Synthetic cancer antigens as targets for treating cancer

Abstract

Synthetic cancer antigens and / or their cognitive binders that do not appear on the surface of non-cancer cells are provided herein. Pharmaceutical compositions comprising synthetic cancer antigens and / or cognitive binders, as well as methods for using them to treat cell proliferation disorders such as cancer, are also provided herein. TIFF2026520446000043.tif104170

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 504,394, filed May 25, 2023, and U.S. Provisional Application No. 63 / 552,061, filed Feb. 9, 2024, each titled "SYNTHETIC CANCER ANTIGENS AS TARGETS FOR TREATING CANCERS", the contents of which are hereby incorporated by reference in their entirety.

[0002] Reference to Electronic Sequence Listing This application is filed with an electronic sequence listing. The sequence listing is provided as a file named 307612000140SeqList.XML, which is 151,676 bytes in size and was created on May 24, 2024. The information in the electronic form of the sequence listing is hereby incorporated by reference in its entirety.

[0003] Field The present disclosure generally relates to synthetic cancer antigens and / or their cognate binders, and the use of such molecules for treating different types of cancers.

Background Art

[0004] Background Adoptive transfer of T lymphocytes carrying modified chimeric antigen receptors (CARs) against tumor-associated antigens (TAAs) has shown promising results for several hematological malignancies (Rossig et al., Mol Ther. (2004) 10(1):5-18 (Non-Patent Literature 1)). Despite its potential, these therapies have not been well adapted for the treatment of solid tumors due to several factors. Key factors include immunosuppression of CAR-Ts in the tumor microenvironment, limited migration of T cells to tumor sites, and, most importantly, "on-target, off-tumor" toxicity (Wang et al., Front Med. (2020) 14(6):726-45 (Non-Patent Literature 2); Tahmasebi et al., Stem Cell Rev Rep. (2019) 15(5):619-36 (Non-Patent Literature 3)). Cell therapies, including those for the treatment of solid tumors, require improvements in systems and methods. Embodiments that satisfy such needs are provided herein. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Rossig et al., Mol Ther. (2004) 10(1):5-18 [Non-Patent Document 2] Wang et al., Front Med. (2020) 14(6):726-45 [Non-Patent Document 3] Tahmasebi et al., Stem Cell Rev Rep. (2019) 15(5):619-36 [Overview of the Initiative]

[0006] overview This disclosure provides synthetic cancer antigens and / or their cognitive binders. Synthetic cancer antigens are not expressed on the surface of non-cancer cells but can be expressed on the surface of cancer cells because they are specifically delivered to cancer cells in a target via oncolytic viruses. Alternatively, cognitive binders can be manipulated as chimeric antigen receptors for expression on the surface of immune effector cells (e.g., T cells), such as for use in adoptive cell therapy. This disclosure redesigns the interaction between immune effector cells and cancer targets. By shifting from traditional approaches that target known cancer overexpression proteins that can also be expressed on the surface of non-cancer cells, this disclosure improves the safety of immunotherapy by eliminating recognition by healthy tissue. Furthermore, because the synthetic cancer antigens disclosed herein can be delivered to different types of cancer, this disclosure provides a universal approach for treating different types of cancer (e.g., solid tumors).

[0007] In some aspects, synthetic cancer antigens comprising a target domain and a transmembrane domain are provided herein. In some embodiments, the target domain is not expressed on the surface of the target non-cancer cells. In some embodiments, the target domain is recognizable by a cognitive binder.

[0008] In some of the embodiments provided, the target domain is an antibody or antibody fragment. In some of the embodiments provided, the cognitive binder binds to the idiotype of the antibody or antibody fragment. In some of the embodiments provided, the cognitive binder is an anti-idiotype antibody or antigen-binding fragment. In certain embodiments, the anti-idiotype antibody is an antigen-binding fragment containing a variable heavy chain and a variable light chain, such as a single-stranded variable chain (scFv). In some embodiments, the anti-idiotype antibody or antigen-binding fragment, which is the cognitive binder, is part of the extracellular domain of a chimeric antigen receptor (CAR). In certain embodiments, the extracellular domain of the CAR contains the variable heavy chain and variable light chain of the anti-idiotype antibody for recognition by binding to the target domain. In some embodiments, the variable heavy chain and variable light chain of the anti-idiotype antibody in the extracellular domain of the CAR exist as scFv.

[0009] In some of the embodiments provided, the antibody or antibody fragment of the target domain does not contain all or part of the heavy chain constant region and optionally does not contain the CH1, CH2, and / or CH3 domains. In some of the embodiments provided, the antibody or antibody fragment of the target domain does not contain the heavy chain CH3 domain. In some embodiments, the target domain is an antibody fragment such as a Fab, scFv, or a fragment of only the variable heavy chain. In some embodiments, the target domain is unable to bind to or substantially bind to the antigen of the antibody from which it is derived, or exhibits reduced (low) binding affinity to it (e.g., a higher KD value). For example, in some embodiments, the antigen-binding domain for recognition of the antigen or epitope is absent, partially removed (e.g., via one or more amino acid substitutions in the CDR), or non-functional. In an exemplary embodiment, the parent antibody is a double-chain antibody containing a variable heavy chain and a variable light chain, with the antigen-binding domain consisting of six CDRs. In some such embodiments, the target domain is an antibody fragment consisting only of a variable heavy chain (VH) made up of only three CDRs.

[0010] In some embodiments, the target domain is derived from the trastuzumab parent antibody, the 4D5 anti-HER2 antibody (also known as Ab1). In some embodiments, the target domain is an antibody fragment of the 4D5 (Ab1) antibody.

[0011] In some embodiments, the antibody fragment is a Fab. In some of the embodiments provided, the target domain comprises a light chain having an amino acid sequence at least 85% identical to SEQ ID NO:7 and a heavy chain having an amino acid sequence at least 85% identical to SEQ ID NO:10. In some of the embodiments provided, the target domain comprises a light chain having an amino acid sequence of SEQ ID NO:7 and a heavy chain having an amino acid sequence of SEQ ID NO:10.

[0012] In some of the embodiments provided, the target domain is a single-stranded variable fragment (scFv). In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:11. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO:11.

[0013] In some of the embodiments provided, the target domain is a variable domain of the antibody heavy chain. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:8. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO:8.

[0014] In certain embodiments, the cognitive binder is an anti-idiotype antibody that binds to an epitope of a 4D5 (Ab1) antibody or antibody fragment. In certain embodiments, the epitope of the target domain recognized by the anti-idiotype antibody is located in the variable heavy chain of the 4D5 (Ab1) antibody or antibody fragment. In some embodiments, the cognitive binder is an antigen-binding fragment of an anti-idiotype antibody that binds to a 4D5 (Ab1) antibody, including a variable heavy chain and a variable light chain, such as a single-stranded variable chain (scFv). In some embodiments, the cognitive binder is a single-domain anti-idiotype antibody that binds to a 4D5 (Ab1) antibody, including a variable heavy chain and a variable light chain, such as a single-stranded variable chain (scFv). In some embodiments, the anti-idiotype antibody, such as the antigen-binding fragment of an anti-idiotype antibody that is the cognitive binder, is part of the extracellular domain of a chimeric antigen receptor (CAR).

[0015] In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:13. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:13. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO:13. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 95% identical to SEQ ID NO:13. In some of the embodiments provided, the cognitive binder contains the amino acid sequence of SEQ ID NO:13. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:14. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO:14. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 95% identical to SEQ ID NO:14. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:14. In some of the embodiments provided, the cognitive binder contains the amino acid sequence of SEQ ID NO:14. In any of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:15. In any of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO:15.In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 95% identical to SEQ ID NO:15. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:15. In some of the embodiments provided, the cognitive binder contains the amino acid sequence of SEQ ID NO:15.

[0016] In some of the embodiments provided, the target domain is a fragment of an intracellular protein. In some of the embodiments provided, the target domain is a non-DNA-binding domain or an inactive leucine zipper domain of a transcription factor. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to one of SEQ ID NO: 17-19 and 21-23. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 17. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO: 17. In some of the embodiments provided, the cognitive binder of the synthetic cancer antigen contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 24 or 25. In some of the embodiments provided, the cognitive binder of the synthetic cancer antigen contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 24 or 25. In some of the embodiments provided, the target domain is the active leucine zipper domain of a transcription factor. In some of the embodiments provided, the target domain is the leucine zipper domain of ATF. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:26. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO:26.

[0017] In some of the embodiments provided, the target domain is the leucine zipper domain of JUN. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:27. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO:27.

[0018] In some of the embodiments provided, the cognitive binder of the synthetic cancer antigen is a leucine zipper domain. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:28. In some of the embodiments provided, the cognitive binder contains the amino acid sequence of SEQ ID NO:28. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:29. In some of the embodiments provided, the cognitive binder contains the amino acid sequence of SEQ ID NO:29.

[0019] In some of the embodiments provided, the cognitive binder of the synthetic cancer antigen is the extracellular domain of the chimeric antigen receptor (CAR). In some of the embodiments provided, the CAR further comprises a transmembrane domain and an intracellular signaling domain. In some of the embodiments provided, the CAR is expressed on the surface of immune effector cells. In some of the embodiments provided, the immune effector cells are cytotoxic T cells. In some of the embodiments provided, the immune effector cells are natural killer cells. In some of the embodiments provided, the membrane targeting domain is a transmembrane domain. In some of the embodiments provided, the transmembrane domain is derived from a transmembrane glycoprotein. In some of the embodiments provided, the transmembrane domain is a CD8 transmembrane domain. In some of the embodiments provided, the transmembrane domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:3. In some of the embodiments provided, the transmembrane domain contains the amino acid sequence of SEQ ID NO:3.

[0020] In some aspects, synthetic cancer antigens comprising a target domain and a tumor targeting binding molecule are provided herein. In some embodiments, the synthetic cancer antigen is soluble and not expressed on a membrane. In some embodiments, the tumor targeting binding molecule specifically binds to tumor-associated antigens expressed on the surface of tumor cells. In any of the embodiments provided, the tumor-associated antigen is selected from the group consisting of EpCAM, CEA (carcinoembryonic antigen), gpA33 (glycoprotein A33 (transmembrane)), mucin, TAG-72 (tumor-associated glycoprotein 72), CAIX (carbonic anhydrase IX), PSMA (prostate-specific membrane antigen), and FBP (folate-binding protein), EGFR / ERBB1 / HER1 (epidermal growth factor receptor 1), ERBB2 / HER2 (epidermal growth factor receptor 2), ERBB3 (epidermal growth factor receptor 3), MET (tyrosine protein kinase IGF1R (insulin-like growth factor 1 receptor)), EPHA3 (EPH receptor A3), TRAILR1 (death receptor 4), RANK-L (nuclear factor kappa-B receptor activator ligand), claudin 6, claudin 182, GPC2, and GPC3.

[0021] In some of the embodiments provided, tumor cells are tumor cells of a solid tumor. In some of the embodiments provided, tumor cells are tumor cells 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, 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, and / or ovarian cancer. In any of the embodiments provided, the tumor cells are tumor cells of cancers 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.

[0022] In some of the embodiments provided, the tumor cell targeting domain comprises an antibody or its antigen-binding fragment. In some of the embodiments provided, the antibody or its antigen-binding fragment is a single-chain variable fragment (scFv). In some of the embodiments provided, the tumor-associated antigen is EpCAM. In some of the embodiments provided, the antibody or its antigen-binding fragment comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH region comprises heavy-chain complementarity-determining regions 1 (CDR-H1), CDR-H2, and CDR-H3, each containing amino acid sequences shown in SEQ ID NO: 42, 43, and 44, respectively, and the VL region comprises light-chain complementarity-determining regions 1 (CDR-L1), CDR-L2, and CDR-L3, each containing amino acid sequences shown in SEQ ID NO: 46, 47, and 48, respectively. In some of the embodiments provided, the antibody or its antigen-binding fragment includes a heavy chain variable (VH) region containing 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 with the amino acid sequence of SEQ ID NO:35, and a light chain variable (VL) region containing 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 with the amino acid sequence of SEQ ID NO:37.

[0023] In some of the embodiments provided, 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 with the amino acid sequence of SEQ ID NO:31. In some of the embodiments provided, 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 with the amino acid sequence of SEQ ID NO:39. In some of the embodiments provided, 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 with the amino acid sequence of SEQ ID NO:39.

[0024] In some of the embodiments provided, 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 with the amino acid sequence of SEQ ID NO:88. In some of the embodiments provided, 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 with the amino acid sequence of SEQ ID NO:89. In some of the embodiments provided, 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 with the amino acid sequence of SEQ ID NO:89.

[0025] In some of the embodiments provided, the target domain is not expressed on the surface of the target non-cancer cells. In some of the embodiments provided, the target domain is recognizable by the cognitive binder. In some of the embodiments provided, the target domain is an antibody or antigen-binding fragment recognized by the cognitive binder. In some of the embodiments provided, the cognitive binder binds to the idiotype of the antibody or antigen-binding fragment. In some of the embodiments provided, the cognitive binder is an anti-idiotype antibody or antigen-binding fragment. In some of the embodiments provided, the antibody or antigen-binding fragment does not contain a heavy chain CH3 domain. In some of the embodiments provided, the target domain includes a light chain containing an amino acid sequence at least 85% identical to SEQ ID NO:7 and a heavy chain containing an amino acid sequence at least 85% identical to SEQ ID NO:10. In some of the embodiments provided, the target domain includes a light chain containing the amino acid sequence of SEQ ID NO:7 and a heavy chain containing the amino acid sequence of SEQ ID NO:10.

[0026] In some of the embodiments provided, the target domain is a single-stranded variable fragment (scFv). In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:11. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO:11. In some of the embodiments provided, the target domain is a variable domain of the antibody heavy chain. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:8. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO:8. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:13. In some of the embodiments provided, the cognitive binder contains the amino acid sequence of SEQ ID NO:13. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:14. In some of the embodiments provided, the cognitive binder contains the amino acid sequence of SEQ ID NO:14. In some of the embodiments provided, the target domain is a fragment of an intracellular protein.

[0027] In some of the embodiments provided, the target domain is a non-DNA-binding domain or an inactive leucine zipper domain of a transcription factor. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to one of SEQ ID NO: 17-19 and 21-23. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 17. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO: 17. In some of the embodiments provided, the cognitive binder of the synthetic cancer antigen contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 24 or 25. In some of the embodiments provided, the cognitive binder of the synthetic cancer antigen contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 24 or 25.

[0028] In some of the embodiments provided, the target domain is the active leucine zipper domain of a transcription factor. In some of the embodiments provided, the target domain is the leucine zipper domain of ATF. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:26. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO:26. In some of the embodiments provided, the target domain is the leucine zipper domain of JUN. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:27. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO:27.

[0029] In some of the embodiments provided, the cognitive binder of the synthetic cancer antigen is a leucine zipper domain. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:28. In some of the embodiments provided, the cognitive binder contains the amino acid sequence of SEQ ID NO:28. In some of the embodiments provided, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:29. In some of the embodiments provided, the cognitive binder contains the amino acid sequence of SEQ ID NO:29.

[0030] In some of the embodiments provided, the cognitive binder of the synthetic cancer antigen is the extracellular domain of the chimeric antigen receptor (CAR). In some of the embodiments provided, the CAR further comprises a transmembrane domain and an intracellular signaling domain. In some of the embodiments provided, the CAR is expressed on the surface of immune effector cells. In some of the embodiments provided, the immune effector cells are cytotoxic T cells. In some of the embodiments provided, the immune effector cells are natural killer cells. In some of the embodiments provided, the target domain and the tumor targeting binding molecule are linked by a linker. In some of the embodiments provided, the linker has a length of 1 to 100 amino acids, 1 to 75 amino acids, 1 to 50 amino acids, 1 to 25 amino acids, 5 to 100 amino acids, 5 to 75 amino acids, 5 to 50 amino acids, 5 to 25 amino acids, 10 to 100 amino acids, 10 to 75 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids. In some of the embodiments provided, the linker comprises one amino acid sequence with SEQ ID NO: 33 and 67-86.

[0031] In some aspects, polynucleotides encoding any synthetic cancer antigen disclosed herein are provided herein.

[0032] In some aspects, vectors comprising polynucleotides encoding any synthetic cancer antigen disclosed herein are provided herein.

[0033] In some of the embodiments provided, the vector is delivered specifically to cancer cells. In some of the embodiments provided, the vector is delivered specifically to hematological cancer cells. In some of the embodiments provided, the vector is delivered specifically to solid tumor cancer cells. In some of the embodiments provided, the vector is a viral vector. In some of the embodiments provided, the viral vector is an oncolytic virus. In some of the embodiments provided, the oncolytic virus is selected from the group consisting of adenovirus, herpes simplex virus, vaccinia virus, mumps virus, Newcastle disease virus, poliovirus, Seneca Valley virus, measles virus, Sindbis virus, parvovirus, coxsackievirus, varicella stomatitis virus, reovirus, and maraba and rhabdovirus. In some of the embodiments provided, the viral vector exhibits directivity to tumor cells. In some of the embodiments provided, the viral vector enters cells by binding to cell surface receptors expressed by tumor cells. In some of the embodiments provided, the viral vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, or a herpes simplex virus (HSV) vector. In some of the embodiments provided, the viral vector is an adenovirus vector. In some of the embodiments provided, the adenovirus vector is an adenovirus vector that binds to CD46 and / or desmoglein-2. In some of the embodiments provided, the adenovirus vector is Ad3, Ad7, Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, or Ad50. In some of the embodiments provided, the adenovirus vector is a chimeric adenovirus vector based on Ad3, Ad7, Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, or Ad50. In some of the embodiments provided, the adenovirus vector is a chimeric adenovirus vector based on Ad11.

[0034] In some of the embodiments provided, the adenovirus vector is a chimeric Ad3 / 11p adenovirus vector. In some of the embodiments provided, the chimeric Ad3 / 11p adenovirus vector binds to CD46. In some embodiments, the chimeric Ad3 / 11p contains a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:41. In some embodiments, the chimeric Ad3 / 11p contains a nucleic acid sequence shown in SEQ ID NO:41.

[0035] In some of the embodiments provided, the adenovirus vector is a chimeric adenovirus vector based on Ad5. In some of the embodiments provided, the adenovirus vector is a chimeric Ad5 / 3 adenovirus vector. In some of the embodiments provided, the chimeric Ad5 / 3 adenovirus vector binds to desmoglein-2.

[0036] In some aspects, cells comprising or manipulated by a vector containing a polynucleotide encoding any synthetic cancer antigen disclosed herein are provided herein.

[0037] In some aspects, a population of cells comprising a vector containing a polynucleotide encoding any synthetic cancer antigen disclosed herein, or at least one cell manipulated by such vector, is provided herein.

[0038] In some aspects, pharmaceutical compositions are provided herein that comprise a synthetic cancer antigen disclosed herein, a polynucleotide encoding any synthetic cancer antigen disclosed herein, or a vector comprising a polynucleotide encoding any synthetic cancer antigen disclosed herein. In some embodiments provided, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments provided, the pharmaceutical composition is intended for use in treating cancer in a subject.

[0039] In some aspects, combinations are provided herein that include any vector provided herein and a population of lymphocytes expressing a chimeric antigen receptor (CAR) that recognizes the target domain of a synthetic cancer antigen. In some of the embodiments provided, the CAR includes an extracellular binding domain containing a cognitive binder of the target domain, a transmembrane domain, and an intracellular signaling domain. In some of the embodiments provided, the target domain is an antibody or antibody fragment, and the cognitive binder binds to the idiotype of the antibody or antibody fragment. In some of the embodiments provided, the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor. In some of the embodiments provided, the target domain is an active leucine zipper domain of a transcription factor. In some of the embodiments provided, the target domain is the leucine zipper domain of ATF. In some of the embodiments provided, the target domain is the leucine zipper domain of JUN. In some of the embodiments provided, the cognitive binder of the synthetic cancer antigen is the leucine zipper domain. In some of the embodiments provided, the lymphocytes include cytotoxic T cells. In some of the embodiments provided, the lymphocytes include natural killer cells.

[0040] In some aspects, kits containing any combination provided herein are provided herein. In some of the embodiments provided, the kit further includes instructions for using the vector and the population of lymphocytes.

[0041] In some aspects, methods for treating cancer in a subject are provided herein, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition disclosed herein to the subject. In some aspects, methods for treatment are provided herein, comprising the steps of administering a therapeutically effective amount of any polynucleotide, any vector, or any pharmaceutical composition provided herein to a subject having cancer, and administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) bound to the target domain of a synthetic cancer antigen.

[0042] In some aspects, a treatment method is provided herein that includes administering a therapeutically effective dose of a population of lymphocytes expressing a chimeric antigen receptor (CAR) to a subject having cancer that has been pre-administered with any polynucleotide, any vector, or any pharmaceutical composition provided herein, the CAR binding to the target domain of a synthetic cancer antigen. In some of the embodiments provided, the lymphocytes are T cells. In some of the embodiments provided, the method includes any vector provided herein and a population of lymphocytes expressing a chimeric antigen receptor (CAR) that recognizes the target domain of a synthetic cancer antigen. In some of the embodiments provided, the CAR includes an extracellular binding domain containing a cognitive binder of the target domain, a transmembrane domain, and an intracellular signaling domain. In some of the embodiments provided, the target domain is an antibody or antibody fragment, and the cognitive binder binds to the idiotype of the antibody or antibody fragment. In some of the embodiments provided, the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor. In some of the embodiments provided, the target domain is the active leucine zipper domain of a transcription factor. In some of the embodiments provided, the target domain is the leucine zipper domain of ATF. In some of the embodiments provided, the target domain is the leucine zipper domain of JUN. In some of the embodiments provided, the cognitive binder of a synthetic cancer antigen is the leucine zipper domain. In some of the embodiments provided, the lymphocytes include cytotoxic T cells. In some of the embodiments provided, the lymphocytes include natural killer cells.

[0043] In some aspects, a method for tagging tumor cells in vivo is provided, comprising the step of contacting the tumor cells with any polynucleotide, any vector, or any pharmaceutical composition provided herein. In some aspects, a method for tagging tumor cells in a subject having cancer is provided, comprising the step of administering a therapeutically effective amount of any polynucleotide, any vector, or any pharmaceutical composition provided herein to the subject. In some aspects, a method for treating cancer in a subject is provided, comprising the step of administering a therapeutically effective amount of any pharmaceutical composition provided herein to the subject.

[0044] In some of the embodiments provided, at least one immune effector cell expresses a CAR on the surface of the immune effector cell. In some of the embodiments provided, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain. In some of the embodiments provided, the extracellular domain of the CAR comprises a cognitive binder of the synthetic cancer antigen. In some of the embodiments provided, the immune effector cell is a cytotoxic T cell. In some of the embodiments provided, the immune effector cell is a natural killer cell. In some of the embodiments provided, the pharmaceutical composition and the immune effector cells are administered simultaneously. In some of the embodiments provided, the pharmaceutical composition is administered before the immune effector cells are administered. In some of the embodiments provided, the cancer is a hematological cancer. In some of the embodiments provided, the cancer is a solid tumor cancer.

[0045] In some aspects, chimeric antigen receptors (CARs) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain are provided herein, wherein the extracellular domain binds to synthetic cancer antigens provided herein. In some embodiments provided, the extracellular domain is a cognitive binder. In some aspects, chimeric antigen receptors (CARs) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain are provided herein, wherein the extracellular domain contains an amino acid sequence at least 85% identical to one of SEQ ID NO: 13, 14, 24, 25, 28, and 29. In some embodiments, the extracellular domain contains the amino acid sequence shown in SEQ ID NO: 13.

[0046] In some embodiments, the extracellular domain of the CAR binds to the target domain of a synthetic oncoantigen, such as any of those described herein. In some embodiments, the synthetic oncoantigen comprises a target domain and a membrane targeting domain. In some embodiments, the synthetic oncoantigen comprises a target domain and a tumor targeting binding molecule. In some of the embodiments provided, the target domain is not expressed on the surface of the target non-cancer cells. In some of the embodiments provided, the target domain is an antibody or a molecule derived from an antibody. In some of the embodiments provided, the target domain is an antibody that does not contain a heavy chain CH3 domain.

[0047] In some embodiments, the target domain is derived from the trastuzumab parent antibody, the 4D5 anti-HER2 antibody (also known as Ab1). In some embodiments, the target domain is an antibody fragment of the 4D5 (Ab1) antibody.

[0048] In some embodiments, the antibody fragment is a Fab. In some of the embodiments provided, the target domain comprises a light chain having an amino acid sequence at least 85% identical to SEQ ID NO:7 and a heavy chain having an amino acid sequence at least 85% identical to SEQ ID NO:10. In some of the embodiments provided, the target domain comprises a light chain having an amino acid sequence of SEQ ID NO:7 and a heavy chain having an amino acid sequence of SEQ ID NO:10.

[0049] In some of the embodiments provided, the target domain is a single-stranded variable fragment (scFv). In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:11. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO:11.

[0050] In some of the embodiments provided, the target domain is a variable domain of the antibody heavy chain. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:8. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO:8.

[0051] In some of the embodiments provided, the extracellular domain of the CAR binds to an idiotype of a target domain of a synthetic cancer antigen, such as a target domain containing one of the antibodies derived from 4D5 (e.g., Fab shown in SEQ ID NO: 7 and 10, scFv shown in SEQ ID NO: 11, or variable heavy chain shown in SEQ ID NO: 8). In some of the embodiments provided, the extracellular domain of the CAR contains the amino acid sequence of SEQ ID NO: 13. In some of the embodiments provided, the extracellular domain of the CAR contains the amino acid sequence of SEQ ID NO: 14.

[0052] In some of the embodiments provided, the target domain is a fragment of an intracellular protein. In some of the embodiments provided, the target domain is a non-DNA-binding domain or an inactive leucine zipper domain of a transcription factor. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to one of SEQ ID NO: 17-19 and 21-23. In some of the embodiments provided, the target domain contains the amino acid sequences of SEQ ID NO: 17-19 and 21-23. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 17. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO: 17. In some of the embodiments provided, the extracellular domain of the CAR contains the amino acid sequence of SEQ ID NO: 24 or 25.

[0053] In some of the embodiments provided, the target domain is the active leucine zipper domain of the transcription factor. In some of the embodiments provided, the target domain is the leucine zipper domain of ATF. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:26. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO:26. In some of the embodiments provided, the target domain is the leucine zipper domain of JUN. In some of the embodiments provided, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:27. In some of the embodiments provided, the target domain contains the amino acid sequence of SEQ ID NO:27. In some of the embodiments provided, the extracellular domain of CAR is the leucine zipper domain. In some of the embodiments provided, the extracellular domain of CAR contains the amino acid sequence of SEQ ID NO:28. In some of the embodiments provided, the extracellular domain of CAR contains the amino acid sequence of SEQ ID NO:29. In some of the embodiments provided, the transmembrane domain of the CAR is the CD8 transmembrane domain. In some of the embodiments provided, the transmembrane domain of the CAR contains an amino acid sequence that is at least 85% identical to SEQ ID NO:3. In some of the embodiments provided, the transmembrane domain of the CAR contains the amino acid sequence of SEQ ID NO:3.

[0054] In some of the embodiments provided, the intracellular signaling domain includes a primary intracellular signaling domain. In some of the embodiments provided, the intracellular signaling domain further includes a co-stimulatory signaling domain. In some of the embodiments provided, the intracellular signaling domain includes a CD3z cytoplasmic signaling domain and a 41BB co-stimulatory signaling domain. In some of the embodiments provided, the intracellular signaling domain includes an amino acid sequence that is at least 85% identical to SEQ ID NO:16. In some of the embodiments provided, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO:16.

[0055] In some aspects, polynucleotides encoding CARs provided herein are provided herein. In some of the embodiments provided, the CARs are expressed on the surface of cells. In some of the embodiments provided, the cells are immune effector cells. In some of the embodiments provided, the immune effector cells are cytotoxic T cells. In some of the embodiments provided, the immune effector cells are natural killer cells. In some aspects, a population of cells comprising at least one of the cells provided herein is provided herein.

[0056] In some aspects, pharmaceutical compositions comprising a CAR provided herein, any polynucleotide provided herein, any vector provided herein, any cell provided herein, or a population of cells provided herein are provided herein. In some embodiments provided herein, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments provided herein, the pharmaceutical composition is for use in treating cancer in a subject. In some aspects herein, a method for treating cancer in a subject is provided herein, comprising the step of administering a therapeutically effective amount of any pharmaceutical composition provided herein to the subject. In some embodiments provided herein, the pharmaceutical composition and the additional pharmaceutical composition are administered simultaneously. In some embodiments provided herein, the additional pharmaceutical composition is administered before the pharmaceutical composition is administered. In some embodiments provided herein, the cancer is a hematological cancer. In some embodiments provided herein, the cancer is a solid tumor cancer. [Brief explanation of the drawing]

[0057] [Figure 1] This is a schematic diagram illustrating the delivery of an exemplary synthetic cancer antigen, such as an antibody, into cancer cells via a gene delivery vector, such as an oncolytic virus. In this way, T cells expressing a binding partner for the antibody's idiotype are targeted to the cancer cells, thereby killing them. [Figure 2A] Figure 2A shows exemplary synthetic cancer antigen designs (Ab1-Fab, Ab1-scFv, Ab1-VH) based on antibody or derivative molecules. [Figure 2B] Figure 2B illustrates the details of the construct design, progressing from a synthetic oncogen containing an antibody without the heavy chain CH3 domain (Ab1-Fab) to a synthetic oncogen simply containing the scFv of Ab1 (Ab1-scFv), and finally to a synthetic oncogen containing only the variable domain of the heavy chain of Ab1 (Ab1-VH). [Figure 3A]Figures 3A to 3C show the expression of Ab1-Fab, Ab1-scFv, and Ab1-VH on the surface of different cancer cells. Ab1-Fab and Ab1-scFv expression was detected via PE-labeled goat anti-human IgG, F(ab')2-specific antibody (Jackson Immunoresearch, Inc). Ab1-VH expression was detected via PE-labeled anti-flag antibody. A375: Malignant melanoma cell line; A549: Lung cancer cell line; PC3: Prostate cancer cell line; HCT-116: Colorectal cancer cell line; SKOV-3: Ovarian adenocarcinoma cell line. [Figure 3B] See the explanation in Figure 3A. [Figure 3C] See the explanation in Figure 3A. [Figure 4] Ab1-Fab and Ab1-scFv still bind to the cognitive antigen of Ab1, while Ab1-VH no longer binds to the cognitive antigen of Ab1 due to the deletion of the variable domain in the light chain of Ab1. [Figure 5A] Figures 5A and 5B show the increase in T cells after transduction of one of three different CAR molecules against the Ab1 idiotype, namely Ab1-Fab, Ab1-scFv, and Ab1-VH. Ab1_CAR1: Lama single-domain anti-idiotype antibody; Ab1_CAR2: human anti-idiotype scFv; and Ab1_CAR3: another human anti-idiotype scFv. [Figure 5B] See the explanation in Figure 5A. [Figure 6A] Figure 6A shows that Ab1_CAR1 and Ab1_CAR2 expression were detected on transduced CAR-T cells, but Ab1_CAR3 expression was not detected. Figures 6B-6C show the expression of Ab1_CAR1 and Ab1_CAR2 after the titer of each lentivirus was gradually reduced. UTD: Untransduced. [Figure 6B] See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 7A]Figures 7A to 7E show the death of different Ab1-Fab-expressing cancer cells by Ab1_CAR1-expressing T cells derived from three donors. A375: Malignant melanoma cell line; A549: Lung cancer cell line; PC3: Prostate cancer cell line; HCT-116: Colorectal cancer cell line; SKOV-3: Ovarian adenocarcinoma cell line. [Figure 7B] See the explanation in Figure 7A. [Figure 7C] See the explanation in Figure 7A. [Figure 7D] See the explanation in Figure 7A. [Figure 7E] See the explanation in Figure 7A. [Figure 8A] Figures 8A to 8E show the death of different Ab1-Fab-expressing cancer cells by Ab1_CAR2-expressing T cells derived from three donors. A375: Malignant melanoma cell line; A549: Lung cancer cell line; PC3: Prostate cancer cell line; HCT-116: Colorectal cancer cell line; SKOV-3: Ovarian adenocarcinoma cell line. [Figure 8B] See the explanation in Figure 8A. [Figure 8C] See the explanation in Figure 8A. [Figure 8D] See the explanation in Figure 8A. [Figure 8E] See the explanation in Figure 8A. [Figure 9A] Figure 9A shows the death of Ab1-scFv-expressing A549 cancer cells by T cells derived from a single donor expressing either Ab1_CAR1 or Ab1_CAR2. [Figure 9B] Figures 9B to 9C show the death of different Ab1-scFv-expressing cancer cells by T cells from another donor expressing Ab1_CAR2. [Figure 9C] See the explanation for Figure 9B. [Figure 10A] Figures 10A and 10B show the killing of different Ab1-VH-expressing cancer cells by T cells expressing Ab1_CAR2. A549: lung cancer cell line; PC3: prostate cancer cell line; HCT-116: colorectal cancer cell line; SKOV-3: ovarian adenocarcinoma cell line. [Figure 10B] See the explanation in Figure 10A. [Figure 11A] Figures 11A and 11B compare the death of Ab1-Fab-expressing cancer cells, Ab1-scFv-expressing cancer cells, and Ab1-VH-expressing cancer cells by T cells expressing Ab1_CAR2. A549: Lung cancer cell line; PC3: Prostate cancer cell line; HCT-116: Colorectal cancer cell line; SKOV-3: Ovarian adenocarcinoma cell line. UTD: Non-transduced. [Figure 11B] See the explanation in Figure 11A. [Figure 12A] Figure 12A shows Ab1-Fab expression via adenovirus transduction. Figures 12B-12C show the killing of different Ab1-Fab-expressing cancer cells by T cells from different donors expressing either Ab1_CAR1 or Ab1_CAR2. The Ab1-Fab construct was transduced into cancer cells using adenovirus instead of lentivirus, as shown in Figures 7A-8E. Figures 12D-12E show the killing of different Ab1-VH-expressing cancer cells by T cells expressing Ab1_CAR2. The Ab1-VH construct was transduced into cancer cells using adenovirus instead of lentivirus, as shown in Figures 10A-10B. Figure 12F shows the killing of Ab1-VH-expressing cancer cells by T cells expressing Ab1_CAR1. The Ab1-VH construct was transduced into cancer cells using the chimeric adenovirus Ad3 / Ad11p. A549: lung cancer cell line; PC3: prostate cancer cell line; HCT-116: colorectal cancer cell line; SKOV-3: ovarian adenocarcinoma cell line. UTD: untransduced. [Figure 12B] See the explanation in Figure 12A. [Figure 12C] See the explanation in Figure 12A. [Figure 12D] See the explanation in Figure 12A. [Figure 12E] See the explanation in Figure 12A. [Figure 12F] See the explanation in Figure 12A. [Figure 13A]Figure 13A is a schematic diagram of in vivo testing of two synthetic cancer antigens (Ab1-VH and Ab1-Fab) and their cognitive binders. [Figure 13B] Figures 13B and 13C show the survival rate and tumor volume of mice harboring HER2-positive HCT-116 tumor cells expressing Ab1-VH, treated with different concentrations of T cells expressing Ab1_CAR2. T cells expressing anti-HER2 (4D5) CAR were included as a control. [Figure 13C] See the explanation in Figure 13B. [Figure 13D] Figure 13D shows an increase in T cells expressing either Ab1_CAR2 or anti-HER2 (4D5) CAR. [Figure 13E] Figure 13E shows the tumor volume of mice containing Ab1-Fab-expressing HER2-positive HCT-116 cells, treated with different concentrations of T cells expressing Ab1_CAR1. [Figure 14] This is a schematic diagram illustrating the delivery of a protein fragment, an exemplary synthetic cancer antigen, into cancer cells via a gene delivery vector such as an oncolytic virus. In this way, T cells expressing molecules that target the protein fragment are directed towards the cancer cells, thereby killing them. [Figure 15A] Figures 15A and 15B show the expression of different candidate protein fragments on the surface of 293T cells. [Figure 15B] See the explanation in Figure 15A. [Figure 16A] Figure 16A shows the expression of IC1 protein fragments on the surface of different cancer cells. [Figure 16B] Figure 16B shows the expression of the binder for IC1 on the surface of T cells. [Figure 17A] Figures 17A to 17C show the killing of different cancer cells expressing IC1 by T cells expressing a binder for IC1. [Figure 17B] See the explanation in Figure 17A. [Figure 17C] See the explanation in Figure 17A. [Figure 18] This is a schematic diagram illustrating the delivery of the leucine zipper domain of transcription factor 1 (TF1), an exemplary synthetic cancer antigen, into cancer cells via a gene delivery vector such as an oncolytic virus. In this way, T cells expressing the binding partner of the leucine zipper domain of transcription factor 1 (e.g., the leucine zipper domain of transcription factor 2, TF2) are targeted to cancer cells, thereby enabling their death. [Figure 19] This document presents construct designs for exemplary synthetic cancer antigens (TF2-LZ and TF4-LZ) and their corresponding cognitive binders (TF1-LZ and TF3-LZ). [Figure 20A] Figure 20A shows the expression of TF2-LZ and TF4-LZ on the surface of cancer cells. [Figure 20B] Figure 20B shows the expression of TF1-LZ (a binder for TF2-LZ) on the surface of T cells. [Figure 20C] Figure 20C shows the expression of TF3-LZ (a binder for TF4-LZ) on the surface of T cells. [Figure 21] This is a schematic diagram illustrating the testing of the binding efficacy of the soluble form of the Ab1-VH(FL1)-anti-EpCAM complex to tumor cells. [Figure 22] This shows surface binding expression of Flare1 after co-culturing with supernatant containing a soluble complex or a tethered form of Ab1-VH. [Figure 23A] Figure 23A is a schematic diagram of testing the soluble form of the Ab1-VH(FL1)-anti-EpCAM complex for targeted tumor cell death. [Figure 23B] Figure 23B shows the killing of different Ab1-VH surface-bound cancer cells by T cells expressing Ab1_CAR2, where the Ab1-VH construct bound to cancer cells by complexing with an anti-EpCAM antibody. [Figure 24A] Figure 24A is a schematic diagram of in vivo testing of a soluble synthetic cancer antigen (Ab1-VH(FL1)-anti-EpCAM complex) and its cognitive binder. [Figure 24B] Figure 24B shows the percentage of FL1+ tumor cells after administering doses of the virus encoding Ab1-VH in tethered or soluble form to mice. [Figure 24C] Figure 24C shows the number of infiltrating T cells in the tumor after treatment with the virus encoding Ab1-VH in tethered or soluble form, and subsequent intravenous injection of CAR-T cells. [Figure 24D] Figure 24D is a schematic diagram of the in vivo time-course testing of a soluble synthetic cancer antigen (Ab1-VH(FL1)-anti-EpCAM complex) and its cognitive binder. [Figure 24E] Figure 24E shows tumor growth in mice after treatment with a virus encoding the Ab1-VH(FL1)-anti-EpCAM complex and CAR-T cells. [Figure 25A] Figure 25A shows the surface binding expression of soluble FL1-anti-HER2 synthetic oncogeneric antigen on HER2-expressing SKOV3 cancer cells. MFI is normalized to the antigen-free condition. [Figure 25B] Figures 25B to 25C show the elimination of cancer cells expressing different HER2s by CAR T cells after culturing with soluble FL1-anti-HER2 synthetic cancer antigen. [Figure 25C] See the explanation in Figure 25B. [Figure 26] The left panel shows surface-bound expression of Ab1-VH(FL1) on A549 cancer cells infected with viruses encoding both tethered synthetic oncoantigen and soluble FL1-anti-EpCAM synthetic oncoantigen. The right panel shows surface-bound expression of FL1 on non-transduction acceptor cancer cells after co-culture with supernatant containing the soluble form of the Ab1-VH complex. Pos Con: Positive control, a cell line engineered to express either tethered or soluble FL1; Sec Con: Control antibody staining to ensure signal specificity. [Modes for carrying out the invention]

[0058] Detailed explanation Synthetic cancer antigens are provided herein for use as targets for the treatment of cancers, such as solid tumors, in combination with CAR therapies, such as CART cell therapy, which recognize the extracellular target domain of synthetic cancer antigens on tagged cancer cells. Accordingly, the embodiments provided relate to CAR therapies, which include cells, such as a population of immune effector cells, that include (e.g., express) a chimeric antigen receptor (CAR) for use in combination with synthetic cancer antigens. Polynucleotides encoding synthetic cancer antigens and vectors, such as viral vectors containing them, are also provided herein. In some embodiments, the viral vector is a tumor-targeting vector capable of specifically targeting and expressing the synthetic cancer antigen in tumor cells. The embodiments provided enable tumor-specific gene delivery, which allows the synthetic cancer antigen to selectively tag various types of tumor cells having targetable antigens.

[0059] In some embodiments, synthetic cancer antigens are expressed as membrane proteins, such as those containing a transmembrane domain. In some embodiments, the disclosure provides synthetic cancer antigens and / or their cognitive binders, each comprising a target domain and a transmembrane domain.

[0060] In other embodiments, the synthetic cancer antigen is expressed as a soluble protein that can be secreted from the cells that express it. In such embodiments, the soluble target domain is linked to a tumor targeting molecule that can bind to the tumor antigen and tag cancer cells.

[0061] In some embodiments, synthetic cancer antigens containing a target domain are delivered only to tumor cells. Therefore, it will be understood that synthetic cancer antigens containing a target domain are not expressed on the surface of target non-cancer cells. This disclosure also provides polynucleotides encoding the synthetic cancer antigens disclosed herein, vectors containing such polynucleotides, and cells containing or manipulated by such vectors. In some embodiments, polynucleotides and / or viral vectors encode both chimeric synthetic cancer antigen membrane proteins and secretible synthetic cancer antigens. This disclosure also provides pharmaceutical compositions containing the synthetic cancer antigens, polynucleotides, or vectors disclosed herein, and methods of using such pharmaceutical compositions to treat cancer.

[0062] In some embodiments, CAR activation directed to a synthetic cancer antigen (i.e., containing an extracellular domain that recognizes or binds to the synthetic cancer antigen) initiates cytotoxic death of tumor cells expressing the synthetic cancer antigen by CAR T cells. In some embodiments, the death activates viral release, which in turn allows the virus to infect adjacent tumor cells and spread the expression of the synthetic cancer antigen to other tumor cells, resulting in recognition by CAR cell therapies. Furthermore, embodiments herein also include those in which infected cells may express the synthetic cancer antigen in membrane form or secrete a soluble version of the synthetic cancer antigen to tag adjacent or nearby tumors. Such aspects further enable improved targeting of tumor cells for recognition by CAR cell therapies. In combination with autologous CAR cell therapies directed to a synthetic cancer antigen (e.g., CAR-T cells), the embodiments provided enable the directing of tumor cell death, including solid tumors.

[0063] Section headings used in this specification are for organizational purposes only and should not be construed as limiting the subjects described herein.

[0064] I. Definition Unless otherwise defined herein, technical and scientific terms used in this description have the meanings that are ordinarily understood by those skilled in the art. For the purposes of interpreting this specification, the following definitions of terms apply, and unless otherwise explicitly indicated in the content, where appropriate, a singular term includes the plural and vice versa. In the event of any conflict between any definition of a term and any document incorporated herein by reference, the following definitions shall prevail.

[0065] The terms “a,” “an,” and “the,” as used herein, include multiple references unless otherwise clearly indicated by the context.

[0066] When used herein, the terms "or" and "and / or" include any and all, or one or more combinations of, the items described herein.

[0067] When the term "approximately" is used herein in relation to a number or a range of numbers, it is understood to mean the number plus ±10% of that number, or, in the case of a range, a value 10% lower than the lower limit to 10% higher than the upper limit to 10% of the upper limit.

[0068] The terms “including,” “includes,” and “included,” and other forms thereof, as used herein, are not limited to these.

[0069] The term “comprise” and its grammatical equivalents, as used herein, identify the presence of the features, components, processes, operations, elements, and / or components described herein, but do not exclude the presence or addition of one or more other features, components, processes, operations, elements, components, and / or groups thereof.

[0070] The terms “administer,” “dosage,” or “administer” as used herein mean the act of injecting or otherwise physically delivering a substance (e.g., a pharmaceutical composition provided herein) to a subject (e.g., a human) by oral, mucosal, topical, intradermal, parenteral, intravenous, intravitreous, intra-articular, subretinal, intramuscular, intrathecal delivery, and / or any other physical delivery method described herein or known in the art. Delivery may be systemic or to specific tissues.

[0071] The terms “antibody,” “immunoglobulin,” or “Ig” are used interchangeably herein and are used in their broadest sense, specifically encompassing, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length monoclonal antibodies, or intact monoclonal antibodies), antibody compositions having polyepitope specificity or monoepitope specificity, polyclonal antibodies or monovalent antibodies, multivalent antibodies, and multispecific antibodies (e.g., bispecific antibodies, provided they exhibit the desired biological activity). Antibodies may be human, humanized, chimeric, and / or affinity-mature, as well as antibodies derived from other species, such as mice and rabbits. The term “antibody” is intended to include B cell polypeptide products belonging to the immunoglobulin class of polypeptides, which are capable of binding to specific molecular antigens and consist of two identical polypeptide chain pairs, each pair having one heavy chain (approximately 50–70 kDa) and one light chain (approximately 25 kDa), with each amino-terminus of each chain containing a variable region of approximately 100–130 or more amino acids, and each carboxy-terminus of each chain containing a constant region. See, for example, 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. “Antibody-derived molecule” refers to a functional antigen-binding fragment of any of the above. It is a portion of the antibody heavy and / or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment originated. Non-limiting examples of functional fragments include single-stranded Fv (scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.Such functional antigen-binding fragments can be found, for example, in Harlow 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 (2nd ed. 1990). The antibodies and antibody-derived molecules provided herein may be of any class of immunoglobulin molecules (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2). As used herein, the term "idiotype" refers to a specific set of antigenic epitopes recognized by the variable region of an antibody. An anti-idiotype antibody is an antibody that specifically binds to the antigen-binding site (e.g., the antigen-binding domain) of another antibody.

[0072] The terms “chimeric antigen receptor” or “CAR,” as used herein, refer to a genetically engineered receptor that can be used to transfer one or more antigen specificities 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, a CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain for one or more synthetic oncoantigens disclosed herein. “CAR-T cell” refers to a T cell expressing a CAR. “CAR-NK cell” refers to an NK cell expressing a CAR.

[0073] The term "coding sequence" or "coding" polynucleotide, as used herein, refers to a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) terminus and a translation termination codon at the 3' (carboxy) terminus. The transcription termination sequence may be located on the 3' side of the coding sequence.

[0074] The term “constant region” or “constant domain,” as used herein, refers to the carboxyl-terminal portions of the light and heavy chains that do not directly participate in antibody binding to the antigen but exhibit various effector functions, such as interaction with the Fc receptor. This region has a more conserved amino acid sequence compared to the variable region. The constant region may include the CH1, CH2, and CH3 regions of the heavy chain, as well as the CL region of the light chain.

[0075] The terms “effective dose” or “therapeutic effective dose,” as used herein, refer to an amount of therapeutic agent (e.g., a pharmaceutical composition provided herein) sufficient to treat, diagnose, prevent, delay the onset of, reduce the severity and / or duration of, and / or induce remission of, a given condition, disorder or disease and / or symptoms associated therewith. The terms also include amounts necessary to reduce, slow, or induce remission of the progression or progression of a given disease, or to reduce, slow, or induce relapse of the recurrence, onset, or occurrence of a given disease, and / or to improve or enhance the prophylactic or therapeutic effect(s) of another treatment, or to act as a bridge to another treatment.

[0076] The terms "Fab" or "Fab region," as used herein, refer to the antibody region that binds to an antigen. Conventional IgG typically contains two Fab regions, each located on one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region of the heavy chain and light chain, respectively. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH region and the CH1 region, and the variable and constant regions of the light chain in the Fab region are the VL region and the CL region. The VH, CH1, VL, and CL regions in the Fab region can be aligned in various ways to confer antigen-binding ability. For example, as with the Fab regions of conventional IgG, the VH and CH1 regions can reside on one polypeptide, and the VL and CL regions can reside on another polypeptide. Alternatively, the VH, CH1, VL, and CL regions can all reside on the same polypeptide and be oriented in different orders.

[0077] The term “fragment,” as used herein, refers to a portion of a polypeptide molecule or polynucleotide molecule that contains less than the entire polypeptide sequence or less than the entire polynucleotide sequence. In some embodiments, a polypeptide or polynucleotide fragment contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the full length of a reference polypeptide or reference polynucleotide. In some embodiments, a polypeptide or polynucleotide fragment contains about 10% to 99%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, 90% to 99%, 95% to 99%, 96% to 99%, 97% to 99%, or 98% to 99% of the full length of a reference polypeptide or reference polynucleotide. In some embodiments, the polypeptide fragment 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.

[0078] When used in relation to antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50–70 kDa, with an amino-terminal region containing a variable region of approximately 120–130 or more amino acids, and a carboxy-terminal region containing a constant region. The constant region can be one of five distinct types (e.g., isotypes) designated 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 antibody classes (e.g., isotypes), including the four IgG subclasses, namely IgG1, IgG2, IgG3, and IgG4, namely IgA, IgD, IgE, IgG, and IgM, respectively.

[0079] When used in relation to antibodies, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, with the amino-terminal portion containing a variable region of approximately 100–110 or more amino acids, and the carboxy-terminal portion containing a constant region. The approximate length of a light chain is 211–217 amino acids. There are two distinct types, called kappa or lambda, based on the amino acid sequence of the constant domain.

[0080] The term "leucine zipper domain," as used herein, refers to an amphiphilic α-helix containing a 7-amino acid repeat Leu residue on one face of the helix, which acts as a dimerization module. In some embodiments, the leucine zipper domain is an active leucine zipper domain. Upon dimerization with another leucine zipper domain, the leucine-zipper α-helix forms a parallel coiled coil based on hydrophobic interface side-chain packing. In some embodiments, the leucine zipper domain is an inactive leucine zipper domain that cannot dimerize with another leucine zipper domain.

[0081] The term "naturally occurring," when applied to nucleic acids, polypeptides, cells, or organisms, refers to nucleic acids, polypeptides, cells, or organisms that are found in nature. For example, a polypeptide sequence or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by a human in a laboratory is considered naturally occurring.

[0082] As used herein, the term "oncolytic virus" refers to a type of virus that preferentially infects cancer cells but not non-cancerous cells. Oncolytic viruses can occur naturally or be produced in the laboratory by modifying and altering existing viruses.

[0083] The term "functionally linked" and similar phrases (e.g., genetically fused) as used herein refer to the functional linking of nucleic acid sequences or amino acid sequences that are arranged in a functional relationship with one another. For example, a functionally linked promoter to a polynucleotide encoding a polypeptide results in the transcription of the polynucleotide, and ultimately, the expression of the polypeptide. As another example, a functionally linked peptide is one in which functional domains are arranged at appropriate distances from one another to give each domain a desired function.

[0084] The term “pharmaceutically acceptable pharmaceutical excipient, carrier, or diluent” as used herein refers to any substance formulated together with the active ingredient of a pharmaceutical composition that enables the active ingredient to retain its biological activity and is non-reactive with the target immune system. Such substances may be included for purposes of long-term stabilization, bulking of solid formulations containing small amounts of potent active ingredient, or to impart therapeutic enhancements to the active ingredient in the final dosage form, such as promoting absorption, reducing viscosity, or increasing solubility. The selection of an appropriate substance may depend on the route of administration and dosage form, as well as the active ingredient and other factors. Compositions containing such substances may be formulated by well-known conventional methods (see, for example, Remington, The Science and Practice of Pharmacy, 23rd edition, A. Adejare, ed., Academic Press, 2020).

[0085] The terms “pharmaceutical composition” or “therapeutic composition,” as used herein, refer to a composition having the ability to be administered to a subject for the treatment of a particular disease or disorder.

[0086] The terms “polynucleotide” or “nucleic acid,” as used herein, refer to polymers of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by synthetic reactions. Polynucleotides may include modified nucleotides such as methylated nucleotides and their analogs. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5' end, and the left-side direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5'-to-3' addition in a nascent RNA transcript is referred to as the transcription direction.

[0087] The terms “polypeptide,” “peptide,” and “protein,” as used herein, refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, or may be fragmented by non-amino acids. The terms also encompass amino acid polymers that are naturally modified or modified by intervention, such as by disulfide bond formation, glycosylation, lipid addition, acetylation, phosphorylation, or any other operation or modification. Polypeptides containing, for example, one or more amino acid analogs, including but not limited to non-natural amino acids, and polypeptides containing other modifications known in the art are also included in the definition.

[0088] When used herein, the term “population” of cells refers to any number of cells greater than one, but preferably at least 1 × 10⁶ 3 A single cell, at least 1 × 10⁶ 4 A single cell, at least 1 × 10⁶ 5 A single cell, at least 1 × 10⁶ 6 A single cell, at least 1 × 10⁶ 7 A single cell, at least 1 × 10⁶ 8 A single cell, at least 1 × 10⁶ 9cells, at least 1×10 10 cells, at least 1×10 11 cells, or more. The population of cells can 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 present in a particular tissue).

[0089] The term "sequence identity," as used herein, refers to the percentage of bases or amino acids present in the same and same relative positions between two polynucleotide or polypeptide sequences. One such polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparisons, typically one sequence serves as the reference sequence against which the test sequence is compared. The term "reference sequence" refers to the molecule against which the test sequence is compared. Methods of sequence alignment for comparing and determining sequence identity percentages and complementarity percentages are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443; by the similarity search 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 Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI); by manual alignment and visual verification (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology); and by Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. This can be done using algorithms known in the art, including the BLAST and BLAST 2.0 algorithms described in 215:403-410. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0090] The term "subject" as used herein refers to mammals such as non-primates (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or primates (e.g., monkeys and humans). In some embodiments, the subject is a mammal, for example, a human diagnosed with a disease or disorder provided herein. In some embodiments, the subject is a mammal, for example, a human at risk of developing a disease or disorder provided herein. In some embodiments, the subject is a human.

[0091] The term "synthetic," when applied to nucleic acids, polypeptides, cells, or organisms, refers to nucleic acids, polypeptides, cells, or organisms that cannot be directly isolated from their natural source. In some embodiments, synthetic nucleic acids, polypeptides, cells, or organisms are modified or altered compared to their corresponding naturally occurring counterparts. In some embodiments, synthetic nucleic acids, polypeptides, cells, or organisms are produced by functionally linking or combining different fragments of nucleic acids, polypeptides, cells, or organisms of origin. For example, a synthetic polypeptide may contain functionally linked polypeptides of different origins.

[0092] The term “target domain,” when applied to synthetic cancer antigens disclosed herein, refers to the extracellular domain of the synthetic cancer antigen. It can be expressed on the surface of cancer cells and recognized by a cognitive binder. It can be bound by a chimeric antigen receptor containing a cognitive binder and can be expressed on the surface of cancer cells.

[0093] As used herein, the term "transduced" refers to the process by which a transgene is introduced from a viral particle into a host cell.

[0094] The term “transgene,” as used herein, refers to any heterologous polynucleotide incorporated into a viral vector for, for example, transcription or expression in target cells. An example of a transgene is a polynucleotide encoding a therapeutic polypeptide or a detectable marker.

[0095] As used herein, the term "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane (e.g., a eukaryotic cell membrane). Transmembrane domains may originate from either natural or synthetic sources.

[0096] The terms “treatment” and “to treat” as used herein refer to a pharmaceutical regimen or other intervention regimen for obtaining a favorable or desired outcome in a recipient. Favorable or desired outcomes include, but are not limited to, therapeutic benefits and / or prophylactic benefits. A therapeutic benefit may refer to the eradication or remission of symptoms of the underlying disorder being treated. A therapeutic benefit may also be achieved with the eradication or remission of one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the subject, regardless of whether the subject may still be susceptible to the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the onset of a disease or condition; delaying or eliminating the onset of symptoms of a disease or condition; slowing, stopping, or improving the progression of a disease or condition; or any combination thereof. For a prophylactic benefit, a subject at risk of developing a particular disease, or a subject reporting one or more physiological symptoms of a disease, may receive treatment, even if a diagnosis of the disease has not been made.

[0097] The terms “variable region,” “variable domain,” “V region,” or “V domain,” as used herein, generally refer to a portion of the light or heavy chain of an antibody, located at the amino terminus of the light or heavy chain, having a length of approximately 120–130 amino acids in the heavy chain and approximately 100–110 amino acids in the light chain, and used in the binding and specificity of each particular antibody to a 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 region have significantly different sequences among antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody to a particular antigen. On the other hand, variability is not uniformly distributed across the 110-amino acid span of the variable region. Instead, the V region consists of less variable (e.g., relatively invariant) segments called framework regions (FRs) of about 15-30 amino acids, separated by shorter, more variable (e.g., extremely variable) regions called "hypervariable regions" or "complementarity-determining regions," each about 9-12 amino acids long. The variable regions of the heavy and light chains each largely adopt a β-sheet configuration, connected by three hypervariable regions that form loop connections and, in some cases, form part of a β-sheet structure, each containing four FRs. The hypervariable regions in each chain are held together in close proximity by FRs, and the hypervariable region from the other chain contributes to the formation of the antibody's antigen-binding site (see, e.g., Rabat et al, Sequences of Proteins of Immunological Interest (5th ed. 1991)).

[0098] The term “viral vector,” as used herein, refers to nucleic acids to be delivered into host cells via recombinantly produced viruses or viral particles. Nucleic acids may be single-stranded or double-stranded, linear or circular, and segmented or unsegmented. Nucleic acids may include DNRs, RNA, or combinations thereof. Non-limiting examples of viruses or viral particles that can deliver viral vectors include retroviruses (e.g., lentiviruses and gamma-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses, and poxviruses. Viral vectors delivered by such viruses or viral particles may be referred to by the type of virus used to deliver the viral vector (e.g., a lentiviral vector is a viral vector delivered by a lentivirus). Viral vectors may contain viral elements (e.g., nucleotide sequences) necessary for packaging the viral vector within a virus or viral particle, replicating the virus, or other desired viral activity. Viruses containing viral vectors may be replicable, replication-deficient, or replication-defective. In some embodiments, the viral vector is delivered via an oncolytic virus.

[0099] General methods in molecular biology and cell biochemistry are found in: 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). This can be found in standard textbooks such as (1998), and the disclosures in these documents are incorporated herein by reference.

[0100] II. Synthetic cancer antigens and cognitive binders Synthetic cancer antigens and / or their cognitive binders are provided herein. In the embodiments provided, the synthetic cancer antigen is not normally expressed on the surface of non-cancer cells, is specifically delivered to cancer cells in a target via an oncolytic virus, and can therefore be expressed only on the surface of cancer cells. Furthermore, the cognitive binder can be manipulated as a chimeric antigen receptor for expression on the surface of immune effector cells. This disclosure redesigns the interaction between immune effector cells and cancer targets. By shifting from traditional approaches that target known cancer overexpression proteins that can also be expressed on the surface of non-cancer cells, this disclosure improves the safety of immunotherapy by eliminating recognition by healthy tissue. In some embodiments, the synthetic cancer antigen comprises a target domain and a transmembrane domain. In some embodiments, the synthetic cancer antigen is membrane-bound. In other embodiments, the synthetic cancer antigen comprises a target domain linked to a tumor targeting binding molecule. In some embodiments, the synthetic cancer antigen is soluble. In some embodiments, one or more forms (e.g., membrane-bound and / or soluble) are recognizable by a cognitive binder. Both or one of the forms of the synthetic cancer antigen can be used in combination with one or more cognitive binders. In some embodiments, the target domain of the synthetic cancer antigen is recognizable by a cognitive binder.

[0101] A. Target domain In embodiments provided herein, the target domain of a synthetic cancer antigen is a target domain that is a protein recognizable by a cognitive binder so that it can be specifically targeted for tagging cancer cells. One advantage of the present invention is that the target domain of the synthetic cancer antigen provided herein is designed to be different from proteins expressed on the surface of target non-cancer cells. In some embodiments, the target domain is not expressed on the surface of target non-cancer cells. In some embodiments, the target domain is recognizable by a cognitive binder. As a result, the cognitive binder of the target domain cannot bind to surface proteins expressed on the surface of non-cancer cells, thereby reducing or eliminating off-target binding.

[0102] The target domain of the synthetic cancer antigen provided herein can be any polypeptide that is not expressed on the surface of the target non-cancer cells.

[0103] The binding of a cognitive binder (e.g., scFv) to a target domain can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a binding complex of particular interest (e.g., a protein-antibody complex) by using a reagent (e.g., antibody or scFv) that is specific to the complex of interest.

[0104] In the embodiments provided, the target domain is a protein-binding molecule. In some embodiments, the size of the target domain is approximately 20 to approximately 700 amino acids. In some embodiments, the size of the target domain is at least approximately 20 amino acids. In some embodiments, the size of the target domain is up to approximately 700 amino acids. In some embodiments, the size of the target domain is approximately 20 to approximately 40 amino acids, approximately 20 to approximately 60 amino acids, approximately 20 to approximately 100 amino acids, approximately 20 to approximately 150 amino acids, approximately 20 to approximately 200 amino acids, approximately 20 to approximately 250 amino acids, approximately 20 to approximately 300 amino acids, approximately 20 to approximately 400 amino acids, and approximately 20 to approximately 500 amino acids. 0 amino acids, approximately 20 amino acids to approximately 600 amino acids, approximately 20 amino acids to approximately 700 amino acids, approximately 40 amino acids to approximately 60 amino acids, approximately 40 amino acids to approximately 100 amino acids, approximately 40 amino acids to approximately 150 amino acids, approximately 40 amino acids to approximately 200 amino acids, approximately 40 amino acids to approximately 250 amino acids, approximately 40 amino acids to approximately 300 amino acids, approximately 40 amino acids to approximately 400 amino acids, approximately 40 amino acids to approximately 500 amino acids 60 amino acids, approximately 40 amino acids to approximately 600 amino acids, approximately 40 amino acids to approximately 700 amino acids, approximately 60 amino acids to approximately 100 amino acids, approximately 60 amino acids to approximately 150 amino acids, approximately 60 amino acids to approximately 200 amino acids, approximately 60 amino acids to approximately 250 amino acids, approximately 60 amino acids to approximately 300 amino acids, approximately 60 amino acids to approximately 400 amino acids, approximately 60 amino acids to approximately 500 amino acids, approximately 60 amino acids to approximately 600 amino acids, approximately 60 amino acids to approximately 700 amino acids, approximately 100 amino acids to approximately 150 amino acids, approximately 100 amino acids to approximately 200 amino acids, approximately 100 amino acids to approximately 250 amino acids, approximately 100 amino acids to approximately 300 amino acids, approximately 100 amino acids to approximately 400 amino acids, approximately 100 amino acids to approximately 500 amino acids, approximately 100 amino acids to approximately 600 amino acids, approximately 100 amino acids to approximately 700 amino acids,Approximately 150 amino acids to approximately 200 amino acids, approximately 150 amino acids to approximately 250 amino acids, approximately 150 amino acids to approximately 300 amino acids, approximately 150 amino acids to approximately 400 amino acids, approximately 150 amino acids to approximately 500 amino acids, approximately 150 amino acids to approximately 600 amino acids, approximately 150 amino acids to approximately 700 amino acids, approximately 200 amino acids to approximately 250 amino acids, approximately 200 amino acids to approximately 300 amino acids, approximately 200 amino acids to approximately 400 amino acids, approximately 200 amino acids to approximately 500 amino acids, approximately 200 amino acids to approximately 600 amino acids, approximately 200 amino acids to approximately 700 amino acids, approximately 250 amino acids to approximately 300 amino acids, approximately 25 The amino acids range from 0 to approximately 400, from approximately 250 to approximately 500, from approximately 250 to approximately 600, from approximately 250 to approximately 700, from approximately 300 to approximately 400, from approximately 300 to approximately 500, from approximately 300 to approximately 600, from approximately 300 to approximately 700, from approximately 400 to approximately 500, from approximately 400 to approximately 600, from approximately 400 to approximately 700, from approximately 500 to approximately 600, or from approximately 600 to approximately 700. In some embodiments, the size of the target domain is approximately 20 amino acids, approximately 40 amino acids, approximately 60 amino acids, approximately 100 amino acids, approximately 150 amino acids, approximately 200 amino acids, approximately 250 amino acids, approximately 300 amino acids, approximately 400 amino acids, approximately 500 amino acids, approximately 600 amino acids, or approximately 700 amino acids.

[0105] 1. Antibody or antibody fragment 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, the molecule derived from an antibody is one or more functional fragments of the antibody. Non-limiting examples of functional fragments include heavy chains, light chains, heavy chain variable domains, light chain variable domains, single-chain Fv (scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In some embodiments, the target domain is an antibody that does not contain a heavy chain CH3 domain. In some embodiments, the target domain is an antibody that does not contain a heavy chain CH2 domain. In some embodiments, the target domain is Fab. In some embodiments, the target domain is an antibody that does not contain heavy chain CH2 and CH3 domains.

[0106] In some embodiments, the target domain is an antibody fragment such as a Fab, scFv, or a fragment consisting only of a variable heavy chain. In some embodiments, the target domain is a single-stranded variable fragment (scFv). In some embodiments, the target domain is the heavy chain of the antibody. In some embodiments, the target domain is the light chain of the antibody. In some embodiments, the target domain is the variable domain of the heavy chain of the antibody. In some embodiments, the target domain is the variable domain of the light chain of the antibody.

[0107] In some embodiments, the functional fragments are part of a single polypeptide and are functionally linked. In some embodiments, the functional fragments are directly linked to one another. In some embodiments, the functional fragments are linked via a linker. Exemplary linkers that can be used herein are disclosed in the Examples section of this application. Additional linkers are known to those skilled in the art. In some embodiments, the functional fragments are separate polypeptides that are linked to each other after being translated to form an antibody-derived molecule. 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 contains a nucleotide sequence encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide separates the functional fragments translated from the single polynucleotide. In some embodiments, the self-cleaving peptide is a P2A peptide. In some embodiments, the self-cleaving peptide contains the amino acid sequence SEQ ID NO:4.

[0108] In some embodiments, the antibodies or antibody-derived molecules provided herein include VL, VH, or CDR having the amino acid sequence of VL, VH, or CDR contained in a particular known antibody. In some embodiments, the antibodies or antibody-derived molecules provided herein include VL, VH, or CDR having the amino acid sequence of 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 a particular known antibody.

[0109] Exemplary known antibodies include ReoPro (absiximab), Humira (adalimumab), Hyrimoz (adalimumab-adaz), Cyltezo (adalimumab-adbm), Abrillada (adalimumab-afzb), Amjevita (adalimumab-atto), Hadlima (adalimumab-bwwd), Campath, Lemtrada (aremutuzumab), Praluent (alirocumab), Tecentriq (atezolizumab), Bavencio (avelumab), Simulect (basiliximab), Benlysta (belimumab), Fasenra (benralizumab), Avastin (bevacizumab), Mvasi (bevacizumab-awwb), and Zirabev. (Bevacizumab-bvzr), Zinplava (Bezlotoxumab), Blincyto (Blinatumomab), Siliq (Brodalumab), Beovu (Borucizumab-dbll), Crysvita (Brosumab-twza), Haris (Canakinumab), Cablivi (Caplacizumab-yhdp), Libtayo (Semiprimab-rwlc), Erbitux (Cetuximab), Adakveo (Cryzanlizumab-tmca), Zenapax (Dacrimumab), Zinbryta (Dacrimumab), Darzalex (Daratumumab), Prolia, Xgeva (Denosumab), Unituxin (Dinutuximab), Dupixent (Dupilumab), Imfinzi (Durvalumab), Soliris (Eculizumab), Empliciti (Elotuzumab), Gamifant (Emapalmab-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 (Ivalizumab-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 (olaratumumab), Xolair (omalizumab), Synagis (palivizumab), Vectibix (panitumumab), Keytruda (pembrolizumab), Perjeta (pertuzumab), Cyramza (ramucirumab), Lucentis (ranivizumab), Ultomiris (ravulizumab-cwvz), raxibacumab (laxibacumab), Cinqair (resulizumab), Skyrizi (risankizumab-rzaa), Rituxan (rituximab), Truxima (rituximab-abbs), Ruxience (rituximab-pvvr), Evenity (romosozumab-aqqg), Kevzara (Sarilumab), Cosentyx (Secukinumab), Sylvant (Siltuximab), Tepezza (Teprotumumab-trbw), Ilumya (Childrakizumab-asmn), Actemra (Tocilizumab), Actemra (Tocilizumab), Herceptin (Trastuzumab), Kanjinti (Trastuzumab-anns), Ogivri (Trastuzumab-dkst), Ontruzant (Trastuzumab-dttb), Herzuma (Trastuzumab-pkrb), Trazimera (Trastuzumab-qyyp), Stellara (Ustekinumab), Stellara (Ustekinumab), and EntyvioThis includes, but is not limited to, vedolizumab.

[0110] In some embodiments, the target domain is an antibody fragment of any of the antibodies described above. In some embodiments, the target domain is a molecule derived from any of the antibodies described above. In some embodiments, the molecule derived from the antibody is one or more fragments of the antibody. Non-limiting examples of fragments include heavy chains, light chains, heavy chain variable domains, light chain variable domains, single-chain Fv (scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In some embodiments, the target domain is an antibody that does not contain a heavy chain CH3 domain. In some embodiments, the target domain is an antibody that does not contain a heavy chain CH2 domain. In some embodiments, the target domain is Fab. In some embodiments, the target domain is an antibody that does not contain heavy chain CH2 and CH3 domains.

[0111] In some embodiments, the target domain is a single-chain variable fragment (scFv) of any of the antibodies described above. In some embodiments, the target domain is the heavy chain of any of the antibodies described above. In some embodiments, the target domain is the light chain of any of the antibodies described above. In certain embodiments, the target domain is a fragment that does not bind to the antibody's target but holds an epitope to be recognized by a cognitive binder (e.g., an anti-idiotype antibody). In some embodiments, the target domain is the variable heavy chain (VH) of the antibody. Thus, in some aspects, it will be understood that the target domain lacks a variable light (VL) chain. In some embodiments, the target domain does not bind to the antigen of its source antibody, is substantially unable to bind to it, or exhibits reduced (low) binding affinity to it (e.g., a higher KD value). One advantage of using an antibody fragment as a target domain that does not bind to or substantially does not bind to the antibody's target antigen, or has low affinity binding to the antibody's target antigen, is that it allows the target domain to be delivered in a soluble form (e.g., as described in Section II.AC) while limiting the possibility of the target domain binding to the target antigen of the complete antibody. For example, in some embodiments, the target domain is an antibody fragment in which the antigen-binding domain for recognition of the antigen or epitope is absent, partially removed (e.g., via one or more amino acid substitutions in the CDR), or non-functional. In an exemplary embodiment, the parent antibody is a double-chain antibody containing a variable heavy chain and a variable light chain, with the antigen-binding domain consisting of six CDRs. In some such embodiments, the target domain is an antibody fragment consisting only of a variable heavy chain (VH) composed of only three CDRs.

[0112] In certain embodiments, the target domain is an antibody or antibody fragment that can be recognized by a cognitive binder, which is an anti-idiotype antibody or antigen-binding fragment. Various anti-idiotype antibodies against antibodies are known. Exemplary anti-idiotype antibodies are described in Section II.D.1.

[0113] In the embodiments provided, the antibody target domain, or antibody fragment target domain such as a variable heavy chain, contains an epitope recognized by a cognitive binder (e.g., an anti-idiotype antibody), but does not specifically bind to a target antigen of a native or primary cell, such as a tumor target antigen. In certain embodiments, the cognitive binder is an anti-idiotype antibody (e.g., one of those described in Section II.D.1), and the variable heavy chain contains the idiotype of the antibody. In some embodiments, the target domain is an antibody or antibody fragment having reduced binding to the target antigen of the antibody compared to the parental antibody from which it is derived. In some embodiments, the target domain is an antibody or antibody fragment having low binding affinity to the target antigen of the parental antibody from which it is derived. In some embodiments, the dissociation constant (Kd) of the antibody target domain or antibody fragment target domain for binding to the target antigen of the parental antibody from which it is derived is 10 -6 Ultra (>1 microM), 10 -5 Ultra (>10 microM), or 10 -4 10 Kd and other values ​​of ultra-high (>100 microM) -7 The Kd is greater than (>100 nM). In some embodiments, the Kd of the antibody target domain or antibody fragment target domain for binding to the target antigen of the source antibody is 10 -4 The size is greater than (>100 microM). Exemplary binding assays for determining binding affinity include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), flow cytometry, Western blot assays, or surface plasmon resonance (e.g., Biacore). In some embodiments, such as determined by a binding assay using flow cytometry, an antibody target domain or antibody fragment target domain does not show detectable binding to the target antigen of its source antibody, even if the cell expresses the antibody's target antigen.

[0114] In some embodiments, the target domain is an antibody directed against HER2.

[0115] In some embodiments, the target domain is derived from the trastuzumab parent antibody, the 4D5 anti-HER2 antibody (also known as Ab1). In some embodiments, the target domain is an antibody fragment of the 4D5 (Ab1) antibody.

[0116] In some embodiments, the target domain is a 4D5 antibody or an antigen-binding fragment of a 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, the molecule derived from a 4D5 antibody is one or more fragments of the antibody. In some embodiments, the target domain is a Fab fragment of a 4D5 antibody. In some embodiments, the target domain is a single-chain Fv (scFv) of a 4D5 antibody. In some embodiments, the target domain does not include a constant heavy chain and / or constant light chain domain. In some embodiments, the target domain includes the variable light chain and variable heavy chain of a 4D5 antibody. In some embodiments, the target domain includes the variable heavy chain and does not include the variable light chain of a 4D5 antibody. In some embodiments, the target domain includes the variable light chain and does not include the variable heavy chain of a 4D5 antibody.

[0117] In some embodiments, the target domain is the Fab of the 4D5 antibody. In some embodiments, the target domain includes a light chain containing the amino acid sequence of SEQ ID NO:7. In some embodiments, the target domain includes a heavy chain containing the amino acid sequence of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7. In some embodiments, the target domain includes a heavy chain containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:10.

[0118] In some embodiments, the target domain includes a light chain containing the amino acid sequence of SEQ ID NO:7 and a heavy chain containing the amino acid sequence of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing the amino acid sequence of SEQ ID NO:7 that is at least 70% identical to that of SEQ ID NO:7 and a heavy chain containing the amino acid sequence of SEQ ID NO:10 that is at least 70% identical to that of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing the amino acid sequence of SEQ ID NO:7 that is at least 75% identical to that of SEQ ID NO:7 and a heavy chain containing the amino acid sequence of SEQ ID NO:10 that is at least 75% identical to that of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing the amino acid sequence of SEQ ID NO:7 that is at least 80% identical to that of SEQ ID NO:10 and a heavy chain containing the amino acid sequence of at least 80% identical to that of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing the amino acid sequence of SEQ ID NO:7 that is at least 85% identical to that of SEQ ID NO:7 and a heavy chain containing the amino acid sequence of at least 85% identical to that of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing an amino acid sequence at least 90% identical to that of SEQ ID NO:7, and a heavy chain containing an amino acid sequence at least 90% identical to that of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing an amino acid sequence at least 91% identical to that of SEQ ID NO:7, and a heavy chain containing an amino acid sequence at least 91% identical to that of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing an amino acid sequence at least 92% identical to that of SEQ ID NO:7, and a heavy chain containing an amino acid sequence at least 92% identical to that of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing an amino acid sequence at least 93% identical to that of SEQ ID NO:7, and a heavy chain containing an amino acid sequence at least 93% identical to that of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing an amino acid sequence at least 94% identical to that of SEQ ID NO:7, and a heavy chain containing an amino acid sequence at least 94% identical to that of SEQ ID NO:10.In some embodiments, the target domain includes a light chain containing an amino acid sequence at least 95% identical to that of SEQ ID NO:7, and a heavy chain containing an amino acid sequence at least 95% identical to that of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing an amino acid sequence at least 96% identical to that of SEQ ID NO:7, and a heavy chain containing an amino acid sequence at least 96% identical to that of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing an amino acid sequence at least 97% identical to that of SEQ ID NO:7, and a heavy chain containing an amino acid sequence at least 97% identical to that of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing an amino acid sequence at least 98% identical to that of SEQ ID NO:7, and a heavy chain containing an amino acid sequence at least 98% identical to that of SEQ ID NO:10. In some embodiments, the target domain includes a light chain containing an amino acid sequence at least 99% identical to that of SEQ ID NO:7, and a heavy chain containing an amino acid sequence at least 99% identical to that of SEQ ID NO:10.

[0119] In some embodiments, the target domain is a single-stranded Fv (scFv) of a 4D5 antibody. In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:11. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:11.

[0120] In some embodiments, the target domain is the variable heavy chain of the 4D5 antibody. In some embodiments, the target domain does not include the variable light chain. In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:8. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:8. In the embodiments provided, such a variable heavy chain antibody fragment contains an idiotype that is recognized by an anti-idiotype antibody (e.g., one shown in any of SEQ ID NO:13-15) but does not specifically bind to HER2. In some embodiments, the variable heavy chain antibody fragment has reduced binding to HER2 compared to the 4D5 (Ab1) parent antibody from which it is derived.In some embodiments, the variable heavy chain antibody fragment has a low binding affinity to HER2. In some embodiments, the dissociation constant (Kd) of the target domain of the variable heavy chain antibody fragment for binding to HER2 is 10. -6 Ultra (>1 microM), 10 -5 Ultra (>10 microM), or 10 -4 10 Kd and other values ​​of ultra-high (>100 microM) -7 The Kd is greater than (>100 nM). In some embodiments, the Kd of a variable heavy chain antibody fragment for binding to HER2 is 10 -4 The size is greater than (>100 microM). Exemplary binding assays for determining binding affinity include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), flow cytometry, Western blot assays, or surface plasmon resonance (e.g., Biacore). In some embodiments, the variable heavy chain antibody fragment target domain does not show detectable binding to HER2-expressing cells, such as tumor cells, as determined, for example, by a binding assay using flow cytometry.

[0121] 2. Intracellular proteins In some embodiments, the target domain is a fragment of an intracellular protein. In some embodiments, the target domain is the non-DNA-binding domain of a transcription factor. In some embodiments, the target domain is the inactive leucine zipper domain of a transcription factor. In some embodiments, the target domain is the N-terminal fragment of a transcription factor. In some embodiments, the target domain is the C-terminal fragment of a transcription factor. Non-limiting examples of transcription factors that can be used herein include cFos, cJun, cMAF, ATF, Nfil3, and Xbp1.

[0122] In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:17. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:17.

[0123] In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:18. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:18.

[0124] In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:19. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:19.

[0125] In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:20. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:20.

[0126] In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:21. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:21.

[0127] In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:22. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:22.

[0128] In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:23. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:23.

[0129] 3. Leucine zipper In some embodiments, the target domain is the active leucine zipper domain of the transcription factor. In some embodiments, the transcription factor is a basic leucine zipper (bZIP). Non-limiting examples of bZIP transcription factors that can be used herein include ATF, JUN, CREB, and Fos. In some embodiments, the target domain is the leucine zipper domain of ATF. In some embodiments, the target domain is the leucine zipper domain of JUN. In some embodiments, the target domain is the leucine zipper domain of CREB. In some embodiments, the target domain is the leucine zipper domain of Fos.

[0130] In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:26. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:26.

[0131] In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:27. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:27.

[0132] In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:28. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:28.

[0133] In some embodiments, the target domain contains the amino acid sequence of SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:29. In some embodiments, the target domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:29.

[0134] B. Membrane-targeting domain In some embodiments, the synthetic oncoantigen is a membrane-bound protein, and the target domain is linked to a membrane targeting domain. In some embodiments, the synthetic oncoantigen comprises a target domain and a membrane targeting domain. The target domain may be any of those described in Section II.A.

[0135] The membrane targeting domains of synthetic cancer antigens provided herein may be transmembrane domains of any known transmembrane protein or may be derived from a synthetic source. The transmembrane domains disclosed herein for synthetic cancer antigens may also be used as transmembrane domains of the CAR molecules disclosed below.

[0136] Transmembrane domains are classified based on their three-dimensional structure. For example, a transmembrane domain can form an alpha-helix, a complex of more than one alpha-helix, a beta-barrel, or any other stable structure capable of penetrating the cellular phospholipid bilayer. Furthermore, transmembrane domains can also be classified based on their transmembrane domain topology, which includes the number of times the transmembrane domain crosses the membrane and the orientation of the protein. For example, a single-pass membrane protein crosses the cell membrane once, while a multi-pass membrane protein crosses the cell membrane at least twice (e.g., two, three, four, five, six, seven, or more times). Membrane proteins can be defined as type I, type II, or type III depending on the topology of their terminal and membrane-penetrating segments (maybe more than one) in relation to the inside and outside of the cell. Type I membrane proteins have a single-pass transmembrane domain and are oriented so that the N-terminus of the protein is on the extracellular side of the cellular lipid bilayer and the C-terminus is on the cytoplasmic side. Type II membrane proteins also have a single transmembrane domain, but they are oriented so that the C-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the N-terminus is on the cytoplasmic side. Type III membrane proteins have multiple transmembrane segments and can be further subdivided based on the number of transmembrane segments and the positions of the N-terminus and C-terminus.

[0137] In some embodiments, the transmembrane domain is derived from a type I single-pass membrane protein. In some embodiments, a transmembrane domain derived from a multi-pass membrane protein may also be suitable. Multi-pass membrane proteins may include a complex (at least two, three, four, five, six, seven, or more) alpha-helix or beta-sheet structure. In some embodiments, the N-terminus and C-terminus of the multi-pass membrane protein are located on opposite sides of the lipid bilayer, for example, the N-terminus of the protein is on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is on the extracellular side.

[0138] The transmembrane domain may also include at least a portion of a synthetic, naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, naturally occurring alpha-helix or beta-sheet. In some embodiments, the protein segment consists of approximately 15 to 100 amino acids. In some embodiments, the protein segment consists of at least approximately 20 amino acids, for example, 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. Patent No. 7,052,906 and PCT Publication No. WO2000 / 032776, the relevant disclosures of these documents are incorporated herein by reference.

[0139] The transmembrane domains provided herein may comprise a transmembrane region and a cytoplasmic region located at the C-terminal end of the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids, which in some embodiments serve 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.

[0140] In some embodiments, the transmembrane region of the transmembrane domain contains hydrophobic amino acid residues. In some embodiments, the transmembrane domains provided herein contain artificial hydrophobic sequences. 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 contains substantially 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 contains a polyleucine-alanine sequence. The hydroxyl or hydrophobic or hydrophilic characteristics of a protein or protein segment can be evaluated by any method known in the art, such as Kyte and Doolittle hydroxyl analysis.

[0141] In some embodiments, the transmembrane domain is derived from a transmembrane glycoprotein. In some embodiments, the transmembrane domain is derived from the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, CD11α, CD18, ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL-2Rbeta, IL-2Rgamma, IL-7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49d, ITGA6, VLA-6, CD49f, ITGAD, CD1D, ITGAE, CD103, ITGAL, ITGAM, CD1B, ITGAX, CD1C, ITGB1, CD29, ITGB2, CD18, 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 It includes a transmembrane domain selected from the transmembrane domains of (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0142] In some embodiments, the transmembrane domain is the CD8 transmembrane domain.

[0143] In some embodiments, the transmembrane domain contains the amino acid sequence of SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:3.

[0144] In some embodiments, the size of the transmembrane domain is approximately 40 to 90 amino acids. In some embodiments, the size of the transmembrane domain is at least approximately 40 amino acids. In some embodiments, the size of the transmembrane domain is up to approximately 90 amino acids. In some embodiments, the size of the transmembrane domain is approximately 40 to 50 amino acids, approximately 40 to 60 amino acids, approximately 40 to 70 amino acids, approximately 40 to 80 amino acids, approximately 40 to 90 amino acids, approximately 50 to 60 amino acids, approximately 50 to 70 amino acids, approximately 50 to 80 amino acids, approximately 50 to 90 amino acids, approximately 60 to 70 amino acids, approximately 60 to 80 amino acids, approximately 60 to 90 amino acids, approximately 70 to 80 amino acids, approximately 70 to 90 amino acids, or approximately 80 to 90 amino acids.

[0145] The synthetic cancer antigens disclosed herein may further include additional elements. In some embodiments, the synthetic cancer antigen includes a tag. In some embodiments, the tag is located at the N-terminus of the synthetic cancer antigen. In some embodiments, the tag is located 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 an HA tag. In some embodiments, the tag includes the amino acid sequence of SEQ ID NO:12. In some embodiments, the tag includes the amino acid sequence of SEQ ID NO:30.

[0146] In some embodiments, different elements of the synthetic cancer antigen are functionally linked. In some embodiments, different elements are directly linked to one another. In some embodiments, different elements are linked via linkers. Exemplary linkers that can be used herein are disclosed in the Examples section of this application. Additional linkers are known to those skilled in the art.

[0147] C. Soluble target domain In some embodiments, the synthetic cancer antigen is a soluble (sol) protein, and the target domain is linked to a tumor targeting molecule that directs the synthetic cancer antigen to tumor cells so that the target domain is exposed to the extracellular space. In some embodiments, the synthetic cancer antigen comprises the target domain and the tumor targeting molecule. The target domain may be any of those described in Section II.A.

[0148] In some embodiments, synthetic cancer antigens are provided herein that comprise a target domain containing at least one of the target domains described in Section II.A and a tumor targeting molecule (e.g., an antibody or antigen-binding fragment specific to a tumor-associated antigen). In some embodiments, the amino acid sequences of the target domain and the tumor targeting molecule are directly linked. In some embodiments, the amino acid sequences of the target domain and the tumor targeting molecule are indirectly linked via a linker such as a peptide linker. In certain embodiments, such synthetic cancer antigens are soluble and not cell-bound membranes. In certain embodiments in which the synthetic cancer antigen is expressed from a cell, the synthetic cancer antigen can be secreted from the cell, and in the aspects provided, other uninfected bystander tumor cells can be tagged to expose the synthetic cancer antigen to the outer surface of the cell surface.

[0149] In some embodiments, the tumor targeting molecule of a synthetic cancer antigen can bind to an antigen expressed on a target cell, such as a tumor cell, and as a result, the coupled target domain is exposed on the outer surface of the cell. In some embodiments, the tumor targeting molecule binds to a tumor-associated antigen (TAA). In some embodiments, the tumor targeting molecule is an antibody or antibody fragment. In some embodiments, the tumor targeting molecule is a tumor-associated antigen (TAA) binding molecule. In some embodiments, the tumor targeting molecule binding molecule is an antibody or antigen-binding fragment that targets a tumor-associated antigen. In some embodiments, the antigen is associated with cancer. In some embodiments, the antigen is associated with solid tumors.

[0150] For example, tumor-associated antigens include antigens expressed on tumors, including but not limited to adenomas, carcinomas, or sarcomas. In some embodiments, tumor-associated antigens are expressed on cancer cells, 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, gastric cancer, thyroid cancer, bile duct 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 aspects, the cancer is bladder cancer. In some aspects, the cancer is colorectal cancer. In some aspects, the cancer is endometrial cancer. In some aspects, the cancer is liver cancer. In some aspects, the cancer is kidney cancer. In some aspects, the cancer is lung cancer. In some aspects, the cancer is melanoma. In some aspects, the cancer is pancreatic cancer. In some aspects, the cancer is prostate cancer. In some aspects, the cancer is thyroid cancer. In some aspects, the cancer is ovarian cancer. In some aspects, the cancer is insensitive or resistant.

[0151] In some embodiments, the tumor targeting molecule binds to a tumor-associated antigen (TAA). In some embodiments, the tumor targeting molecule is a molecule derived from an antibody, and is one or more functional fragments of an antibody that targets a tumor-associated antigen. Non-limiting examples of functional fragments include heavy chains, light chains, heavy chain variable domains, light chain variable domains, single-chain Fv (scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In some embodiments, the tumor targeting molecule is an antibody that does not contain a heavy chain CH3 domain. In some embodiments, the tumor targeting molecule is an antibody that does not contain a heavy chain CH2 domain. In some embodiments, the tumor targeting molecule is a Fab. In some embodiments, the tumor targeting molecule is an antibody that does not contain heavy chain CH2 and CH3 domains. In some embodiments, the tumor targeting molecule is a single-chain variable fragment (scFv). In some embodiments, the tumor targeting molecule is the heavy chain of the antibody. In some embodiments, the tumor targeting molecule is the light chain of the antibody. In some embodiments, the tumor targeting molecule is the variable domain of the heavy chain of the antibody. In some embodiments, the tumor targeting molecule is the variable domain of the light chain of the antibody.

[0152] In some embodiments, the tumor targeting molecule is a single-stranded variable fragment (scFv). As used herein, the term “single-stranded variable fragment” or “scFv” is a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin (e.g., mouse or human) are covalently linked to form a VH:VL heterodimer. The heavy chain (VH) and light chain (VL) are linked either directly or by peptide coding linkers (e.g., 10, 15, 20, or 25 amino acids) that link the N-terminus of VH to the C-terminus of VL, or the C-terminus of VH to the N-terminus of VL.

[0153] In some embodiments, the functional fragments are part of a single polypeptide and are functionally linked. In some embodiments, the functional fragments are directly linked to one another. In some embodiments, the functional fragments are linked via a linker. Exemplary linkers that can be used herein are disclosed in the Examples section of this application. Additional linkers are known to those skilled in the art. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the functional fragments are separate polypeptides that are linked to each other after being translated to form an antibody-derived molecule. 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 a nucleotide sequence encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide separates the functional fragments translated from the 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: 4.

[0154] In some embodiments, the tumor-targeting molecule is a tumor-targeting antibody. In some embodiments, the tumor-targeting antibody binds to an antigen expressed on the surface of tumor cells. In some embodiments, the tumor-targeting antibody or antibody-derived molecule provided herein comprises VL, VH, or CDR having an amino acid sequence of VL, VH, or CDR contained in a particular known antibody. In some embodiments, the tumor-targeting antibody or antibody-derived molecule provided herein comprises VL, VH, or CDR having 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 the VL, VH, or CDR contained in a particular known antibody.

[0155] Exemplary tumor-associated antigens and their exemplary known antibodies include, but are not limited to, those listed in Table 1 below.

[0156] (Table 1) Exemplary tumor-associated antigens and their known antibodies TIFF2026520446000002.tif125160TIFF2026520446000003.tif243160TIFF2026520446000004.tif232160TIFF2026520446000005.tif65160

[0157] In some embodiments, tumor-associated antigens targeted by tumor-targeting molecules are expressed on solid tumors.

[0158] In some embodiments, tumor-associated antigens are expressed on solid tumors. Exemplary solid tumor-associated antigens include EpCAM (epithelial cell adhesion molecule), CEA (carcinoembryonic antigen), gpA33 (glycoprotein A33 (transmembrane), mucin), TAG-72 (tumor-associated glycoprotein 72), CAIX (carbonic anhydrase IX), PSMA (prostate-specific membrane antigen), and FBP (folate-binding protein), EGFR / ERBB1 / HER1 (epidermal growth factor receptor 1), ERBB2 / HER2 (epidermal growth factor receptor 2), ERBB3 (epidermal growth factor receptor 3), MET (tyrosine protein kinase), IGF1R (insulin-like growth factor 1 receptor), EPHA3 (EPH receptor A3), TRAILR1 (death receptor 4), and MMP. (This includes, but is not limited to, matrix metalloproteinases such as 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 claudin 18.2, and RANK-L (nuclear factor kappa-B receptor activator ligand).

[0159] In some embodiments, the tumor-associated antigen targeted by the tumor-targeting molecule is an epithelial cell adhesion / activation molecule (EpCAM), epidermal growth factor receptor (EGFR), or human epidermal growth factor receptor 2 (HER2). In some embodiments, the tumor-associated antigen targeted by the tumor-targeting molecule is EpCAM.

[0160] Epithelial cell adhesion molecules (EpCAMs) are transmembrane glycoproteins that mediate Ca2+-independent isoplastic cell adhesion in epithelium. EpCAMs are also involved in cell signaling, migration, proliferation, and differentiation. Additionally, EpCAMs have oncogenic potential through their ability to upregulate c-myc, e-fabp, and cyclins A and E. Because EpCAMs are exclusively expressed in epithelium and epithelial neoplasms, they can be used as a therapeutic marker for various cancers, including head and neck cancer, ovarian cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, esophageal cancer, and lung cancer.

[0161] In some embodiments, the tumor targeting molecule binds to a tumor-associated antigen that is EpCAM. In some embodiments, the tumor targeting molecule is an antibody or its antigen-binding fragment that binds to a tumor-associated antigen that is EpCAM. In some embodiments, the tumor targeting molecule is a single-chain Fv (scFv) of an anti-EpCAM antibody. In some embodiments, the tumor targeting molecule does not contain constant heavy chain and / or constant light chain domains.

[0162] In some embodiments, the tumor targeting molecule is adekatumumab or an antigen-binding fragment of adekatumumab. In some embodiments, the tumor targeting molecule is the scFv of adekatumumab. In some embodiments, the tumor targeting molecule includes the variable light chain and variable heavy chain of adekatumumab. In some embodiments, the tumor targeting molecule is edrecolomab or an antigen-binding fragment of edrecolomab. In some embodiments, the tumor targeting molecule is the scFv of edrecolomab. In some embodiments, the tumor targeting molecule includes the variable light chain and variable heavy chain of edrecolomab. In some embodiments, the tumor targeting molecule is tucotzumab or an antigen-binding fragment of tucotzumab. In some embodiments, the tumor targeting molecule is the scFv of tucotzumab. In some embodiments, the tumor targeting molecule includes the variable light chain and variable heavy chain of tucotzumab.

[0163] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises heavy chain complementarity-determining regions 1 (CDR-H1), CDR-H2, and CDR-H3 contained in the VH shown in SEQ ID NO:35, and the VL region comprises light chain complementarity-determining regions 1 (CDR-L1), CDR-L2, and CDR-L3 contained in the VL shown in SEQ ID NO:37. In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises heavy chain complementarity-determining regions 1 (CDR-H1), CDR-H2, and CDR-H3, each containing amino acid sequences shown in SEQ ID NO: 42, 43, and 44, respectively, and the VL region comprises light chain complementarity-determining regions 1 (CDR-L1), CDR-L2, and CDR-L3, each containing amino acid sequences shown in SEQ ID NO: 45, 46, and 47, respectively.

[0164] In some embodiments, the tumor targeting molecule includes a variable light chain region containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the tumor targeting molecule includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO:35. In some embodiments, the tumor targeting molecule includes a variable light chain containing 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:37. In some embodiments, the tumor targeting molecule includes a variable heavy chain containing 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:35.

[0165] In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of SEQ ID NO:37 and a variable heavy chain containing the amino acid sequence of SEQ ID NO:35. In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of at least 70% identical to that of SEQ ID NO:37 and a variable heavy chain containing the amino acid sequence of at least 70% identical to that of SEQ ID NO:35. In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of at least 75% identical to that of SEQ ID NO:37 and a variable heavy chain containing the amino acid sequence of at least 75% identical to that of SEQ ID NO:35. In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of at least 80% identical to that of SEQ ID NO:37 and a variable heavy chain containing the amino acid sequence of at least 80% identical to that of SEQ ID NO:35. In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of at least 85% identical to that of SEQ ID NO:37 and a variable heavy chain containing the amino acid sequence of at least 85% identical to that of SEQ ID NO:35. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 90% identical to SEQ ID NO:37 and a variable heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO:35. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 91% identical to SEQ ID NO:37 and a variable heavy chain having an amino acid sequence at least 91% identical to SEQ ID NO:35. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 92% identical to SEQ ID NO:37 and a variable heavy chain having an amino acid sequence at least 92% identical to SEQ ID NO:35. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 93% identical to SEQ ID NO:37 and a variable heavy chain having an amino acid sequence at least 93% identical to SEQ ID NO:35. In some embodiments, the tumor targeting molecule comprises a variable light chain having an amino acid sequence at least 94% identical to SEQ ID NO:37 and a variable heavy chain having an amino acid sequence at least 94% identical to SEQ ID NO:35.In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 95% identical to SEQ ID NO:37 and a variable heavy chain having an amino acid sequence at least 95% identical to SEQ ID NO:35. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 96% identical to SEQ ID NO:37 and a variable heavy chain having an amino acid sequence at least 96% identical to SEQ ID NO:35. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 97% identical to SEQ ID NO:37 and a variable heavy chain having an amino acid sequence at least 97% identical to SEQ ID NO:35. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 98% identical to SEQ ID NO:37 and a variable heavy chain having an amino acid sequence at least 98% identical to SEQ ID NO:35. In some embodiments, the tumor targeting molecule comprises a variable light chain having an amino acid sequence at least 99% identical to SEQ ID NO:37 and a variable heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:35. In some of any such embodiments, the variable light chain and the variable heavy chain are... It is linked by a peptide linker, such as the sequence shown in TIFF2026520446000006.tif4128.

[0166] In some embodiments, the tumor targeting molecule binds to a tumor-associated antigen that is HER2. In some embodiments, the tumor targeting molecule is an antibody or its antigen-binding fragment that binds to a tumor-associated antigen that is HER2. In some embodiments, the tumor targeting molecule is a single-chain Fv (scFv) of an anti-HER2 antibody. In some embodiments, the tumor targeting molecule does not contain constant heavy chain and / or constant light chain domains.

[0167] In some embodiments, the tumor targeting molecule is derived from the 4D5 anti-HER2 antibody (also known as Ab1), which is the parent antibody of trastuzumab. In some embodiments, the tumor targeting molecule is an antibody fragment of the 4D5 (Ab1) antibody.

[0168] In some embodiments, the tumor targeting molecule is a single-stranded Fv (scFv) of a 4D5 antibody.

[0169] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises heavy chain complementarity-determining regions 1 (CDR-H1), CDR-H2, and CDR-H3 contained in the VH shown in SEQ ID NO:8, and the VL region comprises light chain complementarity-determining regions 1 (CDR-L1), CDR-L2, and CDR-L3 contained in the VL shown in SEQ ID NO:5.

[0170] In some embodiments, the tumor targeting molecule includes a variable light chain region containing the amino acid sequence of SEQ ID NO:5. In some embodiments, the tumor targeting molecule includes a variable heavy chain region containing the amino acid sequence of SEQ ID NO:8. In some embodiments, the tumor targeting molecule includes a variable light chain containing 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:5. In some embodiments, the tumor targeting molecule includes a variable heavy chain containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8.

[0171] In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of SEQ ID NO:5 and a variable heavy chain containing the amino acid sequence of SEQ ID NO:8. In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of SEQ ID NO:5 that is at least 70% identical and a variable heavy chain containing the amino acid sequence of SEQ ID NO:8 that is at least 70% identical. In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of SEQ ID NO:5 that is at least 75% identical and a variable heavy chain containing the amino acid sequence of SEQ ID NO:8 that is at least 75% identical. In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of SEQ ID NO:5 that is at least 80% identical and a variable heavy chain containing the amino acid sequence of SEQ ID NO:8 that is at least 80% identical. In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of SEQ ID NO:5 that is at least 85% identical and a variable heavy chain containing the amino acid sequence of SEQ ID NO:8 that is at least 85% identical. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 90% identical to SEQ ID NO:5 and a variable heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO:8. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 91% identical to SEQ ID NO:5 and a variable heavy chain having an amino acid sequence at least 91% identical to SEQ ID NO:8. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 92% identical to SEQ ID NO:5 and a variable heavy chain having an amino acid sequence at least 92% identical to SEQ ID NO:8. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 93% identical to SEQ ID NO:5 and a variable heavy chain having an amino acid sequence at least 93% identical to SEQ ID NO:8. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 94% identical to SEQ ID NO:5 and a variable heavy chain having an amino acid sequence at least 94% identical to SEQ ID NO:8.In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 95% identical to SEQ ID NO:5 and a variable heavy chain having an amino acid sequence at least 95% identical to SEQ ID NO:8. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 96% identical to SEQ ID NO:5 and a variable heavy chain having an amino acid sequence at least 96% identical to SEQ ID NO:8. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 97% identical to SEQ ID NO:5 and a variable heavy chain having an amino acid sequence at least 97% identical to SEQ ID NO:8. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 98% identical to SEQ ID NO:5 and a variable heavy chain having an amino acid sequence at least 98% identical to SEQ ID NO:8. In some embodiments, the tumor targeting molecule includes a variable light chain having an amino acid sequence at least 99% identical to SEQ ID NO:5 and a variable heavy chain having an amino acid sequence at least 99% identical to SEQ ID NO:8.

[0172] In some embodiments, the tumor targeting molecule contains the amino acid sequence of SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 70% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 75% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 80% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 85% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 90% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 91% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 92% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 93% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 94% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 95% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 96% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 97% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 98% identical to SEQ ID NO:11. In some embodiments, the tumor targeting molecule contains an amino acid sequence that is at least 99% identical to SEQ ID NO:11.

[0173] In some embodiments, the tumor-associated antigen targeted by the tumor-targeting molecule is GPC2. In some embodiments, the tumor-targeting molecule is an antibody or antibody-conjugated fragment that targets the tumor-associated antigen, which is GPC2. In some embodiments, the tumor-targeting molecule is a single-chain Fv (scFv) anti-GPC2 antibody. In some embodiments, the tumor-targeting molecule does not contain a constant heavy chain and / or constant light chain domain. In some embodiments, the tumor-associated antigen targeted by the tumor-targeting molecule is mesothelin. In some embodiments, the tumor-targeting molecule is an antibody or antibody-conjugated fragment that targets the tumor-associated antigen, which is mesothelin. In some embodiments, the tumor-targeting molecule is a single-chain Fv (scFv) anti-mesothelin antibody. In some embodiments, the tumor-targeting molecule does not contain a constant heavy chain and / or constant light chain domain. In some embodiments, the tumor-targeting molecule is anetumab or an antigen-conjugated fragment of anetumab. In some embodiments, the tumor targeting molecule is the scFv of anetumab. In some embodiments, the tumor targeting molecule comprises the variable light chain and variable heavy chain of anetumab.

[0174] In some embodiments, the tumor targeting molecule is amatuximab or an antigen-binding fragment of amatuximab. In some embodiments, the tumor targeting molecule is the scFv of amatuximab. In some embodiments, the tumor targeting molecule comprises the variable light chain and variable heavy chain of amatuximab.

[0175] In some embodiments, the tumor-associated antigen targeted by the tumor-targeting molecule is a prostate stem cell antigen (PSCA). In some embodiments, the tumor-targeting molecule is an antibody or antibody-binding fragment that targets the tumor-associated antigen, which is a PSCA. In some embodiments, the tumor-targeting molecule is a single-chain Fv (scFv) anti-PSCA antibody. In some embodiments, the tumor-targeting molecule does not contain constant heavy chain and / or constant light chain domains.

[0176] In some embodiments, the tumor targeting molecule is AGS-PSCA or an antigen-binding fragment of AGS-PSCA. In some embodiments, the tumor targeting molecule is the scFv of AGS-PSCA. In some embodiments, the tumor targeting molecule comprises the variable light chain and variable heavy chain of AGS-PSCA.

[0177] In some embodiments, the tumor-associated antigen targeted by the tumor-targeting molecule is claudin 6. In some embodiments, the tumor-targeting molecule is an antibody or antibody-conjugated fragment that targets the tumor-associated antigen, which is claudin 6. In some embodiments, the tumor-targeting molecule is a single-chain Fv (scFv) anti-claudin 6 antibody. In some embodiments, the tumor-targeting molecule does not contain constant heavy chain and / or constant light chain domains.

[0178] In some embodiments, the tumor-associated antigen targeted by the tumor-targeting molecule is GPC3. In some embodiments, the tumor-targeting molecule is an antibody or antibody-conjugated fragment that targets the tumor-associated antigen, which is GPC3. In some embodiments, the tumor-targeting molecule is a single-chain Fv (scFv) of an anti-GPC3 antibody. In some embodiments, the tumor-targeting molecule does not contain constant heavy chain and / or constant light chain domains.

[0179] In some embodiments, the tumor targeting molecule is codolituzumab or an antigen-binding fragment of codolituzumab. In some embodiments, the tumor targeting molecule is the scFv of codolituzumab. In some embodiments, the tumor targeting molecule comprises the variable light chain and variable heavy chain of codolituzumab.

[0180] In some embodiments, the tumor targeting molecule includes a variable light chain containing the amino acid sequence of SEQ ID NO:61. In some embodiments, the tumor targeting molecule includes a variable heavy chain containing the amino acid sequence of SEQ ID NO:62. In some embodiments, the tumor targeting molecule includes a variable light chain containing 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:61. In some embodiments, the tumor targeting molecule includes a variable heavy chain containing 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:62. In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of SEQ ID NO:61 and a variable heavy chain containing the amino acid sequence of SEQ ID NO:62.

[0181] In some embodiments, the tumor-associated antigen targeted by the tumor-targeting molecule is claudin 18.2. In some embodiments, the tumor-targeting molecule is an antibody or antibody-conjugated fragment that targets the tumor-associated antigen, which is claudin 18.2. In some embodiments, the tumor-targeting molecule is a single-chain Fv (scFv) anti-claudin 18.2 antibody. In some embodiments, the tumor-targeting molecule does not contain constant heavy chain and / or constant light chain domains.

[0182] In some embodiments, the tumor targeting molecule is zolbetuximab or an antigen-binding fragment of zolbetuximab. In some embodiments, the tumor targeting molecule is the scFv of zolbetuximab. In some embodiments, the tumor targeting molecule comprises the variable light chain and variable heavy chain of zolbetuximab.

[0183] In some embodiments, the tumor targeting molecule includes a variable light chain containing the amino acid sequence of SEQ ID NO:63. In some embodiments, the tumor targeting molecule includes a variable heavy chain containing the amino acid sequence of SEQ ID NO:64. In some embodiments, the tumor targeting molecule includes a variable light chain containing 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:63. In some embodiments, the tumor targeting molecule includes a variable heavy chain containing 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:64. In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of SEQ ID NO:63 and a variable heavy chain containing the amino acid sequence of SEQ ID NO:64.

[0184] In some embodiments, the tumor-associated antigen targeted by the tumor-targeting molecule is a tumor-associated antigen (MMP). In some embodiments, the tumor-associated antigen targeted by the tumor-targeting molecule is selected from the group consisting of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, and MMP28. In some embodiments, the tumor-associated antigen targeted by the tumor-targeting molecule is MMP9. In some embodiments, the tumor-targeting molecule is an antibody or antibody-binding fragment that targets the tumor-associated antigen, which is MMP9. In some embodiments, the tumor targeting molecule is a single-chain Fv (scFv) of an anti-MMP9 antibody. In some embodiments, the tumor targeting molecule does not contain constant heavy chain and / or constant light chain domains.

[0185] In some embodiments, the tumor targeting molecule is andecaliximab or an antigen-binding fragment of andecaliximab. In some embodiments, the tumor targeting molecule is the scFv of andecaliximab. In some embodiments, the tumor targeting molecule comprises the variable light chain and variable heavy chain of andecaliximab.

[0186] In some embodiments, the tumor targeting molecule includes a variable light chain containing the amino acid sequence of SEQ ID NO:65. In some embodiments, the tumor targeting molecule includes a variable heavy chain containing the amino acid sequence of SEQ ID NO:66. In some embodiments, the tumor targeting molecule includes a variable light chain containing 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:65. In some embodiments, the tumor targeting molecule includes a variable heavy chain containing 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:66. In some embodiments, the tumor targeting molecule comprises a variable light chain containing the amino acid sequence of SEQ ID NO:65 and a variable heavy chain containing the amino acid sequence of SEQ ID NO:66.

[0187] In some embodiments, different elements of a soluble synthetic cancer antigen are functionally linked. In some embodiments, different elements are directly linked to one another. In some embodiments, different elements are linked via a linker. Exemplary linkers that can be used herein are disclosed in the Examples section of this application. Additional linkers are known to those skilled in the art.

[0188] In some embodiments, the linker may be a mobile linker. The linker may include peptides, polymers, nucleotides, nucleic acids, polysaccharides, and lipid organic species (such as polyethylene glycol). In some embodiments, the linker is a peptide linker. The peptide linker may have an amino acid length of about 2 to 100, 10 to 50, or 15 to 30. In some embodiments, the peptide linker may have an amino acid length of at least 10, at least 15, or at least 20, or an amino acid length of up to 80, 90, or 100. In some embodiments, the linker may have a length of 1 to 100 amino acids, 1 to 75 amino acids, 1 to 50 amino acids, 1 to 25 amino acids, 5 to 100 amino acids, 5 to 75 amino acids, 5 to 50 amino acids, 5 to 25 amino acids, 10 to 100 amino acids, 10 to 75 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids. In some embodiments, the linker may have a length of 5 to 50 amino acids.

[0189] Linkers can be naturally occurring, synthetic, or a combination of both. Particularly preferred linker polypeptides primarily consist of amino acid residues selected from glycine (Gly), serine (Ser), alanine (Ala), and threonine (Thr). For example, a linker may contain at least 75% (calculated based on the total number of residues present in the peptide linker) of amino acid residues selected from Gly, Ser, Ala, and Thr, such as at least 80%, at least 85%, or at least 90%. A linker may also consist only of Gly, Ser, Ala, and / or Thr residues. In some embodiments, a linker contains 1 to 25 glycine residues, 5 to 20 glycine residues, 5 to 15 glycine residues, or 8 to 12 glycine residues. In some aspects, a preferred peptide linker typically contains at least 50% glycine residues, such as at least 75% glycine residues. In some embodiments, a peptide linker contains only glycine residues. In some embodiments, the peptide linker comprises only glycine and serine residues.

[0190] In some embodiments, such linkers are primarily composed of the amino acids glycine and serine, which are represented herein as GS linkers. In some embodiments, the linkers contain (GGS)n (where n is 1 to 10, such as 1 to 5, e.g., 1 to 3), GGS(GGS)n (SEQ ID NO: 74) (where n is 0 to 10), etc. In certain embodiments, the linkers contain the sequence (GGGGS)n (SEQ ID NO: 75), where n is 1 to 10, or where n is 1 to 5, 1 to 3, etc. In further embodiments, the linkers contain (GGGGGS)n (SEQ ID NO: 76), where n is 1 to 4, 1 to 3, etc. The linkers may contain any combination of the above, such as combining two, three, four, or five repeats of GS, GGS, GGGGS, and / or GGGGGS linkers. In some embodiments, such linkers have a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids.

[0191] In some embodiments, the linker is (in single-letter amino acid codes) GGS, GGGGS (SEQ ID NO: 77 and 67), or GGGGGS (SEQ ID NO: 78), or includes the same. In some embodiments, the GS linker is the amino acid sequence GGSGGS, i.e., (GGS)2 (SEQ ID NO: 79); the amino acid sequence GGSGGSGGS, i.e., (GGS)3 (SEQ ID NO: 80); The amino acid sequence TIFF2026520446000007.tif3128, i.e., (GGS)4 (SEQ ID NO:81); The amino acid sequence TIFF2026520446000008.tif3128, i.e., (GGS)5 (SEQ ID NO:82); The amino acid sequence TIFF2026520446000009.tif3128, that is, (G5S)3 (SEQ ID NO:83), The amino acid sequence TIFF2026520446000010.tif4128, and The amino acid sequence is TIFF2026520446000011.tif4128, or contains thereof. In some embodiments, the linker is GGGG (SEQ ID NO: 86), or contains thereof. In some embodiments, the linker is GGGGG (SEQ ID NO: 71), or contains thereof. In some embodiments, the linker is GGGGGGGG (SEQ ID NO: 72), or contains thereof. In some of the above examples, serine can be replaced with alanine (e.g., (Gly4Ala) or (Gly3Ala)).

[0192] In some aspects, the linker, TIFF2026520446000012.tif4128 or including it. In some embodiments, the linker is GSGSGS (SEQ ID NO:68) or including it.

[0193] In some cases, it may be desirable to impart some rigidity to the peptide linker. This can be achieved by including proline residues in the amino acid sequence of the peptide linker. Thus, in some embodiments, the linker includes at least one proline residue in the amino acid sequence of the peptide linker. For example, the peptide linker may have an amino acid sequence in which at least 25% (e.g., at least 50% or at least 75%) of the amino acid residues are proline residues. In a particular embodiment, the peptide linker contains only proline residues. In some embodiments, the linker is or includes PAPAP (SEQ ID NO: 70).

[0194] In some embodiments, the linker is or includes EAAAK (SEQ ID NO:69).

[0195] In some embodiments, the linker includes an amino acid sequence selected from the group consisting of SEQ ID NO:33 (corresponding to the nucleotide sequence shown in SEQ ID NO:32) or 67-86, or includes 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 NO:33 or 67-86. In some embodiments, the linker includes an amino acid sequence selected from the group consisting of SEQ ID NO:33 or 67-86. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO:33. In some embodiments, the linker contains 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:33.

[0196] The synthetic cancer antigens disclosed herein may further include additional elements. In some embodiments, the synthetic cancer antigen includes a tag. In some embodiments, the tag is located at the N-terminus of the synthetic cancer antigen. In some embodiments, the tag is located 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 an HA tag. In some embodiments, the tag includes the amino acid sequence of SEQ ID NO:12. In some embodiments, the tag includes the amino acid sequence of SEQ ID NO:30.

[0197] In some embodiments, different elements of the synthetic cancer antigen are functionally linked. In some embodiments, different elements are directly linked to one another. In some embodiments, different elements are linked via linkers. Exemplary linkers that can be used herein are disclosed in the Examples section of this application. Additional linkers are known to those skilled in the art.

[0198] In some of the embodiments provided, 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 with the amino acid sequence of SEQ ID NO:31. In some of the embodiments provided, 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 with the amino acid sequence of SEQ ID NO:39. In some of the embodiments provided, 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 with the amino acid sequence of SEQ ID NO:39.

[0199] In some of the embodiments provided, 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 with the amino acid sequence of SEQ ID NO:88. In some of the embodiments provided, 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 with the amino acid sequence of SEQ ID NO:89. In some of the embodiments provided, 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 with the amino acid sequence of SEQ ID NO:89.

[0200] D. Cognate Binder Cognite binders, for example in Section II.A, specifically bind to the target domain of any of the synthetic cancer antigens disclosed herein. In some embodiments, cognitive binders cannot bind to proteins naturally expressed on the surface of non-cancer cells. In some embodiments, cognitive binders can be manipulated as chimeric antigen receptors for expression on the surface of immune effector cells.

[0201] In the embodiments provided, the cognitive binder is a protein-binding molecule. In some embodiments, the size of the cognitive binder is approximately 20 to approximately 700 amino acids. In some embodiments, the size of the cognitive binder is at least approximately 20 amino acids. In some embodiments, the size of the cognitive binder is up to approximately 700 amino acids. In some embodiments, the size of the cognitive binder is approximately 20 to approximately 40 amino acids, approximately 20 to approximately 60 amino acids, approximately 20 to approximately 100 amino acids, approximately 20 to approximately 150 amino acids, approximately 20 to approximately 200 amino acids, approximately 20 to approximately 250 amino acids, approximately 20 to approximately 300 amino acids, approximately 20 to approximately 400 amino acids, and approximately 20 to approximately 500 amino acids. 40 amino acids, approximately 20 amino acids to approximately 600 amino acids, approximately 20 amino acids to approximately 700 amino acids, approximately 40 amino acids to approximately 60 amino acids, approximately 40 amino acids to approximately 100 amino acids, approximately 40 amino acids to approximately 150 amino acids, approximately 40 amino acids to approximately 200 amino acids, approximately 40 amino acids to approximately 250 amino acids, approximately 40 amino acids to approximately 300 amino acids, approximately 40 amino acids to approximately 400 amino acids, approximately 40 amino acids to approximately 50 0 amino acids, approximately 40 amino acids ~ approximately 600 amino acids, approximately 40 amino acids ~ approximately 700 amino acids, approximately 60 amino acids ~ approximately 100 amino acids, approximately 60 amino acids ~ approximately 150 amino acids, approximately 60 amino acids ~ approximately 200 amino acids, approximately 60 amino acids ~ approximately 250 amino acids, approximately 60 amino acids ~ approximately 300 amino acids, approximately 60 amino acids ~ approximately 400 amino acids, approximately 60 amino acids ~ approximately 500 amino acids, approximately 60 amino acids ~ Approximately 600 amino acids, approximately 60 to approximately 700 amino acids, approximately 100 to approximately 150 amino acids, approximately 100 to approximately 200 amino acids, approximately 100 to approximately 250 amino acids, approximately 100 to approximately 300 amino acids, approximately 100 to approximately 400 amino acids, approximately 100 to approximately 500 amino acids, approximately 100 to approximately 600 amino acids, approximately 100 to approximately 700 amino acids,Approximately 150 amino acids to approximately 200 amino acids, approximately 150 amino acids to approximately 250 amino acids, approximately 150 amino acids to approximately 300 amino acids, approximately 150 amino acids to approximately 400 amino acids, approximately 150 amino acids to approximately 500 amino acids, approximately 150 amino acids to approximately 600 amino acids, approximately 150 amino acids to approximately 700 amino acids, approximately 200 amino acids to approximately 250 amino acids, approximately 200 amino acids to approximately 300 amino acids, approximately 200 amino acids to approximately 400 amino acids, approximately 200 amino acids to approximately 500 amino acids, approximately 200 amino acids to approximately 600 amino acids, approximately 200 amino acids to approximately 700 amino acids, approximately 250 amino acids to approximately 300 amino acids, approximately 25 The amino acids range from 0 to approximately 400, from approximately 250 to approximately 500, from approximately 250 to approximately 600, from approximately 250 to approximately 700, from approximately 300 to approximately 400, from approximately 300 to approximately 500, from approximately 300 to approximately 600, from approximately 300 to approximately 700, from approximately 400 to approximately 500, from approximately 400 to approximately 600, from approximately 400 to approximately 700, from approximately 500 to approximately 600, or from approximately 600 to approximately 700. In some embodiments, the size of the cognitive binder is approximately 20 amino acids, approximately 40 amino acids, approximately 60 amino acids, approximately 100 amino acids, approximately 150 amino acids, approximately 200 amino acids, approximately 250 amino acids, approximately 300 amino acids, approximately 400 amino acids, approximately 500 amino acids, approximately 600 amino acids, or approximately 700 amino acids.

[0202] In some embodiments, the cognitive binder is an antibody. In some embodiments, the cognitive binder is a molecule derived from an antibody. In some embodiments, the molecule derived from an antibody is one or more functional fragments of an antibody. Non-limiting examples of functional fragments include heavy chains, light chains, heavy chain variable domains, light chain variable domains, single-chain Fv (scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.

[0203] In some embodiments, cognitive binders can be part of antibody therapies to target synthetic cancer antigens displayed on cancer cells. In some embodiments, cognitive binders are part of an antibody-drug conjugate containing a cognitive binder and a cytotoxic payload. In some embodiments, cognitive binders are part of a bispecific antibody. In some embodiments, a bispecific antibody is a bispecific T cell engager (BiTe) containing a cognitive binder and a T cell-targeting antibody, such as an anti-CD3 antibody. In some embodiments, cognitive binders are part of the extracellular domain of a chimeric antigen receptor. Exemplary CARs containing cognitive binders are further described below.

[0204] Cognate binders based on antibodies or antibody-derived molecules may be prepared using the hybridoma method, first described by Kohler, et al., Nature, 1975, 256:495-7, or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567).

[0205] In the hybridoma method, mice, or other suitable host animals such as hamsters, are immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the proteins used for immunization. Alternatively, lymphocytes can be immunized in vitro. After immunization, the lymphocytes are isolated and then fused with myeloma cell lines using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice 59-103 (1986)).

[0206] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that, in some embodiments, contains one or more substances that inhibit the proliferation or survival of non-fusion parent myeloma cells (also called fusion partners). For example, if parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), then a selective culture medium for hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium) that inhibit the proliferation of HGPRT-deficient cells.

[0207] Exemplary fusion partner myeloma cells are sensitive to selective media that efficiently fuse, support stable high-level antibody production by selected antibody-producing cells, and reject non-fusion parent cells. Exemplary myeloma cell lines include mouse myeloma lines such as SP-2 and its derivatives, e.g., X63-Ag8-653 cells available from the American Type Culture Collection (Manassas, VA), and MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA). Human myeloma and mouse-human heterozygous myeloma cell lines have also been described for human monoclonal antibody production (Kozbor, Immunol. 1984, 133:3001-05; and Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, 1987, 51-63).

[0208] The culture medium in which hybridoma cells proliferate is assayed for the production of antigen-targeted monoclonal antibodies. The binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or in vitro binding assays such as RIA or ELISA. The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis described in Munson et al., Anal. Biochem., 1980, 107:220-39.

[0209] When a hybridoma cell producing an antibody with the desired specificity, affinity, and / or activity has been identified, the clone is subcloned by limiting dilution procedures and can be grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 medium. Additionally, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by i.p. injection of the cells into mice.

[0210] Monoclonal antibodies secreted by subclones are preferably isolated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as affinity chromatography (e.g., using Protein A or Protein G-Sepharose), ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

[0211] DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes having the ability to specifically bind to the genes encoding the heavy and light chains of the murine antibody). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA may be placed into an expression vector, which can then be transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which do not normally produce the antibody protein, to obtain synthetic monoclonal antibodies in recombinant host cells. Review articles on the recombinant expression of antibody-encoding DNA in bacteria include Skerra, et al, Curr. Opinion in Immunol., 1993, 5:256-62 and Pluckthun, Immunol. Revs., 1992, 130:151-88.

[0212] Cognate binders based on antibodies or antibody-derived molecules can be made, for example, from antibody phage libraries generated using the techniques described in Antibody Phage Display: Methods and Protocols (O’Brien and Aitken, eds., 2002). In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequence encoding them. Examples of phage display methods that can be used to make the antibodies described herein include Brinkman, et al, J. Immunol. Methods, 1995, 182:41-50; Ames, et al, Immunol. Methods, 1995, 184:177-86; Kettleborough, et al., Eur. J. Immunol., 1994, 24:952-8; Persic, et al, Gene, 1997, 187:9-18; Burton et a / ., Advances in Immunology, 1994, 57:191-280; PCT Application No. PCT / GB91 / 01 134; International Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 1 1236, WO 95 / 15982, WO 95 / 20401, and WO97 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0213] In principle, synthetic antibody clones are selected by screening a phage library containing phages that display various antibody variable region (Fv) fragments fused to phage coat proteins. Such a phage library is screened against a desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen adsorb to the antigen and are therefore separated from unbound clones in the library. The bound clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles.

[0214] The variable domain can be functionally displayed on the phage either as a single-stranded Fv (scFv) fragment in which VH and VL are covalently linked via a short mobile peptide, or as a Fab fragment in which they are fused to a constant domain and interact noncovalently, as described, for example, in Winter et al., 1994, Ann. Rev. Immunol. 12:433-55.

[0215] The VH and VL gene repertoires can be individually cloned by PCR and randomly recombined in a phage library, as described by Winter et al., and then the phage library can be searched for antigen-binding clones. Libraries derived from immunized sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, cloning a naive repertoire, as described by Griffiths et al, EMBO J, 1993, 12:725-34, allows for obtaining a single source of human antibodies against a wide range of non-self and even self-antigens without any immunization. Finally, naive libraries can also be synthetically constructed by cloning a stem cell-derived unreorganized V gene segment encoding a highly variable CDR3 region and using PCR primers containing random sequences to achieve in vitro rearrangement, as described, for example, by Hoogenboom and Winter, J. Mol. Biol., 1992, 227:381-88.

[0216] Library screening can be achieved by various techniques known in the art. For example, the synthetic cancer antigens disclosed herein can be used to coat wells of an adsorption plate, expressed on host cells immobilized on an adsorption plate or used in cell sorting, conjugated with biotin for capture with streptavidin-coated beads, or in any other method for panning a display library. The selection of antibodies with low dissociation kinetics (e.g., good binding affinity) can be facilitated by the use of prolonged washing and monovalent phage display as described in Bass, et al, Proteins, 1990, 8:309-14 and WO 92 / 09690, as well as the use of low-coating-density antigens as described in Marks et al, BiotechnoL, 1992, 10:779-83.

[0217] 1. Antibodies (e.g., anti-idiotype antibodies) In some embodiments, the target domain of a synthetic cancer antigen is recognized by a cognitive binder, which is an antibody or an antigen-binding fragment. In the embodiments provided, the antibody is capable of specifically binding to an epitope of the target domain, such as any of those described in Section II.A. For example, in some embodiments, the antibody cognitive binder is an anti-idiotype antibody that binds to an idiotope of the target domain. In some embodiments, the antibody cognitive binder (e.g., an anti-idiotype antibody) is an antibody or an antigen-binding fragment. In some embodiments, the antibody cognitive binder (e.g., an anti-idiotype antibody) is an antibody-derived molecule, which is one or more functional fragments of an antibody that binds to the target domain. Non-limiting examples of antibody fragments include heavy chains, light chains, heavy chain variable domains, light chain variable domains, single-chain Fv (scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In some embodiments, the antibody cognitive binder (e.g., anti-idiotype antibody) is a single-domain antibody that binds to a target domain. Those skilled in the art are familiar with single-domain antibodies, including camelid antibodies, which contain antibodies derived from llamas or camels. Such antibodies have a single monomeric antigen-binding domain. In some embodiments, the single-domain antibody contains a variable heavy chain and may be referred to as a VHH or VH-only antibody. In some embodiments, the single-domain antibody contains three CDRs and four framework regions named FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0218] In some embodiments, the antibody (e.g., anti-idiotype antibody), such as the antigen-binding fragment of an anti-idiotype antibody, which is a cognitive binder, is part of the extracellular domain of a chimeric antigen receptor (CAR). Typically, due to inclusion as part of a CAR, the antibody cognitive binder (e.g., anti-idiotype antibody) is a single-chain polypeptide. In some embodiments, the antibody cognitive binder (e.g., anti-idiotype antibody) is an scFv. In some embodiments, the antibody cognitive binder (e.g., anti-idiotype antibody) is a single-domain antibody, such as one containing only the variable heavy chain of the antibody, like a camel antibody derived from a llama.

[0219] In some embodiments, the target domain of the synthetic cancer antigen is an antibody or antibody fragment (e.g., those described in Section II.A.1), and the cognitive binder is an anti-idiotype antibody that binds to the target domain of the antibody or antibody fragment. In some embodiments, the cognitive binder binds to the idiotype of the antibody-based synthetic cancer antigen. In some embodiments, the cognitive binder is an anti-idiotype antibody that binds to the idiotype of the antibody target domain or antigen-binding fragment target domain. Thus, exemplary cognitive binders include anti-idiotype antibodies or antigen-binding fragments that bind to the antibody or antibody fragment described in Section II.A.1.

[0220] In some embodiments, the antibody or antigen-binding fragment recognized by the anti-idiotype cognitive binder is absiximab, adalimumab, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brodalumab, brolucizumab, brosumab, canakinumab, caplacizumab, semiprimab, cetuximab, chryzanlizumab, dacrizumab, daratumumab, denosumab, dinutuximab (Dupilumab, durvalumab, eculizumab, elotuzumab, emapalmab, emicizumab, eptinezumab, erenumab, evolocumab, fremanezumab, galcanezumab, golimumab, guselkumab, ivalizumab, idarucizumab, infliximab, ipilimumab, isatuximab, ixekizumab, lanadelmab, mepolizumab, mepolizumab, mogamulizumab, natalizumab, necitumumab, nivolumab, obilutoxaximab, obinutuzumab, ocrelizumab), Arzerra (Ofatumumab), Lartruvo (Olaratumab), Xolair (Omalizumab), Synagis (Palivizumab), Vectibix (Panitumumab), Keytruda (Pembrolizumab), Perjeta (Pertuzumab), Cyramza (Ramucirumab), Lucentis (Ranivizumab), Ultomiris (Labrizumab-cwvz), Laxibakumab (Laxibakumab), 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 (Childrakizumab-asmn), Actemra (Tocilizumab), Actemra (Tocilizumab), Herceptin (Trastuzumab), Kanjinti (Trastuzumab-anns), Ogivri (Trastuzumab-dkst), Ontruzant (Trastuzumab-dttb), HerzumaThese include trastuzumab-pkrb, Trazimera (trastuzumab-qyyp), Stellara (ustekinumab), Stellara (ustekinumab), and Entyvio (vedolizumab).

[0221] In some embodiments, the cognitive binder is any anti-idiotype antibody or antigen-binding fragment comprising means for binding to the antibody-target domain or antibody-fragment-target domain of a synthetic cancer antigen.

[0222] Anti-idiotype antibodies against any of the various known antibody target domains are known and can be used. Exemplary anti-idiotype antibodies include, but are not limited to, anti-4D5 antibodies (e.g., WO2005 / 061546, see, e.g., SEQ ID NO:1 and SEQ ID NO:2 listed therein), anti-adalimumab antibodies, anti-cetuximab antibodies, anti-rituximab antibodies, or anti-bevacizumab antibodies.

[0223] Table 2 shows exemplary anti-idiotype antibodies.

[0224] (Table 2) TIFF2026520446000013.tif219160TIFF2026520446000014.tif60160

[0225] In some embodiments, the cognitive binding is an anti-idiotype antibody, which is an anti-4D5 (Ab1) antibody or antibody fragment. In certain embodiments, the epitope of the target domain recognized by the anti-idiotype antibody is located in the variable heavy chain of the 4D5 (Ab1) antibody or antibody fragment.

[0226] In some embodiments, the cognitive binder is a single-domain anti-idiotype antibody that binds to the idiotope of the 4D5 (Ab1) antibody.

[0227] In some embodiments, the cognitive binder contains the amino acid sequence of SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 70% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 75% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 80% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 91% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 92% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 93% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 94% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 95% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 96% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 97% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 98% identical to SEQ ID NO:13. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 99% identical to SEQ ID NO:13.

[0228] In some embodiments, the cognitive binder is an antigen-binding fragment of an anti-idiotype antibody that binds to a 4D5 (Ab1) antibody. In some embodiments, the antigen-binding fragment is an scFv containing a variable heavy chain and a variable light chain. In some embodiments, the scFv contains a variable heavy chain and a variable light chain presented at SEQ ID NO:14. In some embodiments, the scFv contains a variable heavy chain and a variable light chain presented at SEQ ID NO:15. In some embodiments, the variable heavy chain and variable light chain are linked to a linker sequence. It is linked by peptide linkers such as TIFF2026520446000015.tif4128.

[0229] In some embodiments, the cognitive binder contains the amino acid sequence of SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 70% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 75% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 80% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 91% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 92% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 93% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 94% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 95% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 96% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 97% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 98% identical to SEQ ID NO:14. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 99% identical to SEQ ID NO:14.

[0230] In some embodiments, the cognitive binder contains the amino acid sequence of SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 70% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 75% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 80% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 91% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 92% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 93% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 94% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 95% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 96% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 97% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 98% identical to SEQ ID NO:15. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 99% identical to SEQ ID NO:15.

[0231] In some embodiments, the target domain of the synthetic cancer antigen is an intracellular protein (e.g., as described in Section II.A.2), and the cognate binder is an antibody that binds to the intracellular protein. In some embodiments, the antibody cognate binder binds to an intracellular protein shown in any of SEQ ID NO: 17, 18, 19, 20, 21, 22, or 23, or to an amino acid sequence that is at least 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NO: 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the antibody cognate binder binds to the intracellular protein shown in SEQ ID NO: 17.

[0232] In some embodiments, the antibody cognate binder is an antibody fragment that binds to an intracellular protein target domain. In some embodiments, the antigen-binding fragment is a scFv that includes a variable heavy chain and a variable light chain. In some embodiments, the scFv contains the variable heavy chain and variable light chain presented in SEQ ID NO: 24. In some embodiments, the variable heavy chain and variable light chain are connected by a peptide linker such as TIFF2026520446000016.tif4128.

[0233] In some embodiments, the cognitive binder contains the amino acid sequence of SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 70% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 75% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 80% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 91% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 92% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 93% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 94% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 95% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 96% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 97% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 98% identical to SEQ ID NO:24. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 99% identical to SEQ ID NO:24.

[0234] In some embodiments, the cognitive binder contains the amino acid sequence of SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 70% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 75% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 80% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 91% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 92% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 93% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 94% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 95% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 96% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 97% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 98% identical to SEQ ID NO:25. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 99% identical to SEQ ID NO:25.

[0235] Table 3 shows exemplary combinations of target domains and their cognitive binders.

[0236] (Table 3) TIFF2026520446000017.tif41155

[0237] 2. Leucine zipper domain In some embodiments, the cognitive binder of the synthetic cancer antigen is a leucine zipper domain. In such embodiments, the target domain of the synthetic cancer antigen is also a complementary leucine zipper. In some embodiments, the cognitive binder binds to its complementary leucine zipper domain. In some embodiments, the target domain is the leucine zipper domain of ATF, and consequently, the cognitive binder is the leucine zipper domain of CREB. In some embodiments, the target domain is the leucine zipper domain of CREB, and consequently, the cognitive binder is the leucine zipper domain of ATF. In some embodiments, the target domain is the leucine zipper domain of JUN, and consequently, the cognitive binder is the leucine zipper domain of Fos. In some embodiments, the target domain is the leucine zipper domain of Fos, and consequently, the cognitive binder is the leucine zipper domain of JUN.

[0238] In some embodiments, the cognitive binder contains the amino acid sequence of SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 70% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 75% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 80% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 91% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 92% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 93% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 94% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 95% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 96% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 97% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 98% identical to SEQ ID NO:26. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 99% identical to SEQ ID NO:26.

[0239] In some embodiments, the cognitive binder contains the amino acid sequence of SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 70% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 75% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 80% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 91% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 92% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 93% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 94% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 95% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 96% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 97% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 98% identical to SEQ ID NO:27. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 99% identical to SEQ ID NO:27.

[0240] In some embodiments, the cognitive binder contains the amino acid sequence of SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 70% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 75% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 80% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 91% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 92% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 93% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 94% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 95% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 96% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 97% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 98% identical to SEQ ID NO:28. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 99% identical to SEQ ID NO:28.

[0241] In some embodiments, the cognitive binder contains the amino acid sequence of SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 70% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 75% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 80% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 85% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 90% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 91% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 92% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 93% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 94% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 95% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 96% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 97% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 98% identical to SEQ ID NO:29. In some embodiments, the cognitive binder contains an amino acid sequence that is at least 99% identical to SEQ ID NO:29.

[0242] Table 4 shows exemplary combinations of target domains and their cognitive binders.

[0243] (Table 4) TIFF2026520446000018.tif26155

[0244] 3. CAR containing Cognate Binder In some embodiments, the cognitive binder of the synthetic cancer antigen is the extracellular domain of the chimeric antigen receptor (CAR). In some embodiments, the CAR expressed by genetically engineered cells is capable of binding to the target domain of the synthetic cancer antigen via its extracellular domain. In the embodiments provided, the extracellular domain of the CAR is the cognitive binder described in Section D.1 or D2 and is selected based on a specific 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 immune effector cells. In some embodiments, the immune effector cells are cytotoxic T cells. In some embodiments, the immune effector cells are natural killer cells. In the embodiments provided, engineered cells containing the CAR (e.g., T cells) can be used in immunotherapy to target and destroy cancer cells that have been tagged to express the synthetic cancer antigen recognized by the CAR.

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

[0246] In some embodiments, CARs are typically encoded by a nucleic acid sequence (polynucleotide) containing a leader sequence for guiding CAR expression on the cell membrane. In some embodiments, the antigen receptor (e.g., CAR) is encoded by a polynucleotide encoding a CAR having an NH2-terminal leader sequence. The leader sequence (also known as a signal peptide) allows the expressed CAR construct to enter the endoplasmic reticulum (ER) and target the cell surface. The leader sequence is cleaved in the ER, and mature cell surface CARs do not have a leader sequence. Generally, the length of the leader sequence is in the range of 5 to 30 amino acids, including a hydrophobic amino acid interval. In some embodiments, the leader sequence has a length of 5, 10, 15, 20, or 25 amino acids, or any value of amino acid length between any of the aforementioned. Preferably, the leader sequence contains a sequence derived from any secreted protein. In some embodiments, the leader sequence may be one of the signal peptide sequences described herein, for example, those described in SEQ ID NO:1. An exemplary CD8α signal peptide is shown in SEQ ID NO:2. An exemplary GM-CSFRa signal peptide is shown in SEQ ID NO:48. An exemplary IgK signal peptide is shown in SEQ ID NO:49. An exemplary IgK signal peptide is shown in SEQ ID NO:50.

[0247] The CARs disclosed herein include a transmembrane domain that can be directly or indirectly fused with an extracellular domain. The transmembrane domains disclosed above for synthetic cancer antigens can be used as the transmembrane domains of the CARs disclosed herein.

[0248] In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the CAR includes a transmembrane having an amino acid sequence derived from the CD8 transmembrane domain. In some embodiments, the CAR includes a transmembrane domain having an amino acid sequence derived from the human CD8 alpha transmembrane domain. In some embodiments, the CAR includes a CD8 alpha transmembrane domain having an amino acid sequence shown in SEQ ID NO: 51 or an amino acid sequence showing at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 51. In some embodiments, the transmembrane domain is the one shown in SEQ ID NO: 51.

[0249] In some embodiments, the transmembrane domain contains the amino acid sequence of SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 70% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 75% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 80% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 91% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 92% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 93% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:3. In some embodiments, the transmembrane domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:3.

[0250] In some embodiments, the CAR preferably comprises a transmembrane having an amino acid sequence derived from the transmembrane domain of CD28. Preferably, the CAR comprises a transmembrane domain having an amino acid sequence derived from the transmembrane domain of human CD28. In some embodiments, the CAR comprises a transmembrane domain of CD28 having an amino acid sequence shown in SEQ ID NO: 52 or an amino acid sequence showing at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 52. In some embodiments, the transmembrane domain is as shown in SEQ ID NO: 52.

[0251] In some embodiments, the CAR may also include a spacer region located between the cognitive binder of the extracellular antigen-binding domain and the transmembrane domain. In some embodiments, the spacer region contains hydrophilic amino acids, enabling the mobility of the targeting domain relative to the cell surface. In some embodiments, the spacer region contains more than 5, more than 10, more than 15, more than 20, more than 25, or more than 30 amino acids. In some embodiments, the spacer region contains less than 10, less than 15, less than 20, less than 25, less than 30, or less than 35 amino acids. In some embodiments, the spacer region is a hinge domain.

[0252] The CARs disclosed herein may include a hinge domain located between the extracellular domain and the transmembrane domain. A hinge domain is a commonly found amino acid segment between two domains of a protein that can enable the mobility of the protein and the movement of one or both domains relative to each other. Any amino acid sequence can be used to give such mobility and movement of the extracellular domain relative to the transmembrane domain of the effector molecule.

[0253] In some embodiments, the hinge domain is a naturally occurring hinge domain of a protein. Any hinge domain of a protein known in the art to contain a hinge domain is suitable for use in the chimeric receptor described herein. In some embodiments, the hinge domain is at least a portion of a naturally occurring hinge domain of a protein that confers mobility 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 CD8a hinge domain, for example, a fragment containing about 15 to 100 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the CD8a hinge domain. In some embodiments, the CAR contains a CD8 hinge spacer sequence having the amino acid sequence shown in SEQ ID NO: 53 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 53. In some embodiments, the arrangement of the spacers is as shown in SEQ ID NO:53.

[0254] 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 a hinge domain that connects the constant domains CH1 and CH2 of the antibody. In some embodiments, the hinge domain is of the antibody and includes the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain includes the hinge domain of the antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain includes the hinge domain of the 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 IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region includes the hinge region of the IgG1 antibody and the CH2 and CH3 constant regions. In some embodiments, the hinge region includes the hinge region of the IgG1 antibody and the constant CH3 region.

[0255] In some embodiments, the spacer region comprises all or part of the hinge domain of IgG1 Fc or IgG4 Fc. In some embodiments, the spacer is an IgG4 Fc spacer. In some embodiments, the CAR contains an IgG4 Fc spacer having an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 71 or SEQ ID NO: 54. In some embodiments, the spacer sequence is the one shown in SEQ ID NO: 54. In some embodiments, the spacer sequence is the hinge portion of IgG1 Fc or IgG4 Fc. In some embodiments, the CAR contains an IgG4 hinge spacer. In some embodiments, the IgG4 hinge spacer has an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 55 or SEQ ID NO: 55. In some embodiments, the spacer sequence is the one shown in SEQ ID NO: 55.

[0256] Peptides that do not exist in nature can also be used as hinge domains. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of the Fc receptor is a peptide linker such as a (GxS)n linker, where x and n can independently be integers from 3 to 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater.

[0257] The hinge domain may contain approximately 10 to 100 amino acids, for example, one of approximately 15 to 75 amino acids, approximately 20 to 50 amino acids, or approximately 30 to 60 amino acids. In some embodiments, the hinge domain may have a length of at least approximately 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.

[0258] The intracellular signaling domains in CARs provided herein are responsible for activating at least one of the normal effector functions of immune effector cells expressing the CAR. The term "effector function" refers to a specific function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including cytokine secretion. Therefore, the term "cytoplasmic signaling domain" refers to a portion of a protein that translates the effector function signal and guides the cell to perform the specific function. While the entire cytoplasmic signaling domain can usually be used, it is often not necessary to use the entire chain. Insofar as a truncated portion of the cytoplasmic signaling domain is used, it may be used in place of the intact chain, provided that such truncated portion translates the effector function signal. Therefore, the term "cytoplasmic signaling domain" is intended to include any truncated portion of a cytoplasmic signaling domain that is sufficient to translate the effector function signal.

[0259] In some embodiments, the intracellular signaling domain includes the primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR includes an intracellular signaling domain that is essentially derived from the primary intracellular signaling domain of an immune effector cell. The “primary intracellular signaling domain” refers to a cytoplasmic signaling sequence that acts in a stimulating manner to induce immune effector function. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as an immune receptor tyrosine activation motif, i.e., ITAM. When used herein, “ITAM” is a conserved protein motif commonly present in the tail of signaling molecules expressed in many immune cells. The motif may give the conserved motif YxxL / Ix(6-8)YxxL / I by containing two repeats of the amino acid sequence YxxL / I, separated by 6-8 amino acids, where each x is independently any amino acid. ITAM within the signaling molecule is important for intracellular signaling, which is at least partially mediated by the phosphorylation of tyrosine residues in ITAM after activation of the signaling molecule. ITAMs can 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.

[0260] In some embodiments, the intracellular signaling domain is derived from the 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 the cytoplasmic signaling domain of wild-type CD3z.

[0261] In some embodiments, the CAR contains an intracellular signaling domain containing a CD3 zeta signaling domain having the amino acid sequence shown in SEQ ID NO: 56 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 56. In some embodiments, the CAR contains an intracellular signaling domain containing a CD3 zeta signaling domain having the amino acid sequence shown in SEQ ID NO: 56. In some embodiments, the CAR contains an intracellular signaling domain containing a CD3 zeta signaling domain having the amino acid sequence shown in SEQ ID NO: 57 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 57. In some embodiments, the CAR contains an intracellular signaling domain containing a CD3 zeta signaling domain having the amino acid sequence shown in SEQ ID NO: 57.

[0262] The CARs disclosed herein may further comprise at least one co-stimulatory signaling domain. Thus, in some embodiments, a chimeric antigen receptor (CAR) comprises an intracellular signaling region including (1) a cognitive binder; (2) a spacer such as a hinge sequence; (3) a transmembrane domain; and (4) a first primary intracellular signaling domain such as a CD3 zeta intracellular signaling domain, and a 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 signaling within a cell to induce an immune response, such as effector function. Many immune effector cells require co-stimulation in addition to stimulation with antigen-specific signals, not only to activate the cell’s effector function but also to promote cell proliferation, differentiation, and survival.

[0263] The co-stimulatory signaling domains of chimeric receptors described herein may be cytoplasmic signaling domains derived from co-stimulatory proteins that transform signals and modulate responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils. The "co-stimulatory signaling domain" may 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 two, three, four, or more) co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more identical co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains derived from different co-stimulatory proteins, such as two or more co-stimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3z) and one or more co-stimulatory signaling domains (such as the 4-1BB signaling domain). In some embodiments, one or more co-stimulatory signaling domains (such as the 4-1BB signaling domain) and the primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3z) are fused to each other via an optional peptide linker. The primary intracellular signaling domain and one or more co-stimulatory signaling domains may be arranged in any preferred order. Multiple co-stimulatory signaling domains may provide additive or synergistic stimulatory effects.

[0265] In some embodiments, the co-stimulatory domains may 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 co-stimulatory domains.

[0266] In some embodiments, the CAR contains an intracellular signaling domain containing a CD28 co-stimulatory signaling domain having an amino acid sequence shown in SEQ ID NO: 58 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 58. In some embodiments, the CAR contains an intracellular signaling domain containing a CD28 co-stimulatory signaling domain having an amino acid sequence shown in SEQ ID NO: 58. In some embodiments, the CAR contains an intracellular signaling domain containing a CD28 co-stimulatory signaling domain having an amino acid sequence shown in SEQ ID NO: 59 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 59. In some embodiments, the CAR contains an intracellular signaling domain containing a CD28 co-stimulatory signaling domain having an amino acid sequence shown in SEQ ID NO: 59.

[0267] In some embodiments, the CAR contains an intracellular signaling domain comprising a 4-1BB co-stimulatory signaling domain having an amino acid sequence shown in SEQ ID NO:60 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity with SEQ ID NO:60.

[0268] In some embodiments, the intracellular signaling domains may be domains of CD3 zeta, 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.

[0269] In some embodiments, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO:16. In some embodiments, the intracellular signaling domain includes an amino acid sequence that is at least 70% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain includes an amino acid sequence that is at least 75% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain includes an amino acid sequence that is at least 80% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain includes an amino acid sequence that is at least 85% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain includes an amino acid sequence that is at least 90% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain includes an amino acid sequence that is at least 91% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain includes an amino acid sequence that is at least 92% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain includes an amino acid sequence that is at least 93% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain contains an amino acid sequence that is at least 94% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain contains an amino acid sequence that is at least 95% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain contains an amino acid sequence that is at least 96% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain contains an amino acid sequence that is at least 97% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain contains an amino acid sequence that is at least 98% identical to SEQ ID NO:16. In some embodiments, the intracellular signaling domain contains an amino acid sequence that is at least 99% identical to SEQ ID NO:16.

[0270] Table 5 shows exemplary CARs and their components.

[0271] (Table 5)CAR TIFF2026520446000019.tif60155

[0272] 4. Variants of synthetic cancer antigens and cognitive binders The purpose of this specification is to modify the amino acid sequence(s) of the synthetic cancer antigens and / or cognitive binders provided herein. For example, it may be desirable to improve the binding affinity between the synthetic cancer antigens and / or cognitive binders; it may also be desirable to improve other biological properties of the synthetic cancer antigens and / or cognitive binders, including but not limited to specificity, heat resistance, expression level, or solubility. Accordingly, it is intended that variants can be prepared in addition to the synthetic cancer antigens and / or cognitive binders described herein.

[0273] In some embodiments, the synthetic cancer antigens and / or cognitive binders provided herein are chemically modified, for example, by the covalent addition of any type of molecule to the synthetic cancer antigen and / or cognitive binder. Exemplary, non-limiting modifications include glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, cleavage by proteolysis, and linkage with cellular ligands or other proteins. Additionally, the synthetic cancer antigens and / or cognitive binders may contain one or more non-classical amino acids.

[0274] In some embodiments, the difference may be a substitution, deletion, or insertion of one or more codons encoding a synthetic cancer antigen and / or cognitive binder, resulting in a change in the amino acid sequence compared to the original sequence. Amino acid substitutions may result in the replacement of one amino acid with another amino acid having similar structural and / or chemical properties, such as a conservative amino acid substitution, or a substitution of serine with leucine. Mutations can be introduced into the nucleotide sequences encoding the molecules provided herein by using standard techniques known to those skilled in the art, including site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions.

[0275] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be randomly introduced along all or part of the coding sequence by methods such as saturated mutagenesis. By screening the resulting mutants for biological activity, mutants that retain activity can be identified. After mutational introduction, the encoded protein can be expressed, and its activity can be determined.

[0276] Substantial alteration of the biological properties of an antibody is achieved by selecting substitutions that have a significantly different effect on (a) the structure of the polypeptide backbone in the region of substitution, for example, as a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulkiness of the side chain. Alternatively, conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) may be performed so as to maintain or significantly alter the properties. Amino acids can be grouped according to the similarity of their side chain properties (see, for example, Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) Nonpolar: Ala (A), Val (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Non-charged: 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 can be grouped based on common side-chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0277] Non-conservative substitutions involve replacing one member of one of these classes with one from another. Such substitution residues can also be introduced into a conserved substitution site or into the remaining (non-conservative) site.

[0278] Insertions or deletions are optional and may range from approximately 1 to 5 amino acids. In certain embodiments, substitutions, deletions, or insertions include substitutions of fewer than 25 amino acids, fewer than 20 amino acids, fewer than 15 amino acids, fewer than 10 amino acids, fewer than 5 amino acids, fewer than 4 amino acids, fewer than 3 amino acids, or fewer than 2 amino acids compared to the original molecule. In certain embodiments, substitutions are conservative amino acid substitutions made to one or more predicted non-essential amino acid residues. Acceptable differences can be determined by systematically introducing amino acid insertions, deletions, or substitutions into the sequence and testing the resulting variants for the activity exhibited by the full-length native sequence or the mature native sequence.

[0279] Amino acid insertions include amino and / or carboxyl terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminus insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminus or C-terminus of an antibody with an enzyme (e.g., for antibody-directed enzyme prodrug therapy) or a polypeptide that extends the serum half-life of the antibody.

[0280] III. Polynucleotides Polynucleotides or nucleic acid molecules encoding synthetic cancer antigens are provided herein. Polynucleotides or nucleic acid molecules encoding cognitive binders for synthetic cancer antigens are also provided herein.

[0281] In some embodiments, polynucleotides encoding any synthetic cancer antigen (soluble synthetic cancer antigen) including tumor targeting binding molecules, such as those disclosed in Section II.C, are provided herein. In some embodiments, polynucleotides encoding any synthetic cancer antigen (membrane-bound synthetic cancer antigen) including membrane targeting domains, such as those disclosed in Section II.B, are provided herein. In some embodiments, polynucleotides encoding any membrane-bound synthetic cancer antigen (including tumor targeting binding molecules, such as those disclosed in Section II.C, for example) and any soluble synthetic cancer antigen including membrane targeting domains, such as those disclosed in Section II.B, are provided herein.

[0282] In some embodiments, polynucleotides encoding synthetic cancers are provided herein, including soluble synthetic cancer antigens comprising a target domain and a tumor targeting binding molecule, such as any of those disclosed in Section II.C, and membrane-bound synthetic cancer antigens comprising a target domain and a membrane targeting domain, such as any of those disclosed in Section II.B. In some embodiments, the polynucleotide sequence encodes a protein in which the soluble synthetic cancer antigen comprising the target domain and the tumor targeting binding molecule, and the membrane-bound synthetic cancer antigen comprising the target domain and the membrane targeting domain are separated by a cleavable linker. In some embodiments, the cleavable linker comprises a 2A self-cleaving peptide.

[0283] In some embodiments, polynucleotides are provided herein that comprise a first nucleic acid sequence encoding one of a soluble synthetic cancer antigen including a target domain and a tumor targeting binding molecule, such as any of those disclosed in Section II.C, and a second nucleic acid sequence encoding one of a membrane-bound synthetic cancer antigen including a target domain and a membrane targeting domain, such as any of those disclosed in Section II.B. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a nucleotide sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a self-cleaving linker. In some embodiments, the self-cleaving linker is or comprises a 2A peptide.

[0284] In some embodiments, polynucleotides are deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded and can be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. A polynucleotide is a polymer of nucleotides having a length 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, at least 15000, or more than 15000 nucleotides, and all intermediate lengths. In this context, "intermediate length" will be easily understood to mean any length between the stated values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc.

[0285] In some embodiments, the polynucleotides are codon-optimized. As used herein, the term “codon-optimized” means that codons in the polynucleotide encoding the polypeptide have been substituted to increase the polypeptide’s expression, stability, and / or activity. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) differences in codon bias between two or more organisms or genes or synthetically constructed bias tables; (ii) differences in the degree of codon bias within an organism, gene, or gene set; (iii) systematic differences in codons, including their context of existence; (iv) differences in codons due to their decoded tRNA; (v) differences in codons due to GC% at either the entire triplet or one of its positions; (vi) differences in the degree of similarity to a reference sequence, such as a naturally occurring sequence; (vii) differences in codon frequency cutoffs; (viii) structural characteristics of mRNA transcribed from the DNA sequence; (ix) prior knowledge regarding the function of the DNA sequence underlying the design of the codon substitution set; (x) systematic differences in the codon set for each amino acid; (xi) isolated removal of a false translation initiation site; and / or (xii) elimination of an accidental polyadenylation site that would otherwise result in a truncated RNA transcript.

[0286] As a result of the degeneracy of the genetic code, it will be acknowledged by those skilled in the art that there are many nucleotide sequences that encode fragments of polypeptides or variants thereof as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequences of any natural gene. However, different polynucleotides resulting from differences in codon use, such as polynucleotides optimized for human and / or primate codon selection, are specifically contemplated in certain embodiments. Furthermore, alleles of genes containing the polynucleotide sequences provided herein may also be used. An allele is an endogenous gene that changes as a result of one or more mutations, such as nucleotide deletions, additions, and / or substitutions.

[0287] In some embodiments, the polynucleotides provided herein, regardless of the length of the coding sequence itself, further include other DNA sequences such as promoters and / or enhancers disclosed elsewhere herein or known in the Art, untranslated regions (UTRs), signal sequences, Kossack sequences, polyadenylation signals, additional restriction enzyme sites, multicloning sites, internal ribosome entry sites (IRESs), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaved polypeptides, epitope tags, and as a result, their overall length can vary considerably. Therefore, it is intended that polynucleotide fragments of substantially any length may be used in certain embodiments, and the full length is preferably limited by ease of preparation and the use of the intended recombinant DNA protocol.

[0288] Polynucleotides can be prepared, manipulated, and / or expressed using any of the various well-established techniques known and available in the art.

[0289] IV. Vectors Vectors comprising polynucleotides or nucleic acid molecules encoding any of the synthetic cancer antigens disclosed herein are provided herein. Vectors comprising polynucleotides or nucleic acid molecules encoding any of the cognitive binders of the synthetic cancer antigens disclosed herein are also provided herein.

[0290] Vectors comprising polynucleotides encoding two or more of the synthetic cancer antigens disclosed herein, such as synthetic cancer antigens containing tumor targeting binding molecules and synthetic cancer antigens containing membrane targeting domains, are also provided herein.

[0291] Vectors are also provided herein that comprise a first polynucleotide encoding a soluble synthetic cancer antigen including a target domain and a tumor targeting binding molecule, such as one of those disclosed in Section II.C, and a second polynucleotide encoding a membrane-bound synthetic cancer antigen including a target domain and a membrane targeting domain, such as one of those disclosed in Section II.B. In some embodiments, the first and second polynucleotides are separated by a nucleotide sequence encoding a cleavable linker. In some embodiments, the cleavable linker comprises a 2A self-cleaving peptide.

[0292] In some embodiments, the synthetic cancer antigen is delivered by a viral vector containing a polynucleotide encoding the synthetic cancer antigen, such as one of the polynucleotides disclosed in Section III.

[0293] To express the synthetic cancer antigens and / or cognitive binders described herein in cells, an expression cassette encoding the synthetic cancer antigen or cognitive binder can be inserted into a nucleic acid vector. The “expression cassette” contains the gene of interest, which is the synthetic cancer antigen. The cassette is locologically and sequentially oriented within the vector so that the nucleic acid in the cassette can be transposed to a compartment appropriate for biological activity by being transcribed into RNA, translated into a protein or polypeptide if necessary, undergoing appropriate post-translational modifications necessary for activity in transformed cells, and targeting to an appropriate intracellular compartment or secreted into an extracellular compartment. Preferably, the cassette has 3' and 5' ends suitable for rapid insertion into the vector, for example, having restriction endonuclease sites at each end. The cassette can be extracted and inserted into a plasmid or viral vector as a single unit.

[0294] The term "nucleic acid vector" is used herein to refer to a nucleic acid molecule that has the ability to transfer or transport another nucleic acid molecule. The nucleic acid to be transferred is generally ligated to the vector nucleic acid molecule and, for example, inserted into it. The nucleic acid vector may contain sequences that lead to self-replication in cells or sequences sufficient to enable integration into host cell DNA.

[0295] In some embodiments, vectors include, but are not limited to, plasmids, phagemids, cosmids, transposons, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phages or M13 phages, and animal viruses. In some embodiments, coding sequences of synthetic cancer antigens or cognitive binders disclosed herein can be ligated into such vectors for expression in mammalian cells.

[0296] In some embodiments, the vector is either a viral vector or a non-viral vector.

[0297] In some embodiments, the vector is a nonviral vector. In some embodiments, a nonviral vector is used to deliver one or more polynucleotides intended herein. In some embodiments, a recombinant vector containing a polynucleotide encoding a synthetic cancer antigen or cognitive binder described herein is a plasmid. Numerous suitable plasmid expression vectors are known to those skilled in the art, and many are commercially available. The vectors listed below are provided as examples; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other plasmid vector may also be used, insofar as it is compatible with the host cell.

[0298] In some embodiments, the vector is a viral vector. In some embodiments, a viral vector is used to deliver one or more polynucleotides as intended herein. Suitable viral vectors include vaccinia virus; poliovirus; adenovirus (e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649, WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984; and WO95 / 00655); adeno-associated virus (e.g., U.S. Patent No. 7,078,387; Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al,, PNAS 94:6916 6921 , 1997;Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997;Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999;Ali et al., Hum Mol Genet 5:591 594, 1996;Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828;Mendelson et al,, Virol. (1988) See 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al.Viral vectors based on (see J Virol 73:7812 7816, 1999); retroviral vectors (e.g., mouse leukemia virus, splenic necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and similar vectors, but not limited to these. Examples of vectors include the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST (trademark), pLenti6 / V5-DEST (trademark), and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells.

[0299] In some embodiments, the vectors are non-integrating vectors, including but not limited to episomal vectors or vectors maintained outside the chromosome. As used herein, the term “episome” refers to a vector capable of replicating without integration into the host’s chromosomal DNA or slow loss from the dividing host cell, which also means that the vector replicates outside the chromosome or as an episome. The vectors are engineered to possess a DNA replication origin, i.e., “ori,” derived from lymphotropic herpesvirus or gamma herpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, specifically a sequence encoding the replication origin of a lymphotropic herpesvirus or gamma herpesvirus corresponding to oriP of EBV. In some embodiments, the lymphotropic herpesvirus may be Epstein-Barr virus (EBV), Kaposi’s sarcoma herpesvirus (KSHV), herpesvirus thymili (HS), or Marek’s disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gamma herpesviruses.

[0300] In some embodiments, the vector is an embedding vector. In some embodiments, the polynucleotide is introduced into a target or host cell using a transposon vector system. In some embodiments, the transposon vector system includes a vector comprising a transposable element and the polynucleotide contemplated herein; as well as a transposase. In one embodiment, the transposon vector system is a single transposase vector system; see, for example, WO 2008 / 027384. Exemplary transposases include, but are not limited to, piggyBac, Sleeping Beauty, Mos1, Tc1 / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and their derivatives. The piggyBac transposon and transposase are described, for example, in U.S. Patent 6,962,810, which is incorporated herein by reference in its entirety. The Sleeping Beauty transposon and transposase are described, for example, in Izsvak et al., J. Mol. Biol. 302: 93-102 (2000), which is incorporated herein by reference in its entirety. The Tol2 transposon, first isolated from the medaka fish Oryzias latipes and belonging to the hAT transposon family, is described in Kawakami et al. (2000). Mini-Tol2 is a variant of Tol2 and is described in Balciunas et al. (2006). The Tol2 and Mini-Tol2 transposons, when acting together with the Tol2 transposase, promote the integration of transgenes into the genome of organisms. The Frog Prince transposon and transposase are described, for example, in Miskey et al., Nucleic Acids Res. 31:6873-6881 (2003).

[0301] In some embodiments, polynucleotide sequences encoding synthetic tumor antigens or cognitive binders disclosed herein are functionally linked to one or more regulatory elements that enable polynucleotide expression in both prokaryotic and eukaryotic cells. “Regulatory elements” refer to the untranslated regions of a vector that interact with host cell proteins to perform transcription and translation. Non-limiting examples of regulatory elements include origins of replication, selective cassettes, constitutive and inducible promoters, enhancers, translation initiation signal (Schein-Dalgarno or Kozak sequence) introns, transcriptional terminators, and 5' and 3' untranslated regions. See, for example, Bitter et al. (1987) Methods in Enzymology, 153:516-544). Such elements may differ in their intensity and specificity. Transcriptional regulatory elements may be functional in either eukaryotic cells (e.g., mammalian cells) or prokaryotic cells (e.g., bacterial or archaeal cells).

[0302] In some embodiments, polynucleotides encoding synthetic cancer antigens or cognitive binders described herein are functionally linked to promoters and / or enhancers. The term “promoter,” as used herein, refers to a recognition site of a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes the polynucleotide functionally linked to the promoter. In some embodiments, in mammalian cells, an operative promoter includes an AT-rich region located approximately 25–30 bases upstream from the transcription initiation site, and / or a CNCAAT region, which is another sequence found 70–80 bases upstream from the transcription initiation site, where N may be any nucleotide. The term “enhancer” refers to a segment of DNA containing a sequence capable of providing transcriptional enhancement, which, in some embodiments, can function independently of its orientation toward another regulatory sequence. Enhancers can function cooperatively or additively with promoters and / or other enhancer elements.

[0303] Non-limiting examples of suitable eukaryotic promoters (promoters functional in eukaryotic cells) include: cytomegalovirus (CMV) pre-early, herpes simplex virus (HSV) thymidine kinase, viral simian virus 40 (SV40) (e.g., early and late SV40), spleen focus-forming virus (SFFV) promoter, retroviral long-chain terminal repeat sequences (LTRs) (e.g., Moloney's mouse leukemia virus (MoMLV) LTR promoter or Rous sarcoma virus (RSV) LTR), herpes simplex virus (HSV) (thymidine kinase) promoter, vaccinia virus-derived H5, P7.5, and P11 promoters, elongation factor 1-alpha (EF1α) promoter, early growth response 1 (EGR1) promoter, ferritin H (FerH) promoter, ferritin L (FerL) promoter, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, and eukaryotic translation initiation factor 4A1 Examples include the (EIF4A1) promoter, the heat shock 70 kDa protein 5 (HSPA5) promoter, the heat shock protein 90 kDa beta, member 1 (HSP90B1) promoter, the heat shock protein 70 kDa (HSP70) promoter, the β-kinesin (β-KIN) promoter, the human ROSA26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), the ubiquitin C (UBC) promoter, the phosphoglycerate kinase-1 (PGK) promoter, the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, the β-actin promoter and myeloproliferative sarcoma virus enhancer, the dl587rev primer-binding site substitution (MND) promoter with deletion of the negative regulatory region, and those derived from mouse metallothionein-l. The selection of appropriate vectors and promoters is well within the realm of those skilled in the art.

[0304] In some embodiments, the polynucleotide sequences encoding the synthetic cancer antigens or cognitive binders described herein are functionally linked to a constitutive promoter. In such embodiments, the polynucleotides encoding the synthetic cancer antigens or cognitive binders described herein are constitutively and / or ubiquitously expressed in cells.

[0305] In some embodiments, the polynucleotide sequences encoding the synthetic cancer antigens or cognitive binders described herein are functionally linked to an inducible promoter. In such embodiments, the polynucleotides encoding the synthetic cancer antigens described herein are conditionally expressed. As used herein, “conditional expression” can mean any type of conditional expression, including but not limited to inducible expression; repressive expression; and expression in cells or tissues having a particular physiological, biological, or disease condition (e.g., cell type or tissue-specific expression). Examples of inductive promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters of genes encoding glucocorticoids or estrogen receptors (which can be induced by treatment with the corresponding hormone), metallothione promoters (which can be induced by treatment with various heavy metals), MX-1 promoters (which can be induced by interferon), the "GeneSwitch" mifepristone regulatory system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO 2002 / 088346), and tetracycline-dependent regulatory systems.

[0306] In some embodiments, the vectors described herein further include a transcription termination signal. Elements that lead to the efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In some embodiments, the vector includes a polyadenylation sequence at the 3' end of the polynucleotide encoding the polypeptide to be expressed. The terms “polyA site” or “polyA sequence,” as used herein, refer to a DNA sequence that leads to both termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence and thus contribute to increased translation efficiency. Cleavage and polyadenylation are led by a poly(A) sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNA has a cleavage polyadenylation site and two adjacent recognition elements. Typically, a nearly immutable AAUAAA hexamer is located 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, coupled with the addition of up to 250 adenosines to the 5' cleavage product. In some embodiments, the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA). In some embodiments, the poly(A) sequence is the SV40 polyA sequence, the bovine growth hormone polyA sequence (BGHpA), the rabbit β-globin polyA sequence (rβgpA), its variants, or another suitable heterologous or endogenous polyA sequence known in the art.

[0307] In some embodiments, the vector may also include a sequence encoding a signal peptide (e.g., for nuclear, nucleolar, or mitochondrial localization) fused with a polynucleotide encoding a synthetic tumor antigen or cognitive binder. For example, the vector may include a nuclear localization sequence (e.g., derived from SV40) fused with a polynucleotide encoding a synthetic cancer antigen or cognitive binder. 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 includes the amino acid sequence of SEQ ID NO:1. In some embodiments, the signal peptide includes the amino acid sequence of SEQ ID NO:2.

[0308] In some embodiments, the expression vector further comprises a nucleotide sequence encoding one or more protein tags (e.g., a 6×His tag, a hemagglutinin tag, a green fluorescent protein, etc.) fused with a polynucleotide encoding a synthetic cancer antigen, thereby yielding a synthetic cancer antigen further comprising the protein tags.

[0309] Methods for introducing polynucleotides and recombinant vectors into host cells are known in the art. Preferred methods include, for example, 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 microinjection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al., Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9), microfluidic delivery methods (see, e.g., International PCT Publication WO 2013 / 059343), and similar methods.

[0310] In some embodiments, electroporation-mediated delivery includes the steps of mixing cells with a polynucleotide encoding a synthetic cancer antigen or cognitive binder in a cartridge, chamber, or cuvette, and applying one or more electrical impulses of a specified duration and amplitude. In some embodiments, cells are mixed with a polynucleotide encoding a synthetic cancer antigen or cognitive binder in a container connected to a device (e.g., a pump) that supplies the mixture into the cartridge, chamber, or cuvette, one or more electrical impulses of a specified duration and amplitude are applied, and the cells are subsequently delivered to a second container. Examples of polynucleotide delivery systems suitable for use in the particular embodiments intended include, but are not limited to, those offered by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, Neon® Transfection Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for the efficient lipofection of polynucleotides are described in the literature. See, for example, Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12.

[0311] In some embodiments, polynucleotides encoding synthetic cancer antigens or cognitive binders described herein are introduced into cells via non-viral delivery media such as transposons, nanoparticles (e.g., lipid nanoparticles), liposomes, exosomes, attenuated bacteria, or virus-like particles. In some embodiments, the mediators are attenuated bacteria (e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli, which are naturally invasive or artificially engineered to be invasive but attenuated to inhibit pathogenicity), bacteria that target specific cells with trophic and tissue-specific targeting, and bacteria having surface proteins modified to alter target cell specificity. In some embodiments, the mediators are genetically modified bacteriophages (e.g., engineered phages with large packaging capacity, low immunogenicity, containing mammalian plasmid maintenance sequences, and incorporating targeted ligands). In some embodiments, the mediator is a biological liposome. For example, a biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are erythrocytes derived from the target that have been broken down into spherical structures, and tissue targeting can be achieved by the addition of various tissue or cell-specific ligands), a secretory exosome, or a target-derived membrane-bound nanovesicle (30-100 nm) of endocytosis origin (e.g., which can be produced from various cell types and therefore can be taken up by cells without requiring a targeting ligand).

[0312] In some embodiments, vectors containing polynucleotides encoding synthetic cancer antigens or cognitive binders described herein are introduced into cells by viral delivery methods, such as viral transduction. Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Using techniques known in the art, heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles. The recombinant viruses can then be isolated and delivered to the mammalian cells to be manipulated in vitro or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. Several adenovirus vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivated lentiviral vectors are used. For example, using protocols known in the art, self-inactivated lentiviral vectors containing immunomodulatory (such as immune checkpoint inhibitor) coding sequences and / or self-inactivated lentiviral vectors containing chimeric antigen receptors can be packaged. The resulting lentiviral vector can be used to transduce mammalian cells (such as primary human T cells) using methods known in the art. Retrovirus-derived vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they enable the long-term stable integration of the transgene and its proliferation in progeny cells. Lentiviral vectors also exhibit low immunogenicity and can transduce non-proliferating cells.

[0313] In some embodiments, the vector is a mammalian virus-like particle. Modified viral particles can be generated, for example, by ex vivo assembly of the virus using a desired cargo, following the purification of "empty" particles. The vector can also be manipulated to alter its target tissue specificity by incorporating a targeted ligand.

[0314] A. Tumor-targeting viral vectors In some embodiments, synthetic cancer antigens are delivered by a vector, which is a tumor-targeting viral vector.

[0315] Therefore, in some embodiments, a vector containing a polynucleotide encoding a synthetic cancer antigen is a viral vector that is directional to tumor cells, i.e., a tumor-directing viral vector. By being directional to tumor cells, the viral vector can deliver the polynucleotide encoding the synthetic cancer antigen to targeted tumor cells, such as tumor cells of the target cancer.

[0316] In some embodiments, the viral vector is a non-replicating viral vector, for example, a replication-defective one. In some embodiments, the viral vector is a replication-defective adenovirus vector lacking the E1A gene, which is essential for adenovirus replication.

[0317] In some embodiments, the viral vector is a replicating viral vector.

[0318] In some embodiments, the viral vector is, for example, a viral vector or a modified form thereof disclosed in WO2005 / 118825, WO2015 / 097220, WO2015 / 059303, WO2015 / 155370, WO2016 / 174200, WO2017 / 103291, WO2018 / 041838, WO2018 / 041827, W02017 / 103290, WO2018 / 220207, WO2019 / 043020, WO2022 / 171853, the contents of which are incorporated herein by reference in their entirety.

[0319] In some embodiments, the viral vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, or a herpes simplex virus (HSV) vector.

[0320] Among the many viral vectors, exemplary adenovirus vectors, AAV vectors, and lentiviral vectors are described in particular, for example, Bezeljak, Radiol Oncol., 2022, 56(1): 1-13. Exemplary adenovirus vectors include, for example, 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 those disclosed in WO2014198852 A2. There are more than 50 known human adenovirus serotypes, including Ad2, Ad3, Ad5, Ad7, Ad9, Ad11, Ad12, Ad17, and Ad40.

[0321] In some embodiments, the viral vector is an adenovirus vector. In some embodiments, the viral vector is a non-replicating adenovirus vector, such as those described by Tessarollo et al. and Wold et al. Adenoviruses are non-enveloped viruses having a single double-stranded DNA linear chain inside an icosahedral capsid.

[0322] Adenoviruses of animal origin can be obtained from deposited strains, subsequently amplified in competent cell lines, and optionally modified as desired. Complete genome sequences of adenoviruses are available, for example, human adenovirus type 2 (GenBank accession number J01917; SEQ ID NO: 3), human adenovirus type 5 (GenBank accession number M73260, SEQ ID NO: 4; and GenBank accession number NC). - 001406), Human Adenovirus 12 (GenBank Accession Number NC) - 001460, X73487); Human adenovirus type 17 (GenBank accession number NC)- The following have been determined: 002067, AF108105), and human adenovirus type 40 (GenBank accession number L19443). Techniques for producing, isolating, and modifying adenoviruses are described in the literature. For example, 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 See 88 / 10311 and WO 91 / 11525. Such adenoviruses can be modified, for example, by deletion, addition, and / or substitution.

[0323] In some embodiments, the adenovirus vector belongs to one of subgroups A through G. In some embodiments, the adenovirus vector belongs to subgroup A. In some embodiments, the adenovirus vector belongs to subgroup B. In some embodiments, the adenovirus vector belongs to subgroup B type 1. In some embodiments, the adenovirus vector belongs to subgroup B type 2. In some embodiments, the adenovirus vector belongs to subgroup C. In some embodiments, the adenovirus vector belongs to subgroup D. In some embodiments, the adenovirus vector belongs to subgroup E. In some embodiments, the adenovirus vector belongs to subgroup F. In some embodiments, the adenovirus vector belongs to subgroup G.

[0324] Adenoviruses of subgroup A include, for example, serotypes 12, 18, and 31; adenoviruses of subgroup B type 1 include, for example, serotypes 3, 7, 16, and 21; adenoviruses of subgroup B type 2 include, for example, serotypes 11, 14, 34, and 35; adenoviruses of subgroup C include, for example, serotypes 1, 2, 5, and 6; adenoviruses of subgroup D include, for example, serotypes 8-10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-49; adenoviruses of subgroup E include, for example, serotype 4; adenoviruses of subgroup F include, for example, serotypes 40 and 41; and adenoviruses of subgroup G include, for example, serotype 52. See, for example, Ghebremedhin, Eur. J. Microbiol. Immunol., 2014, 4(1): 26-33.

[0325] For example, most adenoviruses, such as those of subgroups A, C, D, E, and F, enter target cells primarily by binding to the coxsackie-adenovirus receptor. On the other hand, with some exceptions, generally, type 1 adenoviruses of subgroup B primarily bind to CD46, while generally, type 2 adenoviruses of subgroup B primarily bind to desmoglein-2. Ad11, Ad14, Ad16, Ad21, Ad35, and Ad50 of subgroup B, as well as Ad17 and Ad47 of subgroup D, bind to CD46. Ad3, Ad7, and Ad14 of subgroup B bind to desmoglein-2. See, for example, Hensen et al., Int. J. Mol. Sci., 2020, 21(18): 6828.

[0326] Coxsackievirus receptor levels vary by tissue and tumor type, and can be upregulated or downregulated in tumor cells depending on the type of cancer. See, for example, Hensen et al., Int. J. Mol. Sci., 2020, 21(18): 6828.

[0327] Since all nucleated cells express CD46, adenoviruses derived from subgroup B type 1 are desirable due to their broad targeting properties. Furthermore, because CD46 expression is low to moderate in most normal tissues but upregulated in many different types of cancer, adenoviruses that primarily bind to CD46, such as Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, and Ad50, are desirable for use in delivering synthetic cancer antigens to tumor cells. See, for example, Do et al., Int. J. Mol. Sci., 2018, 19: 2694; Su et al., JCI Insight, 2018, 3: e121497; and Hensen et al., Int. J. Mol. Sci., 2020, 21(18): 6828.

[0328] Desmoglein-2 is a transmembrane glycoprotein expressed in various tissue types, including the bladder, colon, kidney, prostate, and stomach, and, like CD46, has been reported to be upregulated in cancer. See, for example, Hensen et al., Int. J. Mol. Sci., 2020, 21(18): 6828; and Brennan et al., Cell Adhes. Migr., 2009, 3: 148-154. For use in delivering synthetic cancer antigens to tumor cells, adenoviruses that primarily bind to desmoglein-2, such as Ad3, Ad7, and Ad14, as well as adenoviruses that primarily bind to CD46, are preferred.

[0329] Therefore, in some embodiments, a viral vector, such as an adenovirus 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, Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, or Ad50.

[0330] In some embodiments, the adenovirus vector is based on Ad3, Ad7, Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, and Ad50, or any combination thereof. In some embodiments, the adenovirus vector is based on Ad2. In some embodiments, the adenovirus vector is based on Ad3. In some embodiments, the adenovirus vector is based on Ad5. In some embodiments, the adenovirus vector is based on Ad7. In some embodiments, the adenovirus vector is based on Ad9. In some embodiments, the adenovirus vector is based on Ad11. In some embodiments, Ad11 is Ad11p. In some embodiments, the adenovirus vector is based on Ad12. In some embodiments, the adenovirus vector is based on Ad14. In some embodiments, the adenovirus vector is based on Ad17. In some embodiments, the adenovirus vector is based on Ad35. In some embodiments, the adenovirus vector is based on Ad40. In some embodiments, the adenovirus vector is based on Ad50.

[0331] In some embodiments, the adenovirus vector is a chimeric virus based on two or more serotypes of adenovirus. In some embodiments, the adenovirus vector is a chimeric virus based on an adenovirus vector of serotype 3 (Ad3) and / or serotype 5 (Ad5). In some embodiments, the adenovirus vector is a chimeric virus based on an adenovirus vector of serotype 3 (Ad3) and / or serotype 11 (Ad11).

[0332] In some embodiments, the adenovirus vector is Ad5. In some embodiments, the adenovirus vector is Ad5 or a variant or derivative thereof. Ad5 adenovirus vectors generally enter cells via the coxsackie-adenovirus receptor.

[0333] In some embodiments, the adenovirus is a chimeric adenovirus. In some embodiments, the chimeric adenovirus is Ad3 / 11p or Ad5 / 3.

[0334] In some embodiments, the adenovirus vector is Ad3 / 11p, a chimeric adenovirus composite of Ad3 and Ad11p. In some embodiments, Ad3 / 11p is ColoAd1. ColoAd1 is a chimeric adenovirus whose major coat protein is derived from Ad11p, and, in contrast to Ad11, contains a nearly complete 2444 base pair deletion of the E3 gene, a small 25 base pair deletion of the E4 gene region, and the chimeric Ad3 / Ad11pm E3B gene region. See Kuhn et al., PLoS ONE, 2008, 3: e2409;WO 2005 / 118825;WO2014198852 A2;US8765463. Ad11 adenoviruses are generally expressed at low levels in all nucleated cells, but typically enter cells via the CD46 receptor, which shows increased surface expression in tumor cells, thereby preferentially infecting tumor cells. Both Ad3 and Ad11p are subgroup B adenoviruses (B1 and B2, respectively). Chimeric adenoviruses such as Ad3 / 11p are typically generated using a method called "directed evolution," such as that described by Kuhn et al. above, where a pool of Ad serotypes corresponding to different Ad subgroups is passaged in human tumor cell lines representing major solid tumor characteristics (e.g., breast cancer, colon cancer, pancreatic cancer, or prostate cancer) to trigger recombination and selection of potent viral variants or serotypes. In some embodiments, the Ad3 / 11p viral vector is ColoAd1. See, for example, Kuhn et al.; US8765463 above. In some embodiments, ColoAd1 has a nucleic acid sequence having 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 about 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 with SEQ ID NO:41. In some embodiments, ColoAd1 has the nucleic acid sequence of SEQ ID NO:41.

[0335] In some embodiments, the adenovirus vector is Ad5 / 3, such as those described in Hemminki et al., Oncotarget, 2015, 6: 4467-81; and Safar et al., Cancer Gene Therapy, 2021, 28: 442-454. The Ad5 / 3 chimeric adenovirus vector contains a serotype 3 fiber knob in the Ad5 capsid, thereby utilizing the Ad3 receptor (desmoglein-2), which is highly expressed in progressive tumors. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV is any serotype of AAV, such as AAV1, AAV2, AAV3, or AAV4. In some embodiments, the AAV is AAV1. In some embodiments, the AAV is AAV2. In some embodiments, the AAV is AAV3. In some embodiments, the AAV is AAV4.

[0336] The genomes of AAVs have been determined. For example, the genome sequence of AAV2 is available under GenBank accession numbers J01901 and NC_001401.

[0337] In some embodiments, the viral vector includes a tumor-specific promoter element that promotes increased expression of an encoded engineered antigen tag when expressed in tumor cells or tumor cells of a particular cancer type compared to when it is expressed in non-tumor cells, such as normal non-cancerous cells of the same cell or tissue type as the tumor cells. In some embodiments, the tumor-specific promoter element is the STAT3 promoter, the Survivin promoter, the cyclooxygenase-2 (COX-2) promoter, the prostate-specific antigen (PSA) promoter, the CXCR4 promoter, or any other promoter that promotes increased expression in tumor cells compared to normal cells of the same cell or tissue type. Examples of tumor-specific promoter elements and methods for modifying a viral vector to include them can be found, for example, in US20190275093A1.

[0338] In some embodiments, the viral vector is an oncolytic virus. Oncolytic viruses have the ability to infect and kill cancer cells. Oncolytic viruses may be, but are not limited to, adenoviruses, herpes simplex virus (HSV), parvoviruses, and poxviruses such as vaccinia virus (VACV) and myxoma virus (MYXV). In some embodiments, the oncolytic virus is selected from the group consisting of adenoviruses, herpes simplex virus, vaccinia virus, mumps virus, Newcastle disease virus, poliovirus, Seneca Valley virus, measles virus, Sindbis virus, parvovirus, coxsackievirus, varicella stomatitis virus, reovirus, and maraba and rhabdoviruses. In some embodiments, the viral vector exhibits weak oncolytic activity.

[0339] In some embodiments, the vector is delivered specifically to cancer cells. In some embodiments, the vector is delivered specifically to hematological cancer cells. In some embodiments, the vector is delivered specifically to solid tumor cancer cells.

[0340] In some embodiments, the vector is specifically delivered to immune effector cells. In some embodiments, the immune effector cells are cytotoxic T cells. In some embodiments, the immune effector cells are natural killer cells.

[0341] V. Compositions and Kits Compositions such as pharmaceutical compositions, comprising one or more syntheti...

Claims

1. Synthetic cancer antigens containing a target domain and a membrane targeting domain.

2. The synthetic cancer antigen according to claim 1, wherein the target domain is not expressed on the surface of target non-cancer cells.

3. The synthetic cancer antigen according to claim 1 or 2, wherein the target domain is recognizable by a cognitive binder.

4. The synthetic cancer antigen according to any one of claims 1 to 3, wherein the target domain is an antibody or antibody fragment recognized by the cognitive binder.

5. The synthetic cancer antigen according to claim 4, wherein the cognitive binder is bound to the idiotype of the antibody or antibody fragment.

6. The synthetic cancer antigen according to claim 4 or 5, wherein the cognitive binder is an anti-idiotype antibody or an antigen-binding fragment.

7. The synthetic cancer antigen according to any one of claims 4 to 6, wherein the target domain of the antibody or antibody fragment does not contain all or part of the heavy chain constant region, optionally does not contain the CH1, CH2, and / or CH3 domains, and more optionally the target domain of the antibody or antibody fragment does not contain the heavy chain CH3 domain.

8. The synthetic cancer antigen according to any one of claims 1 to 7, wherein the target domain is derived from a 4D5 anti-HER2 antibody.

9. The synthetic cancer antigen according to claim 7 or claim 8, wherein the target domain comprises a light chain having an amino acid sequence at least 85% identical to SEQ ID NO: 7, and a heavy chain having an amino acid sequence at least 85% identical to SEQ ID NO:

10.

10. The synthetic cancer antigen according to any one of claims 4 to 9, wherein the target domain comprises a light chain containing the amino acid sequence of SEQ ID NO: 7 and a heavy chain containing the amino acid sequence of SEQ ID NO:

10.

11. The synthetic cancer antigen according to any one of claims 4 to 10, wherein the target domain is a single-stranded variable fragment (scFv).

12. The synthetic cancer antigen according to claim 11, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

11.

13. The synthetic cancer antigen according to claim 11 or 12, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

11.

14. The synthetic cancer antigen according to any one of claims 4 to 8, wherein the target domain is a variable domain of the antibody's heavy chain.

15. The synthetic cancer antigen according to claim 14, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

8.

16. The synthetic cancer antigen according to claim 14 or claim 15, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

8.

17. The synthetic cancer antigen according to any one of claims 8 to 16, wherein the cognitive binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

13.

18. The synthetic cancer antigen according to any one of claims 8 to 17, wherein the cognitive binder comprises the amino acid sequence of SEQ ID NO:

13.

19. The synthetic cancer antigen according to any one of claims 8 to 16, wherein the cognitive binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

14.

20. The synthetic cancer antigen according to any one of claims 8 to 16 and 19, wherein the cognitive binder comprises the amino acid sequence of SEQ ID NO:

14.

21. The synthetic cancer antigen according to any one of claims 1 to 3, wherein the target domain is a fragment of an intracellular protein.

22. The synthetic cancer antigen according to claim 21, wherein the target domain is a non-DNA binding domain of a transcription factor or an inactive leucine zipper domain.

23. The synthetic cancer antigen according to claim 21 or 22, wherein the target domain contains an amino acid sequence that is at least 85% identical to one of SEQ ID NO: 17-19 and 21-23.

24. The synthetic cancer antigen according to any one of claims 21 to 23, wherein the target domain comprises one amino acid sequence from among SEQ ID NO: 17 to 19 and 21 to 23.

25. The synthetic cancer antigen according to claim 21 or claim 22, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

17.

26. The synthetic cancer antigen according to any one of claims 21 to 22 or 25, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

17.

27. The synthetic cancer antigen according to claim 25 or 26, wherein the cognitive binder of the synthetic cancer antigen comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 24 or 25.

28. The synthetic cancer antigen according to any one of claims 25 to 27, wherein the cognitive binder of the synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 24 or 25.

29. The synthetic cancer antigen according to any one of claims 1 to 3, wherein the target domain is the active leucine zipper domain of a transcription factor.

30. The synthetic cancer antigen according to claim 29, wherein the target domain is the leucine zipper domain of ATF.

31. The synthetic cancer antigen according to claim 29 or 30, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

26.

32. The synthetic cancer antigen according to any one of claims 29 to 31, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

26.

33. The synthetic cancer antigen according to claim 29, wherein the target domain is the leucine zipper domain of JUN.

34. The synthetic cancer antigen according to claim 29 or 33, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

27.

35. The synthetic cancer antigen according to any one of claims 29 and 33-34, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

27.

36. The synthetic cancer antigen according to any one of claims 29 to 35, wherein the cognitive binder of the synthetic cancer antigen is a leucine zipper domain.

37. The synthetic cancer antigen according to any one of claims 29 to 32 and 36, wherein the cognitive binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

28.

38. The synthetic cancer antigen according to any one of claims 29 to 32, 36, or 37, wherein the cognitive binder comprises the amino acid sequence of SEQ ID NO:

28.

39. The synthetic cancer antigen according to any one of claims 29 and 33-36, wherein the cognitive binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

29.

40. The synthetic cancer antigen according to any one of claims 29, 33-36, or 39, wherein the cognitive binder comprises the amino acid sequence of SEQ ID NO:

29.

41. The synthetic cancer antigen according to any one of claims 3 to 40, wherein the cognitive binder of the synthetic cancer antigen is the extracellular domain of a chimeric antigen receptor (CAR).

42. The synthetic cancer antigen according to claim 41, wherein the CAR further comprises a transmembrane domain and an intracellular signaling domain.

43. The synthetic cancer antigen according to claim 41 or 42, wherein the CAR is expressed on the surface of immune effector cells.

44. The synthetic cancer antigen according to claim 43, wherein the immune effector cells are T cells, and optionally cytotoxic T cells.

45. The synthetic cancer antigen according to claim 43, wherein the immune effector cells are natural killer cells.

46. The synthetic cancer antigen according to any one of claims 1 to 45, wherein the membrane targeting domain is a transmembrane domain.

47. The synthetic cancer antigen according to claim 46, wherein the transmembrane domain is derived from a transmembrane glycoprotein.

48. The synthetic cancer antigen according to claim 46 or 47, wherein the transmembrane domain is a CD8 transmembrane domain.

49. The synthetic cancer antigen according to any one of claims 46 to 48, wherein the transmembrane domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO:

3.

50. The synthetic cancer antigen according to any one of claims 46 to 49, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

3.

51. Synthetic cancer antigens containing a target domain and tumor targeting binding molecules.

52. The synthetic cancer antigen according to claim 51, which is soluble.

53. The synthetic cancer antigen according to claim 51 or claim 52, wherein the tumor-targeting binding molecule specifically binds to tumor-associated antigens expressed on the surface of tumor cells.

54. The synthetic cancer antigen according to claim 53, wherein the tumor-associated antigen is selected from the group consisting of EpCAM, CEA (carcinoembryonic antigen), gpA33 (glycoprotein A33 (transmembrane)), mucin, TAG-72 (tumor-associated glycoprotein 72), CAIX (carbonic anhydrase IX), PSMA (prostate-specific membrane antigen), and FBP (folate-binding protein), EGFR / ERBB1 / HER1 (epidermal growth factor receptor 1), ERBB2 / HER2 (epidermal growth factor receptor 2), ERBB3 (epidermal growth factor receptor 3), MET (tyrosine protein kinase IGF1R (insulin-like growth factor 1 receptor)), EPHA3 (EPH receptor A3), TRAILR1 (death receptor 4), and RANK-L (nuclear factor kappa-B receptor activator ligand), claudin 6, claudin 182, GPC2, and GPC3.

55. The synthetic cancer antigen according to claim 53 or claim 54, wherein the tumor cells are tumor cells of a solid tumor.

56. The synthetic cancer antigen according to any one of claims 53 to 55, wherein the tumor cells are tumor cells 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, gastric cancer, thyroid cancer, bile duct cancer, bone cancer, skin cancer, colon cancer, rectal cancer, endometrial cancer, or cervical cancer.

57. The synthetic cancer antigen according to any one of claims 53 to 56, wherein the tumor cells are tumor cells of 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.

58. The synthetic cancer antigen according to any one of claims 51 to 57, wherein the tumor targeting binding molecule comprises an antibody or an antigen-binding fragment thereof.

59. The synthetic cancer antigen according to claim 58, wherein the antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv).

60. The synthetic cancer antigen according to any one of claims 53 to 59, wherein the tumor-associated antigen is EpCAM.

61. The antibody or its antigen-binding fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region. The VH region includes heavy chain complementarity determination regions 1 (CDR-H1), CDR-H2, and CDR-H3, which contain amino acid sequences shown in SEQ ID NO: 42, 43, and 44, respectively. The VL region includes light chain complementarity determination regions 1 (CDR-L1), CDR-L2, and CDR-L3, which contain amino acid sequences shown in SEQ ID NO: 46, 47, and 48, respectively. A synthetic cancer antigen according to any one of claims 58 to 60.

62. The synthetic cancer antigen according to any one of claims 58 to 61, wherein the antibody or antigen-binding fragment comprises a heavy chain variable (VH) region containing 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 with the amino acid sequence of SEQ ID NO: 35; and a light chain variable (VL) region containing 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 with the amino acid sequence of SEQ ID NO:

37.

63. The synthetic cancer antigen according to any one of claims 58 to 62, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable (VH) region containing the amino acid sequence of SEQ ID NO: 35 and a light chain variable (VL) region containing the amino acid sequence of SEQ ID NO:

37.

64. The synthetic cancer antigen according to any one of claims 51 to 63, wherein the target domain is not expressed on the surface of the target non-cancer cells.

65. The synthetic cancer antigen according to any one of claims 51 to 64, wherein the target domain is recognizable by a cognitive binder.

66. The synthetic cancer antigen according to any one of claims 51 to 65, wherein the target domain is an antibody or antibody fragment recognized by the cognitive binder.

67. The synthetic cancer antigen according to claim 66, wherein the cognitive binder is bound to the idiotype of the antibody or antibody fragment.

68. The synthetic cancer antigen according to claim 66 or claim 67, wherein the cognitive binder is an anti-idiotype antibody or an antigen-binding fragment.

69. The synthetic cancer antigen according to any one of claims 66 to 68, wherein the target domain is derived from a 4D5 anti-HER2 antibody.

70. The synthetic cancer antigen according to any one of claims 66 to 69, wherein the target domain comprises a light chain having an amino acid sequence identical to at least 85% of SEQ ID NO: 7, and a heavy chain having an amino acid sequence identical to at least 85% of SEQ ID NO:

10.

71. The synthetic cancer antigen according to any one of claims 66 to 70, wherein the target domain comprises a light chain containing the amino acid sequence of SEQ ID NO: 7 and a heavy chain containing the amino acid sequence of SEQ ID NO:

10.

72. The synthetic cancer antigen according to any one of claims 66 to 71, wherein the target domain is a single-stranded variable fragment (scFv).

73. The synthetic cancer antigen according to claim 72, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

11.

74. The synthetic cancer antigen according to claim 72 or claim 73, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

11.

75. The synthetic cancer antigen according to any one of claims 66 to 69, wherein the target domain is a variable domain of the antibody's heavy chain.

76. The synthetic cancer antigen according to claim 75, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

8.

77. The synthetic cancer antigen according to claim 75 or 76, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

8.

78. The synthetic cancer antigen according to any one of claims 69 to 77, wherein the cognitive binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

13.

79. The synthetic cancer antigen according to any one of claims 69 to 78, wherein the cognitive binder comprises the amino acid sequence of SEQ ID NO:

13.

80. The synthetic cancer antigen according to any one of claims 69 to 77, wherein the cognitive binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

14.

81. The synthetic cancer antigen according to any one of claims 69 to 77 and 80, wherein the cognitive binder comprises the amino acid sequence of SEQ ID NO:

14.

82. The synthetic cancer antigen according to any one of claims 51 to 65, wherein the target domain is a fragment of an intracellular protein.

83. The synthetic cancer antigen according to claim 82, wherein the target domain is a non-DNA binding domain of a transcription factor or an inactive leucine zipper domain.

84. The synthetic cancer antigen according to claim 82 or 83, wherein the target domain comprises an amino acid sequence that is at least 85% identical to one of SEQ ID NO: 17-19 and 21-23.

85. The synthetic cancer antigen according to any one of claims 82 to 84, wherein the target domain comprises one amino acid sequence from among SEQ ID NO: 17-19 and 21-23.

86. The synthetic cancer antigen according to any one of claims 82 to 85, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

17.

87. The synthetic cancer antigen according to any one of claims 82 to 86, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

17.

88. The synthetic cancer antigen according to claim 86 or 87, wherein the cognitive binder of the synthetic cancer antigen comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 24 or 25.

89. The synthetic cancer antigen according to any one of claims 86 to 88, wherein the cognitive binder of the synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 24 or 25.

90. The synthetic cancer antigen according to any one of claims 51 to 65, wherein the target domain is the active leucine zipper domain of a transcription factor.

91. The synthetic cancer antigen according to claim 90, wherein the target domain is the leucine zipper domain of ATF.

92. The synthetic cancer antigen according to claim 90 or 91, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

26.

93. The synthetic cancer antigen according to any one of claims 90 to 92, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

26.

94. The synthetic cancer antigen according to claim 90, wherein the target domain is the leucine zipper domain of JUN.

95. The synthetic cancer antigen according to claim 90 or 94, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

27.

96. The synthetic cancer antigen according to any one of claims 90, 94, and 95, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

27.

97. The synthetic cancer antigen according to any one of claims 90 to 96, wherein the cognitive binder of the synthetic cancer antigen is a leucine zipper domain.

98. The synthetic cancer antigen according to any one of claims 90 to 93 and 97, wherein the cognitive binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

28.

99. The synthetic cancer antigen according to any one of claims 90-93, 97, or 98, wherein the cognitive binder comprises the amino acid sequence of SEQ ID NO:

28.

100. The synthetic cancer antigen according to any one of claims 90 and 94-97, wherein the cognitive binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

29.

101. The synthetic cancer antigen according to any one of claims 90, 94-97, or 100, wherein the cognitive binder comprises the amino acid sequence of SEQ ID NO:

29.

102. The synthetic cancer antigen according to any one of claims 65 to 101, wherein the cognitive binder of the synthetic cancer antigen is the extracellular domain of a chimeric antigen receptor (CAR).

103. The synthetic cancer antigen according to claim 102, wherein the CAR further comprises a transmembrane domain and an intracellular signaling domain.

104. The synthetic cancer antigen according to claim 102 or claim 103, wherein the CAR is expressed on the surface of immune effector cells.

105. The synthetic cancer antigen according to claim 104, wherein the immune effector cells are T cells, and optionally cytotoxic T cells.

106. The synthetic cancer antigen according to claim 104, wherein the immune effector cells are natural killer cells.

107. The synthetic cancer antigen according to any one of claims 51 to 106, wherein the target domain and the tumor targeting binding molecule are linked by a linker.

108. The synthetic cancer antigen according to claim 107, wherein the linker has a length of 1 to 100 amino acids, 1 to 75 amino acids, 1 to 50 amino acids, 1 to 25 amino acids, 5 to 100 amino acids, 5 to 75 amino acids, 5 to 50 amino acids, 5 to 25 amino acids, 10 to 100 amino acids, 10 to 75 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids.

109. The synthetic cancer antigen according to claim 107 or claim 108, wherein the linker comprises one amino acid sequence of SEQ ID NO: 33 and 67-86.

110. A polynucleotide encoding a synthetic cancer antigen according to any one of claims 1 to 50.

111. A polynucleotide encoding a synthetic cancer antigen according to any one of claims 51 to 109.

112. A polynucleotide comprising a nucleic acid sequence encoding a synthetic cancer antigen according to any one of claims 1 to 50 and a nucleic acid sequence encoding a synthetic cancer antigen tag according to any one of claims 51 to 109.

113. The polynucleotide according to claim 112, wherein the nucleic acid sequence encoding a synthetic cancer antigen according to any one of claims 1 to 50 and the nucleic acid sequence encoding a synthetic cancer antigen according to any one of claims 51 to 109 are separated by a nucleotide sequence encoding a cleavable linker.

114. The polynucleotide according to claim 113, wherein the cleavable linker is a self-cleaving linker.

115. The polynucleotide according to claim 114, wherein the self-cleaving linker is a 2A peptide or comprises the same.

116. A vector comprising the polynucleotide according to claim 110 or claim 111.

117. A vector comprising a polynucleotide according to any one of claims 112 to 115.

118. A vector comprising the polynucleotide according to claim 110 and the polynucleotide according to claim 111.

119. A vector according to any one of claims 116 to 118, for specific delivery to cancer cells.

120. The vector according to claim 119, for specific delivery to blood cancer cells.

121. The vector according to claim 119, for specific delivery to solid tumor cancer cells.

122. A viral vector, as described in any one of claims 116 to 121.

123. The vector according to claim 122, wherein the viral vector is a tumor-targeting viral vector and optionally a tumor-lytic virus.

124. The vector according to claim 123, wherein the viral vector is selected from the group consisting of adenovirus, herpes simplex virus, vaccinia virus, mumps virus, Newcastle disease virus, poliovirus, Seneca Valley virus, measles virus, Sindbisvirus, parvovirus, coxsackievirus, vesicular stomatitis virus, reovirus, and maraba and rhabdovirus.

125. The vector according to any one of claims 122 to 124, wherein the viral vector exhibits directivity to tumor cells.

126. The vector according to any one of claims 122 to 125, wherein the viral vector enters a cell by binding to a cell surface receptor expressed by a tumor cell.

127. The vector according to any one of claims 122 to 126, wherein the viral vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, or a herpes simplex virus (HSV) vector.

128. The vector according to any one of claims 122 to 127, wherein the viral vector is an adenovirus vector.

129. The vector according to claim 128, wherein the adenovirus vector is an adenovirus vector that binds to CD46 and / or desmoglein-2.

130. The vector according to claim 128 or claim 129, wherein the adenovirus vector is Ad3, Ad7, Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, or Ad50.

131. The vector according to claim 128 or claim 129, wherein the adenovirus vector is a chimeric adenovirus vector based on Ad3, Ad7, Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, or Ad50.

132. The vector according to any one of claims 128, 129, and 131, wherein the adenovirus vector is a chimeric adenovirus vector based on Ad11.

133. The vector according to any one of claims 128, 129, 131, and 132, wherein the adenovirus vector is a chimeric Ad3 / 11p adenovirus vector.

134. The vector according to claim 133, wherein the chimeric Ad3 / 11p adenovirus vector binds to CD46.

135. The vector according to claim 133, wherein the chimeric Ad3 / 11p adenovirus vector comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 41, and optionally, the nucleic acid sequence is the one shown in SEQ ID NO:

41.

136. The vector according to claim 128 or claim 129, wherein the adenovirus vector is a chimeric adenovirus vector based on Ad5.

137. The vector according to any one of claims 128, 129, and 136, wherein the adenovirus vector is a chimeric Ad5 / 3 adenovirus vector.

138. A cell comprising the polynucleotide according to any one of claims 110 to 115.

139. Cells comprising or manipulated by a vector according to any one of claims 116 to 138.

140. A population of cells comprising at least one of the cells described in claim 138 or claim 139.

141. A pharmaceutical composition comprising a synthetic cancer antigen according to any one of claims 1 to 50, a polynucleotide according to any one of claims 51 to 109, a polynucleotide according to any one of claims 110 or 111, or a vector according to any one of claims 116 to 138.

142. The pharmaceutical composition according to claim 141, further comprising a pharmaceutically acceptable carrier.

143. A pharmaceutical composition according to any one of claims 141 to 142 for use in treating cancer in a subject.

144. A combination comprising a vector according to any one of claims 116 to 137, and a population of lymphocytes expressing a chimeric antigen receptor (CAR) that recognizes the target domain of the synthetic cancer antigen.

145. The combination according to claim 144, wherein the CAR comprises an extracellular binding domain containing a cognitive binder of the target domain, a transmembrane domain, and an intracellular signaling domain.

146. The combination according to claim 145, wherein the target domain is an antibody or antibody fragment, and the cognitive binder is bound to the idiotype of the antibody or antibody fragment.

147. The combination according to claim 145, wherein the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor.

148. The combination according to claim 145, wherein the target domain is the active leucine zipper domain of a transcription factor.

149. The combination according to claim 145 or 148, wherein the target domain is the leucine zipper domain of ATF.

150. The combination according to claim 145 or 148, wherein the target domain is the leucine zipper domain of JUN.

151. The combination according to any one of claims 145, 148, 149, or 150, wherein the cognitive binder of the synthetic cancer antigen is a leucine zipper domain.

152. The combination according to any one of claims 145 to 151, wherein the lymphocytes include T cells, optionally cytotoxic T cells.

153. The lymphocytes are a combination according to any one of claims 145 to 151, wherein the lymphocytes include natural killer cells.

154. A kit comprising the combination described in any one of claims 144 to 153.

155. The kit according to claim 154, further comprising instructions for using the vector and the population of lymphocytes.

156. A step of administering a therapeutically effective amount of a polynucleotide according to any one of claims 110 to 115, a vector according to any one of claims 116 to 137, or a pharmaceutical composition according to any one of claims 141 to 143 to a subject having cancer, and The process involves administering a therapeutically effective dose of a population of lymphocytes expressing a chimeric antigen receptor (CAR) that binds to the target domain of the synthetic cancer antigen. Treatment methods, including those mentioned above.

157. A treatment method comprising the step of administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) to a subject having cancer who has been previously administered a polynucleotide according to any one of claims 110 to 115, a vector according to any one of claims 116 to 138, or a pharmaceutical composition according to any one of claims 141 to 143, wherein the CAR binds to the target domain of the synthetic cancer antigen.

158. The method according to claim 156 or 157, wherein the lymphocytes are T cells.

159. The method according to any one of claims 156 to 158, wherein a population of lymphocytes expressing a chimeric antigen receptor (CAR) that recognizes the target domain of a vector and synthetic cancer antigen according to any one of claims 116 to 138 is administered to the subject.

160. The method according to claim 159, wherein the CAR comprises an extracellular binding domain containing a cognitive binder of the target domain, a transmembrane domain, and an intracellular signaling domain.

161. The method according to claim 160, wherein the target domain is an antibody or antibody fragment, and the cognitive binder is bound to the idiotype of the antibody or antibody fragment.

162. The method according to claim 160, wherein the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor.

163. The method according to claim 160, wherein the target domain is the active leucine zipper domain of a transcription factor.

164. The method according to claim 160 or 163, wherein the target domain is the leucine zipper domain of ATF.

165. The method according to claim 160 or 163, wherein the target domain is the leucine zipper domain of JUN.

166. The method according to any one of claims 160, 163, 164, and 165, wherein the cognitive binder of the synthetic cancer antigen is a leucine zipper domain.

167. The method according to any one of claims 160 to 166, wherein the lymphocytes include cytotoxic T cells.

168. The method according to any one of claims 160 to 166, wherein the lymphocytes include natural killer cells.

169. A method for tagging tumor cells in vivo, comprising the step of contacting the tumor cells with a polynucleotide according to any one of claims 110 to 115, a vector according to any one of claims 116 to 137, or a pharmaceutical composition according to any one of claims 141 to 143.

170. A method for tagging tumor cells of a subject having cancer, comprising the step of administering to the subject a therapeutically effective amount of a polynucleotide according to any one of claims 110 to 115, a vector according to any one of claims 116 to 137, or a pharmaceutical composition according to any one of claims 141 to 143.

171. A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 141 or 142.

172. The method according to claim 171, further comprising the step of administering a therapeutically effective amount of immune effector cells to the subject.

173. The method according to claim 172, wherein at least one of the immune effector cells expresses CAR on the surface of the immune effector cell.

174. The method according to claim 173, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

175. The method according to claim 174, wherein the extracellular domain of the CAR comprises a cognitive binder for the target domain of the synthetic cancer antigen.

176. The method according to any one of claims 171 to 175, wherein the immune effector cells are cytotoxic T cells.

177. The method according to any one of claims 171 to 175, wherein the immune effector cells are natural killer cells.

178. The method according to any one of claims 171 to 177, wherein the pharmaceutical composition and the immune effector cells are administered simultaneously.

179. The method according to any one of claims 171 to 177, wherein the pharmaceutical composition is administered before the immunoeffector cells are administered.

180. The method according to any one of claims 156 to 179, wherein the cancer is a blood cancer.

181. The method according to any one of claims 156 to 179, wherein the cancer is a solid tumor carcinoma.

182. A chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain binds to a target domain of a synthetic cancer antigen according to any one of claims 1 to 109.

183. A chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 13, 14, 24, 25, 28, and 29, and optionally, the extracellular domain comprises the amino acid sequences shown in SEQ ID NO: 13, 14, 24, 25, 28, and 29.

184. The CAR according to claim 183, wherein the extracellular domain comprises the amino acid sequence shown in SEQ ID NO:

13.

185. The CAR according to claim 183 or claim 184, wherein the extracellular domain binds to the target domain of a synthetic cancer antigen, and optionally the synthetic cancer antigen comprises (1) the target domain and a membrane targeting domain, or (2) the target domain and a tumor targeting binding molecule.

186. The CAR according to claim 185, wherein the target domain is not expressed on the surface of the target non-cancer cells.

187. The CAR according to claim 185 or 186, wherein the target domain is an antibody or a molecule derived from an antibody.

188. The CAR according to claim 187, wherein the target domain is derived from a 4D5 anti-HER2 antibody.

189. The CAR according to claim 188, wherein the target domain comprises a light chain having an amino acid sequence at least 85% identical to SEQ ID NO: 7, and a heavy chain having an amino acid sequence at least 85% identical to SEQ ID NO:

10.

190. The CAR according to claim 188 or claim 189, wherein the target domain comprises a light chain containing the amino acid sequence of SEQ ID NO: 7 and a heavy chain containing the amino acid sequence of SEQ ID NO:

10.

191. The CAR according to claim 188, wherein the target domain is a single-stranded variable fragment (scFv).

192. The CAR according to claim 191, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

11.

193. The CAR according to claim 191 or 192, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

11.

194. The CAR according to claim 188, wherein the target domain is a variable domain of the antibody's heavy chain.

195. The CAR according to claim 194, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

8.

196. The CAR according to claim 194 or 195, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

8.

197. The CAR according to any one of claims 187 to 196, wherein the extracellular domain of the CAR binds to the idiotype of the target domain of the synthetic cancer antigen.

198. The CAR according to claim 197, wherein the extracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO:

13.

199. The CAR according to claim 197, wherein the extracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO:

14.

200. The CAR according to claim 185 or 186, wherein the target domain is a fragment of an intracellular protein.

201. The CAR according to claim 200, wherein the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor.

202. The CAR according to claim 200 or claim 201, wherein the target domain comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 17-19 and 21-23.

203. The CAR according to any one of claims 200 to 202, wherein the target domain comprises amino acid sequences of SEQ ID NO: 17-19 and 21-23.

204. The CAR according to any one of claims 200 to 202, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

17.

205. The CAR according to any one of claims 200 to 204, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

17.

206. The CAR according to claim 204 or 205, wherein the extracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 24 or 25.

207. The CAR according to claim 185 or 186, wherein the target domain is the active leucine zipper domain of a transcription factor.

208. The CAR according to claim 207, wherein the target domain is the leucine zipper domain of ATF.

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

26.

210. The CAR according to claim 208 or claim 209, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

26.

211. The CAR according to claim 207, wherein the target domain is the leucine zipper domain of JUN.

212. The CAR according to claim 207 or claim 211, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

27.

213. The CAR according to claim 207, 211, or 212, wherein the target domain comprises the amino acid sequence of SEQ ID NO:

27.

214. The CAR according to any one of claims 207 to 213, wherein the extracellular domain of the CAR is a leucine zipper domain.

215. The CAR according to claims 207-210 and 214, wherein the extracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO:

28.

216. The CAR according to claims 207 and 211-214, wherein the extracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO:

29.

217. The CAR according to any one of claims 182 to 216, wherein the transmembrane domain of the CAR is a CD8 transmembrane domain.

218. The CAR according to any one of claims 182 to 217, wherein the transmembrane domain of the CAR contains an amino acid sequence that is at least 85% identical to SEQ ID NO:

3.

219. The CAR according to any one of claims 182 to 217, wherein the transmembrane domain of the CAR comprises the amino acid sequence of SEQ ID NO:

3.

220. The CAR according to any one of claims 182 to 219, wherein the intracellular signaling domain includes a primary intracellular signaling domain.

221. The CAR according to claim 220, wherein the intracellular signaling domain further comprises a co-stimulatory signaling domain.

222. The CAR according to any one of claims 182 to 221, wherein the intracellular signaling domain comprises a CD3z cytoplasmic signaling domain and a 41BB costimulatory signaling domain.

223. The CAR according to any one of claims 182 to 222, wherein the intracellular signaling domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

16.

224. The CAR according to any one of claims 182 to 223, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

16.

225. A polynucleotide encoding a CAR according to any one of claims 182 to 224.

226. A vector comprising the polynucleotide described in claim 225.

227. Cells comprising or manipulated by the vector according to claim 226.

228. A cell comprising the polynucleotide described in claim 225.

229. The cells according to claim 227 or claim 228, which are immune effector cells.

230. The cell according to claim 229, wherein the immune effector cell is a T cell, and optionally a cytotoxic T cell.

231. The cell according to claim 229, wherein the immune effector cell is a natural killer cell.

232. A population of cells comprising at least one cell according to any one of claims 227 to 231.

233. A pharmaceutical composition comprising a CAR according to any one of claims 182 to 224, a polynucleotide according to claim 225, a vector according to claim 226, a cell according to any one of claims 227 to 231, or a population of cells according to claim 232.

234. The pharmaceutical composition according to claim 233, further comprising a pharmaceutically acceptable carrier.

235. A pharmaceutical composition according to claim 233 or 234 for use in treating cancer in a subject.

236. A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 233 or 234.

237. The method according to claim 236, further comprising the step of administering to the subject a therapeutically effective amount of an additional pharmaceutical composition comprising a synthetic cancer antigen, a polynucleotide encoding the synthetic cancer antigen, or a vector comprising the polynucleotide encoding the synthetic cancer antigen.

238. The method according to claim 236 or 237, wherein a pharmaceutical composition and an additional pharmaceutical composition are administered simultaneously.

239. The method according to claim 236 or 237, wherein an additional pharmaceutical composition is administered before the pharmaceutical composition is administered.

240. The method according to any one of claims 236 to 239, wherein the cancer is a blood cancer.

241. The method according to any one of claims 236 to 239, wherein the cancer is a solid tumor carcinoma.

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