Chimeric antigen receptors 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 and cells expressing such chimeric antigen receptors. In certain embodiments, engineered cells expressing the chimeric antigen receptors of the present disclosure can inhibit 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 priority to U.S. Provisional Patent Application Nos. 62 / 878,125, filed July 24, 2019, 63 / 020,177, filed May 5, 2020, and 63 / 021,407, filed May 7, 2020, each of which is incorporated by reference herein in its entirety for all purposes.

[0002] Sequence Listing Reference This application incorporates by reference the Sequence Listing, which was filed in computer readable format as file 10600WO01-Sequence.txt, created on July 24, 2020, and contains 123,650 bytes.

[0003] The present disclosure provides chimeric antigen receptors (CARs) that are specific for melanoma-associated antigen A4 (MAGE-A4), and engineered cells comprising such CARs, 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 cell growth arrest in the G1 phase.

[0005] MAGE-A4 is abundantly expressed by many tumors of various histological types, such as head and neck squamous cell carcinoma, lung cancers including non-small cell lung cancer, esophageal squamous cell carcinoma, colon cancer, bladder cancer, mucosal and cutaneous melanoma, ovarian cancers, e.g., serous carcinoma, and uterine cancer, but in normal healthy adult tissues, MAGE-A4 expression is restricted to the testis.

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

[0007] Adoptive immunotherapy, which involves the transfer of ex vivo generated autoantigen-specific T cells, is a promising strategy for treating viral infections and cancer. T cells used in adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or by redirecting T cells through genetic engineering.

[0008] New specificities in T cells have been successfully generated through gene transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety associated with one or more signaling domains in a single fusion molecule. Generally, the binding portion of a CAR consists of the antigen-binding domain of a single-chain antibody (scFv), which contains the light and heavy chain variable fragments of a monoclonal antibody joined by a flexible linker. The signaling domain for first-generation CARs is derived from the cytoplasmic region of CD3 zeta or the Fc receptor gamma chain. First-generation CARs have been shown to successfully redirect T cell cytotoxicity. However, they failed to provide long-term proliferation and antitumor activity in vivo. Signaling domains derived from costimulatory molecules, as well as transmembrane and hinge domains, have been added to form second- and third-generation CARs, leading to some successful human therapeutic trials. 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 TCR-directed T cells have shown benefit in patients with solid tumors. Stauss et al., for example, describe a strategy for modifying therapeutic CARs and TCRs for use in cancer treatment that enhances antigen-specific effector function and limits the toxicity of engineered T cells (Non-Patent Document 1).

[0009] There is an unmet need for new CAR-based targeting agents that specifically bind to the MAGE-A4 antigen, and methods for producing and using such agents in therapeutic and diagnostic settings. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Current Opinion in Pharmacology 2015,24:113-118 Summary of the Invention

[0011] The present disclosure provides chimeric antigen receptors (CARs) generated against the MAGE-A4 peptide antigen in the context of MHC (HLA-A2). In some embodiments, the CAR sequence specifically binds to the small peptides MAGE-A4 286-294 or MAGE-A4 230-239 presented by HLA-A2.

[0012] In one aspect, the present disclosure provides a melanoma-associated antigen A4 (MAGE-A4)-specific chimeric antigen receptor (CAR), which interacts with amino acids 286-294, or 230-239, or a portion thereof, of SEQ ID NO: 32, and which specifically binds to an HLA-bound MAGE-A4 polypeptide. In some embodiments, the MAGE-A4-specific CAR comprises, from N-terminus to C-terminus, (a) an extracellular ligand-binding domain comprising an anti-MAGE-A4 antigen-binding domain, (b) a hinge, (c) a transmembrane domain, and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain. In some embodiments, the extracellular ligand-binding domain comprises an anti-MAGE-A4 single-chain variable fragment (scFv) domain comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR). In some embodiments, the anti-MAGE-A4 scFv domain comprises a first linker between the LCVR and the HCVR. In some embodiments, the MAGE-A4-specific CAR further comprises a second linker between the extracellular ligand-binding domain and the hinge. In some embodiments, the first linker and the second linker comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-26. In some embodiments, 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.

[0013] In some embodiments, the hinge, transmembrane domain, or both of the MAGE-A4-specific CAR are derived from a CD8α polypeptide. In some embodiments, the costimulatory domain comprises a 4-1BB costimulatory domain. In some embodiments, the hinge, transmembrane domain, or both are derived from a CD28 polypeptide. In some embodiments, the costimulatory domain comprises a CD28 costimulatory domain. In some embodiments, the signaling domain comprises a CD3 zeta signaling domain. In some embodiments, 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: 37. In some embodiments, the LCVR comprises LCDR1-LCDR2-LCDR3 comprising the amino acid sequences of SEQ ID NOs: 12-14-16, respectively. In some embodiments, the HCVR comprises CDRs of the HCVR comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the HCVR comprises HCDR1-HCDR2-HCDR3 comprising the amino acid sequences of SEQ ID NOs: 4-6-8, respectively.

[0014] In some embodiments, a MAGE-A4-specific CAR comprises an LCVR comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 37, or an amino acid sequence having 95% to 99% sequence identity to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 37, and an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, or an amino acid sequence having 95% to 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the LCVR comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 37, and the HCVR comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the LCVR comprises a complementarity-determining region (CDR) of the LCVR comprising the amino acid sequence of SEQ ID NO: 59. In some embodiments, the LCVR comprises LCDR1-LCDR2-LCDR3 comprising the amino acid sequences of SEQ ID NOs: 61-63-65, respectively. In some embodiments, the HCVR comprises the CDRs of the HCVR comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the HCVR comprises HCDR1-HCDR2-HCDR3 comprising the amino acid sequences of SEQ ID NOs: 53-55-57, respectively. In some embodiments, the MAGE-A4-specific CAR comprises an LCVR comprising the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence having 95% to 99% sequence identity to the amino acid sequence of SEQ ID NO: 59, and an HCVR comprising the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence having 95% to 99% sequence identity to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the LCVR comprises the amino acid sequence of SEQ ID NO: 59, and the HCVR comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the hinge comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the hinge comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD28 costimulatory domain comprises the amino acid sequence of SEQ ID NO: 45.In some embodiments, the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the MAGE-A4-specific CAR comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, the MAGE-A4-specific CAR comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, the MAGE-A4-specific CAR comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the chimeric antigen receptor specifically binds to one or more amino acids from positions 286 to 294 of SEQ ID NO: 32. In some embodiments, the MAGE-A4-specific CAR specifically binds to one or more amino acids from positions 230 to 239 of SEQ ID NO: 32. In some embodiments, the HLA is HLA-A2.

[0015] In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding a MAGE-A4-specific CAR described herein. In some embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 38. In some embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 48. In some embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 70. In some embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 72. In one aspect, the present disclosure provides a vector comprising the nucleic acid molecule described herein. In some embodiments, 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.

[0016] In one aspect, the present disclosure provides a cell comprising a nucleic acid molecule described herein or a vector described herein. In some embodiments, the cell is a human T cell. In some embodiments, the engineered cell comprises a chimeric antigen receptor described herein. In some embodiments, the engineered cell is an immune cell such as an immune effector cell or a T lymphocyte (e.g., an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte). In some embodiments, the engineered cell is a CD8+ cytotoxic T lymphocyte. In some embodiments, the engineered cell is for use in treating a cancer that expresses MAGE-A4, such as multiple myeloma or melanoma.

[0017] In one aspect, the disclosure provides engineered human T cells comprising a chimeric antigen receptor comprising, from N-terminus to C-terminus, (a) an extracellular ligand-binding domain comprising an anti-MAGE-A4 single-chain variable fragment (scFv) domain comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR); (b) a hinge; (c) a transmembrane domain; and (d) a cytoplasmic domain comprising a 4-1BB costimulatory domain or a CD28 costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the anti-MAGE-A4 scFv specifically binds to one or more amino acid residues at positions 286-294 of SEQ ID NO: 32. In some embodiments, the scFv domain comprises an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 2 / 10. In some embodiments, the scFv domain comprises an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 2 / 37. In some embodiments, the scFv domain specifically binds to one or more amino acid residues at positions 230-239 of SEQ ID NO: 32. In some embodiments, the scFv domain comprises an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO:51 / 59. In some embodiments, the hinge comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:28. In some embodiments, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO:29. In some embodiments, the hinge comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, the CD28 costimulatory domain comprises the amino acid sequence of SEQ ID NO:45. In some embodiments, the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO:30. In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO:47.In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO: 73.

[0018] In one aspect, the present disclosure provides a pharmaceutical composition comprising a genetically modified human T cell and a pharmaceutically acceptable carrier, wherein the genetically modified human T cell comprises a chimeric antigen receptor described herein. In some embodiments, the pharmaceutical composition comprises an engineered cell or engineered human T cell described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is for use in treating a cancer that expresses MAGE-A4, such as multiple myeloma or melanoma.

[0019] In one aspect, the disclosure provides for the use of a chimeric antigen receptor described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, an engineered cell described herein, or an engineered human T cell described herein in the manufacture of a medicament for the treatment of a cancer that expresses MAGE-A4, such as multiple myeloma or melanoma.

[0020] In one aspect, the present disclosure provides a method for enhancing T lymphocyte activity in a subject, comprising introducing into the subject a T lymphocyte comprising a chimeric antigen receptor described herein. In one aspect, the present disclosure provides a method for treating a subject suffering from cancer, comprising introducing into the subject a therapeutically effective amount of T lymphocytes comprising a chimeric antigen receptor described herein. In one aspect, the present disclosure provides a method for 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 a chimeric antigen receptor described herein. In one aspect, the present disclosure provides a method for providing anti-tumor immunity to a subject, comprising administering to the subject an effective amount of cells genetically modified to express a chimeric antigen receptor described herein. In some embodiments, the subject is 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.

[0021] In one aspect, the present disclosure provides a method of engineering a population of cells to express a chimeric antigen receptor, comprising: (a) introducing into a population of immune cells a nucleic acid molecule encoding a chimeric antigen receptor described herein, (b) culturing the population of immune cells under conditions for expression of the nucleic acid molecule, and (c) isolating the immune cells that express the chimeric antigen receptor on their surface. In some embodiments, the method further comprises obtaining the population of immune cells from a subject prior to introducing the nucleic acid molecule.

[0022] In one aspect, the present disclosure provides a method of treating a MAGE-A4-expressing cancer in a subject, comprising (a) manipulating a population of cells as described herein, and (b) reintroducing into the subject the population of cells that express the chimeric antigen receptor. In some embodiments, the MAGE-A4-expressing cancer is multiple myeloma.

[0023] In one aspect, the present disclosure provides an isolated antigen binding protein that competes for binding with a MAGE-A4-specific CAR described herein. In some embodiments, the isolated antigen binding protein is a CAR. In one aspect, the present disclosure provides an isolated antigen binding protein that binds to the same epitope as a MAGE-A4-specific CAR described herein. In some embodiments, the isolated antigen binding protein is a CAR.

[0024] In one aspect, the present disclosure provides an isolated, recombinant antibody or antigen-binding fragment thereof that specifically binds to a melanoma-associated antigen A4 (MAGE-A4) polypeptide, wherein the antibody has the following characteristics: (a) a concentration of about 10 -9 (b) binds to a MAGE-A4 polypeptide with an EC50 of less than M; (b) exhibits increased survival in animals with cancer after administration to the animal compared to comparable animals not administered; and / or (c) comprises one or more of: (i) three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised 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) comprised 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. In some embodiments, the MAGE-A4 polypeptide is an HLA-A2-bound MAGE-A4 polypeptide.

[0025] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an LCVR having the amino acid sequence of SEQ ID NO: 10. Optionally, the isolated antibody or antigen-binding fragment thereof comprises an HCVR / LCVR amino acid sequence pair of SEQ ID NO: 2 / 10. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an LCVR having the amino acid sequence of SEQ ID NO: 37. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO: 51. Optionally, the isolated antibody or antigen-binding fragment thereof comprises an HCVR / LCVR amino acid sequence pair of SEQ ID NO: 2 / 37.

[0026] In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO: 4; (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO: 6; (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO: 8; (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO: 12; (e) an LCDR2 domain having amino acid 14; and (f) an LCDR3 domain having the amino acid sequence of SEQ ID NO: 16.

[0027] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an LCVR having the amino acid sequence of SEQ ID NO:59.

[0028] In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO: 53; (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO: 55; (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO: 57; (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO: 61; (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO: 63; and (f) an LCDR3 domain having the amino acid sequence of SEQ ID NO: 65.

[0029] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 51 / 59.

[0030] In some embodiments, the isolated antibody or antigen-binding fragment thereof is an IgG1 antibody. In some embodiments, the isolated antibody or antigen-binding fragment thereof is an IgG4 antibody. In some embodiments, the isolated antibody or antigen-binding fragment thereof is a bispecific antibody.

[0031] In one aspect, the present disclosure provides a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof as described above or herein and a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some cases, the second therapeutic agent is selected from the group consisting of an anti-tumor agent, a steroid, and a targeted therapy.

[0032] In one aspect, the disclosure provides a polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR or LCVR of an antibody described above or discussed herein.

[0033] In one aspect, the present disclosure provides a vector comprising the polynucleotide discussed above.

[0034] In one aspect, the present disclosure provides a cell comprising the vector discussed above.

[0035] In one aspect, the present disclosure provides a method for treating a cancer that expresses MAGE-A4, the method comprising administering to a subject an antibody or antigen-binding fragment, or a pharmaceutical composition as described above or herein. In some embodiments, the pharmaceutical composition is administered in combination with a second therapeutic agent. In some cases, the second therapeutic agent is selected from the group consisting of an anti-tumor agent, a steroid, and a targeted therapy. Use of the antibodies, antigen-binding fragments thereof, and pharmaceutical compositions for treating a cancer that expresses MAGE-A4 or in the manufacture of a medicament for treating a cancer that expresses MAGE-A4 is also contemplated within the scope of the present disclosure.

[0036] Other embodiments will become apparent from review of the following detailed description. [Brief explanation of the drawings]

[0037] [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 an IRES:eGFP sequence for tracking CAR-transduced cells. [Figure 2A] Figure 2A shows in vitro cytotoxicity data demonstrating that CAR T cells targeting HLA-A2 / MAGE-A4286-294 exhibit enhanced cytotoxicity against A375 human melanoma tumor cells (open circles, dashed line) and A375 cells overexpressing HLA-A2 / MAGE-A4286-294 (filled circles, solid line, indicated by A375++) compared to control CAR T cells against A375 cells (A375 and A375++ can be seen as the top two curves). [Figure 2B]Figure 2B shows in vitro cytotoxicity data demonstrating that CAR T cells targeting HLA-A2 / MAGE-A4286-294 exhibit enhanced cytotoxicity against IM9 multiple myeloma cells (open circles, dashed line) and IM9 cell target cells overexpressing HLA-A2 / MAGE-A4286-294 (filled circles, solid line, shown as IM9++) compared to control CAR T cells against A375 cells (IM9 and IM9++ can be seen as the top two curves). [Figure 3A] Figure 3A shows tumor volume over time in mice bearing HLA-A2+MAGE-A4+A375 human melanoma tumor cell xenografts treated with control CAR T cells (anti-HLA-A2 / HPV16E7(11-19) CAR T cells). Of the five mice tested, none were tumor-free at the end of the study (animals were sacrificed on day 42). [Figure 3B] Figure 3B shows tumor volume over time in mice bearing HLA-A2+MAGE-A4+A375 human melanoma tumor cell xenografts treated with anti-HLA-A2 / MAGE-A4286-294 CAR T cells. All five mice tested were tumor-free at the end of the study (day 55). [Figure 4A] Figure 4A shows tumor volume over time in mice bearing HLA-A2+MAGE-A4+IM9 human multiple myeloma tumor cell xenografts treated with control CAR T cells (anti-HLA-A2 / HPV16E7(11-19) CAR T cells). Of the five mice tested, none were tumor-free at the end of the study (animals were sacrificed on day 29). [Figure 4B] Figure 4B shows tumor volume over time in mice bearing HLA-A2+MAGE-A4+IM9 human multiple myeloma tumor cell xenografts treated with anti-HLA-A2 / MAGE-A4286-294 CAR T cells. Of the five mice tested, three were tumor-free at the end of the study (day 52). DETAILED DESCRIPTION OF THE INVENTION

[0038] It should be understood that the invention described herein is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims. Any embodiment or feature of an embodiment can be combined with each other, and such combinations are expressly included within the scope of the present invention. Any specific value described above or herein can be combined with another related value described above or herein to enumerate ranges, where those values represent the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those 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 listed numerical value means that the value can vary by 1% or less from the listed value.For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0040] 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.

[0041] 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 set forth 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 or MAGE-A4 230-239) are with respect to SEQ ID NO: 32. As used herein, "MAGE-A4 286-294," "MAGE-A4(286-294)," and "MAGEA4 286-294 " may be used interchangeably. Similarly, "MAGE-A4 230-239," "MAGE-A4(230-239)," and "MAGEA4 230-239 " may be used interchangeably. The polypeptide sequence of MAGE-A4(286-294) (KVLEHVVRV) is given as SEQ ID NO: 33. The polypeptide sequence of MAGE-A4(230-239) (GVYDGREHTV) is given as SEQ ID NO: 49.

[0042] As used herein, "antibodies that bind to MAGE-A4" or "anti-MAGE-A4 antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize MAGE-A4. In some embodiments, antibodies that bind to MAGE-A4 interact with amino acids 286-294 of MAGE-A4 or amino acids 230-239 of MAGE-A4.

[0043] The terms "ligand-binding domain" and "antigen-binding domain" are used interchangeably herein and refer to the portion of a chimeric antigen receptor or corresponding antibody that specifically binds to a predetermined 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 are derived. For example, the chimeric antigen receptor construct discussed in the Examples comprises an scFv having a variable region derived from an anti-MAGE-A4 antibody. This anti-MAGE-A4 antibody is the "corresponding antibody" for the respective chimeric antigen receptor.

[0044] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds to or interacts with a specific 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 or 230-239) presented by HLA-A2. The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain (abbreviated herein as HC) comprises a heavy chain variable region (abbreviated herein as HCVR or V H The heavy chain constant region comprises three domains: C H 1. C H 2, and C H Each light chain (abbreviated herein as LC) comprises a light chain variable region (abbreviated herein as LCVR or V LThe light chain constant region comprises one domain (C L 1) V H Area and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L Each CDR consists of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of an 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 parallel analysis of two or more CDRs.

[0045] The term "antibody" as used herein also includes antigen-binding fragments of complete antibody molecules. "Antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and similar terms, as used herein, include naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind antigens to form complexes. Antibody-binding fragments of antibodies can be derived from complete antibody molecules using any suitable standard techniques, such as, for example, proteolytic or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add, or delete amino acids, etc.

[0046] Non-limiting examples of antibody-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 a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0047] An antigen-binding fragment of an antibody will typically contain at least one variable domain, which 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 Domain is V L In the antigen-binding fragment linked to the domain, V H Domains and V L The domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be a dimer, with the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H Domain or V L It may also include a domain.

[0048] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure 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 -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C LIn any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains 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 link between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or to one or more monomeric V H Domain or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, in which the domains are non-covalently associated (e.g., by disulfide bonds).

[0049] In certain embodiments, the anti-MAGE-A4 antibody from which the antigen-binding fragment is derived is derived from 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-specific 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., a mouse) have been grafted onto human framework sequences.

[0050] The antibody used to generate the anti-MAGE-A4 antigen-binding fragment may, in some embodiments, be a recombinant human antibody. The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (see below), antibodies isolated from a recombinant combinatorial human antibody library (see below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means, including splicing of human immunoglobulin gene sequences into 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 transgenic for human Ig sequences are used), thus improving the V H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but which may not naturally occur within the human antibody germline repertoire in vivo.

[0051] Human antibodies can exist in two forms related to hinge heterogeneity. In the first form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa in which dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, forming approximately 75-80 kDa molecules consisting of covalently linked light and heavy chains (half antibodies). These forms have been extremely difficult to separate, even after affinity purification.

[0052] 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 various intact IgG isotypes. H 2 or C H Antibodies with one or more mutations in the three regions are included, which may be desirable, for example, in production to improve the yield of the desired antibody form.

[0053] An antibody may be an isolated antibody. As used herein, "isolated antibody" 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 purposes of this disclosure. Isolated antibodies also include antibodies in situ within recombinant cells. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0054] The anti-MAGE-A4 antibodies or antigen-binding fragments thereof disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibody was 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 numerous antibodies and antibody-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within a domain 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 the mutated residue is found 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 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 antagonist or agonist 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.

[0055] 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 with, 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 substitution relative to any of the HCVR, LCVR, and / or CDR amino acid sequences described herein.

[0056] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes may include saccharide, phosphoryl, or sulfonyl moieties on an antigen.

[0057] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% nucleotide sequence identity of the nucleotide bases 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 can, 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 identifying a polypeptide 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 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 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 129% or at least 130% of the polypeptide sequence of SEQ ID NO:22 or SEQ ID NO:39, or a portion of SEQ ID NO:22 or SEQ ID NO:39 (e.g., an HCVR such as the sequence of SEQ ID NO:2, or an LCVR such as the sequence of SEQ ID NO:10 or SEQ ID In some embodiments, the disclosure provides a polypeptide comprising a sequence that is 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 any of the sequences of the present invention. In some embodiments, the disclosure provides a polynucleic acid encoding such a polypeptide.In some embodiments, the present disclosure provides a method for determining a 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%, 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 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 1109%, at least 1111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 1211%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 1391%, at least 1392%, at least 1393%, at least 1394%, Polynucleic acids are provided that contain sequences that are 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.

[0058] When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned using a program such as GAP or BESTFIT with a predetermined gap weight, share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. 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 with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter 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 upward 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 substitution groups 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 "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0059] Sequence similarity for polypeptides, also known 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 alignments and percent sequence identity of the best overlapping regions between the query and search sequences (see 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.

[0060] As used herein, the terms "nucleic acid" or "polynucleotide" refer to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by polymerase chain reaction (PCR), and fragments generated by 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), 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 pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. Additionally, the entire sugar moiety can be replaced with sterically 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.

[0061] The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a binding domain 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 domain that activates the T cell receptor to generate a chimeric protein that exhibits specific anti-target cell immune activity. Generally, CARs consist of an extracellular single-chain antibody binding domain (scFv) fused to the intracellular signaling domain 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.

[0062] The term "HLA" refers to the human leukocyte antigen (HLA) system or complex, which is a complex of genes that encode 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 cells.

[0063] 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 a variant HLA-A gene, and the β chain (β2-microglobulin) is an invariant β2-microglobulin molecule.

[0064] The term "HLA-A2" refers to one particular group of class I major histocompatibility complex (MHC) alleles at the HLA-A locus, with the alpha chain encoded by the HLA-A*02 gene and the beta chain encoded by the beta2-microglobulin or B2M locus.

[0065] As used herein, the term "vector" includes, but is not limited to, a viral vector, a plasmid, an RNA vector, or a linear or circular DNA or RNA molecule that may consist of a chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid. In some cases, vectors are capable of autonomous replication (episomal 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-strand RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), positive-strand RNA viruses such as rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), 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 viruses, mammalian C, B, and D viruses, HTLV-BLV complex viruses, and lentiviruses.

[0066] A "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 ligands that specifically bind to 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 CD83. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient immune response.

[0067] A "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, for example, by engagement of the TCR / CD3 complex with 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), intercellular 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 Toll ligand receptors, and ligands that specifically bind B7-H3.

[0068] 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.

[0069] As used herein, the term "extracellular ligand-binding domain" refers to an oligopeptide or polypeptide capable of binding to a ligand, e.g., a cell surface molecule. For example, the extracellular ligand-binding domain can 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 can act as ligands include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells. The extracellular ligand-binding domain can include LCVR and HCVR regions (e.g., formatted as an scFv), optionally joined by a linker.

[0070] As used herein, the term "subject" or "patient" includes all members of the animal kingdom, including non-human primates and humans. In one embodiment, the patient is a human with cancer (e.g., multiple myeloma or melanoma).

[0071] As used herein, the "signal transducing domain" or "signaling domain" of a CAR is involved in intracellular signal transduction following binding of the extracellular ligand-binding domain to a target, resulting in activation of immune cells and immune responses. In other words, the signaling domain is involved in activating 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 cytokine secretion. Thus, the term "signaling domain" 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 domains for use in CARs include the cytoplasmic sequences of T cell receptors and coreceptors, which act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same function. In some cases, the signaling domain 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 can include a signaling motif known as an ITAM immunoreceptor tyrosine-based activation motif. ITAMs are well-defined signal transduction motifs found in the cytoplasmic tails of various receptors that function as binding sites for tyrosine kinases of the syk / zap70 class. Exemplary 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 signal transduction domain of the CAR may comprise the CD3 zeta signal transduction domain (SEQ ID NO: 30).

[0072] Chimeric antigen receptor (CAR) 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 intracellularly and presented, for example, by HLA.

[0073] 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 an HLA, such as HLA-A2. In one embodiment of the present disclosure, the CAR described herein comprises an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (such as a signaling domain derived from CD3 zeta or FcR gamma), and / or one or more costimulatory signaling domains derived from a costimulatory molecule, such as, but not limited to, 4-1BB. In one embodiment, the CAR comprises a hinge or spacer region, such as a CD8 alpha hinge, between the extracellular binding domain and the transmembrane domain.

[0074] The binding domain or extracellular domain of a CAR provides the CAR with the ability to bind to a target antigen of interest. The binding domain (e.g., a ligand-binding domain or antigen-binding domain) can be any protein, polypeptide, oligopeptide, or peptide capable of specifically recognizing and binding to a biomolecule (e.g., a cell surface receptor or tumor protein, or a component thereof). Binding domains include naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partners for a biomolecule of interest. For example, as further described herein, the binding domain can be an antibody light chain and heavy chain variable region, or the light chain and heavy chain variable region can be joined together in a single chain and in either orientation (e.g., VL-VH or VH-VL). Various assays are known for identifying the binding domain of the present disclosure that specifically binds 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 domain of the chimeric antigen receptor is MAGE-A4 protein on the surface of a tumor cell (e.g., HLA-presented MAGE-A4 protein, such as HLA-A2-presented MAGE-A4 protein).

[0075] Exemplary ligand-binding domains include antigen-binding proteins, such as antigen-binding fragments of antibodies, such as scFvs and scTCRs, extracellular domains of receptors, ligands for cell surface molecules / receptors or their receptor-binding domains, and tumor-binding proteins. In certain embodiments, the antigen-binding domain included in the CAR of the present disclosure can be a variable region (Fv), CDR, Fab, scFv, VH, VL, domain antibody variant (dAb), camelid antibody (VHH), fibronectin 3 domain variant, ankyrin repeat variant, and other antigen-specific binding domains derived from other protein scaffolds.

[0076] In one embodiment, the binding domain of the CAR is an anti-MAGE-A4 single-chain antibody (scFv), which can be a mouse, human, or humanized scFv. Single-chain antibodies can be cloned from the V region genes of hybridomas specific to the desired target. Techniques that can be used to clone variable heavy chains (VH) and variable light chains (VL) are described, for example, in Orlandi et al., PNAS, 1989;86:3833-3837. Thus, in certain embodiments, the binding domain includes an antibody-derived binding domain, but can also be a non-antibody-derived binding domain. An antibody-derived binding domain can be an antibody fragment or a genetically engineered product of one or more antibody fragments, which fragments are involved in binding to antigens.

[0077] In certain embodiments, the CAR of the present disclosure may include linkers between various domains, added for proper spacing and conformation of the molecule. For example, in one embodiment, there may be a linker between the VH or VL binding domain, which may be 1 to 20 amino acids in length. In other embodiments, the linker between any of the domains of the chimeric antigen receptor may be 1 to 15 or 15 amino acids in length. 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 in length. In further embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Ranges inclusive of the numbers set forth herein are also included herein, e.g., linkers of 10 to 30 amino acids in length.

[0078] 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 a variety of lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0079] Exemplary flexible linkers include glycine polymers (G), glycine-serine polymers, where n is at least one 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 potentially capable of functioning as neutral tethers between domains 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, those skilled in the art will recognize that CAR designs can include fully or partially flexible linkers, such that the linker can include one or more moieties that provide a flexible linker and a less flexible structure to accommodate the desired CAR structure. Specific linkers include (G4S) as shown in SEQ ID NOs: 23-25. n and a linker, where n=1 to 3. Another exemplary linker is provided as SEQ ID NO: 26. The linker can be present 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.

[0080] The binding domain of a CAR may be followed by a "spacer" or "hinge," which refers to a region that distances the antigen-binding domain from the effector cell surface, allowing 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 located between the transmembrane (TM) and the binding domain. In certain embodiments, the hinge region is an immunoglobulin hinge region, and may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. Other exemplary 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).

[0081] The "transmembrane" region or domain is the portion of the CAR that anchors the extracellular binding moiety to the plasma membrane of immune effector cells and facilitates binding of the binding domain to the target antigen. The transmembrane domain can be a CD3 zeta transmembrane domain, but other transmembrane domains that can 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 domain is that of CD137. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the transmembrane domain is synthetic, in which case it will contain primarily hydrophobic residues such as leucine and valine.

[0082] The term "intracellular signaling domain" or "signaling domain" refers to a portion of a chimeric antigen receptor protein that transmits the message of effective CAR binding to a target antigen to the interior of immune effector cells, thereby inducing effector cell functions, such as cytotoxic activity, including activation, cytokine production, proliferation, and release of cytotoxic factors into CAR-bound target cells, or other cellular responses triggered by antigen binding to the extracellular CAR domain. The term "effector function" refers to a specialized function of a cell. T cell effector functions can be cytotoxic or non-cytotoxic activities, including, for example, cytolytic activity or cytokine secretion. Thus, the terms "intracellular signaling domain" or "signaling domain," used interchangeably herein, refer to the portion of a protein that transmits an effector function signal and instructs a cell to perform a specialized function. Typically, the entire intracellular signaling domain can be employed, although in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used instead of the entire domain, as long as it transmits the effector function signal. The term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal. The intracellular signaling domain, also known as the "signaling domain," is typically derived from a portion of the human CD3 or FcRy chain.

[0083] It is known that signals generated solely through the T cell receptor are insufficient for full T cell activation; secondary or costimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the T cell receptor (primary cytoplasmic signaling sequences), and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). Costimulatory-acting cytoplasmic signaling sequences may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0084] 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 specific embodiment, the intracellular signaling domain of the anti-MAGE-A4 CAR described herein is derived from CD3 zeta. In some embodiments, the signaling domain comprises the amino acid sequence of SEQ ID NO: 30.

[0085] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to a portion of a CAR that contains the intracellular domain 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 ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and CD83. Thus, while the present disclosure provides exemplary costimulatory domains derived from CD3 zeta and 4-1BB, other costimulatory domains are contemplated for use in the CARs described herein. The inclusion of one or more costimulatory signaling domains can enhance the efficacy and proliferation of T cells expressing a CAR receptor. The intracellular signaling and costimulatory signaling domains can be linked in tandem to the carboxyl terminus of the transmembrane domain in any order. In some embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO:29.

[0086] Although scFv-based CARs engineered to contain signaling domains 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 contain binding domains, hinges, transmembrane domains, and signaling domains derived from CD3 zeta or FcR gamma, along with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from CD28, CD137, CD134, and CD278), can more effectively direct anti-tumor activity and increased cytokine secretion, lytic activity, survival, and proliferation in CAR-expressing T cells in vitro, as well as in animal models and cancer patients (Milone et al., Molecular Therapy, 2009; 17:1453-1464; Zhong et al., Molecular Therapy, 2010; 18:413-420; Carpenito et al., PNAS, 2009; 106:3360-3365).

[0087] In various embodiments, an anti-MAGE-A4 CAR of the present disclosure comprises (a) an anti-MAGE-A4 scFv as a binding domain (e.g., an scFv having a binding region (e.g., CDR or variable domain) from an anti-MAGE-A4 antibody identified in Table 1), (b) a hinge region derived from human CD8 alpha, (c) a human CD8 alpha transmembrane domain, and (d) a human T cell receptor CD3 zeta chain (CD3) intracellular signaling domain, and optionally one or more costimulatory signaling domains, e.g., 4-1BB. In one embodiment, the various protein domains are arranged from amino-terminus to carboxyl-terminus in the following order: binding domain, hinge region, and transmembrane domain. The intracellular signaling domain and optional costimulatory signaling domain are linked in tandem to the carboxy-terminus of the transmembrane in any order 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 the coding sequences (5' to 3') 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 the coding sequences (5' to 3') 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.

[0088] 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 are sometimes referred to as "expression vectors." An isolated polynucleotide can be inserted into a vector using any suitable method known in the art, for example, but not limited to, digesting the vector with an appropriate restriction enzyme and then ligating it to an isolated polynucleotide with a matching restriction end. An expression vector can incorporate and express heterologous or modified nucleic acid sequences that encode 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 can contain various control sequences, which refer to nucleic acid sequences necessary for the transcription and, in some cases, 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 can also contain nucleic acid sequences that perform other functions, as discussed below. An expression vector may contain additional elements, for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, human cells for expression and a prokaryotic host for cloning and amplification.

[0089] Expression vectors may contain necessary 5' upstream and 3' downstream regulatory elements, such as promoter sequences such as the 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 the constitutive promoters of the 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. Human gene promoters, including but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter, may also be used. In certain embodiments, inducible promoters are also contemplated as part of vectors expressing chimeric antigen receptors. This provides a molecular switch that can turn expression of the polynucleotide sequence of interest on or off. Examples of inducible promoters include, but are not limited to, the metallothionine promoter, glucocorticoid promoter, progesterone promoter, or tetracycline promoter.

[0090] 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 contain 5' and 3' untranslated regulatory sequences, which may function as enhancer sequences, promoter regions, and / or terminator sequences that can promote 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 specific cell types, cell locations, or tissue types. Expression vectors may contain selectable markers for maintaining the vector in host or recipient cells.

[0091] In various embodiments, vectors are plasmids, autonomously replicating sequences, and transposable elements.Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses.Examples of animal virus categories that are useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). Examples of expression vectors include the Lenti-X™ Bicistronic Expression System (Neo) vector (Clontrch) for expression in mammalian cells, the pClneo vector (Promega), and 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 an expression vector for expression of the chimeric protein in mammalian cells.

[0092] In certain embodiments, the nucleic acid encoding the CAR of the present disclosure is provided in a viral vector. The viral vector can 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 can contain the coding sequence for various chimeric proteins described herein instead of non-essential viral genes. The vector and / or particle can be used to transfer DNA, RNA, or other nucleic acids into cells either in vitro or in vivo. Many forms of viral vectors are known in the art.

[0093] In certain embodiments, the viral vector that comprises the coding sequence of CAR described herein is retroviral vector or lentiviral vector.The term " retroviral vector " refers to the vector that comprises the structural and functional genetic elements that are mainly derived from retrovirus.The term " lentiviral vector " refers to the vector that comprises the structural and functional genetic elements outside LTR that are mainly derived from lentivirus.

[0094] Retroviral vectors as used herein can be derived from any known retrovirus (e.g., c-type retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse 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 viruses, HTLV-1 and HTLV-2, as well as human immunodeficiency viruses, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses.

[0095] As used herein, lentiviral vector refers to vectors derived from lentiviruses, a group (or genus) of retroviruses that cause slowly progressive diseases. Viruses in this group include HIV (including human immunodeficiency virus, HIV types 1 and 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 described by 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).

[0096] 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, F. M. 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 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. al. (1993) J. Immunol 150:4104-4115, U.S. Patent No. 4,868,116, U.S. Patent No. 4,980,286, PCT Application No. 89 / 07136, PCT Application No. 89 / 02468, PCT Application No. 89 / 05345, and PCT Application No. 92 / 07573).

[0097] 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 Type Culture Collection (ATCC), Rockville, Md. Sequences can also be chemically synthesized.

[0098] For the expression of anti-MAGE-A4 CAR, a vector can be introduced into a host cell to allow expression of the polypeptide within the host cell. Expression vectors can 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 can be appropriately selected by those skilled in the art, as described herein. For example, a promoter sequence can be selected to promote transcription of the 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 can be selected to enhance transcription of the polynucleotide. A selectable marker can be selected to allow selection of host cells into which the vector has been inserted from those that do not. For example, a selectable marker can be a gene that confers antibiotic resistance. A signal sequence can be selected to allow the expressed polypeptide to be transported out of the host cell.

[0099] For cloning polynucleotides, a vector can be introduced into a host cell (isolated host cell) to allow the vector itself to replicate, thereby amplifying copies of the polynucleotide contained therein. Cloning vectors generally contain sequence components, including, but not limited to, a replication origin, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements can be appropriately selected by those skilled in the art. For example, a replication origin can be selected to promote the autonomous replication of the vector in a host cell.

[0100] In certain embodiments, the present disclosure provides an isolated host cell comprising a vector provided herein. Host cells comprising the vector may be useful for expressing or cloning polynucleotides contained in the vector. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Prokaryotic cells suitable for this purpose include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Enterobacterehaceae, such as Serratia marcescans and Shigella, as well as Bacilli, e.g., B. subtilis and B. licheniformis, Pseudomonas, e.g., P. aeruginosa, and Streptomyces.

[0101] The CAR of the present disclosure is introduced into host cells using transfection and / or transduction techniques known in the art. As used herein, the terms "transfection" and "transduction" refer to the process by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid can be integrated into the DNA of the host cell or maintained extrachromosomally. The nucleic acid can be transiently maintained or stably introduced. Transfection can be achieved by various means known in the art, including, but not limited to, calcium phosphate-DNA coprecipitation, 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 viral or retroviral vectors by viral infection rather than by transfection. In certain embodiments, retroviral vectors are transduced by packaging the vector into virions before contacting cells. 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.

[0102] 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.

[0103] In particular, the CARs of the present disclosure are introduced into 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.

[0104] The present disclosure provides a method for producing immune effector cells expressing a CAR described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from a subject, such as a subject with tumor cells expressing MAGE-A4, so that the immune effector cells express one or more CARs described herein. In a specific embodiment, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered to the individual. In a further embodiment, the immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express a CAR. In this regard, the immune effector cells can be cultured before or after being genetically modified (i.e., transduced or transfected to express a CAR as described herein).

[0105] Prior to in vitro manipulation or genetic modification of immune effector cells as described herein, a source of cells can be obtained from a subject. In particular, immune effector cells for use with the CARs described herein include T cells. T cells can be obtained from many sources, such as peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL separation. In one embodiment, cells from an individual's circulating blood can be obtained by apheresis. The apheresis product typically contains 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 can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing. In one embodiment of the present disclosure, the cells are washed with PBS. In alternative embodiments, the washed solution may lack calcium, magnesium, or many, if not all, divalent cations. As will be appreciated by those skilled in the art, the washing step may be accomplished by methods known to those skilled in the art, such as using a semi-automated flow-through centrifuge. After washing, the cells may be resuspended in various biocompatible buffers or other saline solutions with or without buffers. In certain embodiments, undesirable components of the apheresis sample may be removed in the culture medium in which the cells are directly resuspended.

[0106] 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, can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be achieved with a combination of antibodies directed against surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. 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 can also be used to isolate cell populations of interest for use in the present disclosure.

[0107] PBMCs can be directly used for genetic modification with CARs using the methods described herein. In certain embodiments, after PBMC isolation, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or expansion. CD8+ cells can be obtained 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 exist 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 T cells 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 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.

[0108] 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- and CD45RO-positive. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-negative.

[0109] 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 (e.g., transduced with a viral vector containing a nucleic acid encoding a CAR) as described herein, and then activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Pat. Nos. 6,905,874, 6,867,041, 6,797,514, and WO 2012 / 079000. Generally, such methods involve contacting PBMCs or isolated T cells with stimulatory and costimulatory 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 beads serve as "surrogate" antigen-presenting cells (APCs). In other embodiments, T cells can be activated and stimulated to expand with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Pat. No. 6,040,177, U.S. Pat. No. 5,827,642, and WO2012 / 129514.

[0110] The present disclosure provides a population of engineered immune effector cells for the treatment of patients having malignant tumors caused by tumors that express MAGE-A4, such as multiple myeloma or melanoma, wherein the engineered immune effector cells comprise an anti-MAGE-A4 CAR disclosed herein.

[0111] The immune effector cells expressing CAR prepared as described herein can be used in methods and compositions for adoptive immunotherapy according to known techniques or variations thereof that will be apparent to those skilled in the art based on this disclosure.See, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al. Also see U.S. Patent No. 4,690,915 to Rosenberg.

[0112] In some embodiments, cells are formulated by first harvesting them from their culture medium, then washing and concentrating the cells in a medium and container system (a "pharmaceutically 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.

[0113] 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 10 2 Over 10 cells, up to 6 Up to 10 8 or 10 9 cells, and 10 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.

[0114] The cells can be autologous or xenogeneic to the patient undergoing therapy. Optionally, the treatment can also include administration of a mitogen (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 the induction of an immune response.

[0115] The CAR-expressing immune effector cell population of the present disclosure can be administered alone or in combination with other components, such as diluents and / or IL-2 or other cytokines or cell populations, as a pharmaceutical composition. Briefly, the pharmaceutical composition of the present disclosure can include a CAR-expressing immune effector cell population, such as T cells, described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers 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.

[0116] The anti-tumor immune response induced in a subject by administering T cells expressing the CAR described herein using the methods described herein or other methods known in the art can include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses, which can kill infected cells. A humoral immune response, primarily mediated by helper T cells, which can activate B cells and lead to antibody production, can also be induced. To analyze the type of immune response induced by the composition of the present disclosure, various techniques can be used, which are fully described in the art, for example, in 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.

[0117] Accordingly, 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.

[0118] In one embodiment, the present disclosure provides a method of treating a subject diagnosed with a MAGE-A4-expressing cancer, comprising depleting immune effector cells from the subject diagnosed with a MAGE-A4-expressing cancer, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor of the present disclosure, thereby producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the subject. In one embodiment, the immune effector cells comprise T cells.

[0119] 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 genetically modified precursor cells of immune effector cells that differentiate into mature immune effector cells expressing a CAR when introduced into the 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.

[0120] Binding characteristics of chimeric antigen receptors and corresponding antibodies As used herein, the term "binding" in the context of binding of a chimeric antigen receptor or corresponding antibody to a predetermined 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 a minimal interaction or association between two entities or molecular structures, such as antigen-binding domains, antigen interaction, etc.

[0121] For example, binding affinities, as determined by surface plasmon resonance (SPR) techniques on, for example, a BIAcore 3000 instrument using an antigen as the ligand and an antibody or chimeric antigen receptor as the analyte (or antiligand), are typically about 10 -8 M or less, about 10 -9 M or less, etc., 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).

[0122] Thus, a chimeric antigen receptor or corresponding antibody of the present disclosure has a K that is at least 10-fold lower than its affinity for binding to a nonspecific antigen (e.g., BSA, casein). D The chimeric antigen receptor or corresponding antibody of the present disclosure binds to a predetermined antigen or cell surface molecule (receptor) with an affinity corresponding to a K value. As described herein, the chimeric antigen receptor or corresponding antibody of the present disclosure can bind to an HLA-presented MAGE-A4 antigen, for example, an HLA-A2-presented MAGE-A4 antigen. According to the present disclosure, the K value 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 can be considered as undetectable binding.

[0123] "K D The term "(M)" refers to the dissociation equilibrium constant of a particular antigen-binding domain-to-antigen interaction, or the dissociation equilibrium constant of the corresponding antibody to the antigen. D There is an inverse relationship between K and binding affinity, and therefore, K D The smaller the value, the higher, i.e., stronger, the affinity. 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 terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction, i.e., a larger K D In some situations, 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) is 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 (higher or stronger affinity), e.g., 5-fold or 10-fold higher binding affinity as the case may be.

[0124] "k dThe term "(sec-1 or 1 / s)" refers to the dissociation rate constant of a particular antigen-binding domain-to-antigen interaction, or the dissociation rate constant of a chimeric antigen receptor or the corresponding antibody. Its value is k off Also called value.

[0125] "k a The term "(M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antigen-binding domain-to-antigen interaction, or the association rate constant of a chimeric antigen receptor or corresponding antibody.

[0126] "K A The term "(M-1 or 1 / M)" refers to the association equilibrium constant of a particular antigen-binding domain-to-antigen interaction, or the association equilibrium constant of a chimeric antigen receptor or the corresponding antibody. The association equilibrium constant is k a k d It is obtained by dividing by

[0127] "EC50" or "EC 50 The term "half-maximal effective concentration" refers to the concentration of chimeric antigen receptor that induces a response midway between baseline and maximum after a specified exposure time. 50 essentially represents the concentration of chimeric antigen receptor or antibody at which 50% of its maximal effect is observed. In certain embodiments, the EC 50 The EC value is equal to the concentration of a chimeric antigen receptor or corresponding antibody of the present disclosure that confers half-maximal binding to cells expressing an antigen (e.g., a tumor-associated antigen such as MAGE-A4) as determined, for example, by a FACS binding assay. Thus, reduced or weak binding is considered to be an EC 50 An increase in the concentration of α-glucan is observed at half-maximal effective concentrations.

[0128] In one embodiment, the reduction in binding is measured by measuring the EC2 concentration that allows half-maximal binding to target cells. 50 It can be defined as an increase in chimeric antigen receptor or corresponding antibody concentration.

[0129] Chimeric antigen receptor sequence variants A chimeric antigen receptor or the present disclosure may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the individual antigen-binding domains of the corresponding antibodies are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. A chimeric antigen receptor of the present disclosure may contain an antigen-binding domain derived from any of the exemplary CDR or variable region 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 corresponding antibody is 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 easily generate many antibodies containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll framework and / or CDR residues within the domain are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues are found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues are found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antigen-binding domain was originally derived). Furthermore, the antigen-binding domain 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 maintained or mutated to the corresponding residue in a different germline sequence.

[0130] Biological characteristics of chimeric antigen receptors and corresponding antibodies The present disclosure provides antibodies that have high affinity (e.g., nanomolar or subnanomolar K) for human MAGE-A4. D The present invention provides a chimeric antigen receptor having an antigen-binding domain derived from an antibody that binds at a specific target site (a specific target site).

[0131] According to certain embodiments, the present disclosure provides a medicament having a K of less than about 5 nM as measured by surface plasmon resonance. D

[0010] The present invention provides a chimeric antigen receptor having an antigen-binding domain derived from a corresponding antibody that binds to human MAGE-A4 at 25°C (e.g., at 25°C). In certain embodiments, the corresponding antibody has a K of less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, or less than about 25 pM as measured by surface plasmon resonance. DIt binds to MAGE-A4.

[0132] The present disclosure also provides chimeric antigen receptors having an antigen-binding domain derived from a corresponding antibody that binds to MAGE-A4 with a dissociation half-life (t) of greater than about 10 minutes or greater than about 125 minutes, as measured by surface plasmon resonance at 25° C. In certain embodiments, the corresponding antibody binds to MAGE-A4 with a t of greater than about 3 minutes, greater than about 4 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 110 minutes, or greater than about 120 minutes, as measured by surface plasmon resonance at 25° C.

[0133] The present disclosure also provides a chimeric antigen receptor having an antigen-binding domain derived from a corresponding antibody that specifically binds to a human cell line expressing endogenous MAGE-A4, as determined by a FACS binding assay.

[0134] The present disclosure also provides engineered cells that express a MAGE-A4-specific chimeric antigen receptor that (i) are activated by cells expressing MAGE-A4 and / or (ii) exhibit inhibition of tumor growth in immunocompromised mice bearing human multiple myeloma or melanoma xenografts.

[0135] Preparation of antigen-binding domains The antigen-binding domain of the chimeric antigen receptor of the present disclosure, which is 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 antibody of the present disclosure are derived from a chimeric antibody, a humanized antibody, or a fully human antibody. Methods for producing 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, selectivity, epitope, etc. As discussed herein, these human variable regions (or CDRs) can then be incorporated into the antigen-binding domain of the chimeric antigen receptor.

[0136] Polynucleotides and Vectors The present disclosure also provides polynucleotides and vectors encoding the chimeric antigen receptors discussed herein.

[0137] 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 insect host cells, or a lentivirus vector for transfection of mammalian host cells).

[0138] In various embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:21, or comprises a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide sequence of SEQ ID NO:22. In various embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:38, or comprises a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide sequence of SEQ ID NO:39. In various embodiments, the polynucleotide and / or vector comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0139] Methods for engineering immune cells to express chimeric antigen receptors The present disclosure provides a method for 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.

[0140] The present disclosure provides immune cells comprising a polynucleotide or lentiviral vector encoding one of the MAGE-A4-specific chimeric antigen receptors discussed herein. In some embodiments, these immune cells are used for immunotherapy (e.g., treating cancer).

[0141] 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), as described in WO2013 / 176915, which may 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 be more active or limit attrition by inactivating genes encoding proteins that act as "immune checkpoints" that act as regulators of T cell activation, such as PD1 or CTLA-4.

[0142] Engineered immune cells The present disclosure also provides immune cells (e.g., engineered immune cells) comprising the 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.

[0143] In some embodiments, the engineered immune cells are engineered human T cells that comprise a chimeric antigen receptor comprising, from N-terminus to C-terminus, (a) an extracellular ligand-binding domain comprising an anti-MAGE-A4 single-chain variable fragment (scFv) domain comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR); (b) a hinge; (c) a transmembrane domain; and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain.

[0144] In some embodiments, the scFv domain 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. In some embodiments, the scFv domain of the engineered human T cell comprises an HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO:2 / 37. Optionally, the hinge comprises the amino acid sequence of SEQ ID NO:27. Optionally, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:28. Optionally, the costimulatory domain is a 4-1BB costimulatory domain. Optionally, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO:29. Optionally, the signaling domain is a CD3 zeta signaling domain. Optionally, the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO:30.

[0145] 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: 39.

[0146] biological equivalent The present disclosure provides chimeric antigen receptors and engineered cells expressing chimeric antigen receptors that have amino acid sequences that differ from those of the exemplary molecules disclosed herein but that retain the ability to bind to MAGE-A4, activate immune cells expressing chimeric antigen receptors in the presence of cells expressing MAGE-A4, or inhibit the growth or proliferation of tumor cells expressing MAGE-A4. Such variant molecules may contain one or more amino acid additions, deletions, or substitutions compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described bispecific antigen binding molecules.

[0147] In one embodiment, two engineered immune cells expressing a chimeric antigen receptor of the present disclosure are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0148] In one embodiment, two engineered immune cells are biologically equivalent if a patient can switch between a reference product and a biological product one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity or diminished efficacy, compared to therapy sustained without switching between the reference product and the biological product.

[0149] In one embodiment, two engineered immune cells are biologically equivalent if they both act by a common mechanism of action for the conditions of use, to the extent such mechanism is known.

[0150] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals in which 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 correlate with and reasonably predict human in vivo bioavailability data, (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effects of the engineered cells are measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the engineered cells.

[0151] Biologically equivalent variants of the exemplary engineered cells described herein can 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.

[0152] Species selectivity and species cross-reactivity According to certain embodiments of the present disclosure, an antigen-binding domain is provided that binds to human MAGE-A4 but not to MAGE-A4 from other species. The present disclosure also provides an antigen-binding domain that binds to human MAGE-A4 and MAGE-A4 from one or more non-human species. In some embodiments, the antigen-binding domain of the present disclosure binds to MAGE-A4 286-294 or 230-239. In some embodiments, the MAGE-A4 to which the antigen-binding domain binds (e.g., MAGE-A4 286-294 or 230-239) is presented on the surface of a cell by HLA, such as HLA-A2.

[0153] According to certain exemplary embodiments of the present disclosure, antigen-binding domains are provided that bind to human MAGE-A4 and, in some cases, 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. Furthermore, binding to MAGE-A4 may be in the context of MHC-presented MAGE-A4, such as HLA-presented MAGE-A4. An exemplary HLA-presented MAGE-A4 is human MAGE-A4 bound by HLA-A2.

[0154] Activation and proliferation of engineered immune cells Whether before or after genetic modification of the engineered cells (e.g., T cells), immune cells, particularly T cells of the present disclosure, can generally be engineered using methods described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, even though the genetically modified immune cells of the present disclosure are activated and proliferate independent of antigen binding mechanisms. The T cells may be further activated and expanded using methods described in U.S. Patent Application Publication Nos. 6, 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.

[0155] Generally, the T cells of the present disclosure proliferate upon contact with agents that stimulate the CD3 TCR complex and costimulatory molecules on the surface of the T cells, generating a T cell activation signal. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or the mitogenic lectin-like phytohemagglutinin (PHA) can be used to generate a T cell activation signal.

[0156] As non-limiting examples, a population of T cells can be stimulated in vitro by contact with an anti-CD3 antibody or its antigen-binding fragment, 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, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate for stimulating T cell proliferation. Suitable conditions for T cell culture include an appropriate medium (e.g., minimal essential medium or RPMI medium 1640, or X-vivo5, (Lonza)), which may contain factors necessary for growth and survival, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp, and TNF-α, or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. Culture media may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo1, X-Vivo20, or Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in amounts sufficient for T cell growth and proliferation. Antibiotics such as penicillin and streptomycin are included only in experimental cultures and are not included in the culture of cells infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% O2). T cells exposed to various stimulation times may exhibit different characteristics.

[0157] In some embodiments, the cells may be expanded by co-culturing with tissue or cells. The cells may also be expanded in vivo in the blood of a subject, for example, after administration of the cells to the subject.

[0158] 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 pharmaceutically acceptable vehicle. In some cases, the engineered cell forms a pharmaceutical product, particularly for immunotherapy. In some cases, the engineered cell is used for the treatment of cancer (e.g., multiple myeloma or melanoma). In some cases, the engineered cell is used in the manufacture of a pharmaceutical product for immunotherapy and / or the treatment of cancer (e.g., a cancer that expresses MAGE-A4).

[0159] The present disclosure provides methods comprising administering to a subject in need thereof a therapeutic composition comprising engineered cells (e.g., T cells) expressing a chimeric antigen receptor as discussed herein. The therapeutic composition may comprise cells expressing any of the chimeric antigen receptors disclosed herein and a pharmaceutically acceptable carrier, diluent, or vehicle. As used herein, the phrase "subject in need thereof" refers to a human or non-human animal exhibiting 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.

[0160] The engineered cells 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 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 can be inhibited or killed using the engineered cells of the present disclosure include, for example, multiple myeloma cells, melanoma cells, or other solid tumor cells.

[0161] The engineered cells of the present disclosure can be used to treat diseases or disorders associated with MAGE-A4 expression, including, for example, cancers including, but not limited to, multiple myeloma, synovial sarcoma, esophageal cancer, head and neck cancer, lung cancer, bladder cancer, ovarian cancer, uterine cancer, gastric cancer, cervical cancer, breast cancer, and melanoma. The engineered cells of the present disclosure can generally be used to treat tumors that express MAGE-A4. According to other related embodiments of the present disclosure, methods are provided that include administering the engineered cells disclosed herein to a patient suffering from a tumor that expresses MAGE-A4, including tumors from the cancers listed above. Analytical / diagnostic methods known in the art, such as tumor scanning, can be used to identify whether the patient has such a tumor, disease, or condition.

[0162] 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.

[0163] According to certain aspects, the present disclosure provides methods for treating a disease or disorder associated with MAGE-A4 expression (e.g., cancer), comprising administering to a subject a population of engineered cells as described elsewhere herein after the subject has been determined to have the disease or disorder. For example, the present disclosure provides methods for treating a disease or disorder comprising administering to a patient engineered immune cells 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.

[0164] The treatments discussed herein can be ameliorative, curative, or preventative.The treatments can be either part of autoimmunotherapy or part of allogeneic immunotherapy.Autologous means that the cells, cell lines, or cell populations used for patient treatment are derived from the patient or human leukocyte antigen (HLA) compatible donors.Allogeneic means that the cells, cell lines, or cell populations used for patient treatment are derived from donors, not from patients.

[0165] Cells that can be used in the disclosed methods are described herein. The treatment can be used to treat patients diagnosed with pre-malignant or malignant cancer conditions characterized by an excess of cells that express MAGE-A4, particularly MAGE-A4. Such conditions can be found in cancer.

[0166] Cancer types that may be treated with the engineered cells 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.

[0167] The compositions and methods of the present disclosure can 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 would 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.

[0168] Administration of cells or populations of cells according to the present disclosure can be carried out in any convenient manner, including aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein can 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.

[0169] The administration of cells or cell populations is 10 per kg of body weight. 4 ~10 9 cells, preferably 10 5 ~10 6 The range may consist of administering 1000 cells / kg body weight or 1000 cells / kg body weight, including all integer values within these ranges. The cells or population of cells 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 patient's clinical condition. The cells or population of cells can be obtained from any source, such as a blood bank or donor. While individual needs vary, determining the effective amount range of a given cell type for a particular disease or condition is within the skill of the art. An effective amount refers to an amount that provides a therapeutic or prophylactic benefit. The dosage administered will depend on the recipient's age, health, and weight, the type and frequency of concurrent treatment, if any, and the nature of the desired effect.

[0170] In one embodiment, an effective amount of cells or a composition comprising the cells is administered parenterally. This administration can be intravenous. Optionally, administration can be by injection into a tumor.

[0171] In certain embodiments of the present disclosure, the cells are administered to a 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 present 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, cytoxin, fludarivine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.

[0172] In a further embodiment, the cell composition of the present disclosure is administered to a patient in conjunction with (e.g., before, simultaneously with, or after) T cell ablative therapy using bone marrow transplantation, chemotherapy 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 ablative therapy, such as an agent reactive 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 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 tumor burden before administration of the expanded immune cells of the present disclosure. In one embodiment, reducing tumor burden before administration of the engineered cells of the present disclosure can reduce or prevent the possibility of cytokine release syndrome or cytokine storm, a side effect that can be associated with CAR T cell therapy.

[0173] Combination therapy The present disclosure provides methods comprising administering engineered cells or populations 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 with or administered in combination with the cells or populations of cells 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 comprises a steroid. In some embodiments, the second therapeutic agent comprises a targeted therapy 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®), or 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 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 small molecule cytokine inhibitors and cytokine inhibitors, including 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. Pharmaceutical compositions of the present disclosure (e.g., pharmaceutical compositions comprising the engineered cells or populations of cells disclosed herein) may also be administered as part of a therapeutic regimen that includes one or more therapeutic combinations selected from monoclonal antibodies other than those described herein that can 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 conjugated 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®).

[0174] The present disclosure also includes therapeutic combinations comprising any of the engineered cells or populations of cells 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, where the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody, or antibody fragment (e.g., Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or other engineered molecules such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units). In some embodiments, the engineered cells or populations of cells of the present disclosure may also be administered as part of a treatment regimen that also includes radiation therapy and / or conventional chemotherapy.

[0175] The additional therapeutically active ingredient 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 administration regimens will be considered administration of the engineered cells "in combination with" the additional therapeutically active ingredient).

[0176] The present disclosure provides pharmaceutical compositions in which the engineered cells or populations of cells of the present disclosure are co-formulated with one or more of the additional therapeutically active ingredients described elsewhere herein.

[0177] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of engineered cells can be administered to a subject over a defined time course. The method according to this aspect includes sequentially administering multiple doses of cells to a subject. As used herein, "sequentially administering" means that each dose is administered to a subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure provides methods that include sequentially administering to a patient a single primary dose, followed by one or more secondary doses, and optionally one or more tertiary doses.

[0178] The terms "primary dose," "secondary dose," and "tertiary dose" refer to the time sequence of administration of engineered cells of the present disclosure. Thus, a "primary 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 primary dose, and a "tertiary dose" is a dose administered after the secondary dose. The primary, secondary, and tertiary doses may all contain engineered cells but generally may differ from one another in terms of frequency of administration. However, in certain embodiments, the amount of engineered cells contained in the primary, secondary, and / or tertiary doses will differ from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., two, three, four, or five) doses are administered as a "loading dose" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0179] 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, The phrase "immediately preceding dose" as used herein means a dose administered to a patient prior to administration of the next dose in the sequence, with no intervening doses, in a series of multiple administrations.

[0180] The method according to this aspect of the invention can 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.

[0181] In embodiments involving multiple secondary doses, each secondary dose may be administered at 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 at 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 administration frequency may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical testing. [Example]

[0182] The following examples are presented to provide those skilled 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.

[0183] Example 1: Generation of anti-MAGE-A4 antibodies Anti-MAGE-A4 antibodies are generated by transfecting genetically modified mice (e.g., engineered mice containing DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions) with human MAGE-A4 antigen (e.g., mAb 31345). hMAGE-A4 286-294 or for mAb33229 hMAGE-A4 230-239 ) and HLA-A2.

[0184] After immunization, splenocytes were harvested from each mouse and either (1) fused with mouse myeloma cells to maintain their viability and form hybridoma cells that were screened for MAGE-A4 specificity, or (2) B cells were sorted using human MAGE-A4 fragments as selection reagents that bind to and identify reactive antibodies (antigen-positive B cells) (as described in U.S. Patent Publication No. 2007 / 0280945A1).

[0185] Chimeric antibodies against MAGE-A4 were first isolated, containing human variable regions and mouse constant regions. The antibodies were characterized and selected for desirable characteristics, including affinity, selectivity, etc. If necessary, the mouse constant regions were replaced with desired human constant regions, such as wild-type or modified IgG1 or IgG4 constant regions, to generate fully human anti-MAGE-A4 antibodies. While the constant region selected can vary depending on the specific application, the high-affinity antigen binding and target specificity characteristics reside in the variable regions.

[0186] 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 mAb31345 and mAb31345* sequences in Table 1 are identical except for one extra C-terminal amino acid in the "called" LCVR sequence of mAb31345* (i.e., when annotating the LCVR region, the full-length antibodies are identical but one additional amino acid has been assigned to the LCVR of mAb31345*). The corresponding nucleic acid sequence identifiers are listed in Table 2. A summary of all sequences contained herein is provided in Table 56. [Table 1] [Table 2]

[0187] Example 2: Generation of MAGE-A4-specific chimeric antigen receptors The anti-MAGE-A4 31345 and 33229 antibodies in Table 1 were reformatted into VL-VH single-chain variable fragments (ScFvs) and placed into chimeric antigen receptor (CAR) constructs 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 antibodies corresponding to SEQ ID NOs: 1 and 36, respectively. The full-length nucleic acid and polypeptide heavy chain sequences of the corresponding 31345 anti-MAGE-A4 antibody correspond to SEQ ID NOs: 17 and 18, respectively. The full-length nucleic acid and polypeptide light chain sequences of the corresponding 31345 anti-MAGE-A4 antibody correspond to SEQ ID NOs: 19 and 20, respectively. Full-length nucleic acid and polypeptide 31345 HLA-A2 / MAGE-A4 286-294The CAR sequences targeting MAGE-A4 correspond to SEQ ID NOs: 38 and 39, respectively. As a non-binding control, a similar CAR was designed using the nucleotide sequence of an unrelated scFv (CAR construct of SEQ ID NO: 34, the polypeptide sequence of the control CAR corresponds to SEQ ID NO: 35). The MAGE-A4-specific CAR was cloned into a lentiviral expression vector (Lenti-X™ Bicistronic Expression System (Neo), Clontech Cat# 632181), and lentiviral particles were generated via the Lenti-X Packaging Single Shot (VSV-G) System (Clontech Cat# 631276) according to the manufacturer's protocol. Jurkat cells (Jurkat / NKFBLuc cl 1C11) engineered to express an NFKB-luciferase reporter were then transduced with the CAR construct using RetroNectin® pre-coated dishes (Clontech, Cat# T110a) according to the manufacturer's protocol. After selection with 500 μg / ml G418 (Gibco, Cat# 11811-098) for at least two weeks, the following CAR T cell line was generated: Jurkat / NKFBLuc cl 1C11 / MAGE-A4(286-294)31345 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.

[0188] Example 3: 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 the MAGE-A4 CAR construct in Jurkat / Jurkat / NKFBLuc cells were assessed by flow cytometry. To stain, cells were seeded at a density of 200,000 cells per well in a 96-well V-bottom plate in a staining buffer of calcium- and magnesium-free PBS (Irving 9240) and 2% FBS (ATCC 30-2020) and stained with 10 μg / ml Protein L (Genscript Biotin Protein L) for 30 minutes at 4°C. After incubation, cells were washed once with staining buffer and stained with 0.5 μg / ml streptavidin-Alexa-647 secondary antibody (Biolegend) for 30 minutes at 4°C. Cells were then washed and fixed using a 50% solution of BD Cytofix (Becton Dickinson) diluted in staining buffer. Samples were run on an Intellicyt iQue flow cytometer and analyzed with FlowJo 10.2 to calculate mean fluorescence intensity (MFI). The percentage of protein L positive cells (Table 3) was calculated by taking the percentage of protein L positive cells with respect to the total number of cells.

[0189] The activity of CAR T cell lines was assessed 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 containing 10% FBS and 1% P / S / G) were added to a Thermo-Nunc 96-well white plate (Thermo Fisher Scientific), followed by 3-fold serial dilutions of APCs (500,000 cells to 685 cells) in 50 μl of assay medium. The following APCs were used: IM9 (which endogenously expresses the MAGE-A4 286-294 peptide) and HEK293 (which is MAGE-A4 286-294 negative). The cell mixture was incubated for 5 hours in a humidified incubator at 37°C and 5% CO2. NFKB-luciferase activity was measured using a Promega One-Glo and Perkin Elmer Envision plate reader. Relative luciferase units (RLU) were generated and plotted in GraphPad Prism using a four-parameter logistic equation on eight-point response curves. A zero APC condition for each dose-response curve was also included in the analysis as a series of three-fold serial dilutions and represented as the lowest dose. Maximum CAR T activity was determined by taking the ratio of the highest to the lowest RLU on the curve and is represented in Table 4 as signal:noise (S:N). Protein L staining and MAGE-A4 CAR T cell activation results are shown in Tables 3 and 4. [Table 3]

[0190] Table 3 shows the percentage of CAR-positive Jurkat / NFKBLuc cells as measured by protein L staining. The MAGE-A4-specific CAR generated from mAb31345 was expressed in 49.9% of cells, whereas the non-targeting control CAR was expressed in 67.2% of cells, with CAR expression only seen in 3.9% of negative control (Jurkat / NFKBLuc cl.1C11) cells. [Table 4]

[0191] Table 4 shows that the HLA-A2:MAGE-A4(286-294)31345 CAR T cell line was activated by IM9 cells with a signal-to-noise ratio of 35.0, but the HLA-A2:MAGE-A4(286-294) CAR T cell line was not activated by the HEK293 negative control cell line.

[0192] Example 4: Generation of CAR T cells expressing a MAGE-A4-specific CAR The CAR of Example 2 (comprising an anti-MAGE-A4 VL-VH scFv, a huCD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain, constructed using the HCVR and LCVR nucleotide sequences of the MAGE-A4 antibody corresponding to SEQ ID NOs: 1 and 36, respectively) and the non-binding control CAR of Example 2 (the CAR construct of SEQ ID NO: 34; the polypeptide sequence of the control CAR corresponds to SEQ ID NO: 35) were cloned into a pLVX lentiviral vector with an EF1a promoter and an IRES:eGFP sequence (to track CAR-transduced cells) to produce VSV-pseudotyped lentivirus. CD3+ T cells were then isolated from human peripheral blood mononuclear cells (PBMCs), stimulated with CD3 / CD28 microbeads and 100 U / ml recombinant human IL-2, and transduced with the lentivirus at an MOI of 5. Transduced cells were grown with CD3 / CD28 microbeads and 100 U / ml recombinant human IL-2 for 19 days and then cryopreserved until use in in vivo experiments. These lines of CAR T cells were used to evaluate their cytotoxicity in vitro and efficacy in reducing tumor burden in vivo.

[0193] Example 5: MAGE-A4-specific CAR T cells mediate cytolysis of cells expressing MAGE-A4 As described above, CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs), stimulated with CD3 / CD28 microbeads and 100 U / ml recombinant human IL-2, and transduced with lentivirus at an MOI of 5. Transduced cells were grown with CD3 / CD28 microbeads and 100 U / ml recombinant human IL-2 for 19 days before setting up a cytolytic assay.

[0194] To measure the cytolytic potential of MAGE-A4-targeting chimeric antigen receptor (CAR) T cells, we performed a cytolytic assay using expanded CAR T cells and various tumor target cell lines expressing various levels of MAGE-A4. On day 19 of expansion, expanded CAR T cells were co-cultured in triplicate with calcein-labeled MAGE-A4+ or control target cell lines at various ratios. Calcein is a cell-permeable fluorescent dye with excitation and emission wavelengths of 495 nm and 515 nm, respectively. After 2.5 hours, the percentage of cytotoxicity was calculated based on the amount of calcein dye released from target cells upon loss of membrane integrity: ((calcein signal - spontaneous calcein release) / (maximum calcein release - spontaneous calcein release)) * 100. To determine maximum calcein release, target cells were treated with a 1% solution of Triton-X-114 detergent over the course of the assay. To measure spontaneous calcein release, MAGE-A4+ target cells were labeled with calcein and cultured in the absence of CAR T cells.

[0195] 2-hour calcein cytotoxicity assay: At the time of harvest, expanding CAR T cells were washed and resuspended in Optmizer cell culture medium. Each target cell line was harvested and 2x10 6 After resuspension at a density of 1 / mL, calcein-AM dye was added at a concentration of 8 μM for 35 minutes at 37°C. After calcein labeling, the target cells were washed twice to remove excess calcein. Subsequently, T cells and target cells were co-cultured at various ratios in a 96-well round-bottom plate and incubated for 2.5 hours at 37°C before harvesting the culture supernatant. For the negative control, HLA-A2-bound MAGE-A4 was used. 286-294Target cells were co-cultured with T cells generated using a similar CAR designed to contain an irrelevant scFv that does not recognize HLA-A2. The CAR scFv control was an anti-HLA-A2 / HPV16E7(11-19) scFv (VL-VH orientation). The scFv was introduced into the cells using a lentivirus vector. As a CAR-negative control, untransduced, expanded T cells from the same normal healthy donor were used. As a control for antigen-specific CAR T cell-mediated killing, the 293T human embryonic kidney cell line, which expresses the SV40 antigen, was used because this cell line is negative for MAGE-A4 expression. To determine whether calcein was spontaneously released from MAGE-A4+ target cell lines, each cell line was cultured in the absence of CAR T cells. To determine the maximum possible release of calcein, target cell lines were cultured and lysed using Optmizer medium supplemented with 1% Triton™ X-114 detergent. Relative calcein levels were measured in the supernatants using a Viktor X4 plate reader, and the percentage of cytotoxicity was calculated as ((calcein signal-spontaneous calcein release) / (maximum calcein release-spontaneous calcein release))*100.

[0196] Cell lines used for this assay included: A375 human melanoma tumor cell line (ATCC®, cell line number: CRL-9068™), MAGE-A4 286-294 A375 human melanoma tumor cell line (ATCC, cell line number: CRL-9068™), IM9 multiple myeloma cell line (DSMZ, CAT#: ACC569), MAGE-A4, engineered to overexpress peptide-loaded HLA-A2. 286-294 The IM9 multiple myeloma cell line (DSMZ, CAT#: ACC569™) engineered to overexpress peptide-loaded HLA-A2, the 293T human embryonic kidney cell line (ATCC®, cell line number: CRL-3216™) expressing the SV40 antigen, and the HPV16E7 11-19CaSKI cervical epidermoid carcinoma (ATCC® CRL-1550™) engineered to overexpress peptide-loaded HLA-A2.

[0197] As shown in Figures 2A and 2B and Tables 5, 6, and 7, HLA-A2 / MAGE-A4 generated using 31345 scFv-induced A375 cells 286-294 Cultures consisting of CAR T cells targeting HLA-A2 / MAGE-A4 (Figure 2A: open circle, dashed line). 286-294 A375 cells (Fig. 2A: filled circle, solid line, indicated by A375++), IM9 cells (Fig. 2B: open circle, dashed line), and HLA-A2 / MAGE-A4 overexpressing 286-294 IM9 cells overexpressing HLA-A2 / MAGE-A4 (Figure 2B: indicated by black circles, solid lines, and IM9++). Higher levels of cytotoxicity were observed against endogenous IM9 cells compared with A375 cells. 286-294 Overexpression of MAGE-A4 did not increase the level of cytotoxicity compared to endogenous A375 cells. Without wishing to be bound by theory, this result may be because IM9 cells express higher levels of MAGE-A4 antigen than A375 cells. [Table 5] [Table 6] [Table 7]

[0198] As seen in Tables 5, 6, and 7, when co-cultured with MAGE-A4+ target cells, untransduced, expanded (MOI 0) T cells and CAR T cells expressing CARs not specific for MAGE-A4 (A375, A375++, IM9, IM9++) were unable to induce significant target cell lysis against MAGE-A4+ tumor cells, even at a maximum ratio of 50 T cells to one target cell. These results support the conclusion that the CAR constructs are HLA-A2 / MAGE-A4286-294 Furthermore, this indicates that cell lysis was observed only when the 31345 scFv, which recognizes HLA-A2 / MAGE-A4, was included. 286-294 CAR T cells targeting MAGE-A4 showed negligible cytotoxicity against 293T cells lacking MAGE-A4 expression, indicating that MAGE-A4 expression is required to observe cell lysis.

[0199] Example 6: CAR T cells targeting MAGE-A4 reduce the growth of MAGE-A4-expressing tumors in vivo in a xenogeneic melanoma model HLA-A2 / MAGE-A4 286-294 To determine the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting NOD.Cg-Prkdc mice, a xenograft study was performed in mice using A375 human melanoma tumor cells expressing MAGE-A4. scid Il2rg(trademark) 1Wjl 5 × 10 6 HLA-A2 + MAGE-A4 + A375 human melanoma tumor cells were injected subcutaneously. After allowing sufficient time for tumor establishment, mice were injected with a control CAR (anti-HLA-A2 / HPV16E7) on day 10 of the experiment. 11-19 scFv, VL-VH orientation) or anti-HLA-A2 / MAGE-A4 286-294 4 x 10 expressing either CAR (as determined by the frequency of cells expressing GFP, a marker for CAR-transduced cells) 6 Specifically, mice (n=5 per group) were intravenously injected with 4×10 T cells. 6 4 × 10 irrelevant scFc CAR T cells (control scFv CAR) or 4 × 10 encoding the 31345 scFv CAR 6 Anti-MAGE-A4 286-294 Patients were administered either CAR T cells or IFN-γ. Tumor growth was assessed by measuring tumor volume up to day 52.

[0200] To determine tumor volume by external caliper, the maximum major axis (length in mm) and maximum transverse axis (width in mm) were determined. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2. [Table 8] [Table 9]

[0201] As shown in Tables 8 and 9 and Figures 3A and 3B, A375 tumors grew progressively in mice receiving irrelevant scFv CAR T cells, but not in mice receiving 31345 anti-HLA-A2 / MAGE-A4 286-294 CAR T cells encoding scFv CAR suppressed the growth of and subsequently eradicated established A375 tumors in vivo.

[0202] Example 7: CAR T cells targeting MAGE-A4 reduce the growth of MAGE-A4-expressing tumors in vivo in a heterogeneous multiple myeloma model HLA-A2 / MAGE-A4 286-294 To determine the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting NOD.Cg-Prkdc mice, a xenogeneic tumor study was performed in mice using IM9 human multiple myeloma tumor cells expressing MAGE-A4. On day 0, immunodeficient NOD.Cg-Prkdc mice were transfected with CAR T cells. scid Il2rg(trademark) 1Wjl 5 × 10 6 HLA-A2 + MAGE-A4 + IM9 human multiple myeloma tumor cells were injected subcutaneously. After allowing sufficient time for tumor establishment, mice were injected with a control CAR (anti-HLA-A2 / HPV16E7(11-19) scFv, VL-VH orientation) or anti-HLA-A2 / MAGE-A4 scFv on day 7 of the experiment. 286-2944 x 10 expressing either CAR (as determined by the frequency of cells expressing GFP, a marker for CAR-transduced cells) 6 Specifically, mice (n=5 per group) were intravenously injected with 4×10 T cells. 6 4 × 10 irrelevant scFc CAR T cells (control scFv CAR) or 4 × 10 encoding the 31345 scFv CAR 6 Anti-MAGE-A4 286-294 Patients were administered either CAR T cells or IFN-γ. Tumor growth was assessed by measuring tumor volume up to day 52.

[0203] To determine tumor volume by external caliper, the maximum major axis (length in mm) and maximum transverse axis (width in mm) were determined. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2. [Table 10] [Table 11]

[0204] As shown in Tables 10 and 11 and Figures 4A and 4B, IM9 tumors grew progressively in mice receiving irrelevant scFv CAR T cells, but not in mice receiving 31345 anti-HLA-A2 / MAGE-A4 286-294 CAR T cells encoding the scFv CAR suppressed the growth of and subsequently eradicated established IM9 tumors in three of five mice in vivo.

[0205] Example 8: CAR T cells targeting MAGE-A4 reduce the growth of MAGE-A4-expressing tumors in vivo in a xenogeneic A375 melanoma model V L -V H Anti-HLA-A2 / MAGEA4 286-294A chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR), or 2) the huCD28 hinge / transmembrane / costimulatory domain and CD3z signaling domain (28 / z CAR) was used in combination with anti-HLA-A2 / MAGEA4 scFv. 286-294 V of antibody, mAb31345 L and V H As a non-binding control, the BB / z CAR was designed using the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain in addition to the scFv. These CARs were cloned into the pLVX lentiviral vector with an EF1a promoter and an IRES:eGFP sequence (to track CAR-transduced cells) to produce VSV-pseudotyped lentivirus.

[0206] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) from two normal donors ("Donor 1" and "Donor 2"), stimulated with CD3 / CD28 microbeads and 100 U / ml recombinant human IL-2, and transduced with lentivirus at an MOI of 5. Transduced cells were grown with CD3 / CD28 microbeads and 100 U / ml recombinant human IL-2 for 19 days and then cryopreserved until use in in vivo experiments.

[0207] HLA-A2 / MAGEA4 286-294 A xenogeneic tumor study was conducted to determine the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting immunodeficient NOD.Cg-Prkdc tumors. scid Il2rg(trademark) 1Wjl 5 × 10 6 HLA-A2 + MAGEA4 + A375 human melanoma tumor cells were injected subcutaneously. Mass spectrometry was used to determine whether A375 melanoma cells express MAGEA4. 286-294The mice were determined to express approximately 424 cell surface copies of the peptide. Thirteen days after tumor establishment, mice (N=5 per group) were injected with a non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 CAR (anti-HLA-A2 / MAGEA4 CAR), and a non-binding control BB / z CAR (control CAR T) from two different donors. 286-294 BB / z CAR, or anti-HLA-A2 / MAGEA4 286-294 4 x 10 expressing either 28 / z CAR (as determined by the frequency of cells expressing GFP, a marker for CAR-transduced cells) 6 T cells were injected intravenously. Tumor growth was assessed by measuring tumor volume up to day 64.

[0208] To determine tumor volume by external caliper, the maximum major axis (length in mm) and maximum transverse axis (width in mm) were determined. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2.

[0209] Donor 1: A375 tumors received control CAR T cells or MAGEA4 286-294 BB / z CAR T cells proliferated in mice receiving either MAGEA4 or BB / z CAR T cells. 286-294 28 / z inhibited the growth of established A375 tumors in vivo, with 1 in 5 mice remaining tumor-free at day 64. 286-294 MAGEA4 for BB / z CAR 286-294 The enhanced efficacy of 28 / z CAR was confirmed, with tumor sizes on days 27, 29, 33, 36, 40, and 44 statistically significant at p<0.0001 by two-way ANOVA test.

[0210] Donor 2: A375 tumors grew progressively in mice that received control CAR T cells. MAGEA4 286-294 BB / z and MAGEA4 286-294 Treatment with both 28 / z CAR T cells inhibited A375 tumor growth, but with different kinetics. 286-294The 28 / z CAR T cells acted with faster kinetics, eradicating tumors in 5 of 5 mice by day 27. 286-294 BB / z CAR T cells acted with slower kinetics, eradicating tumors in 4 of 5 mice by day 44. 286-294 MAGEA4 for BB / z CAR 286-294 The enhanced kinetics of antitumor activity of 28 / z CAR was confirmed, with tumor sizes at days 19 and 22 statistically significant at p=0.0071 and p=0.0008 by two-way ANOVA, respectively. Tables 12-27 provide summaries of these data. [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27]

[0211] These results demonstrate that the MAGE-A4-specific CAR of the present disclosure has potent anti-tumor effects in vivo.

[0212] Example 9: CAR T cells targeting MAGE-A4 reduce the growth of MAGE-A4-expressing tumors in vivo in a xenogeneic SK-MEL-37 melanoma model V L -V H Anti-HLA-A2 / MAGEA4 286-294 A chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR), or 2) the huCD28 hinge / transmembrane / costimulatory domain and CD3z signaling domain (28 / z CAR) was used in combination with anti-HLA-A2 / MAGEA4 scFv. 286-294 V of antibody, mAb31345 L and V H As a non-binding control, BB / z CARs were designed using the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain in addition to different scFvs. These CARs were cloned into the pLVX lentiviral vector with an EF1a promoter and an IRES:eGFP sequence (to track CAR-transduced cells) to produce VSV-pseudotyped lentivirus.

[0213] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) from two normal donors ("Donor 1" and "Donor 2"), stimulated with CD3 / CD28 microbeads and 100 U / ml recombinant human IL-2, and transduced with lentivirus at an MOI of 5. Transduced cells were grown with CD3 / CD28 microbeads and 100 U / ml recombinant human IL-2 for 19 days and then cryopreserved until use in in vivo experiments.

[0214] HLA-A2 / MAGEA4 286-294 A xenogeneic tumor study was conducted to determine the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting immunodeficient NOD.Cg-Prkdc tumors. scid Il2rg(trademark) 1Wjl 5 × 10 6 HLA-A2 + MAGEA4 + SK-MEL-37 human melanoma tumor cells were injected subcutaneously. Mass spectrometry was used to confirm that SK-MEL-37 melanoma cells express MAGEA4. 286-294 The mice were determined to express approximately 1,326 cell surface copies of the peptide. Seven days after tumor establishment, mice (N=5 per group) were injected with a non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 CAR (anti-HLA-A2 / MAGEA4 CAR), and a non-binding control BB / z CAR (control CAR T) from two different donors. 286-294 BB / z CAR, or anti-HLA-A2 / MAGEA4 286-294 4 x 10 expressing either 28 / z CAR (as determined by the frequency of cells expressing GFP, a marker for CAR-transduced cells) 6 T cells were injected intravenously. Tumor growth was assessed by measuring tumor volume up to day 64.

[0215] To determine tumor volume by external caliper, the maximum major axis (length in mm) and maximum transverse axis (width in mm) were determined. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2. [Table 28]

[0216] Donor 1: SK-MEL-37 tumors received control CAR T cells or MAGEA4 286-294 BB / z CAR T cells proliferated in mice receiving either MAGEA4 or BB / z CAR T cells. 286-294 28 / z suppressed the growth of established SK-MEL-37 tumors in vivo. 286-294 MAGEA4 for BB / z CAR 286-294 The efficacy of 28 / z CAR was observed from tumor size on days 31, 35, 40, 47, 55, and 62, which was statistically significant at p<0.0001 by two-way ANOVA test. Donor 2: SK-MEL-37 tumors grew progressively in mice that received control CAR T cells. MAGEA4 286-294 Treatment with BB / z CAR T cells was effective, slowing tumor growth by approximately one week and suppressing MAGEA4 286-294 28 / z CAR T cells potently suppressed SK-MEL-37 tumor growth, rendering tumors undetectable (non-palpable) in 5 of 5 mice by day 20. These tumors remained undetectable until days 62–69, when tumors recurred.

[0217] Taken together, the results demonstrate that the MAGE-A4-specific CARs of the present disclosure exhibit anti-tumor activity and anti-tumor kinetics in vivo (Tables 29-45). [Table 29] [Table 30] [Table 31] [Table 32] [Table 33] [Table 34] [Table 35] [Table 36] [Table 37] [Table 38] [Table 39] [Table 40] [Table 41] [Table 42] [Table 43] [Table 44] [Table 45]

[0218] Example 10: CAR T cells targeting MAGE-A4 (230-239) reduce the growth of MAGE-A4-expressing tumors in vivo in a xenogeneic A375 melanoma model V L -V H Anti-HLA-A2 / MAGEA4 230-239A chimeric antigen receptor containing either 1) the huCD8 hinge / transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (BB / z CAR), or 2) the huCD28 hinge / transmembrane / costimulatory domain and CD3z signaling domain (28 / z CAR) was used as the V-antigen of mAb33229. L and V H As a non-binding control, 28 / z CARs were designed using the huCD28 hinge / transmembrane / co-stimulatory domain and the CD3z signaling domain in addition to an irrelevant scFv. These CARs were cloned into the pLVX lentiviral vector with the EF1a promoter and P2A:eGFP sequence (to track CAR-transduced cells) to produce VSV-pseudotyped lentivirus.

[0219] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) from normal donors, stimulated with CD3 / CD28 microbeads and 100 U / ml recombinant human IL-2, and transduced with lentivirus at an MOI of 5. Transduced cells were grown with CD3 / CD28 microbeads and 100 U / ml recombinant human IL-2 for approximately 14 days and then cryopreserved until use in in vivo experiments.

[0220] Anti-HLA-A2 / MAGEA4 230-239 A xenogeneic tumor study was conducted to determine the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting immunodeficient NOD.Cg-Prkdc tumors. scid Il2rg(trademark) 1Wjl 5 × 10 6 HLA-A2 + MAGEA4 + A375 human melanoma tumor cells were injected subcutaneously. Mass spectrometry was used to determine whether A375 melanoma cells express HLA-A2 / MAGEA4. 230-239The mice were determined to express approximately 553 cell surface copies of the peptide. On day 13 after tumor establishment, mice (N=4 or 5 per group) were injected with either a non-binding control BB / z CAR (control CAR T), anti-HLA-A2 / MAGEA4 230-239 BB / z CAR, or anti-HLA-A2 / MAGEA4 230-239 4 x 10 expressing either 28 / z CAR (as determined by the frequency of cells expressing GFP, a marker for CAR-transduced cells) 6 T cells were injected intravenously. Tumor growth was assessed by measuring tumor volume up to day 28.

[0221] To determine tumor volume by external caliper, the maximum major axis (length in mm) and maximum transverse axis (width in mm) were determined. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2.

[0222] A375 tumors grew progressively in untreated mice and in mice that received control CAR T cells. 230-239 Mice receiving BB / z CAR T cells demonstrated reduced tumor growth and tumor control compared to mice treated with control CAR T on days 19 (p<0.02), 23 (p<0.02), and 26 (p<0.0001) (statistics analyzed by two-way ANOVA). 230-239 28 / z CAR T treatment also resulted in suppression of the growth of established A375 tumors on days 19 (p=0.007), 23 (p<0.0001), and 26 (p<0.0001) (statistics analyzed by two-way ANOVA). See Tables 46-54. [Table 46] [Table 47] [Table 48] [Table 49] [Table 50] [Table 51] [Table 52] [Table 53] [Table 54]

[0223] Example 11: Structural analysis of Fab / soluble TCR binding to HLA-A2-bound MAGE-A4 (230-239) polypeptide To better understand the specific interactions between antibodies or TCRs and HLA-peptide complexes, two X-ray crystal structures and three cryo-electron microscopy (cryo-EM) structures were determined for engineered soluble portions of antibody Fab fragments or TCRs bound to the HLA-A2 and beta-2-microglobulin complex (HLA-A2 / b2m) displaying the MAGE-A4 230-239 polypeptide in the HLA peptide-binding groove (Table 55). The X-ray structures of the complexes containing the two Fabs were determined at 1.4 and 2.5 Å resolution. These two Fabs have very similar sequences and exhibit nearly identical binding modes. The cryo-EM structure of the complex containing the 33229 Fab was determined at 3.7 Å resolution, and the cryo-EM structures of the additional Fab and sTCR were determined at 3.0 Å and 2.9 Å resolution, respectively. Although they encompass a range of resolutions, in each of the structures, the HLA-displayed MAGEA4:230-239 peptide residues are clearly visualized in electron / cryo-EM density maps, allowing precise determination of residue-level interactions between the HLA-displayed peptide and the complementarity-determining regions (CDRs) of the Fab or sTCR.

[0224] The structures show that the Fabs bound to the HLA-peptide complex in an overall similar orientation. The heavy chain CDRs were located proximal to the N-terminus of the peptide, and the light chain CDRs were located proximal to the C-terminus of the peptide. In each of the Fab-bound structures, the solvent-exposed MAGE-A4 polypeptide residue, arginine 235, was located near the central plane separating the heavy and light chain CDRs. The four Fabs recognized and bound three different rotamers of arginine 235. The remainder of the HLA-displayed peptide conformations were very similar across the structures.

[0225] The sTCR bound to the HLA-peptide complex in a typical TCR orientation, with the α chain closer to the N-terminus of the peptide and the β chain closer to the C-terminus. The CDRs of the sTCR were shifted closer to the N-terminus of the peptide compared to the Fab. The sTCR bound a different rotamer of peptide residue arginine 235 than that observed in the four-Fab structure.

[0226] Contacts between Fab or sTCR and peptides are summarized in Table 55. A "contact" here is defined as a Fab / stCR residue with a non-hydrogen atom that is within 3.5 Å of a non-hydrogen atom of the HLA-presented peptide and may involve hydrogen bonding, charge-charge interactions, or hydrophobic / van der Waals interactions. Bound peptides are numbered according to their residue position in the MAGE-A4 polypeptide as follows: [Table 55]

[0227] The peptide contacts made by each Fab were concentrated almost exclusively with the CDRs HCDR3, LCDR1, and LCDR3. HCDR3 of each antibody made multiple contacts with the side chain of MAGE-A4 residue 233. HCDR3 and / or LCDR3 of each Fab contacted the side chain of residue 235. The main-chain carbonyl of peptide residue 236 contacted LCDR1 of each Fab. None of the four Fabs contacted peptide residues 230, 231, 232, 234, or 239, most of which are buried within the HLA groove and therefore inaccessible to Fab binding. Although peptide residue 234 is solvent-exposed, the lack of a side chain as a glycine limits its ability to make CDR contacts. Nevertheless, substitution at glycine 234 may reduce the avidity of these antibodies due to steric clashes between the bulkier peptide residue and the nearby HCDR3 loop.

[0228] The sTCR contacted residue 233 through its α1 and α3 loops and peptide residue 235 through its β1 and β3 loops. Peptide residues 230, 231, 232, 234, 236, 237, 238, and 239 were not contacted by the sTCR. Thus, peptide contact coverage of the sTCR was less complete than that observed for each of the Fabs above or in other structures of TCR bound to decameric peptides (e.g., PDB 3QDG). [Table 56]

[0229] Array Description [Table 57-1] [Table 57-2] [Table 57-3]

[0230] 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.).

[0231] MAGEA4(286-294)31345 VL-VH BBz CAR P2A-GFP (SEQ ID NO: 76) [ka] VL (G4S)3 VH G4S CD8 Hinge / TM 4-1BB costimulatory domain CD3Z P2A / GFP

[0232] MAGEA4(286-294)31345 VL-VH CD28 hinge / TM / cytoCD3z CAR P2A-GFP (SEQ ID NO: 77) [ka] VL (G4S)3 VH G4S CD28 hinge CD28™ CD28 costimulatory domain CD3Z P2A / GFP

[0233] MAGEA4(286-294)31345*VL-VH BBz CAR P2A-GFP (SEQ ID NO: 78) [ka] VL (G4S)3 VH G4S CD8 Hinge / TM 4-1BB costimulatory domain CD3Z P2A / GFP

[0234] MAGEA4(286-294)31345*VL-VH CD28hinge / TM / cytoCD3z CAR P2A-GFP (SEQ ID NO: 79) [ka] VL (G4S)3 VH G4S CD28 hinge CD28™ CD28 costimulatory domain CD3Z P2A / GFP

[0235] MAGEA4(230-239)33229 VL-VH BBz CAR P2A-GFP (SEQ ID NO: 80) [ka] VL (G4S)3 VH G4S CD8 Hinge / TM 4-1BB costimulatory domain CD3Z P2A / GFP

[0236] MAGEA4(230-239)33229 VL-VH CD28hinge / TM / cytoCD3z CAR P2A-GFP (SEQ ID NO: 81) [ka] VL (G4S)3 VH G4S CD28 hinge CD28™ CD28 costimulatory domain CD3Z P2A / GFP

[0237] 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 comprising: From the N-terminus to the C-terminus, (a) an extracellular ligand-binding domain comprising an anti-MAGE-A4 antigen-binding domain; (b) a hinge derived from a CD28 polypeptide; and (c) a transmembrane domain derived from a CD28 polypeptide; and (d) a cytoplasmic domain comprising a costimulatory domain and a CD3 zeta signaling domain derived from a CD28 polypeptide; Including, the extracellular ligand-binding domain is an anti-MAGE-A4 single-chain variable fragment (scFv) domain comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR) joined by a linker; A chimeric antigen receptor, wherein the LCVR comprises LCDR1, LCDR2 and LCDR3 domains set forth in SEQ ID NOs: 12, 14 and 16, respectively, and the HCVR comprises HCDR1, HCDR2 and HCDR3 domains set forth in SEQ ID NOs: 4, 6 and 8, respectively.

2. A melanoma-associated antigen A4 (MAGE-A4)-specific chimeric antigen receptor comprising: From the N-terminus to the C-terminus, (a) an extracellular ligand-binding domain comprising an anti-MAGE-A4 antigen-binding domain; (b) a hinge derived from a CD28 polypeptide; and (c) a transmembrane domain derived from a CD28 polypeptide; and (d) a cytoplasmic domain comprising a costimulatory domain and a CD3 zeta signaling domain derived from a CD28 polypeptide; Including, the extracellular ligand-binding domain is an anti-MAGE-A4 single-chain variable fragment (scFv) domain comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR) joined by a linker; The LCVRs are LCDR1, LCDR2, LCDR3, and LCDR4 shown in SEQ ID NOs: 61, 63, and 65, respectively. A chimeric antigen receptor comprising an HCDR1, HCDR2 and HCDR3 domains as set forth in SEQ ID NOs: 53, 55 and 57, respectively.

3. The chimeric antigen receptor of claim 1 , wherein the LCVR comprises the amino acid sequence of SEQ ID NO: 37 and the HCVR comprises the amino acid sequence of SEQ ID NO:

2.

4. The chimeric antigen receptor of claim 2, wherein the LCVR comprises the amino acid sequence of SEQ ID NO: 59 and the HCVR comprises the amino acid sequence of SEQ ID NO:

51.

5. The chimeric antigen receptor of any one of claims 1 to 4, further comprising a peptide linker between the extracellular ligand-binding domain and the hinge.

6. The chimeric antigen receptor of any one of claims 1 to 5, wherein the linker or the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 26.

7. The chimeric antigen receptor according to any one of claims 1 to 6, (a) the hinge comprises the amino acid sequence of SEQ ID NO: 41; (b) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 43; or (c) the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 45; or (d) the signaling domain comprises the amino acid sequence of SEQ ID NO:

30. Chimeric antigen receptor.

8. The chimeric antigen receptor of claim 1, comprising the amino acid sequence of SEQ ID NO:

47.

9. The chimeric antigen receptor of claim 2, comprising the amino acid sequence of SEQ ID NO:

73.

10. An isolated nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1 to 9.

11. 9. An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 8, comprising the nucleotide sequence of SEQ ID NO:

48.

12. 10. An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 9, comprising the nucleotide sequence of SEQ ID NO:

72.

13. A vector comprising the nucleic acid molecule of any one of claims 10 to 12.

14. The vector of claim 13 , wherein the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

15. A cell comprising the nucleic acid molecule according to any one of claims 10 to 12 or the vector according to claim 13 or 14.

16. An engineered cell comprising the chimeric antigen receptor of any one of claims 1 to 9.

17. 17. The engineered cell of claim 16, which is a T lymphocyte.

18. The T lymphocytes may be inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or 18. The engineered cell of claim 17, which is a helper T lymphocyte or a T lymphocyte.

19. 19. The engineered cell of claim 18, which is a CD8+ cytotoxic T lymphocyte.

20. below: (a) providing a population of immune cells obtained from a subject; (b) introducing a nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 1 to 9 into immune cells; (c) culturing the immune cells under conditions to express the nucleic acid molecule; (d) a population of engineered cells obtained by isolating said immune cells that express said chimeric antigen receptor on their surface.

21. below: (a) a genetically modified human T cell and a pharmaceutically acceptable carrier, wherein the genetically modified human T cell comprises a chimeric antigen receptor according to any one of claims 1 to 9; or (b) the engineered cell of claim 16 and a pharmaceutically acceptable carrier; or (c) the engineered cell of claim 17 and a pharmaceutically acceptable carrier; or (d) the engineered cell of claim 18 and a pharmaceutically acceptable carrier; or (e) the engineered cell of claim 19 and a pharmaceutically acceptable carrier; A pharmaceutical composition comprising one of:

22. 21. A pharmaceutical composition comprising the engineered cells of any one of claims 16 to 19 or the population of engineered cells of claim 20 for treating a cancer that expresses MAGE-A4.

23. The pharmaceutical composition of claim 22, wherein the cancer expressing MAGE-A4 is selected from the group consisting of 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.

24. The pharmaceutical composition according to claim 22 or 23, wherein the cancer expressing MAGE-A4 is multiple myeloma expressing MAGE-A4.

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