Chimeric antigen receptor with MAGE-A4 specificity and uses thereof

Abstract

MAGE-A4, or melanoma associated antigen A4, is a 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 MAGE-A4 specific isolated antibodies. In certain embodiments, engineered cells expressing the chimeric antigen receptors of the present disclosure are capable of inhibiting the growth of tumors expressing MAGE-A4. The engineered cells of the present disclosure are useful for treating diseases and disorders in which an upregulated or induced immune response targeting MAGE-A4 is desired and / or therapeutically beneficial. For example, engineered cells expressing the MAGE-A4 specific chimeric antigen receptors of the present disclosure are useful for treating various cancers.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 184,183, filed May 4, 2021, and U.S. Provisional Application No. 63 / 239,293, filed August 31, 2021, each of which is incorporated by reference in its entirety for all purposes.

[0002] (Reference to sequence listing) This application incorporates by reference a sequence listing, which was submitted in computer readable format as file 10901WO01-Sequence.txt, created on May 3, 2022, and contains 95,050 bytes.

[0003] FIELD OF THEINVENTION The present disclosure provides antibodies, chimeric antigen receptors (CARs), and engineered cells comprising such antibodies and CARs, that are specific for Melanoma-Associated Antigen A4 (MAGE-A4), and methods of their use. [Background technology]

[0004] MAGE-A4, or melanoma associated antigen A4, is a 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, overexpression of MAGE-A4 has been shown to promote the growth of spontaneously transformed oral keratinocytes and inhibit the growth arrest of cells in the G1 phase.

[0005] MAGE-A4 is abundantly expressed by many tumors of different histological types, such as head and neck squamous cell carcinoma, lung cancer including 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 tissues, MAGE-A4 expression is restricted to the testis.

[0006] The restricted expression pattern together with the ability of the MAGE-A4 antigen to elicit an immune response makes it an excellent candidate for cancer immunotherapy.

[0007] Dual targeting antibody strategies applied to complex diseases such as cancer also represent a promising strategy, whereby multifactorial modulation aims to improve therapeutic efficacy. CD3 is a homodimeric or heterodimeric antigen expressed on T cells together with the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed from the dimeric pairing of two of four different chains: epsilon, zeta, delta, and gamma. Bispecific antibodies with MAGE-A4 binding arms and CD3 binding arms may be useful to enhance antitumor activity.

[0008] Adoptive immunotherapy, which involves the transfer of ex vivo generated self-antigen-specific T cells, is another promising strategy for treating viral infections and cancer. The T cells used in adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or by redirecting T cells by genetic engineering.

[0009] New specificities in T cells have been successfully generated through the transduction of transgenic T cell receptors or chimeric antigen receptors (CARs). CARs are synthetic receptors that consist of a targeting moiety associated with one or more signaling domains in a single fusion molecule. In general, the binding portion of a CAR consists of the antigen-binding region of a single-chain antibody (scFv), which includes the light and heavy chain variable fragments of a monoclonal antibody linked by a flexible linker. The signaling domain for first generation CARs is derived from the cytoplasmic domain of CD3 zeta or the gamma chain of the Fc receptor. First generation CARs have been shown to successfully redirect the cytotoxicity of T cells. However, they have failed to provide long-term proliferation and antitumor activity in vivo. Signaling domains and transmembrane and hinge domains from costimulatory molecules have been added to form second and third generation CARs, leading to some successful therapeutic trials in humans. For example, T cells redirected with CARs specific for the B cell differentiation antigen CD19 have shown dramatic efficacy in treating B cell malignancies, and T cell receptor (TCR) redirected T cells have shown benefit in patients suffering from solid tumors. Stauss et al., for example, describe a strategy to modify therapeutic CARs and TCRs for use in the treatment of cancer that enhances antigen-specific effector function and limits the toxicity of engineered T cells (Current Opinion in Pharmacology 2015,24:113-118).

[0010] 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 settings. Summary of the Invention

[0011] In one aspect, the 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 the CDRs of the HCVR comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 83, or SEQ ID NO: 107.

[0012] In some embodiments, the LCVR comprises an amino acid sequence having 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 having at least 95% identity to SEQ ID NO: 2, SEQ ID NO: 83, or SEQ ID NO: 107.

[0013] In some cases, the antigen binding protein interacts with amino acids 286-294 of SEQ ID NO:32, or a portion thereof.

[0014] In one aspect, the disclosure provides a MAGE-A4 specific chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, (a) an extracellular ligand binding region comprising an anti-MAGE-A4 single chain variable fragment (scFv) region comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR); (b) a hinge; (c) a transmembrane region; and (d) a cytoplasmic region comprising a 4-1BB costimulatory region or a CD28 costimulatory region and a CD3 zeta signaling region, wherein the LCVR comprises a complementarity determining region (CDR) of the LCVR comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 115, and a CDR of the HCVR comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 83, or SEQ ID NO: 107.

[0015] In some embodiments, the MAGE-A4 specific CAR comprises, from N-terminus to C-terminus, (a) an extracellular ligand binding region, (b) a hinge, (c) a transmembrane region, and (d) a cytoplasmic region comprising a costimulatory region and a signaling region. Optionally, the anti-MAGE-A4 scFv region comprises a first linker between the LCVR and the HCVR.

[0016] In some embodiments, the MAGE-A4 specific CAR further comprises a second linker between the extracellular ligand binding region and the hinge. Optionally, the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-26, and the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-26. Optionally, 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.

[0017] In various embodiments of the MAGE-A4 specific CAR, the hinge, the transmembrane region, or both, are derived from a CD8α polypeptide.

[0018] In various embodiments of the MAGE-A4 specific CAR, the costimulatory region comprises a 4-1BB costimulatory region.

[0019] In various embodiments of the MAGE-A4 specific CAR, the costimulatory region comprises a CD28 costimulatory region.

[0020] In various embodiments of the MAGE-A4 specific CAR, the hinge, the transmembrane region, or both, are derived from a CD28 polypeptide.

[0021] In various embodiments of the MAGE-A4 specific CAR, the hinge comprises the amino acid sequence of SEQ ID NO:27.

[0022] In various embodiments of the MAGE-A4 specific CAR, the transmembrane region comprises the amino acid sequence of SEQ ID NO:28.

[0023] In various embodiments of the MAGE-A4-specific CAR, the 4-1BB costimulatory region comprises the amino acid sequence of SEQ ID NO:29.

[0024] In various embodiments of the MAGE-A4 specific CAR, the hinge comprises the amino acid sequence of SEQ ID NO:34.

[0025] In various embodiments of the MAGE-A4 specific CAR, the transmembrane region comprises the amino acid sequence of SEQ ID NO:36.

[0026] In various embodiments of the MAGE-A4-specific CAR, the CD28 costimulatory region comprises the amino acid sequence of SEQ ID NO:38.

[0027] In various embodiments of the MAGE-A4 specific CAR, the signaling region comprises a CD3 zeta signaling region. In some cases, the CD3 zeta signaling region comprises the amino acid sequence of SEQ ID NO:30.

[0028] In various embodiments the antigen binding protein is a MAGE-A4 specific antibody or antigen binding fragment thereof.

[0029] In some cases, the antigen binding protein or MAGE-A4 specific CAR discussed above or herein comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) 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 set forth in SEQ ID NO:2. In some cases, the HCVR comprises the amino acid sequence set forth in SEQ ID NO:83. In some cases, the antigen binding protein or MAGE-A4 specific CAR comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) 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 set forth in SEQ ID NO:107.

[0030] In some cases, the antigen binding protein or MAGE-A4 specific CAR discussed above or herein comprises three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an 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 set forth 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 set forth in SEQ ID NO: 115.

[0031] 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.

[0032] 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.

[0033] In various embodiments, the antigen binding protein or MAGE-A4 specific CAR discussed above or herein specifically binds to one or more amino acids 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.

[0034] In one aspect, the disclosure provides an isolated nucleic acid molecule encoding an antigen binding protein or MAGE-A4 specific CAR as described above or discussed herein. Optionally, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 21. Optionally, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 104.

[0035] In one aspect, the present disclosure provides a vector comprising the nucleic acid molecule described above or discussed herein.In some cases, the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.In some embodiments, the vector is a lentiviral vector.

[0036] In one aspect, the disclosure provides a cell comprising the nucleic acid molecule(s) described above or discussed herein. In some cases, the cell is a human T cell.

[0037] In one aspect, the disclosure provides an engineered cell comprising an antigen binding protein or MAGE-A4 specific CAR as described above or discussed herein. Optionally, the engineered cell is an immune cell. Optionally, the immune cell is an immune effector cell. Optionally, the immune effector cell is a T lymphocyte. Optionally, the T lymphocyte is an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte. Optionally, the engineered cell is a CD8+ cytotoxic T lymphocyte. In various embodiments, the engineered cell is for use in treating a cancer that expresses MAGE-A4. Optionally, the cancer that expresses MAGE-A4 is multiple myeloma. Optionally, the cancer that expresses MAGE-A4 is melanoma.

[0038] In one aspect, the disclosure provides an engineered human T cell comprising a chimeric antigen receptor comprising, from N-terminus to C-terminus: (a) an extracellular ligand binding region comprising an anti-MAGE-A4 single chain variable fragment (scFv) region comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR); (b) a hinge; (c) a transmembrane region; and (d) a cytoplasmic region comprising a 4-1BB costimulatory region or a CD28 costimulatory region and a CD3 zeta signaling region, wherein the LCVR comprises a complementarity determining region (CDR) of the LCVR comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 115, and a CDR of the HCVR comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 83, or SEQ ID NO: 107.

[0039] 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. Optionally, the scFv region comprises an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 2 / 10. Optionally, the scFv region comprises an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 2 / 83. Optionally, the scFv region comprises an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 107 / 115. In some embodiments, the HCVR comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and the LCVR comprises three light chain CDRs (LCDR1, LCDR2, and LCDR3), where 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, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:8, 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 HCVR comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and the LCVR comprises three light chain CDRs (LCDR1, LCDR2, and LCDR3), where 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, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 113, 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 hinge comprises the amino acid sequence of SEQ ID NO: 27. In some cases, the transmembrane region comprises the amino acid sequence of SEQ ID NO: 28. In some cases, the 4-1BB costimulatory region 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 region comprises the amino acid sequence of SEQ ID NO: 36. In some cases, the CD28 costimulatory region comprises the amino acid sequence of SEQ ID NO: 38. In some cases, the CD3 zeta signaling region comprises the amino acid sequence of SEQ ID NO:30.

[0040] 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.

[0041] In one aspect, the disclosure provides a pharmaceutical composition comprising a genetically modified human T cell and a pharma- ceutically acceptable carrier, wherein the genetically modified human T cell comprises an antigen binding protein or a MAGE-A4 specific CAR as discussed above or herein. Optionally, the pharmaceutical composition comprises an engineered cell as discussed above or herein and a pharma- ceutically acceptable carrier. Optionally, the pharmaceutical composition comprises an engineered human T cell as discussed above or herein and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition is for use in treating a cancer that expresses MAGE-A4. Optionally, the cancer that expresses MAGE-A4 is multiple myeloma. Optionally, the cancer that expresses MAGE-A4 is melanoma.

[0042] In one aspect, the 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 described above or discussed herein in the manufacture of a medicament for the treatment of a cancer that expresses MAGE-A4. Optionally, the cancer that expresses MAGE-A4 is multiple myeloma. Optionally, the cancer that expresses MAGE-A4 is melanoma.

[0043] In one aspect, the present disclosure provides a method of enhancing T lymphocyte activity in a subject comprising introducing into the subject T lymphocytes comprising an antigen binding protein or a MAGE-A4 specific CAR as described above or herein.

[0044] In one aspect, the disclosure provides a method of treating a subject suffering from cancer, the method comprising introducing into the subject a therapeutically effective amount of T lymphocytes comprising an antigen binding protein or a MAGE-A4 specific CAR as described above or discussed herein.

[0045] In one aspect, the present disclosure provides a method of stimulating a T cell-mediated immune response against a target cell population or tissue in a subject comprising administering to the subject an effective amount of cells genetically modified to express an antigen binding protein or MAGE-A4 specific CAR as described above or discussed herein.

[0046] In one aspect, the disclosure provides a method of providing anti-tumor immunity to a subject, the method comprising administering to the subject an effective amount of a cell genetically modified to express an antigen binding protein or a MAGE-A4 specific CAR as described above or discussed herein.

[0047] 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, gastric cancer, cervical cancer, breast cancer, or melanoma. In some embodiments, the subject has multiple myeloma.

[0048] In one aspect, the disclosure provides a method of engineering a cell population to express an antigen binding protein or MAGE-A4 specific CAR as described above or discussed herein, the method comprising: (a) introducing into an immune cell population a nucleic acid molecule encoding an antigen binding protein or MAGE-A4 specific CAR as described above or discussed herein, (b) culturing the immune cell population under conditions for expression of the nucleic acid molecule, and (c) isolating immune cells expressing the MAGE-A4 specific antigen binding protein on their cell surface. Optionally, the method further comprises obtaining the immune cell population from a subject prior to introducing the nucleic acid molecule.

[0049] In one aspect, the disclosure provides a method of treating a MAGE-A4 expressing cancer in a subject, the method comprising: (a) manipulating a cell population as described above or discussed herein; and (b) reintroducing into the subject a cell population expressing a chimeric antigen receptor. In some cases, the MAGE-A4 expressing cancer is multiple myeloma.

[0050] In one aspect, the disclosure provides an isolated antigen binding protein, the antigen binding protein comprising a first antigen binding region that specifically binds to HLA-bound melanoma associated antigen A4 (MAGE-A4) and a second antigen binding region that specifically binds to human CD3, the first antigen binding region comprising three heavy chain complementarity determining regions (CDRs) (A1-HCDR1, A1-HCDR2, and A1-HCDR3) contained in a heavy chain variable region (A1-HCVR) and three light chain CDRs (A1-LCDR1, A1-LCDR2, and A1-LCDR3) contained in a light chain variable region (A1-LCVR). the first antigen-binding region comprises three heavy chain CDRs (A2-HCDR1, A2-HCDR2, and A2-HCDR3) contained in a heavy chain variable region (A2-HCVR) and three light chain CDRs (A2-LCDR1, A2-LCDR2, and A2-LCDR3) contained in a light chain variable region (A2-LCVR), where A1-HCVR comprises the amino acid sequence of SEQ ID NO:2, A1-LCVR comprises the amino acid sequence of SEQ ID NO:10, A2-HCVR comprises the amino acid sequence of SEQ ID NO:55, and A2-LCVR comprises the amino acid sequence of SEQ ID NO:10.

[0051] In one aspect, the disclosure provides an isolated antigen binding protein, the antigen binding protein comprising a first antigen binding region that specifically binds to HLA-bound melanoma associated antigen A4 (MAGE-A4) and a second antigen binding region that specifically binds to human CD3, the first antigen binding region comprising three heavy chain complementarity determining regions (CDRs) (A1-HCDR1, A1-HCDR2, and A1-HCDR3) contained in a heavy chain variable region (A1-HCVR) and three light chain CDRs (A1-LCDR1, A1-LCDR2, and A1-LCDR3) contained in a light chain variable region (A1-LCVR). the first antigen-binding region comprises three heavy chain CDRs (A2-HCDR1, A2-HCDR2, and A2-HCDR3) contained in a heavy chain variable region (A2-HCVR) and three light chain CDRs (A2-LCDR1, A2-LCDR2, and A2-LCDR3) contained in a light chain variable region (A2-LCVR), where A1-HCVR comprises the amino acid sequence of SEQ ID NO:2, A1-LCVR comprises the amino acid sequence of SEQ ID NO:10, A2-HCVR comprises the amino acid sequence of SEQ ID NO:73, and A2-LCVR comprises the amino acid sequence of SEQ ID NO:10.

[0052] In various embodiments of the isolated antigen binding protein described above or discussed herein, the antigen binding protein interacts with amino acids 286-294 of SEQ ID NO:32, or a portion thereof. Optionally, the isolated antigen binding protein is a CAR. Optionally, the isolated antigen binding protein is a bispecific antibody. In some embodiments, the isolated antigen binding protein interacts with CD3. Optionally, 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. Optionally, 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. Optionally, 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.

[0053] In one aspect, the disclosure provides an isolated antigen binding protein that binds to the same antigenic determinant as an antigen binding protein or MAGE-A4 specific CAR as described above or discussed herein. In one aspect, the disclosure provides an isolated antigen binding protein that competes for binding with an antigen binding protein or MAGE-A4 specific CAR as described above or discussed herein. Optionally, the isolated antigen binding protein is a CAR. Optionally, the isolated antigen binding protein is a bispecific antibody. Optionally, the isolated antigen binding protein interacts with amino acids 286-294 of SEQ ID NO:32, or a portion thereof. Optionally, the isolated antigen binding protein interacts with CD3. In some embodiments, the isolated antigen binding protein comprises a heavy chain variable region (HCVR) comprising three heavy chain CDRs, HCDR1, HCDR2, and HCDR3, comprising the amino acid sequences of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively. In some embodiments, the isolated antigen binding protein comprises a HCVR corresponding to another arm of a bispecific antibody comprising three heavy chain CDRs, HCDR1, HCDR2, and HCDR3, comprising the amino acid sequences of SEQ ID NO:57, SEQ ID NO:59, and SEQ ID NO:61, respectively. In some embodiments, the isolated antigen binding protein comprises a HCVR corresponding to another arm of a bispecific antibody comprising three heavy chain CDRs, HCDR1, HCDR2, and HCDR3, comprising the amino acid sequences of SEQ ID NO:75, SEQ ID NO:77, and SEQ ID NO:79, respectively. In some embodiments, the isolated antigen binding protein comprises a light chain variable region (LCVR) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:10, and / or an LCVR comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:63.

[0054] 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, the antibody having one or more of the following characteristics: (a) a concentration of about 2×10 9(b) binds to a MAGE-A4 polypeptide with an EC50 of less than M; (b) exhibits an 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) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising an amino acid sequence having at least about 90% sequence identity to an HCVR listed in Table 1, and (ii) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising an amino acid sequence having at least about 90% sequence identity to an LCVR listed in Table 1.

[0055] 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 an HLA-A2-binding MAGE-A4 polypeptide. Optionally, 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. Optionally, 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. Optionally, the isolated antibody or antigen-binding fragment thereof comprises a pair of HCVR / LCVR amino acid sequences of SEQ ID NO:2 / 10, or SEQ ID NO:83 / 10, or SEQ ID NO:107 / 115.

[0056] In one aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising: (a) an HCDR1 region having the amino acid sequence of SEQ ID NO: 4; (b) an HCDR2 region having the amino acid sequence of SEQ ID NO: 6; (c) an HCDR3 region having the amino acid sequence of SEQ ID NO: 8; (d) an LCDR1 region having the amino acid sequence of SEQ ID NO: 12; (e) an LCDR2 region having the amino acid sequence of SEQ ID NO: 14; and (f) an LCDR3 region having the amino acid sequence of SEQ ID NO: 16.

[0057] In one aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof comprising: (a) an HCDR1 region having the amino acid sequence of SEQ ID NO: 109; (b) an HCDR2 region having the amino acid sequence of SEQ ID NO: 111; (c) an HCDR3 region having the amino acid sequence of SEQ ID NO: 113; (d) an LCDR1 region having the amino acid sequence of SEQ ID NO: 117; (e) an LCDR2 region having the amino acid sequence of SEQ ID NO: 14; and (f) an LCDR3 region having the amino acid sequence of SEQ ID NO: 119.

[0058] In various embodiments of the isolated antibody or antigen-binding fragment thereof described above or discussed herein, the isolated antibody or antigen-binding fragment thereof is an IgG1 antibody or an IgG4 antibody. Optionally, the isolated antibody or antigen-binding fragment thereof is a bispecific antibody.

[0059] In one aspect, the disclosure provides an isolated recombinant antibody or antigen-binding fragment thereof comprising a first antigen-binding region that specifically binds to a melanoma associated antigen A4 (MAGE-A4) polypeptide and a second antigen-binding region that specifically binds to a CD3 polypeptide, wherein (a) the first antigen-binding region (A1) that binds to MAGE-A4 comprises three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2, and A1-HCDR3) and three light chain complementarity determining regions (A1-LCDR1, A1-LCDR2, and A1-LCDR3), 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, and A1-LCDR2 comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 113. (b) a second antigen-binding region (A2) that binds to CD3 comprises three heavy chain complementarity determining regions (A2-HCDR1, A2-HCDR2, and A2-HCDR3) and three light chain complementarity determining regions (A2-LCDR1, A2-LCDR2, and A2-LCDR3), A2-HCDR1 comprises the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 119; and 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, and A2-LCDR3 comprises the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 67, or SEQ ID NO: 119.

[0060] In one aspect, the disclosure provides a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof as described above or discussed herein and a pharma- ceutically acceptable carrier or diluent. Optionally, the pharmaceutical composition further comprises a second therapeutic agent. Optionally, the second therapeutic agent is selected from the group consisting of an anti-tumor agent, a steroid, and a targeted therapy.

[0061] In one aspect, the disclosure provides polynucleotide molecules comprising a polynucleotide sequence encoding one or more HCVRs and / or one or more LCVRs of an antibody as described above or discussed herein, as well as vectors comprising the polynucleotides, and cells comprising the vectors.

[0062] In one aspect, the disclosure provides a method of treating a cancer expressing MAGE-A4, the method comprising administering to a subject an antibody or antigen-binding fragment as described above or herein, or a pharmaceutical composition as described above or herein. Optionally, the pharmaceutical composition is administered in combination with a second therapeutic agent. Optionally, the second therapeutic agent is selected from the group consisting of an anti-tumor agent, a steroid, and a targeted therapy.

[0063] Other embodiments will be apparent from review of the detailed description that follows. [Brief description of the drawings]

[0064] [Figure 1] 1 shows an exemplary nucleotide construct for expressing a chimeric antigen receptor (CAR) construct, which includes an anti-MAGE-A4 VL-linker-VH scFv, a human CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, a CD3 zeta signaling domain, and optionally an IRES, an eGFP sequence for tracking CAR-transduced cells. [Diagram 2] Illustrated is an exemplary bispecific antibody of the present disclosure. The exemplary bispecific antibody is composed of one HCVR capable of interacting with MAGE-A4 (anti-MAGE-A4 arm) and another HCVR capable of interacting with CD3 (anti-CD3 arm). In an exemplary embodiment, the LCVR is generic (e.g., corresponding to an anti-CD3 antibody or an antibody light chain that is both promiscuous or known to effectively pair with a variety of heavy chain arms). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] It is to be understood that the invention described herein is not limited to the specific methods and experimental conditions described, and thus the methods and conditions may be modified. It is also to be understood that the terminology used herein is intended only to describe specific embodiments, and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims. Any embodiment or feature of an embodiment may be combined with each other, and such combinations are expressly included within the scope of the present invention. Any specific value described above or discussed herein may be combined with another related value described above or discussed herein to recite a range in which those values ​​represent the upper and lower limits of the range, and such ranges are included within the scope of the present disclosure.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.As used herein, the term "about" when used in relation to a specific recited numerical value means that the value may vary by 1% or less from the recited value.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.).

[0067] 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 herein are incorporated by reference in their entirety.

[0068] definition As used herein, the term "MAGE-A4" refers to melanoma associated antigen A4. MAGE-A4 is an intracellular protein expressed by a variety of different tumor cells. As used herein, "MAGE-A4" refers to human MAGE-A4 protein, unless specified as being derived from a non-human species (e.g., "mouse MAGE-A4", "monkey MAGE-A4", etc.). Human MAGE-A4 protein has the amino acid sequence shown in SEQ ID NO: 32 and the polynucleic acid sequence of SEQ ID NO: 31. References to specific regions of the MAGE-A4 polypeptide (e.g., MAGE-A4 286-294) are with respect to SEQ ID NO: 32. As used herein, "MAGE-A4 286-294", "MAGE-A4(286-294)", and "MAGEA4" refer to the human MAGE-A4 protein. 286-294 " may be used interchangeably. The polypeptide sequence of MAGE-A4(286-294) (KVLEHVVRV) is given as SEQ ID NO:33.

[0069] As used herein, an "antibody that binds to MAGE-A4" or an "anti-MAGE-A4 antibody" includes an antibody and antigen-binding fragment thereof that specifically recognizes at least MAGE-A4. In some embodiments, an antibody that binds to MAGE-A4 interacts 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 further be capable of specifically recognizing other MAGE-A4-related peptides (e.g., MAGE-A4-related peptides predicted to form a complex with HLA-A2). Furthermore, an antibody that binds to MAGE-A4 may be further capable of binding to one or more additional ligands, for example, when formatted as a bispecific antibody. In certain embodiments, an anti-MAGE-A4 antibody may be formatted as a bispecific antibody that binds to both MAGE-A4 and CD3.

[0070] The terms "ligand binding region" and "antigen binding region" are used interchangeably herein and refer to the portion of a chimeric antigen receptor or corresponding antibody that specifically binds to a given antigen (e.g., MAGE-A4). Reference to a "corresponding antibody" refers to the antibody from which the CDRs or variable regions (heavy chain variable region (abbreviated HCVR or VH) and light chain variable region (abbreviated LCVR or VL)) used in the chimeric antigen receptor or bispecific antibody are derived. For example, the chimeric antigen receptor construct discussed in the Examples comprises an scFv having variable regions derived from an anti-MAGE-A4 antibody. This anti-MAGE-A4 antibody is the "corresponding antibody" for the respective chimeric antigen receptor.

[0071] As used herein, the term "antibody", including "bispecific antibody", refers to any antigen-binding molecule or molecular complex that comprises at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., MAGE-A4). In some embodiments, an antibody may bind to or interact with an MHC-bound polypeptide, such as an HLA-bound polypeptide. In the context of the present disclosure, an antibody may, in some embodiments, bind to an HLA-A2-bound polypeptide, such as a MAGE-A4 polypeptide (e.g., MAGE-A4 286-294) presented by HLA-A2. The term "antibody", including "bispecific antibody", includes immunoglobulin molecules that comprise four polypeptide chains, two heavy (H) chains, and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM), however, immunoglobulin molecules that consist only of heavy chains (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) contains a heavy chain variable region (abbreviated herein as HCVR or V H The heavy chain constant region is made up of three regions: H 1. C H 2, and CH Each light chain (abbreviated herein as LC) comprises a light chain variable region (abbreviated herein as LCVR or V L The light chain constant region comprises one region (C L 1) is included. H Area and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of the anti-MAGE-A4 antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0072] In certain embodiments of the invention, the antibody is a bispecific antibody. For example, in certain embodiments, an anti-MAGE-A4 antibody (e.g., mAbM31339N2) is reformatted into a bispecific antibody (e.g., anti-MAGE-A4 x anti-CD3) using either a high affinity (7195P) CD3 arm to generate bsb6054, or a medium affinity (7221G) CD3 arm to generate bsAb6043.

[0073] As used herein, the term "antibody" also includes antigen-binding fragments of a complete antibody molecule. An "antigen-binding portion" of an antibody, an "antigen-binding fragment" of an antibody, and similar terms, as used herein, include naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind an antigen to form a complex. As used herein, an "antigen-binding portion" or an "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to MAGE-A4 (or a MAGE-A4 related peptide) and / or CD3. Antigen-binding fragments of an antibody may be derived from a complete antibody molecule using any suitable standard technique, such as, for example, proteolytic or recombinant genetic engineering techniques, including the engineering and expression of DNA-encoded antibody variable and optionally constant regions. Such DNA is well known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or may be synthesized. The DNA may be sequenced and manipulated, either chemically or by using molecular biology techniques, e.g., to place one or more variable and / or constant regions into a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0074] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs), such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Region-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR regions are also encompassed within the expression "antigen-binding fragment" as used herein.

[0075] Antigen-binding fragments of antibodies typically contain at least one variable region. The variable region may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. H The area is V L In the antigen-binding fragment paired with the V H Area and V L The domains may be positioned relative to each other in any suitable configuration. For example, the variable domain may be a dimer and the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of the antibody may comprise a monomeric V H Area or V L It may include regions.

[0076] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable region covalently linked to at least one constant region. Non-limiting exemplary configurations of variable and constant regions that may be found in an antigen-binding fragment of an antibody of the invention include: (i) a 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 -CH 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, and (xiv) V L -C L In any configuration of the variable and constant regions, including any of the representative configurations listed above, the variable and constant regions may be either directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant regions in a single polypeptide molecule. Additionally, antigen-binding fragments of antibodies of the present invention may be linked to each other and / or to one or more monomeric V H Or V L The variable region and constant region configurations may comprise homodimers or heterodimers (or other multimers) of any of the variable region and constant region configurations listed above in non-covalent association with the domain (e.g., via disulfide bonds).

[0077] In certain embodiments, the anti-MAGE-A4 antibody from which the antigen-binding fragment is derived is a human antibody. The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present disclosure may include, for example, amino acid residues in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, 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 sequences.

[0078] In various embodiments, the anti-MAGE-A4x anti-CD3 bispecific antibody of the present disclosure (e.g., BsAb6054 and / or BsAb6043) is a human antibody. In various embodiments, the anti-MAGE-A4x anti-CD3 bispecific antibody is a human IgG antibody. In various embodiments, the anti-MAGE-A4x anti-CD3 bispecific antibody is a human antibody of isotype IgG1, isotype IgG2, isotype IgG3 or isotype IgG4, or mixed isotype. In some embodiments, the anti-MAGE-A4x anti-CD3 bispecific antibody is a human IgG1 antibody (i.e., the antibody comprises a human IgG1 heavy chain constant region attached to the HCVRs of each of the first and second antigen binding regions, respectively). In some embodiments, the anti-MAGE-A4x anti-CD3 bispecific antibody is a human IgG4 antibody (i.e., the antibody comprises a human IgG4 heavy chain constant region attached to the HCVRs of each of the first and second antigen binding regions, respectively). In any of the embodiments described above or discussed herein, the anti-MAGE-A4xanti-CD3 bispecific antibody may comprise a human kappa light chain. In any of the embodiments described above or discussed herein, the anti-MAGE-A4xanti-CD3 bispecific antibody may comprise a human lambda light chain.

[0079] In any embodiment, the bispecific antibody may comprise a modification in one or both heavy chains to facilitate purification of the bispecific antibody (i.e., heterodimer) from homodimeric impurities. 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-CD3 binding arm) that are identical (e.g., both isotype IgG1 or isotype IgG4) except for a modification in the CH3 region of one or the other heavy chain that reduces binding of the bispecific antibody to protein A compared to an antibody lacking the modification. In some cases, the CH3 region of the first heavy chain (e.g., of the anti-MAGE-A4 binding arm) binds protein A and the CH3 region of the second heavy chain (e.g., of the anti-CD3 binding arm) comprises a mutation that reduces or eliminates protein A binding. In some cases, the mutation is an H435R modification (according to IMGT exon numbering, according to EU numbering H95R). In some cases, the mutation is an H435R modification (according to IMGT exon numbering, EU numbering H95R) and an Y436F modification (according to IMGT, EU numbering Y96F). Additional modifications that may be found within the second CH3 region include D356E, L358M, N384S, K392N, V397M, and V422I by EU (D16E, L18M, N44S, K52N, V57M, and V82I by IMGT) for IgG1 CH3 regions, and Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU (Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I by IMGT) for IgG4 CH3 regions.

[0080] In any embodiment, the bispecific antibody may 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, a chimeric hinge, in one embodiment, 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 sequence or "upper hinge" sequence comprises amino acid residues 216-227 according to EU numbering. In some embodiments, the second sequence or "lower hinge" sequence comprises amino acid residues 228-236 according to EU numbering.

[0081] The antibodies of the present disclosure, including those used to generate bispecific antibodies, anti-MAGE-A4 antigen-binding fragments, and / or chimeric antigen receptors, may in some embodiments be recombinant human antibodies. The term "recombinant human antibodies" as used herein is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (discussed further below), antibodies isolated from recombinant combinatorial human antibody libraries (discussed further below), antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means, including splicing of human immunoglobulin gene sequences to 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 are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals that are transgenic for human Ig sequences are used), thus improving the V H Area and V LThe amino acid sequence of the region is H Sequence and human germline V L These are sequences that are derived from and related to the human antibody germline repertoire but may not naturally occur in vivo within the human antibody germline repertoire.

[0082] Human antibodies can exist in two forms related to hinge heterogeneity. In the first form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, forming molecules of approximately 75-80 kDa consisting of covalently linked light and heavy chains (half antibodies). These forms have proven extremely difficult to separate, even after affinity purification.

[0083] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present disclosure provides a method for determining the frequency of occurrence of the second form in the hinge, C H 2nd Area or C H Antibodies with one or more mutations in three regions are included, which may be desirable, for example, in production to improve the yield of the desired antibody form.

[0084] The antibodies disclosed herein, including bispecific antibodies, may be isolated antibodies. An "isolated antibody," as used herein, refers to an antibody that has been identified, separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it naturally occurs or is naturally produced, is an "isolated antibody" for the purposes of this disclosure. An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. Thus, an isolated antibody also includes an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to human MAGE-A4, or human MAGE-A4 and human CD3, is substantially free of antibodies that specifically bind to antigens other than human MAGE-A4, or human MAGE-A4 and human CD3). However, as disclosed herein, an isolated antibody, such as one that specifically binds human MAGE-A4, may in some examples further specifically bind to one or more other MAGE-A4 related proteins or peptides. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0085] "Specifically binds" or similar terms means that an antibody (including a bispecific antibody) or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1×10 -6The specific binding may be characterized by a dissociation constant of 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, an isolated antibody that specifically binds to human MAGE-A4, or human MAGE-A4 and human CD3, may have cross-reactivity to other antigens, such as MAGE-A4 molecules and / or CD3 molecules from other species (orthologs). In the context of the present invention, a monospecific antibody that binds to human MAGE-A4 and one or more additional antigens, including MAGE-A4-related peptides, is considered to "specifically bind" to human MAGE-A4. In the context of the present invention, a multispecific (e.g., bispecific) antibody that binds to human MAGE-A4 and human CD3, as well as one or more additional antigens, is considered to "specifically bind" to human MAGE-A4 and human CD3.

[0086] Bispecific antibodies comprising an anti-MAGE-A4 specific binding region and an anti-CD3 specific binding region may be constructed using standard methods, where the anti-MAGE-A4 antigen binding region and the anti-CD3 antigen binding region each may comprise a different distinct HCVR paired with a common LCVR. In an exemplary bispecific antibody, the molecule was constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MAGE-A4 antibody, and a common light chain from an anti-MAGE-A4 antibody. In other cases, bispecific antibodies may be constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MAGE-A4 antibody, and a light chain from an anti-CD3 antibody, or antibody light chains that are promiscuous or known to pair effectively with a variety of heavy chain arms.

[0087] According to a particular embodiment of the invention, the anti-MAGE-A4xanti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a first antigen-binding region that specifically binds human MAGE-A4 and a second antigen-binding region that specifically binds human CD3, the first antigen-binding region comprising heavy chain CDRs A1-HCDR1, A1-HCDR2 and A1-HCDR3 comprising the amino acid sequences of SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8, respectively, and the second antigen-binding region comprising heavy chain CDRs A2-HCDR1, A2-HCDR2 and A2-HCDR3 comprising the amino acid sequences of SEQ ID NO:57 or SEQ ID NO:75, SEQ ID NO:59 or SEQ ID NO:77 and SEQ ID NO:61 or SEQ ID NO:79, respectively. According to a particular embodiment of the invention, the anti-MAGE-A4xanti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises common (to both the first antigen-binding region and the second antigen-binding region) light chain complementarity determining regions LCDR1-LCDR2-LCDR3, respectively. In one example, the common light chain complementarity determining region comprises the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16. In another example, the common light chain complementarity determining region comprises the amino acid sequences of SEQ ID NO: 65, SEQ ID NO: 14, and SEQ ID NO: 67.

[0088] In certain embodiments, the anti-MAGE-A4 x anti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a first antigen-binding region that specifically binds human MAGE-A4 and a second antigen-binding region that specifically binds human CD3, wherein the first antigen-binding region 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 region comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 73. In certain embodiments, the anti-MAGE-A4 x anti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a common 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, the anti-MAGE-A4 x anti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a first antigen-binding region comprising an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 2 / 10, and a second antigen-binding region comprising an 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, the anti-MAGE-A4 x anti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a first antigen-binding region comprising an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 83 / 10, and a second antigen-binding region comprising an 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, the anti-MAGE-A4 x anti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a first antigen-binding region comprising an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 107 / 115, and a second antigen-binding region comprising an 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, the anti-MAGE-A4 x anti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a first antigen-binding region comprising an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 2 / 63, and a second antigen-binding region comprising an 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, the anti-MAGE-A4x anti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a first antigen-binding region comprising an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 83 / 63, and a second antigen-binding region comprising an 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, the anti-MAGE-A4x anti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a first antigen-binding region comprising an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 107 / 63, and a second antigen-binding region comprising an 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-CD3 bispecific antibody comprises the HCVR / LCVR sequence pair as described above and a human IgG1 heavy chain constant region. In some embodiments the anti-MAGE-A4x anti-CD3 bispecific antibody comprises the HCVR / LCVR sequence pair above and a human IgG4 heavy chain constant region. In some embodiments the anti-MAGE-A4x anti-CD3 bispecific antibody comprises the HCVR / LCVR sequence pair above and a human IgG heavy chain constant region. In some embodiments the anti-MAGE-A4x anti-CD3 bispecific antibody comprises the HCVR / LCVR sequence pair above and a human IgG1 heavy chain constant region or an IgG4 heavy chain constant region.

[0089] The anti-MAGE-A4 antibodies or antigen-binding fragments thereof disclosed herein 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 regions compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody was derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce many antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof. In certain embodiments, the V H Region and / or V LAll framework and / or CDR residues within the region are mutated back to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., the mutated residue is found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or the mutated residue is found only in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, 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., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[0090] Anti-MAGE-A4 antibodies 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, anti-MAGE-A4 antibodies may have HCVR, LCVR and / or CDR amino acid sequences that have, for example, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 conservative amino acid substitutions relative to any of the HCVR, LCVR and / or CDR amino acid sequences described herein.

[0091] The term "antigenic determinant" 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 antigenic determinant. Thus, different antibodies may bind to different regions on the antigen and have different biological effects. Antigenic determinants may be either conformational or linear. Conformational antigenic determinants are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear antigenic determinants are those produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, antigenic determinants may include sugar, phosphoryl, or sulfonyl moieties on the antigen.

[0092] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or a fragment thereof, indicates that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is at least about 95% nucleotide sequence identity of the nucleotide bases, more preferably at least about 96%, 97%, 98%, 99%, 99.5%, or 99.9%, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, 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 method for determining a nucleotide sequence identity of at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77% relative to the sequence of SEQ ID NO:22 or SEQ ID NO:105, or a portion of SEQ ID NO:22 or SEQ ID NO:105 (e.g., an HCVR such as the sequence of SEQ ID NO:2, or SEQ ID NO:83, or an LCVR such as the sequence of SEQ ID NO:10, or a framework region of a polypeptide sequence such as those found in SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:22, or SEQ ID NO:105). , 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 the polypeptide of the present invention. In some embodiments, the disclosure provides polynucleic acids encoding such polypeptides.In some embodiments, the disclosure provides a method for the detection of a nucleotide 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 99%, at least 98%, at least 99%, at least 99%, at least 100%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 109, at least 108, at least 109, at least 109, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109 ... The present invention provides polynucleic acids comprising sequences which are 8%, 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 sequences of the present invention.

[0093] 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 (such as SEQ ID NO:55), or the sequence of SEQ ID NO:71, or a portion thereof (such as SEQ ID NO:63), or the sequence of SEQ ID NO:81, or a portion thereof (such as SEQ ID NO:73). In some embodiments, the disclosure provides polynucleic acids 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 (such as SEQ ID NO:54), or the sequence of SEQ ID NO:70, or a portion thereof (such as SEQ ID NO:62), or the sequence of SEQ ID NO:80, or a portion thereof (such as SEQ ID NO:72).

[0094] As applied to polypeptides, the term "substantial similarity" or "substantially similar" refers to two peptide sequences that share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the program GAP or the program BESTFIT, using predefined gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other in conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with 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 chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: 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 that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference.A "reasonably conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0095] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the best overlapping regions between the query and search sequences (Pearson (2000) (see above)). Sequences can also be compared using the Smith-Waterman homology search algorithm using an affine gap search with a gap opening penalty of 12, a gap extension penalty of 2, and a BLOSUM matrix of 62. Another preferred algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., 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.

[0096] 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 nucleic acid ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have changes in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications include, for example, substitution with one or more hydroxyl groups with halogens, alkyl groups, amine groups, and azide groups, or the sugar can be functionalized as an ether or ester. Additionally, the entire sugar moiety can be replaced with stereochemically and electronically similar structures, such as azasugars 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 substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Nucleic acids can be either single-stranded or double-stranded.

[0097] The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a binding region for a component present on a target cell, e.g., an antibody-based specificity for a desired antigen (e.g., a tumor antigen such as MAGE-A4), with an intracellular region that activates the T cell receptor to generate a chimeric protein that exhibits specific anti-target cell immune activity. In general, CARs consist of an extracellular single-chain antibody binding region (scFv) fused to the intracellular signaling region of the T cell antigen receptor complex zeta chain, and when expressed in T cells, have the ability to redirect antigen recognition based on the specificity of a monoclonal antibody.

[0098] The term "HLA" refers to the Human Leukocyte Antigen (HLA) system or complex, which is a complex of genes that encodes the Major Histocompatibility Complex (MHC) proteins in humans. These cell surface proteins are involved in regulating the immune system in humans. HLA corresponding to MHC class I (A, B, and C) presents peptides from within the cell. In the context of this application, a peptide may be "HLA-bound" if it is bound to an HLA system or complex. In some embodiments, the HLA-bound peptide is present on the surface of a cell.

[0099] The term "HLA-A" refers to a group of human leukocyte antigens (HLA) encoded by the HLA-A locus. HLA-A is one of the three major types of human MHC class I cell surface receptors. The receptor is a heterodimer, consisting of a heavy α chain and a small β chain. The α chain is encoded by the variant HLA-A gene, and the β chain (β2-microglobulin) is an invariant β2 microglobulin molecule.

[0100] The term "HLA-A2" refers to one particular class I major histocompatibility complex (MHC) allele group at the HLA-A locus. * The β chain is encoded by the β2-microglobulin or B2M locus.

[0101] 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 that may consist of chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acids. In some cases, vectors are capable of autonomous replication (epithelial vectors) and / or expression of nucleic acids to which they are linked (expression vectors). Many suitable vectors are known to those of skill in the art and are commercially available. Viral vectors include negative-stranded RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), positive-stranded RNA viruses such as rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), picornaviruses and alphaviruses, as well as double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma, mammalian C, B, D viruses, HTLV-BLV group, and lentivirus.

[0102] "Costimulatory domain" or "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by 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 (CD137) (SEQ ID NO: 29), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind CD83. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or its ligand, that is necessary for an efficient immune response.

[0103] "Costimulatory ligand" refers to 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 engagement of the TCR / CD3 complex with, e.g., a peptide-bearing MHC molecule. Costimulatory ligands may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Inducible Costimulatory Ligand (ICOS-L), Inter Cellular Adhesion Molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind B7-H3.

[0104] A "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up- or down-regulation of key molecules.

[0105] As used herein, the term "extracellular ligand binding region" refers to a ligand, e.g., an oligopeptide or polypeptide, capable of binding to a cell surface molecule. For example, the extracellular ligand binding region may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state (e.g., cancer). Examples of cell surface markers that may act as ligands include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells. The extracellular ligand binding region may include LCVR and HCVR regions (e.g., formatted as an scFv), optionally linked by a linker.

[0106] The term "subject" or "patient" as used herein includes all members of the animal kingdom, including non-human primates and humans. In one embodiment, the patient is a human with cancer (e.g., multiple myeloma or melanoma).

[0107] As used herein, the "signal transducing region" or "signaling region" of a CAR is involved in intracellular signal transduction following binding of the extracellular ligand binding region to a target, resulting in activation of immune cells and immune responses. In other words, the signaling region is involved in the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "signaling region" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Examples of signaling regions for use in CARs can be the cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence with the same function. In some cases, the signaling region includes two different classes of cytoplasmic signaling sequences, those that initiate antigen-dependent primary activation and those that act antigen-independently to provide secondary or costimulatory signals. The primary cytoplasmic signaling sequence may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). ITAMs are distinct signaling motifs found in the cytoplasmic tails of various receptors that act as binding sites for syk / zap70 class tyrosine kinases. Representative ITAMs include those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the signaling region of the CAR may comprise the CD3 zeta signaling region (SEQ ID NO: 30).

[0108] Chimeric antigen receptors (CARs) Chimeric antigen receptors (CARs) can redirect the specificity of T cells to antigens recognized by antibodies on the surface of cells (e.g., cancer cells), whether those antigens are expressed on the cell surface or expressed intracellularly and presented, for example, by HLA.

[0109] One aspect of the present disclosure includes a chimeric antigen receptor (CAR) specific for the MAGE-A4 antigen presented on the surface of a cell, such as a tumor cell. This presentation can be by HLA, such as HLA-A2. In one embodiment of the present disclosure, the CAR described herein includes an extracellular target-specific binding region, a transmembrane region, an intracellular signaling region (such as a signaling region derived from CD3 zeta or FcR gamma), and / or one or more costimulatory signaling regions derived from a costimulatory molecule, such as, but not limited to, 4-1BB. In one embodiment, the CAR includes a hinge or spacer region, such as a CD8 alpha hinge, between the extracellular binding region and the transmembrane region.

[0110] The binding region or extracellular region of a CAR provides the CAR with the ability to bind to a target antigen of interest. A binding region (e.g., a ligand-binding region or an antigen-binding region) can be any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and bind to a biomolecule (e.g., a cell surface receptor or a tumor protein, or a component thereof). A binding region includes naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partners for a biomolecule of interest. For example, as further described herein, a binding region can be the light and heavy chain variable regions of an antibody, or the light and heavy chain variable regions can be linked together in a single chain and in either orientation (e.g., VL-VH or VH-VL). Various assays are known for identifying binding regions of the present disclosure that specifically bind to a particular target, including Western blot, ELISA, flow cytometry, or surface plasmon resonance analysis (e.g., using BIACORE analysis). The target can be an antigen of clinical interest where it is desired to elicit an effector immune response that results in tumor killing. In one embodiment, the target antigen of the binding region 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 an HLA-A2-presented MAGE-A4 protein).

[0111] Exemplary ligand binding regions include antigen binding proteins, such as antigen binding fragments of antibodies, such as scFvs, scTCRs, extracellular regions of receptors, ligands for cell surface molecules / receptors or their receptor binding regions, and tumor binding proteins. In certain embodiments, the antigen binding region included in the CAR of the present disclosure can be a variable region (Fv), CDR, Fab, scFv, VH, VL, domain antibody variants (dAb), camelid antibodies (VHH), fibronectin 3 domain variants, ankyrin repeat variants, and other antigen-specific binding regions derived from other protein scaffolds.

[0112] In one embodiment, the binding region of the CAR is an anti-MAGE-A4 single chain antibody (scFv), which may be a murine, human, or humanized scFv. Single chain antibodies may be cloned from V-region genes of a hybridoma specific for a desired target. Techniques that may be used for cloning the variable heavy (VH) and variable light (VL) chains are described, for example, in Orlandi et al., PNAS, 1989;86:3833-3837. Thus, in certain embodiments, the binding region includes an antibody-derived binding region, but may also be a non-antibody-derived binding region. An antibody-derived binding region may be a fragment of an antibody, or a genetically engineered product of one or more antibody fragments, which fragments are involved in binding to an antigen.

[0113] In certain embodiments, the CAR of the present disclosure may include linkers between the various regions, added for proper spacing and conformation of the molecule. For example, there may be a linker between the binding regions VH or VL, which in one embodiment may be 1-20 amino acids long. In other embodiments, the linker between any of the regions of the chimeric antigen receptor may be 1-15 or 15 amino acids long. In this regard, 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 long. In further embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. Ranges inclusive of the numbers described herein are also included herein, e.g., linkers 10-30 amino acids long.

[0114] In certain embodiments, a linker suitable for use in the CAR described herein is a flexible linker. Suitable linkers can be easily selected and can be any of different lengths, 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 may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0115] Exemplary flexible linkers include glycine polymers (G), glycine-serine polymers (where n is at least an integer), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to function as neutral tethers between regions of fusion proteins such as the CARs described herein. Glycine has access to much more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). In general, one of skill in the art will recognize that CAR designs may include linkers that are all or partially flexible, such that the linker may include one or more moieties that provide a flexible linker and a less flexible structure to provide for the desired CAR structure. A particular linker is (G4S) n linker, where n=1-3. Another representative linker is provided as SEQ ID NO: 26. The linker can be between the LCVR and HCVR regions of the CAR, between the variable region (such as the HCVR) and the hinge region (such as the CD8α hinge), or both. For example, the present disclosure provides a CAR comprising a (G4S)3 linker between the LCVR and HCVR, and a (G4S)1 linker between the HCVR and the CD8α hinge.

[0116] The binding region of a CAR may be followed by a "spacer" or "hinge," which refers to a region that distances the antigen binding region from the effector cell surface to allow for proper cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). The hinge region of a CAR is generally between the transmembrane (TM) and the binding region. In certain embodiments, the hinge region is an immunoglobulin hinge region, which may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. Other representative hinge regions used in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8 alpha, CD4, CD28, and CD7, which may be wild-type hinge regions from these molecules or may be modified. In one embodiment, the hinge region comprises a CD8 alpha hinge (SEQ ID NO: 27).

[0117] A "transmembrane" region or domain is a portion of a CAR that anchors the extracellular binding moiety to the plasma membrane of an immune effector cell and facilitates binding of the binding moiety to a target antigen. The transmembrane region may be a CD3 zeta transmembrane region, but other transmembrane regions that may be employed include those obtained from CD8 alpha, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane region is the transmembrane region of CD137. In some embodiments, the transmembrane region comprises the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the transmembrane region is synthetic, in which case it will comprise primarily hydrophobic residues such as leucine and valine.

[0118] "Intracellular signaling region" or "signaling region" refers to a portion of a chimeric antigen receptor protein that is involved in transmitting the message of effective CAR binding to a target antigen to the interior of an immune effector cell to induce effector cell functions, such as cytotoxic activity, including activation, cytokine production, proliferation, and release of cytotoxic factors to a target cell to which the CAR is bound, or other cellular responses elicited by antigen binding to the extracellular CAR region. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be auxiliary or active, including, for example, cytolytic activity or secretion of cytokines. Thus, the term "intracellular signaling region" or "signaling region", as used interchangeably herein, refers to a portion of a protein that transmits an effector function signal and instructs a cell to perform a specialized function. Usually, the entire intracellular signaling region can be employed, but in many cases it is not necessary to use the entire region. To the extent that a truncated portion of the intracellular signaling region is used, such a truncated portion may be used instead of the entire region, so long as it transmits an effector function signal. The term intracellular signaling region is meant to include any truncated portion of the intracellular signaling region sufficient to transmit an effector function signal. The intracellular signaling region, also known as the "signaling domain," is typically derived from a portion of the human CD3 or FcRy chain.

[0119] It is well known that signals generated only through the T cell receptor are insufficient for the complete activation of T cells, and secondary or costimulatory signals are also required. Therefore, it can be said that the activation of T cells is mediated by two different classes of cytoplasmic signaling sequences, namely, those that initiate antigen-dependent primary activation through the T cell receptor (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). Cytoplasmic signaling sequences that act in a costimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0120] Examples of ITAMs comprising primary cytoplasmic signaling sequences that are particularly useful in the present disclosure include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one particular embodiment, the intracellular signaling region of the anti-MAGE-A4 CAR described herein is derived from CD3 zeta. In some embodiments, the signaling region comprises the amino acid sequence of SEQ ID NO:30.

[0121] As used herein, the term "costimulatory signaling region" or "costimulatory region" refers to a portion of a CAR that includes the intracellular region of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that, upon binding to an antigen, provides a second signal necessary for efficient activation and function of T lymphocytes. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and ligands that specifically bind to CD83. Thus, while the present disclosure provides representative costimulatory regions derived from CD3 zeta and 4-1BB, other costimulatory regions are contemplated for use in the CARs described herein. The inclusion of one or more costimulatory signaling regions may enhance the efficacy and proliferation of T cells expressing a CAR receptor. The intracellular signaling and costimulatory signaling regions may be linked in tandem to the carboxyl terminus of the transmembrane region in any order. In some embodiments, the costimulatory region comprises the amino acid sequence of SEQ ID NO:29.

[0122] Although scFv-based CARs engineered to contain signaling regions from CD3 or FcR gamma have been shown to deliver potent signals for T cell activation and effector function, they are not sufficient to induce signals that promote T cell survival and proliferation in the absence of concomitant costimulatory signals. Other CARs that include a binding region, hinge, transmembrane, and signaling region derived from CD3 zeta or FcR gamma, along with one or more costimulatory signaling regions (e.g., intracellular costimulatory regions derived from CD28, CD137, CD134, and CD278), can more effectively direct anti-tumor activity and increased cytokine secretion, lytic activity, survival, and proliferation in T cells expressing the CAR 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; Zhong et al., Molecular Therapy, 2010; 18:413-420; Carpenito et al., PNAS, 2009; 106:3360-3365).

[0123] In various embodiments, an anti-MAGE-A4 CAR of the present disclosure comprises (a) an anti-MAGE-A4 scFv as a binding region (e.g., an scFv having a binding region (e.g., CDR or variable region) from an anti-MAGE-A4 antibody identified in Table 1), (b) a hinge region from human CD8 alpha, (c) a human CD8 alpha transmembrane region, and (d) a human T cell receptor CD3 zeta chain (CD3) intracellular signaling region, and optionally one or more costimulatory signaling regions, e.g., 4-1BB. In one embodiment, the various protein regions are arranged from amino-terminus to carboxyl-terminus in the following order: binding region, hinge region, and transmembrane region. The intracellular signaling region and optional costimulatory signaling region are linked in tandem in any order to the carboxy terminus of the transmembrane to form a single chain chimeric polypeptide. In one embodiment, the nucleic acid construct encoding the anti-MAGE-A4 CAR is a chimeric nucleic acid molecule comprising various coding sequences, for example, a nucleic acid molecule comprising (5' to 3') the coding sequences of a human anti-MAGE-A4 scFv, a human CD8 alpha hinge, a human CD8 alpha transmembrane domain, and a CD3 zeta intracellular signaling domain. In another embodiment, the nucleic acid construct encoding the anti-MAGE-A4 CAR is a chimeric nucleic acid molecule comprising various coding sequences, for example, a nucleic acid molecule comprising (5' to 3') the coding sequences of a human anti-MAGE-A4 scFv, a human CD8 alpha hinge, a human CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta costimulatory domain.

[0124] In certain embodiments, the polynucleotide encoding the CAR described herein is inserted into a vector. A vector is a vehicle into which a polynucleotide encoding a protein can be covalently inserted to provide for the expression of the protein and / or cloning of the polynucleotide. Such vectors may also be referred to as "expression vectors". An isolated polynucleotide may be inserted into a vector using any suitable method known in the art, including, but not limited to, digesting the vector with an appropriate restriction enzyme and then ligating with the isolated polynucleotide having a matching restriction end. An expression vector may have the ability to incorporate and express heterologous or modified nucleic acid sequences that code for at least a portion of a gene product that can be transcribed in a cell. In most cases, the RNA molecule is translated into a protein. An expression vector may contain various control sequences, which refer to nucleic acid sequences necessary for the transcription, and possibly translation, of an operably linked coding sequence in a particular host organism. In addition to control sequences that regulate transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well, as discussed below. An expression vector may contain additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, a human cell for expression and a prokaryotic host for cloning and amplification.

[0125] The expression vector may have the necessary 5' upstream and 3' downstream regulatory elements, such as promoter sequences, such as CMV, PGK and EF1 alpha promoters, ribosome recognition and binding TATA boxes, and 3'UTR AAUAAA transcription termination sequences for efficient gene transcription and translation in the respective host cells. Other suitable promoters include Simian Virus 40 (SV40) early promoter, Mouse Mammary Tumor Virus (MMTV), HIV LTR promoter, MoMuLV promoter, Avian Leukosis Virus promoter, EBV immediate early promoter, and Rous Sarcoma Virus promoter constitutive 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, inducible promoters are also contemplated as part of the vector expressing the chimeric antigen receptor. This provides a molecular switch that can turn on or off the expression of the polynucleotide sequence of interest. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter.

[0126] Expression vectors may have additional sequences such as 6x-histidine, c-Myc, and FLAG tags that are incorporated into the expressed CAR. Thus, expression vectors may be engineered to include 5' and 3' untranslated regulatory sequences, which may function as enhancer sequences, promoter regions, and / or terminator sequences that may facilitate or enhance efficient transcription of the nucleic acid of interest carried on the expression vector. Expression vectors may also be engineered for replication and / or expression functionality (e.g., transcription and translation) in a particular cell type, cell location, or tissue type. Expression vectors may include selectable markers for maintenance of the vector in the host or recipient cell.

[0127] In various embodiments, the vector is a plasmid, an autonomously replicating sequence, and a transposable element. Additional representative vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of animal virus categories useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). Examples of expression vectors are Lenti-X™ Bicistronic Expression System (Neo) vector (Clontrch), PClneo vector (Promega) for expression in mammalian cells, pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequence of the CAR disclosed herein can be ligated into such expression vectors for expression of chimeric proteins in mammalian cells.

[0128] In certain embodiments, the nucleic acid encoding the CAR of the present disclosure is provided in a viral vector. The viral vector may be derived from a retrovirus, lentivirus, or foamy virus. As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. The viral vector may contain coding sequences for various chimeric proteins described herein in place of non-essential viral genes. The vectors and / or particles may be utilized for the purpose of transferring DNA, RNA, or other nucleic acids into cells either in vitro or in vivo. Many forms of viral vectors are well known in the art.

[0129] In certain embodiments, the viral vector comprising the coding sequence of the CAR described herein is a retroviral vector or a lentiviral vector. The term "retroviral vector" refers to a vector that comprises structural and functional genetic elements that are primarily derived from retroviruses. The term "lentiviral vector" refers to a vector that comprises structural and functional genetic elements outside the LTR that are primarily derived from lentiviruses.

[0130] Retroviral vectors as used herein may be derived from any well-known retrovirus (e.g., c-type retroviruses such as Moloney Murine Sarcoma Virus (MoMSV), Harvey Murine Sarcoma Virus (HaMuSV), Murine Mammary Tumor Virus (MuMTV), Gibbon ape Leukemia Virus (GaLV), Feline Leukemia Virus (FLV), Spumavirus, Friend, Murine Stem Cell Virus (MSCV), and Rous Sarcoma Virus (RSV)). "Retrovirus" of the present disclosure also includes the lentivirus family of retroviruses, such as Human T-cell Leukemia Virus, HTLV-1 and HTLV-2, as well 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.

[0131] As used herein, lentiviral vector refers to a vector derived from lentivirus, a group (or genus) of retroviruses that cause slowly progressive diseases. Viruses in this group include HIV (Human Immunodeficiency Virus, including HIV type 1 and HIV type 2), visna-maedi, caprine arthritis-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 achieved using the methods of 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).

[0132] Retroviral vectors (i.e., both lentiviral and non-lentiviral) for use in the present disclosure can be formed using standard cloning techniques by combining the desired DNA sequences in the order and orientation described herein (Current Protocols in Molecular Biology, Ausubel, FM et al. (eds.) 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 85:3014-3018, and others). 87:6141-6145,Huber et al.(1991)Proc.Natl.Acad.Sci.USA 88:8039-8043,Ferry et al.(1991)Proc.Natl.Acad.Sci.USA 88:8377-8381,Chowdhury et al.(1991)Science 254:1802-1805;van Beusechem et al.(1992)Proc.Natl.Acad.Sci.USA 89:7640-7644;Kay et al.(1992)Human Gene Therapy 3:641-647;Dai et al.(1992)Proc.Natl.Acad.Sci.USA 89:10892-10895,Hwu et (1993) J. Immunol 150:4104-4115; U.S. Patent Nos. 4,868,116, 4,980,286, PCT International Publication Nos. 89 / 07136, 89 / 02468, 89 / 05345, and 92 / 07573).

[0133] Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for use in forming vectors include genomic RNA and cDNA, available from commercial sources including, for example, the American Type Culture Collection (ATCC), Rockville, Md. Sequences can also be chemically synthesized.

[0134] For the expression of anti-MAGE-A4 CAR, a vector may be introduced into a host cell to allow expression of the polypeptide in the host cell. The expression vector 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 appropriately selected by those skilled in the art, as described herein. For example, a promoter sequence may be selected to promote transcription of a polynucleotide in the vector. Suitable promoter sequences include but are not limited to T7 promoter, T3 promoter, SP6 promoter, beta actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. An enhancer sequence may be selected to enhance transcription of a polynucleotide. A selectable marker may be selected to allow selection of host cells into which the vector has been inserted from those that are not, for example, a selectable marker may be a gene that confers antibiotic resistance. A signal sequence may be selected to allow the expressed polypeptide to be transported out of the host cell.

[0135] For cloning of polynucleotides, a vector may be introduced into a host cell (isolated host cell) to allow the vector to replicate itself, thereby amplifying copies of the polynucleotide contained therein. Cloning vectors generally contain sequence components, including, but not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements may be appropriately selected by those skilled in the art. For example, an origin of replication may be selected to promote the autonomous replication of the vector in the host cell.

[0136] In certain embodiments, the disclosure provides an isolated host cell comprising a vector provided herein. A host cell comprising a vector may be useful in expressing or cloning a polynucleotide contained in the vector. Suitable host cells may include, but are not limited to, higher eukaryotic cells, such as prokaryotic cells, fungal cells, yeast cells, or mammalian cells. Prokaryotic cells suitable for this purpose include eubacteria, such as gram-negative or gram-positive organisms, e.g., Enterobacter, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, Examples of suitable microbial organisms include, but are not limited to, Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

[0137] The CAR of the present disclosure may be introduced into a host cell using transfection and / or transduction techniques well known in the art. As used herein, the terms "transfection" and "transduction" refer to the process by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid may be integrated into the DNA of the host cell or maintained extrachromosomally. The nucleic acid may be maintained transiently or may be stably introduced. Transfection may be achieved by various means well 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 biolistic methods. Transduction refers to the delivery of genes using a viral or retroviral vector by viral infection rather than by transfection. In certain embodiments, retroviral vectors are transduced by packaging the vector into a viral particle prior to contacting the cell. For example, a nucleic acid encoding an anti-MAGE-A4 CAR carried by a retroviral vector can be transduced into cells via infection and proviral integration.

[0138] As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of extra 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 "transformed cell" are used interchangeably.

[0139] In particular, the CARs of the present disclosure are introduced and expressed in immune effector cells to redirect their specificity to a target antigen of interest, e.g., malignant MAGE-A4-expressing cells, such as malignant cells that present MAGE-A4 in conjunction with HLA-A2.

[0140] The present disclosure provides a method for generating 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 tumor cells expressing MAGE-A4, 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 ex vivo. Such cells may then be directly re-administered to the individual. In further embodiments, the immune effector cells are first activated and stimulated to expand ex vivo before being genetically modified to express a CAR. In this regard, the immune effector cells may be cultured before or after being genetically modified (i.e., transduced or transfected to express a CAR as described herein).

[0141] Prior to ex vivo manipulation or genetic modification of immune effector cells as described herein, a source of cells may be obtained from a subject. In particular, immune effector cells for use with the CARs described herein include T cells. T cells may be obtained from many sources, such as peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic matter, tissue from a site of infection, ascites, pleural effusion, splenic tissue, tumors, etc. In certain embodiments, T cells may be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL separation. In one embodiment, cells from an individual's circulating blood may be obtained by apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for further processing. In one embodiment of the present disclosure, the cells are washed with PBS. In alternative embodiments, the washed solution may lack calcium, lack magnesium, or lack many, if not all, divalent cations. As will be appreciated by those of skill in the art, the washing step may be accomplished by methods well known to those of skill in the art, such as using a semi-automated flow-through centrifuge. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solutions with or without buffers. In certain embodiments, undesirable components of the apheresis sample may be removed in the culture medium in which the cells are directly resuspended.

[0142] In certain embodiments, T cells are isolated from Peripheral Blood Mononuclear Cells (PBMCs) by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, may be further isolated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection may be achieved with a combination of antibodies directed against surface markers unique to the negatively selected cells. One method for use herein is negative magnetic immunoadhesion using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells, or cell sorting and / or selection by flow cytometry. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate cell populations of interest for use in the present disclosure.

[0143] PBMCs may be used directly for genetic modification with CARs using the methods described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes may be sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion. CD8+ cells may be obtained by using standard methods. In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, expression of phenotypic markers of central memory TCMs includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, 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 are positive for granzyme B and perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

[0144] In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+ CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L positive and CD45RO positive. In some embodiments, effector CD4+ cells are CD62L negative and CD45RO negative.

[0145] Immune effector cells such as T cells can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of precursor cells) in vitro before being genetically modified. In another embodiment, immune effector cells such as T cells are genetically modified with a chimeric antigen receptor as described herein (e.g., transduced with a viral vector containing a nucleic acid encoding a CAR) and then activated and expanded in vitro. Methods for activating and expanding T cells are well known in the art and are described, for example, in U.S. Pat. Nos. 6,905,874, 6,867,041, 6,797,514, and PCT Publication WO 2012079000. In general, such methods include contacting PBMCs or isolated T cells with stimulatory and co-stimulatory agents, typically anti-CD3 and anti-CD28 antibodies bound to beads or other surfaces, in a medium containing appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies bound to the same bead function as "surrogate" antigen presenting cells (APCs). In other embodiments, T cells may be activated and stimulated with feeder cells and appropriate antibodies and cytokines to expand using methods such as those described in U.S. Patent Nos. 6,040,177, 5,827,642, and WO2012129514.

[0146] The present disclosure provides a population of modified immune effector cells for the treatment of patients having malignant tumors caused by tumors expressing MAGE-A4, such as multiple myeloma or melanoma, wherein the modified immune effector cells comprise an anti-MAGE-A4 CAR as disclosed herein.

[0147] The CAR-expressing immune effector cells prepared as described herein can be utilized in methods and compositions for adoptive immunotherapy according to well-known techniques, or variations thereof that will be apparent to those of skill in the art based on this disclosure. See, e.g., U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al. See also U.S. Patent No. 4,690,915 to Rosenberg.

[0148] In some embodiments, the cells are formulated by first harvesting them from their culture medium, then washing and concentrating the cells in a medium and container system (a "pharmaceutical acceptable" carrier) suitable for administration in a therapeutically effective amount. A suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), although 5% dextrose or Ringer's lactate in water can also be utilized. The infusion medium can be supplemented with human serum albumin.

[0149] A therapeutically effective amount of cells in the composition is at least 2 cells (e.g., at least one CD8+ central memory T cell and at least one CD4+ helper T cell subset), or more typically, at least 10 2 More than 10 cells 6 Up to 10 8 Cells or 10 9 up to or including 10 cells 10 The number of cells will depend on the end use for which the composition is intended, as well as the type of cells contained therein.

[0150] The cells may be autologous or xenogeneic to the patient undergoing therapy. Optionally, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance induction of an immune response.

[0151] The CAR-expressing immune effector cell population of the present disclosure may be administered as a pharmaceutical composition, alone or in combination with other components such as diluents and / or IL-2 or other cytokines or cell populations. Briefly, a pharmaceutical composition of the present disclosure may comprise a CAR-expressing immune effector cell population, such as T cells, described herein, in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents or excipients. Such compositions may include a buffer, such as neutral buffered saline, phosphate buffered saline, carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides or amino acids, such as glycine, antioxidants, chelating agents, such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present disclosure are preferably formulated for intravenous administration.

[0152] By administering T cells expressing the CARs described herein using the methods described herein or other methods known in the art, the anti-tumor immune response induced in the subject may include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses capable of killing infected cells. A humoral immune response mediated primarily by helper T cells capable of activating B cells to result in antibody production may also be induced. To analyze the type of immune response induced by the compositions of the present disclosure, various techniques may be used, which are well described in the art, such as Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0153] Thus, the present disclosure provides a method of treating an individual diagnosed with, suspected of having, or at risk of developing a malignancy characterized at least in part by expression of MAGE-A4 by cancer cells (e.g., solid tumor cells that express MAGE-A4), comprising administering to the individual a therapeutically effective amount of immune effector cells expressing a CAR as described herein.

[0154] In one embodiment, the present disclosure provides a method of treating a subject diagnosed with a cancer that expresses MAGE-A4, comprising depleting immune effector cells from a subject diagnosed with a cancer that expresses MAGE-A4, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor of the present disclosure, thereby producing a modified immune effector cell population, and administering the modified immune effector cell population to the same subject. In one embodiment, the immune effector cells comprise T cells.

[0155] Methods for administering the cell compositions described herein include any method effective to result in the reintroduction of ex vivo genetically modified immune effector cells, either directly expressing a CAR of the present disclosure in the subject, or the reintroduction of genetically modified precursor cells of immune effector cells that differentiate into mature immune effector cells expressing a CAR when introduced into a subject. One method involves 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.

[0156] Binding characteristics of chimeric antigen receptors and corresponding antibodies As used herein, the term "binding" is considered in the context of the binding of a chimeric antigen receptor or corresponding antibody (or bispecific antibody) to a given antigen, such as, for example, a cell surface protein or fragment thereof (or to an antigen bound to a cell surface protein, such as an HLA molecule). Binding typically refers to the interaction or association between, at a minimum, two entities or molecular structures, such as an antigen-binding region:antigen interaction.

[0157] For example, binding affinities, as determined by surface plasmon resonance (SPR) techniques, for example on a BIAcore 3000 instrument, using an antigen as the ligand and an antibody or chimeric antigen receptor as the analyte (or antiligand), are typically around 10 -8 M or less, about 10 -9 M or less, about 10 -7 K below M D Cell-based binding strategies such as fluorescence-activated cell sorting (FACS) binding assays are also routinely used, and FACS data correlate well 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).

[0158] Thus, a chimeric antigen receptor or corresponding antibody (or bispecific antibody) of the present disclosure has a K that is at least 10-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). D As described herein, the chimeric antigen receptor or corresponding antibody of the present disclosure can bind to an HLA-presented MAGE-A4 antigen, e.g., HLA-A2-presented MAGE-A4 antigen. According to the present disclosure, the chimeric antigen receptor binds to a given antigen or cell surface molecule (receptor) with an affinity corresponding to a K value that is equal to or less than 10-fold lower than that of a non-specific antigen. D The affinity of a chimeric antigen receptor or corresponding antibody having a value may be considered as undetectable binding.

[0159] "K D The term "M" refers to the dissociation equilibrium constant of a particular antigen-binding region:antigen interaction, or the dissociation equilibrium constant of the corresponding antibody to the antigen. D There is an inverse relationship between the binding affinity and the K D The smaller the value, the higher the affinity, i.e., the stronger. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, i.e., a smaller K D Conversely, the term "lower affinity" or "weaker affinity" refers to a lower ability to form an interaction, i.e., a larger K D In some contexts, the binding affinity (or K) of a particular molecule (e.g., a chimeric antigen receptor or a corresponding antibody) to its interacting partner molecule (e.g., antigen X) may be expressed as D ) relative to the binding affinity of a molecule (e.g., a chimeric antigen receptor or a corresponding antibody) to another interacting partner molecule (e.g., antigen Y), indicates a larger K D A smaller K value (lower or weaker affinity) D The binding affinity may be expressed as a binding ratio determined by dividing by (the higher or stronger affinity), e.g., 5-fold or 10-fold higher binding affinity in some cases.

[0160] "k dThe term "(sec-1 or 1 / sec) refers to the dissociation rate constant of a particular antigen-binding region:antigen interaction, or the dissociation rate constant of a chimeric antigen receptor or corresponding antibody. Its value is k off Also called the value.

[0161] "k a The term "(M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antigen binding region:antigen interaction, or the association rate constant of a chimeric antigen receptor or corresponding antibody.

[0162] "K A The term "(M-1 or 1 / M)" refers to the pairing equilibrium constant of a particular antigen-binding region:antigen interaction, or the pairing equilibrium constant of a chimeric antigen receptor or corresponding antibody. The pairing equilibrium constant is k a k d It is obtained by dividing by.

[0163] "EC50" or "EC 50 The term "half-maximal effective concentration" refers to the concentration of antibody or chimeric antigen receptor that induces a response halfway between the baseline and maximum after a specified exposure time. EC 50 essentially represents the concentration of a chimeric antigen receptor or antibody (e.g., a bispecific antibody) at which 50% of its maximal effect is observed. In certain embodiments, the EC 50 The value is equal to the concentration of a chimeric antigen receptor or corresponding antibody of the present disclosure that gives half-maximal binding to cells expressing an antigen (e.g., a tumor-associated antigen such as MAGE-A4) as determined, for example, by FACS binding assays and / or T cell reporter / antigen presenting cell (APC) bioassays. Thus, reduced or weak binding is an EC 50 An increase in the α-amyloid level or half-maximal effective concentration is observed.

[0164] In one embodiment, the reduction in binding is an EC2 concentration that allows half-maximal binding to target cells. 50 This may be defined as an increase in bispecific antibody concentration, antigen-binding fragment concentration, chimeric antigen receptor concentration, or the corresponding antibody concentration.

[0165] Sequence variants of antibodies and chimeric antigen receptors The antibodies, antigen-binding fragments, and chimeric antigen receptors of the present disclosure may contain one or more amino acid substitutions, insertions, and / or deletions in the framework regions (FR) and / or complementarity determining region (CDR) regions of the heavy and light chain variable regions compared to the corresponding germline sequences from which the respective antigen-binding regions of the corresponding antibodies are derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The antibodies, antigen-binding fragments, and chimeric antigen receptors of the present disclosure may contain antigen-binding regions derived from any of the representative CDR or variable region amino acid sequences disclosed herein, where one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the corresponding antibody is derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (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 skill in the art can readily generate many antibodies that contain one or more individual germline mutations or combinations thereof. H Region and / or V LAll framework and / or CDR residues within the region are mutated back to the residues found in the original germline sequence from which the antigen binding region was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., the mutated residues are found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or the mutated residues are found only in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antigen binding region was originally derived). Furthermore, the antigen binding region may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues in a different germline sequence.

[0166] Biological characterization of chimeric antigen receptors and corresponding antibodies The present disclosure provides antibodies (including bispecific antibodies), antigen-binding fragments, and chimeric antigen receptors (CARs) having an antigen-binding region derived from an antibody that binds to human MAGE-A4 or human MAGE-A4 and CD3. As described herein, bispecific antibodies can be used to generate CARs with similar properties to bispecific antibodies. Thus, the properties described herein and attributed to bispecific antibodies apply to CARs as well. For example, the present disclosure provides antibodies that have an EC of less than 2 nM as assessed by a flow cytometry-based peptide pulse assay described below with respect to Example 2. 50As another example, the disclosure provides an anti-MAGE-A4 antibody that binds to one or more MAGE-A4 related peptides with a signal to noise ratio ranging from about 5 to greater than 300, as assessed by the flow cytometry based peptide pulse assay of Example 2 and detailed in Example 3. As another example, the disclosure provides an anti-MAGE-A4 x anti-CD3 bispecific antibody that binds to one or more MAGE-A4 related peptides with a signal to noise ratio ranging from about 5 to greater than 240, as assessed by the flow cytometry based peptide pulse assay of Example 2 and detailed in Example 3.

[0167] The present disclosure also provides anti-MAGE-A4 x anti-CD3 bispecific antibodies that exhibit substantial activity in a T cell reporter / antigen presenting cell (APC) bioassay, as detailed below in Example 4. Specifically, the anti-MAGE-A4 x anti-CD3 bispecific antibodies of the present disclosure inhibit NF-κB-dependent T cell (e.g., Jurkat cells) gene transcription / translation, as measured by luciferase expression, in the presence of APCs expressing MAGE-A4 and HLA-A2 (e.g., IM9 cells and U266B1 cells), with an EC of less than 3.2 nM (U266B1 cells) or less than 0.75 nM. 50 Inclusion of an anti-CD28 antibody in a T cell reporter / APC bioassay can modulate the activity of an anti-MAGE-A4 x anti-CD3 bispecific antibody as a function of endogenous levels of CD80 and CD86, as detailed in Example 4 below.

[0168] The present disclosure also provides anti-MAGE-A4 x anti-CD3 bispecific antibodies that exhibit substantial activity in the above-described T cell reporter / antigen presenting cell (APC) bioassays using one or more MAGE-A4 related peptides, as detailed below in Example 5. Specifically, the anti-MAGE-A4 x anti-CD3 bispecific antibodies are capable of binding to luciferase reporter-bearing T cells and AOX1 795-803 Subnanomolar EC 50values ​​(e.g., 2.10E-11M for bispecific antibodies with high affinity CD3 arms and 3.30E-10M for bispecific antibodies with intermediate affinity CD3 arms). Furthermore, anti-MAGE-A4 x anti-CD3 bispecific antibodies stimulated T cells with luciferase reporter and SHTN1 198-206 Nanomolar EC 50 values ​​(e.g., 1.30E-9M for a bispecific antibody with a high affinity CD3 arm and 3.5E-9M for a bispecific antibody with a medium affinity CD3 arm).

[0169] The present disclosure also relates to the use of MAGE-A4 presenting tumor cells (e.g., HLA-A2 positive IM9 cells) as described in detail in Example 6 below. 286-294 The present disclosure provides MAGE-A4xanti-CD3 bispecific antibodies, as assessed by an imaging-based multiplexed primary T cell killing assay, that allow for the directing of T cells (e.g., CD8+ T cells isolated from human PBMCs) responding to a specific antigen (antigen). Specifically, certain anti-MAGE-A4xanti-CD3 bispecific antibodies of the present disclosure are capable of reducing tumor cell viability due to antibody-dependent T cell-mediated responses to less than 50% (e.g., 38%) at concentrations less than 10 nM (e.g., 6.6 nM). This reduction in viability can be at least partially prevented by co-culturing the anti-MAGE-A4xanti-CD3 bispecific antibodies with an anti-CD28 antibody in a situation where the tumor cells endogenously express CD80 and CD86, which may be at least partially due to the anti-CD28 antibody blocking the interaction of CD28 with CD80 and CD86.

[0170] The present disclosure also relates to the use of MAGE-A4 presenting tumor cells (e.g., HLA-A2 positive IM9 cells) as described in detail in Example 7 below. 286-294The present disclosure provides anti-MAGE-A4 x anti-CD3 bispecific antibodies and T cells (e.g., CD8+ T cells isolated from human PBMCs) capable of stimulating cytokine release when cultured in the presence of a tumor antigen (e.g., CD8+ T cells isolated from human PBMCs). Specifically, the present disclosure provides anti-MAGE-A4 x anti-CD3 bispecific antibodies capable of stimulating greater than two-fold cytokine release (e.g., IL2, IFN-γ) compared to cytokine release under conditions in which the tumor and T cells lack the anti-MAGE-A4 x anti-CD3 bispecific antibodies.

[0171] The present disclosure also provides a chimeric antigen receptor having an antigen-binding region derived from a corresponding antibody that specifically binds to a human cell line expressing endogenous MAGE-A4 as determined by a FACS binding assay.

[0172] The present disclosure also provides engineered cells that express a MAGE-A4 specific chimeric antigen receptor that (i) is activated by cells expressing MAGE-A4 (see Example 8) and / or (ii) exhibits inhibition of tumor growth in immunocompromised mice bearing human multiple myeloma or melanoma xenografts.

[0173] Preparation of antigen-binding regions Antigen-binding regions of 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 making such antibodies are well known in the art. For example, one or more of the heavy and / or light chains can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generation technique), high affinity chimeric antibodies against a particular antigen (e.g., MAGE-A4) are first isolated with human variable regions and mouse constant regions. The antibodies are characterized and selected for desirable characteristics including affinity, neutralization, selectivity, antigenic determinants, etc. As discussed herein, these human variable regions (or CDRs) can then be incorporated into the antigen-binding region of the chimeric antigen receptor. In other examples, two different antigens (e.g., anti-MAGE-A4 and anti-CD3) can be appropriately positioned relative to each other to generate a bispecific antigen-binding molecule (e.g., an antibody, CAR, or antigen-binding fragment of either) of the disclosure by routine methods. In certain embodiments, one or more individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecule of the disclosure are derived from a chimeric antibody, a humanized antibody, or a fully humanized antibody.

[0174] Polynucleotides and Vectors The present disclosure also provides polynucleotides and vectors encoding the antibodies (or portions thereof) and chimeric antigen receptors discussed herein.

[0175] 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 such as a baculovirus vector for transfection of an insect host cell, or a lentivirus or adeno-associated virus for transfection of a mammalian host cell).

[0176] In various embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:21 or comprises a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide sequence of SEQ ID NO:22. In other embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:104 or comprises a nucleic acid molecule comprising a nucleotide sequence that encodes a 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 comprises a nucleic acid molecule comprising a nucleotide sequence that encodes 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 a nucleotide sequence of SEQ ID NO:9 or comprises a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide sequence of SEQ ID NO:10. In various embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:17 or comprises a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide sequence of SEQ ID NO:18. In various embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:19 or comprises a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide sequence of SEQ ID NO:20. In various embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:54, or comprises a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide sequence of SEQ ID NO:55. In various embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:62, or comprises a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide sequence of SEQ ID NO:63. In various embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:68, or comprises a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide sequence of SEQ ID NO:69. In various embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:70, or comprises a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide sequence of SEQ ID NO:71.In various embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 72, or comprises 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 a nucleotide sequence of SEQ ID NO: 80, or comprises a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 81. With respect to the above SEQ ID NOs, it is within the scope of this disclosure that the polynucleotide and / or vector encompasses any subsequence thereof, such as one or more HCDRs, LCDRs, etc. 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.

[0177] Methods for engineering immune cells to express chimeric antigen receptors The present disclosure provides a method 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.

[0178] 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, the immune cells are used for immunotherapy (e.g., treating cancer).

[0179] The present disclosure provides a method for genetically modifying immune cells to make them more suitable for allogeneic transplantation. According to a first aspect, immune cells can be made allogeneic by inactivating at least one gene expressing one or more components of the T cell receptor (TCR), for example as described in PCT Publication WO 2013 / 176915, which can be combined with inactivation of genes encoding or regulating the expression of HLA or β2m proteins. Thus, the risk of graft-versus-host syndrome and graft rejection is significantly reduced. According to a further aspect of the present disclosure, immune cells can be further engineered to make them more active or limit attrition by inactivating genes encoding proteins acting as "immune checkpoints" that act as regulators of T cell activation, such as PD1 or CTLA-4.

[0180] Engineered immune cells The present disclosure also provides an immune cell (e.g., an engineered immune cell) comprising a chimeric antigen receptor described herein. In some cases, the immune cell is an immune effector cell. In some cases, the immune cell is a T cell. In some cases, the immune cell is a T lymphocyte selected from an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte. In some cases, the immune cell is a CD8+ cytotoxic T lymphocyte.

[0181] In some embodiments, the engineered immune cells are human T cells that comprise a chimeric antigen receptor comprising, from N-terminus to C-terminus, (a) an extracellular ligand binding region comprising an anti-MAGE-A4 single chain variable fragment (scFv) region comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR); (b) a hinge; (c) a transmembrane region; and (d) a cytoplasmic region comprising a costimulatory region and a signaling region.

[0182] In some embodiments, the scFv region of the engineered human T cell comprises an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO:2 / 10. Optionally, the hinge comprises the amino acid sequence of SEQ ID NO:27. Optionally, the transmembrane region comprises the amino acid sequence of SEQ ID NO:28. Optionally, the costimulatory region is a 4-1BB costimulatory region. Optionally, the 4-1BB costimulatory region comprises the amino acid sequence of SEQ ID NO:29. Optionally, the signaling region is a CD3 zeta signaling region. Optionally, the CD3 zeta signaling region comprises the amino acid sequence of SEQ ID NO:30.

[0183] 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.

[0184] biological equivalent The present disclosure provides antibodies and chimeric antigen receptors, as well as engineered cells expressing chimeric antigen receptors, that have amino acid sequences that differ from those of the representative molecules disclosed herein, but that retain the ability to bind MAGE-A4 (and CD3 in the case of bispecific antibodies), activate immune cells expressing the chimeric antigen receptor in the presence of cells expressing MAGE-A4, or inhibit the growth or proliferation of tumor cells expressing MAGE-A4. Such mutant molecules may contain one or more amino acid additions, deletions, or substitutions when compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antigen binding molecules.

[0185] In one embodiment, two engineered immune cells expressing a chimeric antigen receptor of the present disclosure, or two antigen binding proteins of the present disclosure, are biologically equivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0186] In one embodiment, two engineered immune cells or two antigen binding proteins are bioequivalent if a patient can be switched one or more times compared to therapy sustained without switching between the reference product and the biological product without a predicted increased risk of adverse effects, including clinically significant changes in immunogenicity or diminished efficacy.

[0187] In one embodiment, two engineered immune cells, or two antigen binding proteins, are biologically equivalent if they both operate by a common mechanism or mode of action for a condition or condition of use, to the extent that such mechanism is well known.

[0188] In one embodiment, two antigen binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical options that exhibit no significant difference in absorption rate and extent when administered at the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antigen binding proteins will be considered equivalents or pharmaceutical options if their extent of absorption is equivalent but their absorption rate is not, and such differences in absorption rate are considered to be intentional and reflected in the label, are not essential to achieving effective body drug concentrations for, e.g., long-term use, and are not considered medically significant for the particular drug product tested.

[0189] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, for example, (a) in vivo studies in humans or other mammals where the concentration of engineered cells is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro studies that are correlated with and reasonably predictive of human in vivo bioavailability data, (c) in vivo studies in humans or other mammals that measure the relevant acute pharmacological effect of the engineered cells (or their targets) as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the engineered cells.

[0190] Biologically equivalent variants of the representative engineered cells described herein may be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity.

[0191] Biologically equivalent variants of the representative bispecific antigen-binding molecules shown herein may be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity may be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, biologically equivalent antigen-binding proteins may include variants of the representative bispecific antigen-binding molecules described herein that contain amino acid changes that modify the glycosylation characteristics of the molecule, for example, mutations that eliminate or remove glycosylation.

[0192] Species selectivity and species cross-reactivity According to certain embodiments of the present disclosure, antigen binding regions are provided that bind to human MAGE-A4 but not to MAGE-A4 from other species. Also provided are anti-MAGE-A4 antigen binding regions that bind to human AOX1 but not to AOX1 from other species. Also provided are anti-MAGE-A4 antigen binding regions that bind to human SHTN1 but not to SHTN1 from other species. The present disclosure also provides antigen binding regions that bind to human MAGE-A4 and MAGE-A4 from one or more non-human species. The present disclosure also provides anti-MAGE-A4 antigen binding regions that bind to human AOX1 and AOX1 from one or more non-human species. The present disclosure also provides anti-MAGE-A4 antigen binding regions that bind to human SHTN1 and SHTN1 from other species. In some embodiments, the antigen binding regions of the present disclosure bind to MAGE-A4 286-294, and AOX1 795-803, and / or SHTN1 198-206. In some embodiments, MAGE-A4 and / or AOX1 and / or SHTN1 (e.g., MAGE-A4 286-294 or AOX1795-803 or SHTN1 198-206) to which the antigen binding region binds is presented on the surface of a cell by HLA, e.g., HLA-A2.

[0193] According to certain exemplary embodiments of the present disclosure, antigen-binding regions are provided which bind human MAGE-A4 and / or MAGE-A4 related peptides and may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey or chimpanzee MAGE-A4 and / or MAGE-A4 related peptides, as the case may be. Furthermore, binding to MAGE-A4 and / or MAGE-A4 related peptides may be in the context of MHC-presented MAGE-A4 (or MAGE-A4 related peptide), such as HLA-presented MAGE-A4. An exemplary HLA-presented MAGE-A4 is human MAGE-A4 bound by HLA-A2.

[0194] Activation and proliferation of engineered immune cells Whether prior to or after genetic modification of the engineered cells (e.g., T cells), immune cells, particularly T cells, of the present disclosure may generally be engineered to express any of the antigen-binding proteins described in, for example, U.S. Pat. Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,410, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,410, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,410, 6,534,055, 6,905,680, 6,692,964, 6,534,055, 6,534,055, 6,905,680, 6,534,05 ... The T cells may be further activated and expanded using methods described in U.S. Patent Application Publication Nos. 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. 2006 / 0121005. T cells may be expanded in vitro or in vivo.

[0195] Generally, the T cells of the present disclosure may be proliferated and generated an activation signal for the T cells by contact with an agent that stimulates the CD3 TCR complex and costimulatory molecules on the surface of the T cells. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-A acetate (PMA), or the mitogenic lectin-like phytohemagglutinin (PHA) may be used to generate an activation signal for the T cells.

[0196] As non-limiting examples, T cell populations may be stimulated in vitro, such as by contact with an anti-CD3 antibody or an antigen-binding fragment thereof, or a surface-immobilized anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, T cell populations may be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. Suitable conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640, or X-vivo5, (Lonza)), which may contain factors necessary for proliferation and survival, 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 for the growth of cells known to those skilled in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media may include RPMI1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo1, X-Vivo20, 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 cytokines in sufficient amounts for T cell growth and proliferation. Antibiotics such as penicillin and streptomycin are included only in experimental cultures and not in cultures of cells injected into subjects. Target cells are cultured under conditions necessary to support growth, e.g., appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% O 2 ). 2 ) T cells exposed to different stimulation times may display different characteristics.

[0197] In some embodiments, the 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.

[0198] Therapeutic Formulations and Administration As used herein, the terms "effective amount" and "therapeutically effective amount" refer to an amount of active therapeutic agent sufficient to produce a desired therapeutic response without undue adverse side effects such as toxicity, irritation, or allergic reaction. The specific "effective amount" will obviously vary depending on factors such as the particular condition being treated, the physical condition of the patient, the type of animal being treated, the duration of treatment, the nature of the concomitant therapy (if any), and the specific formulation and structure of the compound or its derivatives used. In this case, an amount is considered to be therapeutically effective if it results in one or more of the following, but is not limited to: (a) inhibition of tumor growth (e.g., cancers expressing MAGE-A4); (b) antagonism or stabilization of cancers expressing MAGE-A4.

[0199] The dose of the antigen-binding molecule administered to a patient may vary depending on the age and size of the patient, the target disease, the pathology, the route of administration, and the like. The preferred dose is typically calculated according to body weight or body surface area. When the antibody or bispecific antigen-binding molecule of the present invention is used for the purpose of treating an adult patient, it may be advantageous to administer the bispecific antigen-binding molecule of the present invention intravenously, usually at a single dose of about 0.01 to about 20 mg / kg body weight, more preferably at a single dose of about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted. The effective dosage and schedule for administering the bispecific antigen-binding molecule may be determined empirically. For example, the progress of the patient may be monitored by periodic evaluation, and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages may be performed using methods well known in the art (e.g., M Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0200] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, 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 compositions may be administered by any convenient route, for example, by infusion or bolus injection, absorption through epithelial or mucocutaneous linings (such as oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration may be systemic or local.

[0201] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. It should be noted that for subcutaneous delivery, a pen delivery device facilitates application in delivering the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and easily replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled 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.

[0202] Numerous reusable pen and autoinjector delivery devices have utility for subcutaneous delivery of the pharmaceutical compositions of the present invention, including, for example, the AUTOPEN™ (Owen Mumford, Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Company, Indianapolis, IN), the NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN Examples of disposable pen delivery devices that have application in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), the SURECLICK autoinjector (Amgen, Thousand Oaks, Calif.), PENLET (Haselmeyer, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA pen (Abbott Laboratories, Abbott Park Ill.), to name just a few.

[0203] In certain circumstances, pharmaceutical compositions can be delivered in controlled release systems. In one embodiment, pumps can be used (see Langer, supra, Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, a controlled release system can be placed in the vicinity of the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., G Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0204] The injectable preparations may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, and intramuscular injection, drip infusion, etc. These injectable preparations may be prepared by well-known methods.

[0205] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into a suitable unit dose dosage form to suit the dosage of the active ingredient. Examples of such unit dose dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per unit dose dosage form, and in particular, in the form of an injection, the antibody is preferably contained in an amount of about 5 to about 100 mg, and in other dosage forms, about 10 to about 250 mg.

[0206] Administration of cells or cell populations according to the present disclosure may be performed in any convenient manner, including aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous, or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present disclosure are preferably administered by intravenous injection.

[0207] The administration of cells or cell populations is at 10 / kg body weight. 4 ~10 9 Cells, preferably 10 5 ~10 6 The administration of the effective amount of cells / kg body weight may consist of administration of 100 mg / kg of cells / kg body weight, including all integer values ​​of the number of cells within these ranges. The cells or cell populations may be administered in one or more doses. In some embodiments, an effective amount of cells is administered as a single dose. In some embodiments, an effective amount of cells is administered as two or more doses over a period of time. The timing of administration is within the discretion of the attending physician and depends on the clinical condition of the patient. The cells or cell populations may be obtained from any source, such as a blood bank or a donor. While individual needs vary, the determination of the range of effective amounts of a given cell type for 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 dosage administered will depend on the age, health, and weight of the recipient, the type of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0208] In one embodiment, an effective amount of the cells or a composition comprising those cells is administered parenterally. This administration can be intravenous. In some cases, administration can be by injection directly into a tumor.

[0209] In certain embodiments of the disclosure, the cells are administered to the patient in conjunction with (e.g., before, simultaneously with, or after) any number of relevant therapies, including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or efalizutimab treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the disclosure may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, or other antibody therapies, cytotoxin, fludarivine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.

[0210] In further embodiments, the cell composition of the present disclosure is administered to the patient in conjunction with (e.g., before, simultaneously, or after) bone marrow transplantation, T cell depletion therapy using either chemotherapy agents such as fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell composition of the present disclosure is administered after B cell depletion therapy, such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, the subject may receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following transplantation, the subject receives an infusion of the expanded immune cells of the present disclosure. In additional 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 the tumor burden prior to administration of the expanded immune cells of the present disclosure. In one embodiment, reducing the tumor burden prior to administration of the engineered cells of the present disclosure may reduce or prevent the possibility of cytokine release syndrome or cytokine storm, a side effect that may be associated with CAR T cell therapy.

[0211] Therapeutic Applications The present disclosure provides a composition comprising an engineered cell (e.g., a T cell) expressing a chimeric antigen receptor of the present disclosure and a pharma- ceutically acceptable vehicle. The present disclosure also provides a composition comprising an antibody or an antigen-binding fragment thereof and a pharma- ceutically acceptable vehicle. In some cases, the engineered cell, antibody, or antigen-binding fragment forms a medicament, particularly for immunotherapy. In some cases, the engineered cell, antibody, or antigen-binding fragment is used to treat cancer (e.g., multiple myeloma or melanoma). In some cases, the engineered cell, antibody, or antigen-binding fragment is used to manufacture a medicament for immunotherapy and / or treatment of cancer (e.g., cancer expressing MAGE-A4).

[0212] The present disclosure provides methods that include administering to a subject in need thereof a therapeutic composition that includes an antibody (e.g., a bispecific antibody), or an antigen-binding fragment thereof, and / or an engineered cell (e.g., a T cell) as discussed herein that expresses a chimeric antigen receptor. The therapeutic composition may include a cell expressing any of the chimeric antigen receptors disclosed herein and a pharma- ceutically acceptable carrier, diluent, or vehicle. In additional or alternative examples, the therapeutic composition includes an antibody and / or an antigen-binding fragment as discussed herein. As used herein, the phrase "subject in need thereof" refers to a human or non-human animal that exhibits one or more symptoms or signs of cancer (e.g., a subject having a tumor that expresses MAGE-A4 or a subject suffering from any of the cancers described herein) or who would otherwise benefit from inhibition or reduction of MAGE-A4 activity or depletion of MAGE-A4+ cells.

[0213] The engineered cells and / or antibodies and antigen-binding fragments of the present disclosure may be useful, inter alia, for treating any disease or disorder in which stimulating, activating, and / or targeting an immune response is beneficial. In particular, the engineered cells and / or antibodies and antigen-binding fragments of the present disclosure may be used for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by MAGE-A4 expression or activity, or the proliferation of MAGE-A4+ cells. Cells expressing MAGE-A4 that may be inhibited or killed using the engineered cells and / or antibodies and antigen-binding fragments of the present disclosure include, for example, multiple myeloma cells, melanoma cells, or other solid tumor cells.

[0214] The engineered cells and / or antibodies and antigen-binding fragments of the present disclosure may 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 may be used to treat tumors expressing MAGE-A4 in general. According to other related embodiments of the present disclosure, methods are provided that include administering the engineered cells and / or antibodies and antigen-binding fragments disclosed herein to a patient suffering from a tumor expressing MAGE-A4, including tumors from the cancers listed above. Analytical / diagnostic methods well known in the art, such as tumor scanning, may be used to determine whether the patient has such a tumor, disease, or condition.

[0215] The present disclosure also provides a method for treating residual cancer in a subject. As used herein, the term "residual cancer" refers to the presence or persistence of one or more cancerous cells in a subject after treatment with an anti-cancer therapy.

[0216] According to certain aspects, the disclosure provides methods for treating a disease or disorder associated with MAGE-A4 expression (e.g., cancer) comprising administering to a subject an engineered cell population, and / or antibodies, and antigen-binding fragments as described elsewhere herein, after the subject has been determined to have the disease or disorder. For example, the 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 has received other immunotherapy or chemotherapy.

[0217] The treatments discussed herein may be ameliorative, curative, or preventative. Treatments may be either part of autologous immunotherapy or part of allogeneic immunotherapy. Autologous means that the cells, cell lines, or cell populations used for the treatment of a patient are derived from the patient or a human leukocyte antigen (HLA)-matched donor. Allogeneic means that the cells, cell lines, or cell populations used for the treatment of a patient are derived from a donor, not from the patient.

[0218] Cells that can be used in the disclosed methods are described herein. The treatments can be used to treat patients diagnosed with a pre-malignant or malignant cancer condition characterized by an excess of cells expressing MAGE-A4, particularly cells expressing MAGE-A4. Such conditions can be found in cancers.

[0219] Cancer types that may 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, gastric cancer, cervical cancer, breast cancer, and melanoma.

[0220] The compositions and methods of the present disclosure may be used to treat subjects characterized as having cells or tissues that express MAGE-A4 or suspected of having cells or tissues that express MAGE-A4. For example, subjects that may benefit 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.

[0221] Combination therapy The present disclosure provides methods that include administering an antibody and / or antigen-binding fragment (e.g., bispecific antibody), and / or 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 combined or administered in combination with the antibody / antigen-binding fragment cells and / or cell or cell population of the present disclosure include, for example, anti-tumor agents (e.g., chemotherapeutic agents including melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), obendamustine (Treanda), or any other known to be effective in treating plasma cell neoplasms in a subject). In some embodiments, the second therapeutic agent includes a steroid. In some embodiments, the second therapeutic agent includes targeted therapies including thalidomide, lenalidomide, and bortezomib, which are therapies approved for treating newly diagnosed patients. For example, lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab, and daratumumab are examples of second therapeutic agents effective for treating relapsed myeloma. In certain embodiments, the second therapeutic agent is a regimen including 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 (Velcade®), carfilzomib (Kyprolis®), and ixazomib (Ninlaro®). In certain embodiments, the second therapeutic agent may be a histone deacetylase inhibitor, such as panobinostat (Farydak®). 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 to an anti-tumor 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 their respective receptors. The pharmaceutical compositions of the present disclosure (e.g., pharmaceutical compositions comprising an engineered cell or cell population disclosed herein) may also be administered as part of a treatment regimen that includes one or more therapeutic combinations selected from monoclonal antibodies other than those described herein that may interact with different antigens on the surface of plasma cells, bispecific antibodies having one arm that binds to an antigen on the surface of tumor cells and the other arm that binds to an antigen on T cells, antibody drug conjugates, bispecific antibodies coupled to anti-tumor agents, checkpoint inhibitors, such as those targeting PD-1 or CTLA-4, or combinations thereof. In certain embodiments, the checkpoint inhibitor may be selected from a PD-1 inhibitor, such as pembrolizumab (Keytruda®), nivolumab (Opdivo®), or cemiplimab (Libtayo®). In certain embodiments, the checkpoint inhibitor may be selected from a PD-L1 inhibitor, such as atezolizumab (Tecentriq®), avelumab (Bavencio®), or durvalumab (Imfinzi®). In certain embodiments, the checkpoint inhibitor may be selected from a CTLA-4 inhibitor, such as ipilimumab (Yervoy®).

[0222] The disclosure also includes therapeutic combinations comprising any of the antibodies / antigen-binding fragments and / or engineered cells or cell populations described herein and one or more inhibitors of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, 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 fragments, Fv fragments, scFv, dAb fragments, or other engineered molecules such as diabodies, triabodies, tetrabodies, minibodies and minimal recognition units). In some embodiments, the antibodies / antigen-binding fragments of the present disclosure, and / or engineered cells or cell populations may also be administered as part of a treatment regimen that also includes radiation therapy and / or conventional chemotherapy.

[0223] The additional therapeutically active ingredient(s) may be administered immediately prior to, simultaneously with, or immediately following administration of the engineered cells of the present disclosure (for purposes of this disclosure, such dosing regimens will be considered as administering the genetically engineered cells "in combination" with the additional therapeutically active ingredient).

[0224] The present disclosure provides pharmaceutical compositions in which the engineered cells or cell populations of the present disclosure are co-formulated with one or more of the additional therapeutically active ingredients described elsewhere herein.

[0225] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of the antibody / antigen-binding fragment and / or engineered cells may be administered to the subject over a defined time course. The method according to this aspect includes sequentially administering multiple doses of the antigen-binding molecule and / or cells to the subject. As used herein, "sequentially administering" means that each dose is administered to the subject at different time points, e.g., on different days separated by a predefined interval (e.g., hours, days, weeks, or months). The present disclosure provides a method that includes sequentially administering to the patient a single primary dose, followed by one or more secondary doses, and optionally followed by one or more tertiary doses.

[0226] The terms "primary dose", "secondary dose", and "tertiary dose" refer to the time sequence of administration of the antigen-binding molecules and / or engineered cells of the present disclosure. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"). A "secondary dose" is a dose administered after the initial administration, and a "tertiary dose" is a dose administered after the secondary dose. The primary, secondary, and tertiary doses may all contain the same amount of antigen-binding molecules and / or engineered cells, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of antigen-binding molecules and / or engineered cells contained in the primary, secondary, and / or tertiary doses differ from each other during the course of treatment (e.g., adjusted up or down as appropriate). In certain embodiments, two or more (e.g., two, three, four, or five) doses are administered as a "loading dose" at the beginning of the treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0227] In one exemplary embodiment of the present disclosure, each secondary and / or tertiary dose is 1 to 26 (e.g., 1, 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 The phrase "immediately preceding dose" as used herein means a dose in a series of multiple administrations that is administered to a patient prior to administration of the next dose in the sequence, with no intervening doses.

[0228] Methods according to this aspect of the invention may include administering any number of secondary and / or tertiary doses to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight or more) tertiary doses are administered to the patient.

[0229] In embodiments involving multiple secondary doses, each secondary dose may be administered with the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered with the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical testing. EXAMPLES

[0230] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the disclosed methods and compositions, and are not intended to limit the scope of what the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0231] Example 1: Generation of anti-MAGE-A4 antibodies Anti-MAGE-A4 antibodies were generated by inducing transgenic mice (e.g., engineered mice containing DNA encoding human immunoglobulin heavy and kappa light chain variable regions) to human MAGE-A4 antigen (e.g., hMAGE-A4 286-294 for mAb31339N2) and HLA-A2 (HLA-A * The genetically modified mice were obtained by immunizing mice with MAGE-A4 antigen and HLA-A2 (02:01). More specifically, the genome of the genetically modified mice had nucleotide sequences encoding human HLA-A2 (as well as sequences encoding human immunoglobulin heavy chain variable regions and kappa light chain variable regions) such that the genetically modified mice express human HLA-A2, but where the mice were tolerized to human HLA-A2 such that they would generate a specific B cell response when immunized with MAGE-A4 antigen and HLA-A2.

[0232] Following immunization, splenocytes were harvested from each mouse and either (1) fused with mouse myeloma cells to preserve their viability, forming hybridoma cells, and screened for MAGE-A4 specificity, or (2) B cells were selected using human MAGE-A4 fragments as selection reagents that bind and identify reactive antibodies (antigen-positive B cells) (as described in U.S. Patent Publication No. 2007 / 0280945(A1)).

[0233] Chimeric antibodies against MAGE-A4 with human variable regions and mouse constant regions were first isolated. The antibodies are characterized and selected for desirable characteristics including affinity, selectivity, etc. For example, the mouse constant regions were replaced with the desired human constant regions, e.g., wild-type or modified IgG1 or IgG4 constant regions, to generate fully human anti-MAGE-A4 antibodies (e.g., mAb M31339N2 (containing mouse constant regions) was used to generate mAb H31339N2 (containing human constant regions)). The constant regions selected may vary depending on the specific application, but the high affinity antigen binding and target specificity characteristics reside in the variable regions.

[0234] mAbM31339N2 was also reformatted into a bispecific antibody using either a high affinity (7195P) CD3 arm to generate bsAb6054 (HCVR1, LCVR1, and LCVR2 from mAb31339 and HCVR2 from 7195P) or a medium affinity (7221G) CD3 arm to generate bsAb6043 (HCVR1, LCVR1, and LCVR2 from mAb31339 and HCVR2 from 7221G).

[0235] Amino acid and nucleic acid sequences of heavy and light chain variable regions of anti-MAGE-A4 antibodies: Table 1 lists the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-MAGE-A4 antibodies of the present disclosure. The mAb31339N and mAb31339N2 sequences in Table 1 are identical except for one amino acid difference in HCVR framework region 3. However, the CDRs are the same for both mAb31339N and mAb31339N2. The corresponding nucleic acid sequence identifiers are shown in Table 2. A summary of all sequences contained herein is provided in Table 15.

[0236] [Table 1]

[0237] [Table 2]

[0238] Generation of bispecific antibodies that bind to CD3 and MAGE-A4: Bispecific antibodies comprising an anti-CD3 specific binding region and an anti-MAGE-A4 specific binding region were constructed using the sequences listed in Tables 3 and 4, using a methodology that combines a heavy chain from an anti-CD3 antibody with a heavy chain from an anti-MAGE-A4 antibody and the cognate light chain.

[0239] Thus, the bispecific antibodies generated according to this example comprise two separate antigen-binding regions (i.e., binding arms). The first antigen-binding region comprises a heavy chain variable region from an anti-MAGE-A4 antibody ("MAGE-A4-VH") paired with a cognate light chain variable region from an anti-MAGE-A4 antibody ("MAGE-A4-VL"), and the second antigen-binding region comprises a heavy chain variable region from an anti-CD3 antibody ("CD3-VH") paired with a MAGE-A4-VL. In principle, it is also possible to use the cognate light chain variable region from an anti-CD3 antibody ("CD3-VL") as a light chain variable region 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 comprises a MAGE-A4 binding arm comprising the HCVR / LCVR of SEQ ID NO:2 / 10 and a CD3 binding arm comprising the HCVR / LCVR of SEQ ID NO:73 / 10. The bsAb6043 antibody comprises a MAGE-A4 binding arm comprising the HCVR / LCVR of SEQ ID NO:2 / 10 and a CD3 binding arm comprising the HCVR / LCVR of SEQ ID NO:55 / 10.

[0240] The amino acid sequence identifiers of the heavy and light chain variable regions and CDRs used to construct the anti-CD3 antigen binding arm and the anti-MAGE-A4 binding arm are listed in Table 3. The corresponding nucleic acid sequence identifiers are shown in Table 4. The anti-MAGE / anti-CD3 bispecific antibodies were generated from either a medium affinity CD3 antibody (anti-CD3-A, referred to herein as H4sH7221G or 7221G) or a high affinity CD3 antibody (anti-CD3-B, referred to herein as HpH4sH7195P or 7195P). The first antigen binding region and the second antigen binding region are referred to herein as the "arms" of the bispecific antibody. In the examples, 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, while the other arm comprises a HCVR comprising the amino acid sequence of SEQ ID NO:55, or SEQ ID NO:73, and the LCVR comprises the amino acid sequence of SEQ ID NO:10.

[0241] [Table 3]

[0242] [Table 4]

[0243] Example 2: Binding of anti-HLA-A2:MAGE-A4(286-294) antibodies to T2 cells pulsed with MAGE-A4(286-294) via flow cytometry Anti-HLA-A 2:MAGE-A4 286-294 antibody (mAbM31339N) * Cell surface binding to 02:01 positive T2 (174CEM.T2) cells was assessed in a flow cytometry-based peptide pulse assay. 6T2 cells were cultured with 10 μg / ml human (h)B2M (EMD Millipore, Catalog No. 475828) and 100 μg / ml MAGE-A4 286-294 peptide in 1 ml AIM V medium (Gibco, Catalog No. 31035-025) for 16 hours at 37° C. Cells were washed in staining buffer (calcium and magnesium free PBS (Corning, Ref. No. 21-031-CV) + 2% FBS (Theradigm, Lot No. 238B15)), harvested using cell dissociation buffer (Millipore, Catalog No. S-004-C) and resuspended in staining buffer. Pulsed cells (200,000 cells) were seeded into 96-well V-bottom plates (Axygen, Cat. No. P-96-450V-CS) and stained with 3-fold serial dilutions of mAb M31339N (1.7 pM to 100 nM) or with a non-binding control antibody (mAb 1097) for 30 minutes at 4°C. Cells were then washed once with staining buffer and incubated with 5ug / ml Alexa Fluor 647-conjugated Fab'2 anti-mouse Fc specific secondary antibody (Jackson ImmunoResearch, Cat. No. 115-606-071) for 30 minutes at 4°C. Finally, cells were stained with green fluorescent viability dye (Molecular Probes, Cat. No. L-34970, reconstituted in 50μl DMSO) at a concentration of 1:1000. Cells were then washed and fixed using a 50% solution of BD Cytofix (BD, Cat. No. 554655) diluted in PBS. Samples were run on an Intellicyt iQue flow cytometer (Intellicyt) and results were analyzed using Forecyte analysis software (Intellicyte) to calculate the mean fluorescent intensity (MFI) after gating on live cells. EC 50To calculate values, MFI values ​​were plotted in GraphPad Prism using a four-parameter logistic equation on a 12-point response curve. Secondary antibody alone (i.e., no primary antibody) for each dose-response curve was also included in the analysis as a succession of 3-fold serial dilutions and is represented as the lowest dose. Signal-to-noise (S / N) was determined by taking the ratio of the highest MFI on the dose-response curve to the MFI in the secondary alone wells. EC 50 The values ​​(M) and maximum S / N are shown in Table 5. mAbM31339N bound with an EC50 of 1.7 nM and a maximum S / N of 1933, while no binding of the control antibody (mAb1097) was detected, indicating that mAb31339N has a strong binding affinity for MAGE-A4 (286-294). With reference to Table 5, ND refers to an EC50 value that could not be accurately determined because binding did not reach saturation within the antibody concentration range tested.

[0244] [Table 5]

[0245] Example 3: Binding of anti-HLA-A2:MAGE-A4 antibody and MAGE-A4xCD3 bispecific antibody to T2 cells pulsed with MAGE-A4 related peptides. An in silico computational strategy (Dhanik A et.al. BMC Bioinformatics 2016) identified several peptides associated with MAGE-A4, but these peptides were not associated with HLA-A. * It was predicted to form a complex with 02:01. The identified peptides are summarized in Table 6.

[0246] [Table 6]

[0247] The binding of the parent antibody (mAbH31339N2), the MAGE-A4xCD3(7221G) bispecific antibody (bsAb6043) and the non-binding isotype control antibody (mAb4241) to these relevant peptides shown in Table 6 were assessed in the T2 pulse assay described above. As described above in Example 1, the bsAb6043 antibody comprises a MAGE-A4 binding arm comprising the HCVR / LCVR of SEQ ID NO:2 / 10 and a CD3 binding arm comprising the HCVR / LCVR of SEQ ID NO:55 / 10. As summarized in Table 7, both antibodies bound to the MAGE-A4 peptide with S / N values ​​of 310 for mAbH31339N2 and 244 for bsAb6043, substantially above baseline (no peptide). The antibodies tested showed lower binding to AOX1 with S / N values ​​of 131 for mAbH31339N2 and 110 for bsAb6043, and for both antibodies lower binding to SHTN1 with S / N values ​​of 5. No detectable binding was observed to the remaining peptides, and control antibody binding was ≦3 for all peptides tested.

[0248] [Table 7]

[0249] Example 4: Evaluation of the activity of MAGE-A4xCD3 bispecific antibodies in T cell reporter / antigen presenting cell (APC) bioassays. The activity of the MAGE-A4 x CD3 bispecific antibody was evaluated in a T cell reporter / antigen presenting cell (APC) bioassay. To generate the assay cell line, Jurkat cells were transduced with an NF-KB-dependent κBLuc luciferase lentiviral reporter vector (Qiagen) and single cells were selected for high luciferase activity to generate the Jurkat / NF-κBLuc assay cell line. *IM9 and U266B1 cells, myeloma cell lines that endogenously express 02:01, were utilized as antigen-presenting cells (APCs). Additionally, MAGE-A4 and HLA-A2 negative Raji cells were used as controls. Briefly, 25,000 Jurkat / NF-κBLuc cells were added to a Thermo-Nunc 96-well white plate (Thermo Scientific, Cat. No. 136101) in 25 μl of assay medium (RPMI medium with 10% FBS and 1% P / S / G), followed by 25,000 APCs in 25 μl of assay medium. Three-fold serial dilutions of antibodies from 27.4 pM to 20 nM were added to the plate in 50 μl of assay medium. The cell mixture was incubated at 37 °C, 5% CO 2 The cells were incubated at 4°C for 5 hours in a humidified incubator. NF-κB-luciferase activity was measured using Promega One-Glo (cat. no. E6130) and a Perkin Elmer Envision plate reader according to the manufacturer's instructions. Relative luciferase units (RLU) were generated and EC50 values ​​were determined using a four-parameter logistic equation across an 8-point dose-response curve (GraphPad Prism). The primary antibody zero condition (secondary antibody alone) of each dose-response curve was also included in the analysis as a series of 3-fold serial dilutions and is expressed as the lowest dose. Maximum activity was determined by taking the ratio of the highest to the lowest RLU on the curve and is expressed as signal:noise (S / N). EC50 values ​​and S / N are summarized in Table 8. For Table 8, ND corresponds to an EC50 value that could not be accurately determined because binding did not reach saturation within the antibody concentration range tested.

[0250] [Table 8]

[0251] As described in Example 1, the bsAb6054 antibody comprises a MAGE-A4 binding arm comprising the HCVR / LCVR of SEQ ID NO:2 / 10 and a CD3 binding arm comprising the HCVR / LCVR of SEQ ID NO:73 / 10, and the bsAb6043 antibody comprises a MAGE-A4 binding arm comprising the HCVR / LCVR of SEQ ID NO:2 / 10 and a CD3 binding arm comprising the HCVR / LCVR of SEQ ID NO:55 / 10. The bsAb4241 and bsAb3905 antibodies are non-binding controls (for MAGE-A4) that have anti-CD3 binding arms that contain the same anti-CD3 HCVRs as bsAb6043 and bsAb6054, respectively, and are paired with the cognate light chain variable region from the non-MAGE-A4 binding arm of each antibody.

[0252] As shown in Table 8, the MAGE-A4 x CD3 intermediate affinity bispecific antibody (bsAb6043) had no activity in the Jurkat / NF-κBLuc bioassay in the presence of IM9, U266B1, or RAJI cells. In contrast, the MAGE-A4 x CD3 high affinity bispecific antibody (bsAb6054) had EC50 values ​​of 7.4E-10M and 3.1E-09M against IM9 and U266B1 cells, respectively, and S / N values ​​of 41.9 and 6.3. Non-binding control bispecific antibodies with either an intermediate affinity CD3 arm (mAb4241) or a high affinity CD3 arm (mAb3905) were minimally active with S / N values ​​of ≦2.2. Additionally, there was no activity of either antibody against RAJI cells.

[0253] A similar bioassay was performed, but this time a fixed amount of anti-CD28 antibody was added. Here, the addition of CD28 partially blocked the activity of bsAb6054 on IM9 cells (EC50 2.1E09M, S / N 28.7), but slightly increased the activity on U266B1 cells (EC50 6.1E-09M, S / N 8.6). This is likely due to the levels of CD80 and CD86 costimulatory molecules endogenously expressed on these cell lines. IM9 has high levels of CD80 and CD86 endogenously. Thus, the addition of CD28 antibody in the bioassay blocks 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 stimulates CD28 signaling on Jurkat cells, increasing activity (Table 9).

[0254] [Table 9]

[0255] In addition to the antibodies discussed above in connection with Table 8, mAb5705 is an anti-CD28 antibody that comprises a HCVR of SEQ ID NO:85 and a LCVR of SEQ ID NO:93.

[0256] Example 5: Peptide specificity in the Jurkat / NF-κB-Luc / APC reporter bioassay An engineered T cell / APC functional Jurkat / NF-κBLuc reporter / APC bioassay was also utilized to assess whether the MAGE-A4×CD3 bispecific antibodies retained selectivity for MAGE-A4 peptides over the related peptides identified in Table 7. In this assay, T2 cells were pulsed with the target peptide MAGE-A4 286-294 or sequence-related off-target peptides (Table 7) as previously described. As summarized in Table 10, both bsAb6043 (MAGE-A4×CD3 intermediate affinity) and bsAb6054 (MAGE-A4×CD3 high affinity) bispecific antibodies stimulated NF-κB-dependent reporter activity when cultured with T2 cells pulsed with MAGE-A4 286-294 peptide. Activity was also detected when cultured with T2 cells pulsed with the off-target peptides AOX1795-803 and SHTN1198-206. 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 the MAGE-A4 293-294, AOX1 795-803, and SHTN1 198-206 peptides, respectively. bsAb6054 stimulated reporter activity with EC50 values ​​of 2.0E-12M, 2.1E-11M, and 1.3E-09M and S / N values ​​of 78.7, 36, and 81.1 for the MAGE-A4 286-294, AOX1 795-803, and SHTN1 198-206 peptides, respectively. Non-binding control bispecific antibodies with either a medium affinity CD3 arm (mAb4241) or a high affinity CD3 arm (mAb3905) were minimally active with S / N values ​​of ≦2.9.

[0257] [Table 10]

[0258] Example 6: Primary Imaging-Based Killing Assay To assess the ability of the MAGE-A4 × CD3 bispecific antibody to redirect T cell responses against cancer cells, an imaging-based multiplexed primary T cell killing assay was performed. Briefly, CD8+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) using magnetic bead separation (Miltenyi Biotec, Cat. No. 130-045-201) according to the manufacturer's protocol and were immunized with MAGE-A4 286-294 antigen-presenting HLA-A *02:01 positive IM9 cell line was pre-labeled with CellTrace™ Violet (Thermo Fisher, C34557) according to the manufacturer's instructions. IM9 multiple myeloma cells (10,000) were combined with CD8+ T cells (25,000) in 100ul of stimulation medium (X Vivo 15 + 10% FBS + 1% HEPES + 1% NaPyr + 1% NEAA + 0.01mM BME) in a 96-well imaging plate (PerkinElmer, Cat. No. 6055308). Three-fold serial dilutions of antibodies ranging from 20nM to 27.4pM were added to the cells in an additional 100μl of stimulation medium. After 72 hours of culture, 50 μl of supernatant was removed for cytokine release assay (see below) and 15 μl of a 1:1000 dilution (in PBS) of the nuclear dye DRAQ5 (Thermo Fisher, Cat. No. 62252) was added to the cells. Images of DRAQ5-labeled nuclei and Cell-Trace Violet-labeled APCs were collected on an Opera Phenix (PerkinElmer) and the number of viable IM9 cells in all conditions (populations labeled with both CellTrace™ Violet and DRAQ5) was calculated using Harmony analysis software (PerkinElmer). EC50 values ​​could not be generated from the resulting non-sigmoidal curves, so the % of viable IM9 (normalized to the no antibody condition) cells was reported for the 6.6 nM dose (the highest tested dose at which the non-binding control was inactive). As summarized in Table 11, bsAb6043 (MAGE-A4 x CD3 (7221G) medium affinity bispecific antibody) had no positive effect on the % of viable IM9 cell population, while bsAb6054 (MAGE-A4 x CD3 high affinity bispecific antibody) reduced the IM9 population to 38%. Addition of 2 nM of anti-CD28 agonist antibody doubled the number of viable cells (80%) (Table 9). This may be due to the ability of mAb5705 to block CD28, thereby inhibiting its interaction with CD80 and CD86 endogenously expressed on IM9 cells. Non-binding control x CD3 bispecific antibodies mAb4241 (medium CD28 affinity) and mAb3905 (high CD3 affinity) had no activity in the assay.

[0259] [Table 11]

[0260] Example 7: Primary cytokine release assay IL2 and IFN-γ release were also assessed in cell culture supernatants sampled from the 6.6 nM treatment dose utilized in the imaging-based killing assay described above. Cytokine levels were determined by AlphaLisa (PerkinElmer, Cat. No. AL221F, AL217F) according to the manufacturer's instructions, and RLU values ​​were normalized to untreated wells to determine S / N values. As summarized in Table 12, bsAb6043 (MAGE-A4×CD3(7221G) intermediate affinity bispecific antibody) did not release any IL2 or IFN-γ, while the higher affinity MAGE-A4×CD3 bispecific antibody bsAb6054 induced moderate production of IL2 and IFN-γ with S / N values ​​of 2.1 and 2.4, respectively. Addition of CD28 antibody (mAb5705) did not significantly affect cytokine production (Table 12).

[0261] [Table 12]

[0262] Example 8: Generation of MAGE-A4-specific chimeric antigen receptors The anti-MAGE-A4 31339N2 antibody of Table 1 was reformatted into a VL-VH single chain variable fragment (ScFv) and placed into a chimeric antigen receptor (CAR) configuration using the CD8α hinge and transmembrane domain, 4-1BB costimulatory domain, and CD3 zeta stimulatory domain, or the CD28 hinge, transmembrane, and signaling domain, using the HCVR and LCVR nucleotide sequences of the anti-MAGE-A4 antibody corresponding to SEQ ID NO: 1 and SEQ ID NO: 9, respectively. The full length nucleic acid and polypeptide heavy chain sequences of the corresponding anti-MAGE-A4 antibody (mAbH31339N2) correspond to SEQ ID NO: 17 and SEQ ID NO: 18, respectively. The full length nucleic acid and polypeptide light chain sequences of the corresponding anti-MAGE-A4 antibody (mAbH31339N2) correspond to SEQ ID NO: 19 and SEQ ID NO: 20, respectively. Full length nucleic acid and polypeptide HLA-A2 / MAGE-A4 286-294The CAR sequences targeting MAGE-A4 correspond to SEQ ID NO: 21 and SEQ ID NO: 22, respectively. As a non-binding control, a similar CAR was designed using the nucleotide sequence of an irrelevant scFv. The MAGE-A4-specific CAR was cloned into a lentiviral expression vector (Lenti-X™ Bicistronic Expression System (Neo), Clontech, Cat. No. 632181), and lentiviral particles were generated via the Lenti-X Packaging Single Shot (VSV-G) System (Clontech, Cat. No. 631276) according to the manufacturer's protocol. Jurkat cells engineered to express an NFKB-luciferase reporter (Jurkat / NKFBLuc cl 1C11) were then transduced with the CAR construct using RetroNectin® Precoated Dishes (Clontech, Cat. No. T110a) according to the manufacturer's protocol. After selection with 500 μg / ml G418 (Gibco, Cat. No. 11811-098) for at least 2 weeks, the following CAR T cell line was generated: Jurkat / NKFBLuc c 11 C 11 / MAGE-A4(286-294)31339 VL-VH CART. As a non-binding control, a similar CAR was designed using the nucleotide sequence of an irrelevant scFv. This CAR T cell line was evaluated for cell surface expression and functional activity in response to cells expressing MAGE-A4.

[0263] Example 9: Cell surface expression of MAGE-A4 CAR constructs in Jurkat cells and activation of MAGE-A4 CAR T cells The relative levels of cell surface expression of MAGE-A4 CAR constructs in Jurkat / NF-κB-Luc cells were assessed by flow cytometry. For staining, cells were seeded at a density of 200,000 cells per well of a 96-well V-bottom plate in staining buffer (Irving9240) of calcium- and magnesium-free PBS and 2% BSA (Sigma-Aldrich, Cat. No. A8577) and stained with 10 μg / ml Protein L (GenScript, Biotin Protein L, Cat. No. M00097) for 30 min at 4° C. After incubation, cells were washed once with staining buffer and stained with 0.5 μg / ml of streptavidin Alexa-647 secondary antibody (Biolegend, Cat. No. 405237) for 30 min at 4° C. Finally, cells were stained with green fluorescent viability dye (Molecular Probes, Cat. No. L-34970) at a concentration of 1:1000. Cells were then washed and fixed using a 50% solution of BD Cytofix (Becton Dickinson, Cat. No. 554655) diluted in PBS. Samples were run on an Intellicyt iQue flow cytometer to obtain viable cells, followed by analysis in FlowJo 10.2 to calculate mean fluorescence intensity (MFI). 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 Jurkat / NF-κB-Luc / MAGE-A4 31339N2 VL-VH CAR-T cell line was 33.8% CAR positive as measured by percent protein L staining. The non-targeting control CAR was expressed in 67.2% of the cells.

[0264] The activity of the CAR T cell lines was evaluated in a CAR T / APC (antigen presenting cell) bioassay. To perform the bioassay, 50,000 CAR T cells in 50 μl of assay medium (RPMI medium with 10% FBS and 1% P / S / G) were added to a Thermo-Nunc 96-well white plate (Thermo Fisher Scientific, Cat. No. 136101) followed by 3-fold serial dilutions of APC (500,000 cells to 685 cells) in 50 μl of assay medium. The following APCs were utilized: IM9 (endogenously expressing MAGE-A4 286-294 peptide and HLA-A * 02:01 positive), and HEK293 (MAGE-A4 286-294 negative but HLA-A * The cell mixture was incubated at 37°C, 5% CO 2 The plates were incubated at 4°C for 5 hours in a humidified incubator. NF-κB-luciferase activity was determined using Promega One-Glo (cat. no. E6130) and a PerkinElmer Envision plate reader. Relative luciferase units (RLU) were generated and plotted in GraphPad Prism using a four-parameter logistic equation across the 8-point dose-response curves to generate EC50 values ​​(number of APCs). A zero APC condition for each dose-response curve was also included in the analysis as a continuation of the 3-fold serial dilutions and 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, reported as EC50 (number of APCs), and expressed as signal:noise (S:N) in Table 14. 31339N2 CAR-T was activated upon culture with IM9 cells with an EC50 of 14,669 cells and S / N of 37.4, whereas non-targeting control CAR-T was only activated 4.9-fold. No activation was observed in HEK293 cells (MAGE-A4 negative but HLA-A2 positive).

[0265] [Table 13]

[0266] [Table 14]

[0267] Example 10: Binding of anti-HLA-A2:MAGE-A4 230-239 antibodies to T2 cells pulsed with MAGE-A4 230-239 and related off-target peptides by flow cytometry Anti-HLA-A 2:MAGE-A4 230-239 antibody (mAbM34852N) * Cell surface binding to 02:01 positive T2 (174CEM.T2) cells was assessed in a flow cytometry-based peptide pulse assay. 6T2 cells were cultured with 10 μg / ml human (h)B2M (EMD Millipore, Catalog No. 475828) and 100 μg / ml MAGE-A4 230-239 peptide in 1 ml of AIM V medium (Gibco, Catalog No. 31035-025) for 16 hours at 37° C. Cells were washed in staining buffer (calcium and magnesium free PBS (Irvine Scientific, Catalog No. 9319) + 2% FBS (Theradigm, Lot No. 238B15)), harvested using cell dissociation buffer (Millipore, Catalog No. S-004-C), and resuspended in staining buffer. Pulsed cells (200,000 cells) were seeded into 96-well V-bottom plates (Axygen, Cat. No. P-96-450V-CS) and stained with 3-fold serial dilutions of mAb M34852N (1.7 pM to 100 nM) or with a non-binding control antibody (mAb 1097) for 30 minutes at 4°C. Cells were then washed once with staining buffer and incubated with 5ug / ml Alexa Fluor 647-conjugated Fab'2 anti-mouse Fc specific secondary antibody (Jackson ImmunoResearch, Cat. No. 115-606-071) for 30 minutes at 4°C. Finally, cells were stained with green fluorescent viability dye (Molecular Probes, Cat. No. L-34970, reconstituted in 50μl DMSO) at a concentration of 1:1000. Cells were then washed and fixed using a 50% solution of BD Cytofix (BD, Cat. No. 554655) diluted in PBS. Samples were run on an Intellicyt iQue flow cytometer (Intellicyt) and results were analyzed using Forecyte analysis software (Intellicyt) to calculate the mean fluorescent intensity (MFI) after gating on live cells. EC 50To calculate values, MFI values ​​were plotted in GraphPad Prism using a four-parameter logistic equation on a 12-point response curve. Secondary antibody alone (i.e., no primary antibody) for each dose-response curve was also included in the analysis as a succession of 3-fold serial dilutions and expressed as the lowest dose. Signal-to-noise (S / N) was determined by taking the ratio of the highest MFI on the dose-response curve to the MFI in the secondary alone wells. EC 50 The values ​​(M) and maximum S / N are shown in Table 15. mAb M34852N had an EC 50 and bound with a maximum S / N of 243.3, whereas binding of the control antibody (mAb1097) was only weakly detected.

[0268] [Table 15] ND=EC50 values ​​could not be determined precisely because binding was not saturated within the antibody concentration range tested.

[0269] In silico computational strategies were used to identify HLA-A * Several MAGE-A4-associated peptides were identified that are predicted to form complexes with 02:01. The identified peptides are summarized in Table 16. Binding of the HLA-A2:MAGE-A4 230-239 antibody (mAbM34852N) and a non-binding isotype control antibody (mAb1097) to these associated peptides was assessed at a single concentration of 100 nM in the T2 pulsing assay described above. Binding here is shown in Table 17 as the MFI ratio of binding to peptide-pulsed cells divided by binding to non-pulsed cells. mAbM34852N bound to the MAGE-A4 peptide with a binding ratio of 512.9, whereas the isotype control mAb1097 bound to it with a binding ratio of 4.3. mAbM34852N had similar binding to the MAGE-A8(232-241) peptide with a binding ratio of 870.2. However, no detectable binding was observed to the remaining peptides and control antibody binding was <4.3 for all tested peptides, indicating that mAb M34852N has potential as a therapeutic agent against MAGE-A4.

[0270] [Table 16] Changes from the MAGE-A4(230-239) sequence are underlined.

[0271] [Table 17]

[0272] Example 11: HLA-A2:MAGE-A4 (230-239) Alanine Scanning Anti-HLA-A 2:MAGE-A4 230-239 antibody (mAbM34852N) * Cell surface binding to 02:01 positive T2 (174CEM.T2) cells was assessed in a flow cytometry-based peptide pulse assay. 6T2 cells were cultured with 10 μg / ml human (h)B2M (EMD Millipore, catalog number 475828) and 100 μg / ml MAGE-A4 230-239 peptide (Gibco, catalog number 31035-025) in 1 ml of AIM V medium at 37°C for 16 hours. Cells were washed with staining buffer (calcium and magnesium free PBS (Irvine Scientific, Cat. No. 9319) + 2% FBS (Theradigm, Lot No. 238B15), harvested using cell separation buffer (Millipore, Cat. No. S-004-C), and resuspended in staining buffer. Pulsed cells (200,000) were plated in 96-well V-bottom plates (Axygen, Cat. No. P-96-450V-CS) and stained with 3-fold serial dilutions (1.7 pM-100 nM) of mAb M34852N or non-binding isotype control antibody for 30 minutes at 4°C. Cells were then washed once with staining buffer and stained with 5ug / ml Alexa Fluor 647-conjugated Fab'2 anti-mouse Fc specific secondary antibody (Jackson The cells were then incubated with 100% BD Cytofix (BD, Cat. No. 554655) diluted in PBS for 30 min at 4°C. Finally, the cells were stained with green fluorescent viability dye (Molecular Probes, Cat. No. L-34970) and reconstituted in 50 μl of DMSO at a concentration of 1:1000. The cells were then washed and fixed using a 50% solution of BD Cytofix (BD, Cat. No. 554655) diluted in PBS. The samples were run on an Intellicyt iQue flow cytometer (Intellicyt) and the results were analyzed using the Forecyte analysis software (Intellicyt) to calculate the Mean Fluorescent Intensity (MFI) after gating on live cells. EC 50To calculate values, MFI values ​​were plotted in GraphPad Prism using a four-parameter logistic equation on a 12-point response curve. Secondary antibody alone (i.e., no primary antibody) for each dose-response curve was also included in the analysis as a succession of 3-fold serial dilutions and expressed as the lowest dose. Signal-to-noise (S / N) was determined by taking the ratio of the highest MFI on the dose-response curve to the MFI in the secondary alone wells. EC 50 The values ​​(M) and maximum S / N are shown in Table 19. mAb M34852N had an EC 50 and 243.3, whereas binding of the control antibody was only weakly detected. Results from an alanine scanning assay indicate that mAb M34852 exhibited binding across the MAGE-A4 (230-239) peptide.

[0273] [Table 18]

[0274] [Table 19]

[0275] Array Description

[0276] [Table 20-1]

[0277] [Table 20-2]

[0278] [Table 20-3]

[0279] Annotated sequences In the annotated sequences below, moieties are identified by alternating non-underlined and underlined sections, with the order of the moieties corresponding to the order listed below 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, etc.). MAGE-A4(286-294)31339N2 VL-VH BBz CAR (SEQ ID NO: 22)

[0280] [Table 21] VL (G4S)3 VH G4S CD8 Hinge / TM 4-1BB costimulation area CD3Z MAGE-A4(286-294)31339N2 VL-VH BBz CAR (SEQ ID NO: 120)

[0281] [Table 22] VL (G4S)3 VH G4S CD8 Hinge / TM 4-1BB costimulation area CD3Z MAGE-A4(286-294)31339N2 VL-VH CD28 hinge / TM / cytoCD3z CAR (SEQ ID NO: 105)

[0282] [Table 23] VL (G4S)3 VH G4S CD28 hinge CD28 TM CD28 costimulatory region CD3Z MAGE-A4(286-294)31339N2 VL-VH CD28 hinge / TM / cytoCD3z CAR (SEQ ID NO: 121)

[0283] [Table 24] VL (G4S)3 VH G4S CD28 hinge CD28 TM CD28 costimulatory region CD3Z

[0284] 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 be included within the scope of the appended claims.

Claims

1. A melanoma-associated antigen A4 (MAGE-A4)-specific chimeric antigen receptor (CAR), comprising, 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 costimulatory domain and a signal transduction domain, wherein the extracellular ligand-binding domain comprises: (i) a light chain variable region (LCVR) comprising complementarity determining regions LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 12, 14, and 16, respectively, and a heavy chain variable region (HCVR) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, respectively; or (ii) a light chain variable region (LCVR) comprising complementarity determining regions LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 117, 14, and 119, respectively, and a heavy chain variable region (HCVR) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 109, 111, and 113, respectively; A MAGE-A4-specific CAR comprising an anti-MAGE-A4 single chain variable fragment (scFv) region comprising:

2. (a) the anti-MAGE-A4 scFv region comprises a linker between the LCVR and the HCVR, and / or further comprises a linker between the extracellular ligand binding region and the hinge, optionally said linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-26, further optionally said linker between the LCVR and the HCVR comprising the amino acid sequence of SEQ ID NO: 25, and / or (b) the LCVR comprises the amino acid sequence of SEQ ID NO: 10 and the HCVR comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 83, or the LCVR comprises the amino acid sequence of SEQ ID NO: 115 and the HCVR comprises the amino acid sequence of SEQ ID NO: 107; The MAGE-A4-specific CAR according to claim 1.

3. (a) the hinge, the transmembrane region, or both, are derived from a CD8α polypeptide, the costimulatory region comprises a 4-1BB costimulatory region, and the signaling region comprises a CD3 zeta signaling region; or (b) one or more of the hinge, transmembrane region, and costimulatory region are derived from a CD28 polypeptide, and the signaling region comprises a CD3 zeta signaling region; or (c) the hinge comprises the amino acid sequence of SEQ ID NO:27, the transmembrane region comprises the amino acid sequence of SEQ ID NO:28, the costimulatory region comprises the amino acid sequence of SEQ ID NO:29, and / or the signaling region comprises the amino acid sequence of SEQ ID NO:30; or (d) the hinge comprises the amino acid sequence of SEQ ID NO: 34, the transmembrane region comprises the amino acid sequence of SEQ ID NO: 36, the CD28 costimulatory region comprises the amino acid sequence of SEQ ID NO: 38, and / or the signaling region comprises the amino acid sequence of SEQ ID NO: 30; The MAGE-A4-specific CAR according to claim 1 or 2.

4. The MAGE-A4-specific CAR according to claim 1, comprising an amino acid sequence of SEQ ID NO: 22, 105, 120 or 121.

5. An isolated nucleic acid molecule encoding the MAGE-A4-specific CAR according to any one of claims 1 to 4.

6. 6. A vector comprising the nucleic acid molecule of claim 5, optionally wherein the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector, or a cell comprising the isolated nucleic acid molecule of claim 5, optionally wherein the cell is a human T cell.

7. 5. An engineered cell comprising a MAGE-A4 specific CAR according to any one of claims 1 to 4, optionally wherein said engineered cell is an immune cell, further optionally wherein said immune cell is an immune effector cell, a T lymphocyte, an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, a helper T lymphocyte, or a CD8+ cytotoxic T lymphocyte.

8. An engineered human T cell comprising a chimeric antigen receptor comprising, from N-terminus to C-terminus, (a) an extracellular ligand binding region comprising an anti-MAGE-A4 single chain variable fragment (scFv) region comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR); (b) a hinge; (c) a transmembrane region; and (d) a cytoplasmic region comprising a costimulatory region and a signaling region; (i) the LCVR of the anti-MAGE-A4 scFv region comprises complementarity determining regions LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 12, 14, and 16, respectively, and the HCVR of the anti-MAGE-A4 scFv region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, respectively; or (ii) the LCVR of the anti-MAGE-A4 scFv region comprises complementarity determining regions LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 117, 14, and 119, respectively, and the HCVR of the anti-MAGE-A4 scFv region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 109, 111, and 113, respectively; Engineered human T cells.

9. (a) the scFv region comprises an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO:2 / 10, SEQ ID NO:83 / 10, or SEQ ID NO:107 / 115; or (b) the hinge comprises the amino acid sequence of SEQ ID NO:27, the transmembrane region comprises the amino acid sequence of SEQ ID NO:28, the costimulatory region comprises the amino acid sequence of SEQ ID NO:29, and / or the signaling region comprises the amino acid sequence of SEQ ID NO:30; or (c) the hinge comprises the amino acid sequence of SEQ ID NO: 34 and the transmembrane region comprises the amino acid sequence of SEQ ID NO: 36 the costimulatory region comprises the amino acid sequence of SEQ ID NO: 38 and / or the signalling region comprises the amino acid sequence of SEQ ID NO: 30; or (d) the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 22, the amino acid sequence of SEQ ID NO: 105, the amino acid sequence of SEQ ID NO: 120, or the amino acid sequence of SEQ ID NO: 121; The engineered human T cell of claim 8.

10. A pharmaceutical composition comprising a genetically modified human T cell and a pharma- ceutical acceptable carrier, wherein the genetically modified human T cell comprises a MAGE-A4-specific CAR according to any one of claims 1 to 4.

11. 11. The pharmaceutical composition of claim 10, for use in a method of (i) enhancing T lymphocyte activity in a subject, (ii) treating a subject with cancer, (iii) stimulating a T cell-mediated immune response against a target cell population or tissue in a subject, or (iv) providing anti-tumor immunity in a subject, the method comprising administering to a subject a cell comprising a MAGE-A4-specific CAR.

12. 1. An ex vivo method for engineering a cell population that expresses a MAGE-A4 specific chimeric antigen receptor (CAR), comprising: (a) providing a population of immune cells; (b) introducing a nucleic acid molecule encoding a MAGE-A4-specific CAR according to any one of claims 1 to 4 into the immune cell population; (b) culturing the immune cells under conditions to express the nucleic acid molecule; (c) isolating said immune cells expressing said MAGE-A4-specific CAR on the cell surface; and The method includes:

13. 1. An antigen binding protein that specifically binds to HLA-bound melanoma associated antigen A4 (MAGE-A4), said antigen binding protein comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), said LCVR comprising a complementarity determining region (CDR) of LCVR comprising SEQ ID NO:10 or SEQ ID NO:115, and said HCVR comprising a CDR of HCVR comprising an amino acid of SEQ ID NO:2, SEQ ID NO:83, or 107.

14. 14. The antigen binding protein of claim 13, wherein the antigen binding protein is a bispecific antibody, and optionally, the antigen binding protein interacts with CD3.

15. 15. A pharmaceutical composition comprising the antigen-binding protein of claim 13 or 14 and a pharma- ceutically acceptable carrier or diluent.

16. 15. A polynucleotide molecule comprising a polynucleotide sequence encoding one or more HCVRs and / or one or more LCVRs of the antigen binding protein of claim 13 or 14.

17. 16. The pharmaceutical composition of claim 15 for use in a method of treating a cancer that expresses MAGE-A4, said method comprising administering said antigen binding protein to a subject.

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