Viral vectors for expression of synthetic cancer antigens and chemokine and related methods and uses

Abstract

The present disclosure generally relates to a viral vector carrying a synthetic cancer antigen and a chemokine. Also provided herein are compositions and uses of the viral vector for delivering, such as tagging, a tumor with the synthetic cancer antigen.

Description

307612002440VIRAL VECTORS FOR EXPRESSION OF SYNTHETIC CANCER ANTIGENS AND CHEMOKINE AND RELATED METHODS AND USESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Patent Application No. 63 / 726,230 filed on November 27, 2024, entitled “VIRAL VECTORS FOR EXPRESSION OF SYNTHETIC CANCER ANTIGENS AND CHEMOKINE AND RELATED METHODS AND USES”, the content of which is incorporated by reference in its 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 307612002440SeqList. XML created November 26, 2025, which is 415,919 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 viral vector, e.g., an adenoviral vector, carrying a synthetic cancer antigen. In some embodiments, the viral vector further comprises a chemokine.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) l4i6):726M-5; Tahmasebi et al., Stem Cell Rev Rep. (2019) 15(5):619— 36). Improved 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 adenovirus comprising a sequence of formula [I]: 5’ ITR-BI-BA-B2-BX-BB-BY-B3-3’ ITR (I) wherein: Bi is bond or comprises: E1A, E1B or E1A-E1B; BA comprises-E2B-L1-L2-L3-E2A-L4; B2 is a bond or comprises: E3; Bx is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both; BB comprises L5; By is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both; B3 is a bond or comprises: E4; wherein the adenovirus comprises (i) a first transgene sequence encoding a synthetic cancer antigen307612002440comprising a target domain and a membrane targeting domain or a synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule and (ii) a second transgene sequence encoding CXCL9.

[0006] In some of any of the embodiments, the first transgene sequence and the second transgene sequence are independently encoded in a region selected from El, E3, Bx, By and combinations thereof. In some of any of the embodiments, the first transgene sequence and the second transgene sequence are encoded in position By.

[0007] Provided herein is a adenovirus comprising a polynucleotide located between the virus fibre gene L5 and the virus E4 gene, wherein the polynucleotide comprises (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a membrane targeting domain and (ii) a second transgene sequence encoding CXCL9. In some of any of the embodiments, the first transgene sequence and the second transgene sequence are encoded from the same transgene cassette. In some of any of the embodiments, the first transgene sequence and the second transgene sequence are separated by a nucleotide sequence encoding a cleavable linker or translational skipping sequence in the transgene cassette. In some of any of the embodiments, the adenovirus comprises the first transgene sequence encoding the synthetic cancer antigen comprising the target domain and the membrane targeting domain and (ii) a second transgene sequence encoding CXCL9. In some of any of the embodiments, the adenovirus comprises the first transgene sequence encoding the synthetic cancer antigen comprising the target domain and the tumor-targeting binding molecule and (ii) a second transgene sequence encoding CXCL9. In some of any of the embodiments, the first transgene sequence and the second transgene sequence are independently encoded in a region selected from El, E3, Bx, By and combinations thereof. In some of any of the embodiments, the first transgene sequence and the second transgene sequence are encoded in position BY.

[0008] Provided herein is an adenovirus comprising a polynucleotide located between the virus fibre gene L5 and the virus E4 gene, wherein the polynucleotide comprises (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule and (ii) a second transgene sequence encoding CXCL9. In some of any of the embodiments, the first transgene sequence and the second transgene sequence are encoded from the same transgene cassette. In some of any of the embodiments, the first transgene sequence and the second transgene sequence are separated by a nucleotide sequence encoding a cleavable linker or translational skipping sequence in the transgene cassette. In some of any of the embodiments, the cleavable linker or translational skipping sequence is a self-cleaving linker. In some of any of the embodiments, the self-cleaving linker is or comprises a 2A peptide.

[0009] Provided herein is an adenovirus comprising a sequence of formula [I]: 5’ ITR-B1-BA-B2-BX-BB-BY-B3-3’ ITR (I) wherein: Bi is bond or comprises: E1A, E1B or E1A-E1B; BA comprises-E2B-L1-L2-L3-E2A-L4; B2 is a bond or comprises: E3; Bx is a bond or a DNA sequence comprising:307612002440a restriction site, one or more transgenes or both; BB comprises L5; BY is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both; B3 is a bond or comprises: E4; wherein the adenovirus comprises: (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a membrane targeting domain, (ii) a second transgene sequence encoding a synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule; and (iii) a third transgene sequence encoding CXCL9. In some of any of the embodiments, the first transgene sequence, the second transgene sequence and the third transgene sequence are independently encoded in a region selected from El, E3, BX, BY and combinations thereof. In some of any of the embodiments, the first transgene sequence, the second transgene sequence and the third transgene sequence are encoded in position BY.

[0010] Provided herein is an adenovirus comprising a polynucleotide located between the virus fibre gene L5 and the virus E4 gene, wherein the polynucleotide comprises (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a membrane targeting domain, (ii) a second transgene sequence encoding a synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule; and (iii) a third transgene sequence encoding CXCL9. In some of any of the embodiments, the first transgene sequence, the second transgene sequence and the third transgene sequence are encoded from the same transgene cassette. In some of any of the embodiments, in the transgene cassette, the first and second transgene sequence are separated by a nucleotide sequence encoding a first cleavable linker or translational skipping sequence and the second and third transgene sequence are separated by a nucleotide sequence encoding a second cleavable linker or translational skipping sequence.

[0011] In some of any of the embodiments, the first cleavable linker or translational skipping sequence and the second cleavable linker are different. In some of any of the embodiments, each cleavable linker or translational skipping sequence is a self-cleaving linker. In some of any of the embodiments, the self-cleaving linker independently is or comprises a 2A peptide.

[0012] In some of any of the embodiments, the transgene or transgene cassettes is / are under the control of an endogenous or exogenous promoter. In some of any of the embodiments, the transgene or transgene cassettes is / are under the control of an endogenous promoter that is an E4 promoter or a major late promoter. In some of any of the embodiments, the transgene or transgene cassettes is / are under the control of an exogenous promoter that is a CMV promoter

[0013] In some of any of the embodiments, the adenovirus is replication competent.

[0014] In some of any of the embodiments, the target domain is not expressed on the surface of a non-cancer cell of a subject. In some of any of the embodiments, the target domain is an antibody or antibody fragment that is recognized by a cognate binder. In some of any of the embodiments, the target domain is derived from a 4D5 anti-HER2 antibody. In some of any of the embodiments, the target domain is the variable domain of the heavy chain of an antibody. In some of any of the307612002440embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 28. In some of any of the embodiments, the target domain comprises the amino acid sequence of SEQ ID NO: 28.

[0015] In some of any of the embodiments, the synthetic cancer antigen comprising the target domain and the tumor-targeting binding molecule is soluble. In some of any of the embodiments, the tumor-targeting binding molecule specifically binds to a tumor associated antigen expressed on the surface of a tumor cell. In some of any of the 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 (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.

[0016] In some of any of the embodiments, the tumor cell is a tumor cell of a solid tumor. In some of any of the 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 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.

[0017] In some of any of the embodiments, the tumor-targeting binding molecule comprises an antibody or an antigen-binding fragment thereof. In some of any of the embodiments, the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). In some of any of the embodiments, the tumor associated antigen is EpCAM. In some of any of the 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: 42, 43, and 44, 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: 45, 46, and 47, respectively. In some of any of the embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable307612002440(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: 32; 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: 31. In some of any of the 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: 32, and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 31.

[0018] In some of any of the embodiments, the target domain and the tumor-targeting binding molecule are linked by a linker. In some of any of the 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 embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 33, 38 and 50-69.

[0019] In some of any of the embodiments, the target domain is not expressed on the surface of a non-cancer cell of a subject. In some of any of the embodiments, the target domain is recognizable by a cognate binder. In some of any of the embodiments, the cognate binder binds to the idiotype of the antibody or antibody fragment. In some of any of the embodiments, the cognate binder is an antiidiotype antibody or an antigen binding fragment. In some of any of the embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 71. In some of any of the embodiments, the cognate binder comprises the amino acid sequence of SEQ ID NO:71. In some of any of the embodiments, the cognate binder is encoded by a nucleotide sequence that is at least 85% identical to SEQ ID NO: 70. In some of any of the embodiments, the cognate binder is encoded by the nucleotide sequence of SEQ ID NO:70.

[0020] In some of any of the embodiments, the cognate binder of the synthetic cancer antigen is the extracellular domain of a chimeric antigen receptor (CAR). In some of any of the embodiments, the CAR further comprises a transmembrane domain and an intracellular signaling domain. In some of any of the embodiments, the CAR is expressed on the surface of an immune effector cell.

[0021] In some of any of the embodiments, the immune effector cell is a T cell, optionally a cytotoxic T cell. In some of any of the embodiments, the immune effector cell is a natural killer cell.

[0022] In some of any of the embodiments, the adenovirus comprises a nucleic acid sequence that is at least 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: 1, 9, 16, 20 or 23. In307612002440some of any of the embodiments, the adenovirus comprises the nucleic acid sequence of SEQ ID NOs: 1, 9, 16, 20 or 23.

[0023] Provided herein is a pharmaceutical composition comprising any of the adenovirus vectors provided herein. In some of any of the embodiments, the pharmaceutical composition further comprises a pharmaceutical acceptable carrier. In some of any of the embodiments, the pharmaceutical composition is for use in treating a cancer in a subject.

[0024] Provided herein is a kit comprising any of the pharmaceutical compositions disclosed herein and instructions for using the adenovirus. Provided herein is a method of tagging a tumor cell in vivo, comprising contacting a tumor cell with any of the adenovirus vectors provided herein, or any of the pharmaceutical compositions disclosed herein. In some embodiments, the tumor cell is tagged with a synthetic cancer antigen expressed from the adenovirus vector. 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 adenovirus vectors provided herein, or any of the pharmaceutical compositions disclosed herein, to the subject. In some embodiments, the tumor cell is tagged with a synthetic cancer antigen expressed from the adenovirus vector.

[0025] In some of any of the embodiments, the cancer is a blood cancer. In some of any of the embodiments, the cancer is a solid tumor cancer.

[0026] Provided herein is a method of killing a tumor cell in vivo, comprising administering to a subject having a tumor a therapeutical I y effective amount of any of the adenovirus vectors provided herein, or any of the pharmaceutical compositions provided herein. Provided herein is a method of treating a cancer in a subject, comprising administering to a subject having a cancer a therapeutically effective amount of any of the adenovirus vectors provided herein, or any of the pharmaceutical compositions provided herein.BRIEF DESCRIPTION OF THE FIGURES

[0027] FIG. 1A-1B show oncolytic potency of gene delivery vectors provided herein. FIG. 1A shows oncolytic potency as cell index of non-small cell lung cancer (NSCLC) cell line, A549, across 150 hours. FIG. IB shows a summary of the oncolytic potency of each adenovirus shown in FIG. 1A.

[0028] FIGS. 2A-2C show 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. 2A represents expression as a flow cytometry scatter plot. FIG. 2B represents expression as a histogram. FIG. 2C shows a summary of expression shown in FIGS. 2A-2B.

[0029] FIGS. 3A-3C show expression of a soluble synthetic cancer antigen from non-small cell lung cancer (NSCLC) cell line A549 after infection with the adenoviruses provided herein. FIG. 3A represents expression as a flow cytometry scatter plot. FIG. 3B represents expression as a histogram.FIG. 3C shows a summary of expression shown in FIGS. 3A-3B.307612002440

[0030] FIG. 4 shows expression of CXCL9 secreted from non-small cell lung cancer (NSCLC)cell line A549 after infection with the adenoviruses provided herein.

[0031] FIG. 5 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.

[0032] FIG. 6A show oncolytic potency of gene delivery vectors provided herein.

[0033] FIG. 6B and FIG. 6C show expression of a synthetic cancer antigen. FIG. 6B shows expression of a synthetic cancer antigen comprising a target domain and a membrane targeting domain on the surface of non-small cell lung cancer (NSCLC) cell line A549 after infection with the adenoviruses provided herein. FIG. 6C shows expression of a synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule from A549 after infection with the adenoviruses provided herein.

[0034] FIGS. 7A-7B show tumor killing in mice having non-small cell lung cancer cell (A549) tumors after infection with the adenoviruses provided herein. FIG. 7A shows tumor volume (mm3).FIG. 7B represents tumor volume as area under the curve (AUC).DETAILED DESCRIPTION

[0035] Provided herein is an adenoviral vector, for tagging a cancer, e.g., a solid cancer or tumor. In some embodiments, the adenoviral vector is a tumor-tropic viral vector that is able to specifically target and express an encoded antigen in cancer cells, e.g., solid cancer or tumor cells. In some embodiments, the adenoviral vector is a viral vector described in Section II.

[0036] In particular embodiments, the adenoviral vector encodes a synthetic cancer antigen that is recognized by a cognate binder, e.g., a recombinant receptor such as a chimeric antigen receptor (CAR) expressed by an immune cell, and also encodes CXCL9. In some embodiments, the viral vector, e.g., adenoviral vector, results in the expression of the synthetic cancer antigen on the surface of the cancer, e.g., a solid cancer or tumor, or in the tumor microenvironment, which can then be targeted by the cognate binder.

[0037] 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.

[0038] 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 target domain is linked to a tumor-targeting binding molecule able to bind to a tumor antigen to tag cancer cells. Upon binding of the tumor-targeting binding 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 be307612002440recognized by the cognate binder.

[0039] 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. In some embodiments, 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. B.l.

[0040] In embodiments, the adenoviral vector also encodes a CXCL9 chemokine. In some embodiments, when the adenoviral vector infects the cancer cells, the chemokine is secreted by the cancer cells and attracts the immune cell to the tumor microenvironment. In some embodiments, the chemokine is CXCL9, as described in Section II. B.2.

[0041] The present disclosure also provides pharmaceutical compositions comprising the adenoviral vectors disclosed herein.

[0042] 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. In some cases, this then allows immune cells, such as a cell therapy engineered with a cognate binder (e.g., CAR), to be used to bind the targetable antigen. In addition to allowing targetable recognition of tumor cells by a cognate binder, administration of the adenoviral vector also allows for oncolytic targeting and killing of tumors in tumor-bearing subjects (e.g., subjects with cancer), including in connection with methods of cancer treatment.

[0043] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. DEFINITIONS

[0044] 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.

[0045] The terms “a”, “an”, and “the”, as used herein, include plural references unless the context clearly dictates otherwise.

[0046] The terms “or” and “and / or”, as used herein, include any, and all, combinations of one or more of the associated listed items.307612002440

[0047] 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.

[0048] The terms “including”, “includes”, “included”, and other forms, as used herein, are not limiting.

[0049] 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.

[0050] 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.

[0051] 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 (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,307612002440Harlow and Lane, 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).

[0052] 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.

[0053] 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.

[0054] The term “constant region” or “constant domain”, as used herein, refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. This portion has a more conserved amino acid sequence relative to the variable region. The constant region may contain the CHI, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0055] 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.307612002440

[0056] 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.

[0057] 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.

[0058] The term “heavy chain”, when used in reference to an antibody, refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy -terminal portion includes a constant region. The constant region can be one of five distinct types, (e.g, isotypes) referred to as alpha, delta, epsilon, gamma, and mu, based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: alpha, delta, and gamma contain approximately 450 amino acids, while epsilon and mu contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3, and IgG4.

[0059] 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.

[0060] 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,307612002440a 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.

[0061] 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.

[0062] The term “operatively linked” or “operably 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.

[0063] The term “pharmaceutically acceptable excipient, carrier or diluent”, as used herein, refers to any substance formulated alongside the active ingredient of a pharmaceutical composition that allows the active ingredient to retain biological activity and is non-reactive with the subject’s immune system. Such a substance can be included for the purpose of long-term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating absorption, reducing viscosity, or enhancing solubility. The selection of appropriate substance can depend upon the route of administration and the dosage form, as well as the active ingredient and other factors. Compositions having such substances can 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).

[0064] 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.

[0065] 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.307612002440

[0066] 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, gl ycosy lation, 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.

[0067] The term “population” of cells, as used herein, refers to any number of cells greater than 1, but is preferably at least 1×103cells, at least 1×104cells, at least 1×105cells, at least 1×106cells, at least 1×107cells, at least 1×108cells, at least 1×109cells, at least 1×1010cells, at least 1×1011or 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).

[0068] 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 “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.

[0069] 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.307612002440

[0070] 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.

[0071] 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.

[0072] The term “transduced”, as used herein, refers to a process by which a transgene is introduced into a host cell from a virus particle.

[0073] The term “transgene”, as used herein, refers to any heterologous polynucleotide incorporated in a viral vector, e.g., for transcription or expression in a target cell. An example of a transgene is a polynucleotide encoding a therapeutic polypeptide or a detectable marker.

[0074] 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.

[0075] 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.

[0076] 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 the307612002440heavy 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 0 sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the 0 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)).

[0077] 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.

[0078] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.307612002440IL VIRAL VECTORS

[0079] Provided herein are adenoviral vectors, for tagging a cancer, e.g., a solid cancer or tumor. In some embodiments, the adenoviral vectors encode any of the one or more proteins disclosed herein, such as a synthetic cancer antigen and a CXCL9 chemokine. In particular embodiments, the adenoviral vector encodes one or more copies of any of the one or more proteins disclosed herein. In some embodiments, the adenoviral vector encodes one or more copies of any of the synthetic cancer antigens disclosed herein. In some embodiments, the adenoviral vector encodes one or more copies of any of the CXCL9 chemokine disclosed herein.

[0080] Also provided herein are adenoviral vectors comprising polynucleotides encoding two or more of the synthetic cancer antigens disclosed herein, such as a synthetic cancer antigen comprising a tumor-targeting binding molecule, and a synthetic cancer antigen comprising a membrane targeting domain, and also comprising a polynucleotide encoding a CXCL9 chemokine.

[0081] In some embodiments, provided herein are adenoviral 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. B.1,a.ii., 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. B.l.a.i, and a third polynucleotide encoding a CXCL9 chemokine, e.g., any of those as described in Section II. B.2. In some embodiments, the first polynucleotide and the second polynucleotide are separated by nucleotide sequence encoding a cleavable linker or a translatable skipping sequence. In some embodiments, the second polynucleotide and the third polynucleotide are separated by nucleotide sequence encoding a cleavable linker or a translatable skipping sequence. In some embodiments, the cleavable linker or translatable skipping sequence comprises a 2A self-cleaving peptide.

[0082] In any of the above embodiments, the first, second or third polynucleotide is an expression cassette. 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 adenoviral 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 the adenoviral vector as a single unit.307612002440

[0083] 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.A. Adenoviral Vectors

[0084] In some embodiments, the viral vector is an adenoviral vector.

[0085] 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.

[0086] In some embodiments, the adenoviral vector is a viral vector exhibiting tumor tropism.

[0087] Accordingly, in some embodiments, the vector comprising the polynucleotide encoding the synthetic cancer antigen is a viral vector having tropism to a tumor cell, i.e., is a tumor tropic viral vector. By having tropism for a tumor cell, the viral vector is able to deliver the polynucleotide encoding the synthetic cancer antigen to the tumor cell being targeted, e.g., a tumor cell of a subject’s cancer.

[0088] 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.

[0089] In some embodiments, the viral vector is a replicating viral vector.

[0090] In some embodiments, the viral vector is a viral vector or modified form thereof of a viral vector as disclosed in, e.g., W02005 / 118825, WO2015 / 097220, WO2015 / 059303, WO2015 / 155370, W02016 / 174200, W02017 / 103291, WO2018 / 041838, WO2018 / 041827, W02017 / 103290, W02018 / 220207, W02019 / 043020, WO2022 / 171853, the contents of which are hereby incorporated by reference in their entity.

[0091] 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 nonenveloped viruses with a single linear strand of double-stranded DNA inside of an icoasahedral capsid.

[0092] Adenoviruses of animal original can be obtained from deposited strains, and then amplified in competent cell lines, and, optionally, modified as desired. Complete genome sequences of adenoviruses have been determined for, e.g., human adenovirus type 2 (GenBank Accession No. JO 1917), human adenovirus type 5 (GenBank Accession No. M73260; and GenBank Accession No. NC—001406), human adenovirus type 12 (GenBank Accession No. NC— 001460, X73487); human307612002440adenovirus 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.

[0093] 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.

[0094] 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.

[0095] Most adenoviruses, e.g., those in subgroups A, C, D, E, and F, enter target cells by binding primarily to the coxsackie-adenovirus receptor. However, generally, the subgroup B, type 1 group of adenoviruses bind primarily to CD46, whereas, generally, the subgroup B, type 2 group of adenoviruses bind primarily to desmoglein-2, with some exceptions. Adil, Adl4, Adl6, Ad21, Ad35, and Ad50 of subgroup B bind to CD46, as well as Adi 7 and Ad47 of subgroup D. Ad3, Ad7, and Ad l4 of subgroup B bind to desmoglein-2. See, e.g., Hensen et al., Int. J. Mol. Sci., 2020, 21(18): 6828.

[0096] 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.

[0097] 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 making307612002440adenoviruses that primarily bind CD46, e.g., Adi 1, 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: el 21497; and Hensen et al., Int. J. Mol. Sen, 2020, 21(18): 6828.

[0098] 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 Adi 4, are desirable for use in delivering synthetic cancer antigen s to tumor cells.

[0099] 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.

[0100] In some embodiments, the adenoviral vector is based on Ad3, Ad7, Adi 1, Adl4, Adi 6, Adi 7, 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.

[0101] 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.

[0102] 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.

[0103] In some embodiments, the adenovirus is a chimeric adenovirus. In some embodiments, the chimeric adenovirus is Ad3 / 1 Ip or is Ad5 / 3.307612002440

[0104] In some embodiments, the adenoviral vector is Ad3 / 1 Ip, which is a chimeric adenovirus composite of Ad3 and Adi Ip. In some embodiments, the Ad3 / 1 Ip is 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.

[0105] 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: 49. In some embodiments, ColoAdl has the nucleic acid sequence of SEQ ID NO: 49. 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 is 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 least307612002440about 98%, at least about 99% or 100% sequence identity to SEQ ID NO: 106. In some embodiments, a ColoAdl -derived sequence has the nucleic acid sequence of SEQ ID NO: 106. A plasmid map is shown in FIG. 5. Any of such sequences may be inserted with a transgene sequence as described.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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, such as an intron or intergenic sequence. In some embodiments, the transgene is under the control of a promoter.

[0110] For example, with reference to adenovirus, including a chimeric adenovirus, such as ColoAdl, among regions for insertion include the L5 region, such as between L5 and the E4 region.

[0111] 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.

[0112] In some embodiments, an adenovirus, including a chimeric adenovirus such as ColoAdl, that includes a transgene, has a sequence with the following formula (I): 5’ ITR-BI-BA-B2-BX-BB-BY-B3-3’ ITR.307612002440

[0113] 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: 49 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: 49. In one embodiment, the 5’ITR comprises or consists of the sequence from about 2bp to 137bp of SEQ ID NO: 49 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: 49.1n 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: 49 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: 49. In one embodiment, the 3’ITR comprises or consists of the sequence from about 32189bp to 32324bp of SEQ ID NO:49 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: 49.

[0114] In embodiments of the formula (I), Bi is a bond or comprises El A, E1B or E1A-E1B. In some embodiments, Bi 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 Bi is a bond then E1A and E1B sequences will be omitted from the virus. In one embodiment Bi is a bond and thus the virus is a vector. In one embodiment Bi further comprises a transgene. It is known in the art that the Bi region can accommodate a transgene which may be inserted in a disruptive way into the El region (i.e. in the "middle" of the sequence) or part or all of the El region may be deleted to provide more room to accommodate genetic material.

[0115] 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.307612002440

[0116] 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.

[0117] Thus Bi 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.

[0118] In one embodiment Bi has the sequence from 139bp to 3932bp of SEQ ID NO: 49. In one embodiment, Bl has the sequence from 568bp to 3,902bp of SEQ ID NO: 49.

[0119] 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.

[0120] 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: 49 or a sequence 95%, 96%, 97%, 98% or 99% identical thereto over the whole length. In one embodiment the E2B region has the sequence from 5067bp to 10354bp of SEQ ID NO: 49 or a sequence 95%, 96%, 97%, 98% or 99% identical thereto over the whole length.

[0121] In one embodiment BA has the sequence from 3933bp to 27184bp of SEQ ID NO: 49.

[0122] 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-conservative amino acid changes, such that it has the same function as wildtype (the corresponding unmutated protein); increased function in comparison to wild-type protein; decreased function, such as no function in comparison to wild-type protein or has a new function in comparison to wild-type protein or a combination of the same, as appropriate.307612002440

[0123] 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.

[0124] 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.

[0125] 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.

[0126] In one embodiment the B2 region comprises the sequence from 27185bp to 28165bp of SEQ ID NO: 49. In one embodiment B2 region consists of the sequence from 27185bp to 28165bp of SEQ ID NO: 49.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] In one embodiment Bx has the sequence from 28166bp to 28366bp of SEQ ID NO: 49. 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: 49. In one embodiment Bx has a DNA sequence inserted between a place corresponding to between positions 28192bp and 28193bp of SEQ ID NO: 49.

[0131] In one embodiment Bx is a bond.

[0132] 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 some307612002440flexibility on the exact location of the transgene while minimizing the disruptive effects on virus stability and viability.

[0133] In embodiments of the formula (I), BB as employed herein refers to the DNA sequence encoding the L5 region. As employed herein the L5 region refers to the DNA sequence containing the gene encoding the fibre polypeptide / protein, as appropriate in the context. The fibre gene / region encodes the fibre protein which is a major capsid component of adenoviruses. The fibre functions in receptor recognition and contributes to the adenovirus’ ability to selectively bind and infect cells.

[0134] 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 Adi 1, such as Adi Ip.

[0135] In one embodiment BB has the sequence from 28367bp to 29344bp of SEQ ID NO: 49.

[0136] 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.

[0137] Thus, in one embodiment BY is joined directly to a base of L5 which represents the "end" of a coding sequence.

[0138] 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.

[0139] In one embodiment BY has the sequence from 29345bp to 29379bp of SEQ ID NO: 49. This sequence is a non-coding sequence wherein a DNA sequence, for example comprising a transgene (or transgene cassette), a restriction site or a combination thereof may be inserted. This sequence is advantageous because it acts a buffer in that allows some flexibility on the exact location of the transgene while minimizing the disruptive effects on virus stability and viability. The insert(s) can occur anywhere within the sequence from the 5’ end to the 3’ end, or at any point between bp 1 to 35 of the sequence from 29345bp to 29379bp of SEQ ID NO: 49. In some embodiments, insert(s) occur at about or after position 29356bp of SEQ ID NO: 49 (e.g. corresponding to insert(s) at about or after position 29369 of SEQ ID NO: 106). In one embodiment BY has a DNA sequence inserted between a place corresponding to positions 29356bp and 29357bp in SEQ ID NO: 49. In one embodiment the insert is at about position 29356bp of SEQ ID NO: 49. In one embodiment the insert is a restriction site insert. In one embodiment the insert is a transgene cassette comprising one or more307612002440transgenes, such as a synthetic cancer antigen and CXCL9 as described herein. In some embodiments, the transgene is under the control of a promoter

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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: 49. In one embodiment E4 is present except for the E4orf4 region which is deleted. In one embodiment B3 is a bond, i.e. wherein E4 is absent. In one embodiment B3 has the sequence consisting of from 32188bp to 29380bp of SEQ ID NO: 49.

[0144] 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 synthetic cancer antigen and CXCL9 as described herein. In some embodiments, the transgene is under the control of a promoter.

[0145] 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: 106) having the organization of formula (I): 5' ITR-B1-BA-B2-BX-BB-BY-B3-3' ITR. Exemplary backbones include SEQ ID NO: 49 and SEQ ID NO: 106, 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: 106, including within the 35-nt window spanning nucleotides30761200244029,345-29,379 of SEQ ID NO: 106 (See, e.g., FIG. 5); 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. 5). 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: 49 or SEQ ID NO: 106 using identical or conserved nucleotides as guides, and insertions relative to such sequences at such corresponding positions are contemplated.

[0146] In some embodiments, provided herein is a replication deficient or replication capable oncolytic adenovirus, comprising a transgene cassette encoding any of the a synthetic cancer antigen and CXCL9 herein located between the virus fibre gene L5 and the virus E4 gene and wherein the transgene is under the control of a promoter. In some embodiments, the virus is a group B adenovirus, such as Adi 1. In some embodiments, the virus is a chimeric virus, for example a virus that comprises a modified E2B region. In some embodiments, the virus is ColAdl 1 (enadenotucirev).

[0147] 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.

[0148] 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 the endogenous promoter. That is, where the promoter is generally on the antisense strand, the cassette is inserted, for example in the antisense orientation.

[0149] 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.

[0150] 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.

[0151] 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 promoter307612002440influences 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.

[0152] 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 and sense promoter. That is, the promoter is upstream of the E4 region in the 3’-5’ direction. Therefore any transgene cassette under control of the E4 promoter may need to be oriented appropriately. In one embodiment the cassette under the control of the E4 promoter is in the antisense orientation. In one embodiment the cassette is under the control of the E4 promoter in the sense orientation. The E4 promoter as employed herein refers to the original E4 promoter located in the virus genome.

[0153] 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 a 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.

[0154] 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.

[0155] 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.

[0156] 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 a synthetic cancer antigen 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.307612002440

[0157] 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.

[0158] 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 [3-globin polyA sequence (r[3gpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.

[0159] 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. In some embodiments, the signal peptide is also referred to herein as a signal sequence or a leader sequence. For example, a vector may comprise a nuclear localization sequence (e.g., from SV40) fused to the polynucleotide encoding the synthetic cancer antigens. 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 the amino acid sequence of SEQ ID NO: 34, SEQ ID NO: 36 or SEQ ID NO: 95. In some embodiments, the signal peptide is encoded by the nucleic acid sequence of any of SEQ ID NOS: 92-94 and 96.307612002440

[0160] 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.

[0161] 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.

[0162] In some embodiments, delivery via electroporation comprises mixing the cells with the polynucleotides encoding the synthetic cancer antigens 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 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.B. Polynucleotide Transgenes

[0163] In provided embodiments, the polynucleotide contained in the vector includes a transgene that encodes a membrane-bound synthetic cancer antigen as described herein, a transgene that encodes a soluble synthetic cancer antigen as described herein and a transgene that encodes a C-X-C motif chemokine ligand 9 (CXCL9) chemokine as described herein.

[0164] 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.

[0165] 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 effects and improve ribosome-skipping efficiency without altering the 2A cleavage site. The spacer may be307612002440about 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: 104. In some embodiments, the self-cleaving peptide comprises the amino acid sequence of SEQ ID NO: 73. 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: 35.

[0166] 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: 12. In some embodiments, the selfcleaving peptide comprises the amino acid sequence set forth in SEQ ID NO: 13.1. Synthetic Cancer Antigens

[0167] Provided herein are systems for targeting tumors in a subject comprising a vector that encodes a synthetic cancer antigen.

[0168] 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 are only expressed on the surface of cancer cells. In some embodiments, the synthetic cancer antigens contain a target domain that is recognizable by a cognate binder Specifically, the synthetic cancer antigens are designed to be recognized by a cognate binder, such as cognate binders that can be engineered as chimeric antigen receptors (CARs) 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.

[0169] 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 tumortargeting 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..a. Target Domain ( e.g., Antibody or antibody fragment)

[0170] 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 cancer307612002440antigens 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.

[0171] 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.

[0172] 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.

[0173] 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 100 amino acids to about 400 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 700 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 250 amino acids, about 150307612002440amino 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.

[0174] 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.

[0175] 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, the target 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.

[0176] In some embodiments, the functional fragments are part of a single polypeptide and are operatively linked. In some embodiments, the functional fragments are directly linked to each other. In some embodiments, the functional fragments are linked via linkers. Exemplary linkers that can be307612002440used herein are disclosed in the Example Section (Section VI) 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 the amino acid sequence of SEQ ID NO: 35.

[0177] 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.

[0178] 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 (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),307612002440Ultomiris (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).

[0179] 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.

[0180] 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 (VE) 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 derived. 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. B.1,a.ii) 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.307612002440

[0181] 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.b.

[0182] 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.b) 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 microM), 10-5(> 10 microM) or 10-4(> 100 microM). 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 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 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.

[0183] In some embodiments, the target domain is an antibody directed against HER2.

[0184] 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.

[0185] 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 the307612002440variable 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.

[0186] 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 the amino acid sequence of SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 28.

[0187] 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: 97-102. In some embodiments, the target domain is encoded by the nucleic acid sequence of any of SEQ ID NOS: 97-102.

[0188] In provided embodiments, such a variable heavy chain antibody fragment contains an idiotype recognized by an anti-idiotype antibody 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 binding307612002440HER2 is Kd greater than 10-7(> 100 nM), such as Kd greater than 10'6(> 1 microM), 10-5(> 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.i. Membrane Targeting Domain

[0189] 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. In some embodiments, such a synthetic cancer antigen may also be referred to as a membrane-bound synthetic cancer antigen. The target domain can be any as described in Section II. B.l.a.

[0190] 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. The transmembrane domains disclosed herein for synthetic cancer antigens can also be used as the transmembrane domain of CAR molecules disclosed below.

[0191] 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.307612002440

[0192] 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.

[0193] 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.

[0194] The transmembrane domain provided herein may comprise a transmembrane region and a cytoplasmic region located at the C-terminal side of the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids and, in some embodiments, helps to orient the transmembrane domain in the lipid bilayer. In some embodiments, one or more cysteine residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

[0195] 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.

[0196] In some embodiments, the transmembrane domain is from a transmembrane glycoprotein. In some embodiments, the transmembrane domain comprises a transmembrane domain chosen from307612002440the 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.

[0197] In some embodiments, the transmembrane domain is a CD8 transmembrane domain.

[0198] In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 29. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 29.

[0199] 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 to307612002440about 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.

[0200] 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 HA tag. In some embodiments, the tag comprises the amino acid sequence of SEQ ID NO: 27.

[0201] 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 (Section VI) of the present application. Additional linkers are known to a skilled artisan.

[0202] In some embodiments, 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: 8 or SEQ ID NO: 76. In some embodiments, the membrane-bound synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 76. In some embodiments, the membrane-bound synthetic cancer antigen 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: 7, 19, 26, 76, 85, 87 or 88. In some embodiments, the membrane-bound synthetic cancer antigen is encoded by the nucleic acid sequence of any of SEQ ID NOS: 7, 19, 26, 76, 85, 87, 88, 107, 108 and 109.ii. Tumor-Targeting Binding Molecule or Domain

[0203] In some embodiments, the synthetic cancer antigen is a soluble (sol) protein and the target domain is linked to a tumor-targeting binding 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, such a synthetic cancer antigen may also be referred to as a soluble synthetic cancer antigen. In some307612002440embodiments, the synthetic cancer antigen comprises a target domain and tumor targeting binding molecule. The target domain can be any as described in Section II. B.l.a.

[0204] In some embodiments, provided herein is a synthetic cancer antigen comprising a target domain comprising at least one target domain described in Section II. B.l.a and a tumor-targeting binding 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 binding molecule are directly linked. In some embodiments, the amino acid sequence of the target domain and the tumor-targeting binding 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 noninfected bystander tumor cells to expose the synthetic cancer antigen on outside surface of the cell.

[0205] In some embodiments, the tumor-targeting binding 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 binding molecule binds to a tumor associated antigen (TAA). In some embodiments, the tumortargeting binding molecule is an antibody or antibody fragment. In some embodiments, tumortargeting binding molecule is a tumor associated antigen (TAA) binding molecule. In some embodiments, the tumor-targeting binding 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.

[0206] 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 some307612002440embodiments, 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.

[0207] In some embodiments, the tumor-targeting binding molecule binds to a tumor associated antigen (TAA). In some embodiments, the tumor-targeting binding molecule is a molecule derived from an antibody is one or more f unctional fragments of the antibody that targets a tumor associated antigen. Non-limiting examples of functional fragments include heavy chain, light chain, heavy chain variable domain, light chain variable domain, single-chain Fvs (scFv), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody. In some embodiments, the tumor-targeting binding molecule is an antibody without the heavy chain CH3 domain. In some embodiments, the tumor-targeting binding molecule is an antibody without the heavy chain CH2 domain. In some embodiments, the tumor-targeting binding molecule is a Fab. In some embodiments, the tumortargeting binding molecule is an antibody without the heavy chain CH2 and CH3 domain. In some embodiments, the tumor-targeting binding molecule is a single-chain variable fragment (scFv). In some embodiments, the tumor-targeting binding molecule is the heavy chain of an antibody. In some embodiments, the tumor-targeting binding molecule is the light chain of an antibody. In some embodiments, the tumor-targeting binding molecule is the variable domain of the heavy chain of an antibody. In some embodiments, the tumor-targeting binding molecule is the variable domain of the light chain of an antibody.

[0208] In some embodiments, the tumor-targeting binding 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.

[0209] In some embodiments, the functional fragments are part of a single polypeptide and are operatively linked. In some embodiments, the functional 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 (Section VI) of the present application. Additional linkers are known to a skilled artisan. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the functional fragments are separate pol y peptides 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 single307612002440polynucleotide. 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 the amino acid sequence set forth in SEQ ID NO: 35.

[0210] In some embodiments, the tumor-targeting binding 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.

[0211] 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 cancerCD254 (RANKL) Denosumab Breast and prostate carcinoma307612002440CD261 (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 carcinoma EGFR (c-erbB) Cetuximab, Panitumumab, Solid tumors including glioma,Nimotuzumab, lung, breast, colon, and head and Nectinumumab, neck tumors Tomuzotuximab,Zalutumamab, matuzumab,futuximab, 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 aVf>3 Etaracizumab Tumor vasculatureIntegrin a5Pl 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 NivolumabPembrolizumabCemiplimabCamrelizumab307612002440SerplulimabSintilimabTislelizumabToripalimabRetifanlimabDostarlimabPD-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 vasculature RanibizumabMMP 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

[0212] In some embodiments, the tumor associated antigen targeted by the tumor-targeting binding molecule is expressed on a solid tumor.

[0213] 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), PSMA307612002440(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).

[0214] In some embodiments, the tumor associated antigen targeted by the tumor-targeting binding molecule is epithelial cell adhesion / activating molecule (EpCAM), epidermal growth factor receptor (EGFR), or human epidermal growth factor receptor 2 (HER2). In some embodiments, the tumor associated antigen targeted by the tumor-targeting binding molecule is EpCAM.

[0215] 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.

[0216] In some embodiments, the tumor-targeting binding molecule binds to a tumor associated antigen that is EpCAM. In some embodiments, the tumor-targeting binding 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 binding molecule is a single-chain Fvs (scFv) of an anti-EpCAM antibody. In some embodiments, the tumor-targeting molecule does not comprise a constant heavy chain and / or a constant light chain domain.

[0217] In some embodiments, the tumor-targeting binding molecule is adecatumumab or an antigen-binding fragment of adecatumumab. In some embodiments, the tumor-targeting binding molecule is a scFv of adecatumumab. In some embodiments, the tumor-targeting binding molecule comprises the variable light chain and the variable heavy chain of adecatumumab. In some embodiments, the tumor-targeting binding molecule is edrecolomab or an antigen-binding fragment of edrecolomab. In some embodiments, the tumor-targeting binding molecule is a scFv of edrecolomab. In some embodiments, the tumor-targeting binding molecule comprises the variable light chain and the variable heavy chain of edrecolomab. In some embodiments, the tumor-targeting binding molecule is tucotuzumab or an antigen-binding fragment of tucotuzumab. In some embodiments, the tumor-targeting binding molecule is a scFv of tucotuzumab. In some embodiments, the tumor-307612002440targeting binding molecule comprises the variable light chain and the variable heavy chain of tucotuzumab.

[0218] 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: 32, 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: 31. 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: 42, 43, 44, 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: 45, 46 and 47, respectively.

[0219] In some embodiments, the tumor-targeting binding molecule comprises a variable light chain region comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the tumor-targeting binding molecule comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, the tumor-targeting binding 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: 31. In some embodiments, the tumor-targeting binding 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: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 31 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 32. In some307612002440embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 32. In some embodiments, the tumor-targeting binding molecule comprises a variable light chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 31 and a variable heavy chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 32.

[0220] In some embodiments, the VH set forth in SEQ ID NO: 32 is encoded by the nucleic acid sequence set forth in SEQ ID NO: 40. In some embodiments, the VL set forth in SEQ ID NO: 31 is encoded by the nucleic acid sequence set forth in SEQ ID NO: 39.

[0221] In some embodiments, the tumor-targeting binding molecule is a functional fragment of an antibody that comprises the variable light chain comprising an amino acid sequence of SEQ ID NO: 31 (or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99% sequence identity thereto) and a variable heavy chain comprising an amino acid sequence of SEQ ID307612002440NO: 32 (or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99% sequence identity thereto). In some embodiments, the functional antibody fragment is a Fv, dsFv, scFv, Fab, Fab’, or a scFab. In some embodiments, the functional antibody fragment is an scFv. In some embodiments, the tumor-targeting binding molecule is an scFv comprising the VH set forth in SEQ ID NO: 32 and the VL set forth in SEQ ID NO: 31. In some of any such embodiments, the variable light chain and variable heavy chain of the scFv are linked by a peptide linker, such as by the sequence set forth as GGGGSGGGGSGGGGS (SEQ ID NO: 38).

[0222] In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises an scFv comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 30. In some embodiments, the tumor-targeting binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 30 (encoded by the nucleic acid sequence set forth in SEQ ID NO: 48). In some embodiments, the tumor-targeting binding molecule is the amino acid sequence set forth in SEQ ID NO: 30 (encoded by the nucleic acid sequence set forth in SEQ ID NO: 48).

[0223] In some embodiments, the tumor-targeting binding molecule binds to a tumor associated antigen that is HER2. In some embodiments, the tumor-targeting binding molecule is an antibody or307612002440antigen-binding fragment thereof that binds to a tumor associated antigen that is HER2. In some embodiments, the tumor-targeting binding molecule is a single-chain Fv (scFv) of an anti-HER2 antibody. In some embodiments, the tumor-targeting binding molecule does not comprise a constant heavy chain and / or a constant light chain domain.

[0224] In some embodiments, the tumor-targeting binding 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 binding molecule is an antibody fragment of the 4D5 (Abl) antibody.

[0225] In some embodiments, the tumor-targeting binding molecule is a single-chain Fvs (scFv) of a 4D5 antibody.

[0226] In some embodiments, the tumor-targeting binding molecule comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the tumor-targeting binding 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: 28.

[0227] In some embodiments, the tumor-targeting binding molecule 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 NO: 101. In some embodiments, the target domain is encoded by the nucleic acid sequence of SEQ ID NO: 101.

[0228] In some embodiments, the different elements of the soluble 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 (Section VI) of the present application. Additional linkers are known to a skilled artisan.

[0229] In some embodiments, the linkers may be flexible linkers. Linkers include peptides, polymers, nucleotides, nucleic acids, polysaccharides, and lipid organic species (such as polyethylene glycol). In some embodiments, the linker is a peptide linker. Peptide linkers may be from about 2-100, 10-50, or 15-30 amino acids long. In some embodiments, peptide linkers may be at least 10, at least 15, or at least 20 amino acids long and no more than 80, no more than 90, or no more than 100 amino acids long. In some embodiments, the linker has a length of between 1 and 100 amino acids, between 1 and 75 amino acids, between 1 and 50 amino acids, between 1 and 25 amino acids, between 5 and 100 amino acids, between 5 and 75 amino acids, between 5 and 50 amino acids, between 5 and 25 amino acids, between 10 and 100 amino acids, between 10 and 75 amino acids, between 10 and 50 amino acids, or between 10 and 25 amino acids. In some embodiments, the linker has a length of between 5 and 50 amino acids.307612002440

[0230] The linkers can be naturally-occurring, synthetic or a combination of both. Particularly suitable linker polypeptides predominantly include amino acid residues selected from Glycine (Gly), Serine (Ser), Alanine (Ala), and Threonine (Thr). For example, the linker may contain at least 75% (calculated on the basis of the total number of residues present in the peptide linker), such as at least 80%, at least 85%, or at least 90% of amino acid residues selected from Gly, Ser, Ala, and Thr. The linker may also consist of Gly, Ser, Ala and / or Thr residues only. In some embodiments, the linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues, or 8-12 glycine residues. In some aspects, suitable peptide linkers typically contain at least 50% glycine residues, such as at least 75% glycine residues. In some embodiments, a peptide linker comprises glycine residues only. In some embodiments, a peptide linker comprises glycine and serine residues only.

[0231] In some embodiments, these linkers are composed predominately of the amino acids Glycine and Serine, denoted as GS-linkers herein. In some embodiments, the linker contains (GGS)n, wherein n is 1 to 10, such as 1 to 5, for example 1 to 3, such as GGS(GGS)n (SEQ ID NO: 50), wherein n is 0 to 10. In particular embodiments, the linker contains the sequence (GGGGS)n (SEQ ID NO: 51), wherein n is 1 to 10 or n is 1 to 5, such as 1 to 3. In further embodiments, the linker contains (GGGGGS)n (SEQ ID NO: 52), wherein n is 1 to 4, such as 1 to 3. The linker can include combinations of any of the above, such as repeats of 2, 3, 4, or 5 GS, GGS, GGGGS, and / or GGGGGS linkers may be combined. In some embodiments, such a linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids in length.

[0232] In some embodiments, the linker is or comprises (in one-letter amino acid code): GGS, GGGGS (SEQ ID NO: 53 and 54), or GGGGGS (SEQ ID NO: 55). In some embodiments, the GS-linker is or comprises the amino acid sequence of GGSGGS, i.e., (GGS)2 (SEQ ID NO: 56);GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 57); GGSGGSGGSGGS, i.e., (GGS)4(SEQ ID NO: 58); GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 59); GGGGGSGGGGGSGGGGGS, i.e., (G5S)3(SEQ ID NO: 60), GGSGGGGSGGGGSGGGGS (SEQ ID NO: 61) and GGGGSGGGGSGGGGS (SEQ ID NO: 62). In some embodiments, the linker is or comprises GGGG (SEQ ID NO: 63). In some embodiments, the linker is or comprises GGGGG (SEQ ID NO: 64). In some embodiments, the linker is or comprises GGGGGGGG (SEQ ID NO: 65). In some of any of the above examples, serine can be replaced with alanine (e.g., (Gly4Ala) or (Gly3Ala)).

[0233] In some embodiments, the linker is or comprises GSAGSAAGSGEF (SEQ ID NO: 66). In some embodiments, the linker is or comprises GSGSGS (SEQ ID NO: 67). In some embodiments, the linker is or comprises GGGGSGGGGS (SEQ ID NO: 33).

[0234] In some embodiments, the linker 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 NO: 105. In some embodiments, the linker is encoded by the nucleic acid sequence of SEQ ID NO: 105.307612002440

[0235] In some cases, it may be desirable to provide some rigidity into the peptide linker. This may be accomplished by including proline residues in the amino acid sequence of the peptide linker. Thus, in some embodiments, a linker comprises at least one proline residue in the amino acid sequence of the peptide linker. For example, a peptide linker can have an amino acid sequence wherein at least 25% (e.g., at least 50% or at least 75%) of the amino acid residues are proline residues. In one particular embodiment, the peptide linker comprises proline residues only. In In some embodiments, the linker is or comprises PAPAP (SEQ ID NO: 68).

[0236] In some embodiments, the linker is or comprises EAAAK (SEQ ID NO:69).

[0237] In some embodiments, the linker comprises the amino acid sequence set forth in any of SEQ ID NOs: 33, 38 and 50-69. In some embodiments, the linker comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 38 (corresponding nucleotide sequence set forth in SEQ ID NO: 37) and 50-69, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 50-69. In some embodiments, the linker comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 50-69. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the linker comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38.

[0238] 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 HA tag. In some embodiments, the tag comprises the amino acid sequence of SEQ ID NO: 27.

[0239] 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 (Section VI) of the present application. Additional linkers are known to a skilled artisan.

[0240] In some of any of the provided embodiments, the soluble synthetic cancer antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 41. In some of any of the provided embodiments, the soluble synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 41.307612002440

[0241] In some of any of the provided embodiments, the soluble synthetic cancer antigen comprising a tumor-targeting domain comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some of any of the provided embodiments, the soluble synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the soluble synthetic cancer antigen 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: 4, 84, 86, 89-91 and 103. In some embodiments, the soluble synthetic cancer antigen is encoded by a nucleic acid sequence of any of SEQ ID NOS: 4, 84, 86, 89-91 and 103.b. Cognate Binders

[0242] In some embodiments, the target domains of any of the synthetic cancer antigens is recognized by a cognate binder, e.g., a chimeric antigen receptor (CAR). In some embodiments, the cognate binders cannot bind to a naturally-expressed protein on the surface of a non-cancer cell. In some embodiments, the cognate binders can be engineered as chimeric-antigen-receptors for expressing on the surface of immune effector cells.

[0243] In some embodiments, the cognate binder is an antibody. In some embodiments, the cognate binder is a molecule derived from an antibody. In some embodiments, a molecule derived from an antibody is one or more functional fragments of the antibody. Non-limiting examples of functional fragments include heavy chain, light chain, heavy chain variable domain, light chain variable domain, single-chain Fvs (scFv), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody. In some embodiments, the cognate binder is part of the extracellular domain of a chimeric antigen receptor. Exemplary CAR comprising cognate binders are further described below.

[0244] In some embodiments, the cognate binder of the synthetic cancer antigen is the extracellular domain of a chimeric antigen receptor (CAR). In some embodiments, the CAR that is expressed by genetically engineered cells is able to bind via its extracellular domain to the target domain of the synthetic cancer antigen. In provided embodiments, the extracellular domain of the CAR is chosen based on the particular target domain of the synthetic cancer antigen. In some embodiments, the CAR further comprises a transmembrane domain and an intracellular signaling domain. In some embodiments, the CAR is expressed on the surface of an immune effector cell. In some embodiments, the immune effector cell is a cytotoxic T cell. In some embodiments, the immune effector cell is a natural killer cell. In provided embodiments, the engineered cells (e.g., T cells) that comprise the CAR may be used for immunotherapy to target and destroy cancer cells that have been tagged to express the synthetic cancer antigen recognized by the CAR.307612002440

[0245] In some embodiments, the CAR is a fusion protein that includes the extracellular domain comprising the cognate binder as an extracellular antigen-binding domain for recognizing the target domain; a transmembrane domain; and an intracellular signaling domain. The extracellular domain and transmembrane domains may be linked by a flexible linker (also called a spacer). In some embodiments, the intracellular signaling domain includes an immunoreceptor tyrosine-based activation motif (IT AM). Activation of the CAR fusion protein results in cellular activation in response to recognition by the cognate binder of the target domain of the synthetic cancer antigen tag. When a cell expresses such a CAR, it can recognize and kill target cells that express, or that are tagged to display on their surface, the synthetic cancer antigen.

[0246] In some embodiments, the antibody (e.g,. an anti-idiotype antibody), such as antigenbinding fragment of an anti-idiotype antibody, that is the cognate binder is part of the extracellular domain of a chimeric antigen receptor (CAR). Typically, for inclusion as part of a CAR the antibody cognate binder (e.g., and -idiotype antibody) is a single chain polypeptide. In some embodiments, the antibody cognate binder (e.g., anti -idiotype antibody) is a scFv. In some embodiments, the antibody cognate binder (e.g., anti -idiotype antibody) is a single domain antibody, such as comprising only the variable heavy chain of an antibody, e.g. a camelid antibody from llama.

[0247] In some embodiments, the cognate binder is any anti -idiotype antibody or antigen-binding fragment comprising a means for binding an antibody or antibody fragment target domain of the synthetic cancer antigen. Anti-idiotypic antibodies against any of a variety of known antibody target domains are known and can be used. Exemplary anti-idiotypic antibodies include, but are not limited to, anti-4D5 (e.g., W02005 / 061546, see e.g, SEQ ID NO:1 and SEQ ID NO:2 described therein).

[0248] In some embodiments, the cognate binder is an antigen binding fragment of an antiidiotype antibody that binds the 4D5 (Abl) antibody. In some embodiments, the antigen binding fragment is an scFv comprising a variable heavy chain and a variable light chain. In some embodiments, the scFv contains a variable heavy chain and a variable light chain as present in SEQ ID NO: 72 or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the variable heavy chain and variable light chain as present in SEQ ID NO: 72. In some embodiments, the extracellular domain of the CAR comprises an scFv that has the sequence set forth in SEQ ID NO: 72 or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 72.

[0249] The CARs disclosed herein comprise a transmembrane domain that can be directly or indirectly fused to the extracellular domain. The transmembrane domains disclosed above for the synthetic cancer antigens can be used as the transmembrane domain of CARs disclosed herein.

[0250] In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the CAR comprises a transmembrane with an amino acid sequence derived from307612002440the transmembrane domain of CD8. In some embodiments, the CAR comprises a transmembrane domain with an amino acid sequence derived from the transmembrane domain of human CD8 alpha.

[0251] In some embodiments, suitably, the CAR comprises a transmembrane with an amino acid sequence derived from the transmembrane domain of CD28. Suitably, the CAR comprises a transmembrane domain with an amino acid sequence derived from the transmembrane domain of human CD28.

[0252] In some embodiments, the CARs can also comprise a spacer region located between the cognate binder of the extracellular antigen binding domain and the transmembrane domain. In some embodiments, the spacer region comprises hydrophilic amino acids and allows flexibility of the targeting domain with respect to the cell surface. In some embodiments, the spacer region comprises greater than 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the spacer region comprises less than 10, 15, 20, 25, 30, or 35 amino acids. In some embodiments, the spacer region is a hinge domain.

[0253] The CARs disclosed herein may comprise a hinge domain that is located between the extracellular domain and the transmembrane domain. A hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular domain relative to the transmembrane domain of the effector molecule can be used.

[0254] In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8a. In some embodiments, the hinge domain is a portion of the hinge domain of CD8a, e.g., a fragment containing about 15-100 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8a.

[0255] In some embodiments, the hinge domain is the hinge domain of an antibody, such as an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the hinge domain is the hinge domain that joins the constant domains CHI and CH2 of an antibody. In some embodiments, the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgGl, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3307612002440constant regions of an IgGl antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgGl antibody.

[0256] In some embodiments, the spacer region includes all or a portion containing the hinge domain of an IgGl Fc or an IgG4 Fc. In some embodiments, the spacer is an IgG4 Fc spacer.

[0257] Non-naturally occurring peptides may also be used as hinge domains. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain of an Fc receptor and the N- terminus of the transmembrane domain is a peptide linker, such as a (GxS)n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0258] The hinge domain may contain about 10-100 amino acids, e.g., about any one of 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. In some embodiments, the hinge domain may be at least about any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0259] The intracellular signaling domain in the CARs provided herein is responsible for activation of at least one of the normal effector functions of the immune effector cell expressing the CARs. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term “cytoplasmic signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire cytoplasmic signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector f unction signal. The term cytoplasmic signaling domain is thus meant to include any truncated portion of the cytoplasmic signaling domain sufficient to transduce the effector function signal.

[0260] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. “Primary intracellular signaling domain” refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as immunoreceptor tyrosine-based activation motif, or IT AM. An “IT AM,” as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells. The motif may comprises two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxL / Ix(6-8)YxxL / I. IT AMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues307612002440in the IT AM following activation of the signaling molecule. IT AMs may also function as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3z, FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0261] In some embodiments, the intracellular signaling domain is derived from CD3z chain (CD3z). In some embodiments, the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3z. In some embodiments, the intracellular signaling domain is a cytoplasmic signaling domain of wild-type CD3z.

[0262] The CARs disclosed herein may further comprise at least one co-stimulatory signaling domain. Thus, in some embodiments, the chimeric antigen receptor (CAR) comprises: 1) the cognate binder; 2) a spacer, such as a hinge sequence; 3) a transmembrane domain; and 4) an intracellular signaling region comprising a first primary intracellular signaling domain, such as a CD3 zeta intracellular signaling domain and second co-stimulatory intracellular signaling domain. The term “co-stimulatory signaling domain,” as used herein, refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function. Many immune effector cells require co-stimulation, in addition to stimulation of an antigen-specific signal, to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell.

[0263] The co-stimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. “Co-stimulatory signaling domain” can be the cytoplasmic portion of a co-stimulatory molecule.

[0264] In some embodiments, the intracellular signaling domain comprises a single co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (such as about any of 2, 3, 4, or more) co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same co- stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3z) and one or more co-stimulatory signaling domains (such as 4- IBB signal). In some embodiments, the one or more co-stimulatory signaling domains (such as 4- IBB signal) and the primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3z) are fused to each other via optional peptide linkers. The primary intracellular signaling domain, and the one or more co-stimulatory307612002440signaling domains may be arranged in any suitable order. Multiple co-stimulatory signaling domains may provide additive or synergistic stimulatory effects.

[0265] In some embodiments, a costimulatory domain can be CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, lymphocyte function- associated antigen-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, and / or B7-H3 costimulatory domains.

[0266] In some embodiments, an intracellular signaling domain can be a domain of CD3zeta, CD28 and / or 4-1BB. In some embodiments, the CAR comprises at least two intracellular signaling domains derived from CD3 zeta and 4-1BB. In other embodiments, the CAR comprises at least two intracellular signaling domains derived from CD3 zeta and CD28.

[0267] In some embodiments, the CAR comprises the sequence set forth in SEQ ID NO: 71 or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 71.c. Variants of Synthetic Cancer Antigens

[0268] Amino acid sequence modification(s) of the synthetic cancer antigens provided herein are contemplated. For example, it may be desirable to improve the binding affinity between the synthetic cancer antigens; it may also be desirable to improve other biological properties of the synthetic cancer antigens, including but not limited to specificity, thermostability, expression level, or solubility. Thus, in addition to the synthetic cancer antigens described herein, it is contemplated that variants can be prepared.

[0269] In some embodiments, the synthetic cancer antigens provided herein are chemically modified, for example, by the covalent attachment of any type of molecule to the synthetic cancer antigens. Exemplary non-limiting modifications include glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Additional ly, the synthetic cancer antigens may contain one or more non-classical amino acids.

[0270] In some embodiments, variations may be a substitution, deletion, or insertion of one or more codons encoding the synthetic cancer antigens that results in a change in the amino acid sequence as compared with the original sequence. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions.

[0271] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include307612002440amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains ( e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g, tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined.

[0272] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Alternatively, conservative (e.g, within an amino acid group with similar properties and / or side chains) substitutions may be made, so as to maintain or not significantly change the properties. Amino acids may be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His(H). Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0273] Non-conservative substitutions entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, into the remaining (non-conserved) sites.

[0274] Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In certain embodiments, the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In a specific embodiment, the substitution is a conservative amino acid substitution made at one or more predicted non-essential amino acid residues. The variation allowed may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.307612002440

[0275] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody.2. CXCL9

[0276] Provided herein are systems for targeting tumors in a subject comprising a vector that encodes a cytokine. In some embodiments, cytokine is a chemotactic cytokine or a chemokine.

[0277] In some embodiments, the cytokine is encoded by the vector, e.g., tumor tropic vector, administered to the subject, such that the tumor cell expresses the cytokine. In some embodiments, the encoded cytokine promotes tumor cell killing and clearance. In some embodiments, the encoded or expressed cytokine promotes immune cell activity and function, such as improved tumor killing function and / or expansion.

[0278] In some embodiments, the chemokine is CXCL9. In some embodiments, the CXCL9 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: 15. In some embodiments, the CXCL9 comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the CXCL9 is encoded by a nucleic acid sequence of SEQ ID NO: 14.C. Exemplary Polynucleotides

[0279] In some embodiments, provided herein is a polynucleotide encoding any of the soluble synthetic cancer antigens (containing a tumor-targeting synthetic cancer antigen (ttSCA)) comprising a tumor-targeting binding molecule, e.g., as disclosed in Section II. B.l.a.ii. and also encoding CXCL9, e.g., as disclosed in Section II. B.2. In some embodiments, provided herein is a polynucleotide encoding any of the membrane bound synthetic cancer antigens (containing a membrane-targeting synthetic cancer antigen (mtSCA)) that comprising a membrane targeting domain, e.g., as disclosed in Section II. B.l.a.i and also encoding CXCL9, e.g., as disclosed in Section II. B.2.

[0280] In some embodiments, the polynucleotides encode one copy of the ttSCA, mtSCA and / or CXCL9. In some embodiments, the polynucleotides encode more than one copy of the ttSCA, mtSCA and / or CXCL9. In some embodiments, the polynucleotides encoded two copies of ttSCA, mtSCA and / or CXCL9.

[0281] In some embodiments, the polynucleotide sequence encodes the ttSCA, the mtSCA and / or the CXCL9. In some embodiments, the ttSCA, mtSCA and / or CXCL9 are separated by a307612002440cleavable linker. In some embodiments, the cleavable linker comprises a 2A self-cleaving peptide. In some embodiments, the self-cleaving peptide is P2A or T2A. Exemplary polynucleotides and transgene orientation are characterized in Table 2.Table 2POLYNUCLEOTIDE TRANSGENE COMPONENTSFull Transgene NT SEQ AA SEQ Polynucleotide ComponentNT SEQ ID NO: ID NO: ID NO:Soluble synthetic84 5 cancer antigenGSG-P2A 104 73 Polynucleotidetransgene 1 (Pl) 77 Membrane-boundsynthetic cancer 85 8 antigenT2A 12 13 CXCL9 14 15 Soluble synthetic86 5 cancer antigenGSG-P2A 104 73 Polynucleotide Membrane-boundtransgene 2 (P2) 78 synthetic cancer 87 8 antigenT2A 12 13 CXCL9 14 15 Soluble synthetic4 5 cancer antigenT2A 12 13 PolynucleotideCXCL9 14 15 transgene 3 (P3) 79GSG-P2A 6 73 Membrane-boundsynthetic cancer 88 8 antigenSoluble synthetic89 5 cancer antigenT2A 12 13 PolynucleotideCXCL9 14 15 transgene 4 (P4) 80GSG-P2A 6 73 Membrane-boundsynthetic cancer 87 8 antigenMembrane-boundsynthetic cancer 87 8 Polynucleotide antigentransgene 5 (P5) 81 T2A 12 13 CXCL9 14 15GSG-P2A 104 73307612002440Table 2POLYNUCLEOTIDE TRANSGENE COMPONENTSFull Transgene NT SEQ AA SEQ Polynucleotide ComponentNT SEQ ID NO: ID NO: ID NO:Soluble synthetic90 5 cancer antigenMembrane-boundsynthetic cancer 87 8 antigenPolynucleotide T2A 12 13 transgene 6 (P6) 82CXCL9 14 15 GSG-P2A 104 73 Soluble synthetic91 5 cancer antigenSoluble synthetic4 5 cancer antigenGSG-P2A 6 73 PolynucleotideMembrane-boundtransgene 7 (P7) 83synthetic cancer 76 8 antigenT2A 12 13 CXCL9 14 15 Soluble synthetic4 5 cancer antigenPolynucleotide GSG-P2A 6 732transgene 8 (P8) Membrane-bound7 (w / sp); synthetic cancer 8107antigenPolynucleotide Soluble synthetic4 5 transgene 9 (P9) cancer antigenT2A 12 13 CXCL9 14 15 10GSG-P2A 6 73 Membrane-bound7 (w / sp); synthetic cancer 8107antigenPolynucleotide Membrane-bound19 (w / sp); transgene 10 (PIO) synthetic cancer 810817 antigenT2A 12 13 CXCL9 14 15 Polynucleotide Soluble synthetic4 5 transgene 11 (Pl 1) cancer antigen21T2A 12 13 CXCL9 14 15 Polynucleotide Membrane-bound7 (w / sp); transgene 12 (Pl 2) synthetic cancer 8107antigen24 GSG-P2A 6 73 Membrane-bound26 (w / sp); synthetic cancer 8109antigen307612002440Table 2POLYNUCLEOTIDE TRANSGENE COMPONENTSFull Transgene NT SEQ AA SEQ Polynucleotide ComponentNT SEQ ID NO: ID NO: ID NO:T2A 12 13 CXCL9 14 15 Polynucleotide Soluble synthetic 4 5 transgene 13 (Pl 3) cancer antigenP2A 6 73 75Membrane-bound 76 8 synthetic cancerantigenNT: nucleotide; AA: amino acid

[0282] In some embodiments, the polynucleotide encodes soluble synthetic cancer antigen, membrane-bound synthetic cancer antigen, and CXCL9, wherein the soluble and the membranebound synthetic cancer antigens are separated by GSG-P2A and CXCL9 is separated from the membrane-bound synthetic cancer antigen by T2A. In some 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 NO: 77 (also referred to herein as Polynucleotide Transgene 1 (Pl)). In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 77.

[0283] In some embodiments, the polynucleotide encodes soluble synthetic cancer antigen, membrane-bound synthetic cancer antigen, and CXCL9, wherein the soluble and the membranebound synthetic cancer antigens are separated by GSG-P2A and CXCL9 is separated from the membrane-bound synthetic cancer antigen by T2A. In some 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 NO: 78 (also referred to herein as Polynucleotide Transgene 2 (P2)). In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 78.

[0284] In some embodiments, the polynucleotide encodes soluble synthetic cancer antigen, CXCL9, and membrane-bound synthetic cancer antigen, wherein the soluble synthetic cancer antigen and CXCL9 are separated by T2A and CXCL9 is separated from the membrane-bound synthetic cancer antigen by GSG-P2A. In some 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% identical307612002440to SEQ ID NO: 79 (also referred to herein as Polynucleotide Transgene 3 (P3)). In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 79.

[0285] In some embodiments, the polynucleotide encodes soluble synthetic cancer antigen, CXCL9, and membrane-bound synthetic cancer antigen, wherein the soluble synthetic cancer antigen and CXCL9 are separated by T2A and CXCL9 is separated from the membrane-bound synthetic cancer antigen by GSG-P2A. In some 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 NO: 80 (also referred to herein as Polynucleotide Transgene 4 (P4)). In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 80.

[0286] In some embodiments, the polynucleotide encodes membrane-bound synthetic cancer antigen, CXCL9, and soluble synthetic cancer antigen, wherein the membrane-bound synthetic cancer antigen and CXCL9 are separated by T2A and CXCL9 is separated from the soluble synthetic cancer antigen by GSG-P2A. In some 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 NO: 81 (also referred to herein as Polynucleotide Transgene 5 (P5)). In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 81.

[0287] In some embodiments, the polynucleotide encodes membrane-bound synthetic cancer antigen, CXCL9, and soluble synthetic cancer antigen, wherein the membrane-bound synthetic cancer antigen and CXCL9 are separated by T2A and CXCL9 is separated from the soluble synthetic cancer antigen by GSG-P2A. In some 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 NO: 82 (also referred to herein as Polynucleotide Transgene 6 (P6)). In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 82.

[0288] In some embodiments, the polynucleotide encodes soluble synthetic cancer antigen, membrane-bound synthetic cancer antigen, and CXCL9, wherein the soluble and the membranebound synthetic cancer antigens are separated by GSG-P2A and CXCL9 is separated from the membrane-bound synthetic cancer antigen by T2A. In some 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 NO: 83 (also referred to herein as Polynucleotide Transgene 7 (P7)). In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 83.307612002440

[0289] In some embodiments, the polynucleotide encodes the ttSCA and mtSCA, separated by P2A. In some 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 NO: 2 (also referred to herein as Polynucleotide Transgene 8 (P8)). In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, the polynucleotide of SEQ ID NO: 2 encodes the amino acid sequence of SEQ ID NO: 3.

[0290] In some embodiments, the polynucleotide encodes the ttSCA, the CXCL9 and the mtSCA, wherein the ttSCA is separated from the CXCL9 by T2A and the CXCL9 is separated from the mtSCA by P2A. In some 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 NO: 10 (also referred to herein as Polynucleotide Transgene 9 (P9)). In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, the polynucleotide of SEQ ID NO: 10 encodes the amino acid sequence of SEQ ID NO: 11.

[0291] In some embodiments, the polynucleotide encodes mtSCA and CXCL9 separated by T2A. In some 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 NO: 17 (also referred to herein as Polynucleotide Transgene 10 (P10)). In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 17. In some embodiments, the polynucleotide of SEQ ID NO: 17 encodes the amino acid sequence of SEQ ID NO: 18.

[0292] In some embodiments, the polynucleotide encodes ttSCA and CXCL9 separated by T2A. In some 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 NO: 21 (also referred to herein as Polynucleotide Transgene 11 (Pl 1)). In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, the polynucleotide of SEQ ID NO: 21 encodes the amino acid sequence of SEQ ID NO: 22.

[0293] In some embodiments, the polynucleotide encodes two copies of mtSCA and CXCL9, wherein the two copies of mtSCA are separated by P2A and CXCL9 is separated from mtSCA by T2A. In some 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 NO: 24 (also referred to herein as Polynucleotide Transgene 12 (Pl 2)). In some embodiments, the polynucleotide307612002440comprises the nucleic acid sequence set forth in SEQ ID NO: 24. In some embodiments, the polynucleotide of SEQ ID NO: 24 encodes the amino acid sequence of SEQ ID NO: 25.

[0294] In some embodiments, the polynucleotide encodes soluble synthetic cancer antigen and membrane-bound synthetic cancer antigen, wherein the soluble and membrane-bound cancer antigens are separated by GSG-P2A. In some 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 NO: 75 (also referred to herein as Polynucleotide Transgene 13 (P13)). In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 75.

[0295] In some embodiments, the polynucleotide is a deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded and either recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: premessenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.Polynucleotides refer to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, as well as all intermediate lengths. It will be readily understood that “intermediate lengths, ” in this context, means any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.', 151, 152, 153, etc.-, 201, 202, 203, etc.

[0296] In some embodiments, polynucleotides are codon-optimized. As used herein, the term “codon-optimized” refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, (x) systematic variation of codon sets for each amino acid, (xi) isolated removal of spurious translation i nitiation sites and / or (xii) elimination of fortuitous polyadenylation sites otherwise leading to truncated RNA transcripts.307612002440

[0297] It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide, or fragment of variant thereof, as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and / or primate codon selection. Further, alleles of the genes comprising the polynucleotide sequences provided herein may also be used. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides.

[0298] In some embodiments, the polynucleotides provided herein, regardless of the length of the coding sequence itself, further comprise with other DNA sequences, such as promoters and / or enhancers, untranslatedregions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may be employed in particular embodiments, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0299] In some embodiments, a polynucleotide sequence encoding the synthetic tumor antigens or the CXCL9 chemokine 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 nontranslated 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).

[0300] In some embodiments, polynucleotides encoding the synthetic cancer antigens or the CXCL9 chemokine 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, a307612002440CNCAAT region where N may be any nucleotide. The term “enhancer” refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and / or other enhancer elements.

[0301] 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 [3-kinesin ( -KIN) promoter, the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C (UBC) promoter, a phosphoglycerate kinase- 1 (PGK) promoter, a cytomegalovirus enhancer / chicken P-actin (CAG) promoter, a P-actin promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev 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.

[0302] In some embodiments, a polynucleotide sequence encoding the synthetic cancer antigens or CXCL9 chemokine described herein is operably linked to a constitutive promoter. In such embodiments, the polynucleotides encoding the synthetic cancer antigens described herein are constitutively and / or ubiquitously expressed in a cell.

[0303] In some embodiments, a polynucleotide sequence encoding the synthetic cancer antigens or CXCL9 chemokine 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 by307612002440interferon), 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.

[0304] Polynucleotides can be prepared, manipulated and / or expressed using any of a variety of well-established techniques known and available in the art.D. Exemplary Adenoviral Vectors

[0305] In some embodiments, the adenoviral vectors provided herein can encode any one of the synthetic cancer antigens provided herein and the CXCL9. In some embodiments, the adenoviral vectors provided herein can encode a synthetic cancer antigen comprising any one of the target domains provided herein. In some embodiments, the adenoviral vectors provided herein can encode a synthetic cancer antigen comprising any one of the membrane-targeting or tumor-targeting domains provided herein. In some embodiments, the adenoviral vectors including any of the polynucleotides provided herein.

[0306] In some embodiments, the adenoviral vector is an adenoviral vector with features as described in Section II. A into which is inserted a polynucleotide transgene provided herein, such as the polynucleotide transgenes described in Section II. B or C. Hence, in provided embodiments, an adenoviral vector comprises a sequence that can be modified to encode any of the polynucleotide transgenes provided herein, such as the polynucleotide transgenes described in Section II. B or C. In some embodiments, the adenoviral vector can be modified using any known means in the art. In some embodiments, any of the polynucleotide transgenes described in Section II. B or C can be inserted into the adenoviral vector. Methods of inserting a polynucleotide transgene into the adenoviral vector are known in the art and include, e.g., insertion via restriction digest and ligation using restriction enzymes. In some embodiments, the adenoviral vector that can be modified to encode any of the polynucleotide transgenes provided herein is referred to as an empty adenoviral vector. In some embodiments, a polynucleotide transgene, such as any of the polynucleotide transgenes disclosed in Section II. B or C, can be inserted or incorporated into the empty adenovirus at an appropriate location.

[0307] In some embodiments, any of the provided polynucleotide transgenes is inserted into any of the adenoviruses provided herein between Bx and By. In some embodiments, the polynucleotide transgene is inserted between L5 and E5. In some embodiments, the polynucleotide transgene is inserted between the 3 ’ end of L5 and the 5 ’ end of E4.

[0308] In some embodiments, the polynucleotide transgene is inserted between Bx and By using any of the restriction sites present in the adenoviral vectors provided herein, including the empty adenoviral vector (e.g., SEQ ID NO: 106) and depicted in FIG.5. In some embodiments, the restriction site is about 3bp to about lObp in length. In some embodiments, the restriction site is about 5bp in length. In some embodiments, the restriction site is about 6bp in length.307612002440

[0309] In some embodiments, the polynucleotide transgene can be inserted or incorporated between any two restriction sites present in the adenoviral vectors provided herein, including the empty adenoviral vector. In some embodiments, the polynucleotide transgene is inserted between Bx and By using two restriction sites. In some embodiments, the restriction site at Bx is an AsiSI restriction site. In some embodiments, the restriction site at BY is an Sbfl restriction site. In some embodiments, the polynucleotide transgene can be inserted or incorporated between the AsiSI restriction site and the Sbfl restriction site of any of the adenoviral vectors provided herein.

[0310] In some embodiments, the polynucleotide transgene can be inserted or incorporated at about or after position 29356bp of SEQ ID NO: 49 or 29369bp of SEQ ID NO: 106.

[0311] In some embodiments, the adenoviral vector 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 NO: 1. In some embodiments, the adenoviral vector comprises the nucleic acid sequence set forth in SEQ ID NO: 1.

[0312] In some embodiments, the adenoviral vector 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 NO: 9. In some embodiments, the adenoviral vector comprises the nucleic acid sequence set forth in SEQ ID NO: 9.

[0313] In some embodiments, the adenoviral vector 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 NO: 16. In some embodiments, the adenoviral vector comprises the nucleic acid sequence set forth in SEQ ID NO: 16.

[0314] In some embodiments, the adenoviral vector 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 NO: 20. In some embodiments, the adenoviral vector comprises the nucleic acid sequence set forth in SEQ ID NO: 20.

[0315] In some embodiments, the adenoviral vector 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 NO: 23. In some embodiments, the adenoviral vector comprises the nucleic acid sequence set forth in SEQ ID NO: 23.

[0316] In some embodiments, the adenoviral vector 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%,307612002440about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 74. In some embodiments, the adenoviral vector comprises the nucleic acid sequence set forth in SEQ ID NO: 74.III. COMPOSITIONS AND KITS

[0317] Provided herein are compositions, e.g., pharmaceutical compositions, comprising any of the adenoviral vectors disclosed herein. In some embodiments, the composition, e.g., pharmaceutical composition, further comprises a pharmaceutical acceptable carrier.

[0318] A pharmaceutically acceptable carrier, diluent or excipient includes without li miration any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, and / or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans and / or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations. Except insofar as any conventional media and / or agent is incompatible with the agents of the present disclosure, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into the compositions.

[0319] Pharmaceutically acceptable salt includes both acid and base addition salts.Pharmaceutical ly-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic- acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid,307612002440naphthalene- 1,5 -disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, pal ini tic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, ptoluenesulfonic acid, trifl uoroacetic acid, undecylenic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0320] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0321] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0322] Further guidance regarding formulations that are suitable for various types of administration can be found in Remington’s Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990).

[0323] In some embodiments, the present disclosure provides kits for carrying out a method described herein. In some embodiments, a kit comprises one or more vectors, and / or compositions, e.g., pharmaceutical compositions disclosed here. In some embodiments, a kit further comprises instructions for using the components of the kit to practice the methods of the present disclosure. The instructions for practicing the methods or uses are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert or in the labeling of the container of the kit307612002440or components thereof (i.e., associated with the packaging or sub-packaging). In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0324] In some embodiments, pharmaceutical compositions disclosed herein are for use in treating a cancer in a subject. Details of using pharmaceutical compositions disclosed herein for methods are disclosed in Section IV, below.

[0325] Provided herein are kits comprising any of the adenoviral vectors disclosed herein, e.g., in Section II, or any of the compositions, e.g., pharmaceutical compositions, disclosed herein, e.g., in Section III. In some embodiments, a kit further comprises instructions for using the components of the kit to practice the methods of the present disclosure. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert or in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging). In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.IV. METHODS AND USES OF SYNTHETIC CANCER ANTIGENS

[0326] Also provided are methods of administering any of the provided adenoviral vectors described herein, or compositions containing the same, to a subject, such as a subject that has a disease or disorder. Also provided herein are uses of any of any of the provided adenoviral vectors described herein, or compositions containing the same, for delivery to tumor cells in a subject, such as a subject that has a disease or disorder. In particular embodiments, the subject has a cancer. For example, the adenoviral vectors, or compositions such as pharmaceutical compositions containing the same, are useful for delivery any of the synthetic cancer antigens to tumor cells in a subject, such as for tagging the tumor cell or neighboring tumor cells in the tumor microenvironment with the engineered cancer antigen. In particular embodiments, the subject has a cancer. In some embodiments, the adenoviral vectors, or compositions such as pharmaceutical compositions containing the same, described herein are useful in connection with a variety of therapeutic, diagnostic and prophylactic307612002440indications. In particular, among such methods and uses are therapeutic methods and uses in combination with a immune cell therapy (e.g. CAR T cells) directed against the cognate binder of the synthetic cancer antigen.

[0327] Provided herein are methods of tagging a tumor cell, comprising contacting a tumor cell with the one or more of any of the adenoviral vectors disclosed herein, e.g., in Section II, or any of the compositions, e.g., pharmaceutical compositions, disclosed herein, e.g., in Section III. In some embodiments, the method comprises tagging the tumor cell in vivo. Also provided herein are methods of tagging a tumor cell in a subject having a subject having a cancer comprising administering a therapeutically effective amount of any of the adenoviral vectors disclosed herein, e.g., in Section II, or any of the compositions, e.g., pharmaceutical compositions, disclosed herein, e.g., in Section III. In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a solid tumor cancer.

[0328] Provided herein are methods of delivered an engineered cancer antigen provided herein, or a polynucleotide encoding the same, to a tumor cell in vivo, comprising administering any of the vectors disclosed herein, e.g., in Section II, or any of the compositions, e.g., pharmaceutical compositions, disclosed herein, e.g., in Section III.

[0329] Among the provided methods are methods of administering any of the adenoviral vectors provided herein to a subject that has a cancer for delivering a synthetic cancer antigen provided herein, or a polynucleotide encoding the same, to tumor cells of the cancer. Among such provided methods are methods of administering any of the adenoviral vectors provided herein to a subject that has a cancer for tagging tumor cells of the cancer with the synthetic cancer antigen.

[0330] The provided methods of delivering a synthetic cancer antigen to a tumor cell, such as for tagging a tumor cell, allows display of the synthetic cancer antigen on the surface of tumor cells and results in re-directing immune cells for cell therapy (e.g., CAR T cells) to recognize the tumor cells. The tumor cells that are tagged or for which the synthetic cancer antigen is displayed can be the tumor cells into with the adenovirus infects or can be neighboring tumor cells in the tumor microenvironment. For example, in provided embodiments, upon administration of a provided adenovirus to a subject that has cancer, the adenovirus is able to specifically infect tumor cells in the subject. In such embodiments in which the synthetic cancer antigen contains a membrane targeting domain (e.g. transmembrane domain), it can be expressed on the surface of the infected tumor cells where it can be recognized by a cognate binder. In other embodiments in which the synthetic cancer antigen is soluble it is secretable from the tumor cells where it is able to bind to a tumor antigen recognized by the tumor targeting domain on neighboring tumor cells. In some embodiments, the synthetic cancer antigen that is delivered includes both a membrane synthetic cancer antigen and a soluble synthetic cancer antigen so that both the infected tumor cell and neighboring tumor cells are tagged for display of the synthetic cancer antigen. The provided methods thus address problems307612002440related to use of cell therapy for treating certain tumors, such as due to the heterogenous expression of tumor antigens in solid tumors as well as potential for target antigen expression on healthy cells or tissues.

[0331] Also among the provided methods and uses are those that include therapeutic methods and uses, for example, involving administration of the compositions, to a subject having a disease, condition, or disorder. Among the provided methods are methods of administering a therapeutically effective amount of any of the vectors disclosed herein, e.g., in Section II, or any of the compositions, e.g., pharmaceutical compositions, disclosed herein, e.g., in Section III for treating a cancer in a subject. Among the provided uses are uses of a therapeutically effective amount of any of the vectors disclosed herein, e.g., in Section II, or any of the compositions, e.g., pharmaceutical compositions, disclosed herein, e.g., in Section III in the manufacture of a medicament for use in the treatment of a cancer in a subject. In some embodiments, the compositions are administered in an effective amount to effect treatment of the disease or disorder in combination with a targeted immune cell therapy (e.g. CAR T cells) that expresses a cognate binder of the synthetic cancer antigen. Uses include uses of the compositions in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.

[0332] In some embodiments, the subject has cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is solid tumor cancer. In some embodiments, the cancer is blood cancer. For example, in some embodiments, cancers that may be treated using the engineered cancer antigens, polynucleotides, vectors, compositions, and methods disclosed herein include, but are not limited to, adenoma, carcinoma, sarcoma, leukemia, or lymphoma. In some embodiments, the cancer is 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, ovarian cancer, chronic lymphocytic leukemia (CLL), B cell acute lymphocytic leukemia (B-ALL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), nonHodgkin’s lymphoma (NHL), diffuse large cell lymphoma (DLCL), diffuse large B cell lymphoma (DLBCL), Hodgkin’s lymphoma, or multiple myeloma. 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, the307612002440cancer 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.

[0333] Administration of the pharmaceutical compositions can occur by injection, irrigation, inhalation, consumption, electro-osmosis, hemodialysis, iontophoresis, and other methods known in the art. In some embodiments administration route is intraarterial, intracranial, intradermal, intraduodenal, intrammamary, intrameningeal, intraperitoneal, intrathecal, intratumoral, intravenous, intravitreal, ophthalmic, parenteral, spinal, subcutaneous, ureteral, urethral, vaginal, or intrauterine. In some embodiments, administration route is local or systemic.

[0334] In some embodiments, the administration route is by infusion (e.g., continuous or bolus). Examples of methods for local administration, that is, delivery to the site of injury or disease, include through an Ommaya reservoir, e.g. for intrathecal delivery (See e.g., US Patent Nos. 5,222,982 and 5,385,582, incorporated herein by reference); by bolus injection, e.g. by a syringe, e.g. into a joint; by continuous infusion, e.g. by cannulation, such as with convection (See e.g., US Patent Application Publication No. 2007-0254842, incorporated herein by reference); or by implanting a device upon which the cells have been reversibly affixed (see e.g. US Patent Application Publication Nos. 2008-0081064 and 2009-0196903, incorporated herein by reference). In some embodiments, the administration route is by topical administration or direct injection.

[0335] In some embodiments, the subject is a mammal, e.g., a human. In some embodiments, the subject is a human. In some embodiments, the subject may be a neonate, a juvenile, or an adult.

[0336] In some embodiments, treating refers to the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting disease development or preventing disease progression; (b) relieving the disease, i.e., causing regression of the disease state or relieving one or more symptoms of the disease; and (c) curing the disease, i.e., remission of one or more disease symptoms. In some embodiments, treatment may refer to a short-term (e.g., temporary and / or acute) and / or a long-term (e.g., sustained) reduction in one or more disease symptoms. In some embodiments, treatment results in an improvement or remediation of the symptoms of the disease. The improvement is an observable or measurable improvement, or may be an improvement in the general feeling of well-being of the subject.

[0337] The effective amount of the pharmaceutical compositions administered to a particular subject will depend on a variety of factors, several of which will differ from patient to patient including the disorder being treated and the severity of the disorder; activity of the specific agent(s) employed; the age, body weight, general health, sex and diet of the patient; the timing of administration, route of administration; the duration of the treatment; drugs used in combination; the judgment of the prescribing physician; and like factors known in the medical arts.307612002440

[0338] In some embodiments, a therapeutically effective amount of the pharmaceutical composition comprising the polynucleotides encoding the synthetic cancer antigen, or the vector comprising polynucleotides encoding the synthetic cancer antigen, is administered to a subject having a cancer.

[0339] In provided embodiments, any of the provided adenovirus viral vectors are administered to the subject. In embodiments of any of the methods and uses, the dose of the adenovirus is IxlO10to IxlO14viral particles per dose, such as IxlO10to IxlO12viral particles per dose. In one embodiment the concentration of virus in the composition is in the range 2 x 108to 2 x 1014vp / mL, such as 2 x IO10vp / mL to 2 x 1012vp / ml.

[0340] The number of administrations of treatment to a subject may vary. In some embodiments, introducing the pharmaceutical compositions into the subject may be a one-time event. In some embodiments, such treatment may require an on-going series of repeated treatments. In some embodiments, multiple administrations of the pharmaceutical compositions may be required before an effect is observed. The exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated.

[0341] In some embodiments, the pharmaceutical compositions disclosed herein are administered in combination with additional therapeutic composition. In some embodiments, administration of the pharmaceutical compositions disclosed herein in combination with the additional therapeutic composition results in an enhanced therapeutic effect in a cancer than is observed by treatment with either the pharmaceutical compositions disclosed herein or the additional therapeutic composition alone. In some embodiments, the cancer is resistant, refractory, or insensitive to treatment by the additional therapeutic composition alone. In some embodiments, the cancer is partially resistant, partial I y refractory, or partially insensitive to treatment by the additional therapeutic composition alone.

[0342] In some embodiments, the second therapeutic composition is an immune checkpoint inhibitor. Several immune checkpoint inhibitors are known in the art and have received FDA approval for the treatment of one or more cancers. For example, FDA-approved PD-L1 inhibitors include Atezolizumab (Tecentriq®, Genentech), Avelumab (Bavencio®, Pfizer), and Durvalumab (Imfinzi®, AstraZeneca); FDA-approved PD-1 inhibitors include Pembrolizumab (Keytruda®, Merck) and Nivolumab (Opdivo®, Bristol-Myers Squibb); and FDA-approved CTLA4 inhibitors include Ipilimumab (Yervoy®, Bristol-Myers Squibb). Additional inhibitory immune checkpoint molecules that may be the target of future therapeutics include A2AR, B7-H3, B7-H4, BTLA, IDO, LAG3 (e.g., BMS-986016, under development by BSM), KIR (e.g., Lirilumab, under development by BSM), TIM3, TIGIT, and VISTA.

[0343] In some embodiments, the second therapeutic composition is CAR expressing immune effector cells, where the CAR recognize a target other than the target domain of the synthetic cancer307612002440antigens being administered to the subject. Non-limiting examples of such CARS include CD 171-specific CARs (Parker al., Mol Ther (2007) 15(4):825-833), EGFRvIII-specific CARs (Morgan et al., Hum Gene Ther (2012) 23(10): 1043-1053), EGF-R-specific CARs (Kobold et al., J Nad Cancer Inst (2014) 107(l):364), carbonic anhydrase K-specific CARs (Larners etal., Biochem Soc Trans (2016) 44(3):951-959), FR-a-specific CARs (Kershaw etal., Clin Cancer Res (2006) 12(20):6106-6015), HER2-specific CARs (Ahmed et al., J Clin Oncol (2015) 33(15)1688-1696; Nakazawa et al., Mol Ther (2011) 19(12):2133-2143; Ahmed etal., Mol Ther (2009) 17(10): 1779-1787; Luo et al., Cell Res (2016) 26(7):850-853; Morgan et al., Mol Ther (2010) 18(4):843-851; Grada et al., Mol Ther Nucleic Acids (2013) 9(2):32), CEA-specific CARs (Katz et al., Clin Cancer Res (2015) 21(14):3149-3159), IL13Ra2-specific CARs (Brown et al., Clin Cancer Res (2015) 21(18):4062-4072), GD2-specific CARs (Louis et al., Blood (2011) 118(23):6050-6056; Caruana et al., Nat Med (2015) 21(5):524-529), ErbB2-specific CARs (Wilkie et al., J Clin Immunol (2012) 32(5): 1059-1070), VEGF-R-specific CARs (Chinnasamy et al., Cancer Res (2016) 22(2):436-447), FAP-specific CARs (Wang et al., Cancer Immunol Res (2014) 2(2): 154-166), MSLN-specific CARs (Moon et al, Clin Cancer Res (2011) 17(14):4719-30), NKG2D-specific CARs (VanSeggelen etal., Mol Ther (2015) 23(10): 1600-1610), CD19-specific CARs (Axicabtagene ciloleucel (Yescarta®) and Tisagenlecleucel (Kymriah®). See also^ Li et al., J Hematol and Oncol (2018) 11(22), reviewing clinical trials of tumor-specific CARs.V. EMBODIMENTS

[0344] This invention provides the following non-limiting embodiments.1. An adenovirus comprising a sequence of formula [I]:5’ ITR-BI-BA-B2-BX-BB-BY-B3-3’ ITR (I)wherein:Bi is bond or comprises: El A, E1B or E1A-E1B;BA comprises-E2B -L 1 -L2-L3 -E2 A-L4;B2 is a bond or comprises: E3;Bx is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both;BB comprises L5;BY is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both;B3 is a bond or comprises: E4;wherein the adenovirus comprises (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a membrane targeting domain or a synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule and (ii) a second transgene sequence encoding CXCL9.3076120024402. The adenovirus of embodiment 1, wherein the first transgene sequence and the second transgene sequence are encoded in position BY.3. An adenovirus comprising a polynucleotide located between the virus fibre gene L5 and the virus E4 gene, wherein the polynucleotide comprises (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a membrane targeting domain and (ii) a second transgene sequence encoding CXCL9.4. The adenovirus of any of embodiments 1-3, wherein the first transgene sequence and the second transgene sequence are encoded from the same transgene cassette.5. The adenovirus of any of embodiments 1-4, wherein the first transgene sequence and the second transgene sequence are separated by a nucleotide sequence encoding a cleavable linker or translational skipping sequence in the transgene cassette.6. The adenovirus of any of embodiments 1, 2, 4 and 5, wherein the adenovirus comprises the first transgene sequence encoding the synthetic cancer antigen comprising the target domain and the membrane targeting domain and (ii) a second transgene sequence encoding CXCL9.7. The adenovirus of any of embodiments 1, 2, 4 and 5, wherein the adenovirus comprises the first transgene sequence encoding the synthetic cancer antigen comprising the target domain and the tumor-targeting binding molecule and (ii) a second transgene sequence encoding CXCL9.8. The adenovirus of any of embodiments 1-7, wherein the first transgene sequence and the second transgene sequence are independently encoded in a region selected from El, E3, Bx, BY and combinations thereof.9. The adenovirus of any one of embodiments 1-8, wherein the first transgene sequence and the second transgene sequence are encoded in position BY.10. An adenovirus comprising a polynucleotide located between the virus fibre gene L5 and the virus E4 gene, wherein the polynucleotide comprises (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule and (ii) a second transgene sequence encoding CXCL9.11. The adenovirus of any of embodiments 7-10, wherein the first transgene sequence and the second transgene sequence are encoded from the same transgene cassette.12. The adenovirus of any of embodiments 7-11, wherein the first transgene sequence and the second transgene sequence are separated by a nucleotide sequence encoding a cleavable linker or translational skipping sequence in the transgene cassette.13. The adenovirus of embodiment 6 or embodiment 12, wherein the cleavable linker or translational skipping sequence is a self-cleaving linker.14. The adenovirus of embodiment 13, wherein the self-cleaving linker is or comprises a 2 A peptide.15. An adenovirus comprising a sequence of formula [I]:3076120024405’ ITR-BI-BA-B2-BX-BB-BY-B3-3’ ITR (I)wherein:Bi is bond or comprises: El A, E1B or E1A-E1B;BA comprises-E2B -L 1 -L2-L3 -E2 A-L4;B2is a bond or comprises: E3;Bx is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both;BB comprises L5;BY is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both;B3 is a bond or comprises: E4;wherein the adenovirus comprises: (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a membrane targeting domain, (ii) a second transgene sequence encoding a synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule; and (iii) a third transgene sequence encoding CXCL9.16. The adenovirus of embodiment 15, wherein the first transgene sequence, the second transgene sequence and the third transgene sequence are independently encoded in a region selected from El, E3, Bx, BY and combinations thereof.17. The adenovirus of embodiment 16, wherein the first transgene sequence, the second transgene sequence and the third transgene sequence are encoded in position BY.18. An adenovirus comprising a polynucleotide located between the virus fibre gene L5 and the virus E4 gene, wherein the polynucleotide comprises (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a membrane targeting domain, (ii) a second transgene sequence encoding a synthetic cancer antigen comprising a target domain and a tumortargeting binding molecule; and (iii) a third transgene sequence encoding CXCL9.19. The adenovirus of any of embodiments 15-18, wherein the first transgene sequence, the second transgene sequence and the third transgene sequence are encoded from the same transgene cassette.20. The adenovirus of any of embodiments 15-19, wherein, in the transgene cassette, the first and second transgene sequence are separated by a nucleotide sequence encoding a first cleavable linker or translational skipping sequence and the second and third transgene sequence are separated by a nucleotide sequence encoding a second cleavable linker or translational skipping sequence.21. The adenovirus of embodiment 20, wherein the first cleavable linker or translational skipping sequence and the second cleavable linker are different.22. The adenovirus of embodiment 20 or embodiment 21, wherein each cleavable linker or translational skipping sequence is a self-cleaving linker.30761200244023. The adenovirus of embodiment 22, wherein the self-cleaving linker independently is or comprises a 2A peptide.24. The adenovirus of any of embodiments 5, 6, 11-14 and 19-23, wherein the transgene or transgene cassettes is / are under the control of an endogenous or exogenous promoter.25. The adenovirus of embodiment 24, wherein the transgene or transgene cassettes is / are under the control of an endogenous promoter that is an E4 promoter or a major late promoter.26. The adenovirus of embodiment 24, wherein the transgene or transgene cassettes is / are under the control of an exogenous promoter that is a CMV promoter.27. The adenovirus of any of embodiments 1-26 that is replication competent.28. The adenovirus of any of embodiments 1-26, wherein the target domain is not expressed on the surface of a non-cancer cell of a subject.29. The adenovirus of any of embodiments 1-28, wherein the target domain is an antibody or antibody fragment that is recognized by a cognate binder.30. The adenovirus of any of embodiments 1-29, wherein the target domain is derived from a 4D5 anti-HER2 antibody.31. The adenovirus of embodiment 29 or embodiment 30, wherein the target domain is the variable domain of the heavy chain of an antibody.32. The adenovirus of embodiment 31, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 28.33. The adenovirus of embodiment 31 or embodiment 32, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 28.34. The adenovirus of any of embodiments 7-33, wherein the synthetic cancer antigen comprising the target domain and the tumor-targeting binding molecule is soluble.35. The adenovirus of any of embodiments 7-33 and 34, wherein the tumor-targeting binding molecule specifically binds to a tumor associated antigen expressed on the surface of a tumor cell.36. The adenovirus of embodiment 35, 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), PS MA (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, GPC3.37. The adenovirus of embodiment 35 or embodiment 36, wherein the tumor cell is a tumor cell of a solid tumor.30761200244038. The adenovirus of any of embodiments 35-37, 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.39. The adenovirus of any of embodiments 35-38, 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.40. The adenovirus of any of embodiments 7-33 and 34-39, wherein the tumor-targeting binding molecule comprises an antibody or an antigen-binding fragment thereof.41. The adenovirus of embodiment 40, wherein the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).42. The adenovirus of any of embodiments 35-41, wherein the tumor associated antigen is EpCAM.43. The adenovirus of any of embodiments 40-42, 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: 42, 43, and 44, 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: 45, 46, and 47, respectively.44. The adenovirus of any of embodiments 40-43, wherein the antibody or an antigenbinding 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: 32; 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: 31.45. The adenovirus of any of embodiments 40-44, wherein the antibody or an antigenbinding fragment thereof comprises a heavy chain variable (VH) region comprising the amino acid307612002440sequence of SEQ ID NO: 32, and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 31.46. The adenovirus of any of embodiments 7-33 and 34-45, wherein the target domain and the tumor-targeting binding molecule are linked by a linker.47. The adenovirus of embodiment 46, 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.48. The adenovirus of embodiment 46 or embodiment 47, wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 33, 38 and 50-69.49. The adenovirus of any of embodiments 7-48, wherein the target domain is not expressed on the surface of a non-cancer cell of a subject.50. The adenovirus of any of embodiments 1-49, wherein the target domain is recognizable by a cognate binder.51. The adenovirus of any of embodiments 29-50, wherein the cognate binder binds to the idiotype of the antibody or antibody fragment.52. The adenovirus of embodiment 51, wherein the cognate binder is an and -idiotype antibody or an antigen binding fragment.53. The adenovirus of any of embodiments 29-52, wherein the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 71.54. The adenovirus of any of embodiments 29-53, wherein the cognate binder comprises the amino acid sequence of SEQ ID NO:71.55. The adenovirus of any of embodiments 29-52, wherein the cognate binder is encoded by a nucleotide sequence that is at least 85% identical to SEQ ID NO: 70.56. The adenovirus of any of embodiments 29-52 and 55, wherein the cognate binder is encoded by the nucleotide sequence of SEQ ID NO:70.57. The adenovirus of any of embodiments 50-56, wherein the cognate binder of the synthetic cancer antigen is the extracellular domain of a chimeric antigen receptor (CAR).58. The adenovirus of embodiment 57, wherein the CAR further comprises a transmembrane domain and an intracellular signaling domain.59. The adenovirus of embodiment 57 or embodiment 58, wherein the CAR is expressed on the surface of an immune effector cell.60. The adenovirus of embodiment 59, wherein the immune effector cell is a T cell, optionally a cytotoxic T cell.61. The adenovirus of embodiment 59, wherein the immune effector cell is a natural killer cell.30761200244062. The adenovirus of any of embodiments 1-61, wherein the adenovirus comprises a nucleic acid sequence that is at least 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: 1, 9, 16, 20 or 23.63. The adenovirus of any of embodiments 1-62, wherein the adenovirus comprises the nucleic acid sequence of SEQ ID NOs: 1, 9, 16, 20 or 23.64. A pharmaceutical composition comprising the adenovirus vector of any of embodiments 1-63.65. The pharmaceutical composition of embodiment 64, wherein the pharmaceutical composition further comprises a pharmaceutical acceptable carrier.66. The pharmaceutical composition of embodiment 64 or embodiment 65, for use in treating a cancer in a subject.67. A kit comprising the pharmaceutical composition of any one of embodiments 64-66 and instructions for using the adenovirus.68. A method of tagging a tumor cell in vivo, comprising contacting a tumor cell with the adenovirus vector of any of embodiments 1-63, or the pharmaceutical composition of any of embodiments 64-66, wherein the tumor cell is tagged with a synthetic cancer antigen expressed from the adenovirus vector.69. A method of tagging a tumor cell of a subject having a cancer, comprising administering a therapeutically effective amount of the adenovirus vector of any of embodiments 1-63, or the pharmaceutical composition of any of embodiments 64-66, to the subject, wherein the tumor cell is tagged with a synthetic cancer antigen expressed from the adenovirus vector.70. The method of embodiment 68 or embodiment 69, wherein the cancer is a blood cancer.71. The method of embodiment 68 or embodiment 69, wherein the cancer is a solid tumor cancer.72. The method of any of embodiments 68-71, wherein the synthetic cancer antigen comprises a target domain and a membrane targeting domain.73. The method of any of embodiments 68-71, wherein the synthetic cancer antigen comprises a target domain and a tumor-targeting domain.74. A method of killing a tumor cell in vivo, comprising administering to a subject having a tumor a therapeutically effective amount of the adenovirus vector of any of embodiments 1-63, or the pharmaceutical composition of any of embodiments 64-66.75. A method of treating a cancer in a subject, comprising administering to a subject having a cancer a therapeutically effective amount of the adenovirus vector of any of embodiments 1-63, or the pharmaceutical composition of any of embodiments 64-66.307612002440VI. EXAMPLES

[0345] The following is a description of various methods and materials used in the studies. They are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like associated with the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be accounted for.Example 1 Design of Exemplary Gene Delivery Vectors

[0346] A gene delivery vector, e.g., a viral vector such as an oncolytic adenoviral vector, was developed for delivering a synthetic cancer antigen and a C-X-C motif chemokine ligand 9 (CXCL9) to a tumor cell. The synthetic cancer antigen includes two formats: soluble synthetic cancer antigen and membrane-bound synthetic cancer antigen. In this example, gene delivery vectors comprising polynucleotide sequences encoding soluble synthetic cancer antigen, membrane-bound synthetic cancer antigen and CXCL9 were developed.

[0347] The soluble synthetic cancer antigen contains a heavy chain variable domain of the 4D5 anti-HER2 antibody (hereinafter “Abl”) set forth in SEQ ID NO: 28, which was fused to a tumortargeting antibody or antibody fragment, in this case an anti-EpCAM scFv (SEQ ID NO: 30). The anti-EpCAM scFv antibody contains a variable heavy chain (VH) region (SEQ ID NO: 32) and a variable light (VL) region (SEQ ID NO: 31) separated by a linker (SEQ ID NO: 38 or SEQ ID NO: 62). The soluble synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 5, which is encoded by SEQ ID NO: 4. An IgGl signal sequence (MGWSCIILFLVATATGVHS; SEQ ID NO: 34) was added to the N-terminus for secretion to the culture supernatant. The polynucleotide sequence also included a nucleotide sequence encoding a FLAG tag sequence (DYKDDDDK; SEQ ID NO: 27) to aid in downstream detection of the FL1 (in some embodiments, FL1 is referred to as a synthetic cancer antigen). The polynucleotide sequence encoding the soluble synthetic cancer antigen was inserted into oncolytic virus, enadenotucirev (EnAd). Also referred to herein as Ad3 / 1 Ip or ColoAdl.

[0348] The membrane-bound synthetic cancer antigen (SEQ ID NO: 8) was designed such that Abl (SEQ ID NO: 28) was linked to a heterologous CD8 transmembrane domain (SEQ ID NO: 29). The polynucleotide sequence encoding the membrane-bound synthetic cancer antigen was inserted into oncolytic virus, enadenotucirev (EnAd).

[0349] The exemplary oncolytic adenoviral vectors are shown in Table El.307612002440Table El: Oncolytic Adenoviral VectorsADENOVIRUS COMPONENTFull Transgene AA NT SEQ ID NT SEQAdenovirus Component SEQ ID NT AA NO: ID NO:NO: SEQ SEQ ID NO ID NOSolublesynthetic cancer 4 5antigenAdenovirus 11 GSG-P2A 6 73 2 3 (Advl)Membrane7 (w / sp);bound synthetic 8107cancer antigenSolublesynthetic cancer 4 5antigenT2A 12 13Adenovirus 29 CXCL9 14 15 10 11 (Adv2)GSG-P2A 6 73Membrane7 (w / sp);bound synthetic 8107cancer antigenMembrane19bound synthetic (w / sp); 8Adenovirus 316 cancer antigen 108 17 18 (Adv3)T2A 12 13CXCL9 14 15Soluble 21 22 synthetic cancer 4 5Adenovirus 420 antigen(Adv4)T2A 12 13CXCL9 14 15Membrane7 (w / sp);bound synthetic 8107cancer antigenGSG-P2A 6 73Adenovirus 523 Membrane26 24 25 (Adv5)bound synthetic (w / sp); 8cancer antigen 109T2A 12 13CXCL9 14 15NT: nucleotide; AA: amino acid; w / sp: with signal peptideExample 2 Characterization of Synthetic Cancer Antigen Delivery to a Cancer Cell

[0350] In this example, the ability of the exemplary vectors described in Example 1 to lyse cancer cells was tested. Also tested was the ability of the exemplary vectors to deliver the307612002440polynucleotide sequences encoding the soluble synthetic cancer antigen, the membrane-bound synthetic cancer antigen, and CXCL9 to a cancer cell for expression.A. Adenovirus-mediated Killing Assay

[0351] Oncolytic potency of the exemplary vectors in Example 1 was assessed by a cell killing assay. Cell killing was measured on an xCELLigence microelectronic biosensor system (Agilent). To start the assay, Real-Time Cell Analysis 96-well plates were filled with 50 pL media for blank baseline measurement of impedance. Target cancer cells (non-small cell lung cancer (NSCLC) cell line A549) were then plated in 50 pL at 10,000 cells / well and returned to the xCELLigence to track cell growth overnight. To allow cells to settle, electrical impedance measurements were initiated 30 min after plating. 24 hours later, the cells were infected with Advl, Adv2, Adv3 or Adv5 at MOI 1. As a negative control, cells were infected with an empty oncolytic vector enadenotucirev (EnAd). Measurements were acquired every 15 min for the duration of the assay and the cell index was normalized to the time point at which adenovirus was added to the wells.

[0352] As shown in FIGS. 1A-1B, oncolytic potency of Advl, Adv2, Adv4 and Adv5 was comparable to the oncolytic potency of EnAd. However, Adv2 had slightly better oncolytic potency compared to other viruses.B. Synthetic Cancer Antigen Expression

[0353] Expression of the soluble and membrane-bound synthetic cancer antigens was assessed in A549 cells by flow cytometry.

[0354] Membrane- bound Synthetic Cancer Antigen Expression. A549 cells were plated in a 96-well plate in 50 pL at 10,000 cells / well. 24 hours later, the cells were infected with Advl, Adv2, Adv3 or Adv5 at MOI 1. 72 hours later, for a total infection time of 96 hours, the cells were harvested for assessment. As a negative control, cells were infected with an empty EnAd. As a positive control, A549 cells were transduced with a lentiviral vector encoding the membrane-bound synthetic cancer antigen. The presence of the membrane-bound synthetic cancer antigen was assessed using an antiidiotype antibody labeled with PE that is specific for the synthetic cancer antigen and the cells were subjected to flow cytometry.

[0355] As shown in FIGS. 2A-2C, in cells infected with empty EnAd, membrane-bound synthetic caner antigen was not detected. However, cells infected with Advl or Adv3 had the highest expression of the synthetic cancer antigen, followed by Adv5 and Adv2.

[0356] Soluble Synthetic Cancer Antigen Expression. A549 cells were plated in a 96-well plate in 50 pL at 10,000 cells / well. 24 hours later, the cells were infected with Advl, Adv2, Adv3 or Adv5 at MOI 1. 72 hours later, for a total infection time of 96 hours, the supernatant was transferred to EpCAM expressing HCT116 colorectal cancer cells. The anti-EpCAM scFv (SEQ ID NO: 30) portion of the soluble synthetic cancer antigen can then bind to the EpCAM expressed on the surface of the HCT116 cells. As a negative control, supernatant from A549 cells infected with empty EnAd307612002440was transferred to HCT116 cells. As a positive control, supernatant from A549 cells transduced with a lentivirus encoding the soluble synthetic cancer antigen was transferred to HCT116 cells. HCT116 cells were incubated in the supernatant described herein for 30 minutes and the cells were then harvested fore assessment. The presence of the soluble synthetic cancer antigen bound to HCT116 cells was assessed using an anti-idiotype antibody labeled with PE that is specific for the synthetic cancer antigen and the cells were subjected to flow cytometry.

[0357] As shown in FIGS.3A-3C, soluble synthetic caner antigen was not detected on HCT116 cells cultured in supernatant from A549 cells infected with empty EnAd. However, soluble synthetic caner antigen was detected on HCT116 cells cultured in supernatant from A549 cells infected with Advl or Adv2.C. CXCL9 Expression

[0358] Expression of CXCL9 was assessed in the supernatant of A549 cells.

[0359] A549 cells were plated in a 96-well plate in 50 pL at 10,000 cells / well. 24 hours later, the cells were infected with Advl, Adv2, Adv3 or Adv5 at MOI 1. 72 hours later, for a total infection time of 96 hours, the supernatant was harvested for assessment. As a negative control, cells were infected with empty EnAd. The presence of CXCL9 in the supernatant was assessed using an ELISA assay.

[0360] As shown in FIG.4, cells infected with Adv3 secreted the most CXCL9 (153.3 ng / mL) compared to cells infected with Adv2 (15.11 ng / mL) or Adv5 (34.9 ng / mL).These data demonstrate that the exemplary adenoviral vectors provided herein lead to the efficient tagging and expression of synthetic cancer antigens and CXCL9 in cancer cells.Example 3 In Vitro and In Vivo Characterization of Vector Potency and Synthetic Cancer Antigen Delivery

[0361] In this example, the oncolytic potency of (e.g., tumor cell lysis) and polynucleotide transgene delivery by exemplary vectors was assessed. The polynucleotide transgenes described herein encode a soluble synthetic cancer antigen, a membrane-bound synthetic cancer antigen, and / or a CXCL9. Some of the exemplary vectors tested in the present example include AdVl, AdV2, AdV3 and AdV4 as described in Example 1, and AdV6 (SEQ ID NO: 74), which is further characterized in Table E2.Table E2ADENOVIRUS COMPONENT Full AA Transgene NT SEQ ID NT SEQAdenovirus Component SEQ ID NT SEQ NO: ID NO:NO: ID NO Soluble synthetic4 5Adenovirus 674 cancer antigen75 (Adv6)P2A 6 73307612002440Table E2ADENOVIRUS COMPONENT Full AA Transgene NT SEQ ID NT SEQAdenovirus Component SEQ ID NT SEQ NO: ID NO:NO: ID NO 76 8Membranebound syntheticcancer antigenNT: nucleotide; AA: amino acid

[0362] Other exemplary vectors tested in the present example include vectors containing any of the polynucleotide transgenes described in Table E3, which are referred to herein as polynucleotide transgene 1 (Pl) to 7 (P7).Table E3POLYNUCLEOTIDE TRANSGENE COMPONENTSFull Transgene NT SEQ AA SEQ Polynucleotide ComponentNT SEQ ID NO: ID NO: ID NO:Soluble synthetic84 5 cancer antigenGSG-P2A 104 73 Polynucleotidetransgene 1 (Pl) 77 Membrane-boundsynthetic cancer 85 8 antigenT2A 12 13 CXCL9 14 15 Soluble synthetic86 5 cancer antigenGSG-P2A 104 73 Polynucleotide Membrane-boundtransgene 2 (P2) 78 synthetic cancer 87 8 antigenT2A 12 13 CXCL9 14 15 Soluble synthetic4 5 cancer antigenT2A 12 13 PolynucleotideCXCL9 14 15 transgene 3 (P3) 79GSG-P2A 6 73 Membrane-boundsynthetic cancer 88 8 antigenSoluble syntheticPolynucleotide 89 5 cancer antigentransgene 4 (P4) 80T2A 12 13CXCL9 14 15307612002440Table E3POLYNUCLEOTIDE TRANSGENE COMPONENTSFull Transgene NT SEQ AA SEQ Polynucleotide ComponentNT SEQ ID NO: ID NO: ID NO:GSG-P2A 6 73 Membrane-boundsynthetic cancer 87 8 antigenMembrane-boundsynthetic cancer 87 8 antigenPolynucleotide T2A 12 13 transgene 5 (P5) 81CXCL9 14 15 GSG-P2A 104 73 Soluble synthetic90 5 cancer antigenMembrane-boundsynthetic cancer 87 8 antigenPolynucleotide T2A 12 13 transgene 6 (P6) 82CXCL9 14 15 GSG-P2A 104 73 Soluble synthetic91 5 cancer antigenSoluble synthetic4 5 cancer antigenGSG-P2A 6 73 PolynucleotideMembrane-boundtransgene 7 (P7) 83synthetic cancer 76 8 antigenT2A 12 13 CXCL9 14 15NT: nucleotide; AA: amino acid

[0363] Pl to P7 were inserted into oncolytic virus, enadenotucirev (EnAd), also referred to herein as Ad3 / 1 Ip or ColoAdl. Particularly, Pl to P7 were inserted into the adenoviral vector of SEQ ID NO: 106 between L5 and E4 in the BY region using a 5’ AsiSI restriction site and a 3’ Sbfl restriction site with reference to FIG. 5. In some embodiments, Pl to P7 can be inserted into SEQ ID NO: 49. Vectors containing Pl to P7 are referred to herein as AdVPl, AdVP2, AdVP3, AdVP4, AdVP5, AdVP6 and AdVP7.A. Adenovirus-mediated Cell Killing Assay

[0364] Oncolytic potency was assessed by a cell killing assay. Cell killing was measured on an xCELLigence Real Time Cell Analyzer (Agilent). To start the assay, Polyethylene Terephthalate (PET) 96-well plates (Agilent) were filled with 50 pL of media for blank baseline measurement of impedance. Target cancer cells (non-small cell lung cancer (NSCLC) - A549) were then plated in 50 pL at 10,000 cells / well and returned to the xCELLigence to track cell growth overnight. To allow307612002440cells to settle, electrical impedance measurements were initiated 30 min after plating. 24 hours later, sextet wells were infected with exemplary vectors described above at MOI 1. As a negative control, sextet wells were left uninfected for the duration of the assay. Measurements were acquired every 15 min for the duration of the assay and the cell index was normalized to the time point at which adenovirus was added to the wells. As shown in FIG.6A, the vectors were capable of lysing A549 cells, including a vector that did not contain any of the payloads described above (see, EnAd in FIG.6A).B. Synthetic Cancer Antigen Expression

[0365] Expression of synthetic cancer antigens was assessed in A549 cells by flow cytometry.

[0366] Membrane- bound Synthetic Cancer Antigen Expression. Target cancer cells (nonsmall cell lung cancer (NSCLC) - A549) were plated in a 48-well plate in 200 pL at 100,000 cells / well. 24 hours later, triplicate wells were infected with exemplary vectors described above at MOI 1. At 72 hours post infection the cells were harvested for assessment. As a negative control, triplicate wells were infected with EnAd, which expresses no synthetic cancer antigens. The presence of the membrane-bound synthetic cancer antigen was assessed via flow cytometry using an antibody labeled with Alexa Fluor 647 that is specific for the synthetic cancer antigen. As shown in FIG. 6B, the vectors were capable of expressing membrane-bound synthetic cancer antigen.

[0367] Soluble Synthetic Cancer Antigen Expression. Target cancer cells (non-small cell lung cancer (NSCLC) - A549) were plated in a 48-well plate in 200 pL at 100,000 cells / well. 24 hours later, triplicate wells were infected with exemplary vectors described above at MOI 1. At 72 hours post infection, supernatants were transferred to 100,000 EpCAM expressing HCT116 colorectal cancer cells / well and incubated for 30 minutes. The anti-EpCAM scFv (SEQ ID NO: 30) portion of the soluble synthetic cancer antigen can then bind to the EpCAM expressed on the surface of the HCT116 cells. As a negative control, supernatants from triplicate wells of A549 cells infected with EnAd, which expresses no synthetic cancer antigens, were transferred to HCT116 cells and incubated for 30 minutes. The presence of the soluble synthetic cancer antigen bound to HCT116 cells was assessed via flow cytometry using an antibody labeled with Alexa Fluor 647 that is specific for the synthetic cancer antigen. As shown in FIG. 6C, the vectors were capable of expressing soluble synthetic cancer antigen.C. Adenovirus-mediated Tumor Killing Assay

[0368] Oncolytic potency of exemplary vectors in in vivo tumor models was assessed. Non-small cell lung adenocarcinoma cell line A549 was implanted subcutaneously into NSG mice and mice received 5 x 109virus particles intravenously on day 28, 30 and 32. Tumor growth or volume was measured with calipers over the course of the experiment. As shown in FIG. 7A and FIG. 7B, the vectors were able to reduce tumor volume.307612002440

[0369] These data demonstrate that the exemplary vectors provided herein lead to the efficient tagging and expression of synthetic cancer antigens and CXCL9 in cancer cells. These data also demonstrate that the exemplary vectors have cell killing ability.

[0370] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description.

[0371] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.307612002440SEQUENCES

[0373] Reference to exemplary sequences disclosed herein is provided in the below table. The sequences designated by SEQ ID NOs are also provided in the electronic sequence listing submitted herewith and incorporated by reference in its entirety.# SEQUENCE ANNOTATION 1. [to be inserted in ST. 26 sequence listing] Adenovirus 1(Advl ) 2. ATGGGATGGAGCTGCATCATCCTATTCCTCGTGGCGACGGCCACTGGAG Advl TGCATAGCGAACTGGTGATGACTCAGTCCCCGTCATCCCTGACGGTGAC transgene CGCCGGCGAGAAGGTCACCATGTCGTGCAAGTCCTCGCAAAGCCTGCTT (NT) AAC AGC GGC AAC C AGAAAAAC TACCTCACGTGGTAT C AGC AAAAGC C AG GT C AAC C C C C AAAAC T GC T C AT C T AC T GGGC GAGC AC C C GC GAGT C GGG GGTGCCAGACCGGTTCACCGGCTCCGGGTCAGGAACTGATTTCACCCTA ACCATCAGCTCGGTGCAAGCGGAGGACCTGGCCGTGTACTACTGCCAAA ATGATTACTCGTACCCTCT GAC C T T T GGAGC GGGC AC C AAGC T C GAAAT CAAGGGCGGTGGAGGAAGCGGCGGGGGAGGCTCAGGTGGGGGAGGATCA GAAGT C C AAC T GC T GGAGC AGT CAGGAGC C GAAC TGGTCCGCCC GGGAA CCTCCGTCAAGATTTCCTGTAAGGCTTCCGGCTACGCTTTTACCAATTA CTGGCTGGGCTGGGTCAAGCAAAGACCGGGGCATGGCCTGGAGTGGATC GGC GAC AT C T T C C C AGGGAGC GGC AAC AT C C AC T AC AAC GAGAAGT T C A AGGGGAAAGC GAC T C T GAC T GC C GAC AAAT CATCCAGCACCGCCTACAT GCAGCTGTCGTCGCTCACTTTCGAAGACAGCGCGGTGTACTTTTGTGCT C GGC T C C GGAAC T GGGAT GAAC C AAT GGAC TACT GGGGAC AAGGAAC TA CCGTGACCGTCTCCTCCGGCGGAGGCGGATCCGGGGGTGGGGGATCTGA GGTGCAGCTGGTGGAGAGCGGAGGTGGCCTCGTGCAGCCGGGCGGCAGC C T GAGAC T GAGC TGCGCCGCTAGCGGCTT C AAC AT C AAGGAC AC C T AC A TCCACTGGGTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGCTAG AAT C T AC C C C AC C AAC GGC T AC AC AAGAT AC GC C GAC AGC GT GAAGGGC AGAT TCACCATCAGCGCC GAC AC AAGC AAGAAC AC CGCCTACCT GC AGA T GAAC AGC C T GAGAGC C GAGGAC AC CGCCGTGTACTACT GC AGC AGAT G GGGCGGCGACGGCTTCTACGCCATGGACGTATGGGGACAAGGCACCCTT GTAACGGTCAGCAGCGGCTCCGGCGCTACCAATTTCTCTCTGCTGAAGC AAGCTGGCGACgtcgaagaaaatcccggacccGCCCTGCCCGTGACCGC ACTGCTGCTACCCCTGGCGCTGCTGCTGCATGCCGCTAGACCCGAAGTA CAATTAGTCGAATCAGGGGGCGGGCTGGTTCAACCCGGTGGGTCATTAC GCTTGTCTTGTGCAGCCTCAGGATTTAATATTAAAGATACTTATATTCA TTGGGTCCGGCAGGCTCCGGGAAAAGGACTTGAATGGGTCGCCCGAATA TATCCAACAAATGGGTATACCCGCTATGCTGATTCCGTCAAAGGGAGGT TTACAATTTCAGCTGATACCTC T AAAAAT AC TGCATATCTT C AAAT GAA TTCGCTCCGTGCTGAAGATACTGCTGTTTATTATTGTAGTCGCTGGGGG GGTGATGGGTTTTATGCAATGGATGTGTGGGGCCAGGGTACTCTGGTTA CCGTTTCATCCACCACTACGCCCGCCCCAAGACCACCCACGCCTGCCCC AAC AAT T GC AAGC C AAC CCTTATCCCT GAGAC C C GAAGC C T GC AGAC C C GCCGCGGGAGGCGCCGTGCACACAAGAGGCCTGGACTTCGCCTGCGACA TCTACATCTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTGCTGCTGAG CCTGGTGATCACCCTGTACTGCTAA3. [to be inserted in ST. 26 sequence listing] Advl transgene (AA)4. GAACTGGTGATGACTCAGTCCCCGTCATCCCTGACGGTGACCGCCGGCG Soluble AGAAGGTCACCATGTCGTGCAAGTCCTCGCAAAGCCTGCTTAACAGCGG Flare (NT) C AAC C AGAAAAAC TACCTCACGTGGTAT C AGC AAAAGC C AGGT C AAC C C CCAAAACTGCTCATCTACTGGGCGAGCACCCGCGAGTCGGGGGTGCCAGACCGGTTCACCGGCTCCGGGTCAGGAACTGATTTCACCCTAACCATCAG307612002440C T C GGT GC AAGC GGAGGAC C T GGC C GT GT AC T AC T GC C AAAAT GAT T AC T C GT AC C C T C T GAC C T T T GGAGC GGGC AC C AAGC T C GAAAT C AAGGGC G GTGGAGGAAGCGGCGGGGGAGGCTCAGGTGGGGGAGGATCAGAAGTCCA ACTGCTGGAGCAGTCAGGAGCCGAACTGGTCCGCCCGGGAACCTCCGTC AAGATTTCCTGTAAGGCTTCCGGCTACGCTTTTACCAATTACTGGCTGG GCTGGGTCAAGCAAAGACCGGGGCATGGCCTGGAGTGGATCGGCGACAT C T T C C C AGGGAGC GGC AAC AT C C AC T AC AAC GAGAAGT T C AAGGGGAAA GC GAC T C T GAC T GC C GAC AAAT C AT C C AGC AC C GC C T AC AT GC AGC T GT CGTCGCTCACTTTCGAAGACAGCGCGGTGTACTTTTGTGCTCGGCTCCG GAAC T GGGAT GAAC C AAT GGAC TACT GGGGAC AAGGAAC T AC C GT GAC C GTCTCCTCCGGCGGAGGCGGATCCGGGGGTGGGGGATCTGAGGTGCAGC TGGTGGAGAGCGGAGGTGGCCTCGTGCAGCCGGGCGGCAGCCTGAGACT GAGC T GC GC C GC T AGC GGC T T C AAC AT C AAGGAC AC C T AC AT C C AC T GG GTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGCTAGAATCTACC C C AC C AAC GGC T AC AC AAGAT AC GC C GAC AGC GT GAAGGGC AGAT T C AC CATCAGCGCC GAC AC AAGC AAGAAC AC CGCCTACCT GC AGAT GAAC AGC C T GAGAGC C GAGGAC AC CGCCGTGTACTACT GC AGC AGAT GGGGCGGCG ACGGCTTCTACGCCATGGACGTATGGGGACAAGGCACCCTTGTAACGGT CAGCAGC5. ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQP Soluble PKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDY Flare (also SYPLTFGAGTKLEIKGGGGSGGGGSGGGGSEVQLLEQSGAELVRPGTSV referred to KI SCKASGYAFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGK as Flarel ATLTADKSSSTAYMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVT (ELI ) - VSSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW antibody VRQAP GKGLE WVARI YP TNG YTRYAD S VKGRF T I S AD T S KNT AYLQMN S conjugate LRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS variant ) (AA)6. GGCTCCGGCGCTACCAATTTCTCTCTGCTGAAGCAAGCTGGCGACgtcg P2A linker aagaaaatcccggaccc (NT)7. GCCCTGCCCGTGACCGCACTGCTGCTACCCCTGGCGCTGCTGCTGCATG MembraneCCGCTAGACCCGAAGTACAATTAGTCGAATCAGGGGGCGGGCTGGTTCA bound Flare ACCCGGTGGGTCATTACGCTTGTCTTGTGCAGCCTCAGGATTTAATATT (NT) AAAGATACTTATATTCATTGGGTCCGGCAGGCTCCGGGAAAAGGACTTG AATGGGTCGCCCGAATATATCCAACAAATGGGTATACCCGCTATGCTGA TTCCGTCAAAGGGAGGTTTACAATTTCAGCTGATACCTCTAAAAATACT GCATATCTTCAAATGAATTCGCTCCGTGCTGAAGATACTGCTGTTTATT ATTGTAGTCGCTGGGGGGGTGATGGGTTTTATGCAATGGATGTGTGGGG CCAGGGTACTCTGGTTACCGTTTCATCCACCACTACGCCCGCCCCAAGA CCACCCACGCCTGCCC C AAC AAT T GC AAGC C AAC CCTTATCCCT GAGAC C C GAAGC C T GC AGAC C C GC C GC GGGAGGC GC C GT GC AC AC AAGAGGC C T GGACTTCGCCTGCGACATCTACATCTGGGCCCCCCTGGCCGGCACCTGC GGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGC8. E VQLVE S GGGLVQP GGS LRL S CAAS GFN I KD T Y I HWVRQAP GKGLE WVA MembraneRIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR bound Flare WGGDGFYAMDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEAC (AA) RPAAGGAVHTRGLDFACDI YIWAPLAGTCGVLLLSLVITLYC9. [to be inserted in ST. 26 sequence listing] Adenovirus 2(Adv2 ) 10. ATGGGATGGAGCTGCATCATCCTATTCCTCGTGGCGACGGCCACTGGAG Adv 2TGCATAGCGAACTGGTGATGACTCAGTCCCCGTCATCCCTGACGGTGAC transgene CGCCGGCGAGAAGGTCACCATGTCGTGCAAGTCCTCGCAAAGCCTGCTT (NT) AAC AGC GGC AAC C AGAAAAAC TACCTCACGTGGTAT C AGC AAAAGC C AG GT C AAC C C C C AAAAC T GC T C AT C T AC T GGGC GAGC AC C C GC GAGT C GGG GGTGCCAGACCGGTTCACCGGCTCCGGGTCAGGAACTGATTTCACCCTA ACCATCAGCTCGGTGCAAGCGGAGGACCTGGCCGTGTACTACTGCCAAA ATGATTACTCGTACCCTCT GAC C T T T GGAGC GGGC AC C AAGC T C GAAAT CAAGGGCGGTGGAGGAAGCGGCGGGGGAGGCTCAGGTGGGGGAGGATCA GAAGT C C AAC T GC T GGAGC AGT CAGGAGC C GAAC TGGTCCGCCC GGGAA CCTCCGTCAAGATTTCCTGTAAGGCTTCCGGCTACGCTTTTACCAATTACTGGCTGGGCTGGGTCAAGCAAAGACCGGGGCATGGCCTGGAGTGGATC307612002440GGC GAC AT C T T C C C AGGGAGC GGC AAC AT C C AC T AC AAC GAGAAGT T C A AGGGGAAAGC GAC T C T GAC T GC C GAC AAAT CATCCAGCACCGCCTACAT GCAGCTGTCGTCGCTCACTTTCGAAGACAGCGCGGTGTACTTTTGTGCT C GGC T C C GGAAC T GGGAT GAAC C AAT GGAC TACT GGGGAC AAGGAAC TA CCGTGACCGTCTCCTCCGGCGGAGGCGGATCCGGGGGTGGGGGATCTGA GGTGCAGCTGGTGGAGAGCGGAGGTGGCCTCGTGCAGCCGGGCGGCAGC C T GAGAC T GAGC TGCGCCGCTAGCGGCTT C AAC AT C AAGGAC AC C T AC A TCCACTGGGTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGCTAG AAT C T AC C C C AC C AAC GGC T AC AC AAGAT AC GC C GAC AGC GT GAAGGGC AGAT TCACCATCAGCGCC GAC AC AAGC AAGAAC AC CGCCTACCT GC AGA T GAAC AGC C T GAGAGC C GAGGAC AC CGCCGTGTACTACT GC AGC AGAT G GGGCGGCGACGGCTTCTACGCCATGGACGTATGGGGACAAGGCACCCTT GT AAC GGT C AGC AGC GGAAGC GGAGAGGGAAGGGGAAGC CTCCTCACAT GT GGC GAT GT GGAAGAAAAC C C C GGAC C C AAAAAGAGC GGAGT GC T GT T CCTGCTGGGGATCATCTTGCTGGTCTTGATCGGGGTGCAGGGCACCCCG GTGGTGCGCAAGGGCCGCTGTTCATGCATCTCCACCAATCAGGGCACCA TCCATCTTCAGTCACTGAAAGACCTGAAACAGTTCGCCCCGTCGCCTAG C T GC GAGAAGAT C GAAAT C AT C GC GAC C C T GAAGAAC GGC GT C C AAAC C T GC C T GAAT C C T GAC AGC GC AGAT GT GAAGGAAC T C AT C AAGAAAT GGG AAAAAC AGGT C AGC C AAAAGAAGAAGC AGAAAAAC GGAAAGAAGC AT C A GAAGAAGAAAGT C C T C AAAGT GC GC AAGT C AC AAC GC AGC AGAC AAAAA AAGACTACCGGCTCCGGCGCTACCAATTTCTCTCTGCTGAAGCAAGCTG GCGACgtcgaagaaaatcccggacccGCCCTGCCCGTGACCGCACTGCT GCTACCCCTGGCGCTGCTGCTGCATGCCGCTAGACCCGAAGTACAATTA GTCGAATCAGGGGGCGGGCTGGTTCAACCCGGTGGGTCATTACGCTTGT CTTGTGCAGCCTCAGGATTTAATATTAAAGATACTTATATTCATTGGGT CCGGCAGGCTCCGGGAAAAGGACTTGAATGGGTCGCCCGAATATATCCA ACAAATGGGTATACCCGCTATGCTGATTCCGTCAAAGGGAGGTTTACAA TTTCAGCTGATACCTC T AAAAAT AC TGCATATCTT C AAAT GAAT T C GC T CCGTGCTGAAGATACTGCTGTTTATTATTGTAGTCGCTGGGGGGGTGAT GGGTTTTATGCAATGGATGTGTGGGGCCAGGGTACTCTGGTTACCGTTT C AT C C AC C AC T AC GC C C GC C C C AAGAC C AC C C AC GC C T GC C C C AAC AAT T GC AAGC C AAC C C T TAT C C C T GAGAC C C GAAGC C T GC AGAC C C GC C GC G GGAGGC GC C GT GC AC AC AAGAGGC C T GGAC TTCGCCTGC GAC AT C T AC A TCTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGT GATCACCCTGTACTGCTAA11. [to be inserted in ST. 26 sequence listing] Adv 2transgene (AA)12. GGAAGC GGAGAGGGAAGGGGAAGC CTCCTCACATGTGGC GAT GT GGAAG T2A linker AAAAC C C C GGAC C C (NT)13. GSGEGRGSLLTCGDVEENPGP T2A linker (AA)14. AAAAAGAGCGGAGTGCTGTTCCTGCTGGGGATCATCTTGCTGGTCTTGA CXCL9 (NT)TCGGGGTGCAGGGCACCCCGGTGGTGCGCAAGGGCCGCTGTTCATGCAT C T C C AC C AAT C AGGGC AC C AT C C AT C T T C AGT C AC T GAAAGAC C T GAAA CAGTTCGCCCCGTCGCCTAGCTGCGAGAAGATCGAAATCATCGCGACCC T GAAGAAC GGC GT C C AAAC C T GC C T GAAT C C T GAC AGC GC AGAT GT GAA GGAAC T C AT C AAGAAAT GGGAAAAAC AGGT C AGC C AAAAGAAGAAGC AG AAAAAC GGAAAGAAGC AT C AGAAGAAGAAAGT C C T C AAAGT GC GC AAGT C AC AAC GC AGC AGAC AAAAAAAGAC T AC C15. KKSGVLFLLGIILLVLIGVQGTPWRKGRCSCISTNQGTIHLQSLKDLK CXCL9 (AA)QFAPSPSCEKIEIIATLKNGVQTCLNPDSADVKELIKKWEKQVSQKKKQ KNGKKHQKKKVLKVRKSQRSRQKKTT16. [to be inserted in ST. 26 sequence listing] Adenovirus 3(Adv3 ) 17. ATGGCCCTGCCCGTGACCGCACTGCTGCTACCCCTGGCGCTGCTGCTGC Adv 3ATGCCGCTAGACCCGAGGTGCAGCTGGTGGAGAGCGGAGGTGGCCTCGT transgene GC AGC C GGGC GGC AGC C T GAGAC T GAGC T GC GC C GC T AGC GGC T T C AAC (NT)AT C AAGGAC AC C T AC AT C C AC T GGGT GAGAC AAGC C C C C GGC AAGGGC CTGGAGTGGGTGGCTAGAATCTACCCCACCAACGGCTACACAAGATACGC307612002440C GAC AGC GT GAAGGGC AGAT TCACCATCAGCGCC GAC AC AAGC AAGAAC ACCGCCTACCT GC AGAT GAAC AGC C T GAGAGC C GAGGAC AC CGCCGTGT ACTACTGCAGCAGATGGGGCGGCGACGGCTTCTACGCCATGGACGTATG GGGACAAGGCACCCTTGTAACGGTCAGCAGCACCACTACGCCCGCCCCA AGAC CACCCACGCCTGCCC C AAC AAT T GC AAGC C AAC CCTTATCCCTGA GAC C C GAAGC C T GC AGAC C C GC C GC GGGAGGC GC C GT GC AC AC AAGAGG CCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCCCTGGCCGGCACC TGCGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCGGAAGCG GAGAGGGAAGGGGAAGC CTCCTCACATGTGGC GAT GT GGAAGAAAAC C C CGGACCCAAAAAGAGCGGAGTGCTGTTCCTGCTGGGGATCATCTTGCTG GTCTTGATCGGGGTGCAGGGCACCCCGGTGGTGCGCAAGGGCCGCTGTT C AT GC AT C T C C AC C AAT C AGGGC AC C AT C C AT C T T C AGT C AC T GAAAGA CCTGAAACAGTTCGCCCCGTCGCCTAGCTGCGAGAAGATCGAAATCATC GC GAC C C T GAAGAAC GGC GT C C AAAC C T GC C T GAAT C C T GAC AGC GC AG AT GT GAAGGAAC T C AT C AAGAAAT GGGAAAAAC AGGT C AGC C AAAAGAA GAAGC AGAAAAAC GGAAAGAAGC AT C AGAAGAAGAAAGT C C T C AAAGT G C GC AAGT C AC AAC GC AGC AGAC AAAAAAAGAC T AC C T AA18. [to be inserted in ST. 26 sequence listing] Adv 3transgene (AA)19. ATGGCCCTGCCCGTGACCGCACTGCTGCTACCCCTGGCGCTGCTGCTGC MembraneATGCCGCTAGACCCGAGGTGCAGCTGGTGGAGAGCGGAGGTGGCCTCGT bound Flare GC AGC C GGGC GGC AGC C T GAGAC T GAGC T GC GC C GC T AGC GGC T T C AAC (NT)AT C AAGGAC AC C T AC AT C C AC T GGGT GAGAC AAGC C C C C GGC AAGGGC C TGGAGTGGGTGGCTAGAATCTACCCCACCAACGGCTACACAAGATACGC C GAC AGC GT GAAGGGC AGAT TCACCATCAGCGCC GAC AC AAGC AAGAAC ACCGCCTACCT GC AGAT GAAC AGC C T GAGAGC C GAGGAC AC CGCCGTGT ACTACTGCAGCAGATGGGGCGGCGACGGCTTCTACGCCATGGACGTATG GGGACAAGGCACCCTTGTAACGGTCAGCAGCACCACTACGCCCGCCCCA AGAC CACCCACGCCTGCCC C AAC AAT T GC AAGC C AAC CCTTATCCCTGA GAC C C GAAGC C T GC AGAC C C GC C GC GGGAGGC GC C GT GC AC AC AAGAGG CCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCCCTGGCCGGCACC TGCGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGC20. [to be inserted in ST. 26 sequence listing] Adenovirus 4(Adv4 ) 21. ATGGGATGGAGCTGCATCATCCTATTCCTCGTGGCGACGGCCACTGGAG Adv 4TGCATAGCGAACTGGTGATGACTCAGTCCCCGTCATCCCTGACGGTGAC transgene CGCCGGCGAGAAGGTCACCATGTCGTGCAAGTCCTCGCAAAGCCTGCTT (NT) AAC AGC GGC AAC C AGAAAAAC TACCTCACGTGGTAT C AGC AAAAGC C AG GT C AAC C C C C AAAAC T GC T C AT C T AC T GGGC GAGC AC C C GC GAGT C GGG GGTGCCAGACCGGTTCACCGGCTCCGGGTCAGGAACTGATTTCACCCTA ACCATCAGCTCGGTGCAAGCGGAGGACCTGGCCGTGTACTACTGCCAAA ATGATTACTCGTACCCTCT GAC C T T T GGAGC GGGC AC C AAGC T C GAAAT CAAGGGCGGTGGAGGAAGCGGCGGGGGAGGCTCAGGTGGGGGAGGATCA GAAGT C C AAC T GC T GGAGC AGT CAGGAGC C GAAC TGGTCCGCCC GGGAA CCTCCGTCAAGATTTCCTGTAAGGCTTCCGGCTACGCTTTTACCAATTA CTGGCTGGGCTGGGTCAAGCAAAGACCGGGGCATGGCCTGGAGTGGATC GGC GAC AT C T T C C C AGGGAGC GGC AAC AT C C AC T AC AAC GAGAAGT T C A AGGGGAAAGC GAC T C T GAC T GC C GAC AAAT CATCCAGCACCGCCTACAT GCAGCTGTCGTCGCTCACTTTCGAAGACAGCGCGGTGTACTTTTGTGCT C GGC T C C GGAAC T GGGAT GAAC C AAT GGAC TACT GGGGAC AAGGAAC TA CCGTGACCGTCTCCTCCGGCGGAGGCGGATCCGGGGGTGGGGGATCTGA GGTGCAGCTGGTGGAGAGCGGAGGTGGCCTCGTGCAGCCGGGCGGCAGC C T GAGAC T GAGC TGCGCCGCTAGCGGCTT C AAC AT C AAGGAC AC C T AC A TCCACTGGGTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGCTAG AAT C T AC C C C AC C AAC GGC T AC AC AAGAT AC GC C GAC AGC GT GAAGGGC AGAT TCACCATCAGCGCC GAC AC AAGC AAGAAC AC CGCCTACCT GC AGA T GAAC AGC C T GAGAGC C GAGGAC AC CGCCGTGT AC TACT GC AGC AGAT G GGGCGGCGACGGCTTCTACGCCATGGACGTATGGGGACAAGGCACCCTT GT AAC GGT C AGC AGC GGAAGC GGAGAGGGAAGGGGAAGC CTCCTCACATGT GGC GAT GT GGAAGAAAAC C C C GGAC C C AAAAAGAGC GGAGT GC T GT T307612002440CCTGCTGGGGATCATCTTGCTGGTCTTGATCGGGGTGCAGGGCACCCCG GTGGTGCGCAAGGGCCGCTGTTCATGCATCTCCACCAATCAGGGCACCA TCCATCTTCAGTCACTGAAAGACCTGAAACAGTTCGCCCCGTCGCCTAG C T GC GAGAAGAT C GAAAT C AT C GC GAC C C T GAAGAAC GGC GT C C AAAC C T GC C T GAAT C C T GAC AGC GC AGAT GT GAAGGAAC T C AT C AAGAAAT GGG AAAAAC AGGT C AGC C AAAAGAAGAAGC AGAAAAAC GGAAAGAAGC AT C A GAAGAAGAAAGT C C T C AAAGT GC GC AAGT C AC AAC GC AGC AGAC AAAAA AAGACTACCTAA22. [to be inserted in ST. 26 sequence listing] Adv 4transgene (AA)23. [to be inserted in ST. 26 sequence listing] Adenovirus 5(Adv5 ) 24. ATGGCCCTGCCCGTGACCGCACTGCTGCTACCCCTGGCGCTGCTGCTGC Adv 5ATGCCGCTAGACCCGAAGTACAATTAGTCGAATCAGGGGGCGGGCTGGT transgene TCAACCCGGTGGGTCATTACGCTTGTCTTGTGCAGCCTCAGGATTTAAT (NT) ATTAAAGATACTTATATTCATTGGGTCCGGCAGGCTCCGGGAAAAGGAC TTGAATGGGTCGCCCGAATATATCCAACAAATGGGTATACCCGCTATGC TGATTCCGTCAAAGGGAGGTTTACAATTTCAGCTGATACCTCTAAAAAT ACTGCATATCTTCAAATGAATTCGCTCCGTGCTGAAGATACTGCTGTTT ATTATTGTAGTCGCTGGGGGGGTGATGGGTTTTATGCAATGGATGTGTG GGGCCAGGGTACTCTGGTTACCGTTTCATCCACCACTACGCCCGCCCCA AGAC CACCCACGCCTGCCC C AAC AAT T GC AAGC C AAC CCTTATCCCTGA GAC C C GAAGC C T GC AGAC C C GC C GC GGGAGGC GC C GT GC AC AC AAGAGG CCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCCCTGGCCGGCACC TGCGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCGGCTCCG GCGCTACCAATTTCTCTCTGCT GAAGC AAGC T GGC GAC GT C GAAGAAAA TCCCGGACCCGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTG CTGCTCCACGCGGCCCGCCCGGAGGTGCAGCTGGTGGAGAGCGGAGGTG GCCTCGTGCAGCCGGGCGGCAGCCTGAGACTGAGCTGCGCCGCTAGCGG C T T C AAC AT C AAGGAC AC C T AC AT C C AC T GGGT GAGAC AAGC C C C C GGC AAGGGC C T GGAGT GGGT GGC T AGAAT C T AC C C C AC C AAC GGC T AC AC AA GAT AC GC C GAC AGC GT GAAGGGC AGAT TCACCATCAGCGCC GAC AC AAG C AAGAAC AC CGCCTACCT GC AGAT GAAC AGC C T GAGAGC C GAGGAC AC C GCCGTGTACTACTGCAGCAGATGGGGCGGCGACGGCTTCTACGCCATGG ACGTATGGGGACAAGGCACCCTTGTAACGGTCAGCAGCACGACGACACC T GC C C C AAGGC C T C C AAC AC C AGC T C C T AC AAT AGC AT C AC AGC C T T T G AGCTTGCGGCCGGAGGCATGCAGGCCAGCCGCAGGGGGTGCGGTTCATA CACGAGGGCTTGATTTTGCCTGCGACATATACATATGGGCACCTCTGGC AGGTACGTGCGGAGTCCTCCTCCTGAGCCTTGTCATAACGTTGTACTGT GGAAGC GGAGAGGGAAGGGGAAGC CTCCTCACATGTGGC GAT GT GGAAG AAAAC C C C GGAC C C AAAAAGAGC GGAGT GC TGTTCCTGCTGGGGATCAT CTTGCTGGTCTTGATCGGGGTGCAGGGCACCCCGGTGGTGCGCAAGGGC CGCTGTTCATGCATCTCCACCAATCAGGGCACCATCCATCTTCAGTCAC T GAAAGAC C T GAAAC AGT T CGCCCCGTCGCCTAGCTGC GAGAAGAT C GA AATCATCGCGACCCTGAAGAACGGCGTCCAAACCTGCCTGAATCCTGAC AGC GC AGAT GT GAAGGAAC T C AT C AAGAAAT GGGAAAAAC AGGT C AGC C AAAAGAAGAAGC AGAAAAAC GGAAAGAAGC AT C AGAAGAAGAAAGT C C T C AAAGT GC GC AAGT C AC AAC GC AGC AGAC AAAAAAAGAC T AC C T AA25. [to be inserted in ST. 26 sequence listing] Adv 5transgene (AA)26. GCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACG MembraneCGGCCCGCCCGGAGGTGCAGCTGGTGGAGAGCGGAGGTGGCCTCGTGCA bound Flare GC C GGGC GGC AGC C T GAGAC T GAGC T GC GC C GC T AGC GGC T T C AAC AT C (NT) AAGGAC AC C T AC AT C C AC T GGGT GAGAC AAGC C C C C GGC AAGGGC C T GG AGT GGGT GGC T AGAAT C T AC C C C AC C AAC GGC T AC AC AAGAT AC GC C GA C AGC GT GAAGGGC AGAT TCACCATCAGCGCC GAC AC AAGC AAGAAC AC C GC C T AC C T GC AGAT GAAC AGC C T GAGAGC C GAGGAC AC CGCCGTGTACT ACTGCAGCAGATGGGGCGGCGACGGCTTCTACGCCATGGACGTATGGGGAC AAGGC AC C C T T GT AAC GGT C AGC AGC AC GAC GAC AC C T GC C C C AAGG307612002440CCTCCAACACCAGCTCCTACAATAGCATCACAGCCTTTGAGCTTGCGGC CGGAGGCATGCAGGCCAGCCGCAGGGGGTGCGGTTCATACACGAGGGCT TGATTTTGCCTGCGACATATACATATGGGCACCTCTGGCAGGTACGTGC GGAGTCCTCCTCCTGAGCCTTGTCATAACGTTGTACTGT27. DYKDDDDK FLAG tag sequence (AA)28. E VQLVE S GGGLVQP GGS LRL S CAAS GEN I KD T Y I HWVRQAP GKGLE WVA heavy chain RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR variable WGGDGF YAMD VWGQGT LVT VS S domain of Abl29. TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI YIW CD8APLAGTCGVLLLSLVITLYC transmembran e (AA) 30. ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQP Anti-EpCAM PKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDY scFv SYPLTFGAGTKLEIKGGGGSGGGGSGGGGSEVQLLEQSGAELVRPGTSV KI SCKASGYAFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGK ATLTADKSSSTAYMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVT VSS31. ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQP Anti-EpCAM PKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDY scFv VH SYPLTFGAGTKLEIK32. EVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLEWI Anti-EpCAM GDIFPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLTFEDSAVYFCA scFv VL RLRNWDEPMDYWGQGTTVTVSS33. GGGGSGGGGS 2x GS linker amino acid sequence 34. MGWSCIILFLVATATGVHS IgGl signal sequence 35. ATNF S LLKQAGDVEENPGP P2A linker (AA)36. MALPVTALLLPLALLLHAARP CD8 leader sequence 37. GGCGGTGGAGGAAGCGGCGGGGGAGGCTCAGGTGGGGGAGGATCA Linker nucleic acid sequence 38. GGGGSGGGGSGGGGS Linker amino acid sequence 39. GAAGT C C AAC T GC T GGAGC AGT C AGGAGC C GAAC TGGTCCGCCC GGGAA Anti-EpCAM CCTCCGTCAAGATTTCCTGTAAGGCTTCCGGCTACGCTTTTACCAATTA VL nucleic CTGGCTGGGCTGGGTCAAGCAAAGACCGGGGCATGGCCTGGAGTGGATC acidGGC GAC AT C T T C C C AGGGAGC GGC AAC AT C C AC T AC AAC GAGAAGT T C A sequence AGGGGAAAGC GAC T C T GAC T GC C GAC AAAT CATCCAGCACCGCCTACAT GCAGCTGTCGTCGCTCACTTTCGAAGACAGCGCGGTGTACTTTTGTGCT C GGC T C C GGAAC T GGGAT GAAC C AAT GGAC TACT GGGGAC AAGGAAC TA CCGTGACCGTCTCCTCC40. GAACTGGTGATGACTCAGTCCCCGTCATCCCTGACGGTGACCGCCGGCG Anti-EpCAM AGAAGGTCACCATGTCGTGCAAGTCCTCGCAAAGCCTGCTTAACAGCGG VH nucleic C AAC C AGAAAAAC TACCTCACGTGGTAT C AGC AAAAGC C AGGT C AAC C C acid CCAAAACTGCTCATCTACTGGGCGAGCACCCGCGAGTCGGGGGTGCCAG sequence ACCGGTTCACCGGCTCCGGGTCAGGAACTGATTTCACCCTAACCATCAG CTCGGTGCAAGCGGAGGACCTGGCCGTGTACTACTGCCAAAATGATTAC TCGTACCCTCT GAC C T T T GGAGC GGGC AC C AAGC T C GAAAT C AAG41. MGWSCIILFLVATATGVHSE L VMT Q S P S S L T VT AGE KVTM S C K S S Q S L L Flarel NSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTL (FL1 ) - TISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEIKGGGGSGGGGSGGGGS antibodyEVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLEWI conjugate307612002440GDIFPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLTFEDSAVYFCA variant RLRNWDEPMDYWGQGTTVTVSSGGGGSGGGGSEVQLVESGGGLVQPGGS (with signal LRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARI YPTNGYTRYADSVKG peptide, RFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTL bold and VTVSS underlined) 42. NYWLG Anti-EpCAM CDR-H1 43. DIEP G S GN I H YNE KF KG Anti-EpCAM CDR-H2 44. LRNWDEPMDY Anti-EpCAM CDR-H3 45. KSSQSLLNSGNQKNYLT Anti-EpCAM CDR-L1 46. WASTRES Anti-EpCAM CDR-L2 47. QNDYSYPLT Anti-EpCAM CDR-L3 48. GGATGGAGCTGCATCATCCTATTCCTCGTGGCGACGGCCACTGGAGTGC ATAGCGAACTGGTGATGACTCAGTCCCCGTCATCCCTGACGGTGACCGC CGGCGAGAAGGTCACCATGTCGTGCAAGTCCTCGCAAAGCCTGCTTAAC AGCGGCAACCAGAAAAACTACCTCACGTGGTATCAGCAAAAGCCAGGTC AAC C C C C AAAAC T GC T C AT C T AC T GGGC GAGC AC C C GC GAGT C GGGGGT GCCAGACCGGTTCACCGGCTCCGGGTCAGGAACTGATTTCACCCTAACC ATCAGCTCGGTGCAAGCGGAGGACCTGGCCGTGTACTACTGCCAAAATG AT T AC T C GT AC C C T C T GAC C T T T GGAGC GGGC AC C AAGC T C GAAAT C AA GGGCGGTGGAGGAAGCGGCGGGGGAGGCTCAGGTGGGGGAGGATCAGAA GTCCAACTGCTGGAGCAGTCAGGAGCCGAACTGGTCCGCCCGGGAACCT CCGTCAAGATTTCCTGTAAGGCTTCCGGCTACGCTTTTACCAATTACTG GCTGGGCTGGGTCAAGCAAAGACCGGGGCATGGCCTGGAGTGGATCGGC GAC AT C T T C C C AGGGAGC GGC AAC AT C C AC T AC AAC GAGAAGT T C AAGG GGAAAGC GAC T C T GAC T GC C GAC AAAT CATCCAGCACCGCCTACATGCA GCTGTCGTCGCTCACTTTCGAAGACAGCGCGGTGTACTTTTGTGCTCGG C T C C GGAAC T GGGAT GAAC C AAT GGAC TACT GGGGAC AAGGAAC T AC C G Anti-EpCAM TGACCGTCTCCTCC scFv (NT) 4 9. ctatctatataatataccttatagatggaatggtgccaatatgtaaatg aggtgattttaaaaagtgtggatcgtgtggtgattggctgtggggttaa cggctaaaaggggcggtgcgaccgtgggaaaatgacgttttgtgggggt ggagtttttttgcaagttgtcgcgggaaatgtgacgcataaaaaggctt ttttctcacggaactacttagttttcccacggtatttaacaggaaatga ggtagttttgaccggatgcaagtgaaaattgttgattttcgcgcgaaaa ctgaatgaggaagtgtttttctgaataatgtggtatttatggcagggtg gagtatttgttcagggccaggtagactttgacccattacgtggaggttt cgattaccgtgttttttacctgaatttccgcgtaccgtgtcaaagtctt ctgtttttacgtaggtgtcagctgatcgctagggtatttatacctcagg gtttgtgtcaagaggccactcttgagtgccagcgagaagagttttctcc tctgcgccggcagtttaataataaaaaaatgagagatttgcgatttctg cctcaggaaataatctctgctgagactggaaatgaaatattggagcttgAd3 / llp tggtgcacgccctgatgggagacgatccggagccacctgtgcagctttt(ColoAdl ) tgagcctcctacgcttcaggaactgtatgatttagaggtagagggatcg gaggattctaatgaggaagctgtaaatggcttttttaccgattctatgc ttttagctgctaatgaagggttagaattagatccgcctttggacacttt tgatactccaggggtaattgtggaaagcggtacaggtgtaagaaaatta cctgatttgagttccgtggactgtgatttgcactgctatgaagacgggt ttcctccgagtgatgaggaggaccatgaaaaggagcagtccatgcagac tgcagcgggtgagggagtgaaggctgccaatgttggttttcagttggat tgcccggagcttcctggacatggctgtaagtcttgtgaatttcacagga aaaatactggagtaaaggaactgttatgttcgctttgttatatgagaac gcactgccactttatttacagtaagtgtgtttaagttaaaatttaaagg aatatgctgtttttcacatgtatattgagtgtgagttttgtgcttcttattataggtcctgtgtctgatgctgatgaatcaccatctcctgattctactacctcacctcctgagattcaagcacctgttcctgtggacgtgcgcaag307612002440c...

Claims

307612002440WHAT IS CLAIMED:

1. An adenovirus comprising a sequence of formula [I]:5’ ITR-BI-BA-B2-BX-BB-BY-B3-3’ ITR (I)wherein:Bi is bond or comprises: El A, E1B or E1A-E1B;BA comprises-E2B -L 1 -L2-L3 -E2 A-L4;B2 is a bond or comprises: E3;Bx is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both;BB comprises L5;BY is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both;B3 is a bond or comprises: E4;wherein the adenovirus comprises (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a membrane targeting domain or a synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule and (ii) a second transgene sequence encoding CXCL9.

2. The adenovirus of claim 1, wherein the first transgene sequence and the second transgene sequence are encoded in position BY.

3. An adenovirus comprising a polynucleotide located between the virus fibre gene L5 and the virus E4 gene, wherein the polynucleotide comprises (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a membrane targeting domain and (ii) a second transgene sequence encoding CXCL9.

4. The adenovirus of any of claims 1-3, wherein the first transgene sequence and the second transgene sequence are encoded from the same transgene cassette.

5. The adenovirus of any of claims 1-4, wherein the first transgene sequence and the second transgene sequence are separated by a nucleotide sequence encoding a cleavable linker or translational skipping sequence in the transgene cassette.

6. The adenovirus of any of claims 1, 2, 4 and 5, wherein the adenovirus comprises the first transgene sequence encoding the synthetic cancer antigen comprising the target domain and the membrane targeting domain and (ii) a second transgene sequence encoding CXCL9.3076120024407. The adenovirus of any of claims 1, 2, 4 and 5, wherein the adenovirus comprises the first transgene sequence encoding the synthetic cancer antigen comprising the target domain and the tumor-targeting binding molecule and (ii) a second transgene sequence encoding CXCL9.

8. The adenovirus of any of claims 1-7, wherein the first transgene sequence and the second transgene sequence are independently encoded in a region selected from El, E3, Bx, By and combinations thereof.

9. The adenovirus of any one of claims 1-8, wherein the first transgene sequence and the second transgene sequence are encoded in position By.

10. An adenovirus comprising a polynucleotide located between the virus fibre gene L5 and the virus E4 gene, wherein the polynucleotide comprises (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule and (ii) a second transgene sequence encoding CXCL9.

11. The adenovirus of any of claims 7-10, wherein the first transgene sequence and the second transgene sequence are encoded from the same transgene cassette.

12. The adenovirus of any of claims 7-11, wherein the first transgene sequence and the second transgene sequence are separated by a nucleotide sequence encoding a cleavable linker or translational skipping sequence in the transgene cassette.

13. The adenovirus of claim 6 or claim 12, wherein the cleavable linker or translational skipping sequence is a self-cleaving linker.

14. The adenovirus of claim 13, wherein the self-cleaving linker is or comprises a 2A peptide.

15. An adenovirus comprising a sequence of formula [I]:5’ ITR-BI-BA-B2-BX-BB-BY-B3-3’ ITR (I)wherein:Bi is bond or comprises: El A, E1B or E1A-E1B;BA comprises-E2B -L 1 -L2-L3 -E2 A-L4;B2is a bond or comprises: E3;307612002440Bx is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both;BB comprises L5;By is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both;B3 is a bond or comprises: E4;wherein the adenovirus comprises: (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a membrane targeting domain, (ii) a second transgene sequence encoding a synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule; and (iii) a third transgene sequence encoding CXCL9.

16. The adenovirus of claim 15, wherein the first transgene sequence, the second transgene sequence and the third transgene sequence are independently encoded in a region selected from El, E3, Bx, BY and combinations thereof.

17. The adenovirus of claim 16, wherein the first transgene sequence, the second transgene sequence and the third transgene sequence are encoded in position BY.

18. An adenovirus comprising a polynucleotide located between the virus fibre gene L5 and the virus E4 gene, wherein the polynucleotide comprises (i) a first transgene sequence encoding a synthetic cancer antigen comprising a target domain and a membrane targeting domain, (ii) a second transgene sequence encoding a synthetic cancer antigen comprising a target domain and a tumortargeting binding molecule; and (iii) a third transgene sequence encoding CXCL9.

19. The adenovirus of any of claims 15-18, wherein the first transgene sequence, the second transgene sequence and the third transgene sequence are encoded from the same transgene cassette.

20. The adenovirus of any of claims 15-19, wherein, in the transgene cassette, the first and second transgene sequence are separated by a nucleotide sequence encoding a first cleavable linker or translational skipping sequence and the second and third transgene sequence are separated by a nucleotide sequence encoding a second cleavable linker or translational skipping sequence.

21. The adenovirus of claim 20, wherein the first cleavable linker or translational skipping sequence and the second cleavable linker are different.30761200244022. The adenovirus of claim 20 or claim 21, wherein each cleavable linker or translational skipping sequence is a self-cleaving linker.

23. The adenovirus of claim 22, wherein the self-cleaving linker independently is or comprises a 2A peptide.

24. The adenovirus of any of claims 5, 6, 11-14 and 19-23, wherein the transgene or transgene cassettes is / are under the control of an endogenous or exogenous promoter.

25. The adenovirus of claim 24, wherein the transgene or transgene cassettes is / are under the control of an endogenous promoter that is an E4 promoter or a major late promoter.

26. The adenovirus of claim 24, wherein the transgene or transgene cassettes is / are under the control of an exogenous promoter that is a CMV promoter.

27. The adenovirus of any of claims 1-26 that is replication competent.

28. The adenovirus of any of claims 1-26, wherein the target domain is not expressed on the surface of a non-cancer cell of a subject.

29. The adenovirus of any of claims 1-28, wherein the target domain is an antibody or antibody fragment that is recognized by a cognate binder.

30. The adenovirus of any of claims 1-29, wherein the target domain is derived from a 4D5 anti-HER2 antibody.

31. The adenovirus of claim 29 or claim 30, wherein the target domain is the variable domain of the heavy chain of an antibody.

32. The adenovirus of claim 31, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 28.

33. The adenovirus of claim 31 or claim 32, wherein the target domain comprises the amino acid sequence of SEQ ID NO: 28.30761200244034. The adenovirus of any of claims 7-33, wherein the synthetic cancer antigen comprising the target domain and the tumor-targeting binding molecule is soluble.

35. The adenovirus of any of claims 7-33 and 34, wherein the tumor-targeting binding molecule specifically binds to a tumor associated antigen expressed on the surface of a tumor cell.

36. The adenovirus of claim 35, 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.

37. The adenovirus of claim 35 or claim 36, wherein the tumor cell is a tumor cell of a solid tumor.

38. The adenovirus of any of claims 35-37, 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.

39. The adenovirus of any of claims 35-38, 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.

40. The adenovirus of any of claims 7-33 and 34-39, wherein the tumor-targeting binding molecule comprises an antibody or an antigen-binding fragment thereof.

41. The adenovirus of claim 40, wherein the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).30761200244042. The adenovirus of any of claims 35-41, wherein the tumor associated antigen is EpCAM.

43. The adenovirus of any of claims 40-42, 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: 42, 43, and 44, 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: 45, 46, and 47, respectively.

44. The adenovirus of any of claims 40-43, 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: 32; 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: 31.

45. The adenovirus of any of claims 40-44, 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: 32, and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 31.

46. The adenovirus of any of claims 7-33 and 34-45, wherein the target domain and the tumor-targeting binding molecule are linked by a linker.

47. The adenovirus of claim 46, 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.30761200244048. The adenovirus of claim 46 or claim 47, wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs:33, 38 and 50-69.

49. The adenovirus of any of claims 7-48, wherein the target domain is not expressed on the surface of a non-cancer cell of a subject.

50. The adenovirus of any of claims 1-49, wherein the target domain is recognizable by a cognate binder.

51. The adenovirus of any of claims 29-50, wherein the cognate binder binds to the idiotype of the antibody or antibody fragment.

52. The adenovirus of claim 51, wherein the cognate binder is an anti -idiotype antibody or an antigen binding fragment.

53. The adenovirus of any of claims 29-52, wherein the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 71.

54. The adenovirus of any of claims 29-53, wherein the cognate binder comprises the amino acid sequence of SEQ ID NO:71.

55. The adenovirus of any of claims 29-52, wherein the cognate binder is encoded by a nucleotide sequence that is at least 85% identical to SEQ ID NO: 70.

56. The adenovirus of any of claims 29-52 and 55, wherein the cognate binder is encoded by the nucleotide sequence of SEQ ID NO:70.

57. The adenovirus of any of claims 50-56, wherein the cognate binder of the synthetic cancer antigen is the extracellular domain of a chimeric antigen receptor (CAR).

58. The adenovirus of claim 57, wherein the CAR further comprises a transmembrane domain and an intracellular signaling domain.

59. The adenovirus of claim 57 or claim 58, wherein the CAR is expressed on the surface of an immune effector cell.30761200244060. The adenovirus of claim 59, wherein the immune effector cell is a T cell, optionally a cytotoxic T cell.

61. The adenovirus of claim 59, wherein the immune effector cell is a natural killer cell.

62. The adenovirus of any of claims 1-61, wherein the adenovirus comprises a nucleic acid sequence that is at least 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: 1, 9, 16, 20 or 23.

63. The adenovirus of any of claims 1-62, wherein the adenovirus comprises the nucleic acid sequence of SEQ ID NOs: 1, 9, 16, 20 or 23.

64. A pharmaceutical composition comprising the adenovirus vector of any of claims 1-63.

65. The pharmaceutical composition of claim 64, wherein the pharmaceutical composition further comprises a pharmaceutical acceptable carrier.

66. The pharmaceutical composition of claim 64 or claim 65, for use in treating a cancer in a subject.

67. A kit comprising the pharmaceutical composition of any one of claims 64-66 and instructions for using the adenovirus.

68. A method of tagging a tumor cell in vivo, comprising contacting a tumor cell with the adenovirus vector of any of claims 1-63, or the pharmaceutical composition of any of claims 64-66, wherein the tumor cell is tagged with a synthetic cancer antigen expressed from the adenovirus vector.

69. A method of tagging a tumor cell of a subject having a cancer, comprising administering a therapeutically effective amount of the adenovirus vector of any of claims 1-63, or the pharmaceutical composition of any of claims 64-66, to a subject, wherein the tumor cell is tagged with a synthetic cancer antigen expressed from the adenovirus vector.

70. The method of claim 68 or claim 69, wherein the cancer is a blood cancer.30761200244071. The method of claim 68 or claim 69, wherein the cancer is a solid tumor cancer.

72. The method of any of claims 68-71, wherein the synthetic cancer antigen comprises a target domain and a membrane targe ti ng domain.

73. The method of any of claims 68-71, wherein the synthetic cancer antigen comprises a target domain and a tumor-targeting domain.

74. A method of killing a tumor cell in vivo, comprising administering to a subject having a tumor a therapeutically effective amount of the adenovirus vector of any of claims 1-63, or the pharmaceutical composition of any of claims 64-66.

75. A method of treating a cancer in a subject, comprising administering to a subject having a cancer a therapeutically effective amount of the adenovirus vector of any of claims 1-63, or the pharmaceutical composition of any of claims 64-66.

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