CN117425491A

CN117425491A - Chimeric antigen receptor with MAGE-A4 specificity and uses thereof

Info

Publication number: CN117425491A
Application number: CN202280040562.1A
Authority: CN
Inventors: K·布雷; F·德尔菲诺; D·迪利略
Original assignee: Regeneron Pharmaceuticals Inc
Current assignee: Regeneron Pharmaceuticals Inc
Priority date: 2021-05-04
Filing date: 2022-05-03
Publication date: 2024-01-19

Abstract

MAGE-A4 or melanoma-associated antigen A4 is cancer-testis antigen (CTA) on the X chromosome. The present disclosure provides MAGE-A4 specific chimeric antigen receptors, cells expressing such chimeric antigen receptors, and isolated MAGE-A4 specific antibodies. In certain embodiments, engineered cells expressing these chimeric antigen receptors of the present disclosure are capable of inhibiting the growth of tumors that express MAGE-A4. The engineered cells of the present disclosure are useful in the treatment of diseases and conditions where upregulated or induced MAGE-A4 targeted immune responses are desired and/or therapeutically beneficial. For example, engineered cells expressing the MAGE-A4 specific chimeric antigen receptor of the present disclosure can be used for the treatment of a variety of cancers.

Description

Chimeric antigen receptor with MAGE-A4 specificity and uses thereof

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application No. 63/184,183 filed on day 4 5 of 2021 and U.S. provisional application No. 63/239,293 filed on day 31 of 2021 in 35 USC 119 (e), each of which is incorporated herein by reference in its entirety for any purpose.

Reference to sequence Listing

The sequence listing created by month 3 of 2022 and containing 95,050 bytes in computer readable form as filed in file 10901WO01-sequence. Txt is incorporated by reference.

Technical Field

The present disclosure provides antibodies, chimeric Antigen Receptors (CARs) and engineered cells comprising such antibodies and CARs specific for melanoma-associated antigen A4 (MAGE-A4), and methods of their use.

Background

MAGE-A4 or melanoma-associated antigen A4 is cancer-testis antigen (CTA) on the X chromosome. The function of MAGE-A4 is unknown, but it may be involved in cell cycle progression/regulation, transcriptional control, cell survival and/or apoptosis. For example, MAGE-A4 overexpression has been shown to promote growth of spontaneously transformed oral mucosal cells and to inhibit growth arrest of G1 cells.

Many different histological types of tumors express large amounts of MAGE-A4, such as head and neck squamous cell carcinoma, lung cancer (e.g., non-small cell lung cancer), esophageal squamous cell carcinoma, colon cancer, bladder cancer, mucosal and cutaneous melanoma, ovarian cancer (e.g., serous carcinoma), and uterine cancer, but in normal healthy adult tissue MAGE-A4 expression is limited to testes.

The ability of MAGE-A4 antigen to elicit an immune response and its limited expression pattern makes MAGE-A4 a good candidate for cancer immunotherapy.

The dual targeting antibody strategy applied to complex diseases such as cancer also represents a promising strategy whereby multifactorial modulation aims at improving efficacy. CD3 is a homodimeric or heterodimeric antigen expressed on T cells that binds to the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed by dimeric association of two of four different chains: epsilon, ζ, delta, and gamma. Bispecific antibodies with MAGE-A4 binding arms and CD3 binding arms can be used for enhancing antitumor activity.

Adoptive immunotherapy involving the transfer of ex vivo generated autoantigen-specific T cells is another promising strategy for the treatment of viral infections and cancers. T cells for adoptive immunotherapy can be generated by genetic engineering using expansion of antigen-specific T cells or redirection of T cells.

New specificities in T cells have been successfully created by genetic transfer of transgenic T cell receptors or Chimeric Antigen Receptors (CARs). CARs are synthetic receptors, consisting of targeting moieties associated with one or more signaling domains in a single fusion molecule. Typically, the binding portion of the CAR consists of the antigen binding domain of a single chain antibody (scFv), including the light and heavy chain variable fragments of monoclonal antibodies linked by flexible linkers. The signaling domain of the first generation CAR is derived from the cytoplasmic domain of the cd3ζ or Fc receptor γ chain. First generation CARs have been shown to successfully redirect T cell cytotoxicity. However, CARs fail to provide prolonged amplification and anti-tumor activity in vivo. The signaling domain from co-stimulatory molecules, as well as the transmembrane and hinge domains, have been added to form second and third generation CARs, leading to some successful human therapeutic trials. For example, CAR-redirected T cells specific for the B cell differentiation antigen CD19 have shown significant efficacy in treating B cell malignancies, while TCR-redirected T cells have shown benefit in patients with solid cancer. Stauss et al describe strategies to modify therapeutic CARs and TCRs for the treatment of cancer, e.g., to enhance antigen specific effector function and limit toxicity of engineered T cells (Current Opinion inPharmacology 2015, 24:113-118).

There is an unmet need for new targeting agents based on dual targeting antibody strategies and/or CARs that specifically bind to the MAGE-A4 antigen, as well as methods for producing and using such agents in therapeutic and diagnostic environments.

Disclosure of Invention

In one aspect, the present disclosure provides an antigen binding protein that specifically binds to HLA-bound melanoma-associated antigen A4 (MAGE-A4), wherein the antigen binding protein comprises a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR), wherein the LCVR comprises Complementarity Determining Regions (CDRs) of the LCVR comprising the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:115, and wherein the HCVR comprises CDRs of the HCVR comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:83, or SEQ ID NO: 107.

In some embodiments, the LCVR comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO. 10 or SEQ ID NO. 115. In some embodiments, the HCVR comprises an amino acid sequence that is at least 95% identical to SEQ ID NO. 2, SEQ ID NO. 83, or SEQ ID NO. 107.

In some cases, the antigen binding protein interacts with amino acids 286-294 of SEQ ID NO. 32 or a portion thereof.

In one aspect, the present disclosure provides a MAGE-A4 specific Chimeric Antigen Receptor (CAR) comprising, from N-terminus to C-terminus: (a) An extracellular ligand binding domain comprising an anti-MAGE-A4 single chain variable fragment (scFv) domain, the anti-MAGE-A4 scFv domain comprising a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR); (b) a hinge; (c) a transmembrane domain; and (d) a cytoplasmic domain comprising a 4-1BB co-stimulatory domain or a CD28 co-stimulatory domain and a CD3 zeta signaling domain, wherein the LCVR comprises: a Complementarity Determining Region (CDR) of a LCVR comprising the amino acid sequence of SEQ ID NO. 10 or SEQ ID NO. 115 and a CDR of a HCVR comprising the amino acid sequence of SEQ ID NO. 2, 83 or 107.

In some embodiments, the MAGE-A4 specific CAR comprises, from N-terminus to C-terminus: (a) an extracellular ligand binding domain; (b) a hinge; (c) a transmembrane domain; and (d) a cytoplasmic domain comprising a co-stimulatory domain and a signaling domain. In some cases, the anti-MAGE-A4 scFv domain comprises a first linker between the LCVR and the HCVR.

In some embodiments, the MAGE-A4 specific CAR further comprises a second linker between the extracellular ligand binding domain and the hinge. In some cases, the first linker comprises an amino acid sequence selected from SEQ ID NOS.23-26 and the second linker comprises an amino acid sequence selected from SEQ ID NOS.23-26. In some cases, the first linker comprises the amino acid sequence of SEQ ID NO. 25 and the second linker comprises the amino acid sequence of SEQ ID NO. 23.

In various embodiments of the MAGE-A4 specific CAR, the hinge, the transmembrane domain, or both are from a CD8 a polypeptide.

In various embodiments of the MAGE-A4 specific CAR, the costimulatory domain comprises A4-1 BB costimulatory domain.

In various embodiments of the MAGE-A4 specific CAR, the co-stimulatory domain comprises a CD28 co-stimulatory domain.

In various embodiments of the MAGE-A4 specific CAR, the hinge, the transmembrane domain, or both are from a CD28 polypeptide.

In various embodiments of the MAGE-A4 specific CAR, the hinge comprises the amino acid sequence of SEQ ID NO. 27.

In various embodiments of the MAGE-A4 specific CAR, the transmembrane domain comprises the amino acid sequence of SEQ ID NO. 28.

In various embodiments of the MAGE-A4 specific CAR, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO. 29.

In various embodiments of the MAGE-A4 specific CAR, the hinge comprises the amino acid sequence of SEQ ID NO. 34.

In various embodiments of the MAGE-A4 specific CAR, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 36.

In various embodiments of the MAGE-A4 specific CAR, the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO. 38.

In various embodiments of the MAGE-A4 specific CAR, the signaling domain comprises a CD3 zeta signaling domain. In some cases, the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 30.

In various embodiments, the antigen binding protein is a MAGE-A4 specific antibody, or antigen binding fragment thereof.

In some cases, the antigen binding protein or MAGE-A4 specific CAR discussed above or herein comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) contained within a HCVR comprising the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO: 83. In some cases, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 4, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 6, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 8. In some cases, the HCVR comprises the amino acid sequence shown in SEQ ID NO. 2. In some cases, the HCVR comprises the amino acid sequence shown in SEQ ID NO. 83. In some cases, the antigen binding protein or MAGE-A4 specific CAR comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) contained within a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 107. In some cases, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 109, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 111, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 113. In some cases, the HCVR comprises the amino acid sequence shown in SEQ ID NO. 107.

In some cases, the antigen binding protein or MAGE-A4 specific CAR discussed above or herein comprises three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) contained within a LCVR comprising the amino acid sequence set forth in SEQ ID NO. 10 or SEQ ID NO. 115. In some cases, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 12, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 14, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 16. In some cases, the LCVR comprises the amino acid sequence shown in SEQ ID NO. 10. In some cases, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:117, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:14, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 119. In some cases, the LCVR comprises the amino acid sequence shown in SEQ ID NO. 115.

In some cases, the antigen binding protein or MAGE-A4 specific CAR discussed above or herein comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO. 2 and a LCVR comprising the amino acid sequence set forth in SEQ ID NO. 10. In some cases, the antigen binding protein or MAGE-A4 specific CAR discussed above or herein comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO. 83 and a LCVR comprising the amino acid sequence set forth in SEQ ID NO. 10. In some cases, the antigen binding protein or MAGE-A4 specific CAR discussed above or herein comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO. 107 and a LCVR comprising the amino acid sequence set forth in SEQ ID NO. 115.

In various embodiments, the MAGE-A4 specific CAR comprises the amino acid sequence of SEQ ID NO. 22. In various embodiments, the MAGE-A4 specific CAR comprises the amino acid sequence of SEQ ID NO. 105. In various embodiments, the MAGE-A4 specific CAR comprises the amino acid sequence of SEQ ID NO: 120. In various embodiments, the MAGE-A4 specific CAR comprises the amino acid sequence of SEQ ID NO: 121.

In various embodiments, the antigen binding proteins or MAGE-A4 specific CARs discussed above or herein specifically bind to one or more amino acids at positions 286-294 of SEQ ID NO. 32. In various embodiments, the antigen binding protein or MAGE-A4 specific CAR discussed above or herein interacts with one or more amino acids of HLA. In some cases, the HLA is HLA-A2.

In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding an antigen binding protein or MAGE-A4 specific CAR as discussed above or herein. In some cases, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO. 21. In some cases, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO. 104.

In one aspect, the present disclosure provides a vector comprising a nucleic acid molecule as discussed above or herein. In some cases, the vector is a DNA vector, RNA vector, plasmid, lentiviral vector, adenoviral vector, or retroviral vector. In some embodiments, the vector is a lentiviral vector.

In one aspect, the present disclosure provides a cell comprising a nucleic acid molecule as discussed above or herein. In some cases, the cell is a human T cell.

In one aspect, the present disclosure provides an engineered cell comprising an antigen binding protein or MAGE-A4 specific CAR as discussed above or herein. In some cases, the engineered cell is an immune cell. In some cases, the immune cell is an immune effector cell. In some cases, the immune effector cell is a T lymphocyte. In some cases, the T lymphocyte is an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte. In some cases, the engineered cell is a cd8+ cytotoxic T lymphocyte. In various embodiments, the engineered cells are used to treat MAGE-A4 expressing cancers. In some cases, the MAGE-A4 expressing cancer is multiple myeloma. In some cases, the MAGE-A4 expressing cancer is melanoma.

In one aspect, the present disclosure provides an engineered human T cell comprising a chimeric antigen receptor comprising, from N-terminus to C-terminus: (a) An extracellular ligand binding domain comprising an anti-MAGE-A4 single chain variable fragment (scFv) domain, the anti-MAGE-A4 scFv domain comprising a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR); (b) a hinge; (c) a transmembrane domain; and (d) a cytoplasmic domain comprising a 4-1BB co-stimulatory domain or a CD28 co-stimulatory domain and a CD3 zeta signaling domain, wherein the LCVR comprises: a Complementarity Determining Region (CDR) of a LCVR comprising the amino acid sequence of SEQ ID NO. 10 or SEQ ID NO. 115 and a CDR of a HCVR comprising the amino acid sequence of SEQ ID NO. 2, 83 or 107.

In various embodiments of the engineered human T cells, the anti-MAGE-A4 scFv specifically binds to one or more amino acid residues at positions 286-294 of SEQ ID NO. 32. In some cases, the scFv domain comprises a HCVR/LCVR amino acid sequence pair that includes the amino acid sequences of SEQ ID NO. 2/10. In some cases, the scFv domain comprises a HCVR/LCVR amino acid sequence pair that includes the amino acid sequences of SEQ ID NO. 2/83. In some cases, the scFv domain comprises a HCVR/LCVR amino acid sequence pair comprising the amino acid sequences of SEQ ID NO. 107/115. In some embodiments, the HCVR comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3), and the LCVR comprises three light chain CDRs (LCDR 1, LCDR2, and LCDR 3), wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6, HCDR3 comprises the amino acid sequence shown in SEQ ID NO:8, LCDR1 comprises the amino acid sequence shown in SEQ ID NO:12, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:14, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16. In some cases, the HCVR comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3), and the LCVR comprises three light chain CDRs (LCDR 1, LCDR2, and LCDR 3), wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO. 109, HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 111, HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 113, LCDR1 comprises the amino acid sequence shown in SEQ ID NO. 117, LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 14, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 119. In some cases, the hinge comprises the amino acid sequence of SEQ ID NO. 27. In some cases, the transmembrane domain comprises the amino acid sequence of SEQ ID NO. 28. In some cases, the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO. 29. In some cases, the hinge comprises the amino acid sequence of SEQ ID NO. 34. In some cases, the transmembrane domain comprises the amino acid sequence of SEQ ID NO. 36. In some cases, the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO. 38. In some cases, the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 30.

In various embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO. 22. In various embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO. 105. In various embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO. 120. In various embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO. 121.

In one aspect, the present disclosure provides a pharmaceutical composition comprising a genetically modified human T cell and a pharmaceutically acceptable carrier, wherein the genetically modified human T cell comprises an antigen binding protein or MAGE-A4 specific CAR as discussed above or herein. In some cases, the pharmaceutical composition comprises an engineered cell as discussed above or herein and a pharmaceutically acceptable carrier. In some cases, the pharmaceutical composition comprises an engineered human T cell as discussed above or herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions are used to treat MAGE-A4 expressing cancers. In some cases, the MAGE-A4 expressing cancer is multiple myeloma. In some cases, the MAGE-A4 expressing cancer is melanoma.

In one aspect, the present disclosure provides the use of an antigen binding protein or MAGE-A4 specific CAR, nucleic acid molecule, vector, cell, engineered cell, or engineered human T cell as discussed above or herein in the manufacture of a medicament for the treatment of a MAGE-A4 expressing cancer. In some cases, the MAGE-A4 expressing cancer is multiple myeloma. In some cases, the MAGE-A4 expressing cancer is melanoma.

In one aspect, the present disclosure provides a method of enhancing T lymphocyte activity in a subject, the method comprising introducing into the subject a T lymphocyte comprising an antigen binding protein or MAGE-A4 specific CAR as discussed above or herein.

In one aspect, the present disclosure provides a method for treating a subject having cancer, the method comprising introducing into the subject a therapeutically effective amount of a T lymphocyte comprising an antigen binding protein or MAGE-A4 specific CAR as discussed above or herein.

In one aspect, the present disclosure provides a method for stimulating a T cell-mediated immune response in a subject against a target cell population or tissue, the method comprising administering to the subject an effective amount of a cell genetically modified to express an antigen binding protein or MAGE-A4 specific CAR as discussed above or herein.

In one aspect, the present disclosure provides a method of providing anti-tumor immunity in a subject, the method comprising administering to the subject an effective amount of a cell genetically modified to express an antigen binding protein or MAGE-A4 specific CAR as discussed above or herein.

In any of the various methods discussed above or herein, the subject may be a human. In some embodiments, the subject has multiple myeloma, synovial sarcoma, esophageal cancer, head and neck cancer, lung cancer, bladder cancer, ovarian cancer, uterine cancer, stomach cancer, cervical cancer, breast cancer, or melanoma. In some embodiments, the subject has multiple myeloma.

In one aspect, the present disclosure provides a method of engineering a population of cells to express an antigen binding protein or MAGE-A4 specific CAR as discussed above or herein, the method comprising: (a) Introducing a nucleic acid molecule encoding an antigen binding protein or MAGE-A4 specific CAR as discussed above or herein into an immune cell population; (b) Culturing the population of immune cells under conditions that express the nucleic acid molecule; and (c) isolating immune cells expressing the MAGE-A4 specific antigen binding protein at the cell surface. In some cases, the method further comprises obtaining the population of immune cells from the subject prior to introducing the nucleic acid molecule.

In one aspect, the present disclosure provides a method of treating a MAGE-A4 expressing cancer in a subject, the method comprising: (a) Engineering a population of cells as discussed above or herein; and (b) reintroducing the population of cells expressing the chimeric antigen receptor into the subject. In some cases, the MAGE-A4 expressing cancer is multiple myeloma.

In one aspect, the present disclosure provides an isolated antigen binding protein, wherein the antigen binding protein comprises a first antigen binding domain that specifically binds to HLA-bound melanoma-associated antigen A4 (MAGE-A4) and a second antigen binding domain that specifically binds to human CD3, wherein the first antigen binding domain comprises three heavy chain Complementarity Determining Regions (CDRs) contained in a heavy chain variable region (A1-HCVR) (A1-HCDR 1, A1-HCDR2 and A1-HCDR 3) and three light chain variable regions (A1-LCVR) contained in a light chain CDR1, A1-LCDR2 and A1-LCDR 3), and the second antigen binding domain comprises three heavy chains (A2-HCDR 1, A2-HCDR2 and A2-HCDR 3) contained in a heavy chain variable region (A1-HCVR) and three heavy chain CDRs contained in a light chain variable region (A1-HCDR 2) and light chain CDRs (A1-LCVR) contained in a light chain variable region (A1-LCVR 2) and wherein the amino acid sequence comprising SEQ ID NO comprises SEQ ID NO 2, amino acid sequence comprising SEQ ID NO. 2-amino acid sequence of SEQ ID NO. 10.

In one aspect, the present disclosure provides an isolated antigen binding protein, wherein the antigen binding protein comprises a first antigen binding domain that specifically binds to HLA-bound melanoma-associated antigen A4 (MAGE-A4) and a second antigen binding domain that specifically binds to human CD3, wherein the first antigen binding domain comprises three heavy chain Complementarity Determining Regions (CDRs) contained in a heavy chain variable region (A1-HCVR) (A1-HCDR 1, A1-HCDR2 and A1-HCDR 3) and three light chain variable regions (A1-LCVR) contained in a light chain CDR (A1-LCDR 1, A1-LCDR2 and A1-LCDR 3), and the second antigen binding domain comprises three heavy chains (A2-HCDR 1, A2-HCDR2 and A2-HCDR 3) contained in a heavy chain variable region (A1-HCVR) and three heavy chain CDRs contained in a light chain variable region (A2-HCVR) and three light chain CDRs (A1-LCVR 2) contained in an amino acid sequence comprising SEQ ID NO. 10 and amino acid sequence comprising SEQ ID NO. 73, amino acid sequence 2-LCVR 10.

In various embodiments of the isolated antigen binding proteins discussed above or herein, the antigen binding protein interacts with amino acids 286-294 of SEQ ID NO. 32 or a portion thereof. In some cases, the isolated antigen binding protein is a CAR. In some cases, the isolated antigen binding protein is a bispecific antibody. In some embodiments, the isolated antigen binding protein interacts with CD 3. In some cases, the isolated antigen binding protein comprises a Heavy Chain Variable Region (HCVR) that comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO. 2 or SEQ ID NO. 83. In some cases, the isolated antigen binding protein comprises a Heavy Chain Variable Region (HCVR) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID No. 55. In some cases, the isolated antigen binding protein comprises a Heavy Chain Variable Region (HCVR) that comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO. 73.

In one aspect, the present disclosure provides an isolated antigen binding protein that binds to the same epitope as the antigen binding protein or MAGE-A4 specific CAR discussed above or herein. In one aspect, the present disclosure provides an isolated antigen binding protein that competes for binding with an antigen binding protein or MAGE-A4 specific CAR as discussed above or herein. In some cases, the isolated antigen binding protein is a CAR. In some cases, the isolated antigen binding protein is a bispecific antibody. In some cases, the isolated antigen binding protein interacts with amino acids 286-294 of SEQ ID NO. 32 or a portion thereof. In some cases, the isolated antigen binding protein interacts with CD 3. In some embodiments, the isolated antigen binding protein comprises a Heavy Chain Variable Region (HCVR) comprising three heavy chain CDRs, namely HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs 4, 6, and 8, respectively. In some embodiments, the isolated antigen binding protein comprises a HCVR corresponding to the other arm of the bispecific antibody, the HCVR comprising three heavy chain CDRs, i.e., HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs 57, 59, and 61, respectively. In some embodiments, the isolated antigen binding protein comprises a HCVR corresponding to the other arm of the bispecific antibody comprising three heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs 75, 77 and 79, respectively. In some cases, the isolated antigen binding protein comprises a Light Chain Variable Region (LCVR) comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO. 10 and/or a LCVR comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO. 63.

In one aspect, the present disclosure provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to a melanomA-Associated antigen A4 (MAGE-A4) polypeptide, wherein the antibody has one or more of the following characteristics: (a) In an amount of less than about 2 x 10 ^-9 M binds to the MAGE-A4 polypeptide at its EC 50; (b) Exhibits the ability to reduce tumor cell viability compared to an isolated recombinant antibody that does not specifically bind to a MAGE-A4 polypeptide; and/or (c) comprises: (i) Three heavy chain Complementarity Determining Regions (CDRs) (HCDR 1, HCDR2 and HCDR 3) contained within a Heavy Chain Variable Region (HCVR) comprising an amino acid sequence having at least about 90% sequence identity to a HCVR as set forth in table 1; and (ii) three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) contained within a Light Chain Variable Region (LCVR), the LCVR comprising an amino acid sequence that has at least about 90% sequence identity to a LCVR as set forth in table 1.

In some embodiments of the isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to a MAGE-A4 polypeptide, the MAGE-A4 polypeptide is a HLA-A 2-binding MAGE-A4 polypeptide. In some cases, the isolated antibody or antigen binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO. 2, SEQ ID NO. 83, or SEQ ID NO. 107. In some cases, the isolated antibody or antigen binding fragment thereof comprises a LCVR having the amino acid sequence of SEQ ID NO. 10 or SEQ ID NO. 115. In some cases, the isolated antibody or antigen binding fragment thereof comprises a HCVR/LCVR amino acid sequence pair of SEQ ID NO. 2/10, SEQ ID NO. 83/10, or SEQ ID NO. 107/115.

In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising: (a) An HCDR1 domain having the amino acid sequence of SEQ ID No. 4; (b) An HCDR2 domain having the amino acid sequence of SEQ ID No. 6; (c) An HCDR3 domain having the amino acid sequence of SEQ ID No. 8; (d) An LCDR1 domain having the amino acid sequence of SEQ ID NO. 12; (e) An LCDR2 domain having the amino acid sequence of SEQ ID NO. 14; and (f) an LCDR3 domain having the amino acid sequence of SEQ ID NO. 16.

In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising: (a) An HCDR1 domain having the amino acid sequence of SEQ ID No. 109; (b) An HCDR2 domain having the amino acid sequence of SEQ ID No. 111; (c) An HCDR3 domain having the amino acid sequence of SEQ ID No. 113; (d) An LCDR1 domain having the amino acid sequence of SEQ ID NO. 117; (e) An LCDR2 domain having the amino acid sequence of SEQ ID NO. 14; and (f) an LCDR3 domain having the amino acid sequence of SEQ ID NO: 119.

In various embodiments of the isolated antibodies or antigen binding fragments thereof discussed above or herein, the isolated antibodies or antigen binding fragments thereof are IgG1 antibodies or IgG4 antibodies. In some cases, the isolated antibody or antigen-binding fragment thereof is a bispecific antibody.

In one aspect, the present disclosure provides an isolated recombinant antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to a melanoma-associated antigen A4 (MAGE-A4) polypeptide and a second antigen-binding domain that specifically binds to a CD3 polypeptide, wherein: (a) The first antigen binding domain (A1) that binds MAGE-A4 comprises three heavy chain complementarity determining regions (A1-HCDR 1, A1-HCDR2, and A1-HCDR 3) and three light chain complementarity determining regions (A1-LCDR 1, A1-LCDR2, and A1-LCDR 3), wherein: A1-HCDR1 comprises the amino acid sequence of SEQ ID NO. 4 or SEQ ID NO. 109; A1-HCDR2 comprises the amino acid sequence of SEQ ID NO. 6 or SEQ ID NO. 111; A1-HCDR3 comprises the amino acid sequence of SEQ ID NO. 8 or SEQ ID NO. 113; A1-LCDR1 comprises the amino acid sequence of SEQ ID NO. 12, SEQ ID NO. 65 or SEQ ID NO. 117; A1-LCDR2 comprises the amino acid sequence of SEQ ID NO. 14; A1-LCDR3 comprises the amino acid sequence of SEQ ID NO. 16, SEQ ID NO. 67 or SEQ ID NO. 119; and (b) the second antigen binding domain (A2) that binds CD3 comprises three heavy chain complementarity determining regions (A2-HCDR 1, A2-HCDR2, and A2-HCDR 3) and three light chain complementarity determining regions (A2-LCDR 1, A2-LCDR2, and A2-LCDR 3), wherein: A2-HCDR1 comprises the amino acid sequence of SEQ ID NO. 57 or SEQ ID NO. 75; A2-HCDR2 comprises the amino acid sequence of SEQ ID NO. 59 or SEQ ID NO. 77; A2-HCDR3 comprises the amino acid sequence of SEQ ID NO. 61 or SEQ ID NO. 79; A2-LCDR1 comprises the amino acid sequence of SEQ ID NO. 12, SEQ ID NO. 65 or SEQ ID NO. 117; A2-LCDR2 comprises the amino acid sequence of SEQ ID NO. 14; A2-LCDR3 comprises the amino acid sequence of SEQ ID NO. 16, SEQ ID NO. 67 or SEQ ID NO. 119.

In one aspect, the present disclosure provides a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof as discussed above or herein and a pharmaceutically acceptable carrier or diluent. In some cases, the pharmaceutical composition further comprises a second therapeutic agent. In some cases, the second therapeutic agent is selected from the group consisting of: antineoplastic agents, steroids, and targeted therapies.

In one aspect, the invention provides a polynucleotide molecule comprising a polynucleotide sequence encoding one or more HCVR and/or one or more LCVR of an antibody as described above or herein, a vector comprising the polynucleotide, and a cell comprising the vector.

In one aspect, the present disclosure provides a method of treating a cancer that expresses MAGE-A4, the method comprising administering to a subject an antibody or antigen binding fragment as discussed above or herein or a pharmaceutical composition as discussed above or herein. In some cases, the pharmaceutical composition is administered in combination with a second therapeutic agent. In some cases, the second therapeutic agent is selected from the group consisting of: antineoplastic agents, steroids, and targeted therapies.

Other embodiments will become apparent from a reading of the detailed description that follows.

Drawings

FIG. 1 shows an exemplary nucleotide construct for expressing a Chimeric Antigen Receptor (CAR) construct. The exemplary nucleotide constructs comprise an anti-MAGE-A4 VL-linker-VH scFv, a human CD8 hinge and transmembrane domain, A4-1 BB costimulatory domain, a CD3 zeta signaling domain, and optionally an IRES to eGFP sequence for use in tracking CAR transduced cells.

Fig. 2 shows an exemplary bispecific antibody of the present disclosure. This exemplary bispecific antibody consists of one HCVR capable of interacting with MAGE-A4 (anti-MAGE-A4 arm) and another HCVR capable of interacting with CD3 (anti-CD 3 arm). In representative examples, LCVR is common (e.g., both correspond to anti-CD 3 antibodies or antibody light chains that are known to be scrambled or operatively paired with multiple heavy chain arms).

Detailed Description

It is to be understood that the invention described herein is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Any embodiment or feature of an embodiment may be combined with one another and such combination is expressly contemplated as being within the scope of the present invention. Any particular value discussed above or herein may be combined with another related value discussed above or herein to enumerate a range of values having upper and lower ends representing the range, and such ranges are encompassed within the scope of the present disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a particular recited value, the term "about" means that the value may differ from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are herein incorporated by reference in their entirety.

Definition of the definition

As used herein, the term "MAGE-A4" refers to melanoma-associated antigen A4.MAGE-A4 is an intracellular protein expressed by a number of different tumor cells. As used herein, "MAGE-A4" refers to a human MAGE-A4 protein unless specified as being from a non-human species (e.g., "mouse MAGE-A4", "monkey MAGE-A4", etc.). The human MAGE-A4 protein has the amino acid sequence shown as SEQ ID NO. 32 and the polynucleic acid sequence shown as SEQ ID NO. 31. Reference to a particular region of a MAGE-A4 polypeptide (e.g., MAGE-A4 286-294) is relative to SEQ ID NO: 32. As used herein, the terms "MAGE-A4 286-294", "MAGE-A4 (286-294)" and "MAGEA4 _286-294 "may be used interchangeably. The polypeptide sequence of MAGE-A4 (286-294) (KVLEHVVRV) is given as SEQ ID NO. 33.

As used herein, "an antibody that binds MAGE-A4" or "an anti-MAGE-A4 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize at least MAGE-A4. In some embodiments, antibodies that bind MAGE-A4 interact with amino acids 286-294 of MAGE-A4. As disclosed herein, an "antibody that binds to MAGE-A4" or an "anti-MAGE-A4 antibody" may also be capable of specifically recognizing other MAGE-A4 related peptides (e.g., MAGE-A4 related peptides predicted to form a complex with HLA-A 2). In addition, antibodies that bind MAGE-A4 can also bind one or more additional ligands, for example, if the antibodies are formatted as bispecific antibodies. In certain embodiments, the anti-MAGE-A4 antibody may be formatted as a bispecific antibody that binds to both MAGE-A4 and CD 3.

The terms "ligand binding domain" and "antigen binding domain" are used interchangeably herein and refer to that portion of a chimeric antigen receptor or corresponding antibody that specifically binds to a predetermined antigen (e.g., MAGE-A4). References to "corresponding antibodies" refer to antibodies derived from CDRs or variable regions (heavy chain variable regions (abbreviated as HCVR or VH) and light chain variable regions (abbreviated as LCVR or VL)) used in chimeric antigen receptors or bispecific antibodies. For example, the chimeric antigen receptor constructs discussed in the examples include scFv having variable regions derived from an anti-MAGE-A4 antibody. Such anti-MAGE-A4 antibodies are "corresponding antibodies" to the corresponding chimeric antigen receptor.

As used herein, the term "antibody" (which includes "bispecific antibody") means any antigen binding molecule or molecular complex comprising at least one Complementarity Determining Region (CDR) that specifically binds or interacts with a particular antigen (e.g., MAGE-A4). In some embodiments, the antibody can bind to or interact with an MHC-binding polypeptide (e.g., an HLA-binding polypeptide). In the context of the present disclosure, in some embodiments, antibodies may bind to polypeptides that bind to HLA-A2, such as MAGE-A4 polypeptides presented by HLA-A2 (e.g., MAGE-A4 286-294). The term "antibody" (which includes "bispecific antibodies") includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers (e.g., igM) thereof. However, immunoglobulin molecules consisting of heavy chains only (i.e., lacking light chains) are also encompassed within the definition of the term "antibody". The term "antibody" also includes immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain (abbreviated herein as HC) comprises a heavy chain variable region (abbreviated herein as HCVR or V _H ) And a heavy chain constant region. The heavy chain constant region comprises three domains C _H 1、C _H 2 and C _H 3. Each light chain (abbreviated herein as LC) comprises a light chain variable region (abbreviated herein as LCVR or V _L ) And a light chain constant region. The light chain constant region comprises a domain (C _L 1). V can be set _H Region and V _L The region is further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each V _H And V _L Consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present disclosure, the FR of the anti-MAGE-A4 antibody (or antigen binding portion thereof) may be identical to the human germline sequence, or may be natural or artificially modified. Amino acid consensus sequences can be defined based on parallel analysis of two or more CDRs.

In certain embodiments of the invention, the antibody is a bispecific antibody. For example, in certain embodiments, an anti-MAGE-A4 antibody (e.g., mAbM31339N 2) is reformatted into a bispecific antibody (e.g., anti-MAGE-A4 x anti-CD 3) by generating bsb6054 using a high affinity (7195P) CD3 arm or bsAb6043 using a medium affinity (7221G) CD3 arm.

As used herein, the term "antibody" also includes antigen binding fragments of whole antibody molecules. As used herein, the term "antigen binding portion" of an antibody, an "antigen binding fragment" of an antibody, and the like, includes any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. As used herein, an "antigen binding portion" or "antigen binding fragment" refers to one or more fragments of an antibody that retains the ability to specifically bind to MAGE-A4 (or MAGE-A4 related peptide) and/or CD 3. Antigen binding fragments of antibodies may be derived from an intact antibody molecule, for example, using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Such DNA is known and/or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or may be synthesized. The DNA may be sequenced and manipulated by chemical means or by molecular biological techniques, for example, arranging one or more variable and/or constant domains into a suitable configuration, or introducing codons, producing cysteine residues, modifying, adding or deleting amino acids, etc.

Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) a F (ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragments; (v) a single chain Fv (scFv) molecule; (vi) a dAb fragment; and (vii) minimal recognition units consisting of amino acid residues that mimic an antibody hypervariable region (e.g., an isolated Complementarity Determining Region (CDR), such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small Modular Immunopharmaceuticals (SMIPs), and shark variant IgNAR domains are also encompassed within the expression "antigen-binding fragments" as used herein.

The antigen binding fragment of an antibody typically comprises at least one variable domain. The variable domain may have any size or amino acid composition and will typically include at least one CDR adjacent to or within one or more framework sequences. In the presence of V _L Domain-associated V _H In the antigen binding fragment of the domain, V _H Domain and V _L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and comprise V _H -V _H 、V _H -V _L Or V _L -V _L A dimer. Alternatively, the antigen binding fragment of the antibody may contain monomer V _H Or V _L A domain.

In certain embodiments, the antigen binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found within antigen binding fragments of antibodies of the present disclosure include: (i) V (V) _H -C _H 1；(ii)V _H -C _H 2；(iii)V _H -C _H 3；(iv)V _H -C _H 1-C _H 2；(v)V _H -C _H 1-C _H 2-C _H 3；(vi)V _H -C _H 2-C _H 3；(vii)V _H -C _L ；(viii)V _L -C _H 1；(ix)V _L -C _H 2；(x)V _L -C _H 3；(xi)V _L -C _H 1-C _H 2；(xii)V _L -C _H 1-C _H 2-C _H 3；(xiii)V _L -C _H 2-C _H 3, a step of; (xiv) V _L -C _L . In any configuration of variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. HingeThe region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that result in flexible or semi-flexible linkages between adjacent variable domains and/or constant domains in a single polypeptide molecule. Furthermore, antigen binding fragments of antibodies of the present disclosure may include homodimers or heterodimers (or other multimers) of any of the variable domain and constant domain configurations listed above, with each other and/or with one or more monomers V _H Or V _L The domains are non-covalently associated (e.g., via one or more disulfide bonds).

In certain embodiments, the anti-MAGE-A4 antibody from which the antigen binding fragment is derived is a human antibody. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in CDRs, and specifically CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework region sequences.

In various embodiments, the anti-MAGE-a4×anti-CD 3 bispecific antibodies of the disclosure (e.g., bsAb 6054 and/or bsAb 6043) are human antibodies. In various embodiments, the anti-MAGE-A4X anti-CD 3 bispecific antibody is a human IgG antibody. In various embodiments, the anti-MAGE-A4X anti-CD 3 bispecific antibody is a human antibody of isotype IgG1, igG2, igG3, or IgG4 or mixed isotype. In some embodiments, the anti-MAGE-A4 xcd 3 bispecific antibody is a human IgG1 antibody (i.e., the antibody comprises a human IgG1 heavy chain constant region linked to a HCVR of each of the first and second antigen binding domains, respectively). In some embodiments, the anti-MAGE-a4×anti-CD 3 bispecific antibody is a human IgG4 antibody (i.e., the antibody comprises a human IgG4 heavy chain constant region of HCVR linked to each of the first and second antigen binding domains, respectively). In any of the embodiments discussed above or herein, the anti-MAGE-a4×anti-CD 3 bispecific antibody may comprise a human kappa light chain. In any of the embodiments discussed above or herein, the anti-MAGE-a4×anti-CD 3 bispecific antibody may comprise a human lambda light chain.

In any embodiment, the bispecific antibody may include modifications in one or both heavy chains to facilitate purification of the bispecific antibody from homodimeric impurities (i.e., heterodimers). In some embodiments, the bispecific antibody comprises a first heavy chain and a second heavy chain (i.e., a heavy chain of an anti-MAGE-A4 binding arm and a heavy chain of an anti-CD 3 binding arm), both chains being identical (e.g., both isotypes IgG1 or IgG 4), except that there is a modification in the CH3 domain of one or the other heavy chain that reduces binding of the bispecific antibody to protein a (as compared to an antibody lacking the modification). In some cases, the CH3 domain of the first heavy chain (e.g., of the anti-MAGE-A4 binding arm) binds protein a, and the CH3 domain of the second heavy chain (e.g., of the anti-CD 3 binding arm) contains mutations that reduce or eliminate protein a binding. In some cases, the mutation is an H435R modification (numbering by EU; numbering by IMGT exon as H95R). In some cases, the mutations are an H435R modification (numbering by EU; numbering by IMGT exon H95R) and a Y436F modification (numbering by EU; numbering by IMGT Y96F). Additional modifications that may be found in the second CH3 domain include: in the case of IgG1 CH3 domains, D356E, L358M, N384S, K392N, V397M and V422I (D16E, L18M, N44S, K52N, V57M and V82I by IMGT numbering); in the case of the IgG4 CH3 domain, Q355R, N384S, K392N, V397M, R409K, E Q and V422I (Q15R, N44S, K52N, V57M, R69K, E Q and V82I as IMGT).

In any embodiment, the bispecific antibody can comprise a chimeric hinge. The term "chimeric hinge" is intended to include a chimeric protein comprising a first amino acid sequence derived from the hinge region of one Ig molecule and a second amino acid sequence derived from the hinge region of a different class or subclass of Ig molecule. For example, in one embodiment, the chimeric hinge comprises a first amino acid sequence or "upper hinge" sequence derived from a human IgG1 hinge region or a human IgG4 hinge region, and a second amino acid sequence or "lower hinge" sequence derived from a human IgG2 hinge region. In certain embodiments, the first or "upper hinge" sequence comprises amino acid residues at positions 216 to 227 according to EU numbering. In some embodiments, the second or "lower hinge" sequence comprises amino acid residues at positions 228 to 236 according to EU numbering.

In some embodiments, the antibodies of the present disclosure (which include bispecific antibodies) used to generate anti-MAGE-A4 antigen binding fragments and/or chimeric antigen receptors can be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to encompass all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below), antibodies isolated from recombinant, combinatorial human antibody libraries (described further below), antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., taylor et al, (1992) nucl. AcidsRes.20:6287-6295), or antibodies prepared, expressed, produced or isolated by any other means that involves splicing human immunoglobulin gene sequences onto other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when animals with transgenic human Ig sequences are used, in vivo somatic mutagenesis), and thus, V of the recombinant antibodies _H Region and V _L The amino acid sequence of the region is the following: although derived from human germline V _H Sequence and V _L Sequences related thereto, but may not be naturally present in human antibody germline libraries.

Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of about 150kDa to 160kDa, wherein the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds and form molecules of about 75kDa to 80kDa, consisting of covalently coupled light and heavy chains (half antibodies). These forms are extremely difficult to isolate even after affinity purification.

The frequency of occurrence of the second form in the form of various intact IgG isotypes is based on, but not limited to, structural differences associated with the hinge region isotype of the antibody. Single amino acid substitutions in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al, (1993) Molecular Immunology 30:105) to the levels typically observed with human IgG1 hinges. The present disclosure covers the hinge region, C _H 2 region or C _H Antibodies with one or more mutations in region 3, which may be desirable, for example, in production, to increase the yield of the desired antibody form.

The antibodies disclosed herein (which include bispecific antibodies) can be isolated antibodies. As used herein, "isolated antibody" means an antibody that has been identified and isolated and/or recovered from at least one component of its natural environment. For example, an antibody that has been isolated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally occurs or naturally occurs, is an "isolated antibody" for purposes of this disclosure. Isolated antibodies also include in situ antibodies within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or isolation step. Thus, isolated antibodies also include antibodies that are substantially free of other antibodies having different antigen specificities (e.g., isolated antibodies that specifically bind to human MAGE-A4 or to both human MAGE-A4 and human CD3 are substantially free of antibodies that specifically bind to antigens other than human MAGE-A4 or to both human MAGE-A4 and human CD 3). However, as disclosed herein, an isolated antibody (e.g., an isolated antibody that specifically binds human MAGE-A4) may, in some examples, additionally specifically bind one or more other MAGE-A4 related proteins or peptides. According to certain embodiments, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "specifically binds" and the like means that an antibody (which includes bispecific antibodies) or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding may be through at least about 1X 10 ^-6 M or greater. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. However, isolated antibodies that specifically bind to human MAGE-A4 or both human MAGE-A4 and human CD3 may be cross-reactive with other antigens, such as MAGE-A4 and/or CD3 molecules from other species (orthologs). In the context of the present invention, monospecific antibodies that bind to human MAGE-A4 as well as one or more other antigens, including MAGE-A4 related peptides, are considered to "specifically bind" to human MAGE-A4. In the context of the present invention, multispecific (e.g., bispecific) antibodies that bind to human MAGE-A4 and human CD3 and one or more additional antigens are said to "specifically bind" to human MAGE-A4 and human CD3.

Bispecific antibodies comprising an anti-MAGE-A4 specific binding domain and an anti-CD 3 specific binding domain can be constructed using standard methods, wherein the anti-MAGE-A4 antigen binding domain and the anti-CD 3 antigen binding domain each comprise a different unique HCVR paired with a common LCVR. In an exemplary bispecific antibody, a heavy chain from an anti-CD 3 antibody, a heavy chain from an anti-MAGE-A4 antibody, and a common light chain building molecule from an anti-MAGE-A4 antibody are utilized. In other cases, bispecific antibodies can be constructed using: heavy chains from anti-CD 3 antibodies, heavy chains from anti-MAGE-A4 antibodies, and light chains from anti-CD 3 antibodies, or antibody light chains known to be operably promiscuous or paired with a variety of heavy chain arms.

According to certain embodiments of the invention, the anti-MAGE-A4X anti-CD 3 bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding domain that specifically binds human MAGE-A4 and a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, respectively, namely A1-HCDR1, A1-HCDR2, and A1-HCDR3, and the second antigen-binding domain comprises heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 57 or 75, 59 or 77 and 61 or 79, respectively, namely A2-HCDR1, A2-HCDR2, and A2-HCDR3. According to certain embodiments of the invention, the anti-MAGE-a4×anti-CD 3 bispecific antibody or antigen binding fragment thereof, respectively, comprises a light chain complementarity determining region LCDR1-LCDR2-LCDR3 in common (for both the first antigen binding domain and the second antigen binding domain). In one example, the consensus light chain complementarity determining regions comprise the amino acid sequences of SEQ ID NOS 12, 14 and 16. In other examples, the consensus light chain complementarity determining region comprises the amino acid sequences of SEQ ID NOS 65, 14 and 67.

In certain embodiments, an anti-MAGE-A4X anti-CD 3 bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding domain that specifically binds human MAGE-A4 and a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises a Heavy Chain Variable Region (HCVR) comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:83, or SEQ ID NO:107, and the second antigen-binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO:55 or SEQ ID NO: 73. In certain embodiments, an anti-MAGE-A4X anti-CD 3 bispecific antibody or antigen binding fragment thereof comprises a consensus Light Chain Variable Region (LCVR) comprising the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 63, or SEQ ID NO. 115. In certain embodiments, an anti-MAGE-A4X anti-CD 3 bispecific antibody or antigen binding fragment thereof comprises a first antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 2/10 and a second antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 55/10 or comprising the amino acid sequence of SEQ ID NO. 73/10. In certain embodiments, an anti-MAGE-A4X anti-CD 3 bispecific antibody or antigen binding fragment thereof comprises a first antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 83/10 and a second antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 55/10 or comprising the amino acid sequence of SEQ ID NO. 73/10. In certain embodiments, an anti-MAGE-A4X anti-CD 3 bispecific antibody or antigen binding fragment thereof comprises a first antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 107/115 and a second antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 55/115 or comprising the amino acid sequence of SEQ ID NO. 73/115. In certain embodiments, an anti-MAGE-A4X anti-CD 3 bispecific antibody or antigen binding fragment thereof comprises a first antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 2/63 and a second antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 55/63 or comprising the amino acid sequence of SEQ ID NO. 73/63. In certain embodiments, an anti-MAGE-A4X anti-CD 3 bispecific antibody or antigen binding fragment thereof comprises a first antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 83/63 and a second antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 55/63 or comprising the amino acid sequence of SEQ ID NO. 73/63. In certain embodiments, an anti-MAGE-A4X anti-CD 3 bispecific antibody or antigen binding fragment thereof comprises a first antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 107/63 and a second antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 55/63 or comprising the amino acid sequence of SEQ ID NO. 73/63. In some embodiments, the anti-MAGE-A4X anti-CD 3 bispecific antibody comprises a HCVR/LCVR sequence pair as described above and a human IgG1 heavy chain constant region. In some embodiments, the anti-MAGE-A4X anti-CD 3 bispecific antibody comprises a HCVR/LCVR sequence pair as described above and a human IgG4 heavy chain constant region. In some embodiments, the anti-MAGE-A4X anti-CD 3 bispecific antibody comprises a HCVR/LCVR sequence pair as described above and a human IgG heavy chain constant region. In some embodiments, the anti-MAGE-A4X anti-CD 3 bispecific antibody comprises a HCVR/LCVR sequence pair as described above and a human IgG1 or IgG4 heavy chain constant region.

Pairs of anti-MAGE-A4 antibodies or antigen binding fragments thereof and derived antibodies disclosed hereinThe germline sequence may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, a public antibody sequence database. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to one or more corresponding residues of the germline sequence of the derived antibody, or mutated to one or more corresponding residues of another human germline sequence, or mutated to conservative amino acid substitutions of one or more corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one of ordinary skill in the art can readily generate a number of antibodies and antigen binding fragments that include one or more individual germline mutations or combinations thereof. In certain embodiments, V _H Domain and/or V _L All of the framework and/or CDR residues within the domain are mutated back to the residues found in the original germline sequence of the derived antibody. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR 3. In other embodiments, one or more of the one or more framework and/or CDR residues are mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence that is different from the germline sequence of the originally derived antibody). Still further, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to corresponding residues of a particular germline sequence, while certain other residues that differ from the original germline sequence may be maintained or mutated to corresponding residues of a different germline sequence. Once obtained, one or more desired properties of antibodies and antigen binding fragments containing one or more germline mutations, such as improved binding characteristics, can be readily detected Specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, and the like. Antibodies and antigen binding fragments obtained in this general manner are encompassed within the present disclosure.

An anti-MAGE-A4 antibody may comprise variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, an anti-MAGE-A4 antibody may have HCVR, LCVR, and/or CDR amino acid sequences with, for example, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences set forth herein.

The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. Epitopes may be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include a portion of a sugar, phosphoryl, or sulfonyl group on an antigen.

When referring to a nucleic acid or fragment thereof, the term "substantial identity" or "substantially identical" means that the nucleotide sequence identity is at least about 95%, and more preferably at least about 96%,97%, 98%, 99%, 99.5% or 99.9% of the nucleotide base when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, as measured by any well known sequence identity algorithm, such as FASTA, BLAST or Gap, as discussed below. In certain instances, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule may encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule. In some embodiments, the disclosure provides a polypeptide comprising a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, at least 99.5%, at least 99.9% or 100% identical to the sequence of SEQ ID NO:22 or a portion of SEQ ID NO:105 (e.g., a sequence of HCVR such as SEQ ID NO:2 or SEQ ID NO:83, or a sequence of LCVR such as SEQ ID NO:10, or a framework region of a polypeptide sequence such as those set forth in SEQ ID NO:2, 10, 22 or 105). In some embodiments, the disclosure provides a polynucleic acid encoding such a polypeptide. In some embodiments, the disclosure provides a polynucleic acid comprising a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, at least 99.5%, at least 99.9% or 100% identical to the sequence of SEQ ID NO. 21 or a portion of SEQ ID NO. 104 (e.g., a sequence of HCVR as SEQ ID NO. 1, or a sequence of LCVR as SEQ ID NO. 9, or a framework region of a polynucleotide sequence as SEQ ID NO. 1, 9, 21 or 104).

In some embodiments, the disclosure provides a polypeptide comprising a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the sequence of SEQ ID NO:69 or a portion thereof (e.g., SEQ ID NO: 55) or to the sequence of SEQ ID NO:71 or a portion thereof (e.g., SEQ ID NO: 63) or to the sequence of SEQ ID NO:81 or a portion thereof (e.g., SEQ ID NO: 73). In some embodiments, the disclosure provides a polynucleic acid comprising a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, at least 99.5%, at least 99.9% or 100% identical to the sequence of SEQ ID NO:68 or a portion thereof (e.g., SEQ ID NO: 54) or to the sequence of SEQ ID NO:70 or a portion thereof (e.g., SEQ ID NO: 62) or to the sequence of SEQ ID NO:80 or a portion thereof (e.g., SEQ ID NO: 72).

The term "substantial similarity" or "substantially similar" when applied to polypeptides means that two peptide sequences share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default GAP weights. Preferably, the different residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a side chain (R group) that is similar in chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, for example, pearson, (1994) Methods mol. Biol.24:307-331, which is incorporated herein by reference. Examples of groups of amino acids having side chains of similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chain: lysine, arginine, and histidine; (6) acidic side chain: aspartic acid and glutamic acid, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and asparagine-glutamine. Alternatively, a conservative substitution is any change with positive values in PAM250 log likelihood matrix disclosed in the following documents: gonnet et al, (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change in the PAM250 log likelihood matrix that has a non-negative value.

Sequence analysis software is typically used to measure sequence similarity, also known as sequence identity, of polypeptides. Protein analysis software matches similar sequences using metrics assigned to similarity of various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine closely related polypeptides, such as sequence homology or sequence identity between homologous polypeptides from different organism species or between wild type proteins and their muteins. See, e.g., GCG version 6.1. The polypeptide sequences may also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA 3) provide the alignment and percent sequence identity of the optimal overlap region between the query sequence and the search sequence (Pearson (2000), supra). Sequences can also be compared using the Smith-Waterman homology search algorithm using an affine gap search gap opening penalty of 12 and a gap extension penalty of 2 with a BLOSUM matrix of 62. Another preferred algorithm when comparing sequences of the present disclosure to databases containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, altschul et al, (1990) J.mol.biol.215:403-410 and Altschul et al, (1997) Nucleic Acids Res.25:3389-402, each of which is incorporated herein by reference.

As used herein, the term "nucleic acid" or "polynucleotide" refers to nucleotides and/or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the Polymerase Chain Reaction (PCR), and fragments generated by any of ligation, fragmentation, endonuclease action, and exonuclease action. The nucleic acid molecule can be composed of monomers that are naturally occurring nucleotides (e.g., DNA and RNA) or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides) or a combination of both. Modified nucleotides may have alterations in the sugar moiety and/or in the pyrimidine or purine base moiety. Sugar modifications include, for example, substitution of one or more hydroxyl groups with halogen, alkyl, amine, and azide groups, or the sugar may be functionalized as an ether or ester. In addition, the entire sugar moiety may be replaced by sterically and electronically similar structures (e.g., azasugar and carbocyclic sugar analogs). Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well known heterocyclic substituents. Nucleic acid monomers may be linked by phosphodiester linkages or analogues of such linkages. The nucleic acid may be single-stranded or double-stranded.

The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a binding domain (e.g., antibody-based specificity for a desired antigen (e.g., a tumor antigen such as MAGE-A4)) to a component present on a target cell with an intracellular domain activated by a T cell receptor to produce a chimeric protein that exhibits specific anti-target cell immune activity. Typically, CARs consist of an extracellular single chain antibody binding domain (scFv) fused to an intracellular signaling domain of the zeta chain of the T cell antigen receptor complex and have the ability to redirect antigen recognition based on the specificity of monoclonal antibodies when expressed in T cells.

The term "HLA" refers to a Human Leukocyte Antigen (HLA) system or complex, which is a complex of genes encoding human Major Histocompatibility Complex (MHC) proteins. These cell surface proteins are responsible for regulating the human immune system. HLA corresponding to MHC class I (A, B and C) presents peptides from inside the cell. In the context of the present application, a peptide may be "HLA-bound" if the peptide binds to an HLA system or complex. In some embodiments, the HLA-bound peptide is present on the surface of the cell.

The term "HLA-A" refers to a group of Human Leukocyte Antigens (HLAs) encoded by the HLA-A locus. HLA-A is one of three major types of human MHC class I cell surface receptors. The receptor is a heterodimer and is composed of a heavy alpha chain and a smaller beta chain. The alpha chain is encoded by the variant HLA-A gene and the beta chain (beta 2-microglobulin) is a constant beta 2 microglobulin molecule.

The term "HLA-A2" is a particular class I Major Histocompatibility Complex (MHC) allele group at the HLA-A locus; the alpha chain is encoded by the HLA-A x 02 gene and the beta chain is encoded by the beta 2-microglobulin or B2M locus.

As used herein, the term "vector" includes, but is not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules, which may consist of chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acids. In some cases, vectors are those capable of autonomous replication (episomal vectors) and/or expression of nucleic acids linked thereto (expression vectors). A number of suitable carriers are known to those skilled in the art and are commercially available. Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and sendai), positive strand RNA viruses such as picornaviruses and alphaviruses, and double stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, epstein-Barr virus), cytomegaloviruses, and poxviruses (e.g., vaccinia, chicken pox, and canary pox). Other viruses include, for example, norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include: avian leukemia sarcoma, mammalian type C, type B viruses, type D viruses, HTLV-BLV group and lentiviruses.

"costimulatory domain" or "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD 137) (SEQ ID NO: 29), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, and the like. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which are necessary for effective immune responses.

By "costimulatory ligand" is meant a molecule on an antigen presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses, including but not limited to proliferation activation, differentiation, etc., in addition to the primary signal provided by the binding of, for example, a TCR/CD3 complex to an MHC molecule loaded with a peptide. Co-stimulatory ligands may include, but are not limited to, CD7, B7-1 (CD 80), B7-2 (CD 86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligands (ICOS-L), intercellular adhesion molecules (ICAM), CD30L, CD, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, agonists or antibodies that bind Toll ligand receptors, and ligands that specifically bind to B7-H3.

"costimulatory signal" refers to a signal that, in combination with a primary signal (e.g., TCR/CD3 linkage), results in up-or down-regulation of T cell proliferation and/or a key molecule.

As used herein, the term "extracellular ligand binding domain" refers to an oligopeptide or polypeptide capable of binding a ligand, such as a cell surface molecule. For example, the extracellular ligand binding domain can be selected to recognize a ligand that serves as a cell surface marker on a target cell associated with a particular disease state (e.g., cancer). Examples of cell surface markers that can be used as ligands include those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells. The extracellular ligand binding domain may comprise a LCVR region and a HCVR region (e.g., formatted as an scFv), optionally connected by a linker.

As used herein, the term "subject" or "patient" includes all members of the animal kingdom, including non-human primates and humans. In one embodiment, the patient is a human having cancer (e.g., multiple myeloma or melanoma).

As used herein, the "signaling domain" or "signaling domain" of a CAR is responsible for intracellular signaling following binding of an extracellular ligand binding domain to a target, thereby activating immune cells and immune responses. In other words, the signaling domain is responsible for activating at least one of the normal effector functions of the CAR-expressing immune cells. For example, the effector function of a T cell may be cytolytic activity or helper activity (including secretion of cytokines). Thus, the term "signaling domain" refers to a portion of a protein that transduces effector function signals and directs cells to perform a specialized function. Examples of signal transduction domains for use in CARs can be cytoplasmic sequences of T cell receptors and co-receptors that act synergistically upon antigen receptor binding to initiate signal transduction, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same functional capability. In some cases, the intracellular signaling domain comprises two different types of cytoplasmic signaling sequences: cytoplasmic signaling sequences that initiate antigen dependent primary activation, and cytoplasmic signaling sequences that function in an antigen independent manner to provide secondary or costimulatory signals. The primary cytoplasmic signaling sequence can include a signaling motif known as ITAM based on an activation motif of immune receptor tyrosine. ITAM is a well-defined signaling motif found in the cytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap 70-type tyrosine kinases. Exemplary ITAMs include those derived from TCR ζ, fcrγ, fcrβ, fcrε, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, and CD66 d. In some embodiments, the signaling domain of the CAR can include a CD3 zeta signaling domain (SEQ ID NO: 30).

Chimeric Antigen Receptor (CAR)

Chimeric Antigen Receptors (CARs) can redirect T-cells specifically to antigens recognized by antibodies on the surface of cells (e.g., cancer cells), whether those antigens are expressed on the cell surface or in cells and presented by, for example, HLA.

One aspect of the disclosure includes Chimeric Antigen Receptors (CARs) that are specific for MAGE-A4 antigens presented on the surface of cells, such as tumor cells. For example, this presentation may be performed by HLA, such as HLA-A 2. In one embodiment of the disclosure, a CAR as described herein includes an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (e.g., a signaling domain derived from cd3ζ or fcrγ), and/or one or more costimulatory signaling domains derived from costimulatory molecules, such as, but not limited to, 4-1BB. In one embodiment, the CAR comprises a hinge or spacer region, such as a CD8 a hinge, between the extracellular binding domain and the transmembrane domain.

The binding domain or extracellular domain of the CAR provides the CAR with the ability to bind to a target antigen of interest. The binding domain (e.g., ligand binding domain or antigen binding domain) can be any protein, polypeptide, oligopeptide or peptide that has the ability to specifically recognize and bind a biological molecule (e.g., a cell surface receptor or tumor protein or component thereof). Binding domains include any naturally occurring, synthetic, semisynthetic or recombinantly produced binding partner of a biological molecule of interest. For example, and as described further herein, the binding domains may be antibody light and heavy chain variable regions, or the light and heavy chain variable regions may be linked together in single chain and either orientation (e.g., VL-VH or VH-VL). Various assays for identifying binding domains of the present disclosure that specifically bind to a particular target are known, including western blotting, ELISA, flow cytometry, or surface plasmon resonance analysis (e.g., using BIACORE analysis). The target may be an antigen of clinical interest against which it is desired to trigger an effector immune response leading to tumor killing. In one embodiment, the target antigen of the binding domain of the chimeric antigen receptor is a MAGE-A4 protein on the surface of a tumor cell (e.g., an HLA-presented MAGE-A4 protein such as a HLA-A 2-presented MAGE-A4 protein).

Illustrative ligand binding domains include antigen binding proteins (e.g., antigen binding fragments of antibodies (e.g., scFv)), sctcrs, extracellular domains of receptors, ligands of cell surface molecules/receptors or receptor binding domains thereof, and tumor binding proteins. In certain embodiments, the antigen binding domains included in the CARs of the present disclosure can be variable regions (Fv), CDR, fab, scFv, VH, VL, domain antibody variants (dabs), camelid antibodies (VHHs), fibronectin 3 domain variants, ankyrin repeat variants, and other antigen specific binding domains derived from other protein scaffolds.

In one embodiment, the binding domain of the CAR is an anti-MAGE-A4 single chain antibody (scFv), and may be a murine, human, or humanized scFv. Single chain antibodies can be cloned from the V region gene of a hybridoma that is specific for the desired target. Techniques that can be used to clone the variable region heavy (VH) and variable region light (VL) chains have been described, for example, in Orlandi et al, PNAS,1989;86:3833-3837. Thus, in certain embodiments, the binding domain comprises an antibody-derived binding domain but may be a non-antibody-derived binding domain. The antibody-derived binding domain may be a fragment of an antibody or a genetically engineered product of one or more fragments of an antibody, which fragments are involved in binding to an antigen.

In certain embodiments, the CARs of the present disclosure can include linkers between the different domains added for proper spacing and conformation of the molecules. For example, in one embodiment, the binding domains VH or VL may have a linker between them, which may be between 1 and 20 amino acids in length. In other embodiments, the length of the linker between any of the domains of the chimeric antigen receptor can be between 1 and 15 amino acids or 15 amino acids. In this regard, the length of the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In further embodiments, the length of the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. Ranges including the numbers described herein are also included herein, e.g., 10 to 30 amino acids long linkers.

In certain embodiments, a linker suitable for use in the CARs described herein is a flexible linker. Suitable linkers can be readily selected and can have any suitable different length, such as 1 amino acid (e.g., gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linkers include glycine polymer (G) n, glycine-serine polymer (where n is an integer of at least 1), glycine-alanine polymer, alanine-serine polymer, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and thus may be capable of acting as a neutral tether between domains of fusion proteins such as CARs as described herein. Glycine acquired significantly more phi-psi space than even alanine and was much less restricted than residues with longer side chains (see Scheraga, rev. Computationalchem.11173-142 (1992)). One of ordinary skill will recognize that the design of the CAR may include a fully or partially flexible joint such that the joint may include a flexible joint and one or more portions that impart less flexible structure to provide the desired CAR structure. The specific joint comprises (G4S) _n And a linker, wherein n=1-3, as shown in SEQ ID NO. 23-25. Another exemplary linker is provided as SEQ ID NO. 26. The linker may be present in the LCV of the CARBetween the R region and the HCVR region, between a variable region (e.g., HCVR) and a hinge region (e.g., CD 8. Alpha. Hinge), or between both. For example, the present disclosure provides a CAR that includes a (G4S) 3 linker between the LCVR and the HCVR and a (G4S) 1 linker between the HCVR and the CD8 a hinge.

The binding domain of the CAR may be followed by a "spacer" or "hinge" which refers to a region that moves the antigen binding domain away from the effector cell surface to achieve proper cell/cell contact, antigen binding and activation (Patel et al, gene Therapy,1999; 6:412-419). The hinge region in a CAR is typically located between the Transmembrane (TM) and the binding domain. In certain embodiments, the hinge region is an immunoglobulin hinge region, and may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. Other exemplary hinge regions for use in the CARs described herein include hinge regions derived from extracellular regions of type 1 membrane proteins, such as CD8 a, CD4, CD28, and CD7, which may be wild-type hinge regions from these molecules or may be altered. In one embodiment, the hinge region comprises a CD8 alpha hinge (SEQ ID NO: 27).

A "transmembrane" region or domain is the portion of the CAR that anchors the extracellular binding moiety to the plasma membrane of an immune effector cell and facilitates binding of the binding domain to a target antigen. The transmembrane domain may be a cd3ζ transmembrane domain, but other transmembrane domains that may be employed include those obtained from CD8 a, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137 and CD 154. In one embodiment, the transmembrane domain is the transmembrane domain of CD 137. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO. 28. In certain embodiments, the transmembrane domain is synthetic, in which case the transmembrane domain will predominantly comprise hydrophobic residues such as leucine and valine.

An "intracellular signaling domain" or "signaling domain" refers to a portion of a chimeric antigen receptor protein that is involved in transduction of information about effective CAR binding to a target antigen into immune effector cells to elicit effector cell functions such as activation, cytokine production, proliferation, and cytotoxic activity, including release of a cytotoxic factor to the CAR-bound target cell or other cellular responses elicited by antigen binding to an extracellular CAR domain. The term "effector function" refers to a specific function of a cell. Effector functions of T cells may be, for example, cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" or "signaling domain" as used interchangeably herein refers to a portion of a protein that transduces effector function signals and directs a cell to perform a specialized function. Although it is generally possible to employ the entire intracellular signaling domain, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion can be used in place of the entire domain, so long as it transduces an effector function signal. The term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal. Intracellular signaling domains are also known as "signal transduction domains" and are typically derived from portions of the human CD3 or FcRy chain.

It is known that signals generated by T cell receptors alone are not sufficient to fully activate T cells and that secondary or co-stimulatory signals are also required. Thus, T cell activation can be said to be mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by T cell receptors (primary cytoplasmic signaling sequences) and those that function in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). Cytoplasmic signaling sequences acting in a costimulatory manner can contain signaling motifs known as immune receptor tyrosine-based activation motifs or ITAMs.

Examples of ITAM-containing primary cytoplasmic signaling sequences having particular utility in the present disclosure include those derived from tcrζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, and CD66 d. In a particular embodiment, the intracellular signaling domain of an anti-MAGE-A4 CAR described herein is derived from cd3ζ. In some embodiments, the signaling domain comprises the amino acid sequence of SEQ ID NO. 30.

As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to a portion of a CAR that comprises the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide the second signal required for efficient activation and T lymphocyte function upon binding to an antigen. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD 137), OX40 (CD 134), CD30, CD40, PD-1, ICOS (CD 278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2 and ligands that specifically bind CD 83. Thus, while the present disclosure provides exemplary costimulatory domains derived from cd3ζ and 4-1BB, it is contemplated that other costimulatory domains are used with the CARs described herein. The inclusion of one or more co-stimulatory signaling domains may enhance the efficacy and expansion of T cells expressing the CAR receptor. The intracellular signaling domain and the costimulatory signaling domain can be connected in series with the carboxy-terminal end of the transmembrane domain in any order. In some embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO. 29.

While scFv-based CARs engineered to contain signaling domains from CD3 or fcrγ have been demonstrated to deliver effective signals for T cell activation and effector function, they are insufficient to elicit signals that promote T cell survival and expansion in the absence of concomitant costimulatory signals. Other CARs containing binding domains, hinges, transmembrane and signaling domains derived from CD3 zeta or fcrgamma, and one or more co-stimulatory signaling domains (e.g., intracellular co-stimulatory domains derived from CD28, CD137, CD134 and CD 278) may more effectively direct antitumor activity and increase cytokine secretion, lytic activity, survival and proliferation of CAR-expressing T cells in vitro as well as in animal models and cancer patients (Milone et al Molecular Therapy,2009;17:1453-1464; zhong et al Molecular Therapy,2010;18:413-420; carpentito et al PNAS,2009; 106:3360-3365).

In various embodiments, an anti-MAGE-A4 CAR of the present disclosure comprises: (a) An anti-MAGE-A4 scFv as a binding domain (e.g., a scFv having a binding region (e.g., CDR or variable domain) from an anti-MAGE-A4 antibody identified in table 1); (b) a hinge region derived from human CD8 a; (c) a human CD8 a transmembrane domain; and (d) a human T cell receptor CD3 zeta chain (CD 3) intracellular signaling domain, and optionally one or more costimulatory signaling domains, such as 4-1BB. In one embodiment, the different protein domains are arranged from amino-terminus to carboxy-terminus in the following order: binding domain, hinge region and transmembrane domain. The intracellular signaling domain and optional costimulatory signaling domain are serially connected to the transmembrane carboxy-terminus in any order to form a single chain chimeric polypeptide. In one embodiment, the nucleic acid construct encoding an anti-MAGE-A4 CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule comprising different coding sequences, such as the coding sequences of (5 'to 3') a human anti-MAGE-A4 scFv, a human CD8 a hinge, a human CD8 a transmembrane domain, and a CD3 zeta intracellular signaling domain. In another embodiment, the nucleic acid construct encoding an anti-MAGE-A4 CAR is a chimeric nucleic acid molecule comprising nucleic acid molecules comprising different coding sequences, for example (5 'to 3') the coding sequences of a human anti-MAGE-A4 scFv, a human CD8 a hinge, a human CD8 a transmembrane domain, A4-1 BB costimulatory domain, and a CD3 zeta costimulatory domain.

In certain embodiments, a polynucleotide encoding a CAR described herein is inserted into a vector. Vectors are vehicles into which a polynucleotide encoding a protein can be covalently inserted in order to cause expression of the protein and/or cloning of the polynucleotide. Such vectors may also be referred to as "expression vectors". The isolated polynucleotide may be inserted into the vector using any suitable method known in the art, for example, but not limited to, the vector may be digested with an appropriate restriction enzyme, and then ligated to the isolated polynucleotide having a matched restriction end. Expression vectors may have the ability to incorporate and express heterologous or modified nucleic acid sequences encoding at least a portion of a gene product capable of transcription in a cell. In most cases, the RNA molecules are then translated into proteins. Expression vectors may contain a variety of control sequences, which refer to nucleic acid sequences necessary for transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions and are discussed below. Expression vectors may include additional elements, for example, the expression vector may have two replication systems so that it is maintained in two organisms, for example, expression in human cells and cloning and amplification in a prokaryotic host.

Expression vectors may have the necessary 5' upstream and 3' downstream regulatory elements, such as promoter sequences (e.g., CMV, PGK, and EF 1. Alpha. Promoters), ribosome recognition and binding to the TATA box, and 3' UTRAAUAAA transcription termination sequences, for efficient gene transcription and translation in their corresponding host cells. Other suitable promoters include the simian virus 40 (SV 40) early promoter, the Mouse Mammary Tumor Virus (MMTV), the HIV LTR promoter, the MoMuLV promoter, the avian leukemia virus promoter, the EBV immediate early promoter, and the constitutive promoter of the rous sarcoma virus promoter. Human gene promoters may also be used, including but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In certain embodiments, the inducible promoter is also considered to be part of a vector expressing the chimeric antigen receptor. This provides a molecular switch that can turn on or off expression of a polynucleotide sequence of interest. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, or tetracycline promoters.

The expression vector may have additional sequences such as 6x histidine, c-Myc, and FLAG tags incorporated into the expressed CAR. Thus, expression vectors can be engineered to contain 5 'and 3' untranslated regulatory sequences, which can sometimes act as enhancer sequences, promoter regions, and/or terminator sequences that can facilitate or enhance efficient transcription of one or more nucleic acids of interest carried on the expression vector. Expression vectors can also be engineered for replication and/or expression functions (e.g., transcription and translation) in a particular cell type, cell location, or tissue type. The expression vector may include a selectable marker for maintaining the vector in a host or recipient cell.

In various embodiments, the vector is a plasmid, an autonomously replicating sequence, and a transposable element. Additional exemplary vectors include, but are not limited to: plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC), phages such as lambda phage or M13 phage, and animal viruses. Examples of classifications of animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV 40). Examples of expression vectors are: lenti-X for expression in mammalian cells ^TM A bicistronic expression system (Neo) vector (cloning) and a pClneo vector (Promega); pLenti4/V5-DEST.TM., pLenti6/V5-DEST.TM., and pLenti6.2N5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of the CARs disclosed herein can be ligated into such expression vectors to express the chimeric proteins in mammalian cells.

In certain embodiments, nucleic acids encoding the CARs of the disclosure are provided in a viral vector. The viral vectors may be those derived from retroviruses, lentiviruses or foamy viruses. As used herein, the term "viral vector" refers to a nucleic acid vector construct that includes at least one viral-derived element and has the ability to be packaged into viral vector particles. Viral vectors may contain coding sequences for various chimeric proteins described herein in place of non-essential viral genes. The vector and/or particle may be used for the purpose of transferring DNA, RNA or other nucleic acids into cells in vitro or in vivo. Various forms of viral vectors are known in the art.

In certain embodiments, the viral vector containing the coding sequences of the CARs described herein is a retroviral vector or a lentiviral vector. The term "retroviral vector" refers to a vector containing structural and functional genetic elements derived primarily from a retrovirus. The term "lentiviral vector" refers to a vector outside the LTR that contains structural and functional genetic elements derived primarily from lentiviruses.

Retroviral vectors for use herein may be derived from any known retrovirus (e.g., a c-type retrovirus such as moloney murine sarcoma virus (MoMSV), haven murine sarcoma virus (hamus v), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline Leukemia Virus (FLV), foamy virus, frank virus (Friend), murine Stem Cell Virus (MSCV), and Rous Sarcoma Virus (RSV)). "retrovirus" of the present disclosure also includes human T cell leukemia virus, HTLV-1 and HTLV-2, and the lentivirus family of retroviruses, such as human immunodeficiency virus, HIV-1, HIV-2, simian Immunodeficiency Virus (SIV), feline Immunodeficiency Virus (FIV), equine Immunodeficiency Virus (EIV), and other classes of retroviruses.

Lentiviral vectors, as used herein, refer to vectors derived from lentiviruses (retrovirus (or genus) that cause a slowly evolving disease). Viruses included in this group include: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); weissner-Meidi virus; goat arthritic encephalitis virus; equine infectious anemia virus; feline Immunodeficiency Virus (FIV); bovine Immunodeficiency Virus (BIV); and Simian Immunodeficiency Virus (SIV). Preparation of recombinant lentiviruses can be accomplished using the methods according to Dull et al and Zufferey et al (Dull et al, J. Virol,1998, 72:8463-8471 and Zufferey et al, J. Virol,1998, 72:9873-9880).

Retroviral (i.e., both lentiviral and non-lentiviral) vectors for the present disclosure can be formed using standard cloning techniques by combining the desired DNA sequences in the order and directions described herein (Current Protocols in Molecular Biology, ausubel, f.m. et al (editions), greene Publishing Associates (1989), sections 9.10-9.14 and other standard laboratory manuals; eglitis et al, (1985) Science 230:1395-1398; danos and Mulligan, (1988) Proc.Natl. Acad.Sci.USA,85:6460-6464; wilson et al, (1988) Proc.Natl. Acad.Sci.USA,85:3014-3018; armentano et al, (1990) Proc.Natl. Acad.Sci.USA,87:6141-6145; huber et al, (1991) Proc.Natl.Acad.Sci.USA,88:8039-8043; ferry et al, (1991) Proc.Natl.Acad.Sci.USA, 88:8377-81; chordhury et al, (1991) Science 254:1802-1805;van Beusechem et al, (1992) Proc.Natl.Acad.Sci.USA,89, (1990) Proc.Natl.Sci.Sci.USA, 641-6145; huber et al, (1993) Proc.Natl.Natl.Acad.Sci.Sci.USA, WO 35-6435; PCT patent application No. 35/1994; PCT 5; pr.Natl.Natl.Sci.Sci.Sci.Sci.USA, 37-37, WO 35-8043; PCT/1994) Proc.Natl.Natl.Sci.Sci.Sci.Sci.Sci.Sci.Sci.Sci.Sci.Sci.Sci.USA, WO 35, PCT/4.

Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for vector formation include, for example, genomic RNA and cDNA available from commercially available sources including the classical culture collection (ATCC) of Rockwell, malyland, U.S.A.. The sequences may also be chemically synthesized.

To express the anti-MAGE-A4 CAR, a vector can be introduced into a host cell to allow expression of the polypeptide within the host cell. Expression vectors may contain various elements for controlling expression including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be suitably selected by one of ordinary skill in the art, as described herein. For example, the promoter sequence may be selected to promote transcription of the polynucleotide in the vector. Suitable promoter sequences include, but are not limited to, the T7 promoter, the T3 promoter, the SP6 promoter, the beta-actin promoter, the EF1a promoter, the CMV promoter, and the SV40 promoter. Enhancer sequences may be selected to enhance transcription of the polynucleotide. The selectable marker may be selected to allow selection of host cells for insertion into the vector from host cells not having the vector inserted therein, e.g., the selectable marker may be a gene conferring antibiotic resistance. The signal sequence may be selected to allow the expressed polypeptide to be transported out of the host cell.

To clone a polynucleotide, a vector may be introduced into a host cell (an isolated host cell) to allow the vector to replicate itself, thereby amplifying copies of the polynucleotide contained therein. Cloning vectors may contain sequence components that generally include, but are not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and selectable markers. These elements may be appropriately selected by one of ordinary skill in the art. For example, the origin of replication may be selected to promote autonomous replication of the vector in a host cell.

In certain embodiments, the present disclosure provides isolated host cells comprising the vectors provided herein. Host cells containing the vector may be used to express or clone the polynucleotide contained in the vector. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, e.g., enterobacteriaceae (Enterobacteriaceae), such as Escherichia (e.g., E.coli), enterobacter (Enterobacter), erwinia (Erwinia), klebsiella (Klebsiella), proteus (Proteus), salmonella (Salmonella) (e.g., salmonella typhimurium (Salmonella typhimurium)), serratia (Serratia) (e.g., serratia marcescens (Serratia marcescans)) and Shigella (Shigella); and bacillus (bacillus) such as bacillus subtilis and bacillus licheniformis; pseudomonas (Pseudomonas), such as Pseudomonas aeruginosa (P.aeroginosa) and Streptomyces (Streptomyces).

The CARs of the present disclosure can be introduced into host cells using transfection and/or transduction techniques known in the art. As used herein, the terms "transfection" and "transduction" refer to the process of introducing an exogenous nucleic acid sequence into a host cell. The nucleic acid may be integrated into the host cell DNA or may be maintained extrachromosomally. The nucleic acid may be maintained transiently or may be introduced stably. Transfection may be accomplished by a variety of methods known in the art including, but not limited to, calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and particle gun methods. Transduction refers to the use of viral or retroviral vectors to deliver one or more genes by viral infection rather than by transfection. In certain embodiments, the retroviral vector is transduced by packaging the vector into a virion prior to contact with the cell. For example, nucleic acid encoding an anti-MAGE-A4 CAR carried by a retroviral vector can be transduced into cells by infection and proviral integration.

As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cell", "modified cell" and "redirecting cell" are used interchangeably.

In particular, the CARs of the present disclosure are introduced into immune effector cells and expressed in order to redirect their specificity to a target antigen of interest, e.g., malignant MAGE-A4 expressing cells, such as malignant cells presenting MAGE-A4 and HLA-A 2.

The present disclosure provides methods for making immune effector cells expressing a CAR as described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from a subject, such as a subject having MAGE-A4 expressing tumor cells, such that the immune effector cells express one or more CARs as described herein. In certain embodiments, immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells may then be reapplied directly to the individual. In further embodiments, immune effector cells are first activated and stimulated to proliferate in vitro prior to genetic modification to express a CAR. In this regard, immune effector cells may be cultured either before or after being genetically modified (i.e., transduced or transfected to express a CAR as described herein).

The cell source may be obtained from the subject prior to in vitro manipulation or genetic modification of the immune effector cells described herein. In particular, immune effector cells for use with the CARs described herein include T cells. T cells may be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan (e.g., FICOLL isolation). In one embodiment, cells from the circulating blood of the individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis may be washed to remove plasma fractions and placed in a suitable buffer or medium for subsequent processing. In one embodiment of the disclosure, the cells are washed with PBS. In alternative embodiments, the wash solution lacks calcium, and may lack magnesium, or may lack many, if not all, divalent cations. As will be appreciated by those of ordinary skill in the art, the washing step may be accomplished by methods known to those of ordinary skill in the art, such as by using a semi-automatic flow-through centrifuge. After washing, the cells may be resuspended in various biocompatible buffers or other saline solutions with or without buffers. In certain embodiments, the undesired constituents of the apheresis sample may be removed in a medium in which the cells are directly resuspended.

In certain embodiments, the method is performed by lysing the erythrocytes and depleting monocytes (e.g., by administering a peptide of PERCOL ^TM Gradient centrifugation) to isolate T cells from Peripheral Blood Mononuclear Cells (PBMCs). Specific T cell subsets, such as cd28+, cd4+, cd8+, cd45ra+ and cd45ro+ T cells, may be further isolated by positive or negative selection techniques. Enrichment of the T cell population by negative selection may be accomplished, for example, by a combination of antibodies directed against surface markers specific for the cells that are negatively selected. One method for use herein is by negative magnetic immunoadhesion using a mixture of monoclonal antibodiesOr flow cytometry, which are directed against cell surface markers present on negatively selected cells. For example, to enrich for cd4+ cells by negative selection, monoclonal antibody mixtures typically include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD 8. Flow cytometry and cell sorting may also be used to isolate a population of cells of interest for use in the present disclosure.

PBMCs can be used directly for genetic modification by CARs using the methods as described herein. In certain embodiments, T lymphocytes are further isolated following isolation of PBMCs, and in certain embodiments, cytotoxic and helper T lymphocytes may be classified into naive, memory and effector T cell subsets prior to or after genetic modification and/or expansion. Cd8+ cells can be obtained by using standard methods. In some embodiments, the cd8+ cells are further sorted into primordial intermediate memory cells and effector cells by identifying cell surface antigens associated with each of those types of cd8+ cells. In embodiments, memory T cells are present in the cd62l+ and CD 62L-subsets of cd8+ peripheral blood lymphocytes. PBMCs were sorted into CD62L-cd8+ and cd62l+cd8+ fractions after staining with anti-CD 8 and anti-CD 62L antibodies. In some embodiments, expression of phenotypic markers of central memory TCM includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, the central memory T cells are cd45ro+, cd62l+, cd8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In some embodiments, the naive cd8+ T lymphocytes are characterized by expression of naive T cell phenotype markers including CD62L, CCR7, CD28, CD3, CD127, and CD45 RA.

In certain embodiments, the cd4+ T cells are further sorted into subpopulations. For example, by identifying a population of cells with cell surface antigens, cd4+ T helper cells can be sorted into primordial intermediate memory cells and effector cells. Cd4+ lymphocytes can be obtained by standard methods. In some embodiments, the naive cd4+ T lymphocytes are CD45RO-, cd45ra+, cd62l+ cd4+ T cells. In some embodiments, the central memory cd4+ cells are CD62L positive and CD45RO positive. In some embodiments, effector cd4+ cells are CD62L and CD45RO negative.

The immune effector cells, such as T cells, may be genetically modified after isolation using known methods, or the immune effector cells may be activated and expanded in vitro (or differentiated in the case of progenitor cells) prior to genetic modification. In another embodiment, immune effector cells, such as T cells, are genetically modified (e.g., transduced with a viral vector comprising nucleic acid encoding a CAR) with the chimeric antigen receptor described herein and then activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. patent No. 6,905,874; U.S. patent No. 6,867,041; U.S. patent No. 6,797,514; WO2012079000 describes. Typically, such methods involve contacting PBMCs or isolated T cells with stimulators and co-stimulators such as anti-CD 3 and anti-CD 28 antibodies, typically attached to beads or other surfaces, in a medium with an appropriate cytokine such as IL-2. anti-CD 3 and anti-CD 28 antibodies attached to the same bead act as "replacement" Antigen Presenting Cells (APCs). In other embodiments, T cells may be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described below: U.S. patent No. 6,040,177; U.S. patent No. 5,827,642; and WO2012129514.

The present disclosure provides a modified population of immune effector cells comprising an anti-MAGE-A4 CAR as disclosed herein for use in treating a patient having a malignancy caused by a MAGE-A4 expressing tumor (e.g., multiple myeloma or melanoma).

The CAR-expressing immune effector cells prepared as described herein can be used in methods and compositions for adoptive immunotherapy according to known techniques, or variants thereof that will be apparent to those of skill in the art based on the present disclosure. See, for example, U.S. patent application publication No. 2003/0170238 to Gruenberg et al; see also U.S. Pat. No. 4,690,915 to Rosenberg.

In some embodiments, the cells are formulated by first harvesting the cells from their culture medium, and then washing and concentrating the cells in a medium and container system ("pharmaceutically acceptable" carrier) suitable for administration in a therapeutically effective amount. Suitable infusion media may be any isotonic medium formulation, typically physiological saline, normosol R (Abbott), or plasma-lysate A (Baxter), although 5% dextrose in water or lactated ringer's solution may also be used. Infusion medium may be supplemented with human serum albumin.

The therapeutically effective amount of cells in the composition is at least 2 cells (e.g., at least 1 cd8+ central memory T cell and at least 1 cd4+ helper T cell subset) or more typically more than 10 ² Individual cells up to 10 ⁶ Individual cells, including 10 ⁸ Or 10 ⁹ Individual cells, and can be more than 10 ¹⁰ Individual cells. The number of cells will depend on the desired end use of the composition, as will the type of cells included therein.

These cells may be autologous or heterologous to the patient receiving the therapy. If desired, treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and/or chemokines (e.g., IFN-gamma, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, flt3-L, RANTES, MIP1 alpha, etc.) as described herein to enhance induction of an immune response.

The CAR-expressing immune effector cell populations of the present disclosure can be administered alone or as a pharmaceutical composition in combination with a diluent and/or with other components such as IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present disclosure can comprise a population of immune effector cells (e.g., T cells) expressing a CAR as described herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise: buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids, such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; auxiliaries (e.g. aluminium hydroxide); and a preservative. The compositions of the present disclosure are preferably formulated for intravenous administration.

The anti-tumor immune response induced in a subject by administering the CAR-expressing T cells described herein using the methods described herein or other methods known in the art can include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses capable of killing the infected cells. Humoral immune responses mediated primarily by helper T cells that activate B cells resulting in antibody production may also be induced. The type of immune response induced by the compositions of the present disclosure may be analyzed using a variety of techniques, which are well described in the art; for example, john e.coligan, ada m.kruisbeek, david h.margulies, ethane m.shevach, warren Strober edit Current Protocols in Immunology (2001), john Wiley & Sons, NY, n.y.

Accordingly, the present disclosure provides a method of treating an individual diagnosed with or suspected of having or at risk of having a malignancy characterized at least in part by expression of MAGE-A4 by cancer cells (e.g., solid tumor cells expressing MAGE-A4), comprising administering to the individual a therapeutically effective amount of an immune effector cell expressing a CAR as described herein.

In one embodiment, the present disclosure provides a method of treating a subject diagnosed with a MAGE-A4 expressing cancer, the method comprising removing immune effector cells from a subject diagnosed with a MAGE-A4 expressing cancer, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor of the present disclosure to produce a modified population of immune effector cells, and administering the modified population of immune effector cells to the same subject. In one embodiment, the immune effector cells comprise T cells.

Methods for administering the cell compositions described herein include any method effective to result in reintroduction of ex vivo genetically modified immune effector cells that directly express a CAR of the present disclosure in a subject or genetically modified progenitor cells that reintroduce immune effector cells that differentiate into mature immune effector cells expressing the CAR upon introduction into a subject. One method includes transducing peripheral blood T cells ex vivo with a nucleic acid construct according to the present disclosure, and returning the transduced cells to the subject.

Binding properties of chimeric antigen receptor and corresponding antibody

As used herein, the term "binding" is discussed in the context of binding of a chimeric antigen receptor or a corresponding antibody (or bispecific antibody) to, for example, a predetermined antigen such as a cell surface protein or fragment thereof (or to an antigen that binds to a cell surface protein such as an HLA molecule). Binding generally refers to an interaction or association between a minimum of two entities or molecular structures, such as an antigen binding domain: antigen interactions.

For example, when assayed by, for example, surface Plasmon Resonance (SPR) techniques in a BIAcore 3000 instrument using an antigen as the ligand and an antibody or chimeric antigen receptor as the analyte (or anti-ligand), the binding affinity generally corresponds to about 10 ^-7 M or less (e.g., about 10) ^-8 M or less, e.g. about 10 ^-9 Or lower) K _D Values. Cell-based binding strategies such as Fluorescence Activated Cell Sorting (FACS) binding assays are also routinely used, and FACS data have a strong correlation with other methods such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods,1997, 201 (2): 223-31, geuijen, CA et al J Immunol Methods,2005, 302 (1-2): 68-77).

Thus, the chimeric antigen receptor or corresponding antibody (or bispecific antibody) of the present disclosure binds to a predetermined antigen or cell surface molecule (receptor) with an affinity corresponding to K that is greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) _D K having a value at least ten times lower _D Values. As described herein, the chimeric antigen receptor or corresponding antibody of the present disclosure can bind to HLA-presented MAGE-A4 antigen, e.g., HLA-a 2-presented MAGE-A4 antigen. According to the present disclosure, there is a K equal to or less than ten times that of the non-specific antigen _D Value fittingThe affinity of an antigen receptor or corresponding antibody may be considered to be undetectable binding.

The term "K _D "(M) refers to the dissociation equilibrium constant of a particular antigen binding domain for interaction with an antigen, or the dissociation equilibrium constant of the corresponding antibody with an antigen. K (K) _D There is an inverse relationship with binding affinity, thus K _D The smaller the value, the higher the affinity, i.e. the stronger. Thus, the term "higher affinity" or "stronger affinity" relates to a higher ability to form interactions and thus to a smaller K _D The values, and conversely, the terms "lower affinity" or "weaker affinity" relate to a lower ability to form interactions, and thus K _D The value is larger. In some cases, a particular molecule (e.g., chimeric antigen receptor or corresponding antibody) has a higher binding affinity (or K) for a partner molecule (e.g., antigen X) with which it interacts than for another interacting partner molecule (e.g., antigen Y) _D ) The binding ratio can be expressed as determined as follows: will be larger in K _D Value (lower or weaker affinity) divided by smaller K _D (higher or stronger affinity), for example, may be expressed as 5-fold or 10-fold greater binding affinity, as appropriate.

The term "k _d "(seconds) ^-1 Or 1/second) refers to the dissociation rate constant of the interaction of a particular antigen binding domain with an antigen, or the dissociation rate constant of a chimeric antigen receptor or corresponding antibody. Said value is also called k _off Values.

The term "k _a ”(M ^-1 X seconds ^-1 Or 1/M) refers to the association rate constant of the interaction of a particular antigen binding domain with an antigen, or the association rate constant of a chimeric antigen receptor or corresponding antibody.

The term "K _A ”(M ^-1 Or 1/M) refers to the association equilibrium constant of a particular antigen binding domain interacting with an antigen, or the association equilibrium constant of a chimeric antigen receptor or corresponding antibody. By combining k _a Divided by k _d The association equilibrium constant is obtained.

"EC50" or "EC ₅₀ "means half maximum effective concentration, which includes the concentration of antibody or chimeric antigen receptor that induces a response intermediate between baseline and maximum after a specified exposure time. EC (EC) ₅₀ Essentially represents the concentration of chimeric antigen receptor or antibody (e.g., bispecific antibody) at which 50% of its maximum effect is observed. In certain embodiments, EC ₅₀ The value is equal to the concentration at which the chimeric antigen receptor or corresponding antibody of the present disclosure produces half maximal binding to cells expressing an antigen (e.g., a tumor-associated antigen such as MAGE-A4), as determined by, for example, a FACS binding assay and/or a T cell reporter/Antigen Presenting Cell (APC) bioassay. Thus, increased EC was observed ₅₀ Or a reduced or weaker combination of half maximum effective concentration values.

In one embodiment, reduced binding may be defined as increased EC of a bispecific antibody, antigen binding fragment, chimeric antigen receptor, or corresponding antibody ₅₀ Concentration, which enables binding to half maximum amount of target cells.

Sequence variants of antibodies and chimeric antigen receptors

The antibodies, antigen binding fragments, and chimeric antigen receptors of the present disclosure may comprise one or more amino acid substitutions, insertions, and/or deletions in the Framework Regions (FR) and/or Complementarity Determining Regions (CDRs) of the heavy and light chain variable domains, as compared to the corresponding germline sequences from which the individual antigen binding domains of the corresponding antibodies are derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, a public antibody sequence database. Antibodies, antigen binding fragments, and chimeric antigen receptors of the present disclosure may comprise antigen binding domains derived from any of the exemplary CDR or variable region amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to a corresponding residue of a germline sequence from which the corresponding antibody is derived or mutated to a corresponding residue of another human germline sequence or mutated to a conserved amino acid substitution of a corresponding germline residue (such sequence changes are collectively referred to herein as "amino acid substitutions"; Germline mutations "). Starting from the heavy and light chain variable region sequences disclosed herein, one of ordinary skill in the art can readily generate a number of antibodies that include one or more individual germline mutations or combinations thereof. In certain embodiments, V _H Domain and/or V _L All of the framework and/or CDR residues within the domain are mutated back to residues found in the original germline sequence from which the antigen binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR 3. In other embodiments, one or more of the CDR residues in-frame and/or mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antigen binding domain was originally derived). Furthermore, the antigen binding domains of the invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., where certain individual residues are mutated to corresponding residues of a particular germline sequence, while certain other residues that differ from the original germline sequence may be maintained or mutated to corresponding residues of a different germline sequence.

Biological properties of chimeric antigen receptor and corresponding antibody

The present disclosure provides antibodies (including bispecific antibodies), antigen binding fragments, and Chimeric Antigen Receptors (CARs) having antigen binding domains derived from antibodies that bind to human MAGE-A4 or both human MAGE-A4 and CD 3. As described herein, bispecific antibodies can be used to generate CARs having similar properties as bispecific antibodies. Thus, the properties described herein and attributed to bispecific antibodies are equally applicable to CARs. For example, the disclosure includes ECs of less than 2nM ₅₀ Values and S/N ratios greater than 1900 bind to anti-MAGE-A4 antibodies to human MAGE-A4 as assessed by flow cytometry-based peptide pulse assays described below with respect to example 2. As another example, the present disclosure provides for combining one or more MAGE-A4 correlations at an S/N ratio in the range of about 5 to greater than 300Peptide anti-MAGE-A4 antibodies as assessed by flow cytometry-based peptide pulse assays of example 2, and as detailed in example 3. As another example, the present disclosure provides anti-MAGE-A4 x anti-CD 3 bispecific antibodies that bind one or more of the MAGE-A4 related peptides at an S/N ratio in the range of about 5 to greater than 240, as assessed by flow cytometry-based peptide pulse assays of example 2, and as detailed in example 3.

The present disclosure also provides anti-MAGE-a4×anti-CD 3 bispecific antibodies that exhibit significant activity in T cell reporter/Antigen Presenting Cell (APC) bioassays, as detailed in example 4 below. In particular, the anti-MAGE-A4X anti-CD 3 bispecific antibodies of the present disclosure can activate NF-. Kappa.B dependent T cell (e.g., jurkat cells) gene transcription/translation, as measured by luciferase expression in the presence of MAGE-A4 and HLA-A2 expressing APCs (e.g., IM9 and U266B1 cells), with an EC of less than 3.2nM (U266B 1 cells) or less than 0.75nM ₅₀ Values. The inclusion of anti-CD 28 antibodies in the T cell reporter/APC bioassays can modulate the activity of anti-MAGE-a4×anti-CD 3 bispecific antibodies as a function of endogenous levels of CD80 and CD86, as detailed in example 4 below.

The present disclosure also provides anti-MAGE-a4×anti-CD 3 bispecific antibodies that exhibit significant activity in the T cell reporter/Antigen Presenting Cell (APC) bioassays discussed above using one or more MAGE-A4 related peptides, as detailed in example 5 below. Specifically, when in T cells carrying a luciferase reporter gene and with AOX1 _795-803 anti-MAGE-A4X anti-CD 3 bispecific antibodies exhibit less than one nanomolar EC when incubated in the presence of pulsed T2 cells with respect to stimulated reporter activity ₅₀ Values (e.g., 2.10E-11M for bispecific antibody with high affinity CD3 arm, 3.30E-10M for bispecific antibody with medium affinity CD3 arm). In addition, when in T cells carrying a luciferase reporter gene and with SHTN1 _198-206 The anti-MAGE-A4X anti-CD 3 bispecific antibody may exhibit nanomolar E with respect to stimulated reporter activity when incubated in the presence of pulsed T2 cellsC ₅₀ Values (e.g., 1.30E-9M for bispecific antibody with high affinity CD3 arm, 3.5E-9M for bispecific antibody with medium affinity CD3 arm).

The disclosure also provides methods of inducing T cells (e.g., CD8+ T cells isolated from human PBMC) against tumor cells (e.g., MAGE-A4) _286-294 Antigen presenting HLA-A2 positive IM9 cells) as assessed by an imaging-based multiplex primary T cell killing assay, as detailed in example 6 below. In particular, certain anti-MAGE-a4×anti-CD 3 bispecific antibodies of the disclosure are capable of reducing tumor cell viability to less than 50% (e.g., 38%) at concentrations below 10nM (e.g., 6.6 nM) due to an antibody-dependent T cell mediated response. In the case of endogenous expression of CD80 and CD86 by tumor cells, this decrease in viability may be blocked at least in part by incubating an anti-MAGE-a4×anti-CD 3 bispecific antibody with an anti-CD 28 antibody, which may be due at least in part to the anti-CD 28 antibody blocking the interaction of CD28 with CD80 and CD 86.

The present disclosure also provides methods of treating tumor cells (e.g., MAGE-A4 _286-294 anti-MAGE-A4 x anti-CD 3 bispecific antibodies capable of stimulating cytokine release when incubated in the presence of antigen presenting HLA-A2 positive IM9 cells) and T cells (e.g., cd8+ T cells isolated from human PBMCs) are detailed in example 7 below. In particular, the present disclosure provides anti-MAGE-a4×anti-CD 3 bispecific antibodies that are capable of stimulating cytokine release (e.g., IL2, IFN- γ) by a factor greater than 2-fold compared to cytokine release in tumor and T cell deficient anti-MAGE-a4×anti-CD 3 bispecific antibodies.

The present disclosure also provides chimeric antigen receptors having antigen binding domains derived from corresponding antibodies that specifically bind to human cell lines expressing endogenous MAGE-A4, as determined by FACS binding assays.

The present disclosure also provides engineered cells expressing MAGE-A4 specific chimeric antigen receptor that (i) are activated by MAGE-A4 expressing cells (see example 8) and/or (ii) exhibit inhibition of tumor growth in immunocompromised mice bearing human multiple myeloma or melanoma xenografts.

Preparation of antigen binding domains

The antigen binding domains of the antibodies (including bispecific antibodies) and chimeric antigen receptors of the present disclosure that are specific for a particular antigen (e.g., MAGE-A4) can be prepared by any antibody generation technique known in the art. In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the corresponding antibodies of the present disclosure are derived from chimeric, humanized, or fully human antibodies. Methods for preparing such antibodies are well known in the art. For example, VELOCIMUNE can be used ^TM Techniques produce one or more of the heavy and/or light chains. Using VELOCIMUNE ^TM Techniques (or any other antibody production technique) the high affinity chimeric antibody is initially isolated as a specific antigen (e.g., MAGE-A4) having human variable and mouse constant regions. Antibodies are characterized and selected to obtain desired properties, including affinity, neutralization, selectivity, epitopes, and the like. These human variable regions (or CDRs) can then be incorporated into the antigen binding domains of chimeric antigen receptors, as discussed herein. In other examples, two different antigens (e.g., anti-MAGE-A4 and anti-CD 3) may be suitably arranged relative to each other to produce bispecific antigen binding molecules of the disclosure (e.g., as well as antibodies, CARs, or antigen binding fragments of either) by conventional methods. In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecules of the present disclosure are derived from a chimeric, humanized, or fully humanized antibody.

Polynucleotide and vector

The present disclosure also provides polynucleotides and vectors encoding the antibodies (or portions thereof) and chimeric antigen receptors discussed herein.

In various embodiments, the polynucleotide may comprise an expression cassette or expression vector (e.g., a plasmid for introduction into a bacterial host cell or a viral vector (e.g., a baculovirus vector) for transfection of an insect host cell or a plasmid or viral vector (e.g., a lentivirus or adeno-associated virus) for transfection of a mammalian host cell).

In various embodiments, the polynucleotide and/or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO. 21 or a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO. 22. In other embodiments, the polynucleotide and/or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO. 104 or comprises a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO. 105. In various embodiments, the polynucleotide and/or vector comprises a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO. 1, or a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide sequence of SEQ ID NO. 2 or SEQ ID NO. 83. In various embodiments, the polynucleotide and/or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO. 9, or comprises a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO. 10. In various embodiments, the polynucleotide and/or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO. 17, or a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO. 18. In various embodiments, the polynucleotide and/or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO. 19 or comprises a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO. 20. In various embodiments, the polynucleotide and/or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO. 54, or a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO. 55. In various embodiments, the polynucleotide and/or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO. 62 or comprises a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO. 63. In various embodiments, the polynucleotide and/or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO. 68, or a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO. 69. In various embodiments, the polynucleotide and/or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO. 70, or comprises a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO. 71. In various embodiments, the polynucleotide and/or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:72, or a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 73. In various embodiments, the polynucleotide and/or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO. 80, or a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO. 81. With respect to the above sequence identification numbers, within the scope of the present disclosure, polynucleotides and/or vectors include any subsequence thereof, such as one or more HCDRs, LCDRs, and the like. In various embodiments, the polynucleotide and/or vector comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29 or SEQ ID NO. 30.

Method for engineering immune cells expressing chimeric antigen receptors

The present disclosure provides methods of preparing immune cells for immunotherapy comprising introducing into such immune cells ex vivo a polynucleotide or vector encoding one of the MAGE-A4 specific chimeric antigen receptors described herein. Such immune cells may be autologous or allogeneic.

The present disclosure provides immune cells comprising a polynucleotide or lentiviral vector encoding one of the MAGE-A4 specific chimeric antigen receptors discussed herein. In some embodiments, these immune cells are used in immunotherapy (e.g., treatment of cancer).

The present disclosure provides methods of genetically modifying immune cells to make them more suitable for allogeneic transplantation. According to the first aspect, immune cells may be rendered allogeneic, for example by inactivating at least one gene expressing one or more components of a T Cell Receptor (TCR) as described in WO 2013/176915, which may be combined with inactivation of genes encoding or modulating expression of HLA or β2m proteins. Thus, the risk of graft versus host syndrome and graft rejection is significantly reduced. According to further aspects of the disclosure, immune cells can be further manipulated to render immune cells more active or to limit failure by inactivating genes encoding proteins that act as "immune checkpoints" that act as modulators of T cell activation, such as PD1 or CTLA-4.

Engineered immune cells

The disclosure also provides immune cells (e.g., engineered immune cells) comprising a chimeric antigen receptor as described herein. In some cases, the immune cell is an immune effector cell. In some cases, the immune cells are T cells. In some cases, the immune cell is a T lymphocyte selected from the group consisting of an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte. In some cases, the immune cells are cd8+ cytotoxic T lymphocytes.

In some embodiments, the engineered immune cell is a human T cell comprising a chimeric antigen receptor comprising, from N-terminus to C-terminus: (a) An extracellular ligand binding domain comprising an anti-MAGE-A4 single chain variable fragment (scFv) domain, the anti-MAGE-A4 scFv domain comprising a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR); (b) a hinge; (c) a transmembrane domain; and (d) a cytoplasmic domain comprising a co-stimulatory domain and a signaling domain.

In some embodiments, the scFv domain of an engineered human T cell comprises a HCVR/LCVR amino acid sequence pair comprising the amino acid sequences of SEQ ID NO. 2/10. In some cases, the hinge comprises the amino acid sequence of SEQ ID NO. 27. In some cases, the transmembrane domain comprises the amino acid sequence of SEQ ID NO. 28. In some cases, the costimulatory domain is a 4-1BB costimulatory domain. In some cases, the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO. 29. In some cases, the signaling domain is a CD3 zeta signaling domain. In some cases, the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 30.

In various embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO. 22. In various embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO. 105.

Bioequivalence

The present disclosure provides antibodies and chimeric antigen receptors and engineered cells expressing these chimeric antigen receptors having amino acid sequences that differ from the amino acid sequences of the exemplary molecules disclosed herein but retain the ability to: bind to MAGE-A4 (CD 3 in the case of bispecific antibodies), activate immune cells expressing chimeric antigen receptors in the presence of MAGE-A4 expressing cells, or inhibit growth or proliferation of MAGE-A4 expressing tumor cells. Such variant molecules may include one or more additions, deletions or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity substantially equivalent to that of the described antigen binding molecule.

In one embodiment, two engineered immune cells expressing the chimeric antigen receptor of the present disclosure or two antigen binding proteins of the present disclosure are bioequivalent if they have no clinically significant differences in safety, purity, and potency.

In one embodiment, two engineered immune cells or two antigen binding proteins are bioequivalent if the patient can switch between the reference product and the biologic product one or more times without an expected increase in risk of side effects (including clinically significant changes in immunogenicity or reduced effectiveness) as compared to a sustained treatment without such a switch.

In one embodiment, two engineered immune cells or two antigen binding proteins are bioequivalent if they both function by one or more co-acting mechanisms (as long as these mechanisms are known) for one or more conditions of use.

In one embodiment, two antigen binding proteins or antibodies are considered bioequivalent if, for example, they are drug equivalents or drug substitutes that do not significantly differ in their rate and extent of absorption when administered in the same molar dose (whether single or multiple doses) under similar experimental conditions. Some antigen binding proteins will be considered equivalent or drug substitutes if they are equivalent in extent of absorption but not in rate of absorption, and they may also be considered bioequivalent, as such differences in the rate of absorption, intentional and reflected on the tag, are not necessary to obtain an effective in vivo drug concentration, for example, over a prolonged period of use, and are also considered medically insignificant for the particular drug product under investigation.

Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo experiments in humans or other mammals in which the concentration of engineered cells in blood, plasma, serum or other biological fluids is measured as a function of time; (b) In vitro tests which correlate and reasonably predict in vivo bioavailability data in humans; (c) In vivo experiments in humans or other mammals, in which the appropriate acute pharmacological effects of the engineered cells (or targets thereof) are measured as a function of time; and (d) in clinical trials establishing good control of safety, efficacy or bioavailability or bioequivalence of engineered cells.

Bioequivalent variants of the exemplary engineered cells set forth herein can be constructed by, for example, making various substitutions or deletions of terminal or internal residues or sequences necessary for non-biological activity.

Bioequivalent variants of the exemplary bispecific antigen binding molecules set forth herein may be constructed by, for example, making various substitutions or deletions of terminal or internal residues or sequences necessary for non-biological activity. For example, cysteine residues necessary for non-biological activity may be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bonds upon renaturation. In other contexts, bioequivalent antigen binding proteins can include variants of the exemplary bispecific antigen binding molecules set forth herein that comprise amino acid changes that alter the glycosylation characteristics of the molecule, e.g., mutations that eliminate or remove glycosylation.

Species selectivity sumCross-reactivity of species

According to certain embodiments of the present disclosure, antigen binding domains are provided that bind to human MAGE-A4 but do not bind to MAGE-A4 from other species. Also provided are anti-MAGE-A4 antigen binding domains that also bind to human AOX1 but not to AOX1 from other species. Also provided are anti-MAGE-A4 antigen binding domains that bind to human SHTN1 but not SHTN1 from other species. The present disclosure also provides antigen binding domains that bind to human MAGE-A4 and to MAGE-A4 from one or more non-human species. The present disclosure also provides anti-MAGE-A4 antigen binding domains that bind to human AOX1 and to AOX1 from one or more non-human species. The present disclosure also provides anti-MAGE-A4 antigen binding domains that bind to human SHTN1 and to SHTN1 from other species. In some embodiments, the antigen binding domains of the present disclosure bind to MAGE-A4 286-294 or AOX1 795-803 and/or SHTN1 198-206. In some embodiments, the antigen binding domain-bound MAGE-A4 and/or AOX1 and/or SHTN1 (e.g., MAGE-A4 286-294 or AOX1 795-803 or SHTN1 198-206) is presented on the cell surface via HLA, e.g., HLA-A 2.

According to certain exemplary embodiments of the present invention, antigen binding domains are provided that bind to and may not bind to one or more of human MAGE-A4 and/or MAGE-A4-related peptides, and optionally to mice, rats, guinea pigs, hamsters, gerbils, pigs, cats, dogs, rabbits, goats, sheep, cattle, horses, camels, cynomolgus monkeys, marmosets, rhesus or chimpanzee MAGE-A4 and/or MAGE-A4-related peptides. In addition, binding to MAGE-A4 and/or MAGE-A4 related peptides may be performed in the context of MHC presented MAGE-A4 (or MAGE-A4-related peptides), such as HLA presented MAGE-A4. An exemplary HLA-presented MAGE-A4 is HLA-A 2-bound human MAGE-A4.

Activation and expansion of engineered immune cells

Whether before or after genetic modification of an engineered cell (e.g., T cell), even if the genetically modified immune cells of the present disclosure are activated and proliferated independent of antigen binding mechanisms, the immune cells of the present disclosure (particularly T cells) can be further activated and expanded generally using methods such as described in the following patents: U.S. patent nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. patent application publication No. 20060121005. T cells can be expanded in vitro or in vivo.

In general, T cells of the present disclosure can be expanded by contacting with an agent that stimulates the CD3 TCR complex and costimulatory molecules on the surface of the T cells to generate an activation signal for the T cells. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA) or mitogenic lectins such as Phytohemagglutinin (PHA) may be used to generate activation signals for T cells.

As non-limiting examples, the T cell population may be stimulated in vitro, such as by contact with an anti-CD 3 antibody or antigen-binding fragment thereof or an anti-CD 2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) and a calcium ionophore. For co-stimulation of the helper molecule at the T cell surface, a ligand that binds to the helper molecule is used. For example, a population of T cells may be contacted with an anti-CD 3 antibody and an anti-CD 28 antibody under conditions suitable to stimulate T cell proliferation. Suitable conditions for T cell culture include suitable media (e.g., minimal essential media or RPMI media 1640 or X-vivo 5 (Lonza), inc.) which may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4, 1L-7, GM-CSF, IL-10, IL-2, 1L-15, TGFp and TNF- α or any other additives known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmaleates, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The culture medium may comprise RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 and X-Vivo 20, optimizer, supplemented with amino acids, sodium pyruvate and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and/or one or more cytokines in an amount sufficient to allow T cells to grow and expand. Antibiotics (e.g., penicillin and streptomycin) are included only when cultured experimentally, and are not included when culturing cells to be injected into a subject. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37 ℃) and atmosphere (e.g., air plus 5% O ₂ ). T cells exposed to different stimulation times may exhibit different characteristics.

In some embodiments, cells may be expanded by co-culturing with tissue or cells. The cells may also be expanded in vivo, for example in the blood of a subject after administration of the cells to the subject.

Therapeutic formulations and administration

As used herein, the terms "effective amount" and "therapeutically effective amount" refer to an amount of an active therapeutic agent sufficient to produce a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response). Obviously, the specific "effective amount" will vary depending on such factors as the particular condition being treated, the physical condition of the patient, the type of animal being treated, the duration of the treatment, the nature of the concurrent therapy (if any), the particular formulation employed, and the structure of the compound or derivative thereof. In this case, an amount is considered therapeutically effective if it results in one or more of the following results (but is not limited thereto): (a) Inhibit tumor growth (e.g., MAGE-A4 expressing cancer); and (b) reversal or stabilization of MAGE-A4 expressing cancers.

The dose of antigen binding molecule administered to a patient may vary depending on the age and size of the patient, the disease, condition of interest, route of administration, and the like. The preferred dosage is typically calculated from body weight or body surface area. When the antibodies or bispecific antigen binding molecules of the invention are used for therapeutic purposes in adult patients, it may be advantageous to administer the bispecific antigen binding molecules of the invention intravenously, typically in a single dose of about 0.01mg/kg to about 20mg/kg body weight, more preferably about 0.02mg/kg to about 7mg/kg body weight, about 0.03mg/kg to about 5mg/kg body weight, or about 0.05mg/kg to about 3mg/kg body weight. The frequency and duration of treatment may be adjusted according to the severity of the condition. Effective dosages and regimens for administration of bispecific antigen binding molecules can be determined empirically; for example, patient progress may be monitored by periodic assessment and the dose adjusted accordingly. In addition, the dose can be scaled between species using methods well known in the art (e.g., mornteni et al, 1991, pharmacut. Res. 8:1351).

Various delivery systems are known and may be used to administer the pharmaceutical compositions of the invention, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., wu et al, 1987, J.biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or skin mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other bioactive agents. Administration may be systemic or local.

The pharmaceutical compositions of the present invention may be delivered subcutaneously or intravenously using standard needles and syringes. In addition, for subcutaneous delivery, pen delivery devices are readily applicable for delivering the pharmaceutical compositions of the present invention. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices typically utilize replaceable cartridges containing a pharmaceutical composition. Once the entire pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device may then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Instead, the disposable pen delivery device is preloaded with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

Many reusable pen delivery devices and auto-injector delivery devices are used to subcutaneously deliver the pharmaceutical compositions of the present invention. Examples include, but are not limited to: AUTOPEN ^TM (Owen Mumford, inc., woodstock, UK), dietronic ^TM Pen (Disetronic medical systems Co., of Switzerland (Disetronic Medical Systems, bergdorf, switzerland)), HUMALOG MIX 75/25 ^TM Pen and HUMALOG ^TM Pen, HUMALIN 70/30 ^TM Pen (Gift corporation of Indiana Polis, indianapolis, ind.)), NOVOPEN ^TM I. II and III (Novo Nordisk, copenhagen, denmark), NOVOPEN JUNIOR ^TM (Norand Norde Corp. Copenhagen, denmark), BD ^TM Pen (Bedi medical Co., becton Dickinson) of franklin lake, N.J.) ^TM 、OPTIPEN PRO ^TM 、OPTIPEN STARLET ^TM OPTICLIK ^TM (Sanofi-aventis, frankfurt, germany), to name a few. Examples of disposable pen delivery devices for application to subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to: SOLOSTAR ^TM Pen (Sainophenanthrene Co., ltd.), FLEXPEN ^TM (Norand Norde Co.) and KWIKPEN ^TM (Gift Corp.) SURECICK ^TM Autoinjector (Amgen, thousand Oaks, calif.) PENLET ^TM (Haselmeier, stuttgart, germany), EPIPEN (Dey, L.P.), HUMIRA ^TM Pen (Abbott Labs, abbott Park Ill.) of the Atlantic science and technology Park, illinois, to name a few.

In some cases, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump (see Langer, supra; sefton,1987,CRC Crit.Ref.Biomed.Eng., 14:201) may be used. In another embodiment, a polymeric material may be used; see Medical Applications of Controlled Release, langer and Wise (editions), 1974, crc Pres., boca Raton, fla. In yet another embodiment, the controlled release system may be placed in proximity to the target of the composition, thus requiring only a small portion of the systemic dose (see, e.g., goodson,1984,Medical Applications of Controlled Release, supra, volume 2, pages 115-138). Other controlled release systems are discussed in Langer,1990,Science 249:1527-1533.

Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, instillation, and the like. These injectable formulations can be prepared by known methods.

Advantageously, the pharmaceutical compositions described above for oral or parenteral use are prepared in unit dosage forms suitable for incorporation of a dose of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like. The amount of such antibodies is typically from about 5mg to about 500mg per unit dose of dosage form; in particular, in the form of an injection, it is preferable that the content of the above antibody is about 5mg to about 100mg, and about 10mg to about 250mg for other dosage forms.

Administration of cells or cell populations according to the present disclosure may be by any convenient means, including by aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell composition of the present disclosure is preferably administered by intravenous injection.

The administration of the cell or cell population may consist of: 10 per kg body weight ⁴ From one to 10 ⁹ Individual cells, preferably 10 per kg body weight ⁵ From one to 10 ⁶ Individual cells (including all integer values of the cell numbers within these ranges). The cells or cell populations may be administered in one or more doses. In some embodiments, an effective amount of the cells is administered as a single dose. In some embodiments, an effective amount of cells is administered as more than one dose over a period of time. The time of administration is within the discretion of the attending physician and depends on the clinical condition of the patient. CellsOr the population of cells may be obtained from any source, such as a blood bank or donor. Although individual needs vary, determination of the range of effective amounts for a given cell type for treating a particular disease or condition is within the skill of the art. An effective amount means an amount that provides a therapeutic or prophylactic benefit. The dose administered will depend on the age, health and weight of the recipient, the nature of the concurrent therapy (if any), the frequency of the therapy and the nature of the desired effect.

In one embodiment, an effective amount of cells or a composition comprising those cells is administered parenterally. Such administration may be intravenous administration. In some cases, administration may be directly by injection into the tumor.

In certain embodiments of the present disclosure, cells are administered to a patient in combination with (e.g., the former administered first, both administered simultaneously, or the former administered later) any number of relevant therapeutic regimens including, but not limited to, treatment with agents such as antiviral therapy, cidofovir (cidofovir) and interleukin-2, cytarabine (also known as ARA-C), or natalizumab (natalizumab) treatment for MS patients, or efalizumab (efalizumab) treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, T cells of the present disclosure may be used in combination with chemotherapeutics, radiation, immunosuppressants such as cyclosporine (cycloporin), azathioprine (azathioprine), methotrexate (methotrerate), mycophenolate (mycophenolate), and FK506, antibodies or other immunodepleting agents such as CAMPATH, anti-CD 3 antibodies or other antibody therapies, cytotoxins, fludarabine (fludarabine), cyclosporine, FK506, rapamycin (rapamycin), mycophenolic acid (mycoplienolic acid), steroids, FR901228, cytokines, and radiation.

In further embodiments, the cell compositions of the present disclosure are administered to a patient in combination (e.g., the former administered first, the latter administered simultaneously, or the former administered later) with bone marrow transplantation, T cell ablation therapy using a chemotherapeutic agent (e.g., fludarabine), external beam radiation therapy (XRT), cyclophosphamide, or an antibody (e.g., OKT3 or CAMPATH). In another embodiment, the cell composition of the present disclosure is administered after B cell ablative therapy, such as an agent that reacts with CD20, e.g., rituxan (Rituxan). For example, in one embodiment, the subject may undergo standard treatment, wherein high dose chemotherapy is followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives infusion of the expanded immune cells of the disclosure. In further embodiments, the expanded cells are administered before or after surgery. In certain embodiments, any means (e.g., surgery, chemotherapy, or radiation therapy) may be used to reduce tumor burden prior to administration of the expanded immune cells of the present disclosure. In one embodiment, reducing tumor burden prior to administration of the engineered cells of the present disclosure can reduce the likelihood of or prevent a cytokine release syndrome or cytokine storm, a side effect that may be associated with CAR T cell therapy.

Therapeutic applications

The present disclosure provides compositions comprising engineered cells (e.g., T cells) expressing the chimeric antigen receptors of the present disclosure and a pharmaceutically acceptable vehicle. The present disclosure also provides compositions comprising an antibody or antigen-binding fragment thereof and a pharmaceutically acceptable vehicle. In some cases, the engineered cells, antibodies, or antigen binding fragments form a drug, particularly for immunotherapy. In some cases, the engineered cells, antibodies, or antigen binding fragments are used to treat cancer (e.g., multiple myeloma or melanoma). In some cases, the engineered cells, antibodies, or antigen binding fragments are used to prepare a medicament for immunotherapy and/or treatment of cancer (e.g., MAGE-A4 expressing cancer).

The present disclosure provides methods comprising administering to a subject in need thereof a therapeutic composition comprising an antibody (e.g., a bispecific antibody) or antigen binding fragment thereof, and/or an engineered cell (e.g., a T cell) expressing a chimeric antigen receptor as discussed herein. The therapeutic composition may include cells expressing any of the chimeric antigen receptors as disclosed herein and a pharmaceutically acceptable carrier, diluent or vehicle. In additional or alternative examples, the therapeutic composition comprises an antibody and/or antigen binding fragment as discussed herein. As used herein, the expression "subject in need thereof" means a human or non-human animal that exhibits one or more symptoms or signs of cancer (e.g., a subject with a tumor expressing MAGE-A4 or with any of the cancers mentioned herein) or otherwise benefits from inhibition or reduction of MAGE-A4 activity or depletion of MAGE-a4+ cells.

The engineered cells and/or antibodies and antigen binding fragments of the present disclosure are particularly useful for treating any disease or disorder in which stimulation, activation, and/or targeting of an immune response would be beneficial. In particular, the engineered cells and/or antibodies and antigen binding fragments of the present disclosure can be used to treat, prevent and/or ameliorate any disease or disorder associated with or mediated by MAGE-A4 expression or activity or MAGE-a4+ cell proliferation. The engineered cells and/or antibodies and antigen binding fragments of the present disclosure can be used to inhibit or kill cells expressing MAGE-A4, including, for example, multiple myeloma cells, melanoma cells, or other solid tumor cells.

The engineered cells and/or antibodies and antigen binding fragments of the present disclosure can be used to treat diseases or disorders associated with MAGE-A4 expression including, for example, cancers including, but not limited to, multiple myeloma, synovial sarcoma, esophageal cancer, head and neck cancer, lung cancer, bladder cancer, ovarian cancer, uterine cancer, gastric cancer, cervical cancer, breast cancer, and melanoma. The engineered cells and/or antibodies and antigen binding fragments of the present disclosure can generally be used to treat tumors that express MAGE-A4. According to other related embodiments of the present disclosure, methods are provided that include administering engineered cells and/or antibodies and antigen binding fragments as disclosed herein to a patient suffering from a MAGE-A4 expressing tumor (including tumors from the cancers listed above). Analytical/diagnostic methods known in the art, such as tumor scanning, may be used to determine whether a patient has such a tumor, disease, or condition.

The present disclosure also provides methods for treating residual cancer in a subject. As used herein, the term "residual cancer" means the presence or persistence of one or more cancer cells in a subject following treatment with an anti-cancer therapy.

According to certain aspects, the present disclosure provides methods for treating a disease or disorder associated with MAGE-A4 expression (e.g., cancer), the methods comprising administering to a subject an engineered cell population and/or antibodies and antigen binding fragments described elsewhere herein, after determining that the subject has the disease or disorder. For example, the present disclosure provides methods for treating a disease or disorder comprising administering engineered immune cells to a patient 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year or more after the subject receives other immune therapies or chemotherapy.

The treatment discussed herein may be ameliorative, curative or prophylactic. The treatment may be part of an autoimmune therapy or part of an alloimmunotherapy. Autologous means that the cells, cell lines or cell populations used to treat the patient are derived from a patient or Human Leukocyte Antigen (HLA) -compatible donor. Allogeneic means that the cell, cell line, or cell population used to treat the patient is not derived from the patient but from a donor.

Described herein are cells that can be used with the disclosed methods. Treatment may be used to treat patients diagnosed with a pre-malignant or malignant cancer condition characterized by an excess of MAGE-A4 expressing cells, particularly MAGE-A4 expressing cells. Such conditions can be found in cancer.

Types of cancers to be treated with the engineered cells, antibodies and antigen binding fragments of the present disclosure include, but are not limited to, multiple myeloma, synovial sarcoma, esophageal cancer, head and neck cancer, lung cancer, bladder cancer, ovarian cancer, uterine cancer, stomach cancer, cervical cancer, breast cancer and melanoma.

The compositions and methods of the present disclosure may be used to treat a subject that has been characterized as having cells or tissues that express MAGE-A4 or is suspected of having cells or tissues that express MAGE-A4. For example, subjects benefiting from treatment according to the present disclosure include subjects with multiple myeloma, synovial sarcoma, esophageal cancer, head and neck cancer, lung cancer, bladder cancer, ovarian cancer, uterine cancer, gastric cancer, cervical cancer, breast cancer, or melanoma.

Combination therapy

The present disclosure provides methods comprising administering an antibody/antigen binding fragment (e.g., a bispecific antibody) and/or an engineered cell or population of cells comprising any of the chimeric antigen receptors described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be administered in combination with or in combination with the antibodies/antigen binding fragments and/or cells or cell populations of the present disclosure include, for example, antineoplastic agents (e.g., chemotherapeutic agents, including melphalan (melphalan), vincristine (Oncovin)), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin)), liposomal doxorubicin (Doxil)), obodamustine (Treanda)) or any other chemotherapeutic agent known to be effective in treating a plasma cell tumor in a subject. In some embodiments, the second therapeutic agent comprises a steroid. In some embodiments, the second therapeutic agent comprises targeted therapies including thalidomide (thalidomide), lenalidomide, and bortezomib (bortezomib) approved for treatment of newly diagnosed patients. For example, lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat (panobinostat), ixazomib (ixazomib), erltuzumab (elotuzumab) and darumumab (daratumumab) are examples of second therapeutic agents for the effective treatment of relapsed myeloma. In certain embodiments, the second therapeutic agent is a regimen comprising radiation therapy or stem cell transplantation. In certain embodiments, the second therapeutic agent may be an immunomodulatory agent. In certain embodiments, the second therapeutic agent may be a proteasome inhibitor, including bortezomib Carfilzomib->I Sha Zuo metersIn certain embodiments, the second therapeutic agent may be a histone deacetylase inhibitor, such as panobinostat +.>In certain embodiments, the second therapeutic agent may be a monoclonal antibody, an antibody drug conjugate, a bispecific antibody that may or may not be conjugated with an anti-neoplastic agent, a checkpoint inhibitor, or a combination thereof. Other agents that may be beneficially administered in combination with the antigen binding molecules of the present disclosure include cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or the like, or their respective receptors. The pharmaceutical compositions of the present disclosure (e.g., pharmaceutical compositions comprising an engineered cell or cell population as disclosed herein) can also be administered as part of a therapeutic regimen comprising one or more therapeutic combinations selected from the group consisting of: monoclonal antibodies other than those described herein that can interact with different antigens on the plasma cell surface; bispecific antibodies in which one arm binds to an antigen on the surface of a tumor cell and the other arm binds to an antigen on a T cell; antibody drug conjugates; bispecific antibodies conjugated to antitumor agents; checkpoint inhibitors such as checkpoint inhibitors targeting PD-1 or CTLA-4; or a combination thereof. In certain embodiments, the checkpoint inhibitor may be selected from PD-1 inhibitors, such as pembrolizumab (pembrolizumab) Na Wu Liyou mab (nivolumab)/(N.E.)>Or cimipro Li Shan antibody (cemiplimab)In certain embodiments, the checkpoint inhibitor may be selected from PD-L1 inhibitors, such as atezolizumab (atezolizumab)>Avelumab (avelumab)/(Avelumab)>Or Durvalumab (Durvalumab)>In certain embodiments, the checkpoint inhibitor may be selected from CTLA-4 inhibitors, such as liplimumab (ipilimumab)>

The invention also includes therapeutic combinations comprising any of the antibodies/antigen binding fragments and/or engineered cells or cell populations mentioned herein and an inhibitor of one or more of VEGF, ang2, DLL4, EGFR, erbB2, erbB3, erbB4, EGFRvIII, cMet, IGF R, B-raf, PDGFR-alpha, PDGFR-beta, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, urolysin (uroplakin) or any of the foregoing cytokines, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody or antibody fragment (e.g., fab fragment; F (ab') ₂ Fragments; fd fragment; fv fragments; an scFv; a dAb fragment; or other engineered molecules such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units). In some embodiments, the antibodies/antigen binding fragments and/or engineered cells or cell populations of the present disclosure may also be administered as part of a treatment regimen that also includes radiation therapy and/or conventional chemotherapy.

The additional therapeutically active component may be administered prior to, simultaneously with, or shortly after administration of the engineered cells of the present disclosure; (for the purposes of this disclosure, such an administration regimen is considered to be "combined" administration of the engineered cells with additional therapeutically active components).

The present disclosure provides pharmaceutical compositions in which an engineered cell or cell population of the present disclosure is co-formulated with one or more of the additional therapeutically active components as described elsewhere herein.

Administration protocol

According to certain embodiments of the present disclosure, multiple doses of antibody/antigen binding fragments and/or engineered cells may be administered to a subject over a defined period of time. The method according to this aspect comprises sequentially administering a plurality of doses of the antigen binding molecule and/or the cell to the subject. As used herein, "sequentially administered" means that each dose is administered to a subject at a different point in time, e.g., on a different date separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure provides methods comprising sequentially administering a single initial dose, followed by one or more second doses, and optionally followed by one or more third doses, to a patient.

The terms "initial dose", "second dose" and "third dose" refer to a temporal administration sequence of the antigen binding molecules and/or engineered cells of the present disclosure. Thus, an "initial dose" is the dose administered at the beginning of a treatment regimen (also referred to as the "baseline dose"); a "second dose" is a dose administered after the initial dose; and "third dose" is the dose administered after the second dose. The initial dose, the second dose and the third dose may each contain the same amount of antigen binding molecule and/or engineered cells, but in general they may differ from each other in the frequency of administration. However, in certain embodiments, the amounts of antigen binding molecules and/or engineered cells contained in the initial, second, and/or third doses are different from one another (e.g., are appropriately increased or decreased) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" at the beginning of a treatment regimen, followed by subsequent doses (e.g., a "maintenance dose") on a less frequent basis.

In an exemplary embodiment of the invention, 1 to 26 are followed immediately after the previous dose (e.g., 1 ¹ / ₂ 、2、2 ¹ / ₂ 、3、3 ¹ / ₂ 、4、4 ¹ / ₂ 、5、5 ¹ / ₂ 、6、6 ¹ / ₂ 、7、7 ¹ / ₂ 、8、8 ¹ / ₂ 、9、9 ¹ / ₂ 、10、10 ¹ / ₂ 、11、11 ¹ / ₂ 、12、12 ¹ / ₂ 、13、13 ¹ / ₂ 、14、14 ¹ / ₂ 、15、15 ¹ / ₂ 、16、16 ¹ / ₂ 、17、17 ₁ / ² 、18、18 ¹ / ₂ 、19、19 ¹ / ₂ 、20、20 ¹ / ₂ 、21、21 ¹ / ₂ 、22、22 ¹ / ₂ 、23、23 ¹ / ₂ 、24、24 ¹ / ₂ 、25、25 ¹ / ₂ 、26、26 ¹ / ₂ Or more) each second dose and/or third dose is administered weekly. As used herein, the phrase "immediately preceding dose" means a dose administered to a patient in the order of multiple administrations, without an intermediate dose, before the next dose is administered in the order.

Methods according to this aspect of the disclosure may include administering any number of second and/or third doses to the patient. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to the patient. Likewise, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) third doses are administered to the patient.

In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency of administration of the second dose and/or the third dose to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by the physician according to the needs of the individual patient after the clinical examination.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present disclosure, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees celsius, and pressure is at or near atmospheric pressure.

Example 1: generation of anti-MAGE-A4 antibodies

anti-MAGE-A4 antibodies were obtained by immunizing genetically modified mice (e.g., engineered mice comprising DNA encoding human immunoglobulin heavy and kappa light chain variable regions) with human MAGE-A4 antigen (e.g., hMAGE-A4286-294 against mAb31339N 2) and HLA-A2 (HLA-A 02: 01). More specifically, the genome of the genetically modified mouse has a nucleotide sequence encoding a human HLA-A2 (as well as sequences encoding human immunoglobulin heavy and kappa light chain variable regions) such that the genetically modified mouse expresses a human HLA-A2, wherein the mouse is tolerant to human HLA-A2 such that they produce a specific B cell response upon immunization with the MAGE-A4 antigen and HLA-A 2.

After immunization, spleen cells are collected from each mouse and either (1) fused with mouse myeloma cells to maintain their viability and form hybridoma cells and screened for MAGE-A4 specificity, or (2) B cell-sorted using human MAGE-A4 fragments as a sorting reagent that binds and identifies reactive antibodies (antigen-positive B cells) (as described in U.S. patent publication No. 2007/0280945 A1).

Chimeric antibodies directed against MAGE-A4 were initially isolated with human variable and mouse constant regions. Antibodies are characterized and selected for desired characteristics (including affinity, selectivity, etc.). In embodiments, the mouse constant region is replaced with a desired human constant region (e.g., a wild-type or modified IgG1 or IgG4 constant region) to produce a fully human anti-MAGE-A4 antibody (e.g., mAbM31339N2 (including mouse constant region) is used to produce mAbH31339N2 (including human constant region)). While the constant region selected may vary depending on the particular application, high affinity antigen binding and target specific properties are present in the variable region.

mAbM31339N2 was also reformatted into bispecific antibodies by generating bsAb6054 using high affinity (7195P) CD3 arms (HCVR 1, LCVR1, and LCVR2 from mAb31339 and HCVR2 from 7195P) or bsAb6043 using medium affinity (7221G) CD3 arms (HCVR 1, LCVR1, and LCVR2 from mAb31339 and HCVR2 from 7221G).

Heavy and light chain variable region amino acid and nucleic acid sequences of anti-MAGE-A4 antibodies: table 1 lists the amino acid sequence identifiers of the heavy and light chain variable regions and the heavy and light chain CDRs of selected anti-MAGE-A4 antibodies of the disclosure. The mAb31339N and mAb31339N2 sequences of table 1 are identical except for one amino acid difference in HCVR framework region 3; however, the CDRs for mAb31339N and mAb31339N2 are identical. The corresponding nucleic acid sequence identifiers are listed in Table 2. A summary of all sequences contained herein is provided in table 15.

Table 1: amino acid sequence identifier

Table 2: nucleic acid sequence identifier

Bispecific antibodies that bind CD3 and MAGE-A4 are generated as follows: bispecific antibodies comprising an anti-CD 3 specific binding domain and an anti-MAGE-A4 specific binding domain were constructed by the sequences listed in tables 3 and 4 using methodologies in which the heavy chain from the anti-CD 3 antibody was combined with the heavy chain and homologous light chain from the anti-MAGE-A4 antibody.

Thus, bispecific antibodies produced according to the present examples comprise two separate antigen binding domains (i.e., binding arms). The first antigen-binding domain comprises a heavy chain variable region derived from an anti-MAGE-A4 antibody ("MAGE-A4-VH") paired with a homologous light chain variable region derived from an anti-MAGE-A4 antibody ("MAGE-A4-VL"), and the second antigen-binding domain comprises a heavy chain variable region derived from an anti-CD 3 antibody ("CD 3-VH") paired with a MAGE-A4-VL. In principle, homologous light chain variable regions ("CD 3-VL") from anti-CD 3 antibodies can also be used as light chain variable regions common to both arms of the antibody. The same MAGE-A4-VH was used in all bispecific antibodies generated in this example. The bsAb6054 antibody includes a MAGE-A4 binding arm comprising a HCVR/LCVR of SEQ ID NO. 2/10 and a CD3 binding arm comprising a HCVR/LCVR of SEQ ID NO. 73/10. The bsAb6043 antibody includes a MAGE-A4 binding arm comprising a HCVR/LCVR of SEQ ID NO. 2/10 and a CD3 binding arm comprising a HCVR/LCVR of SEQ ID NO. 55/10.

The amino acid sequence identifiers used to construct the heavy and light chain variable regions of the anti-CD 3 antigen binding arm and the anti-MAGE-A4 binding arm are set forth in Table 3. The corresponding nucleic acid sequence identifiers are listed in Table 4. anti-MAGE/anti-CD 3 bispecific antibodies are generated from medium affinity CD3 antibodies (anti-CD 3-A; referred to herein as H4sH7221G or 7221G) or high affinity CD3 antibodies (anti-CD 3-B; referred to herein as HpH sH7195P or 7195P). The first antigen binding domain and the second antigen binding domain are referred to herein as the "arms" of the bispecific antibody. In an embodiment, one arm comprises a HCVR comprising the amino acid sequence of SEQ ID NO. 2 and a LCVR comprising the amino acid sequence of SEQ ID NO. 10, and the other arm comprises a HCVR comprising the amino acid sequence of SEQ ID NO. 55 or SEQ ID NO. 73 and a LCVR comprising the amino acid sequence of SEQ ID NO. 10.

Table 3: amino acid sequence identifier

Table 4: nucleic acid sequence identifier

Example 2: determination of anti-HLA-A 2 by flow cytometry MAGE-A4 (286-294) antibodies and methods of using MAGE-A4 (286-294) pulsed T2 cell binding

The cell surface binding of the anti-HLA-A2:MAGE-A4 286-294 antibody (mAbM 31339N) to HLA-A02:01 positive T2 (174 CEM. T2) cells was evaluated in a flow cytometry-based peptide pulse assay. For pulsing, 1X 10 at 37℃C ⁶ The T2 cells were incubated with 10. Mu.g/mL human (h) B2M (EMD Millipore catalog No. 475828) and 100. Mu.g/mL MAGE-A4 286-294 peptide in 1mL AIM V medium (Ji Bo company (Gibco) catalog No. 31035-025) for 16 hours. Cells were washed in staining buffer ((calcium and magnesium free PBS (Corning, commercial 21-031-CV) +2% FBS (Seradigm, batch 238B 15)), collected with cell separation buffer (Miybbo Co., catalog S-004-C) and resuspended in staining buffer. Pulsed cells (200,000) were inoculated in 96-well V-bottom plates (Ainsen, catalog P-96-450V-C-S) and stained with mAbM31339N or triple serial dilutions (1.7 pM to 100 nM) of unbound control antibody (mAb 1097) for 30 min at 4 ℃ C.) cells were then washed in staining bufferSecondary and incubated with 5 μg/mL of Alexa Fluor 647 conjugated Fab'2 anti-mouse Fc specific secondary antibody (Jackson ImmunoResearch, cat# 115-606-071) for 30 min at 4 ℃. Finally, the cells were stained with a green fluorescent vital dye (Molecular Probes) catalog number L-34970, reconstituted in 50. Mu.L DMSO at a concentration of 1:1000. Cells were then washed and fixed with 50% BD Cytofix (BD company, cat. No. 554655) solution diluted in PBS. Samples were run on an intellicytique flow cytometer (Intellicyt) and the results were analyzed using forechyte analysis software (Intellicyte) to calculate the Mean Fluorescence Intensity (MFI) after gating on living cells. MFI values were plotted on a 12-point response curve using a four-parameter logistic equation in Graphpad Prism to calculate EC ₅₀ Values. Individual secondary antibodies (i.e., no primary antibody) for each dose-response curve were also included in the analysis as a continuation of the triple serial dilutions and were expressed as the lowest dose. The signal to noise ratio (S/N) was determined by taking the ratio of the highest MFI on the dose response curve to the MFI in the wells of the secondary antibody alone. EC (EC) ₅₀ The value (M) and the maximum S/N are shown in Table 5. mAbM31339N bound with an EC50 of 1.7nM and a maximum S/N of 1933, whereas no binding of the control antibody (mAb 1097) was detected, indicating that mAb31339N has a strong binding affinity for MAGE-A4 (286-294). Referring to table 5, nd refers to EC50 values that cannot be accurately determined due to binding that does not reach saturation over the range of test antibody concentrations.

Table 5: binding of the anti-HLA-A 2 MAGE-A4 (286-294) antibody to T2 cells presenting MAGE-A4 (286-294)

Example 3: anti-HLA-A 2 MAGE-A4 antibodies and MAGE-A4 XCD 3 bispecific antibodies related to use of MAGE-A4 Peptide pulsed T2 cell binding。

A computer calculation strategy (Dhanik et al, BMC Bioinformatics 2016) identified several MAGE-A4 related peptides predicted to form complexes with HLA-A 02:01. The results of the identification are summarized in Table 6.

Table 6: sequences of MAGE-A4 286-294 peptides and predicted off-target peptides

The binding of the parent antibody (mAbH 31339N 2), MAGE-A4 XCD 3 (7221G) bispecific antibody (bsAb 6043) and non-binding isotype control antibody (mAb 4241) to these related peptides as shown in Table 6 was evaluated in the T2 pulse assay described above. As described in example 1 above, the bsAb6043 antibody includes a MAGE-A4 binding arm comprising a HCVR/LCVR of SEQ ID NO. 2/10 and a CD3 binding arm comprising a HCVR/LCVR of SEQ ID NO. 55/10. As summarized in Table 7, both antibodies bound to the MAGE-A4 peptide, with mAbH31339N2 having an S/N value of 310 and bsAb6043 having an S/N value of 244, substantially above baseline (no peptide). The test antibodies showed lower binding to AOX1 with mAbH31339N2 having an S/N value of 131 and bsab6043 having an S/N value of 110 and lower binding to SHTN1, with both antibodies having an S/N value of 5. No detectable binding to the remaining peptide was observed and for all peptides tested, the control antibody binding was ∈3.

Table 7: binding of the anti-HLA-A 2 MAGE-A4 (286-294) antibody to T2 cells which present MAGE-A4-associated peptides

Example 4: evaluation of MAGE-A4 XCD 3 double in T cell reporter/Antigen Presenting Cell (APC) bioassays Activity of specific antibodies

The activity of MAGE-A4 XCD 3 bispecific antibodies was evaluated in a T cell reporter/Antigen Presenting Cell (APC) bioassay. To generate an assay cell line, jurkat cells were usedNF-KB dependent firefly luciferase lentiviral reporter vector (Qiagen) was transduced and single cell sorted for high luciferase activity to generate Jurkat/NF-KBluc assay cell lines. Myeloma cell lines IM9 and U266B1 cells endogenously expressing MAGE-A4 and HLA-A 02:01 were used as Antigen Presenting Cells (APC). In addition, MAGE-A4 and HLA-A2 negative Raji cells were used as controls. Briefly, 25,000 Jurkat/NF-. Kappa.BLuc cells were mixed in 25. Mu.L of assay medium (RPMI medium containing 10% FBS and 1% P/S/G) and added to Thermo-Nunc 96 Kong Baiban (Semer Feishmania technology Co., thermo Scientific catalog No. 136101), followed by 25,000 APCs mixed in 25. Mu.L of assay medium. A3-fold serial dilution of 27.4pM to 20nM antibody was added to the plate containing 50. Mu.L of assay medium. The cell mixture was incubated at 37℃with 5% CO ₂ Incubate for 5 hours in a humidified incubator. NF- κB luciferase activity was measured using Promega One-Glo (catalog number E6130) and Perkin Elmer Envision plate reader according to manufacturer's instructions. Relative Luciferase Units (RLU) were generated on an 8-point dose response curve (GraphPad Prism) using a four-parameter logistic equation and EC50 values were determined. The zero primary antibody condition (secondary antibody alone) for each dose-response curve was also included in the analysis as a continuation of the triple serial dilutions and expressed as the lowest dose. The maximum activity was determined by taking the ratio of the highest RLU to the lowest RLU on the curve and expressed as signal to noise (S: N). The EC50 values and S/N are summarized in Table 8. With respect to table 8, nd corresponds to EC50 values that could not be accurately determined due to binding that did not reach saturation over the range of antibody concentrations tested.

Table 8: MAGE-A4 XCD 3 bispecific antibodies in

Activity in Jurkat/NF- κB-Luc/APC reporter bioassay

As described in example 1, the bsAb6054 antibody includes a MAGE-A4 binding arm comprising a HCVR/LCVR of SEQ ID NO. 2/10 and a CD3 binding arm comprising a HCVR/LCVR of SEQ ID NO. 73/10, and the bsAb6043 antibody includes a MAGE-A4 binding arm comprising a HCVR/LCVR of SEQ ID NO. 2/10 and a CD3 binding arm comprising a HCVR/LCVR of SEQ ID NO. 55/10. The bsAb4241 and bsAb3905 antibodies are non-binding (to MAGE-A4) controls with anti-CD 3 binding arms comprising the same anti-CD 3 HCVR as bsAb6043 and bsAb6054, respectively, paired with a cognate light chain variable region from the non-MAGE-A4 binding arm of each respective antibody.

As shown in Table 8, MAGE-A4 XCD 3 medium affinity bispecific antibody (bsAb 6043) was not active in Jurkat/NF- κBLuc bioassays in the presence of IM9, U266B1, or RAJI cells. In contrast, MAGE-A4 XCD 3 high affinity bispecific antibody (bsAb 6054) had EC50 values of 7.4E-10M and 3.1E-09M and S/N values of 41.9 and 6.3 on IM9 and U266B1 cells, respectively. The non-binding control bispecific antibody with medium affinity CD3 arm (mAb 4241) or high affinity CD3 arm (mAb 3905) had minimal activity with an S/N value of 2.2 or less. In addition, none of the antibodies were active on RAJI cells.

Similar bioassays were performed, but this time with a constant amount of anti-CD 28 antibody added. Here, the addition of CD28 partially blocked the activity of bsAb6054 on IM9 cells (EC 50 of 2.1E09M, S/N of 28.7) while slightly increasing the activity on U266B1 cells (EC 50 of 6.1E-09M, S/N of 8.6). This may be due to the level of endogenously expressed CD80 and CD86 costimulatory molecules on these cell lines. IM9 endogenously has high levels of CD80 and CD86. Thus, the addition of CD28 antibodies in bioassays blocked the natural interaction of CD28 with its ligands CD80 and CD86 on Jurkat cells, thus reducing activity. U266B1 has low levels of CD80 and CD86. Here, the addition of CD28 stimulated CD28 signaling on Jurkat cells, thereby increasing activity (table 9).

Table 9: the combination of MAGE-A4 XCD 3 bispecific antibody with 2nM mAb5705 (anti-CD 28 antibody) was found in Jurkat +. Activity in NF- κB-Luc/APC reporter bioassay

In addition to the antibodies discussed above in connection with Table 8, mAb5705 is an anti-CD 28 antibody comprising the HCVR of SEQ ID NO:85 and the LCVR of SEQ ID NO: 93.

Example 5: peptide specificity in Jurkat/NF- κB-Luc/APC reporter bioassays

Engineered T cell/APC functional Jurkat/NF- κbluc reporter/APC bioassays were also utilized to assess whether MAGE-a4×cd3 bispecific antibodies retained selectivity for MAGE-A4 peptides (relative to the related peptides identified in table 7). In this assay, T2 cells were pulsed with the target peptides MAGE-A4 286-294 or sequence-related off-target peptides (Table 7), as described previously. As summarized in Table 10, both bsAb6043 (MAGE-A4 XCD 3 medium affinity) bispecific antibody and bsAb6054 (MAGE-A4 XCD 3 high affinity) bispecific antibody stimulated NF-. Kappa.B-dependent reporter activity when incubated with T2 cells pulsed with MAGE-A4 286-294 peptides. Activity was also detected when incubated with T2 cells pulsed with the off-target peptides AOX1 795-803 and SHTN1 198-206. In summary, bsAb6043 stimulated reporter activity with EC50 values of 3.0E-10M, 3.3E-10M and 3.5E-09M and S/N values of 75.2, 40.4 and 75.3 for MAGE-A4 293-294, AOX1 795-803 and SHTN1 198-206 peptides, respectively. For MAGE-A4 286-294, AOX1 795-803, and SHTN1 198-206 peptides, bsAb6054 stimulated reporter activity at EC50 values of 2.0E-12M, 2.1E-11M, and 1.3E-09M, and S/N values of 78.7, 36, and 81.1, respectively. The non-binding control bispecific antibody with medium affinity CD3 arm (mAb 4241) or high affinity CD3 arm (mAb 3905) had minimal activity with an S/N value of 2.9 or less.

Table 10: MAGE-A4 XCD 3 bispecific antibodies against MAGE-A4 and related peptides were found in Jurkat/NF-. Kappa.B-Luc- Activity in T2 pulse reporter bioassay

Example 6: image-based primary killing assay

To assess the ability of MAGE-a4×cd3 bispecific antibodies to redirect T cell responses to cancer cells, an imaging-based multiplex primary T cell killing assay was performed. Briefly, CD8+ T cells were isolated from human Peripheral Blood Mononuclear Cells (PBMC) using magnetic bead isolation (Miltenyi Biotec catalog No. 130-045-201) according to the manufacturer's protocol and CellTrace according to the manufacturer's instructions ^TM Violet (Semer Feishan technologies C34557) pre-tags the MAGE-A4 286-294 antigen presenting HLA-A 02:01 positive IM9 cell line. IM9 multiple myeloma cells (10,000) were mixed with cd8+ T cells (25,000) in 100 μl of stimulation medium (X Vivo 15+10%FBS+1%HEPES+1%NaPyr+1%NEAA+0.01mM BME) in 96 well imaging plates (Perkin Elmer) catalog number 6055308). Three-fold serial dilutions of antibodies ranging from 20nM to 27.4pM were added to cells in an additional 100 μl of stimulation medium. After 72 hours incubation, 50 μl of supernatant was removed for cytokine release assay (see below) and 15 μl of 1:1000 dilution (in PBS) of nuclear dye DRAQ5 (zemoer feier technologies catalog No. 62252) was added to the cells. Images of DRAQ 5-labeled nuclei and Cell-Trace device-labeled APCs were collected on an Opera Phenix (Perkin Elmer) and IM9 cells (CellTrace) surviving under all conditions were calculated using Harmony analysis software (Perkin Elmer) ^TM Cell populations that are simultaneously labeled with Violet and DRAQ 5). EC50 values cannot be generated by the resulting non-sigmoidal curve, and therefore the percent IM9 cell survival (normalized to antibody-free conditions) at the 6.6nM dose (highest test dose at which the non-binding control was inactive) is reported. As summarized in Table 11, bsAb6043 (MAGE-A4 XCD 3 (7221)G) Medium affinity bispecific antibody) had no positive effect on the percent survival of the IM9 cell population, but bsAb6054 (MAGE-A4 xcd 3 high affinity bispecific antibody) reduced the IM9 cell population to 38%. The addition of 2nM of anti-CD 28 agonistic antibody doubled the number of surviving cells (80%) (Table 9). This is attributable to the ability of mAb5705 to block CD28 and thereby inhibit its interaction with CD80 and CD86 endogenously expressed on IM9 cells. Non-binding control xcd 3 bispecific antibody mAb4241 (medium CD affinity) and mAb3905 (high CD3 affinity) were inactive in the assay.

Table 11: t cell cytotoxicity image-based assays

Example 7: important cytokine release assay

IL2 and IFN-gamma release was also assessed in cell culture supernatants sampled from the 6.6nM treatment dose utilized in the imaging-based killing assay described above. Cytokine levels were determined by AlphaLisa (perkin elmer catalog No. AL221F, AL 217F) according to manufacturer' S instructions and RLU values were normalized to untreated wells to determine S/N values. As summarized in Table 12, bsAb6043 (MAGE-A4 XCD 3 (7221G) medium affinity bispecific antibody) did not release any IL2 or IFN-gamma, whereas the higher affinity MAGE-A4 XCD 3 bispecific antibody bsAb6054 induced modest IL2 and IFN-gamma production, with S/N values of 2.1 and 2.4, respectively. The addition of CD28 antibody (mAb 5705) did not significantly affect cytokine production (table 12).

Table 12: release of IL2 and IFN-gamma in a three day killing assay

Example 8: generation of MAGE-A4 specific chimeric antigen receptor

The anti-MAGE-A4 31339N2 antibodies of Table 1 were reformatted into VL-VH single chain variable fragments (ScFv) using HCVR and LCVR nucleotide sequences corresponding to the anti-MAGE-A4 antibodies of SEQ ID NOs 1 and 9, respectively, and placed in Chimeric Antigen Receptor (CAR) constructs using the CD8 alpha hinge and transmembrane domain, the 4-1BB costimulatory domain and CD3 zeta stimulation domain, or the CD28 hinge, transmembrane and signaling domain. The full length nucleic acid and polypeptide heavy chain sequences of the corresponding anti-MAGE-A4 antibody (mAbH 31339N 2) correspond to SEQ ID NO. 17 and 18, respectively. The full length nucleic acid and polypeptide light chain sequences of the corresponding anti-MAGE-A4 antibody (mAbH 31339N 2) correspond to SEQ ID NO. 19 and 20, respectively. Full length nucleic acids and polypeptides HLA-A2/MAGE-A4 _286-294 The targeted CAR sequences correspond to SEQ ID NOS 21 and 22, respectively. As a non-binding control, a similar CAR was designed using the nucleotide sequence of the unrelated scFv. MAGE-A4 specific CARs were cloned into lentiviral expression vectors (Lenti-X according to the manufacturer's protocol ^TM The bicistronic expression system (Neo), clone technologies company (Clontech) catalog number 632181), and lentiviral particles were generated by the Lenti-X packaging single shot (VSV-G) system (clone technologies company catalog number 631276). And then according to the manufacturer's scheme, use The precoated dishes (clone technologies company catalog number T110 a) were transduced with CAR constructs into Jurkat cells engineered to express NFKB-luciferase reporter gene (Jurkat/NKFBLuc cl 1C 11). After selecting for at least 2 weeks in 500 μg/mL G418 (Ji Bo company catalog No. 11811-098), the following CAR T cell lines were generated; jurkat/NKBFLUc cl 1C11/MAGE-A4 (286-294) 31339VL-VH CART. As a non-binding control, a similar CAR was designed using the nucleotide sequence of the unrelated scFv. The cell surface expression and functional activity of this CAR T cell line in response to MAGE-A4 expressing cells was evaluated.

Example 9: cell surface expression of MAGE-A4 CAR constructs in Jurkat cells and MAGE-A4 CAR Activation of T cells

The relative level of cell surface expression of the MAGE-A4 CAR construct in Jurkat/NF-. Kappa.B-Luc cells was assessed by flow cytometry. For staining, cells were seeded in 96-well V-bottom plates at a density of 200,000 cells per well in a staining buffer of PBS (Irving 9240) and 2% bsa (Sigma-Aldrich catalog No. a 8577) without calcium and magnesium, and stained with 10 μg/mL protein L (Genscript Biotin Protein L, catalog No. M00097) for 30 min at 4 ℃. After incubation, the cells were washed once in staining buffer and stained with 0.5 μg/mL streptavidin Alexa-647 secondary antibody (Biolegend, cat. No. 405237) for 30 min at 4 ℃. Finally, the cells were stained with a green fluorescent vital dye (molecular probes, cat. No. L-34970, reconstituted in 50. Mu.L DMSO) at a concentration of 1:1000. Cells were then washed and fixed with 50% BD Cytofix (BD company (Becton Dickinson), catalog No. 554655) solution diluted in PBS. Samples were run on an intelllicyt iQue flow cytometer after gating on living cells and analyzed by FlowJo 10.2 to calculate the Mean Fluorescence Intensity (MFI). The percentage of protein L positive cells was calculated by taking the number of protein L positive cells and dividing by the total number of cells. Table 13 shows that the CAR positive rate of the Jurkat/NF-. Kappa.B-Luc/MAGE-A4 31339N2 VL-VHCAR-T cell line was 33.8% as measured by percent protein L staining. The non-targeted control CAR was expressed in 67.2% of the cells.

The activity of the CAR T cell line was evaluated in a CAR T/APC (antigen presenting cell) bioassay. For bioassays, 50,000 CAR T cells were added to 50 μl of assay medium (RPMI medium containing 10% fbs and 1% p/S/G) and to Thermo-Nunc 96 Kong Baiban (sameifeishi technologies, catalog No. 136101), followed by 3-fold serial dilutions of APCs (500,000 cells to 685 cells) mixed in 50 μl of assay medium. The following APCs were utilized: IM9 (which endogenously expresses the MAGE-A4 286-294 peptide and is HLA-A 02:01 positive) and HEK293 (which is MAGE-A4 286-294 negative and is HLA-A 02:01 positive). The cell mixture was incubated at 37℃with 5% CO ₂ Incubate for 5 hours in a humidified incubator. NF- κB luciferase activity was measured using Promega One-Glo (catalog number E6130) and Perkin Elmer Envision plate reader. Dose response at 8 points using four parameter logistic equation in GraphPad PrismRelative Luciferase Units (RLU) were generated and plotted on the curve to generate EC50 values (number of APCs). Zero APC conditions for each dose response curve are also included in the analysis as a continuation of the tripled series succession and are expressed as the lowest dose. CAR-T activity was determined by taking the ratio of the highest RLU to the lowest RLU on the curve and reporting it as EC50 (number of APCs) and expressed in Table 14 as signal to noise (S: N). 31339N2CAR-T was activated when incubated with IM9 cells, with an EC50 of 14,669 cells and S/N of 37.4, whereas the non-targeted control CAR-T was only 4.9 fold activated. No activation was observed on HEK293 cells, which were MAGE-A4 negative but HLA-A2 positive.

Table 13: protein L staining of CAR in Jurkat/NFKBlucc cl 1C11 cells

Table 14: activation of MAGE-A4 CAR T cells in CAR T cell/APC bioassays

Example 10: determination of anti-HLA-A 2 by flow cytometry, MAGE-A4 230-239 antibodies and methods of using MAGE-A4 230-239 and related off-target peptide pulsed T2 cell binding

The cell surface binding of the anti-HLA-A2:MAGE-A4 230-239 antibody (mAbM 34852N) to HLA-A 02:01 positive T2 (174 CEM. T2) cells was evaluated in a flow cytometry-based peptide pulse assay. For pulsing, 1X 10 at 37℃C ⁶ The T2 cells were incubated with 10. Mu.g/mL human (h) B2M (EMD Mibo catalog No. 475828) and 100. Mu.g/mL MAGE-A4 230-239 peptide in 1mL AIM V medium (Ji Bo catalog No. 31035-025) for 16 hours. Cells were washed in staining buffer (PBS without calcium and magnesium (Irvine Scientific catalog number 9319) +2% fbs (Seradigm, lot number 238B 15), with cell separation buffer (millbot,catalog number S-004-C) was collected and resuspended in staining buffer. Pulsed cells (200,000) were seeded in 96-well V-bottom plates (alsin, cat# P-96-450V-C-S) and stained with a three-fold serial dilution (1.7 pM to 100 nM) of mAbM34852N or unbound control antibody (mAb 1097) for 30 min at 4 ℃. Cells were then washed once in staining buffer and incubated with 5 μg/mL Alexa Fluor 647 conjugated Fab'2 anti-mouse Fc specific secondary antibody (Jackson ImmunoResearch, cat# 115-606-071) for 30 minutes at 4 ℃. Finally, the cells were stained with a green fluorescent dye (molecular probes cat. No. L-34970) reconstituted in 50. Mu.L DMSO at a concentration of 1:1000. Cells were then washed and fixed with 50% BD Cytofix (BD company, catalog 554655) solution diluted in PBS. Samples were run on an IntelliCyt iQue flow cytometer (IntelliCyt) and the results were analyzed using forechyte analysis software (IntelliCyt) to calculate the Mean Fluorescence Intensity (MFI) after gating on living cells. MFI values were plotted on a 12-point response curve using a four-parameter logistic equation in Graphpad Prism to calculate EC ₅₀ Values. Individual secondary antibodies (i.e., no primary antibody) for each dose-response curve were also included in the analysis as a continuation of the triple serial dilutions and were expressed as the lowest dose. The signal to noise ratio (S/N) was determined by taking the ratio of the highest MFI on the dose response curve to the MFI in the wells of the secondary antibody alone. EC (EC) ₅₀ The value (M) and the maximum S/N are shown in Table 15. mAbM34852N with an EC of 4.7nM ₅₀ Maximum S/N binding to 243.3, whereas binding of the control antibody (mAb 1097) was only weakly detected.

Table 15: anti-HLA-A 2 MAGE-A4 (230-239) antibodies and T2 cells pulsed with MAGE-A4 (230-239) Combined flow cytometry assay results

mAb	EC ₅₀ (M)	Maximum S/N
			mAbM34852N	4.7E-09	243.3
mAb1097 (isotype control)	ND	13.4

Nd=ec 50 values cannot be accurately determined because binding does not reach saturation within the measured antibody concentration range.

In silico strategies several MAGE-A4 related peptides were identified that were predicted to form complexes with HLA-A 02:01. The results of the identification are summarized in Table 16. Binding of the HLA-A2: MAGE-A4 230-239 antibody (mAbM 34852N) and the non-binding isotype control antibody (mAb 1097) to these related peptides was evaluated in the T2 pulse assay described above at a single concentration of 100 nM. The binding here is shown in table 17 as the MFI ratio of the binding of peptide pulsed cells divided by the binding of non-pulsed cells. mAbM34852N bound the MAGE-A4 peptide at a binding rate of 512.9, while isotype control mAb1097 bound the peptide at a rate of 4.3. mAbM34852N has a similar binding to the MAGE-A8 (232-241) peptide at a binding rate of 870.2; however, no detectable binding to the remaining peptide was observed, and for all peptides tested, the control antibody binding was <4.3, indicating that mAbM34852N has potential as a therapeutic agent against MAGE-A4.

Table 16: sequences of MAGE-A4 (230-239) peptide and predicted off-target peptide

The changes from the MAGE-A4 (230-239) sequence are underlined.

Table 17: binding of anti-HLA-A 2 MAGE-A4 (230-239) antibodies to T2 cells pulsed with related peptides Results of the cytometry assay

Example 11: HLA-A2 MAGE-A4 (230-239) alanine scanning

The cell surface binding of the anti-HLA-A2:MAGE-A4 230-239 antibody (mAbM 34852N) to HLA-A 02:01 positive T2 (174 CEM. T2) cells was evaluated in a flow cytometry-based peptide pulse assay. For pulsing, 1X 10 at 37℃C ⁶ The T2 cells were incubated with 10. Mu.g/mL human (h) B2M (EMD Mibo catalog No. 475828) and 100. Mu.g/mL MAGE-A4 230-239 peptide in 1mL AIM V medium (Ji Bo catalog No. 31035-025) for 16 hours. Cells were washed in staining buffer (PBS without calcium and magnesium (Irvine Scientific catalog number 9319) +2% FBS (Seradigm, lot number 238B 15), collected with cell separation buffer (Migrabo Co., catalog number S-004-C) and resuspended in staining buffer, pulsed cells (200,000) were inoculated in 96-well V-bottom plate (Aishida, catalog number P-96-450V-C-S) and stained with a three-fold serial dilution of mAb 34852N or non-binding isotype control antibody (1.7 pM to 100 nM) at 4℃for 30 min, then washed once in staining buffer and incubated with 5. Mu.g/mL of Alexa Fluor 647 conjugated Fab'2 anti-mouse Fc specific secondary antibody (Jackson ImmunoResearch, catalog number 115-606-071) at 4℃for 30 min, finally stained with a green fluorescent dye (molecular probe catalog number L-34970) which was reconstituted to a dye at a concentration of 1. Mu.g/L in PBS (Cypraecox) and assayed by calculating the average value of the following dilution of the following the flow of the three-serial dilutions in a sample of cell (Cypraecox) at 62. Cypraecox by using a four-dimensional flow control protocol (Cypraecox) for 50 m) and performing a fluorescent analysis on the sample (Cytox-dependent on the sample) MFI values are plotted on a curve to calculate EC ₅₀ Values. Individual secondary antibodies (i.e., no primary antibody) for each dose-response curve were also included in the analysis as a continuation of the triple serial dilutions and were expressed as the lowest dose. The signal to noise ratio (S/N) was determined by taking the ratio of the highest MFI on the dose response curve to the MFI in the wells of the secondary antibody alone. EC (EC) ₅₀ The value (M) and the maximum S/N are shown in Table 19. mAbM34852N with an EC of 4.7nM ₅₀ Maximum S/N binding to 243.3, whereas the binding of the control antibody was only weakly detected. The results of the alanine scan assay showed that mAbM34852 exhibited binding to the MAGE-A4 (230-239) peptide.

Table 18: MAGE-A4 (230-239) peptide alanine scanning peptide sequence

Peptide (Gene name + peptide position)	Peptide sequences	SEQ ID NO.
			MAGE-A4(230-239)	GVYDGREHTV	122
MAGE-A4(230-239)G230A	AVYDGREHTV	132
			MAGE-A4(230-239)V231A	GAYDGREHTV	133
MAGE-A4(230-239)Y232A	GVADGREHTV	134
			MAGE-A4(230-239)D233A	GVYAGREHTV	135
MAGE-A4(230-239)G234A	GVYDAREHTV	136
			MAGE-A4(230-239)R235A	GVYDGAEHTV	137
MAGE-A4(230-239)E236A	GVYDGRAHTV	138
			MAGE-A4(230-239)H237A	GVYDGREATV	139
MAGE-A4(230-239)T238A	GVYDGREHAV	140
			MAGE-A4(230-239)V239A	GVYDGREHTA	141

Table 19: anti-HLA-A 2 MAGE-A4 (230-239) antibody and scanning peptide pulses with alanineBinding of T2 cells of (E) Flow cytometry assay results of (2)

Sequence description

Table 20: sequence identifier

Annotation sequences

In the following annotation sequences, these parts are identified by the non-underlined sections and the underlined sections alternating, and the order of these parts corresponds to the order listed under each sequence (i.e., the first non-underlined section is VL, the next underlined section is (G4S) 3, the next non-underlined section is VH, and so on).

MAGE-A4(286-294)31339N2 VL-VH BBzCAR(SEQ ID NO:22)DIQMTQSPSSLSASVGDRVTITCRASQSIDTFLNWYQQKPGKVPKLLIYAASNLESGVPSRFSGSGSGTVFTLTISRLQPEDFATYYCQQSYSIPPITFGQGTRVEIKRGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSEYYLTWIRQAPGKGLDWIAYISSSGYNIYYADSVKDRFTISRDNGKNSLFLQMNNLRAEDTAVYFCAREGVTDGMDVWGQGTTVTVSSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

VL

(G4S)3

VH

G4S

CD8 hinge/TM

4-1BB costimulatory domains

CD3Z

MAGE-A4(286-294)31339N2 VL-VH BBzCAR(SEQ ID NO:120)DIQMTQSPSSLSASVGDRVTITCRASQSIDTFLNWYQQKPGKVPKLLIYAASNLESGVPSRFSGSGSGTVFTLTISRLQPEDFATYYCQQSYSIPPITFGQGTRVEIKGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSEYYLTWIRQAPGKGLDWIAYISSSGYNIYYADSVKDRFTISRDNGKNSLFLQMNNLRAEDTAVYFCAREGVTDGMDVWGQGTTVTVSSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

VL

(G4S)3

VH

G4S

CD8 hinge/TM

4-1BB costimulatory domains

CD3Z

MAGE-A4 (286-294) 31399N 2 VL-VH CD28 hinge/TM/cytoCD 3z CAR (SEQ ID NO: 105)

DIQMTQSPSSLSASVGDRVTITCRASQSIDTFLNWYQQKPGKVPKLLIYAASNLESGVPSRFSGSGSGTVFTLTISRLQPEDFATYYCQQSYSIPPITFGQGTRVEIKRGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSEYYLTWIRQAPGKGLDWIAYISSSGYNIYYADSVKDRFTISRDNGKNSLFLQMNNLRAEDTAVYFCAREGVTDGMDVWGQGTTVTVSSGGGGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR

VL

(G4S)3

VH

G4S

CD28 hinge

CD28 TM

CD28 costimulatory domain

CD3Z

MAGE-A4 (286-294) 31399N 2 VL-VH CD28 hinge/TM/cytoCD 3zCAR (SEQ ID NO: 121)

DIQMTQSPSSLSASVGDRVTITCRASQSIDTFLNWYQQKPGKVPKLLIYAASNLESGVPSRFSGSGSGTVFTLTISRLQPEDFATYYCQQSYSIPPITFGQGTRVEIKGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSEYYLTWIRQAPGKGLDWIAYISSSGYNIYYADSVKDRFTISRDNGKNSLFLQMNNLRAEDTAVYFCAREGVTDGMDVWGQGTTVTVSSGGGGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR

VL

(G4S)3

VH

G4S

CD28 hinge

CD28 TM

CD28 costimulatory domain

CD3Z

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Sequence listing

<110> regenerator pharmaceutical company (Regeneron Pharmaceuticals, inc.)

<120> anti-MAGE-A4 antibodies and chimeric antigen receptors

Use of the same

<130> 10901WO01

<150> 63/184,183

<151> 2021-05-04

<150> 63/239,293

<151> 2021-08-31

<160> 141

<170> patent in version 3.5

<210> 1

<211> 354

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caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt gaatactacc tgacctggat ccgccaggct 120

ccagggaagg ggctggactg gattgcatac attagtagta gtggttacaa tatatattac 180

gcagactctg tgaaggaccg attcaccatt tccagggaca acggcaagaa ctcactgttt 240

ctgcaaatga acaacctgag agccgaagac acggccgtct atttttgtgc gagagagggt 300

gtaacggacg gtatggacgt ctggggccaa gggaccacgg tcaccgtctc ctca 354

<210> 2

<211> 118

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr

20 25 30

Tyr Leu Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Ile

35 40 45

Ala Tyr Ile Ser Ser Ser Gly Tyr Asn Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Gly Val Thr Asp Gly Met Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 3

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ggattcacct tcagtgaata ctac 24

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<400> 4

Gly Phe Thr Phe Ser Glu Tyr Tyr

1 5

<210> 5

<211> 24

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<400> 5

attagtagta gtggttacaa tata 24

<210> 6

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<400> 6

Ile Ser Ser Ser Gly Tyr Asn Ile

1 5

<210> 7

<211> 33

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<400> 7

gcgagagagg gtgtaacgga cggtatggac gtc 33

<210> 8

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<400> 8

Ala Arg Glu Gly Val Thr Asp Gly Met Asp Val

1 5 10

<210> 9

<211> 324

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<400> 9

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcatcgac acctttttaa attggtatca gcagaagcca 120

gggaaagtcc ctaaactcct gatctatgct gcatccaatt tggaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagtt ttcactctca ccatcagccg gctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ttcctccgat caccttcggc 300

caagggacac gagtggagat taaa 324

<210> 10

<211> 108

<212> PRT

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<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Thr Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Val Glu Ile Lys

100 105

<210> 11

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cagagcatcg acaccttt 18

<210> 12

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<400> 12

Gln Ser Ile Asp Thr Phe

1 5

<210> 13

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gctgcatcc 9

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Ala Ala Ser

1

<210> 15

<211> 30

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caacagagtt acagtattcc tccgatcacc 30

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Gln Gln Ser Tyr Ser Ile Pro Pro Ile Thr

1 5 10

<210> 17

<211> 1335

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<400> 17

caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt gaatactacc tgacctggat ccgccaggct 120

ccagggaagg ggctggactg gattgcatac attagtagta gtggttacaa tatatattac 180

gcagactctg tgaaggaccg attcaccatt tccagggaca acggcaagaa ctcactgttt 240

ctgcaaatga acaacctgag agccgaagac acggccgtct atttttgtgc gagagagggt 300

gtaacggacg gtatggacgt ctggggccaa gggaccacgg tcaccgtctc ctcagcctcc 360

accaagggcc catcggtctt ccccctggcg ccctgctcca ggagcacctc cgagagcaca 420

gccgccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480

tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540

tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcacgaa gacctacacc 600

tgcaacgtag atcacaagcc cagcaacacc aaggtggaca agagagttga gtccaaatat 660

ggtcccccat gcccaccgtg cccagcacca cctgtggcag gaccatcagt cttcctgttc 720

cccccaaaac ccaaggacac tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 780

gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 840

gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 900

agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 960

tccaacaaag gcctcccgtc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 1020

cgagagccac aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtc 1080

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 1140

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1200

ttcttcctct acagcaggct caccgtggac aagagcaggt ggcaggaggg gaatgtcttc 1260

tcatgctccg tgatgcatga ggctctgcac aaccactaca cacagaagtc cctctccctg 1320

tctctgggta aatga 1335

<210> 18

<211> 444

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 18

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr

20 25 30

Tyr Leu Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Ile

35 40 45

Ala Tyr Ile Ser Ser Ser Gly Tyr Asn Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Gly Val Thr Asp Gly Met Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 19

<211> 648

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 19

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcatcgac acctttttaa attggtatca gcagaagcca 120

gggaaagtcc ctaaactcct gatctatgct gcatccaatt tggaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagtt ttcactctca ccatcagccg gctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ttcctccgat caccttcggc 300

caagggacac gagtggagat taaacgaact gtggctgcac catctgtctt catcttcccg 360

ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420

tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480

caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540

acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600

ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttag 648

<210> 20

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Thr Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 21

<211> 1413

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 21

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcatcgac acctttttaa attggtatca gcagaagcca 120

gggaaagtcc ctaaactcct gatctatgct gcatccaatt tggaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagtt ttcactctca ccatcagccg gctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ttcctccgat caccttcggc 300

caagggacac gagtggagat taaacgaggt ggaggcggta gtggcggagg cggaagtggt 360

ggaggaggct cacaggtgca gctggtggag tctgggggag gcttggtcaa gcctggaggg 420

tccctgagac tctcctgtgc agcctctgga ttcaccttca gtgaatacta cctgacctgg 480

atccgccagg ctccagggaa ggggctggac tggattgcat acattagtag tagtggttac 540

aatatatatt acgcagactc tgtgaaggac cgattcacca tttccaggga caacggcaag 600

aactcactgt ttctgcaaat gaacaacctg agagccgaag acacggccgt ctatttttgt 660

gcgagagagg gtgtaacgga cggtatggac gtctggggcc aagggaccac ggtcaccgtc 720

tcctcaggag gtggtggaag tactaccact cctgctcccc gccccccaac acctgctcca 780

actattgcat cccaaccact ctccctcaga cccgaagctt gtcgccccgc cgccggaggt 840

gctgttcaca ctagaggact cgattttgct tgcgacattt atatctgggc cccacttgca 900

ggtacttgcg gagtattgct gctctcactt gttattactc tttattgcaa acggggcaga 960

aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1020

gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1080

aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1140

gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1200

cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1260

cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1320

ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1380

gcccttcaca tgcaggccct gccccctcgc taa 1413

<210> 22

<211> 470

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Thr Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Val Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

115 120 125

Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu

130 135 140

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr Tyr Leu Thr Trp

145 150 155 160

Ile Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Ile Ala Tyr Ile Ser

165 170 175

Ser Ser Gly Tyr Asn Ile Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn

195 200 205

Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg Glu Gly

210 215 220

Val Thr Asp Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

225 230 235 240

Ser Ser Gly Gly Gly Gly Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

245 250 255

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

260 265 270

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

275 280 285

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

290 295 300

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

305 310 315 320

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

325 330 335

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

340 345 350

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

355 360 365

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

370 375 380

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

385 390 395 400

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

405 410 415

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

420 425 430

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

435 440 445

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

450 455 460

Gln Ala Leu Pro Pro Arg

465 470

<210> 23

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 23

Gly Gly Gly Gly Ser

1 5

<210> 24

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 24

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 25

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 25

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 26

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 26

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 27

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 27

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 28

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 28

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 29

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 29

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 30

<211> 112

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 30

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 31

<211> 1724

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 31

agagacaagc gagcttctgc gtctgactcg cagcttgaga ctggcggagg gaagcccgcc 60

caggctctat aaggagacaa ggttctgagc agacaggcca accggaggac aggattccct 120

ggaggccaca gaggagcacc aaggagaaga tctgcctgtg ggtccccatt gcccagcttt 180

tgcctgcact cttgcctgct gccctgacca gagtcatcat gtcttctgag cagaagagtc 240

agcactgcaa gcctgaggaa ggcgttgagg cccaagaaga ggccctgggc ctggtgggtg 300

cacaggctcc tactactgag gagcaggagg ctgctgtctc ctcctcctct cctctggtcc 360

ctggcaccct ggaggaagtg cctgctgctg agtcagcagg tcctccccag agtcctcagg 420

gagcctctgc cttacccact accatcagct tcacttgctg gaggcaaccc aatgagggtt 480

ccagcagcca agaagaggag gggccaagca cctcgcctga cgcagagtcc ttgttccgag 540

aagcactcag taacaaggtg gatgagttgg ctcattttct gctccgcaag tatcgagcca 600

aggagctggt cacaaaggca gaaatgctgg agagagtcat caaaaattac aagcgctgct 660

ttcctgtgat cttcggcaaa gcctccgagt ccctgaagat gatctttggc attgacgtga 720

aggaagtgga ccccgccagc aacacctaca cccttgtcac ctgcctgggc ctttcctatg 780

atggcctgct gggtaataat cagatctttc ccaagacagg ccttctgata atcgtcctgg 840

gcacaattgc aatggagggc gacagcgcct ctgaggagga aatctgggag gagctgggtg 900

tgatgggggt gtatgatggg agggagcaca ctgtctatgg ggagcccagg aaactgctca 960

cccaagattg ggtgcaggaa aactacctgg agtaccggca ggtacccggc agtaatcctg 1020

cgcgctatga gttcctgtgg ggtccaaggg ctctggctga aaccagctat gtgaaagtcc 1080

tggagcatgt ggtcagggtc aatgcaagag ttcgcattgc ctacccatcc ctgcgtgaag 1140

cagctttgtt agaggaggaa gagggagtct gagcatgagt tgcagccagg gctgtgggga 1200

aggggcaggg ctgggccagt gcatctaaca gccctgtgca gcagcttccc ttgcctcgtg 1260

taacatgagg cccattcttc actctgtttg aagaaaatag tcagtgttct tagtagtggg 1320

tttctatttt gttggatgac ttggagattt atctctgttt ccttttacaa ttgttgaaat 1380

gttcctttta atggatggtt gaattaactt cagcatccaa gtttatgaat cgtagttaac 1440

gtatattgct gttaatatag tttaggagta agagtcttgt tttttattca gattgggaaa 1500

tccgttctat tttgtgaatt tgggacataa taacagcagt ggagtaagta tttagaagtg 1560

tgaattcacc gtgaaatagg tgagataaat taaaagatac ttaattcccg ccttatgcct 1620

cagtctattc tgtaaaattt aaaaaatata tatgcatacc tggatttcct tggcttcgtg 1680

aatgtaagag aaattaaatc tgaataaata attctttctg ttaa 1724

<210> 32

<211> 317

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 32

Met Ser Ser Glu Gln Lys Ser Gln His Cys Lys Pro Glu Glu Gly Val

1 5 10 15

Glu Ala Gln Glu Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Thr

20 25 30

Thr Glu Glu Gln Glu Ala Ala Val Ser Ser Ser Ser Pro Leu Val Pro

35 40 45

Gly Thr Leu Glu Glu Val Pro Ala Ala Glu Ser Ala Gly Pro Pro Gln

50 55 60

Ser Pro Gln Gly Ala Ser Ala Leu Pro Thr Thr Ile Ser Phe Thr Cys

65 70 75 80

Trp Arg Gln Pro Asn Glu Gly Ser Ser Ser Gln Glu Glu Glu Gly Pro

85 90 95

Ser Thr Ser Pro Asp Ala Glu Ser Leu Phe Arg Glu Ala Leu Ser Asn

100 105 110

Lys Val Asp Glu Leu Ala His Phe Leu Leu Arg Lys Tyr Arg Ala Lys

115 120 125

Glu Leu Val Thr Lys Ala Glu Met Leu Glu Arg Val Ile Lys Asn Tyr

130 135 140

Lys Arg Cys Phe Pro Val Ile Phe Gly Lys Ala Ser Glu Ser Leu Lys

145 150 155 160

Met Ile Phe Gly Ile Asp Val Lys Glu Val Asp Pro Ala Ser Asn Thr

165 170 175

Tyr Thr Leu Val Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly

180 185 190

Asn Asn Gln Ile Phe Pro Lys Thr Gly Leu Leu Ile Ile Val Leu Gly

195 200 205

Thr Ile Ala Met Glu Gly Asp Ser Ala Ser Glu Glu Glu Ile Trp Glu

210 215 220

Glu Leu Gly Val Met Gly Val Tyr Asp Gly Arg Glu His Thr Val Tyr

225 230 235 240

Gly Glu Pro Arg Lys Leu Leu Thr Gln Asp Trp Val Gln Glu Asn Tyr

245 250 255

Leu Glu Tyr Arg Gln Val Pro Gly Ser Asn Pro Ala Arg Tyr Glu Phe

260 265 270

Leu Trp Gly Pro Arg Ala Leu Ala Glu Thr Ser Tyr Val Lys Val Leu

275 280 285

Glu His Val Val Arg Val Asn Ala Arg Val Arg Ile Ala Tyr Pro Ser

290 295 300

Leu Arg Glu Ala Ala Leu Leu Glu Glu Glu Glu Gly Val

305 310 315

<210> 33

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 33

Lys Val Leu Glu His Val Val Arg Val

1 5

<210> 34

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 34

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro

35

<210> 35

<211> 117

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 35

atcgaagtga tgtacccccc tccatatctg gataacgaga agagcaatgg cacaatcatc 60

cacgtgaagg gcaagcacct gtgcccttct ccactgttcc ccggccctag caagccc 117

<210> 36

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 36

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 37

<211> 81

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 37

ttttgggtgc tggtggtggt gggaggcgtg ctggcctgtt actccctgct ggtgaccgtg 60

gccttcatca tcttttgggt g 81

<210> 38

<211> 41

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 38

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 39

<211> 123

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 39

agaagcaaga gatccaggct gctgcactct gactatatga atatgacccc taggcgccca 60

ggccccacaa gaaagcacta ccagccatat gcaccaccta gggacttcgc agcataccgc 120

agc 123

<210> 40

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 40

Lys Val Arg Glu Glu Val Val Thr Val

1 5

<210> 41

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 41

Lys Val Met Cys His Val Arg Arg Val

1 5

<210> 42

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 42

Lys Val Leu Glu Arg Val Asn Ala Val

1 5

<210> 43

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 43

Lys Val Leu Val Glu Val Val Asp Val

1 5

<210> 44

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 44

Lys Val Leu Glu Lys Cys Asn Arg Val

1 5

<210> 45

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 45

Lys Val Leu Glu His Val Pro Leu Leu

1 5

<210> 46

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 46

Phe Leu Leu Glu Thr Val Val Arg Val

1 5

<210> 47

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 47

Lys Val Leu Gly Ile Val Val Gly Val

1 5

<210> 48

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 48

Met Val Leu Glu His Pro Ala Arg Val

1 5

<210> 49

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 49

Lys Val Leu Glu Gln Pro Val Val Val

1 5

<210> 50

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 50

Lys Val Leu Gln Asn Val Leu Arg Val

1 5

<210> 51

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 51

Lys Val Leu Glu Gly Val Val Ala Ala

1 5

<210> 52

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 52

Ala Val Glu Glu His Val Val Ser Val

1 5

<210> 53

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 53

Lys Val Leu Glu Thr Leu Val Thr Val

1 5

<210> 54

<211> 372

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 54

gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60

agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120

cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag caagggatac 180

gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240

cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaatacggc 300

agtggttatg gcaagtttta tcattatgga ctggacgtgt ggggacaagg gacaacagtg 360

acagtgagta gc 372

<210> 55

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 55

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Asn Ser Gly Ser Lys Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 56

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 56

gggtttacat tcgacgatta cagc 24

<210> 57

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 57

Gly Phe Thr Phe Asp Asp Tyr Ser

1 5

<210> 58

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 58

atatcatgga actcaggaag caag 24

<210> 59

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 59

Ile Ser Trp Asn Ser Gly Ser Lys

1 5

<210> 60

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 60

gcaaaatacg gcagtggtta tggcaagttt tatcattatg gactggacgt g 51

<210> 61

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 61

Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp

1 5 10 15

Val

<210> 62

<211> 324

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 62

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300

caagggacac gactggagat taaa 324

<210> 63

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 63

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 64

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 64

cagagcatta gcagctat 18

<210> 65

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 65

Gln Ser Ile Ser Ser Tyr

1 5

<210> 66

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 66

caacagagtt acagtacccc tccgatcacc 30

<210> 67

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 67

Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr

1 5 10

<210> 68

<211> 1353

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 68

gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60

agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120

cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag caagggatac 180

gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240

cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaatacggc 300

agtggttatg gcaagtttta tcattatgga ctggacgtgt ggggacaagg gacaacagtg 360

acagtgagta gcgccagcac aaaaggtcct agcgtttttc cacttgcccc atgttcaagg 420

tcaacctccg aaagtaccgc cgctcttggc tgtctcgtaa aagattattt tcccgaacct 480

gtaactgtct cctggaactc cggcgcactc acttccggcg tacatacctt ccccgctgtc 540

ctccaatctt ccggtctcta ctccctgtct tctgttgtca ctgttccatc atcctcactc 600

ggcacaaaaa catatacctg caacgttgat cacaagccaa gtaataccaa agttgataag 660

cgcgtcgaat ccaaatacgg tcccccctgc cccccatgtc ccgctccacc tgtggctggt 720

ccctctgttt tcctttttcc ccctaaaccc aaagataccc tcatgatttc cagaaccccc 780

gaggtcacct gcgtcgtcgt tgatgtaagc caagaagatc ccgaagtcca gttcaattgg 840

tatgtagacg gtgttgaagt ccataatgca aaaacaaaac ccagagagga acagtttaat 900

tcaacctatc gtgtcgttag cgtactcacc gttcttcatc aagactggct caatggaaaa 960

gaatataaat gtaaagttag caacaaaggt ctgcccagtt caatcgaaaa aacaattagc 1020

aaagccaaag gccaacctcg cgaaccccaa gtctatacct tgcccccttc tcaggaagaa 1080

atgaccaaaa accaagtttc actcacatgc ctcgtaaaag gattctatcc atcagacatt 1140

gcagtagaat gggaatctaa cggccaacct gaaaataatt acaaaaccac tcctcctgtc 1200

ctcgattctg acggctcttt tttcctttac tccagattga ctgttgataa atcccgctgg 1260

caggaaggta acgttttttc ttgttctgtg atgcacgaag ccctccataa cagattcact 1320

caaaaatctc tttctctctc ccctggcaaa taa 1353

<210> 69

<211> 450

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 69

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Asn Ser Gly Ser Lys Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 70

<211> 648

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 70

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300

caagggacac gactggagat taaacgaact gtggctgcac catctgtctt catcttcccg 360

ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420

tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480

caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540

acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600

ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttag 648

<210> 71

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 71

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 72

<211> 372

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 72

gaagtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggcaggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttgct gattatacca tgcactgggt ccggcaagct 120

ccagggaagg gcctggagtg ggtctcagat attagttgga atagtggtag tatagcctat 180

gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctctat 240

cttcaaatga acagtctgag aactgaggac acggcctttt attactgtgc aaaagatagt 300

aggggctacg gtcactataa gtacctcggt ttggacgtct ggggccaagg gaccacggtc 360

accgtctcct ca 372

<210> 73

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 73

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Ser Trp Asn Ser Gly Ser Ile Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Phe Tyr Tyr Cys

85 90 95

Ala Lys Asp Ser Arg Gly Tyr Gly His Tyr Lys Tyr Leu Gly Leu Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 74

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 74

ggattcacct ttgctgatta tacc 24

<210> 75

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 75

Gly Phe Thr Phe Ala Asp Tyr Thr

1 5

<210> 76

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 76

attagttgga atagtggtag tata 24

<210> 77

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 77

Ile Ser Trp Asn Ser Gly Ser Ile

1 5

<210> 78

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 78

gcaaaagata gtaggggcta cggtcactat aagtacctcg gtttggacgt c 51

<210> 79

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 79

Ala Lys Asp Ser Arg Gly Tyr Gly His Tyr Lys Tyr Leu Gly Leu Asp

1 5 10 15

Val

<210> 80

<211> 1353

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 80

gaagtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggcaggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttgct gattatacca tgcactgggt ccggcaagct 120

ccagggaagg gcctggagtg ggtctcagat attagttgga atagtggtag tatagcctat 180

gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctctat 240

cttcaaatga acagtctgag aactgaggac acggcctttt attactgtgc aaaagatagt 300

aggggctacg gtcactataa gtacctcggt ttggacgtct ggggccaagg gaccacggtc 360

accgtctcct cagcctctac aaagggacct tctgtgtttc ctctggctcc ttgttctaga 420

tctacatctg aatctacagc tgctctggga tgtctggtga aggattattt tcctgaacct 480

gtgacagtgt cttggaattc tggagctctg acatctggag tgcatacatt tcctgctgtg 540

ctgcagtctt ctggactgta ttctctgtct tctgtggtga cagtgccttc ttcttctctg 600

ggaacaaaga catatacatg taatgtggat cataagcctt ctaatacaaa ggtggataag 660

agagtggaat ctaagtatgg acctccttgt cctccttgtc ctgctcctcc tgtggctgga 720

ccttctgtgt ttctgtttcc tcctaagcct aaggatacac tgatgatctc tagaacacct 780

gaagtgacat gtgtggtggt ggatgtgtct caggaagatc ctgaagtgca gtttaattgg 840

tatgtggatg gagtggaagt gcataatgct aagacaaagc ctagagaaga acagtttaat 900

tctacatata gagtggtgtc tgtgctgaca gtgctgcatc aggattggct gaatggaaag 960

gaatataagt gtaaggtgtc taataaggga ctgccttctt ctatcgaaaa gacaatctct 1020

aaggctaagg gacagcctag agaacctcag gtgtatacac tgcctccttc tcaggaagaa 1080

atgacaaaga atcaggtgtc tctgacatgt ctggtgaagg gattttatcc ttctgatatc 1140

gctgtggaat gggaatctaa tggacagcct gaaaataatt ataagacaac acctcctgtg 1200

ctggattctg atggatcttt ttttctgtat tctagactga cagtggataa gtctagatgg 1260

caggaaggaa atgtgttttc ttgttctgtg atgcatgaag ctctgcataa tagatttaca 1320

cagaagtctc tgtctctgtc tcctggaaag tag 1353

<210> 81

<211> 450

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 81

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Ser Trp Asn Ser Gly Ser Ile Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Phe Tyr Tyr Cys

85 90 95

Ala Lys Asp Ser Arg Gly Tyr Gly His Tyr Lys Tyr Leu Gly Leu Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 82

<211> 354

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 82

caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt gaatactacc tgacctggat ccgccaggct 120

ccagggaagg ggctggactg gattgcatac attagtagta gtggttacaa tatatattac 180

gcagactctg tgaaggaccg attcaccatt tccagggaca acggcaacaa ctcactgttt 240

ctgcaaatga acaacctgag agccgaagac acggccgtct atttttgtgc gagagagggt 300

gtaacggacg gtatggacgt ctggggccaa gggaccacgg tcaccgtctc ctca 354

<210> 83

<211> 118

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 83

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr

20 25 30

Tyr Leu Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Ile

35 40 45

Ala Tyr Ile Ser Ser Ser Gly Tyr Asn Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Gly Asn Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Gly Val Thr Asp Gly Met Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 84

<211> 366

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 84

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120

ccagggaagg gactggagtg gattgggtat atctattaca gtgggatcac ccactacaac 180

ccctccctca agagtcgagt caccatatca gtagacacgt ccaagatcca gttctccctg 240

aagctgagtt ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag atggggggtt 300

cggagggact actactacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360

tcctca 366

<210> 85

<211> 122

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 85

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ile Thr His Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Ile Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Trp Gly Val Arg Arg Asp Tyr Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 86

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 86

ggtggctcca tcagtagtta ctac 24

<210> 87

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 87

Gly Gly Ser Ile Ser Ser Tyr Tyr

1 5

<210> 88

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 88

atctattaca gtgggatcac c 21

<210> 89

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 89

Ile Tyr Tyr Ser Gly Ile Thr

1 5

<210> 90

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 90

gcgagatggg gggttcggag ggactactac tactacggta tggacgtc 48

<210> 91

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 91

Ala Arg Trp Gly Val Arg Arg Asp Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 92

<211> 324

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 92

gaaatagttt tgacacagag tcccggcaca ctgtcactct ctcccgggga aagagccacc 60

ttgtcatgta gagcaagtca gtcagtctct agctcttatc tcgcctggta ccagcagaag 120

ccgggacagg cccctagact gctgatctac ggggcaagtt ccagggccac cggaatcccc 180

gaccggttca gtggaagcgg aagcggaacc gattttactt tgacgatttc tagactggag 240

ccagaggatt tcgccgttta ctattgtcaa cagtacggaa gcagcccgtg gacgtttggc 300

cagggcacga aggtagaaat caag 324

<210> 93

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 93

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 94

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 94

cagtcagtct ctagctctta t 21

<210> 95

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 95

Gln Ser Val Ser Ser Ser Tyr

1 5

<210> 96

<211> 9

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 96

ggggcaagt 9

<210> 97

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 97

Gly Ala Ser

1

<210> 98

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 98

caacagtacg gaagcagccc gtggacg 27

<210> 99

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 99

Gln Gln Tyr Gly Ser Ser Pro Trp Thr

1 5

<210> 100

<211> 1347

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 100

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120

ccagggaagg gactggagtg gattgggtat atctattaca gtgggatcac ccactacaac 180

ccctccctca agagtcgagt caccatatca gtagacacgt ccaagatcca gttctccctg 240

aagctgagtt ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag atggggggtt 300

cggagggact actactacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360

tcctcagcct ctacaaaggg accttctgtg tttcctctgg ctccttgttc tagatctaca 420

tctgaatcta cagctgctct gggatgtctg gtgaaggatt attttcctga acctgtgaca 480

gtgtcttgga attctggagc tctgacatct ggagtgcata catttcctgc tgtgctgcag 540

tcttctggac tgtattctct gtcttctgtg gtgacagtgc cttcttcttc tctgggaaca 600

aagacatata catgtaatgt ggatcataag ccttctaata caaaggtgga taagagagtg 660

gaatctaagt atggacctcc ttgtcctcct tgtcctgctc ctcctgtggc tggaccttct 720

gtgtttctgt ttcctcctaa gcctaaggat acactgatga tctctagaac acctgaagtg 780

acatgtgtgg tggtggatgt gtctcaggaa gatcctgaag tgcagtttaa ttggtatgtg 840

gatggagtgg aagtgcataa tgctaagaca aagcctagag aagaacagtt taattctaca 900

tatagagtgg tgtctgtgct gacagtgctg catcaggatt ggctgaatgg aaaggaatat 960

aagtgtaagg tgtctaataa gggactgcct tcttctatcg aaaagacaat ctctaaggct 1020

aagggacagc ctagagaacc tcaggtgtat acactgcctc cttctcagga agaaatgaca 1080

aagaatcagg tgtctctgac atgtctggtg aagggatttt atccttctga tatcgctgtg 1140

gaatgggaat ctaatggaca gcctgaaaat aattataaga caacacctcc tgtgctggat 1200

tctgatggat ctttttttct gtattctaga ctgacagtgg ataagtctag atggcaggaa 1260

ggaaatgtgt tttcttgttc tgtgatgcat gaagctctgc ataatagatt tacacagaag 1320

tctctgtctc tgtctcctgg aaagtag 1347

<210> 101

<211> 448

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 101

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ile Thr His Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Ile Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Trp Gly Val Arg Arg Asp Tyr Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 102

<211> 648

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 102

gaaatagttt tgacacagag tcccggcaca ctgtcactct ctcccgggga aagagccacc 60

ttgtcatgta gagcaagtca gtcagtctct agctcttatc tcgcctggta ccagcagaag 120

ccgggacagg cccctagact gctgatctac ggggcaagtt ccagggccac cggaatcccc 180

gaccggttca gtggaagcgg aagcggaacc gattttactt tgacgatttc tagactggag 240

ccagaggatt tcgccgttta ctattgtcaa cagtacggaa gcagcccgtg gacgtttggc 300

cagggcacga aggtagaaat caagcgaact gtggctgcac catctgtctt catcttcccg 360

ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420

tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480

caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540

acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600

ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttag 648

<210> 103

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 103

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 104

<211> 1398

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 104

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcatcgac acctttttaa attggtatca gcagaagcca 120

gggaaagtcc ctaaactcct gatctatgct gcatccaatt tggaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagtt ttcactctca ccatcagccg gctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ttcctccgat caccttcggc 300

caagggacac gagtggagat taaacgaggt ggaggcggta gtggcggagg cggaagtggt 360

ggaggaggct cacaggtgca gctggtggag tctgggggag gcttggtcaa gcctggaggg 420

tccctgagac tctcctgtgc agcctctgga ttcaccttca gtgaatacta cctgacctgg 480

atccgccagg ctccagggaa ggggctggac tggattgcat acattagtag tagtggttac 540

aatatatatt acgcagactc tgtgaaggac cgattcacca tttccaggga caacggcaag 600

aactcactgt ttctgcaaat gaacaacctg agagccgaag acacggccgt ctatttttgt 660

gcgagagagg gtgtaacgga cggtatggac gtctggggcc aagggaccac ggtcaccgtc 720

tcctcaggag gtggtggaag tatcgaagtg atgtacccac ccccttatct ggataacgag 780

aagagcaatg gcacaatcat ccacgtgaag ggcaagcacc tgtgcccctc tcctctgttc 840

ccaggcccca gcaagccatt ttgggtgctg gtggtggtgg gaggcgtgct ggcctgttac 900

tccctgctgg tgaccgtggc cttcatcatc ttttgggtga gatctaagcg cagccggctg 960

ctgcactctg actatatgaa tatgacccca cggagacctg gcccaacaag aaagcactac 1020

cagccatatg caccaccaag ggacttcgca gcctacagaa gcagggtgaa gttttccagg 1080

tctgccgatg caccagcata ccagcaggga cagaaccagc tgtataacga gctgaatctg 1140

ggcaggcgcg aggagtatga cgtgctggat aagaggagag gccgcgatcc tgagatggga 1200

ggcaagccaa ggcgcaagaa ccctcaggag ggcctgtaca atgagctgca gaaggacaag 1260

atggccgagg cctatagcga gatcggcatg aagggagagc ggagaagggg caagggacac 1320

gatggcctgt accagggcct gtccaccgcc acaaaggaca cctatgatgc cctgcacatg 1380

caggccctgc ctccaagg 1398

<210> 105

<211> 466

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 105

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Thr Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Val Glu Ile Lys Arg Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

115 120 125

Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu

130 135 140

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr Tyr Leu Thr Trp

145 150 155 160

Ile Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Ile Ala Tyr Ile Ser

165 170 175

Ser Ser Gly Tyr Asn Ile Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn

195 200 205

Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg Glu Gly

210 215 220

Val Thr Asp Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

225 230 235 240

Ser Ser Gly Gly Gly Gly Ser Ile Glu Val Met Tyr Pro Pro Pro Tyr

245 250 255

Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys

260 265 270

His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp

275 280 285

Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val

290 295 300

Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu

305 310 315 320

Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr

325 330 335

Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr

340 345 350

Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys

355 360 365

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

370 375 380

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

385 390 395 400

Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

405 410 415

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

420 425 430

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

435 440 445

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

450 455 460

Pro Arg

465

<210> 106

<211> 381

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 106

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

acctgtgcag cctctggatt cacctttaga agctatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcaact attagtggta atagtgatag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaaa attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagacctc 300

cacattacta tggttcgggg agctatcccc gccgatgttt ttgatatctg gggccaaggg 360

acaatggtca ccgtctcttc a 381

<210> 107

<211> 127

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 107

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Gly Asn Ser Asp Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Leu His Ile Thr Met Val Arg Gly Ala Ile Pro Ala Asp

100 105 110

Val Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 108

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 108

ggattcacct ttagaagcta tgcc 24

<210> 109

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 109

Gly Phe Thr Phe Arg Ser Tyr Ala

1 5

<210> 110

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 110

attagtggta atagtgatag caca 24

<210> 111

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 111

Ile Ser Gly Asn Ser Asp Ser Thr

1 5

<210> 112

<211> 60

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 112

gcgaaagacc tccacattac tatggttcgg ggagctatcc ccgccgatgt ttttgatatc 60

<210> 113

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 113

Ala Lys Asp Leu His Ile Thr Met Val Arg Gly Ala Ile Pro Ala Asp

1 5 10 15

Val Phe Asp Ile

20

<210> 114

<211> 324

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 114

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc ttctatttaa attggtatca acagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgagacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300

caagggacac gactggagat taaa 324

<210> 115

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 115

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Phe Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 116

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 116

cagagcatta gcttctat 18

<210> 117

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 117

Gln Ser Ile Ser Phe Tyr

1 5

<210> 118

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 118

caacagagtt acagtacccc tccgatcacc 30

<210> 119

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 119

Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr

1 5 10

<210> 120

<211> 469

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 120

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Thr Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Val Glu Ile Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val

115 120 125

Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser

130 135 140

Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr Tyr Leu Thr Trp Ile

145 150 155 160

Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Ile Ala Tyr Ile Ser Ser

165 170 175

Ser Gly Tyr Asn Ile Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Asn

195 200 205

Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg Glu Gly Val

210 215 220

Thr Asp Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

225 230 235 240

Ser Gly Gly Gly Gly Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

245 250 255

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

260 265 270

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

275 280 285

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

290 295 300

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

305 310 315 320

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

325 330 335

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

340 345 350

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

355 360 365

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

370 375 380

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

385 390 395 400

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

405 410 415

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

420 425 430

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

435 440 445

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

450 455 460

Ala Leu Pro Pro Arg

465

<210> 121

<211> 465

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 121

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Thr Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Val Glu Ile Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val

115 120 125

Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser

130 135 140

Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr Tyr Leu Thr Trp Ile

145 150 155 160

Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Ile Ala Tyr Ile Ser Ser

165 170 175

Ser Gly Tyr Asn Ile Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Asn

195 200 205

Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg Glu Gly Val

210 215 220

Thr Asp Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

225 230 235 240

Ser Gly Gly Gly Gly Ser Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu

245 250 255

Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His

260 265 270

Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val

275 280 285

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

290 295 300

Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu

305 310 315 320

His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg

325 330 335

Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg

340 345 350

Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln

355 360 365

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

370 375 380

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

385 390 395 400

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

405 410 415

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

420 425 430

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

435 440 445

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

450 455 460

Arg

465

<210> 122

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 122

Gly Val Tyr Asp Gly Arg Glu His Thr Val

1 5 10

<210> 123

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 123

Gly Leu Tyr Asp Gly Arg Glu His Ser Val

1 5 10

<210> 124

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 124

Gly Leu Ala Asp Gly Arg Thr His Thr Val

1 5 10

<210> 125

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 125

Gly Val Pro Asp Cys Arg Ile Phe Thr Val

1 5 10

<210> 126

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 126

Ser Val Tyr Asp Ala Arg Glu Phe Ser Val

1 5 10

<210> 127

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 127

Gly Leu Ser Asp Gly Gln Trp His Thr Val

1 5 10

<210> 128

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 128

Gly Val Phe Asp Asn Cys Ser His Thr Val

1 5 10

<210> 129

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 129

Lys Val Ser Asp Gly His Phe His Thr Val

1 5 10

<210> 130

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 130

Gly Leu Tyr Asp Gly Met Glu His Leu Ile

1 5 10

<210> 131

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 131

Phe Leu Cys Asp Pro Arg Glu His Leu Val

1 5 10

<210> 132

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 132

Ala Val Tyr Asp Gly Arg Glu His Thr Val

1 5 10

<210> 133

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 133

Gly Ala Tyr Asp Gly Arg Glu His Thr Val

1 5 10

<210> 134

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 134

Gly Val Ala Asp Gly Arg Glu His Thr Val

1 5 10

<210> 135

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 135

Gly Val Tyr Ala Gly Arg Glu His Thr Val

1 5 10

<210> 136

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 136

Gly Val Tyr Asp Ala Arg Glu His Thr Val

1 5 10

<210> 137

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 137

Gly Val Tyr Asp Gly Ala Glu His Thr Val

1 5 10

<210> 138

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 138

Gly Val Tyr Asp Gly Arg Ala His Thr Val

1 5 10

<210> 139

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 139

Gly Val Tyr Asp Gly Arg Glu Ala Thr Val

1 5 10

<210> 140

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 140

Gly Val Tyr Asp Gly Arg Glu His Ala Val

1 5 10

<210> 141

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 141

Gly Val Tyr Asp Gly Arg Glu His Thr Ala

1 5 10

Claims

1. An antigen binding protein that specifically binds HLA-bound melanoma-associated antigen A4 (MAGE-A4), wherein the antigen binding protein comprises a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR), wherein the LCVR comprises a Complementarity Determining Region (CDR) of a LCVR comprising the amino acid sequence of SEQ ID No. 10 or SEQ ID No. 115, and wherein the HCVR comprises a CDR of a HCVR comprising the amino acid sequence of SEQ ID No. 2, SEQ ID No. 83, or SEQ ID No. 107.

2. The antigen binding protein of claim 1, wherein the LCVR comprises an amino acid sequence with at least 95% sequence identity to SEQ ID No. 10 or SEQ ID No. 115.

3. The antigen binding protein of claim 1 or claim 2, wherein the HCVR comprises an amino acid sequence with at least 95% identity to SEQ ID No. 2, SEQ ID No. 83, or SEQ ID No. 107.

4. The antigen binding protein of any one of claims 1 to 3, wherein the MAGE-A4 specific antigen binding protein interacts with amino acids 286-294 of SEQ ID No. 32 or a portion thereof.

5. A MAGE-A4 specific Chimeric Antigen Receptor (CAR), the MAGE-A4 specific CAR comprising from N-terminus to C-terminus: (a) An extracellular ligand binding domain comprising an anti-MAGE-A4 single chain variable fragment (scFv) domain, said anti-MAGE-A4 scFv domain comprising a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR); (b) a hinge; (c) a transmembrane domain; and (d) a cytoplasmic domain comprising a 4-1BB co-stimulatory domain or a CD28 co-stimulatory domain and a CD3 zeta signaling domain, wherein the LCVR comprises: a Complementarity Determining Region (CDR) of a LCVR comprising the amino acid sequence of SEQ ID NO. 10 or SEQ ID NO. 115 and a CDR of a HCVR comprising the amino acid sequence of SEQ ID NO. 2, 83 or 107.

6. The MAGE-A4 specific CAR of claim 5, wherein said MAGE-A4 specific CAR comprises, from N-terminus to C-terminus: (a) the extracellular ligand-binding domain; (b) a hinge; (c) a transmembrane domain; and (d) a cytoplasmic domain comprising a co-stimulatory domain and a signaling domain.

7. The MAGE-A4 specific CAR of claim 5, wherein the anti-MAGE-A4 scFv domain comprises a first linker between the LCVR and the HCVR.

8. The MAGE-A4 specific CAR of any one of claims 5 to 7, further comprising a second linker between the extracellular ligand binding domain and the hinge.

9. The MAGE-A4 specific CAR of claim 8, wherein the first linker comprises an amino acid sequence selected from SEQ ID NOs 23-26, and wherein the second linker comprises an amino acid sequence selected from SEQ ID NOs 23-26.

10. The MAGE-A4 specific CAR of claim 9, wherein the first linker comprises the amino acid sequence of SEQ ID No. 25 and the second linker comprises the amino acid sequence of SEQ ID No. 23.

11. The MAGE-A4 specific CAR of any one of claims 5 to 10, wherein the hinge, the transmembrane domain, or both are from a CD8 a polypeptide.

12. The MAGE-A4 specific CAR of any one of claims 5 to 11, wherein said co-stimulatory domain comprises A4-1 BB co-stimulatory domain.

13. The MAGE-A4 specific CAR of any one of claims 5 to 11, wherein said co-stimulatory domain comprises a CD28 co-stimulatory domain.

14. The MAGE-A4 specific CAR of any one of claims 5 to 10, 12 and 13, wherein the hinge, the transmembrane domain, or both are from a CD28 polypeptide.

15. The MAGE-A4 specific CAR of any one of claims 5 to 12, wherein the hinge comprises the amino acid sequence of SEQ ID No. 27.

16. The MAGE-A4 specific CAR of any one of claims 5 to 12, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID No. 28.

17. The MAGE-A4 specific CAR of any one of claims 5 to 12, wherein the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 29.

18. The MAGE-A4 specific CAR of any one of claims 5 to 10 and 12 to 14, wherein the hinge comprises the amino acid sequence of SEQ ID NO 34.

19. The MAGE-A4 specific CAR of any one of claims 5 to 10, 12 to 14 and 18, wherein said transmembrane domain comprises the amino acid sequence of SEQ ID NO: 36.

20. The MAGE-A4 specific CAR of any one of claims 5 to 10, 12 to 14, 18 and 19, wherein said CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 38.

21. The MAGE-A4 specific CAR of any one of claims 5 to 20, wherein said signaling domain comprises a CD3 zeta signaling domain.

22. The MAGE-A4 specific CAR of claim 21, wherein said CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID No. 30.

23. The antigen binding protein of any one of claims 1 to 4, wherein the antigen binding protein is a MAGE-A4 specific antibody or antigen binding fragment thereof.

24. The antigen binding protein of any one of claims 1 to 4 and 23 or the MAGE-A4 specific CAR of any one of claims 5 to 22, wherein the antigen binding protein or the MAGE-A4 specific CAR comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) contained within a HCVR comprising the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO: 83.

25. The antigen binding protein or MAGE-A4 specific CAR of claim 24, wherein said HCDR1 comprises the amino acid sequence shown in SEQ ID No. 4, said HCDR2 comprises the amino acid sequence shown in SEQ ID No. 6, and said HCDR3 comprises the amino acid sequence shown in SEQ ID No. 8.

26. The antigen binding protein or MAGE-A4 specific CAR of claim 25, wherein said HCVR comprises the amino acid sequence set forth in SEQ ID No. 2.

27. The antigen binding protein or MAGE-A4 specific CAR of claim 25, wherein said HCVR comprises the amino acid sequence shown in SEQ ID No. 83.

28. The antigen binding protein of any one of claims 1 to 4 and 23 or the MAGE-A4 specific CAR of any one of claims 5 to 22, wherein the antigen binding protein or the MAGE-A4 specific CAR comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) contained within a HCVR comprising the amino acid sequence shown in SEQ ID NO: 107.

29. The antigen binding protein or MAGE-A4 specific CAR of claim 28, wherein said HCDR1 comprises the amino acid sequence shown in SEQ ID No. 109, said HCDR2 comprises the amino acid sequence shown in SEQ ID No. 111, and said HCDR3 comprises the amino acid sequence shown in SEQ ID No. 113.

30. The antigen binding protein or MAGE-A4 specific CAR of claim 28, wherein said HCVR comprises the amino acid sequence set forth in SEQ ID No. 107.

31. The antigen binding protein of any one of claims 1 to 4 and 23, the MAGE-A4 specific CAR of any one of claims 5 to 22, or the antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 29, wherein the antigen binding protein or MAGE-A4 specific CAR comprises three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) contained within an LCVR comprising the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO: 115.

32. The antigen binding protein or MAGE-A4 specific CAR of claim 30, wherein said LCDR1 comprises the amino acid sequence shown in SEQ ID No. 12, said LCDR2 comprises the amino acid sequence shown in SEQ ID No. 14, and said LCDR3 comprises the amino acid sequence shown in SEQ ID No. 16.

33. The antigen binding protein or MAGE-A4 specific CAR of claim 31, wherein said LCVR comprises the amino acid sequence set forth in SEQ ID No. 10.

34. The antigen binding protein or MAGE-A4 specific CAR of claim 30, wherein said LCDR1 comprises the amino acid sequence shown in SEQ ID No. 117, said LCDR2 comprises the amino acid sequence shown in SEQ ID No. 14, and said LCDR3 comprises the amino acid sequence shown in SEQ ID No. 119.

35. The antigen binding protein or MAGE-A4 specific CAR of claim 33, wherein said LCVR comprises the amino acid sequence set forth in SEQ ID No. 115.

36. The antigen binding protein of any one of claims 1 to 4 and 23 or the MAGE-A4 specific CAR of any one of claims 5 to 22, wherein the antigen binding protein or the MAGE-A4 specific CAR comprises a HCVR comprising the amino acid sequence set forth in SEQ ID No. 2 and a LCVR comprising the amino acid sequence set forth in SEQ ID No. 10.

37. The antigen binding protein of any one of claims 1 to 4 and 23 or the MAGE-A4 specific CAR of any one of claims 5 to 22, wherein the antigen binding protein or the MAGE-A4 specific CAR comprises a HCVR comprising the amino acid sequence set forth in SEQ ID No. 83 and a LCVR comprising the amino acid sequence set forth in SEQ ID No. 10.

38. The antigen binding protein of any one of claims 1 to 4 and 23 or the MAGE-A4 specific CAR of any one of claims 5 to 22, wherein the antigen binding protein or the MAGE-A4 specific CAR comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO:107 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 115.

39. The MAGE-A4 specific CAR of claim 5, comprising the amino acid sequence of SEQ ID No. 22.

40. The MAGE-A4 specific CAR of claim 5, comprising the amino acid sequence of SEQ ID No. 105.

41. The MAGE-A4 specific CAR of claim 5, comprising the amino acid sequence of SEQ ID No. 120.

42. The MAGE-A4 specific CAR of claim 5, comprising the amino acid sequence of SEQ ID No. 121.

43. The antigen binding protein of any one of claims 1 to 4 and 23, the MAGE-A4 specific CAR of any one of claims 5 to 22 and 38 to 41, or the antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 37, wherein the antigen binding protein or MAGE-A4 specific CAR specifically binds to one or more amino acids at positions 286-294 of SEQ ID No. 32.

44. The antigen binding protein of any one of claims 1 to 4 and 23, the MAGE-A4 specific CAR of any one of claims 5 to 22 and 38 to 41, or the antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 37 and 42, wherein the antigen binding protein or the MAGE-A4 specific CAR interacts with one or more amino acids of the HLA.

45. The antigen binding protein of any one of claims 1 to 4 and 23, the MAGE-A4 specific CAR of any one of claims 5 to 22 and 38 to 41, or the antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 37, 42 and 43, wherein the HLA is HLA-a2.

46. An isolated nucleic acid molecule encoding the antigen binding protein of any one of claims 1 to 4 and 23, the MAGE-A4 specific CAR of any one of claims 5 to 22 and 38 to 41, or the antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 37 and 42 to 44.

47. The isolated nucleic acid molecule of claim 45 comprising the nucleotide sequence of SEQ ID NO. 21.

48. The isolated nucleic acid molecule of claim 45 comprising the nucleotide sequence of SEQ ID NO. 104.

49. A vector comprising the nucleic acid molecule of any one of claims 45 to 47.

50. The vector of claim 48, wherein the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

51. The vector of claim 49, wherein the vector is a lentiviral vector.

52. A cell comprising the nucleic acid molecule of any one of claims 45 to 47 or the vector of any one of claims 48 to 50.

53. The cell of claim 51, wherein the cell is a human T cell.

54. An engineered cell comprising the antigen binding protein of any one of claims 1 to 4 and 23, the MAGE-A4 specific CAR of any one of claims 5 to 22 and 38 to 41, or the antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 37 and 42 to 44.

55. The engineered cell of claim 53, which is an immune cell.

56. The engineered cell of claim 54, wherein the immune cell is an immune effector cell.

57. The engineered cell of claim 55, wherein the immune effector cell is a T lymphocyte.

58. The engineered cell of claim 56, wherein the T lymphocyte is an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte.

59. The engineered cell of claim 57, which is a cd8+ cytotoxic T lymphocyte.

60. The engineered cell of any one of claims 53-58 for use in treating a MAGE-A4 expressing cancer.

61. The engineered cell of claim 59, wherein the MAGE-A4 expressing cancer is multiple myeloma.

62. The engineered cell of claim 59, wherein the MAGE-A4 expressing cancer is melanoma.

63. An engineered human T cell comprising a chimeric antigen receptor comprising from N-terminus to C-terminus: (a) An extracellular ligand binding domain comprising an anti-MAGE-A4 single chain variable fragment (scFv) domain, said anti-MAGE-A4 scFv domain comprising a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR); (b) a hinge; (c) a transmembrane domain; and (d) a cytoplasmic domain comprising a 4-1BB co-stimulatory domain or a CD28 co-stimulatory domain and a CD3 zeta signaling domain, wherein the LCVR comprises: a Complementarity Determining Region (CDR) of a LCVR comprising the amino acid sequence of SEQ ID NO. 10 or SEQ ID NO. 115 and a CDR of a HCVR comprising the amino acid sequence of SEQ ID NO. 2, 83 or SEQ ID NO. 107.

64. The engineered human T cell of claim 62, wherein the anti-MAGE-A4 scFv specifically binds to one or more amino acid residues at positions 286-294 of SEQ ID No. 32.

65. The engineered human T cell of claim 62 or 63, wherein the scFv domain comprises a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID No. 2/10.

66. The engineered human T cell of claim 62 or 63, wherein the scFv domain comprises a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID No. 2/83.

67. The engineered human T cell of claim 62 or 63, wherein the scFv domain comprises a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID No. 107/115.

68. The engineered human T-cell of claim 64 or 65, wherein the HCVR comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3), and the LCVR comprises three light chain CDRs (LCDR 1, LCDR2, and LCDR 3), wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:8, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:12, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:14, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16.

69. The engineered human T cell of claim 66, wherein the HCVR comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3), and the LCVR comprises three light chain CDRs (LCDR 1, LCDR2, and LCDR 3), wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:109, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:111, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:113, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:117, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:14, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 119.

70. The engineered human T-cell according to any one of claims 62 to 68, wherein said hinge comprises the amino acid sequence of SEQ ID No. 27.

71. The engineered human T-cell according to any one of claims 62 to 69, wherein said transmembrane domain comprises the amino acid sequence of SEQ ID No. 28.

72. The engineered human T-cell of any one of claims 62 to 70, wherein the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 29.

73. The engineered human T-cell according to any one of claims 62 to 68, wherein said hinge comprises the amino acid sequence of SEQ ID No. 34.

74. The engineered human T-cell according to any one of claims 62 to 68 and 72, wherein said transmembrane domain comprises the amino acid sequence of SEQ ID No. 36.

75. The engineered human T-cell of any one of claims 62 to 68, 72 and 73, wherein the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 38.

76. The engineered human T cell of any one of claims 62-74, wherein the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID No. 30.

77. The engineered human T-cell of claim 62, comprising a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 22.

78. The engineered human T-cell of claim 62, comprising a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 105.

79. The engineered human T-cell of claim 62, comprising a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 120.

80. The engineered human T-cell of claim 62, comprising a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 121.

81. A pharmaceutical composition comprising a genetically modified human T cell and a pharmaceutically acceptable carrier, wherein the genetically modified human T cell comprises the antigen binding protein of any one of claims 1 to 4 and 23, the MAGE-A4 specific CAR of any one of claims 5 to 22 and 38 to 41, or the antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 37 and 42 to 44.

82. A pharmaceutical composition comprising the engineered cell of any one of claims 53-58 and a pharmaceutically acceptable carrier.

83. A pharmaceutical composition comprising the engineered human T-cell of any one of claims 62-79 and a pharmaceutically acceptable carrier.

84. The pharmaceutical composition of any one of claims 80-82 for use in treating a MAGE-A4 expressing cancer.

85. The pharmaceutical composition of claim 83, wherein the MAGE-A4 expressing cancer is multiple myeloma.

86. The pharmaceutical composition of claim 83, wherein the MAGE-A4 expressing cancer is melanoma.

87. Use of an antigen binding protein according to any one of claims 1 to 4 and 23, a MAGE-A4 specific CAR according to any one of claims 5 to 22 and 38 to 41, or an antigen binding protein or MAGE-A4 specific CAR according to any one of claims 24 to 37 and 42 to 44, a nucleic acid molecule according to any one of claims 45 to 47, a vector according to any one of claims 48 to 50, a cell according to claim 51 or 52, an engineered cell according to any one of claims 53 to 58, or an engineered human T cell according to any one of claims 62 to 79 in the manufacture of a medicament for the treatment of a MAGE-A4 expressing cancer.

88. The use of claim 86, wherein the MAGE-A4 expressing cancer is multiple myeloma.

89. The use of claim 86, wherein the MAGE-A4 expressing cancer is melanoma.

90. A method of enhancing T lymphocyte activity in a subject, the method comprising introducing into the subject a T lymphocyte comprising the antigen binding protein of any one of claims 1 to 4 and 23, the MAGE-A4 specific CAR of any one of claims 5 to 22 and 38 to 41, or the antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 37 and 42 to 44.

91. A method for treating a subject having cancer, the method comprising introducing into the subject a therapeutically effective amount of T lymphocytes comprising the antigen binding protein of any one of claims 1 to 4 and 23, the MAGE-A4 specific CAR of any one of claims 5 to 22 and 38 to 41, or the antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 37 and 42 to 44.

92. A method for stimulating a T cell-mediated immune response against a target cell population or tissue in a subject, the method comprising administering to the subject an effective amount of cells genetically modified to express an antigen binding protein according to any one of claims 1 to 4 and 23, a MAGE-A4 specific CAR according to any one of claims 5 to 22 and 38 to 41, or an antigen binding protein or MAGE-A4 specific CAR according to any one of claims 24 to 37 and 42 to 44.

93. A method of providing anti-tumor immunity in a subject, the method comprising administering to the subject an effective amount of a cell genetically modified to express an antigen binding protein of any one of claims 1 to 4 and 23, a MAGE-A4 specific CAR of any one of claims 5 to 22 and 38 to 41, or an antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 37 and 42 to 44.

94. The method of any one of claims 89-92 wherein the subject is a human.

95. The method of any one of claims 89-93, wherein the subject has multiple myeloma, synovial sarcoma, esophageal cancer, head and neck cancer, lung cancer, bladder cancer, ovarian cancer, uterine cancer, gastric cancer, cervical cancer, breast cancer, or melanoma.

96. The method of claim 94, wherein the subject has multiple myeloma.

97. A method of engineering a population of cells to express an antigen binding protein that specifically binds to HLA-bound MAGE-A4 or a MAGE-A4 specific Chimeric Antigen Receptor (CAR), the method comprising:

(a) Introducing a nucleic acid molecule encoding the antigen binding protein of any one of claims 1 to 4 and 23, the MAGE-A4 specific CAR of any one of claims 5 to 22 and 38 to 41, or the antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 37 and 42 to 44 into an immune cell population;

(b) Culturing the population of immune cells under conditions that express the nucleic acid molecule; and

(c) Isolating said immune cells expressing said MAGE-A4 specific antigen binding protein at the cell surface.

98. The method of claim 96, further comprising obtaining the population of immune cells from a subject prior to introducing the nucleic acid molecule.

99. A method of treating a MAGE-A4 expressing cancer in a subject, the method comprising:

(a) Engineering a population of cells according to claim 96 or claim 97; and

(b) Reintroducing the population of cells expressing the chimeric antigen receptor into the subject.

100. The method of claim 98, wherein the MAGE-A4 expressing cancer is multiple myeloma.

101. An isolated antigen binding protein, wherein the antigen binding protein comprises a first antigen binding domain that specifically binds to HLA-bound melanoma-associated antigen A4 (MAGE-A4) and a second antigen binding domain that specifically binds to human CD3, wherein the first antigen binding domain comprises three heavy chain Complementarity Determining Regions (CDRs) contained in a heavy chain variable region (A1-HCVR) (A1-HCDR 1, A1-HCDR2 and A1-HCDR 3) and three light chain CDRs (A1-LCDR 1, A1-LCDR2 and A1-LCDR 3) contained in a light chain variable region (A1-LCVR), and the second antigen binding domain comprises three heavy chain CDRs (A2-HCDR 1, A2-HCDR2 and A2-HCDR 3) contained in a heavy chain variable region (A2-LCVR) and three light chain CDRs (A2-LCVR) contained in A1-HCDR 2-LCVR), and the amino acid sequence comprising SEQ ID No. 2-dr 2, amino acid sequence comprising SEQ ID No. 2-3 and amino acid sequence No. 10.

102. An isolated antigen binding protein, wherein the antigen binding protein comprises a first antigen binding domain that specifically binds to HLA-bound melanoma-associated antigen A4 (MAGE-A4) and a second antigen binding domain that specifically binds to human CD3, wherein the first antigen binding domain comprises three heavy chain Complementarity Determining Regions (CDRs) contained in a heavy chain variable region (A1-HCVR) (A1-HCDR 1, A1-HCDR2 and A1-HCDR 3) and three light chain CDRs (A1-LCDR 1, A1-LCDR2 and A1-LCDR 3) contained in a light chain variable region (A1-LCVR), and the second antigen binding domain comprises three heavy chain CDRs (A2-HCDR 1, A2-HCDR2 and A2-HCDR 3) contained in a heavy chain variable region (A2-LCVR) and three light chain CDRs (A2-LCVR) contained in A1-HCDR2 and A2-LCVR), and the amino acid sequence comprising SEQ ID No. 2-dr 2, amino acid sequence comprising SEQ ID No. 2-dr 2 and amino acid sequence comprising SEQ ID No. 2-3 and amino acid sequence SEQ ID NO 10.

103. The isolated antigen binding protein of claim 100 or claim 101, wherein the antigen binding protein interacts with amino acids 286-294 of SEQ ID No. 32 or a portion thereof.

104. The isolated antigen binding protein of claim 100 or 101, wherein the isolated antigen binding protein is a CAR.

105. The isolated antigen binding protein of claim 100 or 101, wherein the isolated antigen binding protein is a bispecific antibody.

106. The isolated antigen binding protein of any one of claims 100-104, wherein the isolated antigen binding protein interacts with amino acids 286-294 of SEQ ID No. 32 or a portion thereof.

107. The isolated antigen binding protein of any one of claims 100-105, wherein the isolated antigen binding protein interacts with CD 3.

108. The isolated antigen binding protein of claim 100 or any one of claims 102-106, wherein the isolated antigen binding protein comprises a Heavy Chain Variable Region (HCVR) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID No. 2 or SEQ ID No. 83.

109. The isolated antigen binding protein of claim 100 or any one of claims 102-106, wherein the isolated antigen binding protein comprises a Heavy Chain Variable Region (HCVR) comprising an amino acid sequence with at least 95% sequence identity to SEQ ID No. 55.

110. The isolated antigen binding protein of any one of claims 101-106, wherein the isolated antigen binding protein comprises a Heavy Chain Variable Region (HCVR) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID No. 73.

111. An isolated antigen binding protein that binds to the same epitope as the antigen binding protein of any one of claims 1 to 4 and 23, the MAGE-A4 specific CAR of any one of claims 5 to 22 and 38 to 41, or the antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 37 and 42 to 44.

112. An isolated antigen binding protein that competes for binding with the antigen binding protein of any one of claims 1 to 4 and 23, the MAGE-A4 specific CAR of any one of claims 5 to 22 and 38 to 41, or the antigen binding protein or MAGE-A4 specific CAR of any one of claims 24 to 37 and 42 to 44.

113. The isolated antigen binding protein of claim 110 or 111, wherein the isolated antigen binding protein is a CAR.

114. The isolated antigen binding protein of claim 110 or 111, wherein the isolated antigen binding protein is a bispecific antibody.

115. The isolated antigen binding protein of any one of claims 111 to 113, wherein the isolated antigen binding protein interacts with amino acids 286-294 of SEQ ID No. 32 or a portion thereof.

116. The isolated antigen binding protein of claim 113 or 114, wherein the isolated antigen binding protein interacts with CD 3.

117. The isolated antigen binding protein of any one of claims 100-115, wherein the isolated antigen binding protein comprises a Heavy Chain Variable Region (HCVR) comprising three heavy chain CDRs, i.e., HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of SEQ ID NOs 4, 6 and 8, respectively.

118. The isolated antigen binding protein of any one of claims 100 or 102-116, wherein the isolated antigen binding protein comprises an HCVR corresponding to the other arm of the bispecific antibody comprising three heavy chain CDRs, i.e., HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences of seq id NOs 57, 59 and 61, respectively.

119. The isolated antigen binding protein of any one of claims 101-116, wherein the isolated antigen binding protein comprises an HCVR corresponding to another arm of the bispecific antibody, the HCVR comprising three heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs 75, 77 and 79, respectively.

120. The isolated antigen binding protein of any one of claims 100-118, wherein the isolated antigen binding protein comprises a Light Chain Variable Region (LCVR) comprising an amino acid sequence with at least 95% sequence identity to SEQ ID No. 10 and/or a LCVR comprising an amino acid sequence with at least 95% sequence identity to SEQ ID No. 63.

121. An isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to a melanoma-associated antigen A4 (MAGE-A4) polypeptide, wherein the antibody has one or more of the following characteristics:

(a) In an amount of less than about 2 x 10 ^-9 M binds to said MAGE-A4 polypeptide with an EC50 of M;

(b) Exhibits the ability to reduce tumor cell viability as compared to an isolated recombinant antibody that does not specifically bind to the MAGE-A4 polypeptide; and/or

(c) Comprising: (i) Three heavy chain Complementarity Determining Regions (CDRs) (HCDR 1, HCDR2 and HCDR 3) contained within a Heavy Chain Variable Region (HCVR) comprising an amino acid sequence having at least about 90% sequence identity to a HCVR as set forth in table 1;

and (ii) three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) contained within a Light Chain Variable Region (LCVR), the LCVR comprising an amino acid sequence having at least about 90% sequence identity to the LCVR shown in table 1.

122. The antibody or antigen-binding fragment of claim 120, wherein the MAGE-A4 polypeptide is a HLA-A 2-bound MAGE-A4 polypeptide.

123. The isolated antibody or antigen-binding fragment thereof of claim 120 or 121, comprising a HCVR having the amino acid sequence of SEQ ID No. 2, SEQ ID No. 83, or SEQ ID No. 107.

124. The isolated antibody or antigen-binding fragment thereof of any one of claims 120-122, comprising a LCVR having the amino acid sequence of SEQ ID No. 10 or SEQ ID No. 115.

125. The isolated antibody or antigen-binding fragment thereof of any one of claims 120 to 123, comprising the HCVR/LCVR amino acid sequence pair of SEQ ID No. 2/10, SEQ ID No. 83/10, or SEQ ID No. 107/115.

126. The isolated antibody or antigen-binding fragment thereof of any one of claims 120 to 124, comprising:

(a) An HCDR1 domain having the amino acid sequence of SEQ ID No. 4;

(b) An HCDR2 domain having the amino acid sequence of SEQ ID No. 6;

(c) An HCDR3 domain having the amino acid sequence of SEQ ID No. 8;

(d) An LCDR1 domain having the amino acid sequence of SEQ ID NO. 12;

(e) An LCDR2 domain having the amino acid sequence of SEQ ID NO. 14; and

(f) An LCDR3 domain having the amino acid sequence of SEQ ID NO. 16.

127. The isolated antibody or antigen-binding fragment thereof of any one of claims 120 to 124, comprising:

(a) An HCDR1 domain having the amino acid sequence of SEQ ID No. 109;

(b) An HCDR2 domain having the amino acid sequence of SEQ ID No. 111;

(c) An HCDR3 domain having the amino acid sequence of SEQ ID No. 113;

(d) An LCDR1 domain having the amino acid sequence of SEQ ID NO. 117;

(e) An LCDR2 domain having the amino acid sequence of SEQ ID NO. 14; and

(f) An LCDR3 domain having the amino acid sequence of SEQ ID NO. 119.

128. The isolated antibody or antigen-binding fragment thereof of any one of claims 120 to 126, which is an IgG1 antibody.

129. The isolated antibody or antigen-binding fragment thereof of any one of claims 120 to 126, which is an IgG4 antibody.

130. The isolated antibody or antigen-binding fragment thereof of any one of claims 120 to 128, which is a bispecific antibody.

131. An isolated recombinant antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to a melanoma-associated antigen A4 (MAGE-A4) polypeptide and a second antigen-binding domain that specifically binds to a CD3 polypeptide, wherein:

(a) The first antigen binding domain (A1) that binds MAGE-A4 comprises three heavy chain complementarity determining regions (A1-HCDR 1, A1-HCDR2, and A1-HCDR 3) and three light chain complementarity determining regions (A1-LCDR 1, A1-LCDR2, and A1-LCDR 3), wherein

The A1-HCDR1 comprises the amino acid sequence of SEQ ID NO. 4 or SEQ ID NO. 109;

the A1-HCDR2 comprises the amino acid sequence of SEQ ID NO. 6 or SEQ ID NO. 111;

the A1-HCDR3 comprises the amino acid sequence of SEQ ID NO. 8 or SEQ ID NO. 113;

the A1-LCDR1 comprises the amino acid sequence of SEQ ID NO. 12, SEQ ID NO. 65 or SEQ ID NO. 117;

the A1-LCDR2 comprises an amino acid sequence of SEQ ID NO. 14;

the A1-LCDR3 comprises the amino acid sequence of SEQ ID NO. 16, SEQ ID NO. 67 or SEQ ID NO. 119; and is also provided with

(b) The second antigen binding domain (A2) that binds CD3 comprises three heavy chain complementarity determining regions (A2-HCDR 1, A2-HCDR2, and A2-HCDR 3) and three light chain complementarity determining regions (A2-LCDR 1, A2-LCDR2, and A2-LCDR 3), where

The A2-HCDR1 comprises the amino acid sequence of SEQ ID NO. 57 or SEQ ID NO. 75;

the A2-HCDR2 comprises the amino acid sequence of SEQ ID NO 59 or SEQ ID NO 77;

the A2-HCDR3 comprises the amino acid sequence of SEQ ID NO. 61 or SEQ ID NO. 79;

the A2-LCDR1 comprises the amino acid sequence of SEQ ID NO. 12, SEQ ID NO. 65 or SEQ ID NO. 117;

the A2-LCDR2 comprises the amino acid sequence of SEQ ID NO. 14;

the A2-LCDR3 comprises the amino acid sequence of SEQ ID NO. 16, SEQ ID NO. 67 or SEQ ID NO. 119.

132. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 120-130 and a pharmaceutically acceptable carrier or diluent.

133. The pharmaceutical composition of claim 131, wherein the pharmaceutical composition further comprises a second therapeutic agent.

134. The pharmaceutical composition of claim 132, wherein the second therapeutic agent is selected from the group consisting of: antineoplastic agents, steroids, and targeted therapies.

135. A polynucleotide molecule comprising a polynucleotide sequence encoding one or more HCVR and/or one or more LCVR of the antibody of any one of claims 120-133.

136. A vector comprising the polynucleotide of claim 134.

137. A cell comprising the vector of claim 135.

138. A method of treating a MAGE-A4 expressing cancer, the method comprising administering to a subject the antibody or antigen binding fragment of any one of claims 120 to 130 or the pharmaceutical composition of any one of claims 131 to 133.

139. The method of claim 137, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

140. The method of claim 138, wherein the second therapeutic agent is selected from the group consisting of: antineoplastic agents, steroids, and targeted therapies.