any of the provided embodiments, the target domain of the mtSCA and the ttSCA are the same.

[0007] Provided herein is a tumor-targeting system, comprising: (a) a first composition comprising a vector encoding (i) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule and (ii) a C-X-C motif chemokine ligand 9 (CXCL9); and (b) a second composition comprising an immune cell expressing (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR), wherein the CAR binds to the target domain of the ttSCA. In some of any of the provided embodiments, the vector further encodes (iii) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain. In some of any of the provided embodiments, the vector encodes one or more copies of the mtSCA and / or ttSCA, each comprising the target domain and the membrane-targeting domain or tumor-targeting binding molecule. In some of any of the provided embodiments, the target domain of the mtSCA and the ttSCA comprise the same epitope for a cognate binder. In some of any of the provided embodiments, the target domain of the mtSCA and the ttSCA are the same.

[0008] Provided herein is a tumor-targeting system, comprising: (a) a first composition comprising a vector encoding (i) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain, (ii) a C-X-C motif chemokine ligand 9 (CXCL9), and (iii) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumortargeting binding molecule; and (b) a second composition comprising an immune cell expressing (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR), wherein the CAR binds to the target domain of the synthetic cancer antigen.2MF-364447969307612002240

[0009] In some of any of the provided embodiments, the vector encodes one or more copies of the mtSCA and / or ttSCA, each comprising the target domain and the membrane-targeting domain or the tumor-targeting binding molecule. In some of any of the provided embodiments, the target domain of the mtSCA and the ttSCA comprise the same epitope for a cognate binder. In some of any of the provided embodiments, the target domain of the mtSCA and the ttSCA are the same. In some of any of the provided embodiments, the heterologous cytokine receptor comprises a synthetic cytokine receptor comprising an extracellular domain, a transmembrane domain, and an interleukin-9 receptor (IL-9R) intracellular domain capable of IL-9R signaling. In some of any of the provided embodiments, the heterologous cytokine comprises interleukin- 18 (IL- 18).

[0010] In some of any of the provided embodiments, the mtSCA and CXCL9 are separated by a nucleotide sequence encoding a cleavable linker. In some of any of the provided embodiments, the ttSCA and CXCL9 are separated by a nucleotide sequence encoding a cleavable linker. In some of any of the provided embodiments, the mtSCA, ttSCA and CXCL9 are separated from each other by a nucleotide sequence encoding a cleavable linker.

[0011] 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. In some of any of the provided embodiments, the cleavable linker is P2A or T2A.

[0012] 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 an antibody or antibody fragment that is recognized by the cognate binder. In some of any of the provided embodiments, the cognate binder binds to the idiotype of the antibody or antibody fragment. In some of any of the provided embodiments, the cognate binder is an anti-idiotype antibody or an antigen binding fragment.

[0013] In some of any of the provided embodiments, the cognate binder is the extracellular domain of a chimeric antigen receptor (CAR). In some of any of the provided embodiments, the extracellular domain of the CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 68. In some of any of the provided embodiments, the extracellular domain of the CAR comprises an amino acid sequence of SEQ ID NO: 68. In some of any of the provided embodiments, the CAR further comprises a transmembrane domain and an intracellular signaling domain. In some of any of the provided embodiments, the transmembrane domain of the CAR is a CD8 transmembrane domain. In some of any of the provided embodiments, the transmembrane domain of the CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 52. In some of any of the provided embodiments, the transmembrane domain of the CAR comprises the amino acid sequence of SEQ ID NO: 52. In some of any of the provided embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain. In some of any of the provided embodiments, the intracellular signaling domain further comprises a co-stimulatory signaling domain.3MF-364447969307612002240In some of any of the provided embodiments, the intracellular signaling domain comprises a CD3z cytoplasmic signaling domain and a 41BB co-stimulatory signaling domain. In some of any of the provided embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 70. In some of any of the provided embodiments, the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 70. In some of any of the provided embodiments, the CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 29. In some of any of the provided embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 29.

[0014] In some of any of the provided embodiments, the antibody or antibody fragment target domain does not contain all or a portion of the heavy chain constant region, optionally does not contain the CHI, CH2 and / or CH3 domain, more optionally wherein the antibody or antibody fragment target domain does not contain the heavy chain CH3 domain. In some of any of the provided embodiments, the target domain is derived from a 4D5 anti-HER2 antibody. In some of any of the provided embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 7. In some of any of the provided embodiments, the target domain comprises a light chain comprising an amino acid sequence of SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 7.

[0015] In some of any of the provided embodiments, the target domain is a single-chain variable fragment (scFv). In some of any of the provided embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 53. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 53.

[0016] In some of any of the provided embodiments, the target domain is the variable domain of the heavy chain of an antibody. In some of any of the provided embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 50. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 50.

[0017] In some of any of the provided embodiments, the membrane-targeting domain is a transmembrane domain. In some of any of the provided embodiments, the transmembrane domain is from a transmembrane glycoprotein. In some of any of the provided embodiments, he transmembrane domain is a CD8 transmembrane domain. In some of any of the provided embodiments, the transmembrane domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 52. In some of any of the provided embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 52. In some of any of the provided embodiments, the transmembrane domain is encoded by a nucleic acid sequence of SEQ ID NO: 302.

[0018] In some of any of the provided embodiments, the tumor-targeting binding molecule specifically binds to a tumor associated antigen expressed on the surface of a tumor cell. In some of 4MF-364447969307612002240any 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 (Prostatespecific 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), Claudin6, Claudinl82, GPC2, GPC3.

[0019] 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 solid tumor originating from epithelial tissue. In some of any of the provided embodiments, the tumor cell is a tumor cell of a pan-epithelial tumor. In some of any of the provided embodiments, the tumor cell is a tumor cell of a gastrointestinal (GI) tract cancer. In some of any of the provided embodiments, the tumor cell of the GI tract cancer is a colorectal cancer, an esophageal cancer or a stomach cancer.

[0020] 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, or 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.

[0021] In some of any of the provided embodiments, the tumor cell is a tumor cell of a non-small cell lung cancer (NSCLC). In some of any of the provided embodiments, the tumor cell is a tumor cell of a prostate cancer.

[0022] In some of any of the provided embodiments, the ttSCA is soluble. In some of any of the provided embodiments, he target domain and the tumor-targeting binding molecule of the ttSCA 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 SEQ ID NO: 59 or SEQ ID5MF-364447969307612002240NO: 14. In some of any of the provided embodiments, the linker is encoded by a nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 13.

[0023] In some of any of the provided embodiments, the tumor-targeting binding 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). 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-H1), a CDR-H2, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NOs: 92, 93 and 94, 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: 95, 96 and 97, respectively. In some of any of the provided embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable (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: 56; and a light chain variable (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: 55. In some of any of the provided embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 55, and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 56.

[0024] In some of any of the provided embodiments, the tumor-targeting binding molecule comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 54 or comprises the amino acid sequence of SEQ ID NO: 54. In some of any of the provided embodiments, the tumortargeting binding molecule is encoded by a nucleic acid sequence of SEQ ID NO: 240. In some of any of the provided embodiments, the CXCL9 comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 16. In some of any of the provided embodiments, the CXCL9 comprises an amino acid sequence of SEQ ID NO: 16. In some of any of the provided embodiments, the CXCL9 is encoded by a nucleic acid sequence of SEQ ID NO: 15. In some of any of the provided embodiments, the IL-18 comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 37. In some of any of the provided embodiments, the IL- 18 comprises an amino acid sequence of SEQ ID NO: 37. In some of any of the provided embodiments, the IL- 18 is encoded by a nucleic acid sequence of SEQ ID NO: 36. In some of any of the provided embodiments, the intracellular domain capable of IL-9R signaling comprises an IL-9R intracellular domain or a variant thereof. In some of any of the provided 6MF-364447969307612002240embodiments, the IL-9R intracellular domain capable of IL-9R signaling comprises a chimeric JAK / STAT fusion domain.

[0025] In some of any of the provided embodiments, the synthetic cytokine receptor is a constitutively active cytokine receptor. In some of any of the provided embodiments, the synthetic cytokine receptor is a multimer. In some of any of the provided embodiments, the synthetic cytokine receptor is a multi mer of identical polypeptide chains each comprising the extracellular domain, the transmembrane domain and the IL-9R intracellular domain or variant thereof. In some of any of the provided embodiments, the multimer is a dimer. In some of any of the provided embodiments, the dimer is a homodimer. In some of any of the provided embodiments, each polypeptide chain is constitutively multimerized. In some of any of the provided embodiments, the synthetic cytokine receptor comprises at least one self-assembly domain. In some of any of the provided embodiments, at least one self-assembly domain is the extracellular domain and / or the transmembrane domain. In some of any of the provided embodiments, the synthetic cytokine receptor is multimerized through the transmembrane domain and / or the extracellular domain. In some of any of the provided embodiments, the synthetic cytokine receptor is multimerized through the transmembrane domain and the extracellular domain.

[0026] In some of any of the provided embodiments, the synthetic cytokine receptor is a homodimer of identical polypeptide chains each comprising an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain or a variant thereof. In some of any of the provided embodiments, the extracellular domain and / or the transmembrane domain are heterologous to the IL-9R. In some of any of the provided embodiments, the transmembrane domain and extracellular domain are the transmembrane domain and extracellular domain from the same protein. In some of any of the provided embodiments, the transmembrane domain and extracellular domain are the transmembrane domain and extracellular domain from different proteins. In some of any of the provided embodiments, the transmembrane domain is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 amino acids in length.

[0027] In some of any of the provided embodiments, the transmembrane domain comprises a transmembrane domain derived from Glycophorin A (GpA) Carnitine palmitoyltransferase 1 (CPT1), a tumor necrosis factor receptor (TNFR), Mucin 24 (Muc24) or Thrombopoietin receptor. In some of any of the provided embodiments, the transmembrane domain promotes alpha-helix dimerization. In some of any of the provided embodiments, the transmembrane domain comprises the motif GXXXG (SEQ ID NO: 241) or LIxxGVxxGVxxT (SEQ ID NO: 242). In some of any of the provided embodiments, the transmembrane domain: (a) is a transmembrane domain derived from Glycophorin A (GpA) or a variant thereof that comprises one or more mutations (e.g, 1, 2, 3, 4, 5 or 6 mutations) compared to a wild-type GpA transmembrane domain, wherein the variant GpA is sufficient to promote alpha-helix dimerization; (b) comprises a transmembrane domain derived from Glycophorin 7MF-364447969307612002240A (GpA); (c) comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 125 or SEQ ID NO: 130; or (d) comprises the amino acid sequence of SEQ ID NO: 125 or SEQ ID NO: 130. In some of any of the provided embodiments, the transmembrane domain comprises: (a) GXXXG (SEQ ID NO: 241) and GXXXA (SEQ ID NO: 243) motifs; (b) a transmembrane domain derived from Carnitine palmitoyltransferase 1 (CPT1); (c) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 132; or (d) the amino acid sequence of SEQ ID NO: 132. In some of any of the provided embodiments, the synthetic cytokine receptor comprises the motif AXXXA (SEQ ID NO: 244), AXXXS (SEQ ID NO: 245), ΦPXΦ (SEQ ID NO: 246) or ΦTXXAΦ (SEQ ID NO: 247). In some of any of the provided embodiments, the transmembrane domain is derived from a TNFR, optionally wherein the TNFR is TACI, DR5, p75NTR, Fas, TNFR1, TNFR2 or 0X40. In some of any of the provided embodiments, the transmembrane domain comprises: (a) a transmembrane domain derived from DR5; (b) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 133; or (c) the amino acid sequence of SEQ ID NO: 133. In some of any of the provided embodiments, the transmembrane domain comprises: (a) a transmembrane domain derived from TACI; (b) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 131; or (c) the amino acid sequence of SEQ ID NO: 131. In some of any of the provided embodiments, the transmembrane domain comprises 1 to 6 cysteine residues. In some of any of the provided embodiments, the transmembrane domain promotes disulfide-linked dimerization, optionally wherein the disulfide-linked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor.

[0028] In some of any of the provided embodiments, the transmembrane domain is a variant transmembrane domain that comprises one or more mutations compared to a wild-type transmembrane domain to promote homodimerization of the receptor polypeptide, optionally wherein: (a) the one or more mutations promote alpha-helix dimerization or disulfide-linked dimerization; (b) the one or more mutations introduces at least one cysteine into the transmembrane domain; (c) the one or more mutations introduces a proline into the transmembrane domain; (d) the one or more mutations introduces a threonine into the transmembrane domain; (e) the one or more mutations introduces a trimer peptide of cysteine, proline, and another amino acid other than cysteine or proline into the transmembrane domain; or (f) the one or more mutations introduces a trimer peptide of cysteine, proline, threonine (CPT or TCP) into the transmembrane domain. In some of any of the provided embodiments, the transmembrane domain is a variant IL-7R transmembrane domain and the one or more mutations is in the wild-type transmembrane sequence PILLTISILSFFSVALLVILACVLW (SEQ ID NO: 248 or SEQ ID NO: 280). In some of any of the provided embodiments, the transmembrane domain comprises: (a) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 40, SEQ ID NO: 101 or SEQ ID NO: 249; or (b) the amino acid sequence of SEQ ID NO: 40, SEQ ID NO: 101 or SEQ ID NO: 249. In some of any of the provided embodiments, the 8MF-364447969307612002240transmembrane domain comprises: (a) a transmembrane domain derived from Muc24; (b) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 124; or (c) the amino acid sequence of SEQ ID NO: 124.

[0029] In some of any of the provided embodiments, the extracellular domain is between about 150 to 260 amino acids in length. In some of any of the provided embodiments, the extracellular domain is a dimerizing domain, optionally wherein the dimerizing domain comprises a hinge region. In some of any of the provided embodiments, the extracellular domain promotes disulf'idedinked dimerization. In some of any of the provided embodiments, the extracellular domain comprises 1 to 6 cysteine residues. In some of any of the provided embodiments, the disulf'idedinked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor. In some of any of the provided embodiments, the extracellular domain is derived from the extracellular domain of CD34, DAP12, Glycophorin A, CD8, Muc24 or Thrombopoietin receptor. In some of any of the provided embodiments, the extracellular domain comprises: (a) an extracellular domain of CD8 or a truncated portion thereof comprising at least one cysteine residue; (b) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 52, SEQ ID NO: 178 or SEQ ID NO: 43; or (c) the amino acid sequence of SEQ ID NO: 52, SEQ ID NO: 178 or SEQ ID NO: 43. In some of any of the provided embodiments, the extracellular domain comprises: (a) an extracellular domain of CD34 or a truncated portion thereof comprising at least one cysteine residue; (b) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 39; or (c) the extracellular domain comprises the amino acid sequence of SEQ ID NO: 39. In some of any of the provided embodiments, the extracellular domain: (a) is an extracellular domain of Muc24 or a truncated portion thereof comprising at least one cysteine residue; (b) comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 113; or (c) comprises the amino acid sequence of SEQ ID NO: 113. In some of any of the provided embodiments, the extracellular domain: (a) is an extracellular domain of DAP12 or a truncated portion thereof comprising at least one cysteine residue; (b) comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 100; or (c) comprises the amino acid sequence of SEQ ID NO: 100. In some of any of the provided embodiments, the extracellular domain: (a) is an extracellular domain of Glycophorin A (GpA) or a truncated portion thereof comprising at least one cysteine residue; (b) comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 111 or SEQ ID NO: 112; or (c) comprises the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO: 112.

[0030] In some of any of the provided embodiments, the IL-9R intracellular domain or variant thereof is about 100 to 260 amino acids in length, optionally wherein the IL-9R intracellular domain or variant thereof is 230 amino acids in length. In some of any of the provided embodiments, the IL-9R intracellular domain or variant thereof comprises a BOX1 motif and / or a BOX2 motif, optionally wherein the IL-9R intracellular domain or variant thereof comprises a BOX2 motif. In some of any of 9MF-364447969307612002240the provided embodiments, the IL-9R intracellular domain or variant thereof is wild-type IL-9R intracellular domain or a variant thereof that comprises one or more mutations compared to the wildtype IL-9R intracellular domain set forth in SEQ ID NO: 102, optionally wherein the one or more mutations comprises one or more amino acid insertions, deletions, and / or substitutions.

[0031] In some of any of the provided embodiments, the one or more mutations promote signaling through STAT1, STAT3, and / or STAT5 pathways. In some of any of the provided embodiments, the IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 33, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 148, SEQ ID NO: 156, SEQ ID NO: 163, SEQ ID NO: 166, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 108, SEQ ID NO: 109 or SEQ ID NO: 110. In some of any of the provided embodiments, the IL-9R intracellular domain or variant thereof comprises an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 33, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 148, SEQ ID NO: 156, SEQ ID NO: 163, SEQ ID NO: 166, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 108, SEQ ID NO: 109 or SEQ ID NO: 110. In some of any of the provided embodiments, the IL-9R intracellular domain or variant thereof comprises one or more amino acid deletions with reference to wild-type IL-9R intracellular domain (SEQ ID NO: 102).

[0032] In some of any of the provided embodiments, the IL-9R intracellular domain or variant thereof is a truncated IL-9R that lacks a contiguous sequence of amino acids at the C-terminus of wild-type IL-9R intracellular domain, optionally wherein the truncated IL-9R intracellular domain or variant thereof is truncated by between 62 and 99 contiguous amino acids from the C-terminus of wild-type IL-9R intracellular domain. In some of any of the provided embodiments, the IL-9R intracellular domain or variant thereof: (a) is a truncated IL-9R that lacks amino acids 132 to 230 of SEQ ID NO: 102 or lacks amino acids 134 to 230 of SEQ ID NO: 102; (b) comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 156, SEQ ID NO: 167, or SEQ ID NO: 168; (c) comprises an amino acid sequence of SEQ ID NO: 156, SEQ ID NO: 167, or SEQ ID NO: 168. In some of any of the provided embodiments, the synthetic cytokine receptor comprises: (a) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187; or (b) an amino acid sequence 10MF-364447969307612002240of SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187. In some of any of the provided embodiments, the IL-9R intracellular domain or variant thereof comprises one or more amino acid substitutions with reference to wild-type IL-9R intracellular domain SEQ ID NO: 102. In some of any of the provided embodiments, the IL-9R intracellular domain or variant thereof comprises a STAT binding motif or a variant thereof, optionally wherein the STAT binding motif comprises: (a) a STAT1, STAT3, and / or STAT5 binding motif; (b) YLPQ (SEQ ID NO: 250); (c) a variant STAT binding motif; (d) YRPQ (SEQ ID NO: 251); (e) YLPL (SEQ ID NO: 252); or (f) YLKQ (SEQ ID NO: 253). In some of any of the provided embodiments, the variant IL-9R intracellular domain or variant thereof comprises: (a) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 171 or SEQ ID NO: 172, or SEQ ID NO: 173; or (b) an amino acid sequence of SEQ ID NO: 171 or SEQ ID NO: 172, or SEQ ID NO: 173.

[0033] In some of any of the provided embodiments, the chimeric JAK / STAT fusion domain comprises a JAK binding domain from a type I cytokine receptor and a STAT binding domain from an IL-9R intracellular domain. In some of any of the provided embodiments, the IL-9R STAT binding domain comprises amino acid residues 73 to 230 of SEQ ID NO: 102. In some of any of the provided embodiments, the STAT binding domain is 59 to 158 amino acids in length and comprises an IL-9R STAT binding motif. In some of any of the provided embodiments, the IL-9R STAT binding motif comprises YLPQ (SEQ ID NO: 250). In some of any of the provided embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the N-terminus of SEQ ID NO: 102. In some of any of the provided embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the C-terminus of SEQ ID NO: 102. In some of any of the provided embodiments, the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks amino acids at positions 1 to 72 and / or 132 to 230 of SEQ ID NO: 102. In some of any of the provided embodiments, the IL-9R STAT binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 282, or SEQ ID NO: 283. In some of any of the provided embodiments, the IL-9R STAT binding domain comprises an amino acid sequence of SEQ ID NO: 254, SEQ ID NO: 255 SEQ ID NO: 282, or SEQ ID NO: 283.

[0034] In some of any of the provided embodiments, the type I cytokine receptor is selected from the group consisting of interleukin 2 receptor (IL-2R), interleukin 4 receptor (IL-4R), interleukin 7 receptor (IL-7R), interleukin 13 receptor (IL-13R), interleukin 15 receptor (IL-15R), and interleukin 2 receptor (IL-21R), optionally wherein the type I cytokine receptor is IL-7R. In some of any of the provided embodiments, the IL-7R JAK binding domain is 65 amino acids in length and comprises a box 1 motif. In some of any of the provided embodiments, the IL-7R JAK binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 256 or SEQ ID NO: 285, or comprises an amino acid sequence of SEQ ID NO: 256 or SEQ ID NO: 285. In some of any of the 11MF-364447969307612002240provided embodiments, the chimeric JAK / STAT fusion domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 41, SEQ ID NO: 181 or SEQ ID NO: 45 or comprises an amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 181, or SEQ ID NO: 45.

[0035] In some of any of the provided embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 31, SEQ ID NO: 239, or SEQ ID NO: 188 or an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 239, or SEQ ID NO: 188. In some of any of the provided embodiments, the synthetic cytokine receptor elicits signaling through STAT1, STAT3, and / or STAT5 pathways. In some of any of the provided embodiments, signaling through STAT1, STAT3, and / or STAT5 is: (a) increased compared to STAT1, STAT3, and / or STAT5 signaling via wild-type IL-9R; (b) sustained for a longer period of time compared to STAT1, STAT3, and / or STAT5 signaling via wild-type IL-9R, optionally wherein sustained STAT1, STAT3, and / or STAT5 signaling is determined by phosphorylation status of STAT1, STAT3, and / or STAT5.

[0036] In some of any of the provided embodiments, the first and second compositions further comprise a pharmaceutically acceptable carrier. In some of any of the provided embodiments, the first and second compositions are for delivery, specifically to a subject. In some of any of the provided embodiments, the subject has a cancer or tumor. In some of any of the provided embodiments, the cancer or tumor is 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, stomach cancer, thyroid cancer, bile duct cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, or cervical cancer.

[0037] In some of any of the provided embodiments, the immune cell is a lymphocyte. In some of any of the provided embodiments, the immune cell is an immune effector cell. In some of any of the provided embodiments, the immune cell is a T cell or a Natural Killer (NK) cell. In some of any of the provided embodiments, the immune cell is a T cell. In some of any of the provided embodiments, the immune cell is a CD4+ T cell or a CD8+ T cell. In some of any of the provided embodiments, the immune cell is a Natural Killer (NK) cell.

[0038] In some of any of the provided embodiments, the vector is a viral vector. In some of any of the provided embodiments, the vector exhibits tropism to a tumor cell. In some of any of the provided embodiments, the vector is a tumor tropic viral vector, optionally an oncolytic virus. In some of any of the provided embodiments, the tumor tropic viral vector 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 rhabdovirus, such as maraba virus. In 12MF-364447969307612002240some of any of the provided embodiments, the tumor tropic viral vector enters a cell by binding to a cell surface receptor expressed by the cancer cell or the solid tumor cancer cell. In some of any of the provided embodiments, the tumor tropic viral vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a lenti viral vector, a retroviral vector, or a herpes simplex viral (HSV) vector.

[0039] In some of any of the provided embodiments, the tumor tropic viral vector is an adenoviral vector. In some of any of the provided embodiments, the adenoviral vector is an adenoviral vector that binds to CD46 and / or desmoglein-2. In some of any of the provided embodiments, the adenoviral vector is Ad3, Ad7, Adil, Adl4, Adl6, Adl7, Ad21, Ad35, Ad47, or Ad50. In some of any of the provided embodiments, the adenoviral vector is a chimeric adenoviral vector based on Ad3, Ad7, Adi 1, Adl4, Adl6, Adl7, Ad21, Ad35, Ad47, or Ad50. In some of any of the provided embodiments, the adenoviral vector is a chimeric adenoviral vector that is based on Adi 1. In some of any of the provided embodiments, the adenoviral vector is a chimeric Ad3 / 1 Ip adenoviral vector. In some of any of the provided embodiments, the chimeric Ad3 / 1 Ip adenoviral vector binds to CD46. In some of any of the provided embodiments, the chimeric Ad3 / 1 Ip adenoviral vector comprises a nucleic acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 257 or SEQ ID NO: 335, optionally wherein the nucleic acid sequence is set forth in SEQ ID NO: 257 or SEQ ID NO: 335.

[0040] Provided herein is a polynucleotide comprising a nucleic acid sequence encoding (i) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membranetargeting domain and (ii) a C-X-C motif chemokine ligand 9 (CXCL9). In some of any of the provided embodiments, the polynucleotide further comprises a nucleic acid sequence encoding (iii) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule. In some of any of the provided embodiments, the nucleic acid sequence encodes one or more copies of the mtSCA and / or ttSCA, each comprising the target domain and the membrane-targeting domain. In some of any of the provided embodiments, the target domain of the mtSCA and the ttSCA comprises the same epitope for a cognate binder. In some of any of the provided embodiments, the target domain of the mtSCA and the ttSCA are the same.

[0041] Provided herein is a polynucleotide comprising a nucleic acid sequence encoding (i) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule and (ii) a C-X-C motif chemokine ligand 9 (CXCL9). In some of any of the provided embodiments, the polynucleotide further comprises a nucleic acid sequence encoding (iii) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membranetar geting domain. In some of any of the provided embodiments, the nucleic acid sequence encodes one or more copies of the mtSCA and / or ttSCA, each comprising the target domain and the membrane-targeting domain or tumor-targeting binding molecule. In some of any of the provided 13MF-364447969307612002240embodiments, the target domain of the mtSCA and the ttSCA comprises the same epitope for a cognate binder. In some of any of the provided embodiments, the target domain of the mtSCA and the ttSCA are the same.

[0042] Provided herein is a polynucleotide comprising a nucleic acid sequence encoding (i) a membrane-targeting synthetic cancer antigen (mtSCA)comprising a target domain and a membranetargeting domain, (ii) a C-X-C motif chemokine ligand 9 (CXCL9), and (iii) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule. In some of any of the provided embodiments, the nucleic acid sequence encodes one or more copies of the mtSCA and / or ttSCA, each comprising the target domain and the membrane-targeting domain or tumor-targeting binding molecule. In some of any of the provided embodiments, the target domain of the mtSCA and the ttSCA comprises the same epitope for a cognate binder. In some of any of the provided embodiments, the target domain of the mtSCA and the ttSCA are the same.

[0043] In some of any of the provided embodiments, the mtSCA and CXCL9 are separated by a nucleotide sequence encoding a cleavable linker. In some of any of the provided embodiments, the ttSCA and CXCL9 are separated by a nucleotide sequence encoding a cleavable linker. In some of any of the provided embodiments, the mtSCA, ttSCA and CXCL9 are separated from each other by a nucleotide sequence encoding 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. In some of any of the provided embodiments, the linker is P2A or T2A.

[0044] In some of any of the provided embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 7. In some of any of the provided embodiments, the target domain comprises a light chain comprising an amino acid sequence of SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 7.

[0045] In some of any of the provided embodiments, the target domain is a single-chain variable fragment (scFv). In some of any of the provided embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 53. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 53.

[0046] In some of any of the provided embodiments, the target domain is the variable domain of the heavy chain of an antibody. In some of any of the provided embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 50. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 50. In some of any of the provided embodiments, the membrane-targeting domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 52. In some of any of the provided embodiments, the membrane-targeting domain comprises an amino acid sequence of SEQ ID NO: 52.14MF-364447969307612002240In some of any of the provided embodiments, the membrane-targeting domain is encoded by a nucleic acid sequence of SEQ ID NO: 302. In some of any of the provided embodiments, the tumor-targeting binding molecule comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 54 or comprises the amino acid sequence of SEQ ID NO: 54. In some of any of the provided embodiments, the tumor-targeting binding molecule is encoded by a nucleic acid sequence of SEQ ID NO: 240. In some of any of the provided embodiments, the polynucleotide comprises a nucleic 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 NOs: 2, 11, 18, 22 or 25. In some of any of the provided embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NOs: 2, 11, 18, 22 or 25.

[0047] Provided herein is a polynucleotide comprising a nucleic acid sequence encoding (i) a synthetic cytokine receptor comprising an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain and (ii) a chimeric antigen receptor (CAR). In some of any of the provided embodiments, the synthetic cytokine receptor and CAR are separated by a nucleotide sequence encoding 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. In some of any of the provided embodiments, the linker is P2A.

[0048] Provided herein is a polynucleotide comprising a nucleic acid sequence encoding (i) interleukin- 18 (IL- 18) and (ii) a chimeric antigen receptor (CAR). In some of any of the provided embodiments, the IL- 18 and CAR are separated by a nucleotide sequence encoding 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. In some of any of the provided embodiments, the linker is P2A. In some of any of the provided embodiments, the target domain is recognizable by the CAR. In some of any of the provided embodiments, the synthetic cytokine receptor is a constitutively active synthetic cytokine receptor. In some of any of the provided embodiments, the synthetic cytokine receptor comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 33, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 148, SEQ ID NO: 156, SEQ ID NO: 163, SEQ ID NO: 166, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 108, SEQ ID NO: 109 or SEQ ID NO: 110. In some of any of the provided embodiments, the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 33, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID 15MF-364447969307612002240NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 148, SEQ ID NO: 156, SEQ ID NO: 163, SEQ ID NO: 166, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 108, SEQ ID NO: 109 or SEQ ID NO: 110.

[0049] In some of any of the provided embodiments, the IL- 18 comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 37. In some of any of the provided embodiments, the IL- 18 comprises an amino acid sequence of SEQ ID NO: 37. In some of any of the provided embodiments, the IL-18 is encoded by a nucleic acid sequence of SEQ ID NO: 36. In some of any of the provided embodiments, the CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 29. In some of any of the provided embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 29. In some of any of the provided embodiments, the CAR is encoded by a nucleic acid sequence of SEQ ID NO: 28. In some of any of the provided embodiments, the polynucleotide comprises a nucleic 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 NOs: 2, 11, 18, 22 or 25. In some of any of the provided embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NOs: 2, 11, 18, 22 or 25. In some of any of the provided embodiments, the polynucleotide comprises a nucleic 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 NOs: 42, 44, 46, or 64. In some of any of the provided embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NOs: 42, 44, 46, or 64.

[0050] Provided herein is a vector, comprising any of the provided polynucleotides. In some of any of the provided embodiments, the vector is for delivery specifically to an immune cell. In some of any of the provided embodiments, the immune cell is an immune effector cell. In some of any of the provided embodiments, the immune effector cell is a T cell, optionally a cytotoxic T cell. In some of any of the provided embodiments, the immune effector cell is a CD8+ T cell and / or CD4+ T cell. In some of any of the provided embodiments, the immune effector cell is a natural killer cell. In some of any of the provided embodiments, the vector is a lend viral vector.

[0051] In some of any of the provided embodiments, the vector is for delivery specifically 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. In some of any of the provided embodiments, the viral vector is a tumor tropic viral vector, optionally 16MF-364447969307612002240an oncolytic virus. In some of any of the provided embodiments, the viral vector 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 rhabdovirus, such as maraba virus. 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 of the provided embodiments, the viral vector is an adenoviral vector. In some of any of the provided embodiments, the vector comprises the nucleic acid sequence of SEQ ID NOs: 1, 10, 17, 21 or 24.

[0052] Provided herein is a cell comprising any of the provided vector or any of the provided polynucleotides. Provided herein is a pharmaceutical composition comprising any of the provided vector or any of the provided cells. In some of any of the provided embodiments, the pharmaceutical composition further comprises a pharmaceutical acceptable carrier. In some of any of the provided embodiments, the provided pharmaceutical composition is for use in treating a cancer in a subject.

[0053] Provided herein is a tumor-targeting system comprising any of the provided vector or any of the provided cells.

[0054] 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 pharmaceutical compositions. 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.

[0055] Provided herein is a method of treating a cancer in a subject, comprising administering to a subject the first composition and the second composition of any of the provided systems, wherein the vector causes the synthetic cancer antigen to be expressed on the surface of or secreted by a cell of the cancer and the CAR of the immune cell binds to the target domain of the synthetic cancer antigen.

[0056] Provided herein is a method of treating a cancer in a subject, comprising administering: (a) a first composition comprising a vector encoding (i) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain and (ii) a C-X-C motif chemokine ligand 9 (CXCL9); and (b) a second composition comprising an immune cell expressing (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR), wherein the vector causes the synthetic cancer antigen to be expressed on the surface of or secreted by a cell of the cancer and the CAR of the immune cell binds to the target domain of the synthetic cancer antigen.17MF-364447969307612002240

[0057] Provided herein is a method of treating a cancer in a subject, comprising administering: (a) a first composition comprising a vector encoding (i) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule and (ii) a C-X-C motif chemokine ligand 9 (CXCL9); and (b) a second composition comprising an immune cell expressing (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR), wherein the vector causes the synthetic cancer antigen to be expressed on the surface of or secreted by a cell of the cancer and the CAR of the immune cell binds to the target domain of the synthetic cancer antigen.

[0058] Provided herein is a method of treating a cancer in a subject, comprising administering: (a) a first composition comprising a vector encoding (i) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain, (ii) a C-X-C motif chemokine ligand 9 (CXCL9), and (iii) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule; and (b) a second composition comprising an immune cell expressing (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR), wherein the vector causes the synthetic cancer antigen to be expressed on the surface of or secreted by a cell of the cancer and the CAR of the immune cell binds to the target domain of the synthetic cancer antigen.

[0059] In some of any of the provided embodiments, the heterologous cytokine receptor comprises a synthetic cytokine receptor comprising an extracellular domain, a transmembrane domain, and an interleukin-9 receptor (IL-9R) intracellular domain. In some of any of the provided embodiments, the heterologous cytokine comprises interleukin- 18 (IL- 18). In some of any of the provided embodiments, the first composition and second composition are administered concurrently to the subject. In some of any of the provided embodiments, the first composition and the second composition are administered sequentially to the subject. In some of any of the provided embodiments, the first composition is administered prior to administration of the second composition. In some of any of the provided embodiments, the first composition is administered to the subject one or more times. In some of any of the provided embodiments, the first composition is administered to the subject a plurality of times. In some of any of the provided embodiments, the first composition is administered three times. In some of any of the provided embodiments, the first composition is administered three times, optionally wherein the three administrations occur every other day. In some of any of the provided embodiments, each administration of the first composition comprises an effective dose of the vector. In some of any of the provided embodiments, the first composition comprises a first effective dose, a second effective dose and a third effective dose. In some of any of the provided embodiments, each effective dose is the same across administrations. In some of any of the provided embodiments, each effective dose is different across administrations. In some of any of the provided embodiments, the second and third effective doses are higher than the first effective 18MF-364447969307612002240dose. In some of any of the provided embodiments, the first effective dose contains 1 x 1012vp, the second effective dose contains 3 x 1012virus particles (vp), and the third effective dose contains 3 x 1012vp. In some of any of the provided embodiments, the first effective dose contains 1 x 1012vp, the second effective dose contains 6 x 1012vp, and the third effective dose contains 6 x 1012vp. In some of any of the embodiments, the method further comprises administering a lymphodepleting therapy to the subject. In some of any of the provided embodiments, the lymphodepleting therapy is administered to the subject prior to administration of the second composition. In some of any of the provided embodiments, the lymphodepleting therapy is administered after the first composition is administered and before administration of the second composition. In some of any of the provided embodiments, the lymphodepleting therapy is administered 2-7 days before administration of the second composition. In some of any of the provided embodiments, the lymphodepleting therapy comprises fludarabine and cyclophosphamide. In some of any of the provided embodiments, prior to administration of the first composition, an apheresis sample is obtained from the subject, to prepare the CAR-expressing immune cells of the second composition. In some of any of the provided embodiments, the apheresis sample is a leukapheresis sample.

[0060] In some of any of the provided embodiments, the cancer is a blood cancer. In some of any of the provided embodiments, the cancer is a solid tumor cancer. In some of any of the provided embodiments, the solid tumor cancer originates from epithelial tissue. In some of any of the provided embodiments, the solid tumor cancer is a pan-epithelial solid tumor cancer. In some of any of the provided embodiments, the solid tumor cancer is a gastrointestinal (GI) tract cancer. In some of any of the provided embodiments, the GI tract cancer is a colorectal cancer, an esophageal cancer or a stomach cancer. In some of any of the provided embodiments, the solid tumor cancer is 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, or cervical cancer. In some of any of the provided embodiments, the solid tumor cancer is 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. In some of any of the provided embodiments, the solid tumor cancer is a non-small cell lung cancer (NSCLC). In some of any of the provided embodiments, the solid tumor cancer is a prostate cancer.BRIEF DESCRIPTION OF THE FIGURES

[0061] FIG.1A is a schematic illustration of delivering an exemplary synthetic cancer antigen, an antibody, into a cancer cell through a gene delivery vector, such as an oncolytic virus. A T-cell 19MF-364447969307612002240expressing binding partners against the idiotype of the antibody can then be directed to the cancer cell, thus killing the cancer cell.

[0062] FIG IB is a schematic depicting a cycle of infection of a solid tumor with a gene delivery vector, such as an oncolytic virus including: (1) virus infection, (2) synthetic cancer antigen (e.g., “Flare”) expression and tumor microenvironment inflammation, (3) targeted CAR T cell cytotoxic activity, and (4) release of virus for re-infection.

[0063] FIG. 1C is a representative plasmid map of adenoviruses provided herein comprising the following formula (I): 5’ ITR-B1-BA-B2-BX-BB-BY-B3-3’ ITR.

[0064] FIG.2A shows the design of exemplary synthetic cancer antigens (Abl-Fab, Abl-scFv, Abl-Vn) based on antibodies or their derivative molecules. FIG.2B illustrates details of the construct design: from a synthetic cancer antigen with an antibody without the heavy chain CH3 domain (Abl-Fab), to a synthetic cancer antigen with simply the scFv of Abl (Abl-scFv), and then to a synthetic¬ cancer antigen with only the variable domain of the heavy chain of Abl (Ab 1 -VH).

[0065] FIG.3A- FIG.3C show the expression of Abl-Fab, Abl-scFv, and Abl-Vu on the surface of different cancer cells. The expression of Abl-Fab and Abl-scFv was detected via a goat and -human IgG, F(ab’)2 specific antibody (Jackson Immunoresearch, Inc) labeled with PE. The expression of Abl-Vu was detected via an anti-flag antibody labeled with PE. A375: malignant melanoma cell line; A549: lung carcinoma cell line; PC-3: prostate cancer cell line; HCT-116: colorectal cancer cell line; SKOV-3: ovarian adenocarcinoma cell line.

[0066] FIG.4 shows that Abl-Fab and Abl-scFv still bind to the cognate antigen of Abl, while Ab 1 -VH no longer binds to the cognate antigen of Abl due to the deletion of the variable domain of the light chain of Abl.

[0067] FIG.5A and FIG.5B show the expansion of T cells after being transduced with one of three different CAR molecules against the idiotype of Abl, thus against Abl-Fab, Abl-scFv, and Abl-Vfl. Abl_CARl: a llama single domain anti-idiotype antibody; Abl_CAR2: a human antiidiotype scFv; and Abl_CAR3: another human anti-idiotype scFv. UTD: un-transduced.

[0068] FIG.6A shows that the expression of Abl_CARl and Abl_CAR2, but not Abl_CAR3, was detected on respectively transduced CAR-T cells. FIG.6B- FIG.6C show the expression of Abl_CARl and Abl_CAR2 after the respective lentivirus has been titrated. UTD: un-transduced.

[0069] FIGS.7A- FIG.7E show the killing of different Abl -Fab-expressing cancer cells by Abl_CARl-expressing T cells from three donors. A375: malignant melanoma cell line; A549: lung carcinoma cell line; PC3: prostate cancer cell line; HCT-116: colorectal cancer cell line; SKOV-3: ovarian adenocarcinoma cell line.

[0070] FIGS.8A- FIG.8E show the killing of different Abl -Fab-expressing cancer cells by Abl_CAR2-expressing T cells from three donors. A375: malignant melanoma cell line; A549: lung20MF-364447969307612002240carcinoma cell line; PC3: prostate cancer cell line; HCT-116: colorectal cancer cell line; SKOV-3: ovarian adenocarcinoma cell line.

[0071] FIG.9A shows the killing of Abl-scFv-expressing A549 cancer cells by T cells from one donor expressing either Abl_CARl or Abl_CAR2. FIG.9B - FIG.9C shows the killing of different Abl-scFv-expressing cancer cells by T cells from another donor expressing Abl_CAR2.

[0072] FIG. 10A - FIG. 10B show the killing of different Abl-VH -expressing cancer cells by T cells expressing Abl_CAR2. A549: lung carcinoma cell line; PC3: prostate cancer cell line; HCT-116: colorectal cancer cell line; SKOV-3: ovarian adenocarcinoma cell line. UTD: un-transduced.

[0073] FIG. 11A - FIG. 11B compares the killing of Ab 1 -Fab, Abl-scFv, and Abl-Vn expressing cancer cells by T cells expressing Abl_CAR2. A549: lung carcinoma cell line; PC3: prostate cancer cell line; HCT-116: colorectal cancer cell line; SKOV-3: ovarian adenocarcinoma cell line. UTD: un-transduced.

[0074] FIG. 12A shows the expression of Ab 1 -Fab via adenovirus transduction. FIG. 12B -FIG. 12C shows the killing of different Ab 1 -Fab-expressing cancer cells by T cells from different donors expressing either Abl_CARl or Abl_CAR2, where the Abl-Fab constructs were transduced into cancer cells using adenovirus instead of lentivirus as shown in FIG.7A - FIG.8E. FIG. 12D -FIG. 12E shows the killing of different Abl-Vn -expressing cancer cells by T cells expressing Abl_CAR2, where the Abl-Vn constructs were transduced into cancer cells using adenovirus instead of lentivirus as shown in FIG. 10A - FIG. 10B. FIG.12F shows the killing of Abl-Vn -expressing cancer cells by T cells from expressing Abl_CARl, where the Abl-Vn constructs were transduced into cancer cells using the chimeric adenovirus Ad3 / Adllp. A549: lung carcinoma cell line; PC3: prostate cancer cell line; HCT-116: colorectal cancer cell line; SKOV-3: ovarian adenocarcinoma cell line. UTD: un-transduced.

[0075] FIG. 13A is a schematic illustration of testing two synthetic cancer antigens (Abl-Vn and Abl-Fab) and their cognate binders in vivo. FIG. 13B and FIG. 13C show the probability of survival and the tumor volume of mice carrying HER2-positive HCT-116 tumor cells expressing Abl-Vn treated with T cells expressing Abl_CAR2 at different concentrations. T cells expressing anti-HER2 (4D5) CAR were included as a control. FIG. 13D shows the expansion of T cells expressing either Abl_CAR2 or anti-HER2 (4D5) CAR. FIG.13E shows the tumor volume of mice carrying HER2-positive HCT-116 cells expressing Abl-Fab treated with T cells expressing Abl_CARl at different concentrations.

[0076] FIG. 14 is a schematic illustration of delivering an exemplary synthetic cancer antigen, a protein fragment, into a cancer cell through a gene delivery vector, such as an oncolytic virus. A T-cell expressing molecule targeting the protein fragment can then be directed to the cancer cell, thus killing the cancer cell.21MF-364447969307612002240

[0077] FIG. 15A- FIG. 15B show the expression of different protein fragment candidates on the surface of 293T cells.

[0078] FIG. 16A shows the expression of IC1 protein fragment on the surface of different cancer cells. A375: malignant melanoma cell line; A549: lung carcinoma cell line; PC3: prostate cancer cell line. FIG. 16B shows the expression of a binder against IC1 on the surface of T cells.

[0079] FIG. 17A- FIG. 17C shows the killing of different cancer cells expressing IC1 by T cells expressing binders against IC1. Tumor cell viability is depicted on the y axis; hours are depicted on the x axis.

[0080] FIG. 18 is a schematic illustration of delivering an exemplary synthetic cancer antigen, the leucine zipper domain of transcription factor 1(TF1), into a cancer cell through a gene delivery vector, such as an oncolytic virus. A T-cell expressing binding partner of the leucine zipper domain of transcription factor 1 (e.g., the leucine zipper domain of transcription factor 2, TF2) can then be directed to the cancer cell, thus killing the cancer cell.

[0081] FIG. 19 shows the construct design of exemplary synthetic cancer antigens (TF2-LZ and TF4-LZ) and their corresponding cognate binders (TF1-LZ and TF3-LZ).

[0082] FIG.20A shows the expression of TF2-LZ and TF4-LZ on the surface of cancer cells. FIG. 20B shows the expression of TF1-LZ (binder of TF2-LZ) on the surface of T cells. FIG.20C shows the expression of TF3-LZ (binder of TF4-LZ) on the surface of T cells.

[0083] FIG. 21 is a schematic illustration of testing the binding effectiveness of the soluble form of the Abl-VH (FLl)-anti-EpCAM conjugate on tumor cells.

[0084] FIG. 22 shows the surface binding expression of the Flare 1 after co-culture with supernatant containing the soluble form of the conjugate or the tethered form of the Abl-VH.

[0085] FIG. 23A is a schematic illustration of testing the soluble form of the Abl-VH (FLl)-anti-EpCAM conjugate for targeted killing of tumor cells. FIG. 23B shows the killing of different Abl-Vn surface bound cancer cells by T cells expressing Abl_CAR2, where the Abl-Vn constructs were bound to the cancer cells by conjugation to the anti-EpCAM antibody. HCT-116: colorectal cancer cell line; SKOV-3: ovarian adenocarcinoma cell line. UTD: un-transduced.

[0086] FIG.24A is a schematic illustration of testing the soluble synthetic cancer antigen (Abl-Vn (FLl)-anti-EpCAM conjugate) and their cognate binders in vivo. FIG. 24B shows the percentage of FL1+ tumor cells after mice received doses of virus encoding the tethered or soluble forms of the Abl-Vn. FIG. 24C shows the number of infiltrating T cells in the tumor after treatment with virus encoding the tethered or soluble forms of the Abl-Vn and subsequent intravenous injections of CAR-T cells. FIG.24D is a schematic illustration of testing the soluble synthetic cancer antigen (Abl-Vn (FLl)-anti-EpCAM conjugate) and their cognate binders in vivo over time. FIG. 24E shows the tumor growth in mice after treatment with virus encoding the Abl-Vn (FLl)-anti-EpCAM conjugate and CAR-T cells.22MF-364447969307612002240

[0087] FIG. 25A shows the surface binding expression of the soluble FLl-anti-HER2 synthetic cancer antigen on HER2-expressing SKOV-3 cancer cells. MFI is normalized to no antigen condition.FIG. 25B - FIG. 25C shows the killing of different HER2-expressing cancer cells by CAR T cells after culture with the soluble FLI -anti- HER2 synthetic cancer antigen.

[0088] FIG. 26, left panel shows the surface binding expression of the Abl-VH (FLI) on A549 cancer cells infected with a virus encoding both the tethered synthetic cancer antigen and the soluble FLI -anti -EpCAM synthetic cancer antigen. FIG. 26, right panel shows the surface binding expression of the FLI on non-transduced acceptor cancer cells after co-culture with supernatant containing the soluble form of the Abl-VH conjugate. Pos Con: Positive Control, cells lines engineered to express the tethered or soluble forms of the FLI; Sec Con: control antibody staining to ensure signal specificity.

[0089] FIG. 27 shows the design of exemplary synthetic cytokine receptors.

[0090] FIG. 28 shows the expression of exemplary synthetic cytokine receptors on the surface of primary human CD3-positive T cells, including CD8+ T cells (left panel) or CD4+ T cells (right panel).

[0091] FIGS. 29A and 29B show the surface expression level of CD27 and CD45RA 5 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing the 9RC synthetic cytokine receptor or in primary human CD3- positive T cells that were untransduced (“UTD”). FIG.29A is a representative flow cytometry dot plot with CD45RA staining on the x-axis and CD27 staining on the y-axis. FIG. 29B depicts the percent of T cells that were positive for CD45RA and CD27 in UTD T cells or in 9RC+ transduced T cells.

[0092] FIG. 30 shows the surface expression level of CD27 and CD45RA 12 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC, 9RC2, or 9RD2) or in primary human CD3-positive T cells that were untransduced.

[0093] FIG. 31 shows the surface expression level of CD27 and Fas 16 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC, 9RC2, or 9RD2) or in primary human CD3-positive T cells that were untransduced.

[0094] FIG. 32A shows the surface expression level of CD27 and Fas (also known as CD95) among all cells 16 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC or 9RC2), the exemplary synthetic cytokine receptor 9RC co-expressed with a CAR separated by a 2A sequence (designated “40bbz-2a-CD34-9RC”), or in untransduced cells. FIG.32B shows the surface expression level of CD27 and Fas among Flag-positive cells 16 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC or 9RC2) or the23MF-364447969307612002240exemplary synthetic cytokine receptor 9RC co-expressed with a CAR separated by a 2A cleavable linker (designated “40bbz-2a-CD34-9RC”).

[0095] FIG.33A shows the surface expression level of CCR7 and CD45RA among all cells 16 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC or 9RC2), the exemplary synthetic cytokine receptor 9RC co-expressed with a CAR separated by a 2A cleavable linker (designated “40bbz-2a-CD34-9RC”), or in untransduced cells. FIG.33B shows the surface expression level of CCR7 and CD45RA among Flag-positive cells 16 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC or 9RC2) or the exemplary synthetic cytokine receptor 9RC co-expressed with a CAR separated by a 2A cleavable linker (designated “40bbz-2a-CD34-9RC”).

[0096] FIG.34 shows the surface expression level of CD27 in 9RC expressing primary human CD3-positive T cells based on geometric mean fluorescence intensity (gMFI) as determined by flow cytometry.

[0097] FIGS.35A-35B show the surface expression level of CD57 in 9RC expressing primary human CD3-positive T cells, as determined by co-staining for the FLAG tag after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing the 9RC synthetic cytokine receptor or in primary human CD3- positive T cells that were untransduced (“UTD”). FIG.35A is a representative flow cytometry dot plot with CD57 staining on the y-axis and staining for FLAG on the x-axis. FIG.35B depicts the percent of CD8+ T cells that were high for CD57 expression (CD57hi) in UTD T cells or in 9RC+ transduced T cells.

[0098] FIG.36 shows the surface expression level of TIM3 and LAG3 in primary human CD3-positive T cells that were co-transduced with lentiviral vectors expressing the 9RC synthetic cytokine receptor and a CAR separated by either a 2A sequence (40bbz-2a-FLAG-9RC”) or a IRES sequence (40bbz-IRES-CD34-9RC), or in primary human CD3- positive T cells that were untransduced (“UTD”). A representative flow cytometry dot plot is shown with TIM3 staining on the y-axis and LAG3 staining on the x-axis.

[0099] FIG.37 shows the surface expression level of CD4 and CD8 in primary human CD3-positive T cells 5 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing an exemplary synthetic cytokine receptor (9RC, 9RC2, or 9RD2) or in primary human CD3-positive T cells that were untransduced.

[0100] FIG.38 illustrates the growth of primary human CD3-positive T cells expressing an exemplary synthetic cytokine receptor (9RC or 9RC2), the exemplary synthetic cytokine receptor 9RC co-expressed with a CAR (designated “40bbz-9RC”), or in untransduced (UTD) cells during extended in vitro culture. The results depict the day of culture post-activation and the fold growth at the respective day compared to the number of cells at the previous count for each condition.24MF-364447969307612002240

[0101] FIGS. 39A-39E show the design and expression of additional exemplary synthetic¬ cytokine receptors comprising an IL7R TMD and an IL-9R ICD. Each additional synthetic receptor construct had a different extracellular domain (ECD) as follows: GpA (construct “G-7-9R”; FIG. 39A), truncated GpA (construct “tG-7-9R”; FIG.39B), CD8 (construct “CD8-7-9R”; FIG. 39C), truncated CD8 (construct “tCD8-7-9R”; FIG. 39D), or Muc24 (construct “M-7-9R”; FIG. 39E).

[0102] FIGS. 40A-40B show the design and expression of additional exemplary syntheticcytokine receptors comprising an IL-9R ICD and a transmembrane domain other than an IL7R TMD.FIG. 40A depicts a synthetic cytokine construct with a Glycophorin A (GpA) ECD, a GpA TMD and an IL-9R ICD (“G-G-9R”). FIG. 40B depicts a synthetic cytokine receptor with a Muc24 ECD, a Muc24 TMD and an IL-9R ICD (“M-M-9R”).

[0103] FIGS. 41A-41H show the designs and expression data of additional exemplary synthetic cytokine receptors comprising an IL9R ICD, and full-length or truncated ECD from GpA, CD8 or Muc24 and a TMD from GpA, truncated GpA (designated GpA*), TACI, CPT1 or DR5. The depicted constructs include an ECD, TMD and ICD as follows: truncated GpA ECD, GpA TMD and IL9R ICD (construct “tGpA-G-9R”; FIG. 41A), CD8 ECD, GpA TMD and IL9R ICD (construct “CD8-G-9R”; FIG. 41B), truncated CD8 ECD, GpA TMD and IL9R ICD (construct “tCD8-G-9R”; FIG. 41C), Muc24 ECD, GpA TMD and IL9R ICD (construct “M-G-9R”; FIG. 41D), truncated CD8 ECD, truncated GpA TMD and IL9R ICD (construct “tCD8-G*-9R”; FIG. 41E), truncated CD7 ECD, TACI TMD and IL9R ICD (construct “tCD8-T-9R”; FIG. 41F), truncated CD8 ECD, CPT1 TMD and IL9R ICD (construct “tCD8-C-9R”; FIG. 41G), and truncated CD8 ECD, DR5 TMD and IL9R ICD (construct “tCD8-D-9R”; FIG. 41H).

[0104] FIG. 42 shows transduction efficiency of exemplary synthetic cytokine receptors in primary human CD3-positive T cells 5 days after the primary human CD3-positi ve T cells were transduced with lentiviral vectors expressing the exemplary synthetic cytokine receptor or in untransduced T cells (UTD). Transduction efficiency was determined by flow cytometry by cell surface staining for cells positive for the Flag tag.

[0105] FIGS. 43A-43C show the surface expression level of CD27 and CD45RA 5 days after the primary human CD3-positive T cells were transduced with lentiviral vectors expressing the exemplary synthetic cytokine receptors. The figure provides representative flow cytometry contour plots with CD45RA staining on the x-axis and CD27 staining on the y-axis.

[0106] FIG. 44 shows in vivo survival of tumor-bearing mice administered primary human CD3-positive T cells co-expressing a CAR (40BBz) and an exemplary synthetic cytokine receptor, as compared to primary human CD3-positi ve T cells expressing the CAR only or that were untransduced (UTD).25MF-364447969307612002240

[0107] FIG.45 shows STAT phosphorylation (pSTAT) in primary human CD3-positive T cells expressing exemplary synthetic cytokine receptors. pSTAT was measured at 0 hours, 20 minutes, 2 hours, and 24 hours post-stimulation with cytokine.

[0108] FIG.46 shows the expression of an exemplary synthetic cytokine receptor on the surface of primary human CD3-positive T cells.

[0109] FIG.47 shows STAT phosphorylation (pSTAT) of STAT1, STAT3, and STAT5 in primary human CD3-positive T cells expressing exemplary synthetic cytokine receptors.

[0110] FIG.48 shows the number of HCT116 tumor cells per field of view (FOV) across time in a T cell-mediated killing assay. T cells were obtained from two healthy donors and cocultured with tumor cells at an effector:target (E: T) ratio of 1:2.

[0111] FIG.49 shows the number of HCT116 tumor cells per field of view (FOV) across time in a T cell-mediated killing assay. T cells were obtained from a healthy donor and cocultured with tumor cells at an effector:target (E: T) ratio of 1:4.

[0112] FIG.50 shows phenotype markers CD27 and CD45RA in primary T cells expressing exemplary synthetic cytokine receptors.

[0113] FIG.51 shows the total number of T cells co-expressing a CAR (40BBz) and a synthetic cytokine receptor at plating and after two rounds of T cell-mediated killing.

[0114] FIG.52 shows the percentage of CD8 T cells positive for cytokine expression after target stimulation. CD8 T cells either co-expressed a CAR (40BBz) and an exemplary synthetic cytokine receptor or a CAR alone. Measured cytokines included IL-2, IFN-y, and TNFa.

[0115] FIG.53 shows the percentage of live T cells after two rounds of stimulation. T cells coexpressing a CAR (40BBz) and synthetic cytokine receptor were cocultured with K562 cells expressing an antigen recognized by the CAR (40BBz).

[0116] FIGS.54A-54B show the percentage of T cells expressing a CAR (40BBz) and exemplary synthetic cytokine receptors post-transduction. 9RC5 = C-7-9R(a); 9RC5.1 = CD8-7-9R(c); 9RC5.2 = tCD8-7-9R(c).

[0117] FIG.55 shows STAT phosphorylation (pSTAT) in primary human CD3-positive T cells co-expressing a CAR (40BBz) and exemplary synthetic cytokine receptors. pSTATl, pSTAT3 and pSTAT5 are represented as a percentage of the highest signal observed for each of the individual STATs.

[0118] FIG.56 shows the percentage of CD8+ T cells expressing cytokines after 4-hour stimulation with target cells. CD8+ T cells either co-expressed a CAR (40BBz) and an exemplary synthetic cytokine receptor or only a CAR. Measured cytokines included IFN-y, IL-2, IL-10, and TNFa.26MF-364447969307612002240

[0119] FIG.57 shows cytokine and effector molecule secretion by CD8+ T cells after 24-hour stimulation with target cells. CD8+ T cells either co-expressed a CAR (40BBz) and an exemplary synthetic cytokine receptor or only a CAR. Cytokine and effector molecule secretion are displayed as a percentage relative to the highest expressor of each analyte.

[0120] FIGS.58A-58B show CD45RA and CD27 staining of CD8+ T cells in a continuous coculture with target cells. CD8+ T cells either co-expressed a CAR (40BBz) and an exemplary synthetic cytokine receptor or only a CAR. FIG.58A depicts CD45RA and CD27 expression across three weeks. FIG.58B depicts summary data for three technical replicates at the week three timepoint.

[0121] FIGS.59A-59C show live CD8+ T cell counts in a continuous co-culture with target cells. CD8+ T cells either co-expressed a CAR (40BBz) and an exemplary synthetic cytokine receptor or only a CAR. FIG.59A depicts live CD8+ T cell counts across three weeks. FIG.59B depicts live CD8+ T cells at week three. FIG.59C depicts CD39 expression in CD8+ T cells at week three.

[0122] FIG.60 shows expansion of CD8+ T cells cultured with or without IL-2 for four weeks. CD8+ T cells either co-expressed a CAR (40BBz) and an exemplary synthetic cytokine receptor or only a CAR.

[0123] FIG.61 shows tumor growth in tumor-bearing mice administered primary human CD3-positive T cells co-expressing a CAR (40BBz) and an exemplary synthetic cytokine receptor, as compared to primary human CD3-positive T cells expressing the CAR only or that were untransduced (UTD). Tumor-bearing mice were administered 1 x 106cells.

[0124] FIG.62 shows tumor growth in tumor-bearing mice administered primary human CD3-positive T cells co-expressing a CAR (40BBz) and an exemplary synthetic cytokine receptor, as compared to primary human CD3-positive T cells expressing the CAR only or that were untransduced (UTD). Tumor-bearing mice were administered 3 x 105cells.

[0125] FIG.63 shows a schematic of a tumor targeting system comprising a gene delivery vector, such as an oncolytic virus, encoding a synthetic cancer antigen (i.e., Flare) and a chemotactic cytokine (e.g., C-X-C motif chemokine ligand 9 (CXCL9)), and a T cell expressing a chimeric antigen receptor (CAR) that recognizes the synthetic cancer antigen (i.e., Flare) and cytokine armoring (e.g., via synthetic cytokine receptor).

[0126] FIGS.64A-64C show T cell-mediated killing of different cancer cell lines. FIG.64A shows killing of a human colorectal adenocarcinoma cell line (HT-29) by chimeric antigen receptor (CAR) T cells at different effector (E): target (T) ratios in media from an A549 producer cell line containing a soluble engineered synthetic cancer (hereinafter “soluble synthetic cancer antigen”), wherein the soluble synthetic cancer antigen contains a target domain targeted by the CAR and a tumor-targeting molecule that binds a molecule expressed on the HT-29 cells. FIG.64B shows killing of a human ovarian cancer cell line (SKOV-3) under the same conditions described in FIG.64A with 27MF-364447969307612002240an added condition containing SKOV-3 cells that expressed a membrane engineered synthetic cancer antigen (hereinafter “membrane-bound synthetic cancer antigen”) and T cells that expressed a chimeric antigen receptor (CAR) specific to the membrane-bound synthetic cancer antigen. FIG.64C shows killing of a human non-small cell lung cancer cell line (A549) under the same conditions described in FIG.64B.

[0127] FIG.65 shows targeted killing of a colorectal cancer cell line (HCT-116) with a soluble engineered synthetic cancer antigen (hereinafter “soluble synthetic cancer antigen”). UTD = untransduced.

[0128] FIGS.66A-66B show targeted killing of a prostate cancer cell line (22rvl) using the tumor targeting system described in FIG.63. Chimeric antigen receptor (CAR) T cells were combined with 22rvl cells at an effector (E): target (T) ratio of 1:1 or 1:8. Virus alone refers to a control where CAR T cells were not combined with the 22rvl cells. FIG.66A shows killing of 22rvl cells that expressed a membrane engineered synthetic cancer antigen (hereinafter “membrane-bound synthetic cancer antigen”) and a soluble engineered synthetic cancer antigen (hereinafter “soluble synthetic cancer antigen”) via tumor-specific adenovirus. FIG.66B shows killing of 22rvl cells that expressed membrane-bound synthetic cancer antigen, soluble synthetic cancer antigen and C-X-C motif chemokine ligand 9 (CXCL9) via tumor-specific adenovirus. FIG.66C shows killing of 22rvl cells that expressed membrane-bound synthetic cancer antigen and CXCL9. FIG.66D shows killing of 22rvl cells that expressed soluble synthetic cancer antigen and CXCL9.

[0129] FIGS.67A-67C show killing efficiency of the tumor targeting system described in FIG.63 in a prostate cancer cell line (22rvl) or a colorectal cancer cell line (HCT-116). FIG.67A shows killing efficiency (EC50) as the time at which 50% of the 22rvl cells have been eliminated as measured in an Xcelligence killing assay. Chimeric antigen receptor (CAR) T cells were combined with 22rvl cells at an effector (E): target (T) ratio of 1:1 or 1:8. Virus alone refers to a control where CAR T cells were not combined with the 22rvl cells. Tumor alone (indicated by a star icon) refers to a control where CAR T cells, not virus, were not combined with the 22rvl cells. FIG.67B shows blue fluorescent protein (BFP) expression of BFP-expressing HCT-116 spheroids after infection with virus, e.g., adenovirus, and co-culture with CAR T cells. FIG.67C represents BFP intensity as area under the curve (AUC).

[0130] FIGS.68A-68B show the immunophenotype of the chimeric antigen receptor (CAR) T cells after a prostate cancer cell (22rvl) killing assay. FIG.68A shows the proportion of activated CD8+ CAR T cells after killing 22rvl cells that expressed a membrane engineered synthetic cancer antigen (hereinafter “membrane-bound synthetic cancer antigen”) and a soluble engineered synthetic cancer antigen (hereinafter “soluble synthetic cancer antigen”) (AdVl) or membrane-bound synthetic cancer antigen and C-X-C motif chemokine ligand 9 (CXCL9) (AdV3). Minimal CD25+ HLA DR-cells were detected in the T cell populations. FIG.68B shows the proportion of activated CD8+ CAR 28MF-364447969307612002240T cells after killing 22rvl cells that expressed soluble synthetic cancer antigen and CXCL9 (AdV4) or membrane-bound synthetic cancer antigen, soluble synthetic cancer antigen and CXCL9 (AdV2).

[0131] FIGS.69A-69B show the immunophenotype of CD8+ chimeric antigen receptor (CAR) T cells after a prostate cancer cell (22rvl) killing assay. FIG.69A shows the proportion of activated (CD25- / HLA-DR+) CD8+ CAR T cells after killing 22rvl cells that expressed a membrane engineered synthetic cancer antigen (hereinafter “membrane-bound synthetic cancer antigen”) and a soluble engineered synthetic cancer antigen (hereinafter “soluble synthetic cancer antigen”) (AdVl) or membrane-bound synthetic cancer antigen and C-X-C motif chemokine ligand 9 (CXCL9) (AdV3).FIG. 69B shows the proportion of activated (CD25- / HLA-DR+) CD8+ CAR T cells after killing 22rvl cells that expressed soluble synthetic cancer antigen and CXCL9 (AdV4) or membrane-bound synthetic cancer antigen, soluble synthetic cancer antigen and CXCL9 (AdV2).

[0132] FIG.70 shows a schematic of in vivo testing of the tumor targeting system described in FIG. 63. Mice are administered 3 doses of a vector comprising membrane engineered synthetic cancer antigen (hereinafter “membrane-bound synthetic cancer antigen”) and a soluble engineered synthetic cancer antigen (hereinafter “soluble synthetic cancer antigen”) (AdVl) or membrane-bound synthetic cancer antigen, soluble synthetic cancer antigen and C-X-C motif chemokine ligand 9 (CXCL9) (AdV2).

[0133] FIG.71 shows the percentage of human non-small cell lung cancer tumor cells positive for a membrane engineered synthetic cancer antigen (hereinafter “membrane-bound synthetic cancer antigen”), a soluble engineered synthetic cancer antigen (hereinafter “soluble synthetic cancer antigen”) and C-X-C motif chemokine ligand 9 (CXCL9) (AdV2), or membrane-bound engineered synthetic cancer antigen and soluble synthetic cancer antigen (AdVl). UTD = untransduced.

[0134] FIG.72 shows the frequency of T cells in a human non-small cell lung cancer tumor expressing a membrane engineered synthetic cancer antigen (hereinafter “membrane-bound synthetic cancer antigen”), a soluble engineered synthetic cancer antigen (hereinafter “soluble synthetic cancer antigen”) and C-X-C motif chemokine ligand 9 (CXCL9) (AdV2), or membrane-bound engineered synthetic cancer antigen and soluble synthetic cancer antigen (AdVl). UTD = untransduced.

[0135] FIG.73A-73B show oncolytic potency of gene delivery vectors provided herein. FIG. 73A shows oncolytic potency as cell index of lung carcinoma cell line, A549, across 150 hours. FIG.73B shows a summary of the oncolytic potency of each adenovirus shown in FIG.73A.

[0136] FIGS.74A-74C show expression of a membrane-bound synthetic cancer antigen on the surface of lung carcinoma cell line A549 after infection with the adenoviruses provided herein. FIG.74A represents expression as a flow cytometry scatter plot. FIG.74B represents expression as a histogram. FIG.74C shows a summary of expression shown in FIGS.74A-74B.

[0137] FIGS.75A-75C show expression of a soluble synthetic cancer antigen on the surface of lung carcinoma cell line A549 after infection with the adenoviruses provided herein. FIG.75A 29MF-364447969307612002240represents expression as a flow cytometry scatter plot. FIG.75B represents expression as a histogram.FIG. 75C shows a summary of expression shown in FIGS.75A-75B.

[0138] FIG.76 shows expression of CXCL9 secreted from lung carcinoma cell line A375 after infection with the adenoviruses provided herein.

[0139] FIG.77 shows expression of membrane-bound synthetic cancer antigen on the surface of A549 NSCLC cells after infection with adenoviruses provided herein. A549 cells infected with an empty adenovirus was included as a negative control.

[0140] FIG.78A shows viability of HT29 colorectal adenocarcinoma cells after infection with the adenoviruses provided herein and co-culture with the CAR T cells provided herein (virus + CAR T). FIG.78B shows viral genome copies in the supernatant of A549 cells after infection with the adenoviruses provided herein and co-culture with the CAR T cells provided herein (virus + CAR T). Controls included: A549 cells not infected with adenovirus or co-cultured with CAR T cells (Tumor Only); A549 cells infected with adenovirus and not co-cultured with CAR T cells (virus); and A549 cells infected with adenovirus co-cultured with untransduced (UTD) primary T cells (virus + UTD).

[0141] FIG.79 shows killing of tumor cells (A549) infected with the adenoviruses provide herein.

[0142] FIG.80 shows T cell expansion in lung carcinoma cell line (A549) spheroids cultures infected with adenoviruses provided herein.

[0143] FIG.81 shows the percentage of synthetic cancer antigen (i.e., Flare or FL1) positive A549 cells in tumors harvested from mice that were administered the adenoviruses and CAR T cells provided herein.

[0144] FIG.82 shows intratumoral expansion of the adenoviruses provided herein.

[0145] FIGS.83A-83C show gene expression profiles, including inflammatory markers, suppressive markers, dendritic cell activation markers and cancer associated fibroblast (CAF) markers in endogenous and exogenous immune cells of mice administered the adenoviruses provided herein (e.g., AdVl). FIG.83A shows gene expression in bystander endogenous mouse cells obtained from mice with A549 NSCLC tumors that were administered the adenoviruses provided herein. FIG.83B shows frequency CD 16+ myeloid cells in PC3 tumors isolated from mice that were administered human monocytes and the adenoviruses provided herein. FIG.83C shows quantity of CD86+ myeloid cells, a marker of activation, in PC3 tumors isolated from mice that were administered human monocytes and an adenovirus provided herein.

[0146] FIG.84A shows oncolytic potency of gene delivery vectors provided herein.

[0147] FIG.84B and FIG.84C show expression of a synthetic cancer antigen. FIG.84B shows expression of a membrane-bound synthetic cancer antigen on the surface of non-small cell lung cancer (NSCLC) cell line A549 after infection with the adenoviruses provided herein. FIG.84C30MF-364447969307612002240shows expression of a soluble synthetic cancer antigen from A549 after infection with the adenoviruses provided herein.

[0148] FIG.85A and FIG.85B show tumor killing in mice having non-small cell lung cancer (NSCLC) cell (A549) tumors after infection with the adenoviruses provided herein. FIG.84A shows tumor volume (mm3). FIG.84B represents tumor volume as area under the curve (AUC).

[0149] FIGS.86A-86F show killing of non-small cell lung carcinoma cell (A549) spheroids after infection with the adenoviruses provided herein at different Particle Per Cell (PPC) ratios and coculture with chimeric antigen receptor (CAR) T cells. FIG.86A shows BFP intensity of BFP-expressing A549 spheroids infected with 1 PPC adenovirus and co-cultured with CAR T cells at an effector:target ratio of 1:1. FIG.86B represents BFP intensity as area under the curve (AUC). FIG.86C shows BFP intensity in BFP-expressing A549 spheroids infected with 0.1 PPC adenovirus and co-cultured with CAR T cells at an E: T of 1:1. FIG.86D represents BFP intensity as AUC. FIG.86E shows BFP intensity in BFP-expressing A549 spheroids infected with 0.01 PPC adenovirus and co-cultured with CAR T cells at an E: T ratio of 1:1. FIG.86F represents BFP intensity in as AUC. Circle = virus only; square = virus + CAR.

[0150] FIG.87A and FIG.87B show T cell expansion in co-cultures containing non-small cell lung carcinoma cell (A549) spheroids infected with adenoviruses provided herein and cultured with chimeric antigen receptor (CAR) T cells. FIG.87A shows GFP intensity of GFP-expressing CAR T cells co-cultured with adenovirus-infected A549 spheroids. FIG.87B represents GFP intensity as area under the curve (AUC).

[0151] FIGS.88A-88C show killing of colorectal cancer cell (HCT-116) spheroids after i nfection with the adenoviruses provided herein at different Particle Per Cell (PPC) ratios and coculture with chimeric antigen receptor (CAR) T cells. FIG.88A shows BFP intensity of BFP-expressing HCT-116 spheroids infected with 100 PPC adenovirus and co-cultured with CAR T cells at an effector:target (E: T) ratio of 1:1. FIG.88B shows BFP intensity in BFP-expressing HCT-116 spheroids infected with 10 PPC adenovirus and co-cultured with CAR T cells at an E: T of 1:1. FIG.88C represents BFP intensity as area under the curve (AUC).

[0152] FIG.89A and FIG.89B show T cell expansion in co-cultures containing colorectal cell (HCT-116) spheroids infected with adenoviruses provided herein and cultured with chimeric antigen receptor (CAR) T cells. FIG.89A shows GFP intensity of GFP-expressing CAR T cells co-cultured with HCT-116 spheroids infected with 10 PPC adenovirus. FIG.89B represents GFP intensity as area under the curve (AUC).

[0153] FIG.90A and FIG.90B show tumor survival in tumor-bearing mice after infection with the adenoviruses provided herein and administration of the chimeric antigen receptor (CAR) T cells provided herein. FIG.90A shows BFP intensity of BFP-expressing prostate cancer cell (22rvl)31MF-364447969307612002240tumors in mice infected with 1000 PPC adenovirus and administered CAR T cells at an effector:target ratio of 1:1. FIG.90B represents BFP-intensity as area under the curve (AUC).

[0154] FIG.91A and FIG.91B show T cell expansion in tumors of mice after infection with the adenoviruses provided herein and administration of the chimeric antigen receptor (CAR) T cells provided herein. FIG.91A shows GFP intensity of GFP-expressing tumors. FIG.91B represents GFP intensity as area under the curve (AUC).DETAILED DESCRIPTION

[0155] Provided herein is a system for targeting a cancer, e.g., a solid cancer or tumor. In some embodiments, the system is intended to be used in a method of treating a subject having a cancer, e.g., a solid cancer or tumor.

[0156] In some aspects, the system comprises one or more biotherapeutic components, such as a viral vector and an immune cell. In some embodiments, the system contains one or more biotherapeutic components, such as an adenoviral vector encoding a synthetic cancer antigen and C-X-C motif chemokine ligand 9 (CXCL9) and an engineered immune cell (such as an engineered T cell) expressing a cognate binder, e.g., a recombinant receptor such as a chimeric antigen receptor (CAR) and a synthetic cytokine receptor or cytokine. In some embodiments, the components of the system are complementary in function. In particular embodiments, the synthetic cancer antigen of the adenoviral vector is recognized by the cognate binder expressed by the engineered immune cell. Thus, in some embodiments, the system results in the expression of the antigen on the surface of the cancer cell, e.g., a solid cancer or tumor, or in the tumor microenvironment, which can then be targeted by the cognate binder. In this way, the system provided herein tags and targets cells of the cancer for subsequent killing. In other particular embodiments, killing of the targeted cancer cells results in cell lysis and release of mature adenoviral vector. The mature adenoviral vector released into the tumor microenvironment can re-infect nearby cells for further expression of the synthetic cancer antigen and recognition by its cognate binder. Thus, in some embodiments, the system provides a positive feedback loop of adenoviral infection, recognition by its cognate binder expressed by the engineered immune cell, cell killing and adenovirus release, tumor re-infection by the released adenovirus and further killing of infected or re-infected tumor cells.

[0157] In some embodiments, the adenoviral vector is a tumor-tropic adenoviral vector that is able to specifically target and express the synthetic cancer antigen in cancer cells, e.g., solid cancer or tumor cells. Any tumor-tropic adenoviral vector known in the art can be used in the provided system. In some embodiments, the viral vector comprises the viral vectors described in Section ILA.

[0158] In some embodiments, the synthetic cancer antigen is expressed as a membrane protein, such as containing a transmembrane domain. In some embodiments, the synthetic cancer antigens comprises a target domain and a transmembrane domain, in which the target domain is a domain recognized by the cognate binder. In some embodiments, the synthetic cancer antigen comprising a 32MF-364447969307612002240target domain and a transmembrane domain is referred to as a membrane-targeting synthetic cancer antigen (mtSCA).

[0159] In other embodiments, the synthetic cancer antigen is expressed as a soluble protein that is secretable from cells in which it is expressed. In such an embodiments, the target domain is not associated with a membrane protein (e.g., transmembrane domain) but instead is expressed as a soluble (not membrane-bound) protein. In embodiments of a soluble synthetic cancer antigen, a soluble target domain is linked to a tumor-targeting molecule able to bind to a tumor antigen to tag cancer cells. Upon binding of the tumor-targeting molecule to the tumor antigen on the cancer cell, the target domain is able to be displayed on the outside surface of the cancer cell where it can be recognized by the cognate binder. In some embodiments, the synthetic cancer antigen comprising a target domain and a tumor-targeting domain is referred to as a tumor-targeting synthetic cancer antigen (ttSCA).

[0160] In some embodiments, the provided viral vectors encoding the synthetic cancer antigen comprising the target domain (e.g., synthetic cancer antigen membrane protein and / or secretable synthetic cancer antigen) is delivered only to or predominantly to the tumor cells. Thus, it is understood that the synthetic cancer antigen comprising the target domain is not targeted to be expressed on the surface of a non-cancer cell of a subject. The present disclosure also provides polynucleotides encoding the synthetic cancer antigens disclosed herein, vectors comprising such polynucleotides, and cells comprising or engineered by such vectors. In some embodiments, polynucleotides and / or the adeno-viral vectors encode both a synthetic cancer antigen membrane protein and a soluble synthetic cancer antigen. In some embodiments, the synthetic cancer antigen comprises any of the synthetic cancer antigens described in Section II. A.l.a.

[0161] In some embodiments, the immune cell is an immune effector cell, e.g., a T cell. In some embodiments, the immune cell expresses a recombinant receptor, such as a chimeric antigen receptor (CAR). In some embodiments, the CAR recognizes the extracellular target domain of the synthetic cancer antigen on tagged cancer or tumor cells. In some embodiments, CAR activation directed against (i.e. contains an extracellular domain that recognizes or binds) the synthetic cancer antigen initiates cytotoxic killing by the CAR T cells against the synthetic cancer antigen-expressing tumor cells. In some embodiments, the killing activates viral release so that the viral vector is able to infect neighboring tumor cells and spread expression of the synthetic cancer antigen to other tumor cells for recognition by the CAR cell therapy. 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 synthetic cancer antigen, provided embodiments allow for directed killing of tumor cells, including solid tumors.

[0162] In some embodiments, the CXCL9 enhances the function (e.g., tumor cell killing) of the overall system. In some embodiments, CXCL9 promotes engineered immune cell (such as engineered 33MF-364447969307612002240T cell) recruitment, activation, expansion and cytotoxic capacity. In some embodiments, the chemokine is CXCL9, as described in Section II. A.2.

[0163] In some embodiments, the enhancements include armoring the immune cell with a constitutively active synthetic cytokine receptor. The present disclosure provides synthetic cytokine receptors capable of driving interleukin-9 (IL-9) receptor (IL-9R) signaling in the absence of its cognate cytokine ligand (i.e., constitutively active). These synthetic cytokine receptors comprise an intracellular domain derived from the intracellular domain of human IL-9 receptor alpha (IL-9Ra), which can activate STAT1, STAT3, and STAT5 and trigger a strong JAK / STAT signal cascade. These synthetic cytokine receptors also comprise various modified intracellular domains, transmembrane domains, and extracellular domains. Immune cells such as T cells expressing these synthetic cytokine receptors are advantageous because such T cells assume characteristics of stem memory T cells without exhibiting signs of malignant transformation. Such T cells can be used in adoptive immunotherapy.

[0164] In some embodiments, the mechanism of constitutive activation comprises multimerization of the extracellular domain. In some embodiments, the mechanism of constitutive activation comprises multimerization of the transmembrane domain. In some embodiments, the mechanism of constitutive activation comprises multimerization of the intracellular domain. In some embodiments, the mechanism of constitutive activation comprises multimerization of at least two domains (e.g., the extracellular domain and the transmembrane domain). In some embodiments, the mechanism of constitutive activation comprises multimerization of three domains (i.e., the extracellular domain, the transmembrane domain, and the intracellular domain).

[0165] Adoptively transferred genetically engineered immune cells (e.g., T cells) have shown substantial anti-tumor activity in patients with hematopoietic malignancies but have limited efficacy in solid tumors. This is due to the immunosuppressive environment of solid tumors, the inefficient infiltration of solid tumors, and chronic antigen stimulation, which leads to lack persistence and / or efficacy of the engineered immune cells. In some cases, multipotency and replicative potential are also diminished. Thus, there is a need for compositions and / or methods that reduce or eliminate immune cell exhaustion, lack persistence, and / or decreased efficacy.

[0166] Another reason for the limited efficacy of adoptively transferred genetically engineered immune cells is that targeting lineage specific markers in solid tumors can lead to the targeting of normal tissue, which is life-threatening. Target antigens expressed on cancer cells but not normal cells of the same type have been reported but are rare and represent the exception rather than the rule. See Young et al., Cancer Discov. (2022) 12:1625-1633; Weber et al., Cell (2020) 181:46-62. Creating cancer-specific targets is therefore desired to create a therapeutic window.34MF-364447969307612002240

[0167] In addition to the challenges of identifying cancer-specific targets, the solid tumor microenvironment can limit the activity of the engineered cells and require further biological modifications to overcome exhaustion and elicit long-term clinical benefit.

[0168] The provided disclosure addresses these needs. The present disclosure demonstrates that the provided synthetic chimeric IL-9 receptors, even in the absence of ligand, have the ability to constitutively activate cells in which they are expressed, such as T cells. In some embodiments, the activation triggers a strong JAK / STAT signal cascade where STAT1, STAT3, and STAT5 can all be activated. In some embodiments, the activation has the ability to result in T cells that overcome T cell exhaustion by polarizing T cells toward a naiver phenotype and skewing the ratio of CD4: CD8, such as to a ratio of 2:1, without triggering malignant transformation of T cells. Thus, the present disclosure demonstrates that the constitutive IL-9R disclosed herein has the potential to reprogram and alter the phenotype of immune cells to overcome exhaustion, which can improve anti-tumor activity in solid tumors.

[0169] In some embodiments, the enhancements include armoring the immune cell with a cytokine. In some embodiments, the immune cell expresses a cytokine. Any cytokine known in the art can be expressed in the immune cell. In some embodiments, expression of the cytokine enhances function and activity of the immune cell. In some embodiments, the cytokine is IL- 18, as described in Section II. B.2b.

[0170] The present disclosure also provides pharmaceutical compositions comprising the synthetic cancer antigens, polynucleotides, or vectors disclosed herein and the methods of using such pharmaceutical compositions for treating cancers. In some embodiments, the one or more components, i.e., the adenoviral vector and immune cell, are formulated as compositions. In some embodiments, the components are formulated as separate compositions. In some embodiments, due to the tumor tropic nature of the adenoviral vector, the adenoviral vector and immune cell are formulated into a single composition. In some embodiments, the pharmaceutical compositions comprise a pharmaceutically acceptable excipient. In some embodiments, the compositions are administered intravenously and / or intratumorally to a subject in need thereof (e.g., having a cancer or tumor).

[0171] As discussed above, the provided embodiments permit tumor specific gene delivery in which the encoded synthetic cancer antigen is able to selectively tag tumor cells of various types with a targetable antigen and the chemotactic cytokine is able to be secreted from the tumor cells to attract immune cells to the tumor cells. This then allows immune cells, such s a cell therapy engineered with a cognate binder (e.g., CAR) to be used to bind the targetable antigen

[0172] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.35MF-364447969307612002240I. DEFINITIONS

[0173] Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa unless the content clearly dictates otherwise. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0174] The terms “a”, “an”, and “the”, as used herein, include plural references unless the context clearly dictates otherwise.

[0175] The terms “or” and “and / or”, as used herein, include any, and all, combinations of one or more of the associated listed items.

[0176] 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.

[0177] The terms “including”, “includes”, “included”, and other forms, as used herein, are not limiting.

[0178] 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.

[0179] 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.

[0180] 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., bispecific antibodies 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 36MF-364447969307612002240(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. The term “idiotype”, as used herein, refers to the unique set of antigenic epitopes that the variable portion of an antibody recognizes. An anti-idiotype antibody is an antibody that specifically binds to the antigen binding site of another antibody (e.g., the antigenbinding domain of the antibody).

[0181] 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 synthetic 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.

[0182] 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.

[0183] 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 37MF-364447969307612002240to 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.

[0184] 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.

[0185] 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 one of 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 VL and CL regions. The VH, CHI, VL, 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.

[0186] 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.

[0187] 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 38MF-364447969307612002240differ 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.

[0188] 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 constant region. 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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 a39MF-364447969307612002240therapeutic 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 be formulated by well-known conventional methods (see, e.g., Remington, The Science and Practice of Pharmacy, 23rd edition, A. Adejare, ed., Academic Press, 2020).

[0194] 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.

[0195] 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 single-stranded 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.

[0196] 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.

[0197] The term “population” of cells, as used herein, refers to any number of cells greater than 1, but is preferably at least 1x103 cells, at least 1x104 cells, at least 1x105 cells, at least 1x106 cells, at least 1x107 cells, at least 1x108 cells, at least 1x109 cells, at least 1x1010 cells, at least 1x1011 or 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).

[0198] 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 certain percentage 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 40MF-364447969307612002240“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. Nat’l. 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.

[0199] 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.

[0200] The term “synthetic”, as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that cannot be directly isolated from a source in nature. In some embodiments, the synthetic nucleic acid, polypeptide, cell, or organism is altered or changed as compared to the corresponding naturally-occurring one. In some embodiments, the synthetic 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, a synthetic polypeptide can comprise different sourcing polypeptides functionally linked together.

[0201] The term “target domain”, as applied to a synthetic cancer antigen disclosed herein, refers to the extracellular domain of the synthetic 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 expressed on the surface of a cancer cell.

[0202] The term “transduced”, as used herein, refers to a process by which a transgene is introduced into a host cell from a virus particle.

[0203] The term “transgene”, as used herein, refers to any heterologous polynucleotide incorporated in a adenoviral 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.41MF-364447969307612002240

[0204] The term “transmembrane domain”, as used herein, refers to any protein structure that is thermodynamically stable in a cell membrane (e.g. an eukaryotic cell membrane). The transmembrane domain may be derived either from a natural or from a synthetic source.

[0205] 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.

[0206] 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 β sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the β 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-binding site of antibodies (see, e.g., Rabat et al, Sequences of Proteins of Immunological Interest (5th ed. 1991)).42MF-364447969307612002240

[0207] 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 single-stranded 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-retro viruses), 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.

[0208] 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 (Kaplitt & 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. SYSTEMS FOR TARGETING TUMORS

[0209] Provided herein are systems for targeting tumors and for treating the targeted tumors with an engineered cell therapy. In some embodiments, the systems include (1) a vector, or a composition containing same, in which the vector comprises a polynucleotide that encodes a synthetic cancer antigen and a C-X-C motif chemokine ligand 9 (CXCL9) chemokine, and an immune cell or populations of such immune cells, or a composition containing the same, in which the immune cell comprises (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR), wherein the CAR binds to the target domain of the mtSCA. In some embodiments, the synthetic cancer antigen is a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain. In some embodiments, the synthetic cancer antigen is a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumortargeting binding molecule. In some embodiments, the synthetic cancer antigen includes both an mtSCA comprising a target domain and a membrane-targeting domain and a ttSCA comprising a target domain and a tumor-targeting binding molecule.43MF-364447969307612002240

[0210] In some embodiments, the tumor-targeting system is depicted in FIG.63. With reference to FIG.63, in some embodiments, the tumor-targeting system comprises a two component system designed to specifically target and eliminate cancers, e.g., cancers of epithelial origin.

[0211] In some embodiments, the first component is a replication-competent, tumor-specific adenovirus expressing a synthetic cancer antigen and CXCL9. In some embodiments, the adenovirus comprises any of the adenoviruses described in Section II. A.4. In some embodiments, the synthetic cancer antigen is designed to be a biologically inert payload not present elsewhere in the body, with the sole function of being targeted by engineered immune cells (e.g., CAR T cells) expressing a receptor that specifically binds to the synthetic cancer antigen. The synthetic cancer antigen was created by truncating trastuzumab, an anti-Her2 humanized monoclonal antibody, to contain only the variable domain of the heavy chain. The truncation abrogates Her2 recognition while retaining the ability to be bound by an anti-idiotype antibody used to create the recognition domain of the an scFv. The synthetic cancer antigen can be membrane-bound by conjugating the synthetic cancer antigen to a membrane-targeting domain (e.g., a transmembrane domain) or soluble by conjugating the synthetic cancer antigen to a tumor-targeting binding molecule (e.g., an scFv specific to an antigen expressed on a tumor cell). In some embodiments, the CXCL9 enhances tumor infiltration of the engineered immune cells. In some embodiments, the adenovirus promotes an inflammatory tumor microenvironment supportive of T cell function.

[0212] In some embodiments, the second component can comprise an autologous or allogeneic engineered immune cell expressing a receptor that recognizes the synthetic cancer antigen. In some embodiments, the receptor contains an anti-synthetic cancer antigen scFv linked to a 4-1BB costimulatory molecule and CD3^ activation domain. In some embodiments, the engineered immune cell also expresses a constitutive, interleukin 9 receptor (IL9R) homodimer to maintain stem cell memory and central memory phenotypes to provide cytokine support for the cell to overcome exhaustion and activation-induced cell death.A. Vectors for Delivery of Transgenes

[0213] In some embodiments, the vectors encode any of the one or more proteins disclosed herein, such as a synthetic cancer antigen, its cognate binder, a synthetic cytokine receptor, and a cytokine. In particular embodiments, the vectors encode one or more copies of any of the one or more proteins disclosed herein.

[0214] Provided herein are vectors comprising the polynucleotides or nucleic acid molecules encoding any of the synthetic cancer antigens disclosed herein. Also provided herein are vectors comprising any of the polynucleotides or nucleic acid molecules encoding the cognate binders of synthetic cancer antigens disclosed herein.44MF-364447969307612002240

[0215] Also provided herein are vectors comprising polynucleotides encoding two or more of the synthetic cancer antigens disclosed herein, such as a synthetic cancer antigen comprising a tumortargeting binding molecule, and a synthetic cancer antigen comprising a membrane targeting domain.

[0216] Also provided herein are vectors comprising a first polynucleotide encoding a soluble synthetic cancer antigen that comprises a target domain and a tumor-targeting binding molecule, e.g., any of those disclosed in Section II. A.l.a.i., and a second polynucleotide encoding a membrane bound synthetic cancer antigen that comprises a target domain and a membrane targeting domain, e.g., any of those disclosed in Section II. A.l.a.ii. In some embodiments, the first polynucleotide and the second polynucleotide are separated by nucleotide sequence encoding a cleavable linker. In some embodiments, the cleavable linker comprises a 2A self-cleaving peptide.

[0217] In some embodiments, the synthetic cancer antigen is delivered by an adenoviral vector comprising a polynucleotide encoding the synthetic cancer antigen, such as any of the polynucleotides disclosed in Section II. A.l and II. B.3.

[0218] In order to express any of the one or more proteins described herein in a cell, an expression cassette encoding the one or more proteins can be inserted into a nucleic acid vector. The “expression cassette” contains the gene of interest, which is any of the one or more proteins described herein. 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 adapted for 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 adenoviral vector as a single unit.

[0219] 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.

[0220] 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 synthetic cancer antigens or the cognate binders disclosed herein can be ligated into such vectors for expression in mammalian cells.

[0221] In some embodiments, the vector is an adenoviral vector or a non-viral vector.45MF-364447969307612002240

[0222] 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 synthetic cancer antigens or the cognate binders 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.

[0223] 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 92:77007704, 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 94:69166921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 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.

[0224] 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 46MF-364447969307612002240herpes virus (KSHV), Herpes virus saimiri (HS), or Marek’s disease virus (MDV). Epstein Barr virus (EB V) and Kaposi’s sarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus.

[0225] 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 described in 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).

[0226] In some embodiments, a polynucleotide sequence encoding the synthetic tumor antigens or the cognate binders 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 Dalgarno 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).

[0227] In some embodiments, polynucleotides encoding the synthetic cancer antigens or the cognate binders described herein are 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 47MF-364447969307612002240which 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.

[0228] 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 Pll 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 β-kinesin (β-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 β-actin (CAG) promoter, a β-actin promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding 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.

[0229] In some embodiments, a polynucleotide sequence encoding the synthetic cancer antigens or the cognate binders described herein is operably linked to a constitutive promoter. In such embodiments, the polynucleotides encoding the synthetic cancer antigens or the cognate binders described herein are constitutively and / or ubiquitously expressed in a cell.

[0230] In some embodiments, a polynucleotide sequence encoding the synthetic cancer antigens or the cognate binders described herein is operably linked to an inducible promoter. In such embodiments, polynucleotides encoding the synthetic cancer antigens 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.48MF-364447969307612002240

[0231] 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 some embodiments, vectors comprise a polyadenylation 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 polyadenylation is 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, ATT AAA, AGTAAA). In some embodiments, the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit β-globin polyA sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.

[0232] 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 synthetic tumor 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 synthetic 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 is an EpCAM signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 66. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 60. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 58. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 98. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 99. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 299. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 300. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 301. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 324. In some embodiments, the signal peptide is encoded by the nucleic acid sequence of any of SEQ ID NOS: 321-323 and 325.49MF-364447969307612002240

[0233] In some embodiments, the expression vector further comprises nucleotide sequences encoding one or more protein tags (e.g., 6xHis tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to polynucleotide encoding the synthetic cancer antigen, thereby resulting in a synthetic cancer antigen that further comprises a protein tag.

[0234] 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.

[0235] In some embodiments, delivery via electroporation comprises mixing the cells with the polynucleotides encoding the synthetic 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 synthetic 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.

[0236] In some embodiments, polynucleotides encoding the synthetic cancer antigens or the cognate binders 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 50MF-364447969307612002240example, 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).

[0237] In some embodiments, vectors comprising polynucleotides encoding the synthetic cancer antigens or the cognate binders 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, lenti virus vectors are used. In some embodiments, self-inactivating lenti viral vectors are used. For example, self-inactivating lenti viral 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.

[0238] 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.

[0239] In some embodiments, the one or more proteins disclosed herein are delivered by a vector that is an adenoviral vector exhibiting tumor tropism.

[0240] Accordingly, in some embodiments, the vector comprising the polynucleotide encoding the synthetic cancer antigen is an adenoviral 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 synthetic cancer antigen to the tumor cell being targeted, e.g., a tumor cell of a subject’s cancer. More specifically, the adenovirus encoding the synthetic cancer antigen(s) (e.g., ColoAdl or Enadenotucirev) binds to cells primarily via cluster of differentiation 46 (CD46), which is expressed at various levels on all nucleated, normal human cells. The receptor is normally 51MF-364447969307612002240hidden on the basolateral surfaces of normal epithelial cells and is thus not available for binding. See Maisner et al., Journal of Biological Chemistry (1997) 272.33:20793-20799. However, CD46 is often upregulated in carcinomas and is expressed on all surfaces of tumor cells. See Shang et al., Arch. Pathol. Lab. Med (2014) 138:910-919; Thorsteinsson et al., APMIS Acta Pathol. Microbiol. Immunol. Scand (1998) 106:869-878. Although CD46 permits virus uptake, it is not sufficient to permit adenovirus replication in non-tumor cells. Enadenotucirev lacks E4orf4, a multifunctional protein that deregulates AMP-activated protein kinase (AMPK) in normal cells. Deletions in E4orf4 have been shown to attenuate oncolytic virus activity in normal cells without impact on tumor cell lines, potentially explaining the tumor selectivity of Ad3 / 11 viruses. See O’Shea et al., EMBO (2005) 24:1211-1221.

[0241] Thus, the viral vectors provided herein, such as those described in Section ILA, target cancer cells for clearance but do not target cells of normal (e.g., non-cancerous) tissue.

[0242] 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 El A gene that is essential for adenovirus replication.

[0243] In some embodiments, the viral vector is a replicating viral vector.

[0244] In some embodiments, the viral vector is an adenoviral vector or modified form thereof of an adenoviral vector as disclosed in, e.g., W02005 / 118825, W02015 / 097220, W02015 / 059303, WO2015 / 155370, W02016 / 174200, W02017 / 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.

[0245] In some embodiments, the viral vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a lenti viral vector, a retroviral vector, or a herpes simplex viral (HSV) vector.

[0246] 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, Adil, Adl2, Adl7, and Ad40.

[0247] 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.

[0248] 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 52MF-364447969307612002240of 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.

[0249] 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.

[0250] 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.

[0251] Most adenoviruses, e.g., those in subgroups A, C, D, E, and F, enter target cells by binding primarily to the coxsackie-adeno virus 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 1, Adl4, Adl6, Ad21, Ad35, and Ad50 of subgroup B bind to CD46, as well as Adl7 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.

[0252] The levels of coxsackie-adenovirus receptor varies 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., Int. J. Mol. Sci., 2020, 21(18): 6828.53MF-364447969307612002240

[0253] 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., Adil, Adl4, Adl6, Adl7, Ad21, Ad35, Ad47, and Ad50, desirable for use in delivering synthetic cancer antigen s 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., Int. J. Mol. Sci., 2020, 21(18): 6828.

[0254] 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., Int. J. Mol. Sci., 2020, 21(18): 6828; 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 Adl4, are desirable for use in delivering synthetic cancer antigen s to tumor cells.

[0255] 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, Adil, Adl4, Adl6, Adl7, Ad21, Ad35, Ad47, or Ad50.

[0256] In some embodiments, the adenoviral vector is based on Ad3, Ad7, Adi 1, 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 1. In some embodiments, the Adi 1 is an Adi Ip. In some embodiments, the adenoviral vector is based on Adi 2. 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.

[0257] 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 1) adenoviral vectors.

[0258] 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.54MF-364447969307612002240

[0259] In some embodiments, the adenovirus is a chimeric adenovirus. In some embodiments, the chimeric adenovirus is Ad3 / 1 Ip or is Ad5 / 3.

[0260] 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 / llp is 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 1, 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; WO 2005 / 118825; 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 Adil 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 / 1 Ip 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. In some embodiments, the Ad3 / 1 Ip viral vector is ColoAdl. See, e.g., Kuhn et al., supra; US8765463.

[0261] In some embodiments, ColoAdl has a nucleic acid sequence that has at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least bout 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to SEQ ID NO: 257. In some embodiments, ColoAdl has the nucleic acid sequence of SEQ ID NO: 257. The nucleic acid sequence may be derived from ColoAdl to include additional sequences, such as sequences that facilitate plasmid-stage propagation and selection and / or, where applicable, posttransduction enrichment of vector-positive cells. For instance, a origin of replication for bacterial propagation (e.g. pl5) and / or antibiotic resistance makers (e.g., KanR) or a neomycin / kanamycin-resistance cassette (Neo / Kan) may be included. In some embodiments, such additional sequences may be flanked by the 5’ and 3’ ITRs. In certain embodiments, elements positioned between the 5' and 3' ITRs (e.g., KanR, Neo / Kan, and p 15) enable bacterial and / or eukaryotic selection and, as needed, provide non-coding spacer sequences that maintain a packaging-competent genome length while being transcriptionally silent in target cells. In some embodiments, a ColoAdl -derived sequence 55MF-364447969307612002240is a nucleic acid sequence that has at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least bout 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to SEQ ID NO: 335. In some embodiments, a ColoAdl -derived sequence has the nucleic acid sequence of SEQ ID NO: 335. A plasmid map is shown in FIG. 1C. Any of such sequences may be inserted with a transgene sequence as described.

[0262] 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 some embodiments, 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.

[0263] 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.

[0264] 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.

[0265] In some embodiments, the viral vector is an oncolytic virus. 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 reo virus, and a rhabdo virus, such as maraba virus. In some embodiments, the viral vector exhibits weak oncolytic activity.

[0266] 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., synthetic cancer antigen and CXCL9) is inserted into a non-coding region of genomic virus DNA,56MF-364447969307612002240such as an intron or intergenic sequence. In some embodiments, the transgene is under the control of a promoter.

[0267] For example, with reference to adenovirus, including a chimeric adenovirus, such as Colo Adi, among regions for insertion include the L5 region, such as between L5 and the E4 region.

[0268] 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 a synthetic cancer antigen and CXCL9 as described herein.

[0269] 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-B1-BA-B2-BX-BB-BY-B3-3’ ITR.

[0270] 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 f unction 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 Ibp to 138bp of SEQ ID NO: 257 or a sequence 90%, 95%, 96%, 97%, 98% or 99% identical thereto along the whole length, in particular the sequence consisting of from about Ibp to 138bp of SEQ ID NO: 257. In one embodiment, the 5’ITR comprises or consists of the sequence from about 2bp to 137bp of SEQ ID NO: 257 or a sequence 90%, 95%, 96%, 97%, 98% or 99% identical thereto along the whole length, in particular the sequence consisting of from about 2bp to 137bp of SEQ ID NO: 257. 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: 257 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: 257. In one embodiment, the 3’ITR comprises or consists of the sequence from about 32189bp to 32324bp of SEQ ID NO: 257 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: 257.

[0271] In embodiments of the formula (I), Bl is a bond or comprises El A, E1B or E1A-E1B. In some embodiments, Bl as employed herein refers to the DNA sequence encoding: part or all of an El A 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 Bl is a bond then E1A and E1B sequences will be omitted from the virus. In one embodiment B 1 is a bond and thus the virus is a vector.57MF-364447969307612002240

[0272] In one embodiment B 1 further comprises a transgene. It is known in the art that the B 1 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.

[0273] 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 pol ypeptide / protein E1A It may be mutated 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 non-mutated 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.

[0274] 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.

[0275] Thus B 1 can be modified or unmodified relative to a wild-type El region, such as a wildtype 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.

[0276] In one embodiment B 1 has the sequence from 139bp to 3932bp of SEQ ID NO: 257. In one embodiment, Bl has the sequence from 568bp to 3,902bp of SEQ ID NO: 257.

[0277] 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, Adil, Adl4, Adl6, Ad21, Ad34, Ad35, Ad51 or a combination thereof, such as Ad3, Adi 1 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.

[0278] 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 1, such as Adi Ip. In one embodiment the E2B region has the sequence from 5068bp to 10355bp of SEQ ID NO: 257 or a sequence 95%, 96%, 97%, 98% or 99% identical thereto over the whole length. In one embodiment 58MF-364447969307612002240the E2B region has the sequence from 5O67bp to 10354bp of SEQ ID NO: 257 or a sequence 95%, 96%, 97%, 98% or 99% identical thereto over the whole length.

[0279] In one embodiment BA has the sequence from 3933bp to 27184bp of SEQ ID NO: 257.

[0280] E3 as employed herein refers to the DNA sequence encoding part or all of an adenovirus E3 region (i.e. protein / polypeptide), it may be mutated such that the protein encoded by the E3 gene has conservative or non-conservati ve 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 wild-type protein or has a new function in comparison to wild-type protein or a combination of the same, as appropriate.

[0281] In one embodiment the E3 region is from an adenovirus serotype, in particular a group B serotype, for example Ad3, Ad7, Adil (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.

[0282] 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.

[0283] 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 includes part or all of the E3 region is replaced with a transgene.

[0284] In one embodiment the B2 region comprises the sequence from 27185bp to 28165bp of SEQ ID NO: 257. In one embodiment B2 region consists of the sequence from 27185bp to 28165bp of SEQ ID NO: 257.

[0285] 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.

[0286] 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.

[0287] 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.59MF-364447969307612002240

[0288] In one embodiment Bx has the sequence from 28166bp to 28366bp of SEQ ID NO: 257. 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: 257. In one embodiment Bx has a DNA sequence inserted between a place corresponding to between positions 28192bp and 28193bp of SEQ ID NO: 257. In one embodiment Bx is a bond.

[0289] 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.

[0290] 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 f unctions in receptor recognition and contributes to the adenovirus’ ability to selectively bind and infect cells.

[0291] 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.

[0292] In one embodiment BB has the sequence from 28367bp to 29344bp of SEQ ID NO: 257.

[0293] 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.

[0294] Thus, in one embodiment BY is joined directly to a base of L5 which represents the "end" of a coding sequence.

[0295] 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.

[0296] In one embodiment, BY has the sequence from 29345bp to 29379bp of SEQ ID NO: 257. 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 60MF-364447969307612002240of 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 to the 3’ end, or at any point between bp 1 to 35 of the sequence from 29345bp to 29379bp of SEQ ID NO: 257. In some embodiments, insert(s) occur at about or after position 29356bp of SEQ ID NO: 257 (e.g. corresponding to insert(s) at about or after position position 29369 of SEQ ID NO: 335). In one embodiment BY has a DNA sequence inserted between a place corresponding to positions 29356bp and 29357bp in SEQ ID NO: 257. In one embodiment the insert is at about position 29356bp of SEQ ID NO: 257. 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 a synthetic cancer antigen and CXCL9 as described herein. In some embodiments, the transgene is under the control of a promoter.

[0297] 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 may occur within sequence positions as described When the sequence is interrupted the virus will still comprise the original sequence, but generally it will be as two fragments sandwiching the insert.

[0298] 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.

[0299] 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 wild-type (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 f unction in comparison to wild-type protein or a combination of the same as appropriate.

[0300] 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: 257.

[0301] In one embodiment E4 is present except for the E4orf4 region which is deleted.

[0302] In one embodiment B3 is a bond, i.e. wherein E4 is absent.

[0303] In one embodiment B3 has the sequence consisting of from 32188bp to 29380bp of SEQ ID NO: 257.

[0304] 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 a synthetic61MF-364447969307612002240cancer antigen and CXCL9 as described herein. In some embodiments, the transgene is under the control of a promoter.

[0305] In particular embodiments, any of the transgene sequences described herein (e.g., a synthetic cancer antigen or CXCL9) is inserted into a region of a ColoAdl or a vector sequence derived therefrom (e.g., SEQ ID NO: 335) having the organization of formula (I): 5' ITR-B1-BA-B2-BX-BB-BY-B3-3' ITR. Exemplary backbones include SEQ ID NO: 257 and SEQ ID NO: 335, which are substantially similar in sequence, as well as sequence variants thereof that are substantially identical and / or functionally equivalent (including variants containing substitutions, insertions, or deletions). For instance, in some embodiments, the transgene is inserted at a position corresponding to about nucleotide 29,369 of SEQ ID NO: 335, including within the 35-nt window spanning nucleotides 29,345-29,379 of SEQ ID NO: 335 (See, e.g., FIG. 1C); in other embodiments, the transgene may be inserted at any position from the 5' end to the 3' end of the vector (e.g., between region L5 and E4 as depicted in FIG. 1C). Positions corresponding to those recited herein in other ColoAdl or ColoAdl -derived backbones, including backbones that differ in sequence, can be identified by alignment to SEQ ID NO: 335 or SEQ ID NO: 257 using identical or conserved nucleotides as guides, and insertions relative to such sequences at such corresponding positions are contemplated.

[0306] In some embodiments, provided herein is a replication deficient or replication capable oncolytic adenovirus, comprising a transgene cassette encoding any of the synthetic cancer antigen and CXCL9 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 1. In some embodiments, the virus is a chimeric virus, for example a virus that comprises a modified E2B region.

[0307] In some embodiments, the virus is ColAdl 1 (enadenotucirev).

[0308] 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.

[0309] 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 of thee endogenous promoter. That is, where the promoter is generally on the antisense strand, the cassette is inserted, for example in the antisense orientation.62MF-364447969307612002240

[0310] 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.

[0311] 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.

[0312] In some embodiments, the promoter is a 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.

[0313] 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 antisense 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.

[0314] 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 synthetic cancer antigen and CXCL9, 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.

[0315] 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.63MF-364447969307612002240

[0316] 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.

[0317] 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 synthetic cancer antigens and CXCL9 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 synthetic cancer antigen and CXCL9 is associated with the L5 region as described elsewhere herein, in particular located between L5 and E4 region.

[0318] Other features of an 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, polyadenylation sequences and other sequences well known to a skilled artisan. Any of such sequences are known and described, for example, in W02005 / 118825, W02015 / 097220, W02015 / 059303, WO2015 / 155370, W02016 / 174200, W02017 / 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.

[0319] In some embodiments, the vector is delivered specifically to a cancer cell. In some embodiments, the vector is delivered specifically to a blood cancer cell. In some embodiments, the vector is delivered specifically to a solid tumor cancer cell. / . Polynucleotide Transgenes

[0320] In provided embodiments, the polynucleotide contained in the vector includes a transgene that encodes a membrane-targeting synthetic cancer antigen (mtSCA), a transgene that encodes a tumor-targeting synthetic cancer antigen (ttSCA), and a transgene that encodes a C-X-C motif chemokine ligand 9 (CXCL9) chemokine. In some embodiments, the polynucleotide includes a transgene that encodes mtSCA. In some embodiments, the polynucleotide includes a transgene that encodes mtSCA and ttSCA. In some embodiments, the polynucleotide includes a transgene that encodes mtSCA, ttSCA and CXCL9.

[0321] In some embodiments, the transgenes encoded by the polynucleotide are separated by a self-cleaving peptide. In some embodiments, the self-cleaving peptide comprises a 2A peptide.

[0322] In some embodiments, the self-cleaving peptide comprises a P2A peptide. In some embodiments, a short flexible spacer is disposed immediately 5' N-terminal to a self-cleaving 2A peptide (e.g., P2A, T2A), for example a Gly-Ser-Gly (GSG) tripeptide, to reduce steric / context effects64MF-364447969307612002240and improve ribosome-skipping efficiency without altering the 2A cleavage site. The spacer may be about 1-10 amino acids, preferably glycine and / or serine residues (e.g., G, GS, GSG, GSSG). For instance, in some embodiments, the self-cleaving peptide is encoded by the nucleic acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 333. In some embodiments, the self-cleaving peptide comprises the amino acid sequence of SEQ ID NO: 298. In some embodiments, the self-cleaving peptide is a P2A peptide. In some embodiments, the self-cleaving peptide comprises the amino acid sequence of SEQ ID NO: 59.

[0323] In some embodiments, the self-cleaving peptide comprises a T2A peptide. In some embodiments, the self-cleaving peptide is a T2A peptide. In some embodiments, the T2A linker is encoded by the nucleic acid sequence set forth in SEQ ID NO: 13. In some embodiments, the self-cleaving peptide comprises the amino acid sequence set forth in SEQ ID NO: 14.a. Synthetic Cancer Antigens

[0324] Provided herein are systems for targeting tumors in a subject comprising a vector that encodes a synthetic cancer antigen.

[0325] In provided embodiments, the synthetic cancer antigens are not normally expressed on the surface of non-cancer cells and can be specifically delivered to cancer cells in a subject via oncolytic viruses, and thus only expressed on the surface of cancer cells. Further, the cognate binders can be engineered as chimeric-antigen-receptors for expressing on the surface of immune effector cells. The present disclosure re-designs the interaction between the immune effector cells and the cancer targets. By shifting away from the traditional approach of targeting known cancer-overexpressed proteins, which can also express on the surface of non-cancer cells, the present disclosure improves the safety of immune cell therapy by eliminating recognition of healthy tissues. In some embodiments, the synthetic cancer antigen comprises a target domain and a transmembrane domain. In some embodiments, the synthetic cancer antigen is membrane-bound. In other embodiments, the synthetic cancer antigen comprises a target domain linked to a tumor-targeting binding molecule. In some embodiments, the synthetic cancer antigen is soluble. In some embodiments, one or more formats (e.g., membrane bound and / or soluble) are recognizable by a cognate binder. Both or either of the formats of the synthetic cancer antigen can be used in combination with one or more cognate binders. In some embodiments, the target domain of the synthetic cancer antigen is recognizable by a cognate binder.

[0326] In some embodiments, an exemplary synthetic cancer antigen described herein is any of the synthetic cancer antigens described in WO 2024 / 243578, which is incorporated herein by reference in its entirety.i. Target Domain65MF-364447969307612002240

[0327] In embodiments provided herein, the target domain of the synthetic cancer antigen is a target domain that is a protein that is recognizable by a cognate binder to be specifically targetable to tag cancer cells. One advantage of the present invention is that the target domain of synthetic cancer antigens provided herein is designed to be different from proteins expressed on the surface of a noncancer cell of a subject. In some embodiments, the target domain is not expressed on the surface of a non-cancer cell of a subject. In some embodiments, the target domain is recognizable by a cognate binder. As a result, the cognate binder of the target domain cannot bind to a surface protein expressed on the surface of a non-cancer cell, reducing or eliminating off-target binding.

[0328] The target domain of synthetic cancer antigens provided herein can be any polypeptide that is not expressed on the surface of a non-cancer cell of a subject.

[0329] Binding of a cognate binder (for example, an scFv) to a target domain 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.

[0330] In provided embodiments, the target domain is a protein binding molecule. In some embodiments, the size of the target domain is about 20 amino acids to about 700 amino acids. In some embodiments, the size of the target domain is at least about 20 amino acids. In some embodiments, the size of the target domain is at most about 700 amino acids. In some embodiments, the size of the target domain 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 250 amino acids, about 100 amino acids to about 300 amino acids, about 10066MF-364447969307612002240amino 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 target domain 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.a) Antibody or antibody fragment

[0331] In some embodiments, the target domain is an antibody or antibody fragment. In some embodiments, the target domain 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 target domain is an antibody without the heavy chain CH3 domain. In some embodiments, the target domain is an antibody without the heavy chain CH2 domain. In some embodiments, the target domain is a Fab. In some embodiments, the target domain is an antibody without the heavy chain CH2 and CH3 domain.

[0332] In some embodiments, the target domain is an antibody fragment, such as a Fab, an scFv or a variable heavy chain only fragment. In some embodiments, the target domain is a single-chain variable fragment (scFv). In some embodiments, the target domain is the heavy chain of an antibody. In some embodiments, the target domain is the light chain of an antibody. In some embodiments, the67MF-364447969307612002240target domain is the variable domain of the heavy chain of an antibody. In some embodiments, the target domain is the variable domain of the light chain of an antibody.

[0333] In some embodiments, the functional fragments are part of a single polypeptide and are operatively linked. In some embodiments, the f unctional fragments are directly linked to each other. In some embodiments, the functional fragments 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. In some embodiments, the functional fragments are separate polypeptides and bind with each other after being translated to form a molecule derived from an antibody. In some embodiments, the functional fragments are translated from multiple polynucleotides. In some embodiments, the functional fragments are translated from a single polynucleotide. In some embodiments, the single polynucleotide comprises nucleotide sequences encoding self-cleaving peptides. In some embodiments, the self-cleaving peptides separate the functional fragments translated from a single polynucleotide. In some embodiments, the self-cleaving peptide is a P2A peptide. In some embodiments, the self-cleaving peptide comprises an amino acid sequence of SEQ ID NO: 59. In some embodiments, the T2A linker is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 14. In some embodiments, the self-cleaving peptide is a T2A peptide. In some embodiments, the self-cleaving peptide comprises an amino acid sequence of SEQ ID NO: 14. In some embodiments, the T2A linker is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 13.

[0334] In some embodiments, the 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 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.

[0335] Exemplary known antibodies include but not limited to ReoPro (abciximab), Humira (adalimumab), Hyrimoz (adalimumab-adaz), Cyltezo (adalimumab-adbm), Abrilada (adalimumab-afzb), Amjevita (adalimumab-atto), Hadlima (adalimumab-bwwd), Campath, Lemtrada (alemtuzumab), Praluent (alirocumab), Tecentriq (atezolizumab), Bavencio (avelumab), Simulect (basiliximab), Benlysta (belimumab), Benlysta (belimumab), Fasenra (benralizumab), A vastin (bevacizumab), Mvasi (bevacizumab-awwb), Zirabev (bevacizumab-bvzr), Zinplava (bezlotoxumab), Blincyto (blinatumomab), Siliq (brodalumab), Beovu (brolucizumab-dbll), Crysvita (burosumab-twza), Haris (canakinumab), Cablivi (caplacizumab-yhdp), Libtayo (cemiplimab-rwlc), Erbitux (cetuximab), Adakveo (crizanlizumab-tmca), Zenapax (daclizumab), Zinbryta (daclizumab), Darzalex (daratumumab), Prolia, Xgeva (denosumab), Unituxin (dinutuximab), Dupixent (dupilumab), Imfinzi (durvalumab), Soliris (eculizumab), Empliciti (elotuzumab), Gamifant (emapalumab-lzsg), Hemlibra 68MF-364447969307612002240(emicizumab-kxwh), Vyepti (eptinezumab-jjmr), Aimovig (erenumab-aooe), Repatha (evolocumab), Ajovy (fremanezumab-vfrm), Emgality (galcanezumab-gnlm), Simponi (golimumab), Simponi Aria (golimumab), Tremfya (guselkumab), Trogarzo (ibalizumab-uiyk), Praxbind (idarucizumab), Remicade (infliximab), Renflexis (infliximab-abda), Avsola (infliximab-axxq), Inflectra (infliximab-dyyb), Ixifi (infliximab-qbtx), Yervoy (ipilimumab), Sarclisa (isatuximab-irfc), Taltz (ixekizumab), Takhzyro (lanadelumab-flyo), Nucala (mepolizumab), Nucala (mepolizumab), Poteligeo (mogamulizumab-kpkc), Tysabri (natalizumab), Portrazza (necitumumab), Opdivo (nivolumab), Anthim (obiltoxaximab), Gazyva (obinutuzumab), Ocrevus (ocrelizumab), Arzerra (ofatumumab), Lartruvo (olaratumab), Xolair (omalizumab), Synagis (palivizumab), Vectibix (panitumumab), Keytruda (pembrolizumab), Perjeta (pertuzumab), Cyramza (ramucirumab), Lucentis (ranibizumab), Ultomiris (ravulizumab-cwvz), raxibacumab (raxibacumab), Cinqair (reslizumab), Skyrizi (risankizumab-rzaa), Rituxan (rituximab), Truxima (rituximab-abbs), Ruxience (rituximab-pvvr), Evenity (romosozumab-aqqg), Kevzara (sarilumab), Cosentyx (secukinumab), Sylvant (siltuximab), Tepezza (teprotumumab-trbw), Ilumya (tildrakizumab-asmn), Actemra (tocilizumab), Actemra (tocilizumab), Herceptin (trastuzumab), Kanjinti (trastuzumab-anns), Ogivri (trastuzumab-dkst), Ontruzant (trastuzumab-dttb), Herzuma (trastuzumab-pkrb), Trazimera (trastuzumab-qyyp), Stelara (ustekinumab), Stelara (ustekinumab) and Entyvio (vedolizumab).

[0336] In some embodiments, the target domain is an antibody fragment of any of the above antibodies. In some embodiments, the target domain is a molecule derived from any of the above antibodies. In some embodiments, a molecule derived from an antibody is one or more fragments of the antibody. Non-limiting examples of 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 target domain is an antibody without the heavy chain CH3 domain. In some embodiments, the target domain is an antibody without the heavy chain CH2 domain. In some embodiments, the target domain is a Fab. In some embodiments, the target domain is an antibody without the heavy chain CH2 and CH3 domain.

[0337] In some embodiments, the target domain is a single-chain variable fragment (scFv) of any of the above antibodies. In some embodiments, the target domain is the heavy chain of any of the above antibodies. In some embodiments, the target domain is the light chain of any of the above antibodies. In particular embodiments, the target domain is a fragment that does not bind to the target of the antibody but that retains an epitope to be recognized by a cognate binder (e.g., anti-idiotypic antibody). In some embodiments, the target domain is the variable heavy chain (VH) of an antibody. Thus, in some aspects, it is understood that the target domain is devoid of the variable light (VL) chain. In some embodiments, the target domain is not able to bind or substantially bind or exhibits reduced (low) binding affinity (e.g., a higher KD value) to the antigen of the antibody from which it is 69MF-364447969307612002240derived. One advantage of using an antibody fragment as a target domain that does not bind or substantially bind to a target antigen of the antibody or has low affinity binding for a target antigen of the antibody is that it allows the target domain to be delivered in a soluble format (e.g., as described in Section II. A.l.iii) while limiting the possibility of the target domain binding to the target antigen of the full antibody. For instance, in some embodiments, the target domain is an antibody fragment in which the antigen-binding domain for recognition of an antigen or epitope is not present or is partially removed or is non-functional (e.g., via one or more amino acid substitutions in a CDR). In an exemplary embodiment, the parental antibody is a two chain antibody containing a variable heavy and variable light chain in which the antigen-binding domain is composed of 6 CDRs. In some such embodiments, the target domain is an antibody fragment that is the variable heavy chain (VH) only composed of only 3 CDRs.

[0338] In particular embodiments, the target domain is an antibody or antibody fragment that is able to be recognized by a cognate binder that is an anti-idiotype antibody or antigen-binding fragment. Various anti-idiotype antibodies to antibodies are known. Exemplary anti-idiotype antibodies are described in Section II. B.l.a.

[0339] In provided embodiments, the antibody or antibody fragment target domain, such as a variable heavy chain, contains an epitope recognized by a cognate binder (e.g., anti-idiotype antibody) but does not specifically bind to a target antigen of a native or primary cells, such as a tumor target antigen. In particular embodiments, the cognate binder is an anti-idiotype antibody (e.g., such as any described in Section II. B.l) and the variable heavy chain contains an idiotype of the antibody. In some embodiments, the target domain is an antibody or antibody fragment that has reduced binding for the target antigen of the antibody compared to the parental antibody from which it has been derived. In some embodiments, the target domain is an antibody or antibody fragment that has low binding affinity for a target antigen of a parental antibody from which it was derived. In some embodiments, dissociation constant (Kd) of the antibody or antibody fragment target domain for binding a target antigen of an antibody from which it is derived is Kd greater than 10-7 (> 100 nM), such as Kd greater than 10-6 (> 1 pM), 10-5 (> 10 pM) or 10-4 (> 100 pM). In some embodiments, the Kd of the antibody or antibody fragment target domain for binding a target antigen of an antibody from which it is derived is greater than 10-4 (> 100 pM). Exemplary binding assays for determining binding affinity include, but are not limited to, enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, Western Blot assay, or surface plasmon resonance (e.g., Biacore). In some embodiments, the antibody or antibody fragment target domain does not exhibit detectable binding to the target antigen of the antibody from which it is derived with a cell expressing the target antigen of the antibody, such as determined by a binding assay for example by flow cytometry.

[0340] In some embodiments, the target domain is an antibody directed against HER2.70MF-364447969307612002240

[0341] In some embodiments, the target domain is derived from the 4D5 anti-HER2 antibody (also known as Abl), which is the parental antibody of trastuzumab. In some embodiments, the target domain is an antibody fragment of the 4D5 (Abl) antibody.

[0342] In some embodiments, the target domain is a 4D5 antibody or an antigen-binding fragment of 4D5 antibody. In some embodiments, the target domain is an antibody fragment of a 4D5 antibody. In some embodiments, the target domain is a molecule derived from a 4D5 antibody. In some embodiments, a molecule derived from a 4D5 antibody is one or more fragments of the antibody. In some embodiments, the target domain is an Fab fragment of a 4D5 antibody. In some embodiments, the target domain is a single-chain Fvs (scFv) of a 4D5 antibody. In some embodiments, the target domain does not comprise a constant heavy chain and / or a constant light chain domain. In some embodiments, the target domain comprises the variable light chain and the variable heavy chain of a 4D5 antibody. In some embodiments, the target domain comprises the variable heavy chain of the 4D5 antibody and does not comprise the variable light chain. In some embodiments, the target domain comprises the variable light chain of the 4D5 antibody and does not comprise the variable heavy chain. In some embodiments, the light chain constant domain comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the heavy chain constant domain comprises the amino acid sequence of SEQ ID NO: 51.

[0343] In some embodiments, the target domain is a Fab of a 4D5 antibody. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence of SEQ ID NO: 47. In some embodiments, the target domain comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 7. In some embodiments, the target domain 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: 47. In some embodiments, the target domain 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: 7.

[0344] In some embodiments, the target domain comprises a light chain comprising an amino acid sequence of SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID 71MF-364447969307612002240NO: 47 and a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 7. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 7.

[0345] In some embodiments, the target domain is a single-chain Fvs (scFv) of a 4D5 antibody. In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is 72MF-364447969307612002240at least 91% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 53. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 53.

[0346] In some embodiments, the target domain is a variable heavy chain of a 4D5 antibody. In some embodiments, the target domain does not comprise a variable light chain. In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 50. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 50.

[0347] In some embodiments, the target domain comprises a nucleic 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 any of SEQ ID NOS:73MF-364447969307612002240326-331. In some embodiments, the target domain is encoded by the nucleic acid sequence of any of SEQ ID NOS: 326-331.

[0348] In provided embodiments, such a variable heavy chain antibody fragment contains an idiotype recognized by an anti-idiotype antibody (e.g., set forth in any of SEQ ID NOS: 67-69) but does not specifically bind to HER2. In some embodiments, the variable heavy chain antibody fragment has reduced binding for HER2 compared to the 4D5 (Abl) parental antibody from which it has been derived. In some embodiments, the variable heavy chain antibody fragment has low binding affinity for HER2. In some embodiments, dissociation constant (Kd) of the variable heavy chain antibody fragment target domain for binding HER2 is Kd greater than IO-7(> 100 nM), such as Kd greater than IO-6(> 1 microM), IO-5(> 10 microM) or 10'4(> 100 microM). In some embodiments, the Kd of the variable heavy chain antibody fragment for binding HER2 is greater than 10'4(> 100 microM). Exemplary binding assays for determining binding affinity include, but are not limited to, enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, Western Blot assay, or surface plasmon resonance (e.g., Biacore). In some embodiments, the variable heavy chain antibody fragment target domain does not exhibit detectable binding to a cell expressing HER2, for example a tumor cell, such as determined by a binding assay for example by flow cytometry.b)Intracellular Protein

[0349] In some embodiments, the target domain is a fragment of an intracellular protein. In some embodiments, the target domain is a non-DNA binding domain of a transcription factor. In some embodiments, the target domain is an inactive leucine zipper domain of a transcription factor. In some embodiments, the target domain is a N-terminal fragment of a transcription factor. In some embodiments, the target domain is a C-terminal fragment of a transcription factor. Non-limiting examples of transcription factors that can be used herein include cFos, eJun, cMAF, ATF, Nfil3, and Xbpl.

[0350] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to74MF-364447969307612002240SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 71. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 71.

[0351] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 72. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 72.

[0352] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 73. In some embodiments, the target 75MF-364447969307612002240domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 73. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 73.

[0353] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 74. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 74.

[0354] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at 76MF-364447969307612002240least 90% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 75. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 75.

[0355] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 76% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 76. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 76.

[0356] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 77. In some77MF-364447969307612002240embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 77. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 77.c) Leucine Zipper

[0357] In some embodiments, the target domain is an active leucine zipper domain of a transcription factor. In some embodiments, the transcription factor is a basic leucine zippers (bZIP). Non-limiting examples of bZIP transcription factors that can be used herein include ATF, JUN, CREB, Fos. In some embodiments, the target domain is the leucine zipper domain of ATF. In some embodiments, the target domain is the leucine zipper domain of JUN. In some embodiments, the target domain is the leucine zipper domain of CREB. In some embodiments, the target domain is the leucine zipper domain of Fos.

[0358] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino78MF-364447969307612002240acid sequence that is at least 95% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 80. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 80.

[0359] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 81. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 81.

[0360] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to 79MF-364447969307612002240SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 82. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 82.

[0361] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 83. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 83.ii. Membrane Targeting Domain

[0362] In some embodiments, the synthetic cancer antigen is a membrane bound protein and the target domain is linked to a membrane targeting domain. In some embodiments, the synthetic cancer antigen comprises a target domain and a membrane targeting domain. The target domain can be any as described in Section II. A.l.a.i.

[0363] The membrane targeting domain of synthetic cancer antigens provided herein can be the transmembrane domain of any known transmembrane protein or from a synthetic source. The80MF-364447969307612002240transmembrane domains disclosed herein for synthetic cancer antigens can also be used as the transmembrane domain of CAR molecules disclosed below.

[0364] 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 membranepassing 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.

[0365] In some embodiments, the transmembrane domain is derived from a Type I single-pass 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.

[0366] 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.

[0367] 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 81MF-364447969307612002240region 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.

[0368] 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.

[0369] In some embodiments, the transmembrane domain is from a transmembrane glycoprotein. In some embodiments, the 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, 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, and / or NKG2C.

[0370] In some embodiments, the transmembrane domain is a CD8 transmembrane domain.

[0371] In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino 82MF-364447969307612002240acid sequence that is at least 85% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 52. In some embodiments, the transmembrane domain of SEQ ID NO: 52 is encoded the nucleic acid sequence set forth in SEQ ID NO: 302.

[0372] 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 40 amino 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.

[0373] The synthetic cancer antigens disclosed herein can further comprise additional elements. In some embodiments, the synthetic cancer antigen comprises a tag. In some embodiments, the tag is at the N-terminus of the synthetic cancer antigen. In some embodiments, the tag is at the C -terminus of the synthetic cancer antigen. In some embodiments, the tag is a flag tag. In some embodiments, the tag is a c-Myc tag. In some embodiments, the tag is a His tag. In some embodiments, the tag is a HA83MF-364447969307612002240tag. In some embodiments, the tag comprises an amino acid sequence of SEQ ID NO: 38. In some embodiments, the tag comprises an amino acid sequence of SEQ ID NO: 84.

[0374] In some embodiments, the synthetic cancer antigen comprising a membrane-targeting domain (i.e., the membrane-bound synthetic cancer antigen) 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: 9 or SEQ ID NO: 305. In some embodiments, the synthetic cancer antigen comprising a membrane-targeting domain (i.e., the membrane-bound synthetic cancer antigen) comprises an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 305. In some embodiments, the synthetic cancer antigen comprising the membrane-targeting domain (i.e., the membrane-bound synthetic cancer antigen) is encoded by a nucleic acid sequence of SEQ ID NO: 8, SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 305, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 336, SEQ ID NO: 337 or SEQ ID NO: 338.

[0375] In some embodiments, the different elements of the synthetic 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.Hi. Tumor-Targeting Domain

[0376] In some embodiments, the synthetic cancer antigen is a soluble (sol) protein and the target domain is linked to a tumor targeting molecule that directs the synthetic cancer antigen to a tumor cells to expose the target domain on the outside of the cell. In some embodiments, the synthetic cancer antigen comprises a target domain and tumor targeting molecule. The target domain can be any as described in Section II. A.l.a.i.

[0377] In some embodiments, provided herein is a synthetic cancer antigen comprising a target domain comprising at least one target domain described in Section II. A.l.a.i and a tumor targeting molecule (e.g., antibody or antigen binding fragment specific to a tumor associated antigen). 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 a synthetic cancer antigen is soluble and is not membrane associated with a cell. In certain embodiments in which the synthetic cancer antigen is expressed from a cell, the synthetic cancer antigen can be secreted from the cell, which, in provided aspects, can tag other non-infected bystander tumor cells to expose the synthetic cancer antigen on outside surface of the cell.84MF-364447969307612002240

[0378] In some embodiments, the tumor targeting molecule of the synthetic 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 binds to a tumor associated antigen (TAA). 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 binding molecule is an antibody or antigen binding fragment targeting a tumor associated antigen. In some embodiments, the antigen is associated with a cancer. In some embodiments, the antigen is associated with a solid tumor.

[0379] 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.

[0380] In some embodiments, the tumor targeting molecule binds to a tumor associated antigen (TAA). In some embodiments, the tumor targeting molecule is a molecule derived from an antibody is one or more functional fragments of the antibody that targets a tumor associated antigen. Non-limiting examples of f unctional 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.85MF-364447969307612002240In 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.

[0381] 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 (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH: VL heterodimer. The heavy (VH) and light chains (VL) 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-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL.

[0382] In some embodiments, the functional fragments are part of a single polypeptide and are operatively linked. In some embodiments, the f unctional fragments are directly linked to each other. In some embodiments, the functional fragments 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. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 88. In some embodiments, the functional fragments are separate polypeptides and bind with each other after being translated to form a molecule derived from an antibody. In some embodiments, the functional fragments are translated from multiple polynucleotides. In some embodiments, the functional fragments are translated from a single polynucleotide. In some embodiments, the single polynucleotide comprises nucleotide sequences encoding self-cleaving peptides. In some embodiments, the self-cleaving peptides separate the functional fragments translated from a single polynucleotide. In some embodiments, the self-cleaving peptide is a P2A peptide. In some embodiments, the self-cleaving peptide comprises an amino acid sequence of SEQ ID NO: 59.

[0383] 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, 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.86MF-364447969307612002240

[0384] Exemplary tumor associated antigens and their exemplary known antibodies include but are not limited to those described in Table 1 below.Table 1. Exemplary tumor associated antigens and their known antibodiesAntigen Antibody Cancer Indication CD2 Siplizumab Non-Hodgkin’s Lymphoma CD3 UCHT1 Peripheral or Cutaneous T-cell LymphomaCD4 HuMax-CD4CD20 Rituximab, Veltuzumab, B cell malignancies (NonTositumomab, Ofatumumab, Hodgkin’s lymphoma, Chronic Ibritumomab, Obinutuzumab, lymphocytic leukemia) CD22 (SIGLEC2) Inotuzumab, tetraxetan, CAT Chemotherapy-resistant hairy cell 8015, DCDT29805, leukemia, Hodgkin’s lymphoma BectumomabCD30 Brentuximab vedotinCD33 Gemtuzumab ozogamicin Acute myeloid leukemia (Mylotarg)CD37 TRU - 016 Chronic lymphocytic leukemia CD38 Daratumumab Multiple myeloma, hematological tumorsCD40 Lucatumumab Non-Hodgkin’s lymphoma CD52 Alemtuzumab (Campath) Chronic lymphocytic leukemia CD56 (NCAM1) Lorvotuzumab Small Cell Lung Cancer CD66e (CEA) Labetuzumab Breast, colon and lung tumors CD70 SGN-75 Non-Hodgkin’s lymphoma CD74 Milatuzumab Non-Hodgkin’s lymphoma CD138 (SYND1) BT062 Multiple MyelomaCD 152 (CTLA-4) Ipilimumab, Tremelimumab Metastatic melanoma CD221 (IGF1R) AVE 1642, IMC - A 12, MK - Glioma, lung, breast, head and 0646, R150, CP 75187 neck, prostate and thyroid cancer CD254 (RANKL) Denosumab Breast and prostate carcinoma CD261 (TRAILR1) Mapatumumab Colon, lung and pancreas tumors CD262 (TRAILR2) HGS-ETR2, CS-1008 and haematological malignancies CD326 (Epcam) Solitomab, Tucotuzumab, Colon and rectal cancer,Edrecolomab, 17-1A, malignant ascites, epithelial IGN 101, Catumaxomab, tumors (breast, colon, lung) AdecatumumabCD309 (VEGFR2) IM-2C6, CDP791 Epithelium-derived solid tumors CD319 (SLAMF7) HuLuc63 Multiple myelomaCD340 (HER2) Trastuzumab, Pertuzumab, Breast cancer Adotrastuzumab emtansine,Margetuximab,TimigutuzumabCAIX (CA9) cG250 Renal cell carcinomaEGFR (c-erbB) Cetuximab, Panitumumab, Solid tumors including glioma,Nimotuzumab, lung, breast, colon, and head and Nectinumumab, neck tumorsTomuzotuximab,Zalutumamab, matuzumab,MF-364447969307612002240futuximab, Amivantamab,Imgatuzumab,Depatuxizumab, and 806EPHA3 (HEK) KB004, IIIA4 Lung, kidney and colon tumors, melanoma, glioma and haematological malignancies Episialin Epitumomab Epithelial ovarian tumors FAP Sibrotuzumab and Fl 9 Colon, breast, lung, pancreas, and head and neck tumorsHLA-DR beta Apolizumab Chronic lymphocytic leukemia, non-Hodkin’s lymphoma FOLR-1 Farletuzumab Ovarian tumors5T4 Anatumomab Non-small cell lung cancer GD3 / GD2 3F8, chl4.18, KW-2871 Neuroectodermal and epithelial tumorsgpA33 huA33 Colorectal carcinoma GPNMB Glembatumumab Breast cancerHER3 (ERBB3) MM-121, Patritumab Breast, colon, lung, ovarian, and Seribantumab prostate tumors LumretuzumabElgemtumabIntegrin aV03 Etaracizumab Tumor vasculatureIntegrin a501 Volociximab Tumor vasculatureLewis-Y antigen hu3S193, IgN311 Breast, colon, lung and prostate tumorsMET (HGFR) AMG 102, METMAB, Breast, ovary and lung tumors SCH900105Emibetuzumab (LY- 2875358)Mucin-l / CanAg Pemtumomab, oregovomab, Breast, colon, lung and ovarian Cantuzumab tumorsPD-1 NivolumabPembrolizumabCemiplimabCamrelizumabSerplulimabSintilimabTislelizumabToripalimabRetifanlimabDostarlimabPD-L1 AtezolizumabAvelumabDurvalumabSugemalimabCosibelimabMXD-1105PSMA ADC, J591 Prostate Cancer Phosphatidylserine Bavituximab Solid tumorsTAG-72 Minretumomab Breast, colon and lung tumors Tenascin 81C6 Glioma, breast and prostate tumorsVEGF Bevacizumab Tumor vasculature88MF-364447969307612002240RanibizumabMMP Andecaliximab Colon, lung, head and neck, basal SDS3 cell, breast, thyroid, prostate, SDS4 ovarian, and gastric carcinomas REGA-3G12GPC2 D3-GPC2 Neuroblastoma, malignant brain tumor, and small-cell lung cancerGPC3 Codrituzumab Hepatocellular carcinoma (HCC), embryonal tumors, melanoma, hepatoblastoma, and testicular germ-cell tumors Mesothelin Anetumab lung adenocarcinomas, ovarian Amatuximab carcinomas, acute myeloid leukemia (AML)PSCA AGS-PSCAClaudin6 IMAB362 germ cell tumors, epithelial IMAB027 ovarian cancer,endometrial carcinoma, testicular Claudinl8.2 Zolbetuximab gastric cancer, hepatocellular ASKB589 carcinoma, biliary tract cancer, breast cancer, renal cell Osemitamab carcinoma, pancreatic cancer, non-small cell lung cancer, andmesothelioma

[0385] In some embodiments, the tumor associated antigen targeted by the tumor targeting molecule is expressed on a solid tumor.

[0386] In some embodiments, the tumor associated antigen is expressed on the solid tumor. Exemplary solid tumor associated antigens include but are not limited to EpCAM (Epithelial cell adhesion molecule), 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), ERBB2 / HER2 (epidermal growth factor receptor 2), ERBB3 (epidermal growth factor receptor 3), MET (Tyrosine-Protein Kinase IGF1R (insulin-like growth factor 1 receptor), EPH A3 (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).

[0387] In some embodiments, the tumor associate antigen includes CD20.

[0388] In some embodiments, the tumor associated antigen targeted by the tumor targeting molecule is epithelial cell adhesion / activating molecule (EpCAM), epidermal growth factor receptor 89MF-364447969307612002240(EGFR), or human epidermal growth factor receptor 2 (HER2). In some embodiments, the tumor associated antigen targeted by the tumor targeting molecule is EpCAM.

[0389] 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.

[0390] 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. In some embodiments, the tumor targeting molecule is a single-chain Fvs (scFv) of an anti-EpCAM antibody.

[0391] In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv 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: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 80%, or 99% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 90MF-36444796930761200224097% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 54. In some embodiments, the tumor targeting molecule comprises an anti-EpCAM scFv comprising an amino acid sequence of SEQ ID NO: 54.

[0392] In some embodiments, the anti-EpCAM scFv is encoded by SEQ ID NO: 240.

[0393] In some embodiments, the tumor targeting molecule does not comprise a constant heavy chain and / or a constant light chain domain.

[0394] In some embodiments, the tumor targeting molecule is adecatumumab or an antigenbinding fragment of adecatumumab. In some embodiments, the tumor targeting molecule is a scFv of adecatumumab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of adecatumumab. In some embodiments, the tumor targeting molecule is edrecolomab or an antigen-binding fragment of edrecolomab. In some embodiments, the tumor targeting molecule is a scFv of edrecolomab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of edrecolomab. In some embodiments, the tumor targeting molecule is tucotuzumab or an antigen-binding fragment of tucotuzumab. In some embodiments, the tumor targeting molecule is a scFv of tucotuzumab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of tucotuzumab.

[0395] 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: 56 (encoded by SEQ ID NO: 90), 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 VL set forth in SEQ ID NO: 55 (encoded by SEQ ID NO: 89). 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 comprising the amino acid sequence set forth in SEQ ID NOs: 92, 93, 94, 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: 95, 96 and 97, respectively.

[0396] In some embodiments, the tumor targeting molecule comprises a variable light chain region comprising an amino acid sequence of SEQ ID NO: 55. In some embodiments, the tumor targeting molecule comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain 91MF-364447969307612002240comprising 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: 55. In some embodiments, the tumor targeting molecule comprises a variable 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: 56.

[0397] In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 56. In some embodiments, the 92MF-364447969307612002240tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 56. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 55 and a variable heavy chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 56. In some of any such embodiments, the variable light chain and variable heavy chain are linked by a peptide linker, such as by the sequence set forth as GGGGSGGGGSGGGGS (SEQ ID NO: 88).

[0398] In some embodiments, the tumor targeting molecule binds to a tumor associated antigen that is HER2. In some embodiments, the tumor targeting molecule is an antibody or antigen-binding fragment thereof that binds to a tumor associated antigen that is HER2. In some embodiments, the tumor targeting molecule is a single-chain Fv (scFv) of an anti-HER2 antibody. In some embodiments, the tumor targeting molecule does not comprise a constant heavy chain and / or a constant light chain domain.

[0399] In some embodiments, the tumor targeting molecule is derived from the 4D5 anti-HER2 antibody (also known as Abl), which is the parental antibody of trastuzumab. In some embodiments, the tumor targeting molecule is an antibody fragment of the 4D5 (Abl) antibody.

[0400] In some embodiments, the tumor targeting molecule is a single-chain Fvs (scFv) of a 4D5 antibody.

[0401] 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: 50, 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 VL set forth in SEQ ID NO: 48.

[0402] In some embodiments, the tumor targeting molecule comprises a variable light chain region comprising an amino acid sequence of SEQ ID NO: 48. In some embodiments, the tumor targeting molecule comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable 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: 48. In some embodiments, the tumor93MF-364447969307612002240targeting molecule comprises a variable 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: 50.

[0403] In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 48 and a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 48 and a variable heavy chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 48 and a variable heavy chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 48 and a variable heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 48 and a variable heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 48 and a variable heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 48 and a variable heavy chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 48 and a variable heavy chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 48 and a variable heavy chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 48 and a variable heavy chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 48 and a variable heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 48 and a variable heavy chain comprising an amino acid 94MF-364447969307612002240sequence that is at least 96% identical to SEQ ID NO: 50. In some embodiments, the tumor targeting molecule comprises a variable light ch...

Claims

AMENDED CLAIMSreceived by the International Bureau on 01 August 2026 (01.08.2026) WHAT IS CLAIMED:

1. A tumor-targeting system, comprising:(a) a first composition comprising a vector comprising a polynucleotide encoding (i) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membranetargeting domain and (ii) a C-X-C motif chemokine ligand 9 (CXCL9); and(b) a second composition comprising an immune cell comprising (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR), wherein the CAR binds to the target domain of the mtSCA.

2. The system of claim 1, wherein the vector further encodes (iii) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule.

3. The system of claim 2, wherein the vector encodes one or more copies of the mtSCA and / or ttSCA, each comprising the target domain and the membrane-targeting domain or tumortargeting binding molecule.

4. The system of claim 2 or claim 3, wherein the target domain of the mtSCA and the ttSCA comprise the same epitope for a cognate binder.

5. The system of any one of claims 2-4, wherein the target domain of the mtSCA and the ttSCA are the same.

6. A tumor-targeting system, comprising:(a) a first composition comprising a vector comprising a polynucleotide encoding (i) a tumortargeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule and (ii) a C-X-C motif chemokine ligand 9 (CXCL9); and(b) a second composition comprising an immune cell comprising (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR), wherein the CAR binds to the target domain of the ttSCA.

7. The system of claim 6, wherein the vector further encodes (iii) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain.

8. The system of claim 7, wherein the vector encodes one or more copies of the mtSCA and / or ttSCA, each comprising the target domain and the membrane-targeting domain or tumortargeting binding molecule.

9. The system of claim 7 or claim 8, wherein the target domain of the mtSCA and the ttSCA comprise the same epitope for a cognate binder.

10. The system of any one of claims 7-9, wherein the target domain of the mtSCA and the ttSCA are the same.

11. A tumor-targeting system, comprising:(a) a first composition comprising a vector comprising a polynucleotide encoding (i) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain, (ii) a C-X-C motif chemokine ligand 9 (CXCL9), and (iii) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule; and(b) a second composition comprising an immune cell comprising (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR), wherein the CAR binds to the target domain of the synthetic cancer antigen.

12. The system of claim 11, wherein the vector encodes one or more copies of the mtSCA and / or ttSCA, each comprising the target domain and the membrane-targeting domain or the tumor-targeting binding molecule.

13. The system of claim 11 or claim 12, wherein the target domain of the mtSCA and the ttSCA comprise the same epitope for a cognate binder.

14. The system of any one of claims 11-13, wherein the target domain of the mtSCA and the ttSCA are the same.

15. The system of any one of claims 1-14, wherein the heterologous cytokine receptor comprises a synthetic cytokine receptor comprising an extracellular domain, a transmembrane domain, and an interleukin-9 receptor (IL-9R) intracellular domain capable of IL-9R signaling.

16. The system of any one of claims 1-15, wherein the heterologous cytokine comprises interleukin- 18 (IL- 18).

17. The system of any one of claims 1-5, wherein the mtSCA and CXCL9 are separated by a nucleotide sequence encoding a cleavable linker.

18. The system of any one of claims 6-10, wherein the ttSCA and CXCL9 are separated by a nucleotide sequence encoding a cleavable linker.

19. The system of any one of claims 11-14, wherein the mtSCA, ttSCA and CXCL9 are separated from each other by a nucleotide sequence encoding a cleavable linker.

20. The system of claim 19, wherein the cleavable linker is a self-cleaving linker.

21. The system of claim 20, wherein the self-cleaving linker is or comprises a 2 A peptide.

22. The system of any one of claims 19-21, wherein the cleavable linker is P2A or T2A.

23. The system of any one of claims 1-22, wherein the target domain is recognizable by a cognate binder.

24. The system of claim 23, wherein the target domain is an antibody or antibody fragment that is recognized by the cognate binder.

25. The system of claim 23 or claim 24, wherein the cognate binder binds to the idiotype of the antibody or antibody fragment.

26. The system of any one of claims 23-25, wherein the cognate binder is an anti-idiotype antibody or an antigen binding fragment.

27. The system of any one of claims 23-26, wherein the cognate binder is the extracellular domain of a chimeric antigen receptor (CAR).

28. The system of claim 27, wherein the extracellular domain of the CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 68.

29. The system of claim 27 or claim 28, wherein the extracellular domain of the CAR comprises an amino acid sequence of SEQ ID NO: 68.

30. The system of any one of claims 27-29, wherein the CAR further comprises a transmembrane domain and an intracellular signaling domain.

31. The system of claim 30, wherein the transmembrane domain of the CAR is a CD8 transmembrane domain.

32. The system of claim 30 or claim 31, wherein the transmembrane domain of the CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 52.

33. The system of any one of claims 30-32, wherein the transmembrane domain of the CAR comprises the amino acid sequence of SEQ ID NO: 52.

34. The system of any one of claims 30-33, wherein the intracellular signaling domain comprises a primary intracellular signaling domain.

35. The system of any one of claims 30-34, wherein the intracellular signaling domain further comprises a co-stimulatory signaling domain.

36. The system of any one of claims 30-35, wherein the intracellular signaling domain comprises a CD3z cytoplasmic signaling domain and a 41BB co-stimulatory signaling domain.

37. The system of any one of claims 30-36, wherein the intracellular signaling domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 70.

38. The system of any one of claims 30-37, wherein the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 70.

39. The system of any one of claims 1-38, wherein the CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 29.

40. The system of any one of claims 1-39, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 29.

41. The system of any one of claims 24-40, wherein the antibody or antibody fragment target domain does not contain all or a portion of the heavy chain constant region, optionally does not contain the CHI, CH2 and / or CH3 domain, more optionally wherein the antibody or antibody fragment target domain does not contain the heavy chain CH3 domain.

42. The system of any one of claims 1-41, wherein the target domain is derived from a 4D5 anti-HER2 antibody.

43. The system of any one of claims 1-42, wherein the target domain comprises a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 7.

44. The system of any one of claims 1-43, wherein the target domain comprises a light chain comprising an amino acid sequence of SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 7.

45. The system of any one of claims 1-44, wherein the target domain is a single-chain variable fragment (scFv).

46. The system of claim 45, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 53.

47. The system of claim 45 or claim 46, wherein the target domain comprises an amino acid sequence of SEQ ID NO: 53.

48. The system of any one of claims 1-44, wherein the target domain is the variable domain of the heavy chain of an antibody.

49. The system of claim 48, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 50.

50. The system of claim 48 or claim 49, wherein the target domain comprises an amino acid sequence of SEQ ID NO: 50.

51. The system of any one of claims 1-5 and 7-50, wherein the membrane-targeting domain is a transmembrane domain.

52. The system of claim 51, wherein the transmembrane domain is from a transmembrane glycoprotein.

53. The system of claim 51 or claim 52, wherein the transmembrane domain is a CD8 transmembrane domain.

54. The system of any one of claims 51-53, wherein the transmembrane domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 52.

55. The system of any one of claims 51-54, wherein the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 52.

56. The system of any one of claims 51-55, wherein the transmembrane domain is encoded by a nucleic acid sequence of SEQ ID NO: 302.

57. The system of any one of claims 2-56, wherein the tumor-targeting binding molecule specifically binds to a tumor associated antigen expressed on the surface of a tumor cell.

58. The system of claim 57, 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), Claudin6, Claudinl82, GPC2, GPC3.

59. The system of claim 57 or claim 58 wherein the tumor cell is a tumor cell of a solid tumor.

60. The system of claim 59, wherein the tumor cell is a tumor cell of a solid tumor originating from epithelial tissue.

61. The system of claim 59 or claim 60, wherein the tumor cell is a tumor cell of a pan-epithelial tumor.

62. The system of any one of claims 59-61, wherein the tumor cell is a tumor cell of a gastrointestinal (GI) tract cancer.

63. The system of claim 62, wherein the tumor cell of the GI tract cancer is a colorectal cancer, an esophageal cancer or a stomach cancer.

64. The system of any one of claims 59-63, 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, or cervical cancer.

65. The system of any one of claims 59-64, 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.

66. The system of any one of claims 59-61, 64 and 65, wherein the tumor cell is a tumor cell of a non-small cell lung cancer (NSCLC).

67. The system of any one of claims 59-61, 64 and 65, wherein the tumor cell is a tumor cell of a prostate cancer.

68. The system of any one of claims 2-67, wherein the ttSCA is soluble.

69. The system of any one of claims 2-68, wherein the target domain and the tumortargeting binding molecule of the ttSCA are linked by a linker.

70. The system of claim 69, 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.I. The system of claim 69 or claim 70, wherein the linker comprises the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 14.

72. The system of any one of claims 69-71, wherein the linker is encoded by a nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 13.

73. The system any one of claims 2-72, wherein the tumor-targeting binding molecule comprises an antibody or an antigen-binding fragment thereof.

74. The system of claim 73, wherein the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

75. The system of any one of claims 57-74, wherein the tumor associated antigen is EpCAM.

76. The system of any one of claims 73-75, 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-H1), a CDR-H2, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NOs: 92, 93 and 94, respectively, andthe 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: 95, 96 and 97, respectively.

77. The system of any one of claims 73-76, wherein the antibody or an antigen-binding fragment thereof comprises a heavy chain variable (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: 55; and a light chain variable (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: 56.

78. The system of any one of claims 73-77, wherein the antibody or an antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 55, and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 56.

79. The system of any one of claims 2-78, wherein the tumor-targeting binding molecule comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 54 or comprises the amino acid sequence of SEQ ID NO: 54.

80. The system of any one of claims 2-79, wherein the tumor-targeting binding molecule is encoded by a nucleic acid sequence of SEQ ID NO: 240.

81. The system of any one of claims 1-80, wherein the CXCL9 comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 16.

82. The system of any one of claims 1-81, wherein the CXCL9 comprises an amino acid sequence of SEQ ID NO: 16.

83. The system of any one of claims 1-82, wherein the CXCL9 is encoded by a nucleic acid sequence of SEQ ID NO: 15.

84. The system of any one of claims 16-83, wherein the IL-18 comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 37.

85. The system of any one of claims 16-84, wherein the IL-18 comprises an amino acid sequence of SEQ ID NO: 37.

86. The system of any one of claims 16-85, wherein the IL-18 is encoded by a nucleic acid sequence of SEQ ID NO: 36.

87. The system of any one of claims 15-86, wherein the intracellular domain capable of IL-9R signaling comprises an IL-9R intracellular domain or a variant thereof.

88. The system of claim 87, wherein the IL-9R intracellular domain capable of IL-9R signaling comprises a chimeric JAK7STAT fusion domain.

89. The system of any one of claims 15-88, wherein the synthetic cytokine receptor is a constitutively active cytokine receptor.

90. The system of any one of claims 15-89, wherein the synthetic cytokine receptor is a multimer.

91. The system of any one of claims 15-90, wherein the synthetic cytokine receptor is a multimer of identical polypeptide chains each comprising the extracellular domain, the transmembrane domain and the IL-9R intracellular domain or variant thereof.

92. The system of claim 90 or claim 91, wherein the multimer is a dimer.

93. The system of claim 92, wherein the dimer is a homodimer.

94. The system of any one of claims 91-93, wherein each polypeptide chain is constitutively multimerized.

95. The system of any one of claims 15-94, wherein the synthetic cytokine receptor comprises at least one self-assembly domain.

96. The system of claim 95, wherein the at least one self-assembly domain is the extracellular domain and / or the transmembrane domain.

97. The system of any one of claims 15-96, wherein the synthetic cytokine receptor is multimerized through the transmembrane domain and / or the extracellular domain.

98. The system of any one of claims 15-97, wherein the synthetic cytokine receptor is multimerized through the transmembrane domain and the extracellular domain.

99. The system of any one of claims 15-98, wherein the synthetic cytokine receptor is a homodimer of identical polypeptide chains each comprising an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain or a variant thereof.

100. The system of any one of claims 15-99, wherein the extracellular domain and / or the transmembrane domain are heterologous to the IL-9R.

101. The system of any one of claims 15-100, wherein the transmembrane domain and extracellular domain are the transmembrane domain and extracellular domain from the same protein.

102. The system of any one of claims 15-100, wherein the transmembrane domain and extracellular domain are the transmembrane domain and extracellular domain from different proteins.

103. The system of any one of claims 15-102, wherein the transmembrane domain is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 amino acids in length.

104. The system of any one of claims 15-103, wherein the transmembrane domain comprises a transmembrane domain derived from Glycophorin A (GpA) Carnitine palmitoyltransferase 1 (CPT1), a tumor necrosis factor receptor (TNFR), Mucin 24 (Muc24) or Thrombopoietin receptor.

105. The system of any one of claims 15-104, wherein the transmembrane domain promotes alpha-helix dimerization.

106. The system of any one of claims 15-105, wherein the transmembrane domain comprises the motif GXXXG (SEQ ID NO: 241) or LIxxGVxxGVxxT (SEQ ID NO: 242).

107. The system of any one of claims 15-106, wherein the transmembrane domain: (a) is a transmembrane domain derived from Glycophorin A (GpA) or a variant thereof that comprises one or more mutations (e.g, 1, 2, 3, 4, 5 or 6 mutations) compared to a wild-type GpA transmembrane domain, wherein the variant GpA is sufficient to promote alpha-helix dimerization; (b) comprises a transmembrane domain derived from Glycophorin A (GpA); (c) comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 125 or SEQ ID NO: 130; or (d) comprises the amino acid sequence of SEQ ID NO: 125 or SEQ ID NO: 130.

108. The system of any one of claims 15-106, wherein the transmembrane domain comprises: (a) GXXXG (SEQ ID NO: 241) and GXXXA (SEQ ID NO: 243) motifs; (b) a transmembrane domain derived from Carnitine palmitoyltransferase 1 (CPT1); (c) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 132; or (d) the amino acid sequence of SEQ ID NO: 132.

109. The system of any one of claims 15-105, wherein the synthetic cytokine receptor comprises the motif AXXXA (SEQ ID NO: 244), AXXXS (SEQ ID NO: 245), <bPX (SEQ ID NO: 246) or OTXXAO) (SEQ ID NO: 247).

110. The system of any one of claims 15-106, wherein the transmembrane domain is derived from a TNFR, optionally wherein the TNFR is TACI, DR5, p75NTR, Fas, TNFR1, TNFR2 or 0X40.

111. The system of any one of claims 15-106 and 110, wherein the transmembrane domain comprises: (a) a transmembrane domain derived from DR5; (b) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 133; or (c) the amino acid sequence of SEQ ID NO: 133.

112. The system of any one of claims 15-106 and 110, wherein the transmembrane domain comprises: (a) a transmembrane domain derived from TACI; (b) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 131; or (c) the amino acid sequence of SEQ ID NO: 131.

113. The system of any one of claims 15-104, wherein the transmembrane domain comprises 1 to 6 cysteine residues.

114. The system of any one of claims 15-104 and 113, wherein the transmembrane domain promotes disulfide-linked dimerization, optionally wherein the disulfide-linked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor.

115. The system of any one of claims 15-104, 113 and 114, wherein the transmembrane domain is a variant transmembrane domain that comprises one or more mutations compared to a wildtype transmembrane domain to promote homodimerization of the receptor polypeptide, optionally wherein: (a) the one or more mutations promote alpha-helix dimerization or disulfide-linked dimerization; (b) the one or more mutations introduces at least one cysteine into the transmembrane domain; (c) the one or more mutations introduces a proline into the transmembrane domain; (d) the one or more mutations introduces a threonine into the transmembrane domain; (e) the one or more mutations introduces a trimer peptide of cysteine, proline, and another amino acid other than cysteine or proline into the transmembrane domain; or (f) the one or more mutations introduces a trimer peptide of cysteine, proline, threonine (CPT or TCP) into the transmembrane domain.

116. The system of claim 115, wherein the transmembrane domain is a variant IL-7R transmembrane domain and the one or more mutations is in the wild-type transmembrane sequence PILLTISILSFFSVALLVILACVLW (SEQ ID NO: 248 or SEQ ID NO: 280).

117. The system of any one of claims 15-104 and 113-116, wherein the transmembrane domain comprises: (a) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 40, SEQ ID NO: 101 or SEQ ID NO: 249; or (b) the amino acid sequence of SEQ ID NO: 40, SEQ ID NO: 101 or SEQ ID NO: 249.

118. The system of any one of claims 15-104, wherein the transmembrane domain comprises: (a) a transmembrane domain derived from Muc24; (b) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 124; or (c) the amino acid sequence of SEQ ID NO: 124.

119. The system of any one of claims 15-118, wherein the extracellular domain is between about 150 to 260 amino acids in length.

120. The system of any one of claims 15-119, wherein the extracellular domain is a dimerizing domain, optionally wherein the dimerizing domain comprises a hinge region.

121. The system of any one of claims 15-120, wherein the extracellular domain promotes disulfide-linked dimerization.

122. The system of any one of claims 15-121, wherein the extracellular domain comprises 1 to 6 cysteine residues.

123. The system of claim 115 or claim 122, wherein the disulfide-linked dimerization forms 1 to 4 disulfide bridges between polypeptide chains of the synthetic cytokine receptor.

124. The system of any one of claims 15-123, wherein the extracellular domain is derived from the extracellular domain of CD34, DAP12, Glycophorin A, CD8, Muc24 or Thrombopoietin receptor.

125. The system of any one of claims 15-124, wherein the extracellular domain comprises: (a) an extracellular domain of CD8 or a truncated portion thereof comprising at least one cysteine residue; (b) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 52, SEQ ID NO: 178 or SEQ ID NO: 43; or (c) the amino acid sequence of SEQ ID NO: 52, SEQ ID NO: 178 or SEQ ID NO: 43.

126. The system of any one of claims 15-124, wherein the extracellular domain comprises: (a) an extracellular domain of CD34 or a truncated portion thereof comprising at least one cysteine residue; (b) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 39; or (c) the extracellular domain comprises the amino acid sequence of SEQ ID NO: 39.

127. The system of any one of claims 15-124, wherein the extracellular domain: (a) is an extracellular domain of Muc24 or a truncated portion thereof comprising at least one cysteine residue; (b) comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 113; or (c) comprises the amino acid sequence of SEQ ID NO: 113.

128. The system of any one of claims 15-124, wherein the extracellular domain: (a) is an extracellular domain of DAP12 or a truncated portion thereof comprising at least one cysteine residue; (b) comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 100; or (c) comprises the amino acid sequence of SEQ ID NO: 100.

129. The system of any one of claims 15-124, wherein the extracellular domain: (a) is an extracellular domain of Glycophorin A (GpA) or a truncated portion thereof comprising at least one cysteine residue; (b) comprises an amino acid sequence that is at least about 85% identical to SEQ IDNO: 111 or SEQ ID NO: 112; or (c) comprises the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO: 112.

130. The system of any one of claims 15-129, wherein the IL-9R intracellular domain or variant thereof is about 100 to 260 amino acids in length, optionally wherein the IL-9R intracellular domain or variant thereof is 230 amino acids in length.

131. The system of any one of claims 15-130, wherein the IL-9R intracellular domain or variant thereof comprises a BOX1 motif and / or a BOX2 motif, optionally wherein the IL-9R intracellular domain or variant thereof comprises a BOX2 motif.

132. The system of any one of claims 15-131, wherein the IL-9R intracellular domain or variant thereof is wild-type IL-9R intracellular domain or a variant thereof that comprises one or more mutations compared to the wild-type IL-9R intracellular domain set forth in SEQ ID NO: 102, optionally wherein the one or more mutations comprises one or more amino acid insertions, deletions, and / or substitutions.

133. The system of claim 132, wherein the one or more mutations promote signaling through STAT1, STAT3, and / or STAT5 pathways.

134. The system of any one of claims 15-133, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 33, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 148, SEQ ID NO: 156, SEQ ID NO: 163, SEQ ID NO: 166, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 108, SEQ ID NO: 109 or SEQ ID NO: 110.

135. The system of any one of claims 87-134, wherein the IL-9R intracellular domain or variant thereof comprises an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 33, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 148, SEQ ID NO: 156, SEQ ID NO: 163, SEQ ID NO: 166, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 108, SEQ ID NO: 109 or SEQ ID NO: 110.

136. The system of any one of claims 15-135, wherein the IL-9R intracellular domain or variant thereof comprises one or more amino acid deletions with reference to wild-type IL-9R intracellular domain (SEQ ID NO: 102).

137. The system of any one of claims 15-136, wherein the IL-9R intracellular domain or variant thereof is a truncated IL-9R that lacks a contiguous sequence of amino acids at the C-terminus of wild-type IL-9R intracellular domain, optionally wherein the truncated IL-9R intracellular domain or variant thereof is truncated by between 62 and 99 contiguous amino acids from the C-terminus of wild-type IL-9R intracellular domain.

138. The system of any one of claims 15-137, wherein the IL-9R intracellular domain or variant thereof: (a) is a truncated IL-9R that lacks amino acids 132 to 230 of SEQ ID NO: 102 or lacks amino acids 134 to 230 of SEQ ID NO: 102; (b) comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 156, SEQ ID NO: 167, or SEQ ID NO: 168; (c) comprises an amino acid sequence of SEQ ID NO: 156, SEQ ID NO: 167, or SEQ ID NO: 168.

139. The system of any one of claims 15-138, wherein the synthetic cytokine receptor comprises: (a) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187; or (b) an amino acid sequence of SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, or SEQ ID NO: 187.

140. The system of any one of claims 15-135, wherein the IL-9R intracellular domain or variant thereof comprises one or more amino acid substitutions with reference to wild-type IL-9R intracellular domain SEQ ID NO: 102.

141. The system of claim 140, wherein the IL-9R intracellular domain or variant thereof comprises a STAT binding motif or a variant thereof, optionally wherein the STAT binding motif comprises: (a) a STAT1, STAT3, and / or STAT5 binding motif; (b) YLPQ (SEQ ID NO: 250); (c) a variant STAT binding motif; (d) YRPQ (SEQ ID NO: 251); (e) YLPL (SEQ ID NO: 252); or (f) YLKQ (SEQ ID NO: 253).

142. The system of any one of claims 15-135, 140 and 141, wherein the variant IL-9R intracellular domain or variant thereof comprises: (a) an amino acid sequence that is at least about 85% identical to SEQ ID NO: 171 or SEQ ID NO: 172, or SEQ ID NO: 173; or (b) an amino acid sequence of SEQ ID NO: 171 or SEQ ID NO: 172, or SEQ ID NO: 173.

143. The system of any one of claims 88-142, wherein the chimeric JAK7STAT fusion domain comprises a JAK binding domain from a type I cytokine receptor and a STAT binding domain from an IL-9R intracellular domain.

144. The system of claim 143, wherein the IL-9R STAT binding domain comprises amino acid residues 73 to 230 of SEQ ID NO: 102.

145. The system of claim 143 or claim 144, wherein the STAT binding domain is 59 to 158 amino acids in length and comprises an IL-9R STAT binding motif.

146. The system of claim 145, wherein the IL-9R STAT binding motif comprises YLPQ (SEQ ID NO: 250).391147. The system of any one of claims 143-146, wherein the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the N-terminus of SEQ ID NO: 102.

148. The system of any one of claims 143-147, wherein the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks a contiguous sequence of amino acids at the C-terminus of SEQ ID NO: 102.

149. The system of any one of claims 143-148, wherein the IL-9R STAT binding domain is a truncated IL-9R STAT binding domain that lacks amino acids at positions 1 to 72 and / or 132 to 230 of SEQ ID NO: 102.

150. The system of any one of claims 143-149, wherein the IL-9R STAT binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 282, or SEQ ID NO: 283.

151. The system of any one of claims 143-150, wherein the IL-9R STAT binding domain comprises an amino acid sequence of SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 282, or SEQ ID NO: 283.

152. The system of any one of claims 143-151, wherein the type I cytokine receptor is selected from the group consisting of interleukin 2 receptor (IL-2R), interleukin 4 receptor (IL-4R), interleukin 7 receptor (IL-7R), interleukin 13 receptor (IL-13R), interleukin 15 receptor (IL-15R), and interleukin 2 receptor (IL-21R), optionally wherein the type I cytokine receptor is IL-7R.

153. The system of any one of claims 143-152, wherein the IL-7R JAK binding domain is 65 amino acids in length and comprises a BOX1 motif.

154. The system of any one of claims 143-153, wherein the IL-7R JAK binding domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 256 or SEQ ID NO: 285, or comprises an amino acid sequence of SEQ ID NO: 256 or SEQ ID NO: 285.

155. The system of any one of claims 88-154, wherein the chimeric JAK7STAT fusion domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 41, SEQ ID NO: 181 or SEQ ID NO: 45 or comprises an amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 181, or SEQ ID NO: 45.

156. The system of any one of claims 1-138 and 140-155, wherein the synthetic cytokine receptor comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 31, SEQ ID NO: 239, or SEQ ID NO: 188 or an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 239, or SEQ ID NO: 188.

157. The system of any one of claims 15-156, wherein the synthetic cytokine receptor elicits signaling through STAT1, STAT3, and / or STAT5 pathways.

158. The system of claim 157, wherein signaling through STAT1, STAT3, and / or STAT5 is: (a) increased compared to STAT1, STAT3, and / or STAT5 signaling via wild-type IL-9R; (b)392sustained for a longer period of time compared to STAT1, STAT3, and / or STAT5 signaling via wildtype IL-9R, optionally wherein sustained STAT1, STAT3, and / or STAT5 signaling is determined by phosphorylation status of STAT1, STAT3, and / or STAT5.

159. The system of any one of claims 1-158, wherein the first and second compositions further comprise a pharmaceutically acceptable carrier.

160. The system of any one of claims 1-159, wherein the first and second compositions are for delivery, specifically to a subject.

161. The system of claim 160, wherein the subject has a cancer or tumor.

162. The system of claim 161, wherein the cancer or tumor is 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, stomach cancer, thyroid cancer, bile duct cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, or cervical cancer.

163. The system of any one of claims 1-162, wherein the immune cell is a lymphocyte.

164. The system of any one of claims 1-163, wherein the immune cell is an immune effector cell.

165. The system of any one of claims 1-164, wherein the immune cell is a T cell or a Natural Killer (NK) cell.

166. The system of any one of claims 1-164, wherein the immune cell is a T cell.

167. The system of any one of claims 1-166, wherein the immune cell is a CD4+ T cell or a CD8+ T cell.

168. The system of any one of claims 1-165 wherein the immune cell is a Natural Killer (NK) cell.

169. The system of any one of claims 1-168, wherein the vector is a viral vector.

170. The system of claim 169, wherein the vector exhibits tropism to a tumor cell.

171. The system of any one of claims 1-170, wherein the vector is a tumor tropic viral vector, optionally an oncolytic virus.

172. The system of claim 171, wherein the tumor tropic viral vector 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 rhabdovirus, such as maraba virus.

173. The system of claim 171 or claim 172, wherein the tumor tropic viral vector enters a cell by binding to a cell surface receptor expressed by the cancer cell or the solid tumor cancer cell.393174. The system of any one of claims 171-173, wherein the tumor tropic 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.

175. The system of any one of claims 171-174, wherein the tumor tropic viral vector is an adenoviral vector.

176. The system of claim 175, wherein the adenoviral vector is an adenoviral vector that binds to CD46 and / or desmoglein-2.

177. The system of claim 175 or claim 176, wherein the adenoviral vector is Ad3, Ad7, Adil, Adl4, Adl6, Adl7, Ad21, Ad35, Ad47, or Ad50.

178. The system of any one of claims 175-177, wherein the adenoviral vector is a chimeric adenoviral vector based on Ad3, Ad7, Adil, Adl4, Adl6, Adl7, Ad21, Ad35, Ad47, or Ad50.

179. The system of any one of claims 175-178, wherein the adenoviral vector is a chimeric adenoviral vector that is based on Adil.

180. The system of any one of claims 175-179, wherein the adenoviral vector is a chimeric Ad3 / 1 Ip adenoviral vector, optionally Enadenotucirev.

181. The system of claim 180, wherein the chimeric Ad3 / 1 Ip adenoviral vector binds to CD46.

182. The system of claim 180 or claim 181, wherein the chimeric Ad3 / 1 Ip adenoviral vector comprises a nucleic acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 257 or SEQ ID NO: 335, optionally wherein the nucleic acid sequence is set forth in SEQ ID NO: 257 or SEQ ID NO: 335.

183. A polynucleotide comprising a nucleic acid sequence encoding (i) a membranetargeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain and (ii) a C-X-C motif chemokine ligand 9 (CXCL9).

184. The polynucleotide of claim 183, wherein the polynucleotide further comprises a nucleic acid sequence encoding (iii) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule.

185. The polynucleotide of claim 184, wherein the nucleic acid sequence encodes one or more copies of the mtSCA and / or ttSCA, each comprising the target domain and the membranetargeting domain or tumor-targeting binding molecule.

186. The polynucleotide of any one of claims 183-185, wherein the target domain of the mtSCA and the ttSCA comprises the same epitope for a cognate binder.

187. The polynucleotide of any one of claims 183-186, wherein the target domain of the mtSCA and the ttSCA are the same.394188. A polynucleotide comprising a nucleic acid sequence encoding (i) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule and (ii) a C-X-C motif chemokine ligand 9 (CXCL9).

189. The polynucleotide of claim 188, wherein the polynucleotide further comprises a nucleic acid sequence encoding (iii) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain.

190. The polynucleotide of claim 188 or claim 189, wherein the nucleic acid sequence encodes one or more copies of the mtSCA and / or ttSCA, each comprising the target domain and the membrane-targeting domain or tumor-targeting binding molecule.

191. The polynucleotide of any one of claims 188-190, wherein the target domain of the mtSCA and the ttSCA comprises the same epitope for a cognate binder.

192. The polynucleotide of any one of claims 188-191, wherein the target domain of the mtSCA and the ttSCA are the same.

193. A polynucleotide comprising a nucleic acid sequence encoding (i) a membranetargeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain, (ii) a C-X-C motif chemokine ligand 9 (CXCL9), and (iii) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule.

194. The polynucleotide of claim 193, wherein the nucleic acid sequence encodes one or more copies of the mtSCA and / or ttSCA, each comprising the target domain and the membranetargeting domain or tumor-targeting binding molecule.

195. The polynucleotide of claim 193 or claim 194, wherein the target domain of the mtSCA and the ttSCA comprises the same epitope for a cognate binder.

196. The polynucleotide of any one of claims 193-195, wherein the target domain of the mtSCA and the ttSCA are the same.

197. The polynucleotide of any one of claims 183-187, wherein the mtSCA and CXCL9 are separated by a nucleotide sequence encoding a cleavable linker.

198. The polynucleotide of any one of claims 188-192, wherein the ttSCA and CXCL9 are separated by a nucleotide sequence encoding a cleavable linker.

199. The polynucleotide of any one of claims 193-196, wherein the mtSCA, ttSCA and CXCL9 are separated from each other by a nucleotide sequence encoding a cleavable linker.

200. The polynucleotide of any one of claims 197-199, wherein the cleavable linker is a self-cleaving linker.

201. The polynucleotide of claim 200, wherein the self-cleaving linker is or comprises a 2 A peptide.

202. The polynucleotide of any one of claims 197-201, wherein the cleavable linker is P2A or T2A.395203. The polynucleotide of any one of claims 183-202, wherein the target domain comprises a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 7.

204. The polynucleotide of any one of claims 183-203, wherein the target domain comprises a light chain comprising an amino acid sequence of SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 7.

205. The polynucleotide of any one of claims 183-204, wherein the target domain is a single-chain variable fragment (scFv).

206. The polynucleotide of claim 205, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 53.

207. The polynucleotide of claim 205 or claim 206, wherein the target domain comprises an amino acid sequence of SEQ ID NO: 53.

208. The polynucleotide of any one of claims 183-202, wherein the target domain is the variable domain of the heavy chain of an antibody.

209. The polynucleotide of claim 208, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 50.

210. The polynucleotide of claim 208 or claim 209, wherein the target domain comprises an amino acid sequence of SEQ ID NO: 50.

211. The polynucleotide of any one of claims 183-187 and 189-210, wherein the membrane-targeting domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 52.

212. The polynucleotide of any one of claims 183-187 and 189-211, wherein the membrane-targeting domain comprises an amino acid sequence of SEQ ID NO: 52.

213. The polynucleotide of any one of claims 183-187 and 189-212, wherein the membrane-targeting domain is encoded by a nucleic acid sequence of SEQ ID NO: 302.

214. The polynucleotide of any one of claims 184-213, wherein the tumor-targeting binding molecule comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 54 or comprises the amino acid sequence of SEQ ID NO: 54.

215. The polynucleotide of any one of claims 184-214, wherein the tumor-targeting binding molecule is encoded by a nucleic acid sequence of SEQ ID NO: 240.

216. The polynucleotide of any one of claims 183-215, wherein the polynucleotide comprises a nucleic 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 NOs: 2, 11, 18, 22 or 25.396217. The polynucleotide of any one of claims 183-216, wherein the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NOs: 2, 11, 18, 22 or 25.

218. A polynucleotide comprising a nucleic acid sequence encoding (i) a synthetic cytokine receptor comprising an extracellular domain, a transmembrane domain, and an interleukin 9 receptor (IL-9R) intracellular domain and (ii) a chimeric antigen receptor (CAR).

219. The polynucleotide of claim 218, wherein the synthetic cytokine receptor and CAR are separated by a nucleotide sequence encoding a cleavable linker.

220. The polynucleotide of claim 219, wherein the cleavable linker is a self-cleaving linker.

221. The polynucleotide of claim 219 or claim 220, wherein the self-cleaving linker is or comprises a 2 A peptide.

222. The polynucleotide of any one of claims 219-221, wherein the linker is P2A.

223. A polynucleotide comprising a nucleic acid sequence encoding (i) interleukin- 18 (IL-18) and (ii) a chimeric antigen receptor (CAR).

224. The polynucleotide of claim 223, wherein the IL- 18 and CAR are separated by a nucleotide sequence encoding a cleavable linker.

225. The polynucleotide of claim 224, wherein the cleavable linker is a self-cleaving linker.

226. The polynucleotide of claim 224 or claim 225, wherein the self-cleaving linker is or comprises a 2 A peptide.

227. The polynucleotide of any one of claims 224-226, wherein the linker is P2A.

228. The polynucleotide of any one of claims 218-227, wherein a target domain is recognizable by the CAR.

229. The polynucleotide of any one of claims 218-228, wherein the synthetic cytokine receptor is a constitutively active synthetic cytokine receptor.

230. The polynucleotide of any one of claims 218-229, wherein the synthetic cytokine receptor comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 33, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 148, SEQ ID NO: 156, SEQ ID NO: 163, SEQ ID NO: 166, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 108, SEQ ID NO: 109 or SEQ ID NO: 110.

231. The polynucleotide of any one of claims 218-230, wherein the synthetic cytokine receptor comprises an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 33,397SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 148, SEQ ID NO: 156, SEQ ID NO: 163, SEQ ID NO: 166, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 108, SEQ ID NO: 109 or SEQ ID NO: 110.

232. The polynucleotide of any one of claims 223-231, wherein the IL- 18 comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 37.

233. The polynucleotide of any one of claims 223-232, wherein the IL-18 comprises an amino acid sequence of SEQ ID NO: 37.

234. The polynucleotide of any one of claims 223-233, wherein the IL-18 is encoded by a nucleic acid sequence of SEQ ID NO: 36.

235. The polynucleotide of any one of claims 218-234, wherein the CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 29.

236. The polynucleotide of any one of claims 218-235, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 29.

237. The polynucleotide of any one of claims 218-236, where the CAR is encoded by a nucleic acid sequence of SEQ ID NO: 28.

238. The polynucleotide of any one of claims 183-237, wherein the polynucleotide comprises a nucleic 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 NOs: 2, 11, 18, 22 or 25.

239. The polynucleotide of any one of claims 183-238, wherein the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NOs: 2, 11, 18, 22 or 25.

240. The polynucleotide of any one of claims 183-237, wherein the polynucleotide comprises a nucleic 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 NOs: 42, 44, 46, or 64.

241. The polynucleotide of any one of claims 183-237 and 240, wherein the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NOs: 42, 44, 46, or 64.

242. A vector, comprising the polynucleotide of any one of claims 218-241.

243. The vector of claim 242, wherein the vector is for delivery specifically to an immune cell.

244. The vector of claim 243, wherein the immune cell is an immune effector cell.

245. The vector of claim 244, wherein the immune effector cell is a T cell, optionally a cytotoxic T cell.398246. The vector of claim 244 or claim 245, wherein the immune effector cell is a CD8+ T cell and / or CD4+ T cell.

247. The vector of claim 244, wherein the immune effector cell is a Natural Killer (NK) cell.

248. The vector of any one of claims 242-247, wherein the vector is a lentiviral vector.

249. A vector, comprising the polynucleotide of any one of claims 183-241.

250. The vector of claim 249, wherein the vector is for delivery specifically to a cancer cell.

251. The vector of claim 250, wherein the vector is for delivery specifically to a blood cancer cell.

252. The vector of claim 250, wherein the vector is for delivery specifically to a solid tumor cancer cell.

253. The vector of any one of claims 249-252, wherein the vector is a viral vector.

254. The vector of any one of claims 249-253, wherein the viral vector is a tumor tropic viral vector, optionally an oncolytic virus.

255. The vector of claim 253 or claim 254, wherein the viral vector 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 rhabdovirus, such as maraba virus.

256. The vector of any one of claims 253-255, wherein the viral vector exhibits tropism to a tumor cell.

257. The vector of any one of claims 253-256, wherein the viral vector enters a cell by binding to a cell surface receptor expressed by a tumor cell.

258. The vector of any one of claims 253-257, 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.

259. The vector of any one of claims 253-258, wherein the viral vector is an adenoviral vector.

260. The vector of claim 259, wherein the adenoviral vector is an adenoviral vector that binds to CD46 and / or desmoglein-2.

261. A cell comprising the polynucleotide of any one of claims 183-241 or the vector of any one of claims 242-260.

262. A pharmaceutical composition comprising the vector of any one of claims 242-260 and the cell of claim 261.399263. The pharmaceutical composition of claim 262, wherein the pharmaceutical composition further comprises a pharmaceutical acceptable carrier.

264. The pharmaceutical composition of claim 262 or claim 263, for use in treating a cancer in a subject.

265. A tumor-targeting system comprising a vector of any one of claims 242-260 and the cell of claim 261.

266. A method of tagging a tumor cell in vivo, comprising contacting the tumor cell with the polynucleotide of any one of claims 183-241, the vector of any one of claims 242-260, or the pharmaceutical composition of any one of claims 262-264.

267. 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 183-241, the vector of any one of claims 242-260, or the pharmaceutical composition of any one of claims 262-264, to the subject.

268. A method of treating a cancer in a subject, comprising administering to a subject the first composition and the second composition of the system of any one of claims 1-182, wherein the vector causes the synthetic cancer antigen to be expressed on the surface of or secreted by a cell of the cancer and the CAR of the immune cell binds to the target domain of the synthetic cancer antigen.

269. A method of treating a cancer in a subject, comprising administering:(a) a first composition comprising a vector encoding (i) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain and (ii) a C-X-C motif chemokine ligand 9 (CXCL9); and(b) a second composition comprising an immune cell expressing (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR),wherein the vector causes the synthetic cancer antigen to be expressed on the surface of or secreted by a cell of the cancer and the CAR of the immune cell binds to the target domain of the synthetic cancer antigen.

270. A method of treating a cancer in a subject, comprising administering:(a) a first composition comprising a vector encoding (i) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule and (ii) a C-X-C motif chemokine ligand 9 (CXCL9); and(b) a second composition comprising an immune cell expressing (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR),wherein the vector causes the synthetic cancer antigen to be expressed on the surface of or secreted by a cell of the cancer and the CAR of the immune cell binds to the target domain of the synthetic cancer antigen.

271. A method of treating a cancer in a subject, comprising administering:400(a) a first composition comprising a vector encoding (i) a membrane-targeting synthetic cancer antigen (mtSCA) comprising a target domain and a membrane-targeting domain, (ii) a C-X-C motif chemokine ligand 9 (CXCL9), and (iii) a tumor-targeting synthetic cancer antigen (ttSCA) comprising a target domain and a tumor-targeting binding molecule; and(b) a second composition comprising an immune cell expressing (i) a heterologous cytokine receptor or heterologous cytokine and (ii) a chimeric antigen receptor (CAR),wherein the vector causes the synthetic cancer antigen to be expressed on the surface of or secreted by a cell of the cancer and the CAR of the immune cell binds to the target domain of the synthetic cancer antigen.

272. The method of any one of claims 269-271, wherein the heterologous cytokine receptor comprises a synthetic cytokine receptor comprising an extracellular domain, a transmembrane domain, and an interleukin-9 receptor (IL-9R) intracellular domain.

273. The methodof any one of claims 269-272, wherein the heterologous cytokine comprises interleukin- 18 (IL- 18).

274. The method of any one of claims 269-273, wherein the first composition and the second composition are administered concurrently to the subject.

275. The method of any one of claims 269-274, wherein the first composition and the second composition are administered sequentially to the subject.

276. The method of any one of claims 269-273 and 275, wherein the first composition is administered prior to administration of the second composition.

277. The method of any one of claims 269-276, wherein the first composition is administered to the subject one or more times.

278. The method of claim 277, wherein the first composition is administered to the subject a plurality of times.

279. The method of claim 277 or claim 278, wherein the first composition is administered three times.

280. The method of any one of claims 277-279, wherein the first composition is administered three times, optionally wherein the three administrations occur every other day.

281. The method of any one of claims 277-280, wherein each administration of the first composition comprises an effective dose of the vector.

282. The method of any one of claims 277-281, wherein the first composition comprises a first effective dose, a second effective dose and a third effective dose.

283. The method of claim 281 or claim 282, wherein each effective dose is the same across administrations.

284. The method of claim 281 or claim 282, wherein each effective dose is different across administrations.401285. The method of claim 282 or claim 284, wherein the second and third effective doses are higher than the first effective dose.

286. The method of any one of claims 282, 284 and 285, wherein:(a) the first effective dose contains 1 x 1012virus particles (vp), the second effective dose contains 3 x 1012vp, and the third effective dose contains 3 x 1012vp; or(b) the first effective dose contains 1 x 1012vp, the second effective dose contains 6 x 1012vp, and the third effective dose contains 6 x 1012vp.

287. The method of any one of claims 268-286, further comprising administering a lymphodepleting therapy to the subject.

288. The method of claim 287, wherein the lymphodepleting therapy is administered to the subject prior to administration of the second composition.

289. The method of claim 287 or claim 288, wherein the lymphodepleting therapy is administered after the first composition is administered and before administration of the second composition.

290. The method of any one of claims 287-289, wherein the lymphodepleting therapy is administered 2-7 days before administration of the second composition.

291. The method of any one of claims 287-290, wherein the lymphodepleting therapy comprises fludarabine and cyclophosphamide.

292. The method of any one of claims 268-291, wherein prior to administration of the first composition, an apheresis sample is obtained from the subject, to prepare the CAR-expressing immune cells of the second composition.

293. The method of claim 292, wherein the apheresis sample is a leukapheresis sample.

294. The method of any one of claims 268-293, wherein the cancer is a blood cancer.

295. The method of any one of claims 268-293, wherein the cancer is a solid tumor cancer.

296. The method of claim 295, wherein the solid tumor cancer originates from epithelial tissue.

297. The method of claim 295 or claim 296, wherein the solid tumor cancer is a pan-epithelial solid tumor cancer.

298. The method of any one of claims 295-297, wherein the solid tumor cancer is a gastrointestinal (GI) tract cancer.

299. The method of claim 298, wherein the GI tract cancer is a colorectal cancer, an esophageal cancer or a stomach cancer.

300. The method of any one of claims 295-297, wherein the solid tumor cancer is 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,402mesothelioma, 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.

301. The method of any one of claims 295-297, wherein the solid tumor cancer is 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.

302. The method of any one of claims 295-297, 300 and 301, wherein the solid tumor cancer is a non-small cell lung cancer (NSCLC).

303. The method of any one of claims 295-297, 300 and 301, wherein the solid tumor cancer is a prostate cancer.403