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WO2024243578A1 - Antigènes synthétiques de cancers en tant que cibles pour le traitement de cancers - Google Patents

Antigènes synthétiques de cancers en tant que cibles pour le traitement de cancers Download PDF

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Publication number
WO2024243578A1
WO2024243578A1 PCT/US2024/031157 US2024031157W WO2024243578A1 WO 2024243578 A1 WO2024243578 A1 WO 2024243578A1 US 2024031157 W US2024031157 W US 2024031157W WO 2024243578 A1 WO2024243578 A1 WO 2024243578A1
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acid sequence
amino acid
domain
target domain
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PCT/US2024/031157
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Lexus R. Johnson
Tatiana M. GARCIA-BATES
David Heo
Raymond Liu
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Dispatch Biotherapeutics, Inc.
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Publication of WO2024243578A1 publication Critical patent/WO2024243578A1/fr

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Definitions

  • the present disclosure generally relates to synthetic cancer antigens and/or cognate binders thereof and the use of such molecules for treating different types of cancers.
  • CAR modified chimeric-antigen-receptor
  • TAAs tumor associated antigens
  • the primary factors include CAR-T immunosuppression in the tumor microenvironment, limited migration of T cells to the tumor site, and most significantly, “on-target, off-tumor” toxicity (Wang et al., Front Med. (2020) 14(6):726-45; Tahmasebi et al., Stem Cell Rev Rep.
  • the present disclosure re-designs the interaction between the immune effector cells and the cancer targets.
  • the present disclosure improves the safety of immune cell therapy by eliminating recognition of healthy tissues.
  • the synthetic cancer antigens disclosed herein can be delivered to different types of cancers, the present disclosure provides a universal approach to treat different types of cancers (e.g., solid tumors).
  • a synthetic cancer antigen comprising a target domain and a transmembrane domain.
  • the target domain is not expressed on the surface of a non-cancer cell of a subject.
  • the target domain is recognizable by a cognate binder.
  • the target domain is an antibody or antibody fragment.
  • the cognate binder binds to the idiotype of the antibody or antibody fragment.
  • the cognate binder is an anti-idiotype antibody or an antigen binding fragment.
  • the anti-idiotype antibody is an antigen binding fragment comprising a variable heavy chain and a variable light chain, such as a single chain variable chain (scFv).
  • the anti-idiotype antibody or antigen-binding fragment that is the cognate binder is part of the extracellular domain of a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the extracellular domain of the CAR comprises a variable heavy chain and variable light chain of the anti-idiotype antibody for recognizing by binding the target domain.
  • the variable heavy chain and variable light chain of the anti-idiotype antibody of the extracellular domain of the CAR is present as a scFv.
  • the antibody or antibody fragment of the target domain does not contain all or a portion of the heavy chain constant region, optionally does not contain the CH1, CH2 and/or CH3 domain. In some of any of the provided embodiments, the antibody or antibody fragment of the target domain does not contain the heavy chain CH3 domain.
  • the target domain is an antibody fragment, such as a Fab, an scFv or a variable heavy chain only fragment.
  • the target domain is not able to bind or substantially bind or exhibits reduced (low) binding affinity (e.g., a higher KD value) to the antigen of the antibody from which it is derived.
  • the antigen-binding domain for recognition of an antigen or epitope is not present or is partially removed or is non-functional (e.g., via one or more amino acid substitutions in a CDR).
  • the parental antibody is a two chain antibody containing a variable heavy and variable light chain in which the antigen-binding domain is composed of 6 CDRs.
  • the target domain is an antibody fragment that is the variable heavy chain (VH) only composed of only 3 CDRs.
  • the target domain is derived from the 4D5 anti-HER2 antibody (also known as Ab1), which is the parental antibody of trastuzumab.
  • the target domain is an antibody fragment of the 4D5 (Ab1) antibody.
  • the antibody fragment is a Fab.
  • the target domain comprises a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 10.
  • the target domain comprises a light chain comprising an amino acid sequence of SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 10.
  • the target domain is a single-chain variable fragment (scFv).
  • the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 11.
  • the target domain comprises an amino acid sequence of SEQ ID NO: 11.
  • the target domain is the variable domain of the heavy chain of an antibody.
  • the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 8. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 8.
  • the cognate binder is an anti-idiotype antibody that binds to an epitope of the 4D5 (Ab1) antibody or antibody fragment. In particular embodiments, the epitope of the target domain recognized by the anti-idiotype antibody is present in the variable heavy chain of the 4D5 (Ab1) antibody or antibody fragment.
  • the cognate binder comprises 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 any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 13. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 90 % identical to SEQ ID NO: 13. n some of any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13.
  • the cognate binder comprises an amino acid sequence of SEQ ID NO: 13. In some of any of the provided embodiments, the cognate binder comprises 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 any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 90 % identical to SEQ ID NO: 14. n some of any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 14.
  • the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 14. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence of SEQ ID NO: 14. In some of any of the provided embodiments, the cognate binder comprises 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 some of any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 90 % identical to SEQ ID NO: 15.
  • the cognate binder comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 15. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 15. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence of SEQ ID NO: 15. [0015] In some of any of the provided embodiments, the target domain is a fragment of an intracellular protein. In some of any of the provided embodiments, the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor.
  • the target domain comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 17-19 and 21-23. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of any one of SEQ ID NOs: 17-19 and 21-23. In some of any of the provided embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 17. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 17. In some of any of the provided embodiments, the cognate binder of the synthetic cancer antigen comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 24 or 25.
  • the cognate binder of the synthetic cancer antigen comprises an amino acid sequence of SEQ ID NO: 24 or 25.
  • the target domain is an active leucine zipper domain of a transcription factor.
  • the target domain is the leucine zipper domain of ATF.
  • the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 26.
  • the target domain comprises an amino acid sequence of SEQ ID NO: 26. [0016] In some of any of the provided embodiments, the target domain is the leucine zipper domain of JUN.
  • the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 27. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 27.
  • the cognate binder of the synthetic cancer antigen is a leucine zipper domain. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 28. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence of SEQ ID NO: 28. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 29.
  • the cognate binder comprises an amino acid sequence of SEQ ID NO: 29.
  • the cognate binder of the synthetic cancer antigen is the extracellular domain of a chimeric antigen receptor (CAR).
  • the CAR further comprises a transmembrane domain and an intracellular signaling domain.
  • the CAR is expressed on the surface of an immune effector cell.
  • the immune effector cell is a cytotoxic T cell.
  • the immune effector cell is a natural killer cell.
  • the membrane targeting domain is a transmembrane domain. In some of any of the provided embodiments, the transmembrane domain is from a transmembrane glycoprotein. In some of any of the provided embodiments, the transmembrane domain is a CD8 transmembrane domain. In some of any of the provided embodiments, the transmembrane domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3. In some of any of the provided embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 3. [0019] In some aspects, provided herein is a synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule.
  • the synthetic cancer antigen is soluble and is not expressed on the membrane.
  • the tumor-targeting binding molecule specifically binds to a tumor associated antigen expressed on the surface of a tumor cell.
  • the tumor associated antigen is selected from the group consisting of EpCAM, CEA (Carcinoembryonic antigen), gpA33 (Glycoprotein A33 (Transmembrane)), mucins, TAG-72 (Tumor-associated glycoprotein 72), CAIX (Carbonic anhydrase IX), PSMA (Prostate-specific membrane antigen), and FBP (Folate-binding protein), EGFR/ERBB1/HER1 (epidermal growth factor receptor 1), 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 (
  • the tumor cell is a tumor cell of a solid tumor.
  • the tumor cell is a tumor cell of a cancer selected from the group consisting of multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, medulloblastoma, liver cancer, stomach cancer, thyroid cancer, bile duct cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, endometrial cancer, or cervical cancer.
  • RRCC renal cell carcinoma
  • neuroblastoma colorectal cancer
  • bladder cancer breast cancer
  • ovarian cancer melanoma
  • sarcoma prostate cancer
  • lung cancer esophageal cancer
  • pancreatic cancer astrocytoma, me
  • 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.
  • the tumor cell is a tumor cell of a cancer selected from the group consisting of bladder cancer, breast cancer, skin cancer, head and neck cancer, colorectal cancer, endometrial cancer, liver cancer, kidney cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, thyroid cancer, and ovarian cancer.
  • the tumor cell targeting domain comprises an antibody or an antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).
  • the tumor associated antigen is EpCAM.
  • the antibody or antigen- binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NOs: 42, 43, and 44, respectively, and the VL region comprises a light chain complementarity determining region 1 (CDR-L1), a CDR-L2, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NOs: 46, 47, and 48, respectively.
  • VH heavy chain variable
  • VL light chain variable
  • the antibody or an antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 35; and a light chain variable (VL) region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 37.
  • VH heavy chain variable
  • VL light chain variable
  • the antibody or an antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 35, and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 37.
  • the soluble synthetic cancer antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 31.
  • the soluble synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 31. In some of any of the provided embodiments, the soluble synthetic cancer antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 39. In some of any of the provided embodiments, the soluble synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 39.
  • the soluble synthetic cancer antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 88.
  • the soluble synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 88.
  • the soluble synthetic cancer antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 89.
  • the soluble synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 89.
  • the target domain is not expressed on the surface of a non-cancer cell of a subject. In some of any of the provided embodiments, the target domain is recognizable by a cognate binder.
  • the target domain is an antibody or an antigen-binding fragment that is recognized by a cognate binder.
  • the cognate binder binds to the idiotype of the antibody or antigen-binding fragment.
  • the cognate binder is an anti-idiotype antibody or an antigen binding fragment.
  • the antibody or antigen binding fragment does not contain the heavy chain CH3 domain.
  • the target domain comprises a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 10.
  • the target domain comprises a light chain comprising an amino acid sequence of SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 10.
  • the target domain is a single-chain variable fragment (scFv).
  • the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 11.
  • the target domain comprises an amino acid sequence of SEQ ID NO: 11.
  • the target domain is the variable domain of the heavy chain of an antibody.
  • the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 8. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 8. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 13. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence of SEQ ID NO: 13. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 14. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence of SEQ ID NO: 14.
  • the target domain is a fragment of an intracellular protein.
  • the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor.
  • the target domain comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 17-19 and 21-23.
  • the target domain comprises an amino acid sequence of any one of SEQ ID NOs: 17-19 and 21-23.
  • the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 17.
  • the target domain comprises an amino acid sequence of SEQ ID NO: 17.
  • the cognate binder of the synthetic cancer antigen comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 24 or 25.
  • the cognate binder of the synthetic cancer antigen comprises an amino acid sequence of SEQ ID NO: 24 or 25.
  • the target domain is an active leucine zipper domain of a transcription factor.
  • the target domain is the leucine zipper domain of ATF.
  • the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 26.
  • the target domain comprises an amino acid sequence of SEQ ID NO: 26. In some of any of the provided embodiments, the target domain is the leucine zipper domain of JUN. In some of any of the provided embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 27. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 27. [0028] In some of any of the provided embodiments, the cognate binder of the synthetic cancer antigen is a leucine zipper domain. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 28.
  • the cognate binder comprises an amino acid sequence of SEQ ID NO: 28. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 29. In some of any of the provided embodiments, the cognate binder comprises an amino acid sequence of SEQ ID NO: 29. [0029] In some of any of the provided embodiments, the cognate binder of the synthetic cancer antigen is the extracellular domain of a chimeric antigen receptor (CAR). In some of any of the provided embodiments, the CAR further comprises a transmembrane domain and an intracellular signaling domain. In some of any of the provided embodiments, the CAR is expressed on the surface of an immune effector cell.
  • CAR chimeric antigen receptor
  • the immune effector cell is a cytotoxic T cell. In some of any of the provided embodiments, the immune effector cell is a natural killer cell. In some of any of the provided embodiments, the target domain and the tumor-targeting binding molecule are linked by a linker. In some of any of the provided embodiments, the linker has a length of between 1 and 100 amino acids, between 1 and 75 amino acids, between 1 and 50 amino acids, between 1 and 25 amino acids, between 5 and 100 amino acids, between 5 and 75 amino acids, between 5 and 50 amino acids, between 5 and 25 amino acids, between 10 and 100 amino acids, between 10 and 75 amino acids, between 10 and 50 amino acids, or between 10 and 25 amino acids.
  • the linker comprises the amino acid sequence of any one of SEQ ID NOs: 33 and 67-86.
  • a polynucleotide encoding any synthetic cancer antigen disclosed herein is a polynucleotide encoding any synthetic cancer antigen disclosed herein.
  • a vector comprising a polynucleotide encoding any synthetic cancer antigen disclosed herein.
  • the vector is delivered specifically to a cancer cell. In some of any of the provided embodiments, the vector is delivered specifically to a blood cancer cell. In some of any of the provided embodiments, the vector is delivered specifically to a solid tumor cancer cell.
  • the vector is a viral vector.
  • the viral vector is an oncolytic virus.
  • the oncolytic virus is selected from the group consisting of an adenovirus, a herpes simplex virus, a vaccinia virus, a mumps virus, a newcastle disease virus, a poliovirus, a seneca valley virus, a measles virus, a Sindbis virus, a parvovirus, a coxsackie virus, a vesicular stomatitis virus, a reovirus, and a maraba and rhabdovirus.
  • the viral vector exhibits tropism to a tumor cell. In some of any of the provided embodiments, the viral vector enters a cell by binding to a cell surface receptor expressed by a tumor cell. In some of any of the provided embodiments, the viral vector is an adenoviral vector, an adeno- associated virus (AAV) vector, a lentiviral vector, a retroviral vector, or a herpes simplex viral (HSV) vector. In some of any of the provided embodiments, the viral vector is an adenoviral vector. In some of any of the provided embodiments, the adenoviral vector is an adenoviral vector that binds to CD46 and/or desmoglein-2.
  • AAV adeno- associated virus
  • HSV herpes simplex viral
  • the adenoviral vector is Ad3, Ad7, Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, or Ad50. In some of any of the provided embodiments, the adenoviral vector is a chimeric adenoviral vector based on Ad3, Ad7, Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, or Ad50. In some of any of the provided embodiments, the adenoviral vector is a chimeric adenoviral vector that is based on Ad11. [0033] In some of any of the provided embodiments, the adenoviral vector is a chimeric Ad3/11p adenoviral vector.
  • the chimeric Ad3/11p adenoviral vector binds to CD46.
  • the chimeric Ad3/11p comprises a nucleic acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:41.
  • the chimeric Ad3/11p comprises the nucleic acid sequence is set forth in SEQ ID NO: 41.
  • the adenoviral vector is a chimeric adenoviral vector based on Ad5.
  • the adenoviral vector is a chimeric Ad5/3 adenoviral vector. In some of any of the provided embodiments, the chimeric Ad5/3 adenoviral vector binds to desmoglein-2.
  • a cell comprising or engineered by a vector comprising a polynucleotide encoding any synthetic cancer antigen disclosed herein.
  • a population of cells comprising at least one cell that comprises or is engineered by a vector comprising a polynucleotide encoding any synthetic cancer antigen disclosed herein.
  • a pharmaceutical composition comprising the 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.
  • the pharmaceutical composition further comprises a pharmaceutical acceptable carrier.
  • the pharmaceutical composition is for use in treating a cancer in a subject.
  • a combination comprising any vector provided herein and a population of lymphocytes expressing a chimeric antigen receptor (CAR) that recognizes the target domain of the synthetic cancer antigen.
  • CAR chimeric antigen receptor
  • the CAR comprises an extracellular binding domain comprising a cognate binder of the target domain, a transmembrane domain and an intracellular signaling domain.
  • target domain is an antibody or antibody fragment and the cognate binder binds to the idiotype of the antibody or antibody fragment.
  • the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor.
  • the target domain is an active leucine zipper domain of a transcription factor.
  • the target domain is the leucine zipper domain of ATF.
  • the target domain is the leucine zipper domain of JUN.
  • the cognate binder of the synthetic cancer antigen is a leucine zipper domain.
  • the lymphocytes comprise cytotoxic T cell. In some of any of the provided embodiments, the lymphocytes comprise natural killer cell. [0039] In some aspects, provided herein is a kit comprising any combination provided herein. In some of any of the provided embodiments, the kit further comprises instructions for using the vector and the population of lymphocytes. [0040] In some aspects, provided herein is method of treating a cancer in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein.
  • a method of treatment comprising: administering a therapeutically effective amount of any polynucleotide provided herein, any vector provided herein, or any pharmaceutical composition provided herein, to a subject having a cancer, and administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) that bind to the target domain of the synthetic cancer antigen.
  • CAR chimeric antigen receptor
  • a method of treatment comprising administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) to a subject having a cancer who was previously administered any polynucleotide provided herein, any vector provided herein, or any pharmaceutical composition provided herein, wherein the CAR binds to the target domain of the synthetic cancer antigen.
  • the lymphocytes are T cells.
  • the method comprises any vector provided herein and a population of lymphocytes expressing a chimeric antigen receptor (CAR) that recognizes the target domain of the synthetic cancer antigen.
  • the CAR comprises an extracellular binding domain comprising a cognate binder of the target domain, a transmembrane domain and an intracellular signaling domain.
  • target domain is an antibody or antibody fragment and the cognate binder binds to the idiotype of the antibody or antibody fragment.
  • the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor.
  • the target domain is an active leucine zipper domain of a transcription factor.
  • the target domain is the leucine zipper domain of ATF.
  • the target domain is the leucine zipper domain of JUN.
  • the cognate binder of the synthetic cancer antigen is a leucine zipper domain.
  • the lymphocytes comprise cytotoxic T cell. In some of any of the provided embodiments, the lymphocytes comprise natural killer cell. [0042] In some aspects, provided herein is a method of tagging a tumor cell in vivo, comprising contacting the tumor cell with any polynucleotide provided herein, any vector provided herein, or any pharmaceutical composition provided herein.
  • provided herein is a method of tagging a tumor cell of a subject having a cancer, comprising administering a therapeutically effective amount of any polynucleotide provided herein, any vector provided herein, or any pharmaceutical composition provided herein, to the subject.
  • a method of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of any pharmaceutical composition provided herein.
  • at least one of the immune effector cells expresses a CAR on the surface of the immune effector cell.
  • the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.
  • the extracellular domain of the CAR comprises the cognate binder of the synthetic cancer antigen.
  • the immune effector cell is a cytotoxic T cell. In some of any of the provided embodiments, the immune effector cell is a natural killer cell.
  • the pharmaceutical composition and the immune effector cells are administered concurrently. In some of any of the provided embodiments, the pharmaceutical composition is administered before the immune effector cells being administered.
  • the cancer is a blood cancer. In some of any of the provided embodiments, the cancer is a solid tumor cancer.
  • a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain binds to the synthetic cancer antigen provided herein.
  • the extracellular domain is a cognate binder.
  • a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 13, 14, 24, 25, 28, and 29.
  • the extracellular domain comprise the amino acid sequence set forth in SEQ ID NO:13.
  • the extracellular domain of the CAR binds to a target domain of a synthetic cancer antigen, such as any described herein.
  • the synthetic cancer antigen comprises the target domain and a membrane targeting domain.
  • the synthetic cancer antigen comprises the target domain and a tumor- targeting binding molecule.
  • the target domain is not expressed on the surface of a non-cancer cell of a subject.
  • the target domain is an antibody or a molecule derived from an antibody.
  • the target domain is an antibody without the heavy chain CH3 domain.
  • the target domain is derived from the 4D5 anti-HER2 antibody (also known as Ab1), which is the parental antibody of trastuzumab.
  • the target domain is an antibody fragment of the 4D5 (Ab1) antibody.
  • the antibody fragment is a Fab.
  • the target domain comprises a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 10.
  • the target domain comprises a light chain comprising an amino acid sequence of SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 10.
  • the target domain is a single-chain variable fragment (scFv).
  • the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 11.
  • the target domain comprises an amino acid sequence of SEQ ID NO: 11.
  • the target domain is the variable domain of the heavy chain of an antibody.
  • the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 8. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 8. [0050] In some of any of the provided embodiments, the extracellular domain of the CAR binds to the idiotype of the target domain of the synthetic cancer antigen, such as a target domain that comprises any of the antibodies derived from 4D5 (e.g., Fab set forth in SEQ ID NOS: 7 and 10, scFv set forth in SEQ ID NO: 11 or variable heavy chain set forth in SEQ ID NO:8).
  • 4D5 e.g., Fab set forth in SEQ ID NOS: 7 and 10, scFv set forth in SEQ ID NO: 11 or variable heavy chain set forth in SEQ ID NO:8.
  • the extracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 13. In some of any of the provided embodiments, the extracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 14. [0051] In some of any of the provided embodiments, the target domain is a fragment of an intracellular protein. In some of any of the provided embodiments, the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor. In some of any of the provided embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 17-19 and 21-23.
  • the target domain comprises an amino acid sequence of SEQ ID NOs: 17-19 and 21-23. In some of any of the provided embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 17. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 17. In some of any of the provided embodiments, the extracellular domain of the CAR comprises an amino acid sequence of SEQ ID NO: 24 or 25. [0052] In some of any of the provided embodiments, the target domain is an active leucine zipper domain of a transcription factor. In some of any of the provided embodiments, the target domain is the leucine zipper domain of ATF.
  • the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 26. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 26. In some of any of the provided embodiments, the target domain is the leucine zipper domain of JUN. In some of any of the provided embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 27. In some of any of the provided embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 27. In some of any of the provided embodiments, the extracellular domain of the CAR is a leucine zipper domain.
  • the extracellular domain of the CAR comprises an amino acid sequence of SEQ ID NO: 28. In some of any of the provided embodiments, the extracellular domain of the CAR comprises an amino acid sequence of SEQ ID NO: 29. In some of any of the provided embodiments, the transmembrane domain of the CAR is a CD8 transmembrane domain. In some of any of the provided embodiments, the transmembrane domain of the CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3. In some of any of the provided embodiments, the transmembrane domain of the CAR comprises an amino acid sequence of SEQ ID NO: 3.
  • the intracellular signaling domain comprises a primary intracellular signaling domain. In some of any of the provided embodiments, the intracellular signaling domain further comprises a co-stimulatory signaling domain. In some of any of the provided embodiments, the intracellular signaling domain comprises a CD3z cytoplasmic signaling domain and a 41BB co-stimulatory signaling domain. In some of any of the provided embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 16. In some of any of the provided embodiments, the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 16.
  • provided herein is a polynucleotide encoding the CAR provided herein.
  • the CAR is expressed on the surface of the cell.
  • the cell is an immune effector cell.
  • the immune effector cell is a cytotoxic T cell.
  • the immune effector cell is a natural killer cell.
  • provided herein is a population of cells comprising at least one cell provided herein.
  • a pharmaceutical composition comprising the CAR provided herein, any polynucleotide provided herein, any vector provided herein, any cell provided herein, or the population of cells provided herein.
  • the pharmaceutical composition further comprises a pharmaceutical acceptable carrier.
  • the pharmaceutical composition is for use in treating a cancer in a subject.
  • a method of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of any pharmaceutical composition provided herein.
  • the pharmaceutical composition and the additional pharmaceutical composition are administered concurrently.
  • FIG. 2A shows the design of exemplary synthetic cancer antigens (Ab1-Fab, Ab1-scFv, Ab1-V H ) based on antibodies or their derivative molecules.
  • FIG. 2B illustrates details of the construct design: from a synthetic cancer antigen with an antibody without the heavy chain CH3 domain (Ab1-Fab), to a synthetic cancer antigen with simply the scFv of Ab1 (Ab1-scFv), and then to a synthetic cancer antigen with only the variable domain of the heavy chain of Ab1 (Ab1-V H ).
  • FIG. 3A- FIG. 3C show the expression of Ab1-Fab, Ab1-scFv, and Ab1-V H on the surface of different cancer cells.
  • Ab1-Fab and Ab1-scFv were detected via a goat anti-human IgG, F(ab’)2 specific antibody (Jackson Immunoresearch, Inc) labeled with PE.
  • the expression of Ab1-V H was detected via an anti-flag antibody labeled with PE.
  • A375 malignant melanoma cell line
  • A549 lung carcinoma cell line
  • PC3 prostate cancer cell line
  • HCT-116 colorectal cancer cell line
  • SKOV-3 ovarian adenocarcinoma cell line.
  • FIG. 5A and FIG. 5B show the expansion of T cells after being transduced with one of three different CAR molecules against the idiotype of Ab1, thus against Ab1-Fab, Ab1-scFv, and Ab1-V H .
  • Ab1_CAR1 a llama single domain anti-idiotype antibody
  • Ab1_CAR2 a human anti-idiotype scFv
  • Ab1_CAR3 another human anti-idiotype scFv.
  • FIG. 6A shows that the expression of Ab1_CAR1 and Ab1_CAR2, but not Ab1_CAR3, was detected on respectively transduced CAR-T cells.
  • FIG. 6B- FIG. 6C show the expression of Ab1_CAR1 and Ab1_CAR2 after the respective lentivirus has been titrated. UTD: un-transduced.
  • FIG. 7A- FIG. 7E show the killing of different Ab1-Fab-expressing cancer cells by Ab1_CAR1-expressing T cells from three donors.
  • FIG. 8A- FIG. 8E show the killing of different Ab1-Fab-expressing cancer cells by Ab1_CAR2-expressing T cells from three donors.
  • A375 malignant melanoma cell line; A549: lung carcinoma cell line; PC3: prostate cancer cell line; HCT-116: colorectal cancer cell line; SKOV-3: ovarian adenocarcinoma cell line.
  • FIG. 8A- FIG. 8E show the killing of different Ab1-Fab-expressing cancer cells by Ab1_CAR2-expressing T cells from three donors.
  • A375 malignant melanoma cell line
  • A549 lung carcinoma cell line
  • PC3 prostate cancer cell line
  • HCT-116 colorectal cancer cell line
  • SKOV-3 ovarian adenocarcinoma cell line.
  • FIG. 9A shows the killing of Ab1-scFv-expressing A549 cancer cells by T cells from one donor expressing either Ab1_CAR1 or Ab1_CAR2.
  • FIG. 9B – FIG. 9C shows the killing of different Ab1-scFv-expressing cancer cells by T cells from another donor expressing Ab1_CAR2.
  • FIG. 10A – FIG. 10B show the killing of different Ab1-V H -expressing cancer cells by T cells expressing Ab1_CAR2.
  • A549 lung carcinoma cell line
  • PC3 prostate cancer cell line
  • HCT-116 colorectal cancer cell line
  • SKOV-3 ovarian adenocarcinoma cell line.
  • FIG. 12A shows the expression of Ab1-Fab via adenovirus transduction.
  • FIG. 12C shows the killing of different Ab1-Fab-expressing cancer cells by T cells from different donors expressing either Ab1_CAR1 or Ab1_CAR2, where the Ab1-Fab constructs were transduced into cancer cells using adenovirus instead of lentivirus as shown in FIG. 7A – FIG.8E.
  • FIG. 12D – FIG. 12E shows the killing of different Ab1-V H - expressing cancer cells by T cells expressing Ab1_CAR2, where the Ab1-V H constructs were transduced into cancer cells using adenovirus instead of lentivirus as shown in FIG. 10A – FIG. 10B.
  • FIG. 10A – FIG. 10B shows the killing of different Ab1-Fab-expressing cancer cells by T cells from different donors expressing either Ab1_CAR1 or Ab1_CAR2, where the Ab1-Fab constructs were transduced into cancer cells using adenovirus instead of lentivirus as shown in FIG. 7A – FIG.8E.
  • FIG. 12F shows the killing of Ab1-V H -expressing cancer cells by T cells from expressing Ab1_CAR1, where the Ab1-V H constructs were transduced into cancer cells using the chimeric adenovirus Ad3/Ad11p.
  • A549 lung carcinoma cell line
  • PC3 prostate cancer cell line
  • HCT-116 colorectal cancer cell line
  • SKOV-3 ovarian adenocarcinoma cell line.
  • UTD un-transduced.
  • FIG. 13A is a schematic illustration of testing two synthetic cancer antigens (Ab1-V H and Ab1-Fab) and their cognate binders in vivo.
  • FIG. 13C show the probability of survival and the tumor volume of mice carrying HER2-positive HCT-116 tumor cells expressing Ab1-V H treated with T cells expressing Ab1_CAR2 at different concentrations.
  • T cells expressing anti-HER2 (4D5) CAR were included as a control.
  • FIG. 13D shows the expansion of T cells expressing either Ab1_CAR2 or anti-HER2 (4D5) CAR.
  • FIG. 13E shows the tumor volume of mice carrying HER2-positive HCT-116 cells expressing Ab1-Fab treated with T cells expressing Ab1_CAR1 at different concentrations.
  • FIG. 14 is a schematic illustration of delivering an exemplary synthetic cancer antigen, a protein fragment, into a cancer cell through a gene delivery vector, such as an oncolytic virus.
  • FIG. 15A- FIG. 15B show the expression of different protein fragment candidates on the surface of 293T cells.
  • FIG. 16A shows the expression of IC1 protein fragment on the surface of different cancer cells.
  • FIG. 16B shows the expression of a binder against IC1 on the surface of T cells.
  • FIG. 17A- FIG. 17C shows the killing of different cancer cells expressing IC1 by T cells expressing binders against IC1.
  • FIG. 18 is a schematic illustration of delivering an exemplary synthetic cancer antigen, the leucine zipper domain of transcription factor 1(TF1), into a cancer cell through a gene delivery vector, such as an oncolytic virus.
  • a T-cell expressing binding partner of the leucine zipper domain of transcription factor 1 e.g., the leucine zipper domain of transcription factor 2, TF2
  • FIG. 19 shows the construct design of exemplary synthetic cancer antigens (TF2- LZ and TF4-LZ) and their corresponding cognate binders (TF1-LZ and TF3-LZ).
  • FIG. 20A shows the expression of TF2-LZ and TF4-LZ on the surface of cancer cells.
  • FIG. 20A shows the expression of TF2-LZ and TF4-LZ on the surface of cancer cells.
  • FIG. 20B shows the expression of TF1-LZ (binder of TF2-LZ) on the surface of T cells.
  • FIG. 20C shows the expression of TF3-LZ (binder of TF4-LZ) on the surface of T cells.
  • FIG. 21 is a schematic illustration of testing the binding effectiveness of the soluble form of the Ab1-V H (FL1)-anti-EpCAM conjugate on tumor cells.
  • FIG. 22 shows the surface binding expression of the Flare1 after co-culture with supernatant containing the soluble form of the conjugate or the tethered form of the Ab1-V H .
  • FIG. 21 is a schematic illustration of testing the binding effectiveness of the soluble form of the Ab1-V H (FL1)-anti-EpCAM conjugate on tumor cells.
  • FIG. 22 shows the surface binding expression of the Flare1 after co-culture with supernatant containing the soluble form of the conjugate or the tethered form of the Ab1-V H .
  • FIG. 23A is a schematic illustration of testing the soluble form of the Ab1-V H (FL1)-anti-EpCAM conjugate for targeted killing of tumor cells.
  • FIG. 23B the killing of different Ab1-V H surface bound cancer cells by T cells expressing Ab1_CAR2, where the Ab1-V H constructs were bound to the cancer cells by conjugation to the anti-EpCAM antibody.
  • FIG. 24A is a schematic illustration of testing the soluble synthetic cancer antigen (Ab1-V H (FL1)-anti-EpCAM conjugate) and their cognate binders in vivo.
  • FIG. 24B shows the percentage of FL1+ tumor cells after mice received doses of virus encoding the tethered or soluble forms of the Ab1-V H .
  • FIG. 24A is a schematic illustration of testing the soluble form of the Ab1-V H (FL1)-anti-EpCAM conjugate for targeted killing of tumor cells.
  • FIG. 23B the killing of different Ab1-V H surface bound cancer cells by T
  • FIG. 24C shows the number of infiltrating T cells in the tumor after treatment with virus encoding the tethered or soluble forms of the Ab1-V H and subsequent intravenous injections of CAR-T cells.
  • FIG. 24D is a schematic illustration of testing the soluble synthetic cancer antigen (Ab1-V H (FL1)-anti-EpCAM conjugate) and their cognate binders in vivo over time.
  • FIG. 24E shows the tumor growth in mice after treatment with virus encoding the Ab1-V H (FL1)-anti-EpCAM conjugate and CAR-T cells.
  • FIG. 25A shows the surface binding expression of the soluble FL1-anti-HER2 synthetic cancer antigen on HER2-expressing SKOV3 cancer cells.
  • FIG. 25B – FIG. 25C shows the killing of different HER2-expressing cancer cells by CAR T cells after culture with the soluble FL1-anti-HER2 synthetic cancer antigen.
  • FIG. 26 left panel shows the surface binding expression of the Ab1-V H (FL1) on A549 cancer cells infected with a virus encoding both the tethered synthetic cancer antigen and the soluble FL1-anti-EpCAM synthetic cancer antigen.
  • FIG. 26, right panel shows the surface binding expression of the FL1 on non-transduced acceptor cancer cells after co- culture with supernatant containing the soluble form of the Ab1-V H conjugate.
  • a CAR therapy including a cell, e.g., a population of immune effector cells, including (e.g., expressing) a chimeric antigen receptor (CAR) for use in combination with synthetic cancer antigen.
  • CAR chimeric antigen receptor
  • the viral vector is a tumor-tropic vector that is able to specifically target and express the synthetic cancer antigen in tumor cells.
  • the provided embodiments permit tumor specific gene delivery in which the synthetic cancer antigen is able to selectively tag tumor cells of various types with a targetable antigen.
  • the synthetic cancer antigen is expressed as a membrane protein, such as containing a transmembrane domain.
  • the present disclosure provides synthetic cancer antigens and/or cognate binders thereof, where each of the synthetic cancer antigens comprises a target domain and a transmembrane domain.
  • the synthetic cancer antigen is expressed as a soluble protein that is secretable from cells in which it is expressed.
  • a soluble target domain is linked to a tumor-targeting molecule able to bind to a tumor antigen to tag cancer cells.
  • the synthetic cancer antigen comprising the target domain is delivered only to the tumor cells.
  • the synthetic cancer antigen comprising the target domain is not expressed on the surface of a non-cancer cell of a subject.
  • the present disclosure also provides polynucleotides encoding the synthetic cancer antigens disclosed herein, vectors comprising such polynucleotides, and cells comprising or engineered by such vectors.
  • polynucleotides and/or viral vectors encode both a chimeric synthetic cancer antigen membrane protein and a secretable synthetic cancer antigen.
  • the present disclosure also provides pharmaceutical compositions comprising the synthetic cancer antigens, polynucleotides, or vectors disclosed herein and the methods of using such pharmaceutical compositions for treating cancers.
  • CAR activation directed against i.e. contains an extracellular domain that recognizes or binds
  • the synthetic cancer antigen initiates cytotoxic killing by the CAR T cells against the synthetic cancer antigen-expressing tumor cells.
  • administer refers to the act of injecting or otherwise physically delivering a substance (e.g., a pharmaceutical composition provided herein) to a subject (e.g., human), such as by oral, mucosal, topical, intradermal, parenteral, intravenous, intravitreal, intraarticular, subretinal, intramuscular, intrathecal delivery and/or any other method of physical delivery described herein or known in the art.
  • a substance e.g., a pharmaceutical composition provided herein
  • a subject e.g., human
  • the delivery can be systemic or to a specific tissue.
  • antibody immunoglobulin
  • Ig immunoglobulin
  • monoclonal antibodies including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies
  • antibody compositions with polyepitopic or monoepitopic specificity polyclonal or monovalent antibodies
  • multivalent antibodies multispecific antibodies (e.g ., bispecific antibodies so long as they exhibit the desired biological activity).
  • An antibody can be human, humanized, chimeric and/or affinity matured, as well as an antibody from other species, for example, mouse and rabbit, etc.
  • Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies or their humanized variants, and intrabodies.
  • a “molecule derived from an antibody” refers to a functional antigen-binding fragment of any of the above. It is a portion of an antibody heavy and/or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • Non-limiting examples of functional fragments include single- chain Fvs (scFv), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody.
  • scFv single-chain Fvs
  • Fab fragments fragments
  • F(ab’) fragments fragments
  • F(ab)2 fragments F(ab’)2 fragments
  • dsFv disulfide-linked Fvs
  • the antibodies and molecules derived from antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.
  • idiotype refers to the unique set of antigenic epitopes that the variable portion of an antibody recognizes.
  • An anti-idiotype antibody is an antibody that specifically binds to the antigen binding site of another antibody (e.g., the antigen-binding domain of the antibody).
  • chimeric antigen receptor refers to a genetically engineered receptor, which can be used to graft one or more antigen specificity onto immune effector cells, such as T cells and NK cells. CARs are also known as “artificial T-cell receptors,” “chimeric T cell receptors,” or “chimeric immune receptors.” In some embodiments, the CAR comprises an extracellular antigen binding domain specific for one or more synthetic cancer antigens disclosed herein, a transmembrane domain, and an intracellular signaling domain of a T cell and/or other receptors. “CAR-T cell” refers to a T cell that expresses a CAR.
  • CAR-NK cell refers to an NK cell that expresses a CAR.
  • coding sequence or a polynucleotide which “encodes” a polypeptide, as used herein, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus.
  • a transcription termination sequence may be located 3’ to the coding sequence.
  • constant region refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. This portion has a more conserved amino acid sequence relative to the variable region.
  • the constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.
  • an effective amount refers to an amount of a therapeutic (e.g., a pharmaceutical composition provided herein) which is sufficient to treat, diagnose, prevent, delay the onset of, reduce and/or ameliorate the severity and/or duration of a given condition, disorder or disease and/or a symptom related thereto.
  • the term also encompasses an amount necessary for the reduction, slowing, or amelioration of the advancement or progression of a given disease, reduction, slowing, or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect (s) of another therapy or to serve as a bridge to another therapy.
  • Fab refers to an antibody region that binds to antigens.
  • a conventional IgG usually comprises two Fab regions, each residing on one of the two arms of the Y-shaped IgG structure.
  • Each Fab region is typically composed of one variable region and one constant region of each of the heavy and the light chain. More specifically, the variable region and the constant region of the heavy chain in a Fab region are VH and CH1 regions, and the variable region and the constant region of the light chain in a Fab region are VL and CL regions.
  • the VH, CH1, VL, and CL in a Fab region can be arranged in various ways to confer an antigen binding capability.
  • a fragment of a polypeptide or polynucleotide comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire length of the reference polypeptide or polynucleotide. In some embodiments, a fragment of a polypeptide or polynucleotide comprises about 10%-99%, 20%-99%, 30%- 99%, 40%-99%, 50%-99%, 60%-99%, 70%-99%, 80%-99%, 90%--99%, 95%--99%, 96%-- 99%, 97%--99%, or 98%-99%, of the entire length of the reference polypeptide or polynucleotide.
  • a polypeptide or polynucleotide fragment may contain 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more nucleotides or amino acids.
  • the constant region can be one of five distinct types, (e.g, isotypes) referred to as alpha, delta, epsilon, gamma, and mu, based on the amino acid sequence of the heavy chain constant region.
  • the distinct heavy chains differ in size: alpha, delta, and gamma contain approximately 450 amino acids, while epsilon and mu contain approximately 550 amino acids.
  • these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3, and IgG4.
  • the approximate length of a light chain is 211 to 217 amino acids.
  • the leucine zipper domain is an active leucine zipper domain. On dimerization with another leucine zipper domain, the leucine-zipper ⁇ helices form a parallel-coiled coil based on hydrophobic interfacial side-chain packing. In some embodiments, the leucine zipper domain is an inactive leucine zipper domain that cannot form a dimer with another leucine zipper domain.
  • a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is naturally occurring.
  • Such a substance can be included for the purpose of long- term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating absorption, reducing viscosity, or enhancing solubility.
  • the selection of appropriate substance can depend upon the route of administration and the dosage form, as well as the active ingredient and other factors.
  • Compositions having such substances can be formulated by well-known conventional methods (see, e.g., Remington, The Science and Practice of Pharmacy, 23rd edition, A. Adejare, ed., Academic Press, 2020).
  • composition refers to a composition capable of being administered to a subject for the treatment of a particular disease or disorder.
  • polynucleotide or “nucleic acid”, as used herein, refers to polymers of nucleotides of any length and includes DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • the left-hand end of any single- stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction.
  • the direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction.
  • the terms “polypeptide” and “peptide” and “protein”, as used herein, refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art.
  • the term “population” of cells refers to any number of cells greater than 1, but is preferably at least 1x10 3 cells, at least 1x10 4 cells, at least 1x10 5 cells, at least 1x10 6 cells, at least 1x10 7 cells, at least 1x10 8 cells, at least 1x10 9 cells, at least 1x10 10 cells, at least 1x10 11 or more cells.
  • a population of cells may refer to an in vitro population (e.g., a population of cells in culture) or an in vivo population (e.g., a population of cells residing in a particular tissue).
  • sequence identity refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared.
  • reference sequence refers to a molecule to which a test sequence is compared. Methods of sequence alignment for comparison and determination of percent sequence identity and percent complementarity are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat’l. Acad. Sci.
  • the term “subject”, as used herein, refers to a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkey and human).
  • the subject is a mammal, e.g., a human, diagnosed with a disease or disorder provided herein.
  • the subject is a mammal, e.g., a human, at risk of developing a disease or disorder provided herein.
  • the subject is human.
  • synthetic refers to a nucleic acid, polypeptide, cell, or organism that cannot be directly isolated from a source in nature.
  • the synthetic nucleic acid, polypeptide, cell, or organism is altered or changed as compared to the corresponding naturally-occurring one.
  • the synthetic nucleic acid, polypeptide, cell, or organism is produced by functionally linking or combining different fragments of sourcing nucleic acids, polypeptides, cells, or organisms together.
  • a synthetic polypeptide can comprise different sourcing polypeptides functionally linked together.
  • target domain refers to the extracellular domain of the synthetic cancer antigen. It can be expressed on the surface of a cancer cell and be recognized by a cognate binder. It can be bound by a chimeric antigen receptor comprising the cognate binder and expressed on the surface of a cancer cell.
  • transduced refers to a process by which a transgene is introduced into a host cell from a virus particle.
  • transgene refers to any heterologous polynucleotide incorporated in a viral vector, e.g., for transcription or expression in a target cell.
  • An example of a transgene is a polynucleotide encoding a therapeutic polypeptide or a detectable marker.
  • transmembrane domain refers to any protein structure that is thermodynamically stable in a cell membrane (e.g. an eukaryotic cell membrane).
  • the transmembrane domain may be derived either from a natural or from a synthetic source.
  • treatment refers to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient.
  • beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated.
  • a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • a prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
  • variable region refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen.
  • the variable region of the heavy chain may be referred to as “VH.”
  • the variable region of the light chain may be referred to as “VL.”
  • variable refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen.
  • variable regions consist of less variable (e.g ., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” or “complementarity determining regions” that are each about 9-12 amino acids long.
  • FRs framework regions
  • hypervariable regions or “complementarity determining regions” that are each about 9-12 amino acids long.
  • the variable regions of heavy and light chains each comprise four FRs, largely adopting a ⁇ sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the ⁇ sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen- binding site of antibodies (see, e.g., Rabat et al, Sequences of Proteins of Immunological Interest (5th ed. 1991)).
  • viral vector refers to a nucleic acid to be delivered into a host cell via a recombinantly produced virus or viral particle.
  • the nucleic acid may be single-stranded or double stranded, linear or circular, segmented or non-segmented.
  • the nucleic acid may comprise DNA, RNA, or a combination thereof.
  • viruses or viral particles that can deliver a viral vector include retroviruses (e.g., lentiviruses and ⁇ -retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses.
  • retroviruses e.g., lentiviruses and ⁇ -retroviruses
  • adenoviruses e.g., lentiviruses and ⁇ -retroviruses
  • AAVs adeno-associated viruses
  • a viral vector delivered by such viruses or viral particles may be referred to by the type of virus to deliver the viral vector (e.g., a lentiviral vector is a viral vector that is to be delivered by a lentivirus).
  • a viral vector can contain viral elements (e.g., nucleotide sequences) necessary for packaging of the viral vector into the virus or viral particle, replicating the virus, or other desired viral activities.
  • a virus containing a viral vector may be replication competent, replication deficient or replication defective.
  • the viral vector is delivered via an oncolytic virus.
  • synthetic cancer antigens and/or cognate binders thereof are not normally expressed on the surface of non-cancer cells and can be specifically delivered to cancer cells in a subject via oncolytic viruses, and thus only expressed on the surface of cancer cells.
  • the cognate binders can be engineered as chimeric-antigen-receptors for expressing on the surface of immune effector cells.
  • the present disclosure re-designs the interaction between the immune effector cells and the cancer targets. By shifting away from the traditional approach of targeting known cancer-overexpressed proteins, which can also express on the surface of non-cancer cells, the present disclosure improves the safety of immune cell therapy by eliminating recognition of healthy tissues.
  • the synthetic cancer antigen comprises a target domain and a transmembrane domain. In some embodiments, the synthetic cancer antigen is membrane-bound. In other embodiments, the synthetic cancer antigen comprises a target domain linked to a tumor-targeting binding molecule. In some embodiments, the synthetic cancer antigen is soluble. In some embodiments, one or more formats (e.g., membrane bound and/or soluble) are recognizable by a cognate binder. Both or either of the formats of the synthetic cancer antigen can be used in combination with one or more cognate binders. In some embodiments, the target domain of the synthetic cancer antigen is recognizable by a cognate binder.
  • the target domain of the synthetic cancer antigen is a target domain that is a protein that is recognizable by a cognate binder to be specifically targetable to tag cancer cells.
  • the target domain of synthetic cancer antigens provided herein is designed to be different from proteins expressed on the surface of a non-cancer cell of a subject. In some embodiments, the target domain is not expressed on the surface of a non-cancer cell of a subject. In some embodiments, the target domain is recognizable by a cognate binder.
  • the target domain of synthetic cancer antigens provided herein can be any polypeptide that is not expressed on the surface of a non-cancer cell of a subject.
  • Binding of a cognate binder (for example, an scFv) to a target domain can be confirmed by, for example, enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g. , growth inhibition), or Western Blot assay.
  • the target domain is a protein binding molecule.
  • the size of the target domain is about 20 amino acids to about 700 amino acids. In some embodiments, the size of the target domain is at least about 20 amino acids. In some embodiments, the size of the target domain is at most about 700 amino acids.
  • the size of the target domain is about 20 amino acids to about 40 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 250 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 700 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 250 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 700 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 150 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 250
  • the size of the target domain is about 20 amino acids, about 40 amino acids, about 60 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, or about 700 amino acids.
  • the target domain is an antibody or antibody fragment.
  • the target domain is a molecule derived from an antibody.
  • a molecule derived from an antibody is one or more functional fragments of the antibody.
  • Non-limiting examples of functional fragments include heavy chain, light chain, heavy chain variable domain, light chain variable domain, single-chain Fvs (scFv), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody.
  • the target domain is an antibody without the heavy chain CH3 domain.
  • the target domain is an antibody without the heavy chain CH2 domain.
  • the target domain is a Fab.
  • the target domain is an antibody without the heavy chain CH2 and CH3 domain.
  • the target domain is an antibody fragment, such as a Fab, an scFv or a variable heavy chain only fragment. In some embodiments, the target domain is a single-chain variable fragment (scFv). In some embodiments, the target domain is the heavy chain of an antibody. In some embodiments, the target domain is the light chain of an antibody. In some embodiments, the target domain is the variable domain of the heavy chain of an antibody. In some embodiments, the target domain is the variable domain of the light chain of an antibody. [0131] In some embodiments, the functional fragments are part of a single polypeptide and are operatively linked. In some embodiments, the functional fragments are directly linked to each other.
  • the functional fragments are linked via linkers. Exemplary linkers can be used herein are disclosed in the Example Section of the present application. Additional linkers are known to a skilled artisan.
  • the functional fragments are separate polypeptides and bind with each other after being translated to form a molecule derived from an antibody.
  • the functional fragments are translated from multiple polynucleotides.
  • the functional fragments are translated from a single polynucleotide.
  • the single polynucleotide comprises nucleotide sequences encoding self-cleaving peptides.
  • the self-cleaving peptides separate the functional fragments translated from a single polynucleotide.
  • the self-cleaving peptide is a P2A peptide.
  • the self-cleaving peptide comprises an amino acid sequence of SEQ ID NO: 4.
  • the antibody or the molecule derived from an antibody provided herein comprises VL, VH or CDRs having amino acid sequences of the VL, VH or CDR contained in certain known antibodies.
  • the antibody or the molecule derived from an antibody provided herein comprises VL, VH or CDRs having amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91/%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the VL, VH or CDR contained in certain known antibodies.
  • Exemplary known antibodies include but not limited to ReoPro (abciximab), Humira (adalimumab), Hyrimoz (adalimumab-adaz), Cyltezo (adalimumab-adbm), Abrilada (adalimumab-afzb), Amjevita (adalimumab-atto), Hadlima (adalimumab-bwwd), Campath, Lemtrada (alemtuzumab), Praluent (alirocumab), Tecentriq (atezolizumab), Bavencio (avelumab), Simulect (basiliximab), Benlysta (belimumab), Benlysta (belimumab), Fasenra (benralizumab), Avastin (bevacizumab), Mvasi (bevacizumab-awwb), Zirabev
  • the target domain is an antibody fragment of any of the above antibodies. In some embodiments, the target domain is a molecule derived from any of the above antibodies. In some embodiments, a molecule derived from an antibody is one or more fragments of the antibody. Non-limiting examples of fragments include heavy chain, light chain, heavy chain variable domain, light chain variable domain, single-chain Fvs (scFv), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide- linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody.
  • scFv single-chain Fvs
  • the target domain is an antibody without the heavy chain CH3 domain. In some embodiments, the target domain is an antibody without the heavy chain CH2 domain. In some embodiments, the target domain is a Fab. In some embodiments, the target domain is an antibody without the heavy chain CH2 and CH3 domain. [0135] In some embodiments, the target domain is a single-chain variable fragment (scFv) of any of the above antibodies. In some embodiments, the target domain is the heavy chain of any of the above antibodies. In some embodiments, the target domain is the light chain of any of the above antibodies.
  • scFv single-chain variable fragment
  • the target domain is a fragment that does not bind to the target of the antibody but that retains an epitope to be recognized by a cognate binder (e.g., anti-idiotypic antibody).
  • the target domain is the variable heavy chain (VH) of an antibody.
  • VH variable heavy chain
  • the target domain is devoid of the variable light (VL) chain.
  • the target domain is not able to bind or substantially bind or exhibits reduced (low) binding affinity (e.g., a higher KD value) to the antigen of the antibody from which it is derived.
  • an antibody fragment as a target domain that does not bind or substantially bind to a target antigen of the antibody or has low affinity binding for a target antigen of the antibody is that it allows the target domain to be delivered in a soluble format (e.g., as described in Section II.A.C) while limiting the possibility of the target domain binding to the target antigen of the full antibody.
  • the target domain is an antibody fragment in which the antigen-binding domain for recognition of an antigen or epitope is not present or is partially removed or is non-functional (e.g., via one or more amino acid substitutions in a CDR).
  • the parental antibody is a two chain antibody containing a variable heavy and variable light chain in which the antigen-binding domain is composed of 6 CDRs.
  • the target domain is an antibody fragment that is the variable heavy chain (VH) only composed of only 3 CDRs.
  • VH variable heavy chain
  • the target domain is an antibody or antibody fragment that is able to be recognized by a cognate binder that is an anti-idiotype antibody or antigen- binding fragment.
  • Various anti-idiotype antibodies to antibodies are known. Exemplary anti- idiotype antibodies are described in Section II.D.1.
  • the antibody or antibody fragment target domain such as a variable heavy chain, contains an epitope recognized by a cognate binder (e.g., anti- idiotype antibody) but does not specifically bind to a target antigen of a native or primary cells, such as a tumor target antigen.
  • a cognate binder e.g., anti- idiotype antibody
  • the cognate binder is an anti- idiotype antibody (e.g., such as any described in Section II.D.1) and the variable heavy chain contains an idiotype of the antibody.
  • the target domain is an antibody or antibody fragment that has reduced binding for the target antigen of the antibody compared to the parental antibody from which it has been derived.
  • the target domain is an antibody or antibody fragment that has low binding affinity for a target antigen of a parental antibody from which it was derived.
  • dissociation constant (Kd) of the antibody or antibody fragment target domain for binding a target antigen of an antibody from which it is derived is Kd greater than 10 -7 (> 100 nM), such as Kd greater than 10 -6 (> 1 microM), 10 -5 (> 10 microM) or 10 -4 (> 100 microM).
  • the Kd of the antibody or antibody fragment target domain for binding a target antigen of an antibody from which it is derived is greater than 10 -4 (> 100 microM).
  • Exemplary binding assays for determining binding affinity include, but are not limited to, enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, Western Blot assay, or surface plasmon resonance (e.g., Biacore).
  • ELISA enzyme- linked immunosorbent assay
  • RIA radioimmunoassay
  • flow cytometry Western Blot assay
  • surface plasmon resonance e.g., Biacore
  • the antibody or antibody fragment target domain does not exhibit detectable binding to the target antigen of the antibody from which it is derived with a cell expressing the target antigen of the antibody, such as determined by a binding assay for example by flow cytometry.
  • the target domain is an antibody directed against HER2.
  • the target domain is derived from the 4D5 anti-HER2 antibody (also known as Ab1), which is the parental antibody of trastuzumab. In some embodiments, the target domain is an antibody fragment of the 4D5 (Ab1) antibody. [0140] In some embodiments, the target domain is a 4D5 antibody or an antigen-binding fragment of 4D5 antibody. In some embodiments, the target domain is an antibody fragment of a 4D5 antibody. In some embodiments, the target domain is a molecule derived from a 4D5 antibody. In some embodiments, a molecule derived from a 4D5 antibody is one or more fragments of the antibody. In some embodiments, the target domain is an Fab fragment of a 4D5 antibody.
  • the target domain is a single-chain Fvs (scFv) of a 4D5 antibody. In some embodiments, the target domain does not comprise a constant heavy chain and/or a constant light chain domain. In some embodiments, the target domain comprises the variable light chain and the variable heavy chain of a 4D5 antibody. In some embodiments, the target domain comprises the variable heavy chain of the 4D5 antibody and does not comprise the variable light chain. In some embodiments, the target domain comprises the variable light chain of the 4D5 antibody and does not comprise the variable heavy chain. [0141] In some embodiments, the target domain is a Fab of a 4D5 antibody. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence of SEQ ID NO: 7.
  • the target domain comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 10.
  • the target domain comprises a light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7.
  • the target domain comprises a heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10.
  • the target domain comprises a light chain comprising an amino acid sequence of SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 10. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 10. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 10.
  • the target domain comprises a light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 10. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 10. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 10.
  • the target domain comprises a light chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 10. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 10. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 10.
  • the target domain comprises a light chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 10. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 10. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 10.
  • the target domain comprises a light chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 10. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 10. In some embodiments, the target domain comprises a light chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 10.
  • the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 11. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 11. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 11. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 11. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 11. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 11. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 11.
  • the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 11. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 11. [0144] In some embodiments, the target domain is a variable heavy chain of a 4D5 antibody. In some embodiments, the target domain does not comprise a variable light chain. In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 8. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 8.
  • the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 8. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 8. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 8. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 8. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 8. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 8.
  • the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 8. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 8. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 8. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 8. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 8. In provided embodiments, such a variable heavy chain antibody fragment contains an idiotype recognized by an anti-idiotype antibody (e.g., set forth in any of SEQ ID NOS: 13-15) but does not specifically bind to HER2.
  • an anti-idiotype antibody e.g., set forth in any of SEQ ID NOS: 13-15
  • variable heavy chain antibody fragment has reduced binding for HER2 compared to the 4D5 (Ab1) parental antibody from which it has been derived. In some embodiments, the variable heavy chain antibody fragment has low binding affinity for HER2.
  • dissociation constant (Kd) of the variable heavy chain antibody fragment target domain for binding HER2 is Kd greater than 10 -7 (> 100 nM), such as Kd greater than 10 -6 (> 1 microM), 10 -5 (> 10 microM) or 10 -4 (> 100 microM). In some embodiments, the Kd of the variable heavy chain antibody fragment for binding HER2 is greater than 10 -4 (> 100 microM).
  • Exemplary binding assays for determining binding affinity include, but are not limited to, enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, Western Blot assay, or surface plasmon resonance (e.g., Biacore).
  • ELISA enzyme- linked immunosorbent assay
  • RIA radioimmunoassay
  • flow cytometry Western Blot assay
  • surface plasmon resonance e.g., Biacore
  • the variable heavy chain antibody fragment target domain does not exhibit detectable binding to a cell expressing HER2, for example a tumor cell, such as determined by a binding assay for example by flow cytometry.
  • the target domain is a fragment of an intracellular protein.
  • the target domain is a non-DNA binding domain of a transcription factor.
  • the target domain is an inactive leucine zipper domain of a transcription factor. In some embodiments, the target domain is a N-terminal fragment of a transcription factor. In some embodiments, the target domain is a C-terminal fragment of a transcription factor.
  • transcription factors include cFos, cJun, cMAF, ATF, Nfil3, and Xbp1.
  • the target domain comprises an amino acid sequence of SEQ ID NO: 17. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 17. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 17.
  • the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 17. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 17. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 17. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 17. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 17. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 17. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 17.
  • the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 17. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 17. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 17. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 17. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 17. [0147] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 18. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 18.
  • the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 18. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 18. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 18. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 18. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 18. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 18. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 18.
  • the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 18. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 18. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 18. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 18. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 18. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 18. [0148] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 19.
  • the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 19. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 19. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 19. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 19. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 19. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 19.
  • the target domain comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 20. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 20. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 20. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 20. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 20. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 20.
  • the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 20. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 20. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 20. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 20. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 20. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 20.
  • the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 20. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 20. [0150] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 21. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 21. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 21. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 21. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 21.
  • the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 21. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 21. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 21. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 21. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 21. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21.
  • the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 21. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 21. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 21. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 21. [0151] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 22. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 22. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 22.
  • the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 22. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 22. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 22. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 22. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 22. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 22.
  • the target domain comprises an amino acid sequence of SEQ ID NO: 23. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 23. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 23. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 23. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 23. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 23. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 23.
  • the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 23. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 23. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 23. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 23. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 23. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 23.
  • the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 23. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 23. 3.
  • Leucine Zipper [0153] In some embodiments, the target domain is an active leucine zipper domain of a transcription factor. In some embodiments, the transcription factor is a basic leucine zippers (bZIP). Non-limiting examples of bZIP transcription factors that can be used herein include ATF, JUN, CREB, Fos. In some embodiments, the target domain is the leucine zipper domain of ATF. In some embodiments, the target domain is the leucine zipper domain of JUN.
  • the target domain is the leucine zipper domain of CREB. In some embodiments, the target domain is the leucine zipper domain of Fos. [0154] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 26. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 26. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 26. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 26. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 26.
  • the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 26. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 26. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 26. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 26. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 26.
  • the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 26. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 26. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 26. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 26. [0155] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 27. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 27.
  • the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 27. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 27. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 27. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 27. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 27. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 27.
  • the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 27. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 27. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 27. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 27. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 27. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 27.
  • the target domain comprises an amino acid sequence of SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 28.
  • the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 28.
  • the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 28. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 28. [0157] In some embodiments, the target domain comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the target domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 29. In some embodiments, the target domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 29. In some embodiments, the target domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 29. In some embodiments, the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 29.
  • the target domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29. In some embodiments, the target domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 29. In some embodiments, the target domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 29. In some embodiments, the target domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 29. In some embodiments, the target domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 29. In some embodiments, the target domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 29.
  • the target domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 29. In some embodiments, the target domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 29. In some embodiments, the target domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 29. In some embodiments, the target domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 29.
  • B. MEMBRANE TARGETING DOMAIN [0158]
  • the synthetic cancer antigen is a membrane bound protein and the target domain is linked to a membrane targeting domain. In some embodiments, the synthetic cancer antigen comprises a target domain and a membrane targeting domain.
  • the target domain can be any as described in Section II.A.
  • the membrane targeting domain of synthetic cancer antigens provided herein can be the transmembrane domain of any known transmembrane protein or from a synthetic source.
  • the transmembrane domains disclosed herein for synthetic cancer antigens can also be used as the transmembrane domain of CAR molecules disclosed below.
  • Transmembrane domains are classified based on the three dimensional structure of the transmembrane domain. For example, transmembrane domains may form an alpha helix, a complex of more than one alpha helix, a beta-barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell.
  • transmembrane domains may also or alternatively be classified based on the transmembrane domain topology, including the number of passes that the transmembrane domain makes across the membrane and the orientation of the protein. For example, single-pass membrane proteins cross the cell membrane once, and multipass membrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 or more times).
  • Membrane proteins may be defined as Type I, Type II or Type III depending upon the topology of their termini and membrane-passing segment(s) relative to the inside and outside of the cell.
  • Type I membrane proteins have a single membrane-spanning region and are oriented such that the N-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is present on the cytoplasmic side.
  • Type II membrane proteins also have a single membrane- spanning region but are oriented such that the C-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the N-terminus of the protein is present on the cytoplasmic side.
  • Type III membrane proteins have multiple membrane- spanning segments and may be further sub-classified based on the number of transmembrane segments and the location of N- and C-termini.
  • the transmembrane domain is derived from a Type I single-pass membrane protein.
  • transmembrane domains from multi- pass membrane proteins may also be compatible.
  • Multi-pass membrane proteins may comprise a complex (at least 2, 3, 4, 5, 6, 7 or more) alpha helices or a beta sheet structure.
  • the N-terminus and the C-terminus of a multi-pass membrane protein are present on opposing sides of the lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side.
  • Transmembrane domains can also comprise at least a portion of a synthetic, non- naturally occurring protein segment.
  • the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet.
  • the protein segment is about 15-100 amino acids.
  • the protein segment is at least approximately 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in U.S. Pat. No. 7,052,906 and PCT Publication No. WO 2000/032776, the relevant disclosures of which are incorporated by reference herein.
  • the transmembrane domain provided herein may comprise a transmembrane region and a cytoplasmic region located at the C-terminal side of the transmembrane domain.
  • the cytoplasmic region of the transmembrane domain may comprise three or more amino acids and, in some embodiments, helps to orient the transmembrane domain in the lipid bilayer.
  • one or more cysteine residues are present in the transmembrane region of the transmembrane domain.
  • one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain.
  • the cytoplasmic region of the transmembrane domain comprises positively charged amino acids.
  • the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.
  • the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues.
  • the transmembrane domain provided herein comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan and valine may be present at the C terminus of the transmembrane domain.
  • the transmembrane region comprises mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine.
  • the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine- alanine sequence.
  • the hydropathy, or hydrophobic or hydrophilic characteristics of a protein or protein segment can be assessed by any method known in the art, for example the Kyte and Doolittle hydropathy analysis. [0165] In some embodiments, the transmembrane domain is from a transmembrane glycoprotein.
  • the transmembrane domain comprises a transmembrane domain chosen from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, CD1 l ⁇ , CD18, ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4, CD49d, ITGA6, VLA-6, CD49f, ITGAD, CD1D, ITGAE, CD103, ITGAL, ITG
  • the transmembrane domain is a CD8 transmembrane domain.
  • the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 3.
  • the transmembrane domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 3.
  • the transmembrane domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 3.
  • the transmembrane domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 3.
  • the transmembrane domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3.
  • the transmembrane domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 3.
  • the transmembrane domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 3. [0168] In some embodiments, the size of the transmembrane domain is about 40 amino acids to about 90 amino acids. In some embodiments, the size of the transmembrane domain is at least about 40 amino acids.
  • the size of the transmembrane domain is at most about 90 amino acids. In some embodiments, the size of the transmembrane domain is about 40 amino acids to about 50 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 90 amino acids, about 50 amino acids to about 60 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 90 amino acids, about 60 amino acids to about 70 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 90 amino acids, about 70 amino acids to about 80 amino acids, about 70 amino acids to about 90 amino acids, or about 80 amino acids to about 90 amino acids.
  • the size of the transmembrane domain is about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, or about 90 amino acids.
  • the synthetic cancer antigens disclosed herein can further comprise additional elements.
  • the synthetic cancer antigen comprises a tag.
  • the tag is at the N-terminus of the synthetic cancer antigen.
  • the tag is at the C-terminus of the synthetic cancer antigen.
  • the tag is a flag tag.
  • the tag is a c-Myc tag.
  • the tag is a His tag.
  • the tag is a HA tag.
  • the tag comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the tag comprises an amino acid sequence of SEQ ID NO: 30. [0170] In some embodiments, the different elements of the synthetic cancer antigen are operatively linked. In some embodiments, the different elements are directly linked to each other. In some embodiments, the different elements are linked via linkers. Exemplary linkers can be used herein are disclosed in the Example Section of the present application. Additional linkers are known to a skilled artisan. C.
  • the synthetic cancer antigen is a soluble (sol) protein and the target domain is linked to a tumor targeting molecule that directs the synthetic cancer antigen to a tumor cells to expose the target domain on the outside of the cell.
  • the synthetic cancer antigen comprises a target domain and tumor targeting molecule.
  • the target domain can be any as described in Section II.A.
  • a synthetic cancer antigen comprising a target domain comprising at least one target domain described in Section II.A and a tumor targeting molecule (e.g., antibody or antigen binding fragment specific to a tumor associated antigen).
  • the amino acid sequence of the target domain and the tumor targeting molecule are directly linked. In some embodiments, the amino acid sequence of the target domain and the tumor targeting molecule are indirectly linked via a linker, such as a peptide linker.
  • a linker such as a peptide linker.
  • such a synthetic cancer antigen is soluble and is not membrane associated with a cell. In certain embodiments in which the synthetic cancer antigen is expressed from a cell, the synthetic cancer antigen can be secreted from the cell, which, in provided aspects, can tag other non-infected bystander tumor cells to expose the synthetic cancer antigen on outside surface of the cell.
  • the tumor targeting molecule of the synthetic cancer antigen is able to bind to an antigen expressed on a target cell, e.g., a tumor cell, so that the coupled target domain is exposed on the outside surface of the cell.
  • the tumor targeting molecule binds to a tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • the tumor targeting molecule is an antibody or antibody fragment.
  • tumor targeting molecule is a tumor associated antigen (TAA) binding molecule.
  • the tumor targeting molecule binding molecule is an antibody or antigen binding fragment targeting a tumor associated antigen.
  • the antigen is associated with a cancer.
  • the antigen is associated with a solid tumor.
  • tumor associated antigens include antigens expressed on tumors including, but are not limited to, adenoma, carcinoma, or sarcoma.
  • the tumor associated antigen is expressed on a cancer cell, including but not limited to multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, medulloblastoma, liver cancer, stomach cancer, thyroid cancer, bile duct cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, endometrial cancer, or cervical cancer.
  • a cancer cell including but not limited to multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, bladder cancer, breast cancer, ovarian cancer,
  • 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.
  • the cancer is breast cancer.
  • the cancer is bladder cancer.
  • the cancer is colorectal cancer.
  • the cancer is endometrial cancer.
  • the cancer is liver cancer.
  • the cancer is kidney cancer.
  • the cancer is lung cancer.
  • the cancer is melanoma.
  • the cancer is pancreatic cancer.
  • the cancer is prostate cancer.
  • the cancer is thyroid cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is insensitive or resistant. [0175] In some embodiments, the tumor targeting molecule binds to a tumor associated antigen (TAA). In some embodiments, the tumor targeting molecule is a molecule derived from an antibody is one or more functional fragments of the antibody that targets a tumor associated antigen.
  • TAA tumor associated antigen
  • Non-limiting examples of functional fragments include heavy chain, light chain, heavy chain variable domain, light chain variable domain, single-chain Fvs (scFv), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody.
  • the tumor targeting molecule is an antibody without the heavy chain CH3 domain.
  • the tumor targeting molecule is an antibody without the heavy chain CH2 domain.
  • the tumor targeting molecule is a Fab.
  • the tumor targeting molecule is an antibody without the heavy chain CH2 and CH3 domain. In some embodiments, the tumor targeting molecule is a single-chain variable fragment (scFv). In some embodiments, the tumor targeting molecule is the heavy chain of an antibody. In some embodiments, the tumor targeting molecule is the light chain of an antibody. In some embodiments, the tumor targeting molecule is the variable domain of the heavy chain of an antibody. In some embodiments, the tumor targeting molecule is the variable domain of the light chain of an antibody. [0176] In some embodiments, the tumor targeting molecule is a single-chain variable fragment (scFv).
  • scFv single-chain variable fragment
  • single-chain variable fragment is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH:VL heterodimer.
  • the heavy (VH) and light chains (VL) are either joined directly or joined by a peptide- encoding linker (e.g., 10, 15, 20, 25 amino acids), which connects the N-terminus of the VH with the C- ⁇ terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL.
  • the functional fragments are part of a single polypeptide and are operatively linked. In some embodiments, the functional fragments are directly linked to each other. In some embodiments, the functional fragments are linked via linkers. Exemplary linkers can be used herein are disclosed in the Example Section of the present application. Additional linkers are known to a skilled artisan. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO:33. In some embodiments, the functional fragments are separate polypeptides and bind with each other after being translated to form a molecule derived from an antibody. In some embodiments, the functional fragments are translated from multiple polynucleotides.
  • the functional fragments are translated from a single polynucleotide.
  • the single polynucleotide comprises nucleotide sequences encoding self-cleaving peptides.
  • the self-cleaving peptides separate the functional fragments translated from a single polynucleotide.
  • the self-cleaving peptide is a P2A peptide.
  • the self-cleaving peptide comprises an amino acid sequence of SEQ ID NO: 4.
  • the tumor targeting molecule is a tumor targeting antibody.
  • the tumor targeting antibody binds to an antigen expressed on the surface of a tumor cell.
  • the tumor targeting antibody or the molecule derived from an antibody provided herein comprises VL, VH or CDRs having amino acid sequences of the VL, VH or CDR contained in certain known antibodies. In some embodiments, the tumor targeting antibody or the molecule derived from an antibody provided herein comprises VL, VH or CDRs having amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91/%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the VL, VH or CDR contained in certain known antibodies. [0179] Exemplary tumor associated antigens and their exemplary known antibodies include but are not limited to those described in Table 1 below. Table 1. Exemplary tumor associated antigens and their known antibodies
  • the tumor associated antigen targeted by the tumor targeting molecule is expressed on a solid tumor.
  • the tumor associated antigen is expressed on the solid tumor.
  • Exemplary solid tumor associated antigens include but are not limited to EpCAM (Epithelial cell adhesion molecule), CEA (Carcinoembryonic antigen), gpA33 (Glycoprotein A33 (Transmembrane), mucins, TAG-72 (Tumor-associated glycoprotein 72), CAIX (Carbonic anhydrase IX), PSMA (Prostate-specific membrane antigen), and FBP (Folate- binding protein), EGFR/ERBB1/HER1 (epidermal growth factor receptor 1), ERBB2/HER2 (epidermal growth factor receptor 2), ERBB3 (epidermal growth factor receptor 3), MET (Tyrosine-Protein Kinase IGF1R (insulin-like growth factor 1 receptor), EPHA3 (EPH Receptor A3),
  • the tumor associated antigen targeted by the tumor targeting molecule is epithelial cell adhesion/activating molecule (EpCAM), epidermal growth factor receptor (EGFR), or human epidermal growth factor receptor 2 (HER2).
  • EpCAM epithelial cell adhesion/activating molecule
  • EpCAM epidermal growth factor receptor
  • HER2 human epidermal growth factor receptor 2
  • EpCAM EpCAM
  • Epithelial cell adhesion molecule is a transmembrane glycoprotein mediating Ca2+-independent homotypic cell-cell adhesion in epithelia. EpCAM is also involved in cell signaling, migration, proliferation, and differentiation. Additionally, EpCAM has oncogenic potential via its capacity to upregulate c-myc, e-fabp, and cyclins A and E.
  • EpCAM Since EpCAM is expressed exclusively in epithelia and epithelial-derived neoplasms, EpCAM can be used as therapeutic marker for various cancers, such as head and neck cancer, ovarian cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, esophageal cancer, and lung cancer.
  • the tumor targeting molecule binds to a tumor associated antigen that is EpCAM.
  • the tumor targeting molecule is an antibody or antigen-binding fragment thereof that binds to a tumor associated antigen that is EpCAM.
  • the tumor targeting molecule is a single-chain Fvs (scFv) of an anti- EpCAM antibody.
  • the tumor targeting molecule does not comprise a constant heavy chain and/or a constant light chain domain.
  • the tumor targeting molecule is adecatumumab or an antigen-binding fragment of adecatumumab.
  • the tumor targeting molecule is a scFv of adecatumumab.
  • the tumor targeting molecule comprises the variable light chain and the variable heavy chain of adecatumumab.
  • the tumor targeting molecule is edrecolomab or an antigen-binding fragment of edrecolomab.
  • the tumor targeting molecule is a scFv of edrecolomab.
  • the tumor targeting molecule comprises the variable light chain and the variable heavy chain of edrecolomab. In some embodiments, the tumor targeting molecule is tucotuzumab or an antigen-binding fragment of tucotuzumab. In some embodiments, the tumor targeting molecule is a scFv of tucotuzumab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of tucotuzumab.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3 as contained in the VH set forth in SEQ ID NO:35, and the VL region comprises a light chain complementarity determining region 1 (CDR-L1), a CDR-L2, and a CDR-L3 as contained in the VL set forth in SEQ ID NO:37.
  • VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3 as contained in the VH set forth in SEQ ID NO:35
  • the VL region comprises a light chain complementarity determining region 1 (CDR-L1), a CDR-L2, and a CDR-L3 as contained in the VL set forth in SEQ ID NO:37
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region
  • the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NOs: 42, 43, 44, respectively
  • the VL region comprises a light chain complementarity determining region 1 (CDR-L1), a CDR-L2, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NOs: 45, 46 and 47, respectively.
  • the tumor targeting molecule comprises a variable light chain region comprising an amino acid sequence of SEQ ID NO: 37. In some embodiments, the tumor targeting molecule comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 35. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37.
  • the tumor targeting molecule comprises a variable heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 35.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 35.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 35.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 35. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 35. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 35.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 35. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 35. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 35.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 35. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 35. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 35.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 35. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 35. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 35.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 37 and a variable heavy chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 35.
  • the variable light chain and variable heavy chain are linked by a peptide linker, such as by the sequence set forth as GGGGSGGGGSGGGGS (SEQ ID NO:33).
  • the tumor targeting molecule binds to a tumor associated antigen that is HER2.
  • the tumor targeting molecule is an antibody or antigen-binding fragment thereof that binds to a tumor associated antigen that is HER2.
  • the tumor targeting molecule is a single-chain Fv (scFv) of an anti-HER2 antibody. In some embodiments, the tumor targeting molecule does not comprise a constant heavy chain and/or a constant light chain domain.
  • the tumor targeting molecule is derived from the 4D5 anti- HER2 antibody (also known as Ab1), which is the parental antibody of trastuzumab. In some embodiments, the tumor targeting molecule is an antibody fragment of the 4D5 (Ab1) antibody. [0191] In some embodiments, the tumor targeting molecule is a single-chain Fvs (scFv) of a 4D5 antibody.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3 as contained in the VH set forth in SEQ ID NO:8, and the VL region comprises a light chain complementarity determining region 1 (CDR-L1), a CDR-L2, and a CDR-L3 as contained in the VL set forth in SEQ ID NO:5.
  • the tumor targeting molecule comprises a variable light chain region comprising an amino acid sequence of SEQ ID NO: 5.
  • the tumor targeting molecule comprises a variable heavy chain region comprising an amino acid sequence of SEQ ID NO: 8.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5.
  • the tumor targeting molecule comprises a variable heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 8. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 8. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 8.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 8. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 8.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 91% identical to SEQ ID NO: 8. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 8. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 93% identical to SEQ ID NO: 8.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 94% identical to SEQ ID NO: 8. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 8. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 8.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 8. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 8. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 5 and a variable heavy chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 8.
  • the tumor targeting molecule comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, the tumor targeting molecule comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 11. In some embodiments, the tumor targeting molecule comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 11. In some embodiments, the tumor targeting molecule comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the tumor targeting molecule comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 11. In some embodiments, the tumor targeting molecule comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 11.
  • the tumor targeting molecule comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 11. In some embodiments, the tumor targeting molecule comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 11. In some embodiments, the tumor targeting molecule comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 11. In some embodiments, the tumor targeting molecule comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 11. In some embodiments, the tumor targeting molecule comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 11. In some embodiments, the tumor targeting molecule comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 11.
  • the tumor targeting molecule comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 11. In some embodiments, the tumor targeting molecule comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 11. In some embodiments, the tumor targeting molecule comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 11. [0196] 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-binding fragment targeting the tumor associated antigen that is GPC2. In some embodiments, the tumor targeting molecule is a single-chain Fvs (scFv) of an anti- GPC2 antibody.
  • scFv single-chain Fvs
  • the tumor targeting molecule does not comprise a constant heavy chain and/or a constant light chain domain.
  • the tumor associated antigen targeted by the tumor targeting molecule is mesothelin.
  • the tumor targeting molecule is an antibody or antibody-binding fragment targeting the tumor associated antigen that is mesothelin.
  • the tumor targeting molecule is a single-chain Fvs (scFv) of an anti-mesothelin antibody.
  • the tumor targeting molecule does not comprise a constant heavy chain and/or a constant light chain domain.
  • the tumor targeting molecule is Anetumab or an antigen-binding fragment of Anetumab.
  • the tumor targeting molecule is a scFv of Anetumab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of Anetumab. [0197] In some embodiments, the tumor targeting molecule is Amatuximab or an antigen- binding fragment of Amatuximab. In some embodiments, the tumor targeting molecule is a scFv of Amatuximab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of Amatuximab. [0198] In some embodiments, the tumor associated antigen targeted by the tumor targeting molecule is prostate stem cell antigen (PSCA).
  • PSCA prostate stem cell antigen
  • the tumor targeting molecule is an antibody or antibody-binding fragment targeting the tumor associated antigen that is PSCA.
  • the tumor targeting molecule is a single-chain Fvs (scFv) of an anti-PSCA antibody.
  • the tumor targeting molecule does not comprise a constant heavy chain and/or a constant light chain domain.
  • the tumor targeting molecule is AGS-PSCA or an antigen- binding fragment of AGS-PSCA.
  • the tumor targeting molecule is a scFv of AGS-PSCA.
  • the tumor targeting molecule comprises the variable light chain and the variable heavy chain of AGS-PSCA.
  • the tumor associated antigen targeted by the tumor targeting molecule is Claudin6.
  • the tumor targeting molecule is an antibody or antibody-binding fragment targeting the tumor associated antigen that is Claudin6.
  • the tumor targeting molecule is a single-chain Fvs (scFv) of an anti- Claudin6 antibody.
  • the tumor targeting molecule does not comprise a constant heavy chain and/or a constant light chain domain.
  • the tumor associated antigen targeted by the tumor targeting molecule is GPC3.
  • the tumor targeting molecule is an antibody or antibody-binding fragment targeting the tumor associated antigen that is GPC3.
  • the tumor targeting molecule is a single-chain Fvs (scFv) of an anti- GPC3 antibody. In some embodiments, the tumor targeting molecule does not comprise a constant heavy chain and/or a constant light chain domain.
  • the tumor targeting molecule is codrituzumab or an antigen-binding fragment of codrituzumab. In some embodiments, the tumor targeting molecule is a scFv of codrituzumab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of codrituzumab. [0203] In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 61.
  • the tumor targeting molecule comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 62. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 61. In some embodiments, the tumor targeting molecule comprises a variable heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 62.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 61 and a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 62.
  • the tumor associated antigen targeted by the tumor targeting molecule is Claudin18.2.
  • the tumor targeting molecule is an antibody or antibody-binding fragment targeting the tumor associated antigen that is Claudin18.2.
  • the tumor targeting molecule is a single-chain Fvs (scFv) of an anti-Claudin18.2 antibody.
  • the tumor targeting molecule does not comprise a constant heavy chain and/or a constant light chain domain.
  • the tumor targeting molecule is Zolbetuximab or an antigen-binding fragment of Zolbetuximab. In some embodiments, the tumor targeting molecule is a scFv of Zolbetuximab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of Zolbetuximab. [0206] In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 63. In some embodiments, the tumor targeting molecule comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 64.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 63.
  • the tumor targeting molecule comprises a variable heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 64.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 63 and a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 64.
  • the tumor associated antigen targeted by the tumor targeting molecule is MMP.
  • 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, MMP28.
  • the tumor associated antigen targeted by the tumor targeting molecule is MMP9.
  • the tumor targeting molecule is an antibody or antibody-binding fragment targeting the tumor associated antigen that is MMP9.
  • the tumor targeting molecule is a single-chain Fvs (scFv) of an anti-MMP9 antibody. In some embodiments, the tumor targeting molecule does not comprise a constant heavy chain and/or a constant light chain domain [0208] In some embodiments, the tumor targeting molecule is Andecaliximab or an antigen-binding fragment of Andecaliximab. In some embodiments, the tumor targeting molecule is a scFv of Andecaliximab. In some embodiments, the tumor targeting molecule comprises the variable light chain and the variable heavy chain of Andecaliximab. [0209] In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 65.
  • the tumor targeting molecule comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 66. In some embodiments, the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 65. In some embodiments, the tumor targeting molecule comprises a variable heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 66.
  • the tumor targeting molecule comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 65 and a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 66.
  • the different elements of the soluble synthetic cancer antigen are operatively linked.
  • the different elements are directly linked to each other.
  • the different elements are linked via linkers. Exemplary linkers can be used herein are disclosed in the Example Section of the present application. Additional linkers are known to a skilled artisan.
  • the linkers may be flexible linkers.
  • Linkers include peptides, polymers, nucleotides, nucleic acids, polysaccharides, and lipid organic species (such as polyethylene glycol).
  • the linker is a peptide linker.
  • Peptide linkers may be from about 2-100, 10-50, or 15-30 amino acids long.
  • peptide linkers may be at least 10, at least 15, or at least 20 amino acids long and no more than 80, no more than 90, or no more than 100 amino acids long.
  • the linker has a length of between 1 and 100 amino acids, between 1 and 75 amino acids, between 1 and 50 amino acids, between 1 and 25 amino acids, between 5 and 100 amino acids, between 5 and 75 amino acids, between 5 and 50 amino acids, between 5 and 25 amino acids, between 10 and 100 amino acids, between 10 and 75 amino acids, between 10 and 50 amino acids, or between 10 and 25 amino acids. In some embodiments, the linker has a length of between 5 and 50 amino acids. [0212]
  • the linkers can be naturally-occurring, synthetic or a combination of both. Particularly suitable linker polypeptides predominantly include amino acid residues selected from Glycine (Gly), Serine (Ser), Alanine (Ala), and Threonine (Thr).
  • the linker may contain at least 75% (calculated on the basis of the total number of residues present in the peptide linker), such as at least 80%, at least 85%, or at least 90% of amino acid residues selected from Gly, Ser, Ala, and Thr.
  • the linker may also consist of Gly, Ser, Ala and/or Thr residues only.
  • the linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues, or 8-12 glycine residues.
  • suitable peptide linkers typically contain at least 50% glycine residues, such as at least 75% glycine residues.
  • a peptide linker comprises glycine residues only. In some embodiments, a peptide linker comprises glycine and serine residues only. [0213] In some embodiments, these linkers are composed predominately of the amino acids Glycine and Serine, denoted as GS-linkers herein.
  • the linker contains (GGS)n, wherein n is 1 to 10, such as 1 to 5, for example 1 to 3, such as GGS(GGS)n (SEQ ID NO:74), wherein n is 0 to 10. In particular embodiments, the linker contains the sequence (GGGGS)n (SEQ ID NO: 75), wherein n is 1 to 10 or n is 1 to 5, such as 1 to 3.
  • the linker contains (GGGGGS)n (SEQ ID NO:76), wherein n is 1 to 4, such as 1 to 3.
  • the linker can include combinations of any of the above, such as repeats of 2, 3, 4, or 5 GS, GGS, GGGGS, and/or GGGGGS linkers may be combined. In some embodiments, such a linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids in length. [0214]
  • the linker is or comprises (in one-letter amino acid code): GGS, GGGGS (SEQ ID NO: 77 and 67), or GGGGGS (SEQ ID NO: 78).
  • the GS-linker is or comprises an amino acid sequence of GGSGGS, i.e., (GGS)2 (SEQ ID NO: 79); GGSGGSGGS, i.e., (GGS)3 (SEQ ID NO: 80); GGSGGSGGSGGS, i.e., (GGS)4 (SEQ ID NO: 81); GGSGGSGGSGGSGGS, i.e., (GGS)5 (SEQ ID NO: 82); GGGGGSGGGGGSGGGGGS, i.e., (G5S) 3 (SEQ ID NO: 83), GGSGGGGSGGGGSGGGGS (SEQ ID NO: 84) and GGGGSGGGGSGGGGS (SEQ ID NO:85).
  • GGSGGS i.e., (GGS)2 (SEQ ID NO: 79)
  • GGSGGSGGS i.e., (GGS)3 (SEQ ID NO: 80
  • GGSGGSGGSGGS i.e.
  • the linker is or comprises GGGG (SEQ ID NO:86). In some embodiments, the linker is or comprises GGGGG (SEQ ID NO:71). In some embodiments, the linker is or comprises GGGGGGGG (SEQ ID NO:72). In some of any of the above examples, serine can be replaced with alanine (e.g., (Gly4Ala) or (Gly3Ala)). [0215] In some embodiments, the linker is or comprises GSAGSAAGSGEF (SEQ ID NO:73). In some embodiments, the linker is or comprises GSGSGS (SEQ ID NO:68). [0216] In some cases, it may be desirable to provide some rigidity into the peptide linker.
  • a linker comprises at least one proline residue in the amino acid sequence of the peptide linker.
  • a peptide linker can have an amino acid sequence wherein at least 25% (e.g., at least 50% or at least 75%) of the amino acid residues are proline residues.
  • the peptide linker comprises proline residues only.
  • the linker is or comprises PAPAP (SEQ ID NO:70). [0217] In some embodiments, the linker is or comprises EAAAK (SEQ ID NO:69).
  • the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33 (corresponding nucleotide sequence set forth in SEQ ID NO:32) or 67-86, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33 or 67-86.
  • the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33 or 67-86.
  • the linker comprises an amino acid sequence of SEQ ID NO: 33.
  • the linker comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:33.
  • the synthetic cancer antigens disclosed herein can further comprise additional elements.
  • the synthetic cancer antigen comprises a tag.
  • the tag is at the N-terminus of the synthetic cancer antigen.
  • the tag is at the C-terminus of the synthetic cancer antigen.
  • the tag is a flag tag.
  • the tag is a c-Myc tag.
  • the tag is a His tag.
  • the tag is a HA tag. In some embodiments, the tag comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the tag comprises an amino acid sequence of SEQ ID NO: 30.
  • the different elements of the synthetic cancer antigen are operatively linked. In some embodiments, the different elements are directly linked to each other. In some embodiments, the different elements are linked via linkers. Exemplary linkers can be used herein are disclosed in the Example Section of the present application. Additional linkers are known to a skilled artisan.
  • the soluble synthetic cancer antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 31.
  • the soluble synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 31.
  • the soluble synthetic cancer antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 39. In some of any of the provided embodiments, the soluble synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 39.
  • the soluble synthetic cancer antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 88. In some of any of the provided embodiments, the soluble synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 88.
  • the soluble synthetic cancer antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 89.
  • the soluble synthetic cancer antigen comprises the amino acid sequence of SEQ ID NO: 89.
  • D. COGNATE BINDERS [0223] The cognate binders specifically bind to the target domains of any of the synthetic cancer antigens disclosed herein, for example in Section II.A.
  • the cognate binders cannot bind to a naturally-expressed protein on the surface of a non-cancer cell. In some embodiments, the cognate binders can be engineered as chimeric-antigen- receptors for expressing on the surface of immune effector cells. [0224] In provided embodiments, the cognate binder is a protein binding molecule. In some embodiments, the size of the cognate binder is about 20 amino acids to about 700 amino acids. In some embodiments, the size of the cognate binder is at least about 20 amino acids. In some embodiments, the size of the cognate binder is at most about 700 amino acids.
  • the size of the cognate binder is about 20 amino acids to about 40 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 250 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 700 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 250 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 700 amino acids, about 60 amino acids to about 100 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to
  • the size of the cognate binder is about 20 amino acids, about 40 amino acids, about 60 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, or about 700 amino acids.
  • the cognate binder is an antibody.
  • the cognate binder is a molecule derived from an antibody.
  • a molecule derived from an antibody is one or more functional fragments of the antibody.
  • Non-limiting examples of functional fragments include heavy chain, light chain, heavy chain variable domain, light chain variable domain, single-chain Fvs (scFv), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody.
  • the cognate binder can be part of an antibody therapy for targeting to the synthetic cancer antigen displayed on the cancer cell.
  • the cognate binder is part of an antibody drug conjugate that contains the cognate binder and a cytotoxic payload.
  • the cognate binder is part of a bispecific antibody.
  • the bispecific antibody is a bispecific T cell engager (BiTe) containing the cognate binder and an antibody targeting a T cells, such as an anti-CD3 antibody.
  • the cognate binder is part of the extracellular domain of a chimeric antigen receptor. Exemplary CAR comprising cognate binders are further described below. [0227] Cognate binders based on antibodies or molecules derived from antibodies may be made using the hybridoma method first described by Kohler, et al., Nature, 1975, 256:495-7, or may be made by recombinant DNA methods (see, e.g., U.S. Pat.
  • lymphocytes In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice 59-103 (1986)).
  • a suitable fusing agent such as polyethylene glycol
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium, which, in some embodiments, contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner).
  • a suitable culture medium which, in some embodiments, contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner).
  • the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • HAT medium thymidine
  • Exemplary fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells.
  • Exemplary myeloma cell lines are murine myeloma lines, such as SP-2 and derivatives, for example, X63-Ag8-653 cells available from the American Type Culture Collection (Manassas, VA), and those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA).
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, Immunol.
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as RIA or ELISA.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson etal, Anal. Biochem., 1980, 107:220-39.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells can serve as a source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells, such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein.
  • Cognate binders based on antibodies or molecules derived from antibodies may be made from antibody phage libraries generated using the techniques described in, for example, Antibody Phage Display: Methods and Protocols (O’Brien and Aitken, eds., 2002).
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. Examples of phage display methods that can be used to make the antibodies described herein include those disclosed in Brinkman, et al, J. Immunol. Methods , 1995, 182:41-50; Ames, et al, Immunol.
  • synthetic antibody clones are selected by screening phage libraries containing phages that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are screened against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library.
  • Fv antibody variable region
  • Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described, for example, in Winter etal, 1994, Ann. Rev. Immunol. 12:433- 55.
  • scFv single-chain Fv
  • naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described, for example, by Hoogenboom and Winter, J. Mol. Biol., 1992, 227:381-88. [0239] Screening of the libraries can be accomplished by various techniques known in the art. For example, the synthetic cancer antigens disclosed herein can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning display libraries.
  • the selection of antibodies with slow dissociation kinetics can be promoted by use of long washes and monovalent phage display as described in Bass, et al, Proteins, 1990, 8:309-14 and WO 92/09690, and by use of a low coating density of antigen as described in Marks et al, BiotechnoL, 1992, 10:779-83.
  • Antibody e.g., Anti-Idiotype Antibody
  • the target domain of the synthetic cancer antigen is recognized by a cognate binder that is an antibody or an antigen-binding fragment.
  • the antibody is able to specifically bind to an epitope of a target domain, such as any described in Section II.A.
  • the antibody cognate binder is an anti-idiotype antibody that binds to the idiotope of the target domain.
  • the antibody cognate binder e.g., anti-idiotype antibody
  • the antibody cognate binder is an antibody or antigen-binding fragment.
  • the antibody cognate binder e.g., anti-idiotype antibody
  • Non-limiting examples of antibody fragments include heavy chain, light chain, heavy chain variable domain, light chain variable domain, single-chain Fvs (scFv), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody.
  • the antibody cognate binder e.g., anti-idiotype antibody
  • the antibody cognate binder is a single domain antibody that binds to the target domain.
  • camelized antibodies including antibodies derived from llama or camels.
  • a single domain antibody comprises a variable heavy chain, and in some cases may be referred to as a VHH or VH only antibody.
  • a single domain antibody comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the antibody e.g,. an anti-idiotype antibody
  • an anti-idiotype antibody such as antigen-binding fragment of an anti-idiotype antibody, that is the cognate binder is part of the extracellular domain of a chimeric antigen receptor (CAR).
  • the antibody cognate binder e.g., anti-idiotype antibody
  • the antibody cognate binder is a single chain polypeptide.
  • the antibody cognate binder e.g., anti-idiotype antibody
  • the antibody cognate binder is a scFv.
  • the antibody cognate binder is a single domain antibody, such as comprising only the variable heavy chain of an antibody, e.g. a camelid antibody from llama.
  • the target domain of the synthetic cancer antigen is an antibody or antibody fragment (e.g., as described in Section II.A.1) and the cognate binder is an anti-idiotype antibody that binds the antibody or antibody fragment target domain.
  • the cognate binder binds to the idiotype of the antibody-based synthetic cancer antigen.
  • the cognate binder is an anti-idiotype antibody that binds to the idiotype of an antibody or antigen-binding fragment target domain.
  • exemplary cognate binders include an anti-idiotype antibody or antigen-binding fragment that binds to a antibody or antibody fragment described in Section II.A.1.
  • the antibody or antigen-binding fragment recognized by an anti-idiotype cognate binder is abciximab, adalimumab, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brodalumab, brolucizumab, burosumab, canakinumab, caplacizumab, cemiplimab, cetuximab, crizanlizumab, daclizumab, daratumumab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emapalumab, emicizumab, eptinezumab, eren
  • the cognate binder is any anti-idiotype antibody or antigen- binding fragment comprising a means for binding an antibody or antibody fragment target domain of the synthetic cancer antigen.
  • Anti-idiotypic antibodies against any of a variety of known antibody target domains are known and can be used. Exemplary anti-idiotypic antibodies include, but are not limited to, anti-4D5 (e.g., WO2005/061546, see e.g, SEQ ID NO:1 and SEQ ID NO:2 described therein), anti-adalimumab, anti-cetuximab, anti-rituximab, or anti-bevacizumab antibodies. [0246] Exemplary anti-idiotypic antibodies are shown in Table 2.
  • the cognate binding is an anti-idiotype antibody that is an anti-4D5 (Ab1) antibody or antibody fragment.
  • the epitope of the target domain recognized by the anti-idiotype antibody is present in the variable heavy chain of the 4D5 (Ab1) antibody or antibody fragment.
  • the cognate binder is an anti-idiotype antibody single domain antibody that binds an idiotope of the 4D5 (Ab1) antibody.
  • the cognate binder comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 13.
  • the cognate binder comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 13. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 13. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 13. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 13. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 13.
  • the cognate binder comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 13. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 13. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 13. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 13. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 13.
  • the cognate binder comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 13.
  • the cognate binder is an antigen binding fragment of an anti-idiotype antibody that binds the 4D5 (Ab1) antibody.
  • the antigen binding fragment is an scFv comprising a variable heavy chain and a variable light chain.
  • the scFv contains a variable heavy chain and a variable light chain as present in SEQ ID NO:14.
  • the scFv contains a variable heavy chain and a variable light chain as present in SEQ ID NO:15.
  • variable heavy chain and variable light chain are connected by a peptide linker, such as the linker sequence GGGGSGGGGSGGGGS (SEQ ID NO:33).
  • the cognate binder comprises an amino acid sequence of SEQ ID NO: 14. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 14. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 14. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 14. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 14.
  • the cognate binder comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 14. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 14. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 14. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 14. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 14.
  • the cognate binder comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 14. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 14. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 14. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 14. [0252] In some embodiments, the cognate binder comprises an amino acid sequence of SEQ ID NO: 15. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 15.
  • the cognate binder comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 15. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 15. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 15. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 15. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 15. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 15.
  • the cognate binder comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 15. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 15. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 15. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 15. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 15.
  • the cognate binder comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 15.
  • 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.
  • the antibody cognate binder binds an intracellular protein set forth in any of SEQ ID NOS: 17, 18, 19, 20, 21, 22 or 23 or a sequence of amino acids 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 NOS: 17, 18, 19, 20, 21, 22 or 23.
  • the antibody cognate binder binds an intracellular protein set forth in SEQ ID NO: 17. [0254]
  • the antibody cognate binder is an antibody fragment that binds the intracellular protein target domain.
  • the antigen binding fragment is an scFv comprising a variable heavy chain and a variable light chain.
  • the scFv contains a variable heavy chain and a variable light chain as present in SEQ ID NO:24.
  • the variable heavy chain and variable light chain are connected by a peptide linker, such as the linker sequence GGGGSGGGGSGGGGS (SEQ ID NO:33).
  • the cognate binder comprises an amino acid sequence of SEQ ID NO: 24.
  • the cognate binder comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 24.
  • the cognate binder comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 24. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 24. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 24. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 24. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 24. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 24.
  • the cognate binder comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 24. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 24. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 24. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 24. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 24. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 24.
  • the cognate binder comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 24. [0256] In some embodiments, the cognate binder comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 25. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 25. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 25. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 25.
  • the cognate binder comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 25. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 25. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 25. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 25. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 25.
  • the cognate binder comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 25. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 25. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 25. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 25. [0257] Exemplary combinations of target domains and their cognate binders are set forth in Table 3. 2. Leucine Zipper Domain [0258] In some embodiments, the cognate binder of the synthetic cancer antigen is a leucine zipper domain.
  • the target domain of the synthetic cancer antigen also is a complementary leucine zipper.
  • the cognate binder binds to its complementary leucine zipper domain.
  • the target domain is the leucine zipper domain of ATF, then the cognate binder is the leucine zipper domain of CREB.
  • the target domain is the leucine zipper domain of CREB, then the cognate binder is the leucine zipper domain of ATF.
  • the target domain is the leucine zipper domain of JUN, then the cognate binder is the leucine zipper domain of Fos.
  • the target domain is the leucine zipper domain of Fos, then the cognate binder is the leucine zipper domain of JUN.
  • the cognate binder comprises an amino acid sequence of SEQ ID NO: 26. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 26. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 26. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 26. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 26. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26.
  • the cognate binder comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 26. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 26. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 26. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 26. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 26. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 26.
  • the cognate binder comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 26. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 26. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 26. [0260] In some embodiments, the cognate binder comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 27. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 27.
  • the cognate binder comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 27. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 27. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 27. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 27. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 27. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 27.
  • the cognate binder comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 27. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 27. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 27. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 27. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 27. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 27.
  • the cognate binder comprises an amino acid sequence of SEQ ID NO: 28. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 28. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 28. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 28. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 28. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 28.
  • the cognate binder comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 28. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 28. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 28. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 28. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 28. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 28.
  • the cognate binder comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 28. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 28. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 28. [0262] In some embodiments, the cognate binder comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 29. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 29.
  • the cognate binder comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 29. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 29. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 29. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 29. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 29.
  • the cognate binder comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 29. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 29. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 29. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 29. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 29. In some embodiments, the cognate binder comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 29.
  • the cognate binder of the synthetic cancer antigen is the extracellular domain of a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the CAR that is expressed by genetically engineered cells is able to bind via its extracellular domain to the target domain of the synthetic cancer antigen.
  • the extracellular domain of the CAR is a cognate binder as described in Section D.1 or D2, and is chosen based on the particular target domain of the synthetic cancer antigen.
  • the CAR further comprises a transmembrane domain and an intracellular signaling domain.
  • the CAR is expressed on the surface of an immune effector cell.
  • the immune effector cell is a cytotoxic T cell.
  • the immune effector cell is a natural killer cell.
  • the engineered cells (e.g., T cells) that comprise the CAR may be used for immunotherapy to target and destroy cancer cells that have been tagged to express the synthetic cancer antigen recognized by the CAR.
  • the CAR is a fusion protein that includes the extracellular domain comprising the cognate binder as an extracellular antigen-binding domain for recognizing the target domain; a transmembrane domain; and an intracellular signaling domain.
  • the extracellular domain and transmembrane domains may be linked by a flexible linker (also called a spacer).
  • the extracellular domain of the CAR is any of the cognate binders as described above.
  • the intracellular signaling domain includes an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • Activation of the CAR fusion protein results in cellular activation in response to recognition by the cognate binder of the target domain of the synthetic cancer antigen tag.
  • a cell expresses such a CAR, it can recognize and kill target cells that express, or that are tagged to display on their surface, the synthetic cancer antigen.
  • the CAR is typically encoded by a nucleic acid sequence (polynucleotide) that includes a leader sequence for directing expression of the CAR at the membrane of a cell.
  • the antigen receptor e.g. CAR
  • CAR is encoded by a polynucleotide that encodes a CAR with an NH2-terminal leader sequence.
  • the leader sequence also known as the signal peptide
  • the leader sequence allows the expressed CAR construct to enter the endoplasmic reticulum (ER) and target the cell surface.
  • the leader sequence is cleaved in the ER and the mature cell surface CAR does not possess a leader sequence.
  • the leader sequence length will be in the range of 5 to 30 amino acids, and comprise a stretch of hydrophobic amino acids. In some embodiments, the leader sequence is 5, 10, 15, 20, or 25 amino acids in length, or any value between any of the foregoing.
  • the leader sequence comprises a sequence derived from any secretory protein.
  • the leader sequence can be any of the signal peptide sequences described herein, for example in SEQ ID NO:1.
  • An exemplary CD8 ⁇ signal peptide is set forth in SEQ ID NO:2.
  • An exemplary GM-CSFRa signal peptide is set forth in SEQ ID NO:48.
  • An exemplary IgK signal peptide is set forth in SEQ ID NO:49.
  • the CARs disclosed herein comprise a transmembrane domain that can be directly or indirectly fused to the extracellular domain.
  • the transmembrane domains disclosed above for the synthetic cancer antigens can be used as the transmembrane domain of CARs disclosed herein.
  • the transmembrane domain is a CD8 transmembrane domain.
  • the CAR comprises a transmembrane with an amino acid sequence derived from the transmembrane domain of CD8.
  • the CAR comprises a transmembrane domain with an amino acid sequence derived from the transmembrane domain of human CD8 alpha.
  • the CAR contains a transmembrane domain of CD8 alpha that has the sequence of amino acids set forth in SEQ ID NO:51 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:51.
  • the transmembrane domain is set forth in SEQ ID NO:51.
  • the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 3.
  • the transmembrane domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 3.
  • the transmembrane domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 3.
  • the transmembrane domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 3. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 3.
  • the transmembrane domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 3.
  • the CAR comprises a transmembrane with an amino acid sequence derived from the transmembrane domain of CD28.
  • the CAR comprises a transmembrane domain with an amino acid sequence derived from the transmembrane domain of human CD28.
  • the CAR contains a transmembrane domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO:52 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:52.
  • the transmembrane domain is set forth in SEQ ID NO:52.
  • the CARs can also comprise a spacer region located between the cognate binder of the extracellular antigen binding domain and the transmembrane domain.
  • the spacer region comprises hydrophilic amino acids and allows flexibility of the targeting domain with respect to the cell surface.
  • the spacer region comprises greater than 5, 10, 15, 20, 25, or 30 amino acids.
  • the spacer region comprises less than 10, 15, 20, 25, 30, or 35 amino acids.
  • the spacer region is a hinge domain.
  • the CARs disclosed herein may comprise a hinge domain that is located between the extracellular domain and the transmembrane domain.
  • a hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular domain relative to the transmembrane domain of the effector molecule can be used. [0273] In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8a.
  • the hinge domain is a portion of the hinge domain of CD8a, e.g., a fragment containing about 15-100 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8a.
  • the CAR contains a CD8 hinge spacer sequence that has the sequence of amino acids set forth in SEQ ID NO:53 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:53.
  • the sequence of the spacer is set forth in SEQ ID NO:53.
  • the hinge domain is the hinge domain of an antibody, such as an IgG, IgA, IgM, IgE, or IgD antibody.
  • the hinge domain is the hinge domain that joins the constant domains CH1 and CH2 of an antibody.
  • the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody.
  • the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody.
  • the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody.
  • the antibody is an IgG, IgA, IgM, IgE, or IgD antibody.
  • the antibody is an IgG antibody.
  • the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.
  • the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.
  • the spacer region includes all or a portion containing the hinge domain of an IgG1 Fc or an IgG4 Fc. In some embodiments, the spacer is an IgG4 Fc spacer. In some embodiments, the CAR contains an IgG4 Fc spacer that has the sequence of amino acids set forth in SEQ ID NO:71 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:54.
  • the sequence of the spacer is set forth in SEQ ID NO:54. In some embodiments, the sequence of the spacer is the hinge portion of the IgG1 Fc or IgG4 Fc. In some embodiments, the CAR contains an IgG4 hinge spacer. In some embodiments, the IgG4 hinge spacer has the sequence of amino acids set forth in SEQ ID NO: 55 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:55. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO:55. [0276] Non-naturally occurring peptides may also be used as hinge domains.
  • the hinge domain between the C-terminus of the extracellular ligand-binding domain of an Fc receptor and the N- terminus of the transmembrane domain is a peptide linker, such as a (GxS)n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.
  • the hinge domain may contain about 10-100 amino acids, e.g., about any one of 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids.
  • the hinge domain may be at least about any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • cytoplasmic signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire cytoplasmic signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term cytoplasmic signaling domain is thus meant to include any truncated portion of the cytoplasmic signaling domain sufficient to transduce the effector function signal.
  • the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.
  • the CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell.
  • Primary intracellular signaling domain refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions.
  • the primary intracellular signaling domain contains a signaling motif known as immunoreceptor tyrosine-based activation motif, or ITAM.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITAM immunoreceptor tyrosine-based activation motif
  • the motif may comprises two repeats of the amino acid sequence YxxL/I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxL/Ix(6-8)YxxL/I.
  • IT AMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the IT AM following activation of the signaling molecule. ITAMs may also function as docking sites for other proteins involved in signaling pathways.
  • Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3z, FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • the intracellular signaling domain is derived from CD3z chain (CD3z).
  • the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3z.
  • the intracellular signaling domain is a cytoplasmic signaling domain of wild-type CD3z.
  • the CAR contains an intracellular signaling domain that contains a signaling domain of CD3zeta that has the sequence of amino acids set forth in SEQ ID NO:56 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:56. In some embodiments, the CAR contains an intracellular signaling domain that contains the signaling domain of CD3zeta that has the sequence of amino acids set forth in SEQ ID NO:56.
  • the CAR contains an intracellular signaling domain that contains a signaling domain of CD3zeta that has the sequence of amino acids set forth in SEQ ID NO:57 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:57. In some embodiments, the CAR contains an intracellular signaling domain that contains a signaling domain of CD3zeta that has the sequence of amino acids set forth in SEQ ID NO:57. [0282] The CARs disclosed herein may further comprise at least one co-stimulatory signaling domain.
  • the chimeric antigen receptor comprises: 1) the cognate binder; 2) a spacer, such as a hinge sequence; 3) a transmembrane domain; and 4) an intracellular signaling region comprising a first primary intracellular signaling domain, such as a CD3 zeta intracellular signaling domain and second co- stimulatory intracellular signaling domain.
  • co-stimulatory signaling domain refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function.
  • the co-stimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils.
  • a co-stimulatory protein which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils.
  • “Co-stimulatory signaling domain” can be the cytoplasmic portion of a co-stimulatory molecule.
  • the intracellular signaling domain comprises a single co- stimulatory signaling domain.
  • the intracellular signaling domain comprises two or more (such as about any of 2, 3, 4, or more) co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same co- stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains from different co- stimulatory proteins, such as any two or more co-stimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3z) and one or more co- stimulatory signaling domains (such as 4-1BB signal).
  • a primary intracellular signaling domain such as cytoplasmic signaling domain of CD3z
  • co- stimulatory signaling domains such as 4-1BB signal
  • the one or more co-stimulatory signaling domains (such as 4-1BB signal) and the primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3z) are fused to each other via optional peptide linkers.
  • the primary intracellular signaling domain, and the one or more co- stimulatory signaling domains may be arranged in any suitable order. Multiple co-stimulatory signaling domains may provide additive or synergistic stimulatory effects.
  • a costimulatory domain can be CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, lymphocyte function- associated antigen-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, and/or B7-H3 costimulatory domains.
  • the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO:58 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:58.
  • the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO:58. In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO:59 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:59. In some embodiments, the CAR contains an intracellular signaling domain that contains the costimulatory signaling domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO:59.
  • the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of 4-1BB that has the sequence of amino acids set forth in SEQ ID NO:60 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:60.
  • the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of 4-1BB that has the sequence of amino acids set forth in SEQ ID NO:60.
  • an intracellular signaling domain can be a domain of CD3zeta, CD28 and/or 4-1BB.
  • the CAR comprises at least two intracellular signaling domains derived from CD3 zeta and 4-lBB. In other embodiments, the CAR comprises at least two intracellular signaling domains derived from CD3 zeta and CD28. [0289] In some embodiments, the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16.
  • the intracellular signaling domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 91% identical to SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 16.
  • the intracellular signaling domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 16. [0290] Exemplary CARs and their components are set forth in Table 5. 4.
  • Variants of Synthetic Cancer Antigens and Cognate Binders are contemplated. For example, it may be desirable to improve the binding affinity between the synthetic cancer antigens and/or cognate binders; it may also be desirable to improve other biological properties of the synthetic cancer antigens and/or cognate binders, including but not limited to specificity, thermostability, expression level, or solubility. Thus, in addition to the synthetic cancer antigens and/or cognate binders described herein, it is contemplated that variants can be prepared.
  • the synthetic cancer antigens and/or cognate binders provided herein are chemically modified, for example, by the covalent attachment of any type of molecule to the synthetic cancer antigens and/or cognate binders.
  • exemplary non-limiting modifications include glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
  • the synthetic cancer antigens and/or cognate binders may contain one or more non-classical amino acids.
  • variations may be a substitution, deletion, or insertion of one or more codons encoding the synthetic cancer antigens and/or cognate binders that results in a change in the amino acid sequence as compared with the original sequence.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements.
  • Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR- mediated mutagenesis which results in amino acid substitutions.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge.
  • Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g ., aspartic acid, glutamic acid), uncharged polar side chains (e.g ., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g, tyrosine, phenylalanine, tryp
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined. [0295] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • amino acids may be grouped according to similarities in the properties of their side chains (see, e.g. , Lehninger, Biochemistry 73-75 (2d ed.
  • Naturally occurring residues may be divided into groups 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 that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • Non-conservative substitutions entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, into the remaining (non-conserved) sites.
  • Insertions or deletions may optionally be in the range of about 1 to 5 amino acids.
  • the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule.
  • the substitution is a conservative amino acid substitution made at one or more predicted non- essential amino acid residues.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue.
  • insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody.
  • an enzyme e.g., for antibody-directed enzyme prodrug therapy
  • a polypeptide which increases the serum half-life of the antibody.
  • a polynucleotide encoding any of the synthetic cancer antigens that comprises a tumor-targeting binding molecule, e.g., as disclosed in Section II.C.
  • a polynucleotide encoding any of the synthetic cancer antigens membrane bound synthetic cancer antigens that comprises a membrane targeting domain, e.g., as disclosed in Section II.B.
  • a polynucleotide encoding any of the membrane bound synthetic cancer antigens (comprising a tumor-targeting binding molecule, e.g., as disclosed in Section II.C, and any of the soluble synthetic cancer antigens that comprises a membrane targeting domain, e.g., as disclosed in Section II.B.
  • a polynucleotide encoding a synthetic cancer antigen that comprises a soluble synthetic cancer antigen comprising a target domain and a tumor-targeting binding molecule, e.g., any of those disclosed in Section II.C., and a membrane bound synthetic cancer antigen that comprises a target domain and a membrane targeting domain, e.g., any of those disclosed in Section II.B.
  • the polynucleotide sequence encodes a protein in which the soluble synthetic cancer antigen that comprises a target domain and a tumor-targeting binding molecule and the membrane bound synthetic cancer antigen that comprises a target domain and a membrane targeting domain are separated by a cleavable linker.
  • the cleavable linker comprises a 2A self-cleaving peptide.
  • a polynucleotide comprising a first nucleic acid sequence encoding any of the soluble synthetic cancer antigens that comprise a target domain and a tumor-targeting binding molecule, e.g., any of those disclosed in Section II.C., and a second nucleic acid sequence encoding any of the membrane bound synthetic cancer antigens that comprise a target domain and a membrane targeting domain, e.g., any of those disclosed in Section II.B.
  • the first nucleic acid sequence and the second nucleic acid sequence are separated by a nucleotide sequence encoding a cleavable linker.
  • the cleavable linker is a self-cleaving linker.
  • the self-cleaving linker is or comprises a 2A peptide.
  • the polynucleotide is a deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids. Polynucleotides may be single-stranded or double-stranded and either recombinant, synthetic, or isolated.
  • Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.
  • pre-mRNA pre-messenger RNA
  • mRNA messenger RNA
  • gDNA genomic DNA
  • cDNA complementary DNA
  • synthetic DNA synthetic DNA
  • Polynucleotides refer to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, as well as all intermediate lengths.
  • polynucleotides are codon-optimized.
  • codon-optimized refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide.
  • Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, (x) systematic variation of codon sets for each amino acid, (xi) isolated removal of spurious translation initiation sites and/or (xii) elimination of fortuitous polyadeny
  • nucleotide sequences that encode a polypeptide, or fragment of variant thereof, as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and/or primate codon selection. Further, alleles of the genes comprising the polynucleotide sequences provided herein may also be used.
  • the polynucleotides provided herein regardless of the length of the coding sequence itself, further comprise with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably.
  • promoters and/or enhancers such as promoters and/or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombina
  • polynucleotide fragment of almost any length may be employed in particular embodiments, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • Polynucleotides can be prepared, manipulated and/or expressed using any of a variety of well-established techniques known and available in the art.
  • IV. VECTORS [0308] Provided herein are vectors comprising the polynucleotides or nucleic acid molecules encoding any of the synthetic cancer antigens disclosed herein. Also provided herein are vectors comprising any of the polynucleotides or nucleic acid molecules encoding the cognate binders of synthetic cancer antigens disclosed herein.
  • vectors comprising polynucleotides encoding two or more of the synthetic cancer antigens disclosed herein, such as a synthetic cancer antigen comprising a tumor-targeting binding molecule, and a synthetic cancer antigen comprising a membrane targeting domain.
  • vectors comprising a first polynucleotide encoding a soluble synthetic cancer antigen that comprises a target domain and a tumor-targeting binding molecule, e.g., any of those disclosed in Section II.C., and a second polynucleotide encoding a membrane bound synthetic cancer antigen that comprises a target domain and a membrane targeting domain, e.g., any of those disclosed in Section II.B.
  • the first polynucleotide and the second polynucleotide are separated by nucleotide sequence encoding a cleavable linker.
  • the cleavable linker comprises a 2A self-cleaving peptide.
  • the synthetic cancer antigen is delivered by a viral vector comprising a polynucleotide encoding the synthetic cancer antigen, such as any of the polynucleotides disclosed in Section III.
  • an expression cassette encoding the synthetic cancer antigens or the cognate binders 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 positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergo appropriate post-translational modifications required for activity in the transformed cell, and be translocated to the appropriate compartment for biological activity by targeting to appropriate intracellular compartments or secretion into extracellular compartments.
  • the cassette has its 3' and 5' ends adapted for ready insertion into a vector, e.g., it has restriction endonuclease sites at each end.
  • the cassette can be removed and inserted into a plasmid or viral vector as a single unit.
  • nucleic acid vector is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule.
  • the transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule.
  • a nucleic acid vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA.
  • vectors include, without limitation, plasmids, phagemids, cosmids, transposons, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • the coding sequences of the synthetic cancer antigens or the cognate binders disclosed herein can be ligated into such vectors for expression in mammalian cells.
  • the vector is a viral vector or a non-viral vector.
  • the vector is a non-viral vector.
  • non-viral vectors are used to deliver one or more polynucleotides contemplated herein.
  • the recombinant vector comprising a polynucleotide encoding the synthetic cancer antigens or the cognate binders described herein is a plasmid.
  • suitable plasmid expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).
  • the vector is a viral vector.
  • viral vectors are used to deliver one or more polynucleotides contemplated herein.
  • Suitable viral vectors include, but are not limited to, viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:25432549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5:10881097, 1999; WO 94/12649, WO 93/03769; WO 93/19191 ; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., U.S.
  • Patent No. 7,078,387 Ali et al., Hum Gene Ther 9:8186, 1998, Flannery et al satisfy PNAS 94:69166921 , 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683690, 1997, Rolling et al., Hum Gene Ther 10:641648, 1999; Ali et al., Hum Mol Genet 5:591594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al satisfy Virol.
  • SV40 herpes simplex virus
  • human immunodeficiency virus see, e.g., Miyoshi et al., PNAS 94:1031923, 1997; Takahashi et al., J Virol 73:78127816, 1999
  • a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus
  • retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloprol
  • vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DESTTM, pLenti6/V5-DESTTM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
  • the vector is a non-integrating vector, including but not limited to, an episomal vector or a vector that is maintained extrachromosomally.
  • the term “episomal” refers to a vector that is able to replicate without integration into host’s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally.
  • the vector is engineered to harbor the sequence coding for the origin of DNA replication or “ori” from a lymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast, specifically a replication origin of a lymphotrophic herpes virus or a gamma herpesvirus corresponding to oriP of EBV.
  • the lymphotrophic herpes virus may be Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV), Herpes virus saimiri (HS), or Marek's disease virus (MDV).
  • Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus.
  • the vector is an integrating vector.
  • a polynucleotide is introduced into a target or host cell using a transposon vector system.
  • the transposon vector system comprises a vector comprising transposable elements and a polynucleotide contemplated herein; and a transposase.
  • the transposon vector system is a single transposase vector system, see, e.g., WO 2008/027384.
  • Exemplary transposases include, but are not limited to: piggyBac, Sleeping Beauty, Mos1, Tc1/mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and derivatives thereof.
  • the piggyBac transposon and transposase are described, for example, in U.S. Patent 6,962,810, which is incorporated herein by reference in its entirety.
  • the Sleeping Beauty transposon and transposase are described, for example, in Izsvak et al., J. Mol. Biol. 302: 93-102 (2000), which is incorporated herein by reference in its entirety.
  • the Tol2 transposon which was first isolated from the medaka fish Oryzias latipes and belongs to the hAT family of transposons is described in Kawakami et al. (2000).
  • Mini-Tol2 is a variant of Tol2 and is described in Balciunas et al. (2006).
  • the Tol2 and Mini-Tol2 transposons facilitate integration of a transgene into the genome of an organism when co-acting with the Tol2 transposase.
  • a polynucleotide sequence encoding the synthetic tumor antigens or the cognate binders disclosed herein is operably linked to one or more control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells.
  • Control elements refer those non-translated regions of the vector which interact with host cellular proteins to carry out transcription and translation.
  • Non-limiting examples of control elements include origin of replication, selection cassettes, constitutive and inducible promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, transcription terminators, 5' and 3' untranslated regions. See e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544) Such elements may vary in their strength and specificity.
  • the transcriptional control element may be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., bacterial or archaeal cell).
  • polynucleotides encoding the synthetic cancer antigens or the cognate binders described herein are operably linked to a promoter and/or an enhancer.
  • promoter refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter.
  • promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.
  • the term “enhancer” refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence.
  • An enhancer can function cooperatively or additively with promoters and/or other enhancer elements.
  • Non-limiting examples of suitable eukaryotic promoters include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, a viral simian virus 40 (SV40) (e.g., early and late SV40), a spleen focus forming virus (SFFV) promoter, long terminal repeats (LTRs) from retrovirus (e.g., a Moloney murine leukemia virus (MoMLV) LTR promoter or a Rous sarcoma virus (RSV) LTR), a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, an elongation factor 1-alpha (EF1 ⁇ ) promoter, early growth response 1 (EGR1) promoter, a ferritin H (FerH) promoter
  • CMV cytomegalovirus
  • HSV40 viral
  • a polynucleotide sequence encoding the synthetic cancer antigens or the cognate binders described herein is operably linked to a constitutive promoter.
  • the polynucleotides encoding the synthetic cancer antigens or the cognate binders described herein are constitutively and/or ubiquitously expressed in a cell.
  • a polynucleotide sequence encoding the synthetic cancer antigens or the cognate binders described herein is operably linked to an inducible promoter.
  • inducible promoters/systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the “GeneSwitch” mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), the cumate inducible gene switch (WO 2002/088346), tetracycline-dependent regulatory systems, etc.
  • steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the “GeneSwitch” m
  • the vectors described herein further comprise a transcription termination signal. Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increases heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal.
  • vectors comprise a polyadenylation sequence 3′ of a polynucleotide encoding a polypeptide to be expressed.
  • polyA site or “polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II.
  • Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3′ end of the coding sequence and thus, contribute to increased translational efficiency.
  • Cleavage and polyadenylation is directed by a poly(A) sequence in the RNA.
  • the core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage- polyadenylation site.
  • an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues.
  • Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5' cleavage product.
  • the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA).
  • the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit ⁇ -globin polyA sequence (r ⁇ gpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.
  • a vector may also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, mitochondrial localization), fused to the polynucleotide encoding the synthetic tumor antigens or the cognate binders.
  • a vector may comprise a nuclear localization sequence (e.g., from SV40) fused to the polynucleotide encoding the synthetic cancer antigens or the cognate binders.
  • the signal peptide is an Igk signal peptide.
  • the signal peptide is a CD8 signal peptide.
  • the signal peptide comprises an amino acid sequence of SEQ ID NO: 1.
  • the signal peptide comprises an amino acid sequence of SEQ ID NO: 2.
  • the expression vector further comprises nucleotide sequences encoding one or more protein tags (e.g., 6xHis tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to polynucleotide encoding the synthetic cancer antigen, thereby resulting in a synthetic cancer antigen that further comprises a protein tag.
  • protein tags e.g., 6xHis tag, hemagglutinin tag, green fluorescent protein, etc.
  • Suitable methods include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al., Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9), microfluidics delivery methods (See e.g., International PCT Publication No. WO 2013/059343), and the like.
  • PKI polyethyleneimine
  • delivery via electroporation comprises mixing the cells with the polynucleotides encoding the synthetic cancer antigens or the cognate binders in a cartridge, chamber, or cuvette and applying one or more electrical impulses of defined duration and amplitude.
  • cells are mixed with polynucleotides encoding the synthetic cancer antigens or the cognate binders in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber, or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.
  • a device e.g., a pump
  • polynucleotides encoding the synthetic cancer antigens or the cognate binders described herein are introduced to a cell in a non-viral delivery vehicle, such as a transposon, a nanoparticle (e.g., a lipid nanoparticle), a liposome, an exosome, an attenuated bacterium, or a virus-like particle.
  • a non-viral delivery vehicle such as a transposon, a nanoparticle (e.g., a lipid nanoparticle), a liposome, an exosome, an attenuated bacterium, or a virus-like particle.
  • Vectors derived from retroviruses such as lentivirus are suitable tools to achieve long-term gene transfer, because they allow long-term, stable integration of a transgene and its propagation in progeny cells. Lentiviral vectors also have low immunogenicity, and can transduce nonproliferating cells.
  • the vehicle is a mammalian virus-like particle.
  • modified viral particles can be generated (e.g., by purification of the “empty” particles followed by ex vivo assembly of the virus with the desired cargo).
  • the vehicle can also be engineered to incorporate targeting ligands to alter target tissue specificity.
  • the viral vector is a non-replicating viral vector, e.g., is replication defective. In some embodiments, the viral vector is a replication defective adenoviral vector that lacks the E1A gene that is essential for adenovirus replication. [0336] In some embodiments, the viral vector is a replicating viral vector.
  • the viral vector is a viral vector or modified form thereof of a viral vector as disclosed in, e.g., 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 hereby incorporated by reference in their entity.
  • the viral vector is an adenoviral vector, an adeno- associated virus (AAV) vector, a lentiviral vector, a retroviral vector, or a herpes simplex viral (HSV) vector.
  • AAV adeno-associated virus
  • HSV herpes simplex viral
  • Exemplary adenoviral vectors include those disclosed, e.g., in Engelhardt et al., Hum Gene Ther 5.1217- 1229, 1994; US 5,756,283; US 5,707,618; Tessarollo et al., Cancers (Basel), 2021, 13(8): 1863; Wold et al., Curr. Gene Ther., 2013, 13(6): 421-433; and WO2014198852 A2.
  • the viral vector is an adenoviral vector.
  • the viral vector is a non-replicating adenoviral vector, such as any of those as described in Tessarollo et al., supra; and Wold et al., supra.
  • Adenoviruses are non-enveloped viruses with a single linear strand of double-stranded DNA inside of an icoasahedral capsid.
  • Adenoviruses of animal original can be obtained from deposited strains, and then amplified in competent cell lines, and, optionally, modified as desired. Complete genome sequences of adenoviruses have been determined for, e.g., human adenovirus type 2 (GenBank Accession No.
  • the adenoviral vector is of any one of subgroups A-G. In some embodiments, the adenoviral vector is of subgroup A. In some embodiments, the adenoviral vector is of subgroup B. In some embodiments, the adenoviral vector is of subgroup B, type 1. In some embodiments, the adenoviral vector is of subgroup B, type 2. In some embodiments, the adenoviral vector is of subgroup C.
  • the adenoviral vector is of subgroup D. In some embodiments, the adenoviral vector is of subgroup E. In some embodiments, the adenoviral vector is of subgroup F. In some embodiments, the adenoviral vector is of subgroup G.
  • Subgroup A adenoviruses includes, e.g., serotypes 12, 18, and 31; subgroup B, type 1 adenoviruses includes, e.g., serotypes 3, 7, 16, and 21; subgroup B, type 2 adenoviruses includes, e.g., serotypes 11, 14, 34, and 35; subgroup C adenoviruses includes, e.g., serotypes 1, 2, 5, and 6; subgroup D adenoviruses includes, e.g., serotypes 8-10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-49; subgroup E adenoviruses includes, e.g., serotype 4; subgroup F adenoviruses includes, e.g., serotypes 40 and 41; and subgroup G adenoviruses includes, e.g., serotype 52.
  • subgroup E adenoviruses includes, e.
  • adenoviruses e.g., those in subgroups A, C, D, E, and F, enter target cells by binding primarily to the coxsackie-adenovirus receptor.
  • the subgroup B, type 1 group of adenoviruses bind primarily to CD46
  • the subgroup B, type 2 group of adenoviruses bind primarily to desmoglein-2, with some exceptions.
  • Ad11, Ad14, Ad16, Ad21, Ad35, and Ad50 of subgroup B bind to CD46, as well as Ad17 and Ad47 of subgroup D.
  • Ad3, Ad7, and Ad14 of subgroup B bind to desmoglein-2.
  • the levels of coxsackie-adenovirus receptor varies by tissue and by tumor type, and can be upregulated or downregulated in tumor cells, depending on the type of cancer. See, e.g., Hensen et al., Int. J. Mol. Sci., 2020, 21(18): 6828.
  • CD46 All nucleated cells express CD46, thereby making adenoviruses from subgroup B, type 1 desirable for its broad tropism. Moreover, the expression of CD46 is reported as being low or moderate in most normal tissues, but upregulated in many different types of cancer, thereby making adenoviruses that primarily bind CD46, e.g., Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, and Ad50, desirable for use in delivering synthetic cancer antigen s to tumor cells. See, e.g., Do et al., Int. J. Mol.
  • Desmoglein-2 is a transmembrane glycoprotein that is expressed in a variety of tissue types, including bladder, colon, kidney, prostate, and stomach, and, like CD46, has been report to be upregulated in cancers. See, e.g., Hensen et al., Int. J. Mol. Sci., 2020, 21(18): 6828; and Brennan et al., Cell Adhes. Migr., 2009, 3: 148-154.
  • the viral vector e.g., adenoviral vector
  • the viral vector that binds to CD46 and/or desmoglein-2 is Ad3, Ad7, Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, or Ad50.
  • the adenoviral vector is based on Ad3, Ad7, Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, and Ad50, or any combination thereof. In some embodiments, the adenoviral vector is based on Ad2. In some embodiments, the adenoviral vector is based on Ad3. In some embodiments, the adenoviral vector is based on Ad5. In some embodiments, the adenoviral vector is based on Ad7. In some embodiments, the adenoviral vector is based on Ad9. In some embodiments, the adenoviral vector is based on Ad11. In some embodiments, the Ad11 is an Ad11p.
  • the adenoviral vector is based on Ad12. In some embodiments, the adenoviral vector is based on Ad14. In some embodiments, the adenoviral vector is based on Ad17. In some embodiments, the adenoviral vector is based on Ad35. In some embodiments, the adenoviral vector is based on Ad40. In some embodiments, the adenoviral vector is based on Ad50. [0350] In some embodiments, the adenoviral vector is a chimeric virus based on any two or more serotypes of adenovirus.
  • the adenoviral vector is a chimeric virus based on serotype 3 (Ad3) and/or serotype 5 (Ad5) adenoviral vectors. In some embodiments, the adenoviral vector is a chimeric virus based on serotype 3 (Ad3) and/or serotype 11 (Ad11) adenoviral vectors. [0351] In some embodiments, the adenoviral vector is Ad5. In some embodiments, the adenoviral vector is Ad5 or a variant or derivative thereof. The Ad5 adenoviral vector generally enters the cell through the coxsackie-adenovirus receptor.
  • the adenovirus is a chimeric adenovirus. In some embodiments, the chimeric adenovirus is Ad3/11p or is Ad5/3. [0353] In some embodiments, the adenoviral vector is Ad3/11p, which is a chimeric adenovirus composite of Ad3 and Ad11p. In some embodiments, the Ad3/11p is ColoAd1.
  • ColoAd1 is a chimeric adenovirus in which the major coat proteins are derived from Ad11p, and, relative to Ad11, includes an almost complete 2444 base pair deletion of the E3 gene, a smaller 25-base pair deletion of the E4 gene region, and a chimeric Ad3/Ad11pm E3B gene region. See Kuhn et al., PLoS ONE, 2008, 3: e2409; WO 2005/118825; WO2014198852 A2; US8765463.
  • the Ad11 adenovirus generally enters a cell through the CD46 receptor, which is expressed at low levels in all nucleated cells, but typically exhibits increased surface expression in tumor cells, thereby preferentially infecting tumor cells.
  • 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 described in, e.g., Kuhn et al., supra, in which pools of Ad serotypes, representing the different Ad subgroups, are passaged on human tumor cell lines representative of major solid tumor indications (e.g., breast cancer, colon cancer, pancreatic cancer, or prostate cancer) to invite recombination and selection of potent viral variants or serotypes.
  • the Ad3/11p viral vector is ColoAd1.
  • ColoAd1 has a nucleic acid sequence that has at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least bout 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to SEQ ID NO: 41.
  • ColoAd1 has the nucleic acid sequence of SEQ ID NO: 41.
  • the adenoviral vector is Ad5/3, such as described in, e.g., Hemminki et al., Oncotarget, 2015, 6: 4467-81; and Safar et al., Cancer Gene Therapy, 2021, 28: 442-454.
  • the Ad5/3 chimeric adenoviral vector includes the serotype 3 fiber knob in an Ad5 capsid, thereby using the Ad3 receptor (desmoglein-2), which is highly expressed in advanced tumors.
  • the vector is an adeno-associated viral (AAV) vector.
  • the AAV is an AAV of any serotype, such as AAV1, AAV2, AAV3, or AAV4.
  • the AAV is an AAV1. In some embodiments, the AAV is an AAV2. In some embodiments, the AAV is an AAV3. In some embodiments, the AAV is an AAV4. [0355] The genomes of AAVs have been determined. For instance, the genomic sequences of AAV2 are provided in GenBank Accession No. J01901 and GenBank No. NC_001401.
  • the viral vector comprises a tumor-specific promoter element that promotes increased expression of the encoded engineered antigen tag when expressed in a tumor cell, or a tumor cell of a specific cancer type, as compared to its expressed in a non-tumor cell, e.g., a normal non-cancerous cell of the same cell or tissue type as the tumor cell.
  • the tumor-specific promoter element is a STAT3 promoter, a survivin promoter, a cyclooxygenase-2 (COX-2) promoter, a prostate specific antigen (PSA) promoter, a CXCR4 promoter, or any other promoter that promoters increased expression in a tumor cell as compared to a normal cell of the same cell or tissue type.
  • Examples of tumor-specific promoter elements and methods for modifying viral vectors to include the same can be found in, e.g., US20190275093A1.
  • the viral vector is an oncolytic virus. Oncolytic viruses are capable of infecting and killing cancer cells.
  • the oncolytic virus can be, but is not limited to, adenoviruses, herpes simplex virus (HSV), parvoviruses, and poxviruses such as vaccinia virus (VACV) and myxoma virus (MYXV).
  • HSV herpes simplex virus
  • VACV vaccinia virus
  • MYXV myxoma virus
  • the oncolytic virus is selected from the group consisting of an adenovirus, a herpes simplex virus, a vaccinia virus, a mumps virus, a newcastle disease virus, a poliovirus, a seneca valley virus, a measles virus, a Sindbis virus, a parvovirus, a coxsackie virus, a vesicular stomatitis virus, a reovirus, and a maraba and rhabdovirus.
  • the viral vector exhibits weak oncolytic activity.
  • the vector is delivered specifically to a cancer cell. In some embodiments, the vector is delivered specifically to a blood cancer cell.
  • the vector is delivered specifically to a solid tumor cancer cell.
  • the vector is delivered specifically to an immune effector cell.
  • the immune effector cell is a cytotoxic T cell.
  • the immune effector cell is a natural killer cell.
  • compositions e.g., pharmaceutical compositions, comprising one or more of the synthetic cancer antigens and/or the cognate binders disclosed herein.
  • compositions e.g., pharmaceutical compositions, comprising any of the polynucleotides or any of the vectors disclosed herein.
  • compositions e.g., pharmaceutical compositions, comprising one or more immune effector cells or a population of cells disclosed herein.
  • the composition e.g., pharmaceutical composition, further comprises a pharmaceutical acceptable carrier.
  • compositions comprising: (i) one or more of any of the vectors disclosed herein, e.g., any of the vectors comprising one or more polynucleotides encoding one or more of the synthetic cancer antigens disclosed herein; and (ii) any of the CAR-T cell therapies disclosed herein, e.g., any of the CAR-T cell therapies comprising CAR-T cells that specifically bind to the target domain of any of the synthetic cancer antigens disclosed herein.
  • kits comprising: (i) a first composition, e.g., a first pharmaceutical composition, comprising one or more of any of the vectors disclosed herein, e.g., any of the vectors comprising one or more polynucleotides encoding one or more of the synthetic cancer antigens disclosed herein; and (ii) a second composition, e.g., a second pharmaceutical composition, comprising any of the CAR-T cell therapies disclosed herein, e.g., any of the CAR-T cell therapies comprising CAR-T cells that specifically bind to the target domain of any of the synthetic cancer antigens disclosed herein.
  • a first composition e.g., a first pharmaceutical composition
  • a second composition e.g., a second pharmaceutical composition
  • the pharmaceutical composition comprises both the synthetic cancer antigens and the cognate binders disclosed herein. In some embodiments, the pharmaceutical composition comprises both polypeptides encoding the synthetic cancer antigens and polypeptides encoding the cognate binders. In some embodiments, the pharmaceutical composition comprises both vectors comprising polypeptides encoding the synthetic cancer antigens and vectors comprising polypeptides encoding the cognate binders. In some embodiments, the pharmaceutical composition comprises both vectors comprising polypeptides encoding the synthetic cancer antigens and immune effector cells comprising or engineered by vectors comprising polypeptides encoding the cognate binders or population of cells comprising at least one such immune effector cell.
  • the first pharmaceutical composition comprises the synthetic cancer antigens, the polypeptides encoding the synthetic cancer antigens, or the vectors comprising the polypeptides encoding the synthetic cancer antigens.
  • the second pharmaceutical composition comprises the cognate binders, the polypeptides encoding the cognate binders, the vectors comprising the polypeptides encoding the cognate binders, the immune effector cells comprising or engineered by vectors comprising the polynucleotides encoding the cognate binders or population of cells comprising at least one such immune effector cell.
  • the first pharmaceutical composition comprises the synthetic cancer antigens
  • the second pharmaceutical composition comprises the cognate binders.
  • the first pharmaceutical composition comprises the polypeptides encoding the synthetic cancer antigens
  • the second pharmaceutical composition comprises the polypeptides encoding the cognate binders.
  • the first pharmaceutical composition comprises the vectors comprising the polypeptides encoding the synthetic cancer antigens
  • the second pharmaceutical composition comprises the vectors comprising the polypeptides encoding the cognate binders.
  • the first pharmaceutical composition comprises the vectors comprising the polypeptides encoding the synthetic cancer antigens
  • the second pharmaceutical composition comprises immune effector cells comprising or engineered by vectors comprising the polynucleotides encoding the cognate binders or population of cells comprising at least one such immune effector cell.
  • at least one of the immune effector cells expresses a CAR comprising a cognate binder disclosed herein on the surface of the immune effector cell.
  • the first composition e.g., the first pharmaceutical composition and the second composition, e.g., the second pharmaceutical composition are administered concurrently.
  • the first composition e.g., the first pharmaceutical composition is administered before the second composition, e.g., the second pharmaceutical composition being administered. In some embodiments, the first composition, e.g., the first pharmaceutical composition is administered after the second composition, e.g., the second pharmaceutical composition being administered. [0368] In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the first composition and the second composition are each a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carrier, diluent or excipient includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, and/or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans and/or domestic animals.
  • Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water
  • compositions include both acid and base addition salts.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4- acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglu
  • organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
  • Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • kits for carrying out a method described herein comprises one or more synthetic cancer antigens, cognate binders, polynucleotides, vectors, immune effector cells, populations of cells, and/or compositions, e.g., pharmaceutical compositions disclosed here.
  • a kit further comprises instructions for using the components of the kit to practice the methods of the present disclosure.
  • the instructions for practicing the methods or uses are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert or in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging).
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided.
  • kits that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
  • pharmaceutical compositions disclosed herein are for use in treating a cancer in a subject. Details of using pharmaceutical compositions disclosed herein for treating cancer are disclosed in Section VI, below. A.
  • compositions comprising one or more of any of the vectors disclosed herein, e.g., any of the vectors comprising one or more polynucleotides encoding one or more of the synthetic cancer antigens disclosed herein.
  • the composition is a pharmaceutical composition comprising one or more of any of the vectors disclosed herein, e.g., any of the vectors comprising one or more polynucleotides encoding one or more of the synthetic cancer antigens disclosed herein, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises a vector comprising a polynucleotide encoding any of the synthetic cancer antigens comprising a tumor-targeting binding molecule. [0379] In some embodiments, the pharmaceutical composition comprises a vector comprising a polynucleotide encoding any of the synthetic cancer antigens comprising a membrane targeting domain.
  • B. CAR-T CELL COMPOSITIONS Provided herein are compositions, e.g., pharmaceutical compositions, comprising a population of lymphocytes expressing a CAR, such as any of the CARs disclosed in Section II.D.3. [0381] In some embodiments, the lymphocytes are T cells.
  • kits comprising: (i) a first composition, e.g., a first pharmaceutical composition, comprising one or more of any of the vectors disclosed herein, e.g., any of the vectors comprising one or more polynucleotides encoding one or more of the synthetic cancer antigens disclosed herein; and (ii) a second composition, e.g., a second pharmaceutical composition, comprising any of the CAR-T cell therapies disclosed herein, e.g., any of the CAR-T cell therapies comprising CAR-T cells that specifically bind to the target domain of any of the synthetic cancer antigens disclosed herein.
  • a first composition e.g., a first pharmaceutical composition
  • a second composition e.g., a second pharmaceutical composition
  • the kit further comprises instructions for use of the first composition and the second composition, e.g., in the treatment of a cancer in a subject.
  • compositions e.g., pharmaceutical compositions, comprising: (i) one or more of any of the vectors disclosed herein, e.g., any of the vectors comprising one or more polynucleotides encoding one or more of the synthetic cancer antigens disclosed herein; and (ii) any of the CAR-T cell therapies disclosed herein, e.g., any of the CAR-T cell therapies comprising CAR-T cells that specifically bind to the target domain of any of the synthetic cancer antigens disclosed herein. VI.
  • RESULTS AND USES OF SYNTHETIC CANCER ANTIGENS comprising administering a therapeutically effective amount of one or more of any of the polynucleotide disclosed herein, e.g., in Section III, any of the vectors disclosed herein, e.g., in Section IV, or any of the compositions, e.g., pharmaceutical compositions, disclosed herein, e.g., in Section V, to a subject having a cancer.
  • the method further comprises administering a therapeutically effective amount of a therapy targeting the target domain of the synthetic cancer antigens, such as any of those disclosed in Section II.D.
  • Also provided herein are methods of treatment comprising: (i) administering a therapeutically effective amount of one or more of any of the polynucleotide disclosed herein, e.g., in Section III, any of the vectors disclosed herein, e.g., in Section IV, or any of the compositions, e.g., pharmaceutical compositions, disclosed herein, e.g., in Section V; and (ii) administering a therapeutically effective amount of a therapy targeting the target domain of the synthetic cancer antigen, such as any of those disclosed in Section II.D.
  • any of the synthetic cancer antigens disclosed herein e.g., in Section II, any of the polynucleotides disclosed herein, e.g., in Section III, any of the vectors disclosed herein, e.g., in Section IV, or any of the compositions, e.g., pharmaceutical compositions, disclosed herein, e.g., in Section V, in the manufacture of a medicament for the treatment of a cancer in a subject.
  • the treatment of a cancer is in further combination with a therapeutically effective amount of a therapy targeting the target domain of the synthetic cancer antigen, such as any of those disclosed in Section II.D.
  • the therapeutically effective amounts are amounts that when administered to a cancer subject deliver a synthetic cancer antigen comprising a target domain to a tumor cell (e.g. via administration of any of the provided polynucleotides, vectors or compositions comprising the same) and deliver a cognate binder that recognizes the target domain (e.g., via administration of cognate binder molecules such as a CAR or cells expressing the same) for directed therapeutic targeting of tumor cells.
  • a tumor tropic viral vector e.g., an oncolytic virus
  • a polynucleotide encoding the synthetic cancer antigen is administered to a cancer subject to deliver the synthetic cancer antigen in combination with engineered immune effector cells (e.g. T cells) expressing a CAR comprising the cognate binder as part of its extracellular binding domain for recognition of the target domain of the synthetic cancer antigen.
  • engineered immune effector cells e.g. T cells
  • the synthetic cancer antigen that is delivered is a membrane bound synthetic cancer antigen that is able to be expressed on the surface of infected tumor cells.
  • the synthetic cancer antigen that is delivered is a soluble synthetic cancer antigen that is secretable from infected tumor cells where it is able to bind to adjacent or bystander tumor cells in the tumor microenvironment.
  • a tumor tropic viral vector e.g. oncolytic virus
  • administering a cognate binder to the subject allows specific targeting to cancer cells within a tumor.
  • engineered immune effector cells e.g. T cells
  • a CAR comprising the cognate binder as part of its extracellular binding domain
  • CAR chimeric antigen receptor
  • synthetic cancer antigens disclosed herein e.g., in Section II, polynucleotides disclosed herein, e.g., in Section III, vectors disclosed herein, e.g., in Section IV, or compositions, e.g., pharmaceutical compositions, disclosed herein, e.g., in Section V, for use in the treatment of a cancer in a subject.
  • synthetic cancer antigens disclosed herein e.g., in Section II, polynucleotides disclosed herein, e.g., in Section III, vectors disclosed herein, e.g., in Section IV, or compositions, e.g., pharmaceutical compositions, disclosed herein, e.g., in Section V, for the treatment of a cancer in a subject.
  • the vector comprises a polynucleotide encoding a synthetic cancer antigen that comprises a target domain and a tumor-targeting binding molecule, e.g., any of those disclosed in Section II.C., and a polynucleotide encoding a synthetic cancer antigen that comprises a target domain and a membrane targeting domain, e.g., any of those disclosed in Section II.B.
  • the composition comprises a vector comprising a polynucleotide encoding a synthetic cancer antigen that comprises a target domain and a tumor-targeting binding molecule, e.g., any of those disclosed in Section II.C., and a polynucleotide encoding a synthetic cancer antigen that comprises a target domain and a membrane targeting domain, e.g., any of those disclosed in Section II.B.
  • the composition comprises: (i) a vector comprising a polynucleotide encoding a synthetic cancer antigen that comprises a target domain and a tumor-targeting binding molecule, e.g., any of those disclosed in Section II.C., and (ii) a vector comprising a polynucleotide encoding a synthetic cancer antigen that comprises a target domain and a membrane targeting domain, e.g., any of those disclosed in Section II.B.
  • the one or more polynucleotides comprise a first polynucleotide encoding a synthetic cancer antigen that comprises a target domain and a tumor-targeting binding molecule, e.g., any of those disclosed in Section II.C., and a second polynucleotide encoding a synthetic cancer antigen that comprises a target domain and a membrane targeting domain, e.g., any of those disclosed in Section II.B.
  • the one or more vectors comprise a first vector comprising a polynucleotide encoding a synthetic cancer antigen that comprises a target domain and a tumor-targeting binding molecule, e.g., any of those disclosed in Section II.C., and (ii) a second vector comprising a polynucleotide encoding a synthetic cancer antigen that comprises a target domain and a membrane targeting domain, e.g., any of those disclosed in Section II.B.
  • the one or more compositions comprise a first composition comprising a vector comprising a polynucleotide encoding a synthetic cancer antigen that comprises a target domain and a tumor-targeting binding molecule, e.g., any of those disclosed in Section II.C., and (ii) a second composition comprising a vector comprising a polynucleotide encoding a synthetic cancer antigen that comprises a target domain and a membrane targeting domain, e.g., any of those disclosed in Section II.B.
  • the therapy targeting the target domain of the synthetic cancer antigen is a CAR-T cell therapy, such as any of those disclosed in Section V.
  • the method described herein may be used in the treatment of a cell-proliferative disorder, such as a cancer.
  • the cancer is a solid cancer.
  • the cancer is blood cancer.
  • the cancer is solid tumor cancer.
  • cancers that may be treated using the synthetic cancer antigens, polynucleotides, vectors, compositions and methods disclosed herein include, but are not limited to, adenoma, carcinoma, sarcoma, leukemia or lymphoma.
  • the cancer is chronic lymphocytic leukemia (CLL), B cell acute lymphocytic leukemia (B-ALL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), diffuse large cell lymphoma (DLCL), diffuse large B cell lymphoma (DLBCL), Hodgkin’s lymphoma, multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, bladder cancer, breast cancer, colorectal cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, medulloblastoma, liver cancer, stomach cancer, thyroid cancer, bile duct cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, end
  • 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.
  • the cancer is bladder cancer.
  • the cancer is colorectal cancer.
  • the cancer is endometrial cancer.
  • the cancer is liver cancer.
  • the cancer is kidney cancer.
  • the cancer is lung cancer.
  • the cancer is melanoma.
  • the cancer is pancreatic cancer.
  • the cancer is prostate cancer.
  • the cancer is thyroid cancer.
  • the cancer is ovarian cancer. In some embodiments, the cancer is insensitive or resistant.
  • Administration of the pharmaceutical compositions can occur by injection, irrigation, inhalation, consumption, electro-osmosis, hemodialysis, iontophoresis, and other methods known in the art.
  • administration route is intraarterial, intracranial, intradermal, intraduodenal, intrammamary, intrameningeal, intraperitoneal, intrathecal, intratumoral, intravenous, intravitreal, ophthalmic, parenteral, spinal, subcutaneous, ureteral, urethral, vaginal, or intrauterine.
  • administration route is local or systemic.
  • the administration route is by infusion (e.g., continuous or bolus).
  • infusion e.g., continuous or bolus
  • methods for local administration that is, delivery to the site of injury or disease, include through an Ommaya reservoir, e.g. for intrathecal delivery (See e.g., US Patent Nos. 5,222,982 and 5,385,582, incorporated herein by reference); by bolus injection, e.g. by a syringe, e.g. into a joint; by continuous infusion, e.g. by cannulation, such as with convection (See e.g., US Patent Application Publication No.
  • the administration route is by topical administration or direct injection.
  • the engineered immune effector cells described herein may be provided to the subject alone or with a suitable substrate or matrix, e.g. to support their growth and/or organization in the tissue to which they are being transplanted.
  • the subject is a mammal, e.g., a human. In some embodiments, the subject is a human.
  • the subject may be a neonate, a juvenile, or an adult.
  • treating refers to the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting disease development or preventing disease progression; (b) relieving the disease, i.e., causing regression of the disease state or relieving one or more symptoms of the disease; and (c) curing the disease, i.e., remission of one or more disease symptoms.
  • treatment may refer to a short-term (e.g., temporary and/or acute) and/or a long-term (e.g., sustained) reduction in one or more disease symptoms.
  • treatment results in an improvement or remediation of the symptoms of the disease.
  • the improvement is an observable or measurable improvement, or may be an improvement in the general feeling of well-being of the subject.
  • the effective amount of the pharmaceutical compositions administered to a particular subject will depend on a variety of factors, several of which will differ from patient to patient including the disorder being treated and the severity of the disorder; activity of the specific agent(s) employed; the age, body weight, general health, sex and diet of the patient; the timing of administration, route of administration; the duration of the treatment; drugs used in combination; the judgment of the prescribing physician; and like factors known in the medical arts.
  • the effective amount of the pharmaceutical compositions may be the number of cells required to result in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more fold decrease in tumor mass or volume, decrease in the number of tumor cells, or decrease in the number of metastases. In some embodiments, the effective amount of the pharmaceutical compositions may be the number of cells required to achieve an increase in life expectancy, an increase in progression-free or disease-free survival, or amelioration of various physiological symptoms associated with the disease being treated.
  • a therapeutically effective amount of the pharmaceutical composition comprising the synthetic cancer antigens, polynucleotides encoding the synthetic cancer antigens, or vectors comprising polynucleotides encoding the synthetic cancer antigens are administered to a subject having a cancer.
  • a therapeutically effective amount of the pharmaceutical composition comprising the CAR-T cell therapy is administered to a subject having a cancer.
  • a therapeutically effective amount of the pharmaceutical composition comprising the polynucleotides encoding the synthetic cancer antigen, or the vector comprising polynucleotides encoding the synthetic cancer antigen, and a therapeutically effective amount of the pharmaceutical composition comprising the CAR-T cell therapy is administered to a subject having a cancer.
  • a therapeutically effective amount of the pharmaceutical composition comprising the cognate binders, polynucleotides encoding the cognate binders, vectors comprising polynucleotides encoding the cognate binders, or immune effector cells comprising or engineered by vectors comprising polynucleotides encoding the cognate binders or population of cells comprising at least one such immune effector cell are administered to a subject.
  • at least 1 x 10 3 immune effector cells expressing a CAR comprising a cognate binder disclosed herein on the surface of the cells are administered to a subject.
  • At least 5 x 10 3 such cells, 1 x 10 4 such cells, 5 x 10 4 such cells, 1 x 10 5 such cells, 5 x 10 5 such cells, 1 x 10 6 , 2 x 10 6 , 3 x 10 6 , 4 x 10 6 , 5 x 10 6 , 1 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , 5 x 10 10 , 1 x 10 11 , 5 x 10 11 , 1 x 10 12 , 5 x 10 12 , or more such cells are administered to a subject.
  • between about 1 x 10 7 and about 1 x 10 12 such cells are administered to a subject. In some embodiments, between about 1 x 10 8 and about 1 x 10 12 such cells are administered to a subject. In some embodiments, between about 1 x 10 9 and about 1 x 10 12 such cells are administered to a subject. In some embodiments, between about 1 x 10 10 and about 1 x 10 12 such cells are administered to a subject. In some embodiments, between about 1 x 10 11 and about 1 x 10 12 such cells are administered to a subject. In some embodiments, between about 1 x 10 7 and about 1 x 10 11 such cells are administered to a subject.
  • between about 1 x 10 7 and about 1 x 10 10 such cells are administered to a subject. In some embodiments, between about 1 x 10 7 and about 1 x 10 9 such cells are administered to a subject. In some embodiments, between about 1 x 10 7 and about 1 x 10 8 such cells are administered to a subject. [0410]
  • the number of administrations of treatment to a subject may vary. In some embodiments, introducing the pharmaceutical compositions into the subject may be a one- time event. In some embodiments, such treatment may require an on-going series of repeated treatments. In some embodiments, multiple administrations of the pharmaceutical compositions may be required before an effect is observed. The exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated.
  • the pharmaceutical compositions disclosed herein are administered in combination with additional therapeutic composition.
  • administration of the pharmaceutical compositions disclosed herein in combination with the additional therapeutic composition results in an enhanced therapeutic effect in a cancer than is observed by treatment with either the pharmaceutical compositions disclosed herein or the additional therapeutic composition alone.
  • the cancer is resistant, refractory, or insensitive to treatment by the additional therapeutic composition alone.
  • the cancer is partially resistant, partially refractory, or partially insensitive to treatment by the additional therapeutic composition alone.
  • the second therapeutic composition is an immune checkpoint inhibitor. Several immune checkpoint inhibitors are known in the art and have received FDA approval for the treatment of one or more cancers.
  • FDA- approved PD-L1 inhibitors include Atezolizumab (Tecentriq ® , Genentech), Avelumab (Bavencio®, Pfizer), and Durvalumab (Imfinzi®, AstraZeneca); FDA-approved PD-1 inhibitors include Pembrolizumab (Keytruda ® , Merck) and Nivolumab (Opdivo ® , Bristol- Myers Squibb); and FDA-approved CTLA4 inhibitors include Ipilimumab (Yervoy®, Bristol-Myers Squibb).
  • Additional inhibitory immune checkpoint molecules that may be the target of future therapeutics include A2AR, B7-H3, B7-H4, BTLA, IDO, LAG3 (e.g., BMS- 986016, under development by BSM), KIR (e.g., Lirilumab, under development by BSM), TIM3, TIGIT, and VISTA.
  • the second therapeutic composition is CAR expressing immune effector cells, where the CAR recognize a target other than the target domain of the synthetic cancer antigens being administered to the subject.
  • Non-limiting examples of such CARS include CD171-specific CARs (Park et al., Mol Ther (2007) 15(4):825-833), EGFRvIII-specific CARs (Morgan et al., Hum Gene Ther (2012) 23(10):1043-1053), EGF- R-specific CARs (Kobold et al., J Natl Cancer Inst (2014) 107(1):364), carbonic anhydrase K-specific CARs (Lamers et al., Biochem Soc Trans (2016) 44(3):951-959), FR- ⁇ -specific CARs (Kershaw et al., Clin Cancer Res (2006) 12(20):6106-6015), HER2-specific CARs (Ahmed et al., J Clin Oncol (2015) 33(15)1688-1696;Nakazawa et al., Mol Ther (2011) 19(12):2133-2143; Ahmed et al., Mol Ther (2009) 17(10):1779-1787; Luo e
  • engineered cells expressing any of the provided cognate binders or any of the provided synthetic cancer antigens.
  • A. Cells Expressing the cognate binder of Synthetic Cancer Antigen Provided herein are immune effector cells comprising or engineered by vectors comprising the polynucleotides or nucleic acid molecules encoding the cognate binders of synthetic cancer antigens provided herein. Also provided herein are population of cells comprising at least one such immune effector cell.
  • immune effector cells comprise or are engineered by vectors comprising the polynucleotides or nucleic acid molecules encoding the cognate binders
  • these immune effector cells can express the cognate binders on the cell surface.
  • Such immune effector cells can be used to kill cancer cells expressing the synthetic cancer antigens provided herein.
  • the term “immune effector cell” refers to cells involved in mounting innate and adaptive immune responses, including but not limited to lymphocytes, natural killer (NK) cells, NKT cells, macrophages, monocytes, eosinophils, basophils, neutrophils, dendritic cells, and mast cells.
  • the immune effector cell is a lymphocyte.
  • the immune effector cell is a cytotoxic T cell, such as a CD4+ T cell, a CD8+ T cell (also referred to as a cytotoxic T cell or CTL), a regulatory T cell (Treg), a Th1 cell, a Th2 cell, or a Th17 cell.
  • the immune effector cell is a natural killer cell.
  • the immune effector cell is a myeloid cell.
  • the immune effector cell is a monocyte.
  • immune effector cell is a macrophage.
  • the immune effector cell is an animal cell or is derived from an animal cell, including invertebrate animals and vertebrate animals (e.g., fish, amphibian, reptile, bird, or mammal).
  • the immune effector cell is a mammalian cell or is derived from a mammalian cell (e.g., a pig, a cow, a goat, a sheep, a rodent, a non-human primate, a human, etc.).
  • the immune effector cell is a human cell or is derived from a human cell.
  • the population of unengineered immune effector cells are autologous.
  • autologous in this context refers to cells that have been derived from the same subject to which they are administered.
  • immune effector cells may be obtained from a subject, engineered ex vivo, and then administered to the same subject in order to treat a disease.
  • the population of unengineered immune effector cells are allogeneic.
  • allogenic in this context refers to cells that have been derived from one subject and are administered to another subject.
  • immune effector cells may be obtained from a first subject, engineered ex vivo, and then administered to a second subject in order to treat a disease such as a cancer.
  • the engineered immune effector cells described herein further comprise an exogenous transgene encoding a detectable tag.
  • detectable tags include but are not limited to, FLAG tags, poly-histidine tags (e.g. 6xHis), SNAP tags, Halo tags, cMyc tags, glutathione-S-transferase tags, avidin, enzymes, fluorescent proteins, luminescent proteins, chemiluminescent proteins, bioluminescent proteins, and phosphorescent proteins.
  • the fluorescent protein is selected from the group consisting of blue/UV proteins (such as BFP, TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, and T-Sapphire); cyan proteins (such as CFP, eCFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, and mTFP1); green proteins (such as: GFP, eGFP, meGFP (A208K mutation), Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, and mNeonGreen); yellow proteins (such as YFP, eYFP, Citrine, Venus, SYFP2, and TagYFP); orange proteins (such as Monomeric Kusabira-Orange, mKO ⁇ , mKO2, mOrange, and mOrange2); red proteins (such as
  • Safety-switch systems (also referred to in the art as suicide gene systems) comprise exogenous transgenes encoding for one or more proteins that enable the elimination of an engineered immune effector cell after the cell has been administered to a subject.
  • Examples of safety-switch systems are known in the art.
  • safety-switch systems include genes encoding for proteins that convert non-toxic pro-drugs into toxic compounds such as the Herpes simplex thymidine kinase (Hsv-tk) and ganciclovir (GCV) system (Hsv-tk/GCV). Hsv-tk converts non-toxic GCV into a cytotoxic compound that leads to cellular apoptosis.
  • Hsv-tk Herpes simplex thymidine kinase
  • GCV ganciclovir
  • administering to a subject that has been treated with the engineered immune effector cells comprising a transgene encoding the Hsv-tk protein can selectively eliminate the engineered immune effector cells while sparing endogenous immune effector cells.
  • the engineered immune effector cells comprising a transgene encoding the Hsv-tk protein
  • Additional safety-switch systems include transgenes encoding pro-apoptotic molecules comprising one or more binding sites for a chemical inducer of dimerization (CID), enabling elimination of engineered immune effector cells by administration of a CID which induces oligomerization of the pro-apoptotic molecules and activation of the apoptosis pathway.
  • CID chemical inducer of dimerization
  • the pro-apoptotic molecule is Fas (also known as CD95) (Thomis et al., Blood, 2001, 97(5), 1249-1257).
  • the pro-apoptotic molecule is caspase-9 (Straathof et al., Blood, 2005, 105(11), 4247-4254).
  • the engineered immune effector cells described herein further comprise an exogenous transgene encoding a chimeric switch receptor.
  • Chimeric switch receptors are engineered cell-surface receptors comprising an extracellular domain from an endogenous cell-surface receptor and a heterologous intracellular signaling domain, such that ligand recognition by the extracellular domain results in activation of a different signaling cascade than that activated by the wild type form of the cell-surface receptor.
  • the engineered immune effector cells described herein comprise a transgene encoding a PD1-CD28 switch receptor, wherein the extracellular domain of PD1 is fused to the intracellular signaling domain of CD28 (See e.g., Liu et al., Cancer Res 76:6 (2016), 1578-1590 and Moon et al., Molecular Therapy 22 (2014), S201).
  • the engineered immune effector cells described herein comprise a transgene encoding the extracellular domain of CD200R and the intracellular signaling domain of CD28 (See Oda et al., Blood 130:22 (2017), 2410-2419).
  • cells comprising one or more of any of the polynucleotides disclosed herein, e.g., in Section III, or any of the vectors disclosed herein, e.g., in Section IV.
  • the cell is a tumor cell.
  • the cell is a tumor cell of a cancer that is non-Hodgkin’s lymphoma (NHL), diffuse large cell lymphoma (DLCL), diffuse large B cell lymphoma (DLBCL), Hodgkin’s lymphoma, multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, skin cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, medulloblastoma, liver cancer, stomach cancer, thyroid cancer, bile duct cancer, liver cancer, bone cancer, colon cancer, rectal cancer, endometrial cancer, or cervical cancer.
  • NHL non-Hodgkin’s lymphoma
  • DLCL diffuse large cell lymphoma
  • DLBCL diffuse large B cell lymphoma
  • the cell is a tumor cell of a cancer that 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.
  • the cell e.g., tumor cell
  • the cell is ex vivo.
  • the cell e.g., tumor cell
  • the cell is in vivo.
  • the cell, e.g., tumor cell is in vitro. VIII. EMBODIMENTS [0428]
  • This invention provides the following non-limiting embodiments. 1.
  • the target domain comprises a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 10.
  • the synthetic cancer antigen of embodiment 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 of embodiment 14 or 15, wherein the target domain comprises an amino acid sequence of SEQ ID NO: 8. 17.
  • the synthetic cancer antigen of any one of embodiments 14-16, wherein the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 13. 18.
  • the synthetic cancer antigen of any one of embodiments 14-17, wherein the cognate binder comprises an amino acid sequence of SEQ ID NO: 13. 19.
  • the synthetic cancer antigen of any one of embodiments 14-16, wherein the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 14. 20.
  • 21. The synthetic cancer antigen of any one of embodiments 1-3, wherein the target domain is a fragment of an intracellular protein. 22.
  • the synthetic cancer antigen of embodiment 25 or 26 wherein the cognate 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 of any one of embodiments 29-35, wherein the cognate binder of the synthetic cancer antigen is a leucine zipper domain. 37.
  • the synthetic cancer antigen of any one of embodiments 16-20, 27, 28, and 36-40, wherein the cognate binder of the synthetic cancer antigen is the extracellular domain of a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the synthetic cancer antigen of embodiment 43, wherein the immune effector cell is a cytotoxic T cell.
  • the synthetic cancer antigen of embodiment 43, wherein the immune effector cell is a natural killer cell.
  • the synthetic cancer antigen of any one of embodiments 1-45, wherein the membrane targeting domain is a transmembrane domain.
  • 47. The synthetic cancer antigen of embodiment 46, wherein the transmembrane domain is from a transmembrane glycoprotein.
  • the synthetic cancer antigen of embodiment 46 or 47, wherein the transmembrane domain is a CD8 transmembrane domain.
  • the synthetic cancer antigen of embodiment 51 that is soluble.
  • the synthetic cancer antigen of embodiment 51 or embodiment 52, wherein the tumor-targeting binding molecule specifically binds to a tumor associated antigen expressed on the surface of a tumor cell. 54.
  • the synthetic cancer antigen of embodiment 53 wherein the tumor associated antigen is selected from the group consisting of EpCAM, CEA (Carcinoembryonic antigen), gpA33 (Glycoprotein A33 (Transmembrane)), mucins, TAG-72 (Tumor-associated glycoprotein 72), CAIX (Carbonic anhydrase IX), PSMA (Prostate-specific membrane antigen), and FBP (Folate-binding protein), EGFR/ERBB1/HER1 (epidermal growth factor receptor 1), ERBB2/HER2 (epidermal growth factor receptor 2), ERBB3 (epidermal growth factor receptor 3), MET (Tyrosine-Protein Kinase IGF1R (insulin-like growth factor 1 receptor), EPHA3 (EPH Receptor A3), TRAILR1 (Death receptor 4), and RANK-L (Receptor activator of nuclear factor kappa-B ligand).
  • RCC renal cell carcinoma
  • neuroblastoma colorectal cancer
  • bladder cancer breast cancer
  • ovarian cancer melanoma
  • the synthetic cancer antigen of any of embodiments 52-56, wherein the tumor cell is a tumor cell of a cancer selected from the group consisting of bladder cancer, breast cancer, skin cancer, head and neck cancer, colorectal cancer, endometrial cancer, liver cancer, kidney cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, thyroid cancer, and ovarian cancer.
  • the tumor cell targeting domain comprises an antibody or an antigen-binding fragment thereof.
  • the synthetic cancer antigen of embodiment 58, wherein the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). 60.
  • VH heavy chain complementarity determining region 1
  • CDR-H2 heavy chain complementarity determining region 1
  • CDR-H3 comprising the amino acid sequence set forth in SEQ ID
  • the synthetic cancer antigen of any of embodiments 58-61, wherein the antibody or an antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 35; and a light chain variable (VL) region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 37.
  • VH heavy chain variable
  • VL light chain variable
  • the synthetic cancer antigen of any of embodiments 58-62, wherein the antibody or an antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 35, and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 37.
  • VH heavy chain variable
  • VL light chain variable
  • the synthetic cancer antigen of embodiment 68, wherein the antibody or antigen binding fragment does not contain the heavy chain CH3 domain. 70.
  • the synthetic cancer antigen of any one of embodiments 51-71, wherein the target domain is a single-chain variable fragment (scFv).
  • the synthetic cancer antigen of embodiment 75, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 8.
  • the synthetic cancer antigen of any one of embodiments 63-66, wherein the target domain is a fragment of an intracellular protein. 83.
  • the synthetic cancer antigen of embodiment 82 wherein the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor.
  • the synthetic cancer antigen of embodiment 90 wherein the target domain is the leucine zipper domain of ATF. 92.
  • the synthetic cancer antigen of embodiment 90, wherein the target domain is the leucine zipper domain of JUN. 95.
  • the synthetic cancer antigen of embodiment 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 of embodiment 97, wherein the cognate binder comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 29. 101.
  • CAR chimeric antigen receptor
  • the synthetic cancer antigen of embodiment 104, wherein the immune effector cell is a cytotoxic T cell.
  • the synthetic cancer antigen of embodiment 104 wherein the immune effector cell is a natural killer cell.
  • the linker has a length of between 1 and 100 amino acids, between 1 and 75 amino acids, between 1 and 50 amino acids, between 1 and 25 amino acids, between 5 and 100 amino acids, between 5 and 75 amino acids, between 5 and 50 amino acids, between 5 and 25 amino acids, between 10 and 100 amino acids, between 10 and 75 amino acids, between 10 and 50 amino acids, or between 10 and 25 amino acids.
  • the linker has a length of between 1 and 100 amino acids, between 1 and 75 amino acids, between 1 and 50 amino acids, between 1 and 25 amino acids, between 5 and 100 amino acids, between 5 and 75 amino acids, between 5 and 50 amino acids, between 5 and 25 amino acids, between 10 and 100 amino acids, between 10 and 75 amino acids, between 10 and 50 amino acids, or between 10 and 25 amino acids.
  • the polynucleotide of embodiment 112 wherein the nucleic acid sequence encoding the synthetic cancer antigen of any one of embodiments 1-50 and the nucleic acid sequence encoding the synthetic cancer antigen of any one of embodiments 51-109 are separated by a cleavable linker 114.
  • the vector of any one of embodiments 116-118, wherein the vector is delivered specifically to a cancer cell.
  • the vector of embodiment 119, wherein the vector is delivered specifically to a blood cancer cell.
  • the vector of embodiment 119, wherein the vector is delivered specifically to a solid tumor cancer cell.
  • the vector of any one of embodiments 119-121, wherein the vector is a viral vector.
  • the vector of embodiment 123 wherein the oncolytic virus is selected from the group consisting of an adenovirus, a herpes simplex virus, a vaccinia virus, a mumps virus, a newcastle disease virus, a poliovirus, a seneca valley virus, a measles virus, a Sindbis virus, a parvovirus, a coxsackie virus, a vesicular stomatitis virus, a reovirus, and a maraba and rhabdovirus.
  • an adenovirus a herpes simplex virus, a vaccinia virus, a mumps virus, a newcastle disease virus, a poliovirus, a seneca valley virus, a measles virus, a Sindbis virus, a parvovirus, a coxsackie virus, a vesicular stomatitis virus, a reovirus, and a maraba
  • the viral vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, or a herpes simplex viral (HSV) vector.
  • AAV adeno-associated virus
  • HSV herpes simplex viral
  • the vector of any one of embodiments 122-127, wherein the viral vector is an adenoviral vector.
  • the vector of embodiment 128 or embodiment 129, wherein the adenoviral vector is Ad3, Ad7, Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, or Ad50.
  • the vector of embodiment 128 or embodiment 129, wherein the adenoviral vector is a chimeric adenoviral vector based on Ad3, Ad7, Ad11, Ad14, Ad16, Ad17, Ad21, Ad35, Ad47, or Ad50.
  • 132 The vector of any one of embodiments 128, 129, and 131, wherein the adenoviral vector is a chimeric adenoviral vector that is based on Ad11.
  • the pharmaceutical composition of embodiment 141 wherein the pharmaceutical composition further comprises a pharmaceutical acceptable carrier.
  • the pharmaceutical composition of embodiment 141 or 142 for use in treating a cancer in a subject.
  • a combination comprising a vector of any one of embodiments 116-138 and a population of lymphocytes expressing a chimeric antigen receptor (CAR) that recognizes the target domain of the synthetic cancer antigen 145.
  • CAR chimeric antigen receptor
  • the combination of embodiment 144, wherein the CAR comprises an extracellular binding domain comprising a cognate binder of the target domain, a transmembrane domain and an intracellular signaling domain.
  • target domain is an antibody or antibody fragment and the cognate binder binds to the idiotype of the antibody or antibody fragment.
  • the combination of embodiment 145, wherein the target domain is a non- DNA binding domain or an inactive leucine zipper domain of a transcription factor. 148.
  • the combination of embodiment 145, wherein the target domain is an active leucine zipper domain of a transcription factor.
  • the combination of embodiment 145 or 148, wherein the target domain is the leucine zipper domain of ATF. 150.
  • the combination of embodiment 145 or 148, wherein the target domain is the leucine zipper domain of JUN. 151.
  • the combination of embodiment 145, 148, 149, 150, wherein the cognate binder of the synthetic cancer antigen is a leucine zipper domain.
  • the combination of embodiment 145, wherein the lymphocytes comprise cytotoxic T cell. 153.
  • lymphocytes comprise natural killer cell 154.
  • a kit comprising the combination of any one of embodiments 144-153 155.
  • the kit of embodiment 154 further comprising instructions for using the vector and the population of lymphocytes.
  • a method of treatment comprising: administering a therapeutically effective amount of the polynucleotide of any one of embodiments 110-115, the vector of any one of embodiments 116-138, or the pharmaceutical composition of any one of embodiments 141- 143, to a subject having a cancer, and administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) that bind to the target domain of the synthetic cancer antigen 157.
  • CAR chimeric antigen receptor
  • a method of treatment comprising administering a therapeutically effective amount of a population of lymphocytes expressing a chimeric antigen receptor (CAR) to a subject having a cancer who was previously administered the polynucleotide of any one of embodiments 110-115, the vector of any one of embodiments 116-138, or the pharmaceutical composition of any one of embodiments 141-143, wherein the CAR binds to the target domain of the synthetic cancer antigen.
  • CAR chimeric antigen receptor
  • the method of any one of embodiments 156-158 comprising a vector of any one of embodiments 116-138 and a population of lymphocytes expressing a chimeric antigen receptor (CAR) that recognizes the target domain of the synthetic cancer antigen.
  • CAR chimeric antigen receptor
  • target domain is an antibody or antibody fragment and the cognate binder binds to the idiotype of the antibody or antibody fragment.
  • 162 The method of embodiment 160, wherein the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor. 163.
  • the method of embodiment 160 wherein the target domain is an active leucine zipper domain of a transcription factor. 164. The method of embodiment 160 or 163, wherein the target domain is the leucine zipper domain of ATF. 165. The method of embodiment 160 or 163, wherein the target domain is the leucine zipper domain of JUN. 166. The method of any one of embodiments 160, 163, 164, and 165, wherein the cognate binder of the synthetic cancer antigen is a leucine zipper domain. 167. The method of any one of embodiments 160-166, wherein the lymphocytes comprise cytotoxic T cell. 168. The method of embodiment 167, wherein the lymphocytes comprise natural killer cell. 169.
  • a method of tagging a tumor cell in vivo comprising contacting the tumor cell with the polynucleotide of any one of embodiments 110-115, the vector of any one of embodiments 116-138, or the pharmaceutical composition of any one of embodiments 141- 143 170.
  • a method of tagging a tumor cell of a subject having a cancer comprising administering a therapeutically effective amount of the polynucleotide of any one of embodiments 110-115, the vector of any one of embodiments 116-138, or the pharmaceutical composition of any one of embodiments 141-143, to the subject 171.
  • a method of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 141 or 142.
  • a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain binds to the synthetic cancer antigen of any one of embodiments 1-50.
  • a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 13, 14, 24, 25, 28, and 29.
  • the CAR of embodiment 183, wherein the extracellular domain binds to a synthetic cancer antigen.
  • the synthetic cancer antigen comprises a target domain and a membrane targeting domain.
  • the CAR of embodiment 184, wherein the target domain is not expressed on the surface of a non-cancer cell of a subject. 187.
  • the CAR of embodiment 184 or 185, wherein the target domain is an antibody or a molecule derived from an antibody.
  • the CAR of embodiment 187, wherein the target domain is an antibody without the heavy chain CH3 domain.
  • the CAR of embodiment 188, wherein the target domain comprises a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 10. 190.
  • the CAR of embodiment 188, wherein the target domain comprises a light chain comprising an amino acid sequence of SEQ ID NO: 7 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 10. 191.
  • the CAR of embodiment 187, wherein the target domain is a single-chain variable fragment (scFv). 192.
  • the CAR of embodiment 191, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 11. 193.
  • the CAR of embodiment 191, wherein the target domain comprises an amino acid sequence of SEQ ID NO: 11. 194.
  • the CAR of embodiment 187, wherein the target domain is the variable domain of the heavy chain of an antibody. 195.
  • the CAR of embodiment 197, wherein the extracellular domain of the CAR comprises an amino acid sequence of SEQ ID NO: 13.
  • the CAR of embodiment 197, wherein the extracellular domain of the CAR comprises an amino acid sequence of SEQ ID NO: 14. 200.
  • the CAR of embodiment 184 or 185, wherein the target domain is a fragment of an intracellular protein.
  • the CAR of embodiment 200, wherein the target domain is a non-DNA binding domain or an inactive leucine zipper domain of a transcription factor.
  • the CAR of embodiment 201, wherein the target domain comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 17-19 and 21-23.
  • the CAR of embodiment 201, wherein the target domain comprises an amino acid sequence of SEQ ID NOs: 17-19 and 21-23.
  • the CAR of embodiment 201, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 17.
  • the CAR of embodiment 201, wherein the target domain comprises an amino acid sequence of SEQ ID NO: 17.
  • the CAR of embodiment 184 or 185, wherein the target domain is an active leucine zipper domain of a transcription factor.
  • the CAR of embodiment 207, wherein the target domain is the leucine zipper domain of ATF. 209.
  • the CAR of embodiment 208, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 26. 210.
  • the CAR of embodiment 208, wherein the target domain comprises an amino acid sequence of SEQ ID NO: 26. 211.
  • the CAR of embodiment 207, wherein the target domain is the leucine zipper domain of JUN. 212.
  • the CAR of embodiment 211, wherein the target domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 27. 213.
  • the CAR of embodiment 211, wherein the target domain comprises an amino acid sequence of SEQ ID NO: 27. 214.
  • the CAR of embodiment 214, wherein the extracellular domain of the CAR comprises an amino acid sequence of SEQ ID NO: 28. 216.
  • the CAR of embodiment 214, wherein the extracellular domain of the CAR comprises an amino acid sequence of SEQ ID NO: 29.
  • the CAR of any one of embodiments 182-216, wherein the transmembrane domain of the CAR is a CD8 transmembrane domain. 218.
  • the CAR of embodiment 217, wherein the transmembrane domain of the CAR comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3. 219.
  • the CAR of embodiment 217, wherein the transmembrane domain of the CAR comprises an amino acid sequence of SEQ ID NO: 3. 220.
  • the CAR of any one of embodiments 182-219, wherein the intracellular signaling domain comprises a primary intracellular signaling domain. 221.
  • the CAR of embodiment 220, wherein the intracellular signaling domain further comprises a co-stimulatory signaling domain. 222.
  • the CAR of embodiment 221, wherein the intracellular signaling domain comprises a CD3z cytoplasmic signaling domain and a 41BB co-stimulatory signaling domain. 223.
  • the CAR of embodiment 222, wherein the intracellular signaling domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 16.
  • the CAR of embodiment 222, wherein the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 16.
  • a cell comprising or engineered by the vector of embodiment 226. 228.
  • the cell of embodiment 228, wherein the cell is an immune effector cell.
  • the cell of embodiment 229, wherein the immune effector cell is a cytotoxic T cell. 231.
  • the cell of embodiment 229, wherein the immune effector cell is a natural killer cell.
  • a population of cells comprising at least one cell of any one of embodiments 227-231.
  • a pharmaceutical composition comprising the CAR of any one of embodiments 182-224, the polynucleotide of embodiment 225, the vector of embodiment 226, the cell of any one of embodiments 227-231, or the population of cells of embodiment 232. 234.
  • the pharmaceutical composition of embodiment 233 wherein the pharmaceutical composition further comprises a pharmaceutical acceptable carrier.
  • 236 A method of treating a cancer in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 233 or 234. 237.
  • 238 The method of embodiment 236 or 237, wherein the pharmaceutical composition and the additional pharmaceutical composition are administered concurrently.
  • Example 1 Design and Generation of Antibody-derived Synthetic cancer antigens
  • the first category of synthetic cancer antigens i.e., flares
  • the polynucleotide encoding an antibody-derived synthetic cancer antigen was delivered into a cancer cell through a gene delivery vector, such as an oncolytic virus.
  • the synthetic cancer antigen was then expressed on the surface of the cancer cell by linking it to a transmembrane domain.
  • a T-cell expressing binding partner against the idiotype of the antibody can then be directed to the cancer cell, thus killing the cancer cell.
  • FIG.2A The design of antibody-derived synthetic cancer antigens is shown in FIG.2A.
  • a synthetic cancer antigen based on the complete Fab of the Ab1 antibody was generated that could be used in combination with a CAR binder. The goal was to begin testing the flare concept using a CAR binder to the complete Fab of the Ab1 antibody, and subsequently reducing its size without affecting the idiotype for the CAR binder.
  • FIG. 2B The details of the construct design are shown in FIG. 2B.
  • the initial and largest construct a membrane-bound Ab1-Fab, was designed by using the nucleotide sequences of the light chain and the heavy chain of the Ab1 antibody (Table 1), which was then linked to a transmembrane domain.
  • the construct had a size of 2001bp.
  • An IgG kappa leader sequence was used as a signal peptide.
  • the sequence of the light chain (Ab1 LC-VL, set forth in SEQ ID NO:5 and Ab1 LC-CL, set forth in SEQ ID NO:6; the full sequence of the light chain set forth in SEQ ID NO:7) was introduced upstream, followed by a 2A self-cleaving peptide (P2A), and then the sequence of a truncated heavy chain composed of the variable domain (Ab1 HC VH, set forth in SEQ ID NO:8) and two constant domains (Ab1 HC-CH1 and Ab1 HC-CH2, set forth in SEQ ID NO:9, the full sequence of the heavy chain set forth in SEQ ID NO:10).
  • P2A 2A self-cleaving peptide
  • a CD8 transmembrane domain was used to tether the protein on the surface of the cell (SEQ ID NO:3). Note that the signal peptide is cut off from the nascent synthetic cancer antigen to form the mature synthetic cancer antigen once the nascent synthetic cancer antigen has been translocated across the cell membrane.
  • Ab1-scFv A smaller version of the membrane-bound antibody-derived synthetic cancer antigen, Ab1-scFv, was generated by replacing the Fab antibody in Ab1-Fab with the scFv of the Ab1 antibody via subcloning.
  • the resulting Ab1-scFv included an scFv containing the VH and VL of Ab1 separated by a linker (scFv set forth in SEQ ID NO:11) linked to the CD8 transmembrane domain (SEQ ID NO:3). Similar to above, the polynucleotide construct included a signal peptide, which is cut off once the synthetic cancer antigen has been translocated across the cell membrane. The construct had a size of 1005bp. [0435] A membrane-bound Ab1-V H was developed to further minimize the size of the antibody-derived synthetic cancer antigen. The scFv of the Ab1-scFv construct was replaced with the variable region of the heavy chain of the Ab1 antibody.
  • the resulting membrane bound Ab1-V H included the heavy chain variable domain of Ab1 (SEQ ID NO:8) linked to the CD8 transmembrane domain (SEQ ID NO:3).
  • the heavy chain variable domain of Ab1 may be linked to the transmembrane domain directly or through use of a linker or spacer.
  • the polynucleotide construct included a signal peptide. The construct had a size of 654bp. [0436] A Flag-tag was added at the N-terminus for detection purposes.
  • the IgGk leader sequence used in Ab1-Fab and Ab1- scFv was replaced with a human CD8 leader sequence.
  • leader sequences that can be used that translocate the expressed nascent synthetic cancer antigen to the membrane where it is cut-off to form the mature synthetic cancer antigen without the signal peptide.
  • All these sequences were introduced into the backbone of a lentiviral vector comprising a EF1a promoter and a WPRE segment.
  • Mini-prep DNA was sequenced to confirm the insertion of the designed sequences and one mini-prep was selected for further maxi-prep DNA preparation of each design.
  • Related sequences are listed below in Table E1.
  • FIG. 3A- FIG. 3C the expression of Ab1-Fab (FIG. 3A), Ab1-ScFv (FIG. 3B), and Ab1- V H (FIG. 3C) were shown to be between 90-100% for different cancer cells after being sorted for positivity.
  • Example 2 Design and Generate CAR-T Cells Targeting Antibody-derived Synthetic cancer antigens
  • CAR molecules that can recognize the antibody-derived synthetic cancer antigens disclosed in Example 1 were designed and generated.
  • Three anti-Ab1 molecules were tested: 1) a llama single domain anti-idiotype antibody (for Ab1_CAR1, SEQ ID NO:13), 2) a human anti-idiotype scFv (for Ab1_CAR2, SEQ ID NO:14), and 3) another human anti- idiotype scFv (for Ab1_CAR3, SEQ ID NO:15).
  • the CAR backbone polypeptide from the N-terminus to the C-terminus comprises the following elements: an Igk leader sequence as a signal peptide, anti-Ab1 molecule, a CD8 transmembrane domain, a 41BB co- stimulatory cytoplasmic domain, and a CD3z cytoplasmic domain.
  • the sequences of the Igk leader sequence and the CD8 transmembrane domain are the same as shown in Table E1. Additional related sequences are listed below in Table E2.
  • Table E2 Sequences Related to CARs against Antibody-derived Synthetic cancer antigens [0443] Lentivirus was produced with the LV-Max system (Thermo) according to the manufacturer’s instructions.
  • the calculated titer was used to transduce T cells at specific MOIs for further testing and comparison between CAR molecules.
  • All T cells were grown in RPMI-1640 with 10% FBS, 1% pen-strep, 1% NEAA, 1% GlutaMAX, and 1% sodium pyruvate.
  • HDs primary T cells from healthy donors (HDs) were incubated with 5 ng/mL IL-7 and 5 ng/mL IL-15 for 3-4 hrs before overnight activation with anti-CD3/anti-CD28 beads at a ratio of 3:1 (beads:cells).
  • T cells were transduced with CAR-carrying LVs on day 1, de-beaded and fed on day 3 to double the previous culture volume with media containing 5 ng/mL IL-7 and 5 ng/mL IL-15.
  • T cells were fed to 8E5 cells/mL with media without IL-7 and IL-15.
  • T cells were assessed by flow cytometry for CAR expression and baseline phenotyping before being frozen in FBS with 10% DMSO.
  • FIG. 5A and FIG. 5B all transduced T cells properly expanded.
  • Two cancer cell lines (A549 and PC3) were transduced with adenovirus expressing Ab1-Fab and co-cultured with T cells expressing either Ab1_CAR1 or Ab1_CAR2 at different E:T ratios. As shown in FIG. 12B – FIG. 12C, CAR-T cells successfully killed both cancer cell lines expressing Ab1-Fab in a dose-dependent and time- dependent manner.
  • An adenovirus carrying Ab1-V H was also generated.
  • Four cancer cell lines (A549, HCT-116, SKOV3, and PC3) were transduced with adenovirus expressing Ab1-V H and co- cultured with Ab1_CAR2-expressing T cells at different E:T ratios. As shown in FIG.
  • a cancer cell line, HT29 was infected with Ad3/11 expressing Ab1- V H at 0.1 particles per cell (PPC). Then, infected cancer cells were co-cultured with T cells expressing Ab1_CAR1 at a 1:64 CAR T cell ratio. Tumor viability was measured over a span of 150 hours by CellCyteX fluorescent detection. In this assay, the HT29 tumor cells were engineered to express a fluorescent tag, which was utilized as a readout for the frequency of viable tumor cells in a given well. As shown in FIG. 12F, CAR-T cells successfully killed the cancer cell line expressing Ab1-V H synthetic antigen in a time-dependent manner.
  • HER2-positive HCT-116 expressing Ab1- V H were engrafted subcutaneously in immunocompromised mice. 10 days after the engraftment, the tumor grew to the size of around 100 mm 3 . The mice were then treated with T cells expressing either Ab1_CAR2 or anti-HER2 CAR. The probability of survival, tumor volume, and the expansion of T cells were measured. The process is illustrated in FIG. 13A. [0460] As shown in FIG. 13B, the probability of survival is positively correlated with the concentration of T cells expressing Ab1_CAR2.
  • T cells expressing Ab1_CAR2 had a better killing efficacy than T cells expressing anti-HER2 CAR.
  • the tumor volume was negatively correlated with the concentration of T cells expressing Ab1_CAR2.
  • T cells expressing Ab1_CAR2 also expanded better than T cells expressing anti-HER2 CAR.
  • the similar in vivo test was performed for mice carrying HER2-positive HCT-116 expressing Ab1-Fab treated with T cells expressing Ab1_CAR1 at different concentrations.
  • FIG. 13E the combination of Ab1-Fab and Ab1_CAR1 leds to no significant killing of cancer cells in the tested xenograft mouse model.
  • Example 4 Design, Generate, and Test Protein-fragment Based Synthetic cancer antigens
  • synthetic cancer antigens i.e., flares
  • fragments of intracellular proteins e.g., fragments of transcription factors that are non-DNA binding domains and/or dead leucine zippers tethered to transmembrane localization domains.
  • the polynucleotide encoding the protein-fragment based synthetic cancer antigen was delivered into a cancer cell through a gene delivery vector, such as an oncolytic virus. The synthetic cancer antigen was then expressed on the surface of the cancer cell.
  • a T-cell expressing binding partner against the protein fragment (e.g., a scFv) can then be directed to the cancer cell, thus killing the cancer cell.
  • a scFv binding partner against the protein fragment
  • a list of protein fragment candidates, as well as the cognate binder of one of the candidates, is shown in Table E3 below. Table E3 Sequences Related to Protein-fragment Based Synthetic Cancer Antigens and Cognate Binders
  • Lentiviral vectors were prepared with the top candidate molecule and used to transduce three different cancer cell lines (A549, A375, and PC3) were transduced with lentivirus carrying IC1. As shown in FIG. 16A, IC1 expressed at high levels on the surface of the three cancer cell lines. Cell lines that were not 95-100% positive for IC1 were further sorted to get a 98-100% positivity.
  • scFv CAR molecules comprising the VL and VH of an anti-IC1 antibody connected by a linker (SEQ ID NO:24), a CD8 TM domain (SEQ ID NO:3), and intracellular co-stimulatory molecules 41BB and CD3z (SEQ ID NO:16) were designed and generated.
  • SEQ ID NO:24 a linker
  • CD8 TM domain SEQ ID NO:3
  • CD3z intracellular co-stimulatory molecules 41BB and CD3z
  • T cells expressing either binder 1 or binder 2 against IC1 successfully killed cancer cells expressing IC1 in a time-dependent manner, with binder 1 showing a better killing capacity than binder 2.
  • T cells expressing binder 1 successfully killed different cancer cells expressing IC1 in a dose-dependent and time-dependent manner.
  • Example 5 Design, Generate, and Test Leucine Zipper Based Synthetic cancer antigens [0471]
  • synthetic cancer antigens i.e., flares
  • LZ partner leucine zipper
  • the polynucleotide encoding the leucine zipper based synthetic cancer antigen was delivered into a cancer cell through a gene delivery vector, such as an oncolytic virus.
  • the synthetic cancer antigen was then expressed on the surface of the cancer cell.
  • a T-cell expressing a complementary leucine zipper domain can then be directed to the cancer cell, thus killing the cancer cell.
  • CAR molecules comprising either TF1- LZ (SEQ ID NO:28) or TF3-LZ (SEQ ID NO:29), a CD8 TM domain (SEQ ID NO:3), and intracellular co-stimulatory molecules 41BB and CD3z (SEQ ID NO:16) were designed and produced;
  • synthetic cancer antigens comprising either TF2-LZ (SEQ ID NO:26; partner of TF1-LZ) and TF4-LZ (SEQ ID NO:27; partner of TF3-LZ), a c-Myc tag (SEQ ID NO:30), and a CD8 TM domain were designed and produced.
  • a synthetic cancer antigen can be generated without the tag.
  • Related sequences are listed below in Table E4.
  • Table E4 Sequences Related to Protein-fragment Based Synthetic Cancer Antigens and Cognate Binders [0474] As shown in FIG. 20A, both TF2-LZ and TF4-LZ expressed on the surface of cancer cells. As shown in FIG. 20B and FIG. 20C, both TF1-LZ and TF3LZ expressed on the surface of T cells. [0475] T cell-mediated killing of cancer cells is evaluated. The methods are the same as described in Example 3 above. T cells expressing TF1-LZ successfully kill different cancer cells expressing TF2-LZ in a dose-dependent and time-dependent manner.
  • T cells expressing TF3-LZ successfully kill different cancer cells expressing TF4-LZ in a dose- dependent and time-dependent manner.
  • Example 6 Effect of Soluble Synthetic Cancer Antigens on Tumor Cell Killing
  • Ab1-V H FL1; SEQ ID NO:8 was linked via a linker (SEQ ID NO: 38) to anti- EpCAM scFv, a tumor-targeted antibody fragment.
  • the anti-EpCAM scFv antibody contains a variable heavy chain (VH) region (SEQ ID NO:35) and a variable light (VL) region (SEQ ID NO:37) separated by a linker (SEQ ID NO: 33), with sequences as set forth in Table E5.
  • VH variable heavy chain
  • VL variable light
  • SEQ ID NO: 33 linker
  • An IgG1 signal sequence (MGWSCIILFLVATATGVHS; SEQ ID NO:87) was added to the N-terminus for secretion to the culture supernatant.
  • a FLAG tag sequence DYKDDDDK; SEQ ID NO:12
  • the full sequence of the FL1-antibody conjugate is set forth in SEQ ID NO:31.
  • a variant of the FL1-antibody conjugate without the addition of the FLAG is set forth in SEQ ID NO: 39 (encoded protein without signal peptide comprising SEQ ID NO:89).
  • Table E5 Sequences Related to soluble Antibody-derived Synthetic cancer antigens [0478] The soluble FL1-antibody synthetic cancer antigen conjugated with the anti- EpCam scFv (this experiment included the Flag tag and signal peptide, polynucleotide encoding SEQ ID NO: 31; encoded protein without signal peptide comprises SEQ ID NO: 88) or the membrane-bound Ab1-V H (FL1) format described in Example 1 was delivered to tumor cells in vitro by the chimeric adenovirus Ad3/Ad11p..
  • Chimeric adenovirus Ad3/Ad11p (also known as ColoAd1) is a chimeric adenovirus with coat proteins derived from Ad11p and with a deletion of the E3 and E4 gene and a chimeric Ad3/Ad11p E3B gene (US8765463 and WO2014198852; e.g., SEQ ID NO:41).
  • the membrane bound Ab1-V H included the heavy chain variable domain of Ab1 (SEQ ID NO:8) linked to the CD8 transmembrane domain (SEQ ID NO:3).
  • the viral vector encoding the soluble and secreted synthetic cancer antigen or the membrane bound synthetic cancer antigen was delivered at varying concentrations (10 PPC, 100 PPC, and 1000 PPC, schematic shown in FIG.
  • the soluble FL1-antibody synthetic cancer antigen was delivered to tumor cells by the chimeric adenovirus Ad3/Ad11p (FIG. 23A). After 48 hours, supernatant from the cell cultures was collected and added to wells containing untransduced HCT116 or SKOV3 cancer cells lines at a 1:10 dilution. Instead of detecting FL1 labeling as described above, the tumor cells were then incubated with anti-FL1 CAR-T cells (T cells expressing Ab1_CAR2 containing the scFv set forth in SEQ ID NO:14, as described in Example 2). T cell-mediated killing was measured on an xCELLigence microelectronic biosensor system (Agilent).
  • mice were then treated with three doses of Ad3/Ad11p adenovirus (three doses of 5 x 10 9 viral particles/dose) expressing the soluble FL1-antibody synthetic cancer antigen (polynucleotide encoding SEQ ID NO: 31, encoded protein without signal peptide comprising SEQ ID NO: 90) or the membrane bound Ab1-V H (FL1), described in Example 1. 7 days after the doses, the mice were treated with 1 x 10 6 T cells expressing Ab1_CAR2. Tumors were harvested 13 days later for assessment. The process is illustrated in FIG. 24A. [0483] As seen in FIG.
  • mice that did not receive the CAR-T cells approximately 70% of the tumor cells were positive for FL1 binding after infection of tumor cells with Ad3/Ad11p adenovirus carrying the soluble FL1-antibody synthetic antigen as determined by staining with anti-flag antibody, demonstrating effective distribution in vivo of the soluble synthetic cancer antigen.
  • the number of infiltrating T cells in the tumor increased in mice given doses of either the soluble FL1-antibody synthetic cancer antigen or the membrane-bound Ab1-V H (FL1) (FIG. 24C).
  • An additional experiment was performed, except instead of harvesting the tumor the mice were monitored, as illustrated by FIG. 24D.
  • A549 non-small cell lung cancer cells were engrafted subcutaneously in immunocompromised mice. 15 days after the engraftment, the tumor grew to the size of around 100-150 mm 3 . The mice were then treated with three Ad3/Ad11p adenovirus doses (three doses of 5 x 10 9 viral particles/dose) expressing the soluble FL1-antibody synthetic cancer antigen, as described above. 7 days after the doses, the mice were treated with 1 x 10 6 T cells expressing Ab1_CAR2. Tumor growth was then measured periodically over the next 50 days. [0485] As seen in FIG.
  • tumors in mice that received doses of both the FL1-antibody synthetic cancer antigen and the CAR-T cells grew less than tumors in mice that received only the FL1-antibody from the administered Ad3/Ad11p adenovirus or non-specific T cells. Further, the growth of the tumors in mice that received the FL1-antibody synthetic cancer antigen and the CAR-T cells was comparable to the tumors in mice that received a control tumor cell line with stable expression of the FL1 antigen and the CAR-T cells. [0486] This demonstrates the effective use of the soluble FL1-antibody synthetic cancer antigen in targeted tumor cell killing.
  • Example 7 Assess the Universality of Soluble Synthetic Cancer Antigens on Tumor Cell Killing
  • Ab1-V H FL1; SEQ ID NO:8
  • SEQ ID NO: 38 linker
  • anti-HER2 scFv 4D5 sequence contains a variable heavy chain (VH) region set forth in SEQ ID NO:8 and a variable light (VL) region set forth in SEQ ID NO:5, separated by a linker.
  • the sequence of the anti-HER2 scFv has the sequence set forth in SEQ ID NO:11.
  • SKOV3 HER2-expressing tumor cells were cultured with purified protein of the soluble FL1-antibody synthetic cancer antigen at varying concentrations. FL1-antibody surface binding on the tumor cells was subsequently measured by flow cytometry. As seen in FIG. 25A, cells cultured with the FL1-anti-HER2 synthetic cancer antigen showed detection of the FL1 on the surface in a dose-dependent manner.
  • T cell-mediated killing of cancer cells expressing antibody-derived synthetic cancer antigens was measured on an xCELLigence microelectronic biosensor system (Agilent).
  • xCELLigence microelectronic biosensor system Agilent
  • Real-Time Cell Analysis 96- well plates were filled with 50 ⁇ L media for blank baseline measurement of impedance.
  • Target cancer cells expressing HER2 either SKOV3 cells or A549 cells
  • electrical impedance measurements were initiated 30 min after plating.
  • anti-FL1 CAR-T cells T cells expressing Ab1_CAR2 containing the scFv set forth in SEQ ID NO:14, as described in Example 2
  • anti-FL1 CAR-T cells T cells expressing Ab1_CAR2 containing the scFv set forth in SEQ ID NO:14, as described in Example 2
  • Measurements were acquired every 15 min for the duration of the assay and the cell index was normalized to the time point at which T cells were added to the co-culture.
  • CAR-T cells successfully killed both SKOV3 and A549 cancer cell lines with surface bound FL1-anti-HER2 in a dose-dependent and time- dependent manner.
  • This demonstrates that the soluble synthetic cancer antigen can utilize other antibodies to target different cancer antigens for tumor cell killing.
  • Example 8 Effect of the Combination of Membrane-bound and Soluble Synthetic Cancer Antigens on Tumor Cell Killing [0492] The effect of utilizing both the membrane-bound format (i.e., tethered format) of the synthetic cancer antigen, as described in Example 1, and the soluble synthetic cancer antigen, described in Example 6, was assessed for targeted tumor cell killing in vitro.
  • the FL1-anti-EpCAM synthetic cancer antigen (this experiment included the Flag tag and the signal peptide, polynucleotide encoding SEQ ID NO: 31; encoded protein without the signal peptide comprises SEQ ID NO: 88) and the membrane-bound Ab1-V H (FL1; SEQ ID NO:8) format described in Example 1 was delivered to A549 tumor cells in vitro by the chimeric adenovirus Ad3/Ad11p to result in varying concentrations (0 MOI (control), 0.1 MOI, 1 MOI). After 72 hours, cell surface binding and display of the FL1-antibody synthetic cancer antigen was detected on infected cells using an anti-flag antibody labeled with PE, as measured by flow cytometry.
  • the initial culture of A549 tumor cells expressed high levels of the Ab1-V H (FL1). Additionally, as shown in the right panel of FIG. 26, HCT- 116 cells cultured with supernatant from the infected A549 cells showed detection of the FL1 on the surface, indicating the soluble FL1-anti-EpCAM synthetic cancer antigen was secreted from the infected A549 cells. This demonstrates the effectiveness of utilizing a combination of the tethered and the soluble synthetic cancer antigen formats in targeted tumor cell killing, in which both infected tumor cells as well as bystander tumor cells can be selectively tagged with a targetable antigen.

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Abstract

L'invention concerne des antigènes synthétiques de cancers non exprimés sur la surface de cellules non cancéreuses et/ou de liants apparentés de celles-ci. L'invention concerne également une composition pharmaceutique comprenant les antigènes synthétiques de cancers et/ou les liants apparentés et des méthodes d'utilisation de celle-ci pour traiter un trouble de prolifération cellulaire, tel qu'un cancer.
PCT/US2024/031157 2023-05-25 2024-05-24 Antigènes synthétiques de cancers en tant que cibles pour le traitement de cancers WO2024243578A1 (fr)

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