T cell receptors with MAGE-B2 specificity and uses thereof

Abstract

To provide T cell receptors with MAGE-B2 specificity and uses thereof.SOLUTION: The present disclosure provides methods for generating MAGE-B2 specific T cells, and compositions comprising engineered MAGE-B2-specific T cell receptors. Further provided are methods of treating cancer comprising administering the MAGE-B2-specific T cells. A particular embodiment of the present disclosure provides a T cell receptor (TCR) capable of binding an antigenic peptide derived from the melanoma-associated antigen B2 (MAGE-B2).SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 660,083, filed April 19, 2018, the entire contents of which are incorporated herein by reference.

[0002] The Sequence Listing contained in the file named "UTFCP1372WO_ST25.txt", which is 29 KB (measured in Microsoft Windows®) created on April 19, 2019, was filed herewith by electronic submission and is hereby incorporated by reference.

[0003] The present invention relates generally to the fields of medicine and immunology, and more particularly to a T cell receptor that specifically recognizes melanoma-associated antigen B2 (MAGE-B2). [Background technology]

[0004] T cell-based therapies have shown remarkable promise as a method for treating many cancers. Unfortunately, this approach has also been hampered by a lack of immunogenic antigen targets for common cancers and potential toxicity to non-cancerous tissues. These T cell-based therapies can include adoptive cell therapy (ACT) and / or vaccination approaches to induce anti-tumor T cell responses. Cancer vaccination approaches can include delivery of specific antigens via peptide, protein, DNA, or RNA vaccines, or the induction of anti-cancer responses using dendritic cell (DC) vaccines.

[0005] ACT generally involves the infusion of activated autologous tumor-specific T cells into patients to treat cancer, for example. ACT has resulted in therapeutic clinical responses in melanoma patients. In general, generating an effective anti-tumor T cell response typically requires three steps: priming and activating antigen-specific T cells, mobilizing the activated T cells to the tumor site, and recognizing and killing the tumor by the antigen-specific T cells.

[0006] The selection of target antigens is important for the induction of effective antigen-specific T cells. Although several tumor-associated antigens have been identified for melanoma and a few other solid tumor malignancies, immunogenic targets for pancreatic cancer, ovarian cancer, gastric cancer, lung cancer, cervical cancer, breast cancer, and head and neck cancer are scarce. Target antigens that are both immunogenic and tumor-specific in their expression patterns and characteristics necessary for effective cancer treatment and to avoid substantial off-target side effects are lacking. Therefore, there is an unmet medical need for novel T cell-based therapies targeting additional target antigens for these malignancies. Summary of the Invention [Means for solving the problem]

[0007] Certain embodiments of the present disclosure provide a T cell receptor (TCR) capable of binding an antigenic peptide derived from melanoma-associated antigen B2 (MAGE-B2). In one embodiment, the TCR comprises a TCR alpha polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 3, and a TCR beta polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 5. In another embodiment, a TCR is provided that comprises a TCR alpha polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 9), and CDR3 (SEQ ID NO: 11), and a TCR beta polypeptide comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15), and CDR3 (SEQ ID NO: 17). In a specific aspect, the TCR comprises a TCR alpha polypeptide having the sequence of SEQ ID NO: 3 and a TCR beta polypeptide having the sequence of SEQ ID NO: 5.

[0008] In another embodiment, the TCR comprises a TCR alpha polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 19, and a TCR beta polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 22. In another embodiment, a TCR is provided that comprises a TCR alpha polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to CDR1 (SEQ ID NO: 23), CDR2 (SEQ ID NO: 25), and CDR3 (SEQ ID NO: 27), and a TCR beta polypeptide comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 31), and CDR3 (SEQ ID NO: 33). In a specific aspect, the TCR comprises a TCR alpha polypeptide having the sequence of SEQ ID NO: 19 and a TCR beta polypeptide having the sequence of SEQ ID NO: 22.

[0009] In some embodiments, the antigenic peptide is HLA-A2 restricted. In some embodiments, the antigenic peptide is HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, or HLA-A*0205 restricted. In certain embodiments, the antigenic peptide is HLA-A*0201 restricted.

[0010] In some embodiments, the TCR is a soluble TCR lacking a transmembrane domain. In certain embodiments, the TCR further comprises a detectable label and / or a therapeutic agent.

[0011] In another embodiment, a multivalent TCR complex is provided comprising a plurality of TCRs according to an embodiment (e.g., TCRs capable of binding antigenic peptides derived from MAGE-B2). In some aspects, the multivalent TCR comprises two, three, four or more TCRs. In certain aspects, the multivalent TCR is present in a lipid bilayer or attached to a particle. In certain aspects, the TCRs are conjugated via a linker molecule.

[0012] Further embodiments provide polypeptides comprising a TCR alpha polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:3, and / or a TCR beta polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:5. Another embodiment provides polypeptides comprising a TCR alpha polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 9), and CDR3 (SEQ ID NO: 11), and a TCR beta polypeptide comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15), and CDR3 (SEQ ID NO: 17). In certain aspects, the polypeptide comprises a TCR alpha polypeptide of SEQ ID NO: 3 and a TCR beta polypeptide of SEQ ID NO: 5. In some aspects, the polypeptide comprises a TCR alpha polypeptide of SEQ ID NO: 3. In certain aspects, the polypeptide comprises a TCR beta polypeptide of SEQ ID NO: 5. Further provided herein are polynucleotides encoding the polypeptides of the embodiments.

[0013] Further embodiments provide polypeptides comprising a TCR alpha polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 19 and / or a TCR beta polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 22. Another embodiment provides polypeptides comprising a TCR alpha polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to CDR1 (SEQ ID NO: 23), CDR2 (SEQ ID NO: 25), and CDR3 (SEQ ID NO: 27), and a TCR beta polypeptide comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 31), and CDR3 (SEQ ID NO: 33). In certain aspects, the polypeptide comprises a TCR alpha polypeptide of SEQ ID NO: 19 and a TCR beta polypeptide of SEQ ID NO: 22. In some aspects, the polypeptide comprises a TCR alpha polypeptide of SEQ ID NO: 19. In certain aspects, the polypeptide comprises a TCR beta polypeptide of SEQ ID NO: 22. Further provided herein are polynucleotides encoding the polypeptides of the embodiments.

[0014] In another embodiment, an expression vector is provided comprising a TCR of the embodiment (e.g., a TCR capable of binding an antigenic peptide derived from MAGE-B2). In some aspects, the expression vector is a viral vector. In certain aspects, the viral vector is a retroviral vector or a lentiviral vector. In additional aspects, the TCR comprises a linker domain. In some aspects, the linker domain is between the TCR alpha polypeptide and the TCR beta polypeptide. In certain aspects, the linker domain comprises one or more cleavage sites. In some aspects, the one or more cleavage sites are furin cleavage sites and / or P2A cleavage sites. In some aspects, the one or more cleavage sites are separated by a spacer. In certain aspects, the spacer is SGSG or GSG. In some aspects, the TCR alpha polypeptide and the TCR beta polypeptide are linked by an IRES sequence.

[0015] Further provided herein are host cells engineered to express a TCR of the embodiments (eg, a TCR capable of binding an antigenic peptide derived from MAGE-B2).

[0016] In some embodiments, the cells are immune cells. In certain embodiments, the cells are isolated from the umbilical cord or blood. In some embodiments, the immune cells are T cells or peripheral blood lymphocytes. In certain embodiments, the T cells are CD8 + T cells, CD4 + The cells are T cells, or γδ T cells. In some aspects, the associated signaling molecule can attach to the TCR and transmit an activation signal in the non-T cell immune effector cell upon TCR engagement. In certain aspects, the cells are NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs), or induced pluripotent stem (iPS) cells. In some aspects, the cells are allogeneic or autologous. Further provided herein is a pharmaceutical composition comprising a population of MAGE-B2 TCR-specific cells of embodiments.

[0017] Further provided herein is a method for engineering MAGE-B2-specific immune cells, comprising contacting the immune cells with an expression vector of an embodiment. In some aspects, the immune cells are T cells, peripheral blood lymphocytes, NK cells, invariant NK cells, or NKT cells. In some aspects, contacting is further defined as transfecting or transducing. In certain aspects, the peripheral blood lymphocytes are stimulated with OKT3 and IL-2. In additional aspects, the method further comprises sorting the immune cells to isolate TCR-engineered T cells, performing T cell cloning by serial dilution, and expanding the T cell clones by a rapid expansion protocol.

[0018] In another embodiment, there is provided a use of a therapeutically effective amount of MAGE-B2-specific TCR-expressing cells according to an embodiment for the treatment of cancer. Also provided herein is a composition comprising an effective amount of MAGE-B2-specific cells according to an embodiment for the treatment of cancer in a subject. In a particular aspect, the MAGE-B2-specific TCR-expressing cells are T cells.

[0019] In another embodiment, a method of treating cancer in a subject is provided, comprising administering to the subject a therapeutically effective amount of a MAGE-B2-specific cell (e.g., expressing a TCR capable of binding an antigenic peptide derived from MAGE-B2) of an embodiment. In some aspects, the MAGE-B2-specific cell is a T cell.

[0020] In certain embodiments, the subject is identified as having an HLA-A2 allele, such as an HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, or HLA-A*0205 allele. In certain embodiments, the subject is identified as having an HLA-A*0201 allele. In additional embodiments, the method further comprises performing lymphodepletion on the subject prior to administering a therapeutically effective amount of MAGE-B2-specific T cells. In some embodiments, the therapeutically effective amount of MAGE-B2-specific T cells is derived from a sample of autologous tumor-infiltrating lymphocytes (TILs) having anti-tumor activity. In some embodiments, the MAGE-B2-specific cells are administered to the subject intravenously, intraperitoneally, or intratumorally. In certain embodiments, the subject is human. In some embodiments, the method further comprises administering at least one additional therapeutic agent to the subject. In certain embodiments, the at least one additional therapeutic agent is selected from the group consisting of chemotherapy, radiation therapy, and immunotherapy. In some embodiments, the at least one additional therapeutic agent is immunotherapy. In some embodiments, the immunotherapy is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor inhibits an immune checkpoint protein or its ligand selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or adenosine A2a receptor (A2aR). In some embodiments, the immune checkpoint inhibitor inhibits PD-1 or CTLA-4.

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. In an embodiment of the present invention, for example, the following items are provided: (Item 1) An isolated T cell receptor (TCR) capable of binding an antigenic peptide derived from melanoma-associated antigen B2 (MAGE-B2), said T cell receptor comprising a TCR alpha polypeptide having at least 90% identity to the sequence of SEQ ID NO: 3 or 19 and a TCR beta polypeptide having at least 90% identity to the sequence of SEQ ID NO: 5 or 22. (Item 2) 2. The TCR according to item 1, wherein the antigenic peptide is HLA-A2 restricted. (Item 3) 3. The TCR according to item 2, wherein the antigenic peptide is HLA-A*0201 restricted. (Item 4) 2. The TCR of item 1, wherein the TCR alpha polypeptide comprises a sequence having at least 95% identity to CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 9), and CDR3 (SEQ ID NO: 11), and the TCR beta polypeptide comprises a sequence having at least 95% identity to CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15), and CDR3 (SEQ ID NO: 17). (Item 5) 2. The TCR of item 1, wherein the TCR alpha polypeptide comprises a sequence having at least 99% identity to CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 9), and CDR3 (SEQ ID NO: 11), and the TCR beta polypeptide comprises a sequence having at least 99% identity to CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15), and CDR3 (SEQ ID NO: 17). (Item 6) The TCR of item 1, wherein the TCR alpha polypeptide comprises the sequences of CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 9), and CDR3 (SEQ ID NO: 11), and the TCR beta polypeptide comprises the sequences of CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15), and CDR3 (SEQ ID NO: 17). (Item 7) 2. The TCR of item 1, wherein the polypeptide has at least 95% identity to the sequence of SEQ ID NO: 3 and the TCR beta polypeptide has at least 95% identity to the sequence of SEQ ID NO: 5. (Item 8) 2. The TCR of item 1, wherein the TCR alpha polypeptide has at least 99% identity to the sequence of SEQ ID NO: 3 and the TCR beta polypeptide has at least 99% identity to the amino acid sequence of SEQ ID NO: 5. (Item 9) The TCR of item 1, wherein the TCR alpha polypeptide has the sequence of SEQ ID NO: 3 and the TCR beta polypeptide has the sequence of SEQ ID NO: 5. (Item 10) 2. The TCR of item 1, wherein the TCR alpha polypeptide comprises a sequence having at least 95% identity to CDR1 (SEQ ID NO: 23), CDR2 (SEQ ID NO: 25), and CDR3 (SEQ ID NO: 27), and the TCR beta polypeptide comprises a sequence having at least 95% identity to CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 31), and CDR3 (SEQ ID NO: 33). (Item 11) 2. The TCR of item 1, wherein the TCR alpha polypeptide comprises a sequence having at least 99% identity to CDR1 (SEQ ID NO: 23), CDR2 (SEQ ID NO: 25), and CDR3 (SEQ ID NO: 27), and the TCR beta polypeptide comprises a sequence having at least 99% identity to CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 31), and CDR3 (SEQ ID NO: 33). (Item 12) The TCR of item 1, wherein the TCR alpha polypeptide comprises the sequences of CDR1 (SEQ ID NO: 23), CDR2 (SEQ ID NO: 25), and CDR3 (SEQ ID NO: 27), and the TCR beta polypeptide comprises the sequences of CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 31), and CDR3 (SEQ ID NO: 33). (Item 13) 2. The TCR of item 1, wherein the polypeptide has at least 95% identity to the sequence of SEQ ID NO: 19 and the TCR beta polypeptide has at least 95% identity to the sequence of SEQ ID NO: 22. (Item 14) 2. The TCR of item 1, wherein the TCR alpha polypeptide has at least 99% identity to the sequence of SEQ ID NO: 19 and the TCR beta polypeptide has at least 99% identity to the amino acid sequence of SEQ ID NO: 22. (Item 15) 2. The TCR of item 1, wherein the TCR alpha polypeptide has the sequence of SEQ ID NO: 19 and the TCR beta polypeptide has the sequence of SEQ ID NO: 22. (Item 16) 2. The TCR of item 1, which is a soluble TCR lacking a transmembrane domain. (Item 17) 17. The TCR of item 16, further comprising a detectable label. (Item 18) 18. The TCR of item 16 or item 17, further comprising a therapeutic agent. (Item 19) 19. A multivalent TCR complex comprising a plurality of TCRs according to any of items 1 to 18. (Item 20) 20. The conjugate of item 19, wherein the multivalent TCR comprises two, three, four or more TCRs. (Item 21) 21. The complex of item 20, wherein the multivalent TCR is present in a lipid bilayer or attached to a particle. (Item 22) 21. The conjugate of item 20, wherein the TCR is conjugated via a linker molecule. (Item 23) A polypeptide comprising a TCR alpha polypeptide comprising the sequences of CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 9), and CDR3 (SEQ ID NO: 11), and / or a TCR beta polypeptide comprising the sequences of CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15), and CDR3 (SEQ ID NO: 17). (Item 24) 24. The polypeptide of item 23, comprising a TCR alpha polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 3 and / or a TCR beta polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 5. (Item 25) 24. The polypeptide of item 23, comprising a TCR alpha polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO: 3 and / or a TCR beta polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO: 5. (Item 26) 24. The polypeptide of item 23, comprising a TCR alpha polypeptide of SEQ ID NO: 3 and a TCR beta polypeptide of SEQ ID NO: 5. (Item 27) 24. The polypeptide of item 23, comprising the TCR alpha polypeptide of SEQ ID NO: 3. (Item 28) 24. The polypeptide of item 23, comprising a TCR beta polypeptide of SEQ ID NO: 5. (Item 29) A polypeptide comprising a TCR alpha polypeptide comprising the sequences of CDR1 (SEQ ID NO: 23), CDR2 (SEQ ID NO: 25), and CDR3 (SEQ ID NO: 27), and / or a TCR beta polypeptide comprising the sequences of CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 31), and CDR3 (SEQ ID NO: 33). (Item 30) 24. The polypeptide of item 23, comprising a TCR alpha polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 19 and / or a TCR beta polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 22. (Item 31) 24. The polypeptide of item 23, comprising a TCR alpha polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO: 19 and / or a TCR beta polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO: 22. (Item 32) 24. The polypeptide of item 23, comprising a TCR alpha polypeptide of SEQ ID NO: 19 and a TCR beta polypeptide of SEQ ID NO: 22. (Item 33) 24. The polypeptide of item 23, comprising the TCR alpha polypeptide of SEQ ID NO: 19. (Item 34) 24. The polypeptide of item 23, comprising a TCR beta polypeptide of SEQ ID NO: 22. (Item 35) 35. A polynucleotide encoding the polypeptide of any one of items 23 to 34. (Item 36) 19. An expression vector comprising the TCR according to any one of items 1 to 18. (Item 37) 37. The expression vector according to item 36, which is a viral vector. (Item 38) 38. The expression vector of item 37, wherein the viral vector is a retroviral vector or a lentiviral vector. (Item 39) 39. The expression vector of any of items 36 to 38, further comprising a linker domain. (Item 40) 40. The expression vector of item 39, wherein the linker domain is between the TCR alpha polypeptide and the TCR beta polypeptide. (Item 41) 41. The expression vector of claim 39 or 40, wherein the linker domain comprises one or more cleavage sites. (Item 42) 42. The expression vector of item 41, wherein the one or more cleavage sites are furin cleavage sites and / or P2A cleavage sites. (Item 43) Item 41 or Item 39, wherein the one or more cleavage sites are separated by a spacer. (Item 44) 44. The expression vector of item 43, wherein the spacer is SGSG or GSG. (Item 45) 40. The expression vector of item 39, wherein the TCR alpha polypeptide and the TCR beta polypeptide are linked by an IRES sequence. (Item 46) 19. A host cell engineered to express the TCR of any of items 1 to 18. (Item 47) 47. The host cell of item 46, which is an immune cell. (Item 48) 47. The host cell of item 46, which is an NK cell, an invariant NK cell, an NKT cell, a mesenchymal stem cell (MSC), or an induced pluripotent stem (iPS) cell. (Item 49) 47. The host cell of item 46, isolated from umbilical cord or blood. (Item 50) 47. The host cell of item 46, wherein the immune cell is a T cell or a peripheral blood lymphocyte. (Item 51) The T cells are CD8 + 51. The host cell of item 50, which is a T cell, a CD4+ T cell, or a γδ T cell. (Item 52) 51. The host cell of item 50, which is allogeneic or autologous. (Item 53) 53. A pharmaceutical composition comprising a population of MAGE-B2 TCR-specific cells according to any of items 46 to 52. (Item 54) 45. A method for manipulating MAGE-B2-specific immune cells, comprising contacting said immune cells with an expression vector according to any one of items 36 to 44. (Item 55) 55. The method of claim 54, wherein the immune cells are T cells, peripheral blood lymphocytes, NK cells, invariant NK cells, or NKT cells. (Item 56) 56. The method of claim 54 or 55, wherein contacting is further defined as transfecting or transducing. (Item 57) 56. The method of item 55, wherein the peripheral blood lymphocytes are stimulated with OKT3 and IL-2. (Item 58) 58. The method of any of items 54 to 57, further comprising sorting the immune cells to isolate TCR-engineered T cells, performing T cell cloning by serial dilution, and expanding the T cell clones by a rapid expansion protocol. (Item 59) 53. Use of a therapeutically effective amount of MAGE-B2 TCR-specific cells according to any one of items 46 to 52 for the treatment of cancer. (Item 60) 60. The use of item 59, wherein the MAGE-B2 TCR-specific cells are T cells. (Item 61) 53. A composition comprising a therapeutically effective amount of MAGE-B2 specific cells according to any one of items 46 to 52 for the treatment of cancer in a subject. (Item 62) 60. The composition of item 59, wherein the MAGE-B2 TCR-specific cells are T cells. (Item 63) 53. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of MAGE-B2 specific cells according to any one of items 46 to 52. (Item 64) 64. The method of claim 63, wherein the MAGE-B2-specific cells are T cells. (Item 65) 64. The method of claim 63, wherein the subject is identified as having an HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, or HLA-A*0205 allele. (Item 66) 64. The method of claim 63, further comprising the step of performing lymphodepletion on the subject prior to administration of the therapeutically effective amount of MAGE-B2-specific T cells. (Item 67) 65. The method of item 64, wherein the therapeutically effective amount of MAGE-B2-specific T cells is derived from a sample of autologous tumor-infiltrating lymphocytes (TILs) with anti-tumor activity. (Item 68) 64. The method of claim 63, wherein the MAGE-B2-specific cells are administered to the subject intravenously, intraperitoneally, or intratumorally. (Item 69) Item 64. The method of item 63, wherein the subject is a human. (Item 70) 64. The method of claim 63, further comprising administering to the subject at least one additional therapeutic agent. (Item 71) 71. The method of claim 70, wherein the at least one additional therapeutic agent is selected from the group consisting of chemotherapy, radiation therapy, and immunotherapy. (Item 72) 71. The method of claim 70, wherein the at least one additional therapeutic agent is an immunotherapy. (Item 73) 73. The method of claim 72, wherein the immunotherapy is an immune checkpoint inhibitor. (Item 74) 74. The method of claim 73, wherein the immune checkpoint inhibitor inhibits an immune checkpoint protein or its ligand selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or adenosine A2a receptor (A2aR). (Item 75) 75. The method of claim 74, wherein the immune checkpoint inhibitor inhibits PD-1 or CTLA-4. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows Western blot detection of MAGE-B2 expression in lung cancer cell lines and immortalized normal human small airway epithelial cells (HSAEC1-KT and HSAEC2-KT).

[0023] [Figure 2] Figure 2 shows the generation of MAGE-B2 HLA-A2-restricted peptide-specific cytotoxic T lymphocytes (CTLs). Detection of a T cell population containing tetramers bearing the HLA-A2-restricted MAGE-B2 epitope (GVYDGEEHSV) (left). Sorting of the CD8+ tetramer+ population and expansion by the Rapid Expansion Protocol (REP) (center). Generation of CTL clones using limiting dilution (right).

[0024] [Figure 3-1] Figures 3A-3D show the detection of the killing ability of MAGE-B2 CTL clones. (Figure 3A) Peptide titration assay of T2 cells pulsed with MAGE-B2 peptide. (Figure 3B) Cytotoxicity of the CTL clones against the lung cancer cell line H2023 (HLA-A*0201) and the normal lung cell line HSAEC2-KT (HLA-A*0201) by 51Cr release assay. (Figure 3C) Cytotoxicity of the CTL clones against the lung cancer cell lines H522, H1355, H1755, and DFC-1032. (Figure 3D) Cytotoxicity of the MAGE-B2 CTL clones against the parent lung cancer cell lines PC-9 and H1573, and against HLA-A2-enforced expression in both cell lines. [Figure 3-2] Figures 3A-3D show the detection of the killing ability of MAGE-B2 CTL clones. (Figure 3A) Peptide titration assay of T2 cells pulsed with MAGE-B2 peptide. (Figure 3B) Cytotoxicity of the CTL clones against the lung cancer cell line H2023 (HLA-A*0201) and the normal lung cell line HSAEC2-KT (HLA-A*0201) by 51Cr release assay. (Figure 3C) Cytotoxicity of the CTL clones against the lung cancer cell lines H522, H1355, H1755, and DFC-1032. (Figure 3D) Cytotoxicity of the MAGE-B2 CTL clones against the parent lung cancer cell lines PC-9 and H1573, and against HLA-A2-enforced expression in both cell lines.

[0025] [Figure 4]Figure 4 shows the generation of MAGE-B2 T cell receptor-engineered T cells (TCR-T). Activated allogeneic PBMCs (left) were infected with retrovirus, resulting in the emergence of CD8+tetramer+ cells after infection (center). TCR-T cell lines were generated by sorting and expanding the CD8+tetramer+ population (right).

[0026] [Figure 5] Figures 5A-B show MAGE-B2 TCR-T killing assays. (Figure 5A) Peptide titration assay. T2 cells were pulsed with different concentrations of MAGE-B2 peptide. (Figure 5B) Cytotoxicity of MAGE-B2 TCR-T against the lung cancer cell line H2023 (HLA-A*0201) and the normal lung cell line HSAEC2-KT (HLA-A*0201) as detected by a standard 51Cr release assay.

[0027] [Figure 6-1] Figure 6 shows MAGE-B2 TCR-T functional detection by intracellular cytokine staining (ICS). [Figure 6-2] Figure 6 shows MAGE-B2 TCR-T functional detection by intracellular cytokine staining (ICS). [Figure 6-3] Figure 6 shows MAGE-B2 TCR-T functional detection by intracellular cytokine staining (ICS).

[0028] [Figure 7-1] Figure 7 shows a representative generation of MAGE-B2-specific T cell products from a dendritic cell-T cell (DC-T) coculture system with healthy donor PBMCs. After two rounds of stimulation with MB2-231 peptide-pulsed DCs, a small population of CD8+ / tetramer+ cells was observed in three wells of a single 48-well plate. Three positive wells were separately sorted using tetramer-guided sorting technology and expanded once or twice by REP. CD8 and tetramer staining of the final product is shown. [Figure 7-2]Figure 7 shows a representative generation of MAGE-B2-specific T cell products from a dendritic cell-T cell (DC-T) coculture system with healthy donor PBMCs. After two rounds of stimulation with MB2-231 peptide-pulsed DCs, a small population of CD8+ / tetramer+ cells was observed in three wells of a single 48-well plate. Three positive wells were separately sorted using tetramer-guided sorting technology and expanded once or twice by REP. CD8 and tetramer staining of the final product is shown.

[0029] [Figure 8-1] Figures 8A-8E show the functional avidity of MAGE-B2-specific T cells. (Figure 8A) Lysis of T2 cells pulsed with various concentrations of MB2-231 peptide by three MAGE-B2 CTL cell lines at an effector-to-target (E:T) ratio of 20:1. (Figure 8B) Lysis of the MAGE-B2-expressing tumor cell line H2023 (HLA-A2+) by three MAGE-B2 CTL cell lines at various E:T ratios. The normal lung cell line HSAEC2-KT (MAGE-B2-, HLA-A2+) serves as a negative control. (Figure 8C) Lysis of the MAGE-B2-expressing and HLA-A2-enforced tumor cell line H1299-A2 by three MAGE-B2 CTL cell lines at various E:T ratios. The parental cell line H1299 (HLA-A2-) serves as a negative control. (Figures 8D-8E) The three MAGE-B2 CTL cell lines lysed a larger number of tumor cell lines: H1395 (MAGE-B2+, HLA-A2+), H522 (MAGE-B2+, HLA-A2+), H1355 (MAGE-B2+, HLA-A2+), H1755 (MAGE-B2+, HLA-A2+), and DFC-1032 (MAGE-B2+, HLA-A2+). [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above. [Figure 8-5] Same as above.

[0030] [Figure 9-1]Figures 9A-9E show the generation and functional avidity of MAGE-B2 TCR-T. (Figure 9A) Tetramer detection of TCR-T before and after infection with a retrovirus containing the TCR-T gene from the highly functional CTL cell line MB2-231 C5, and after tetramer-induced selection and expansion. (Figure 9B) Lysis of T2 cells pulsed with various concentrations of MB2-231 peptide by MB2-231 C5 TCR-T using an effector-to-target (E:T) ratio of 20:1. (Figure 9C) Lysis of the MAGE-B2-expressing tumor cell line H2023 (HLA-A2+) by MB2-231 C5 TCR-T. The normal lung cell line HSAEC2-KT (MAGE-B2-, HLA-A2+) served as a negative control. (Figure 9D) Lysis of the MAGE-B2-expressing and HLA-A2-enforced tumor cell line H1299-A2 by MB2-231 C5 TCR-T at various E:T ratios. The parental cell line H1299 (HLA-A2-) served as a negative control. (Figure 9E) Lysis of more tumor cell lines, including H1395 (MAGE-B2+, HLA-A2+), H522 (MAGE-B2+, HLA-A2+), H1355 (MAGE-B2+, HLA-A2+), H1755 (MAGE-B2+, HLA-A2+), and DFC-1032 (MAGE-B2+, HLA-A2+), by MB2-231 C5 TCR-T at various E:T ratios. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above.

[0031] [Figure 10-1]Figures 10A-10C show functional detection of MB2-231 C5 TCR-T by intracellular cytokine staining (ICS) assay. TCR-T cells were co-cultured with MB2-231 / M26 peptide-pulsed T2 cells, tumor cell line H2023 (MAGE-B2+, HLA-A2+), normal lung cell line HSAEC2-KT (MAGE-B2-, HLA-A2+), tumor cell lines H1395 (MAGE-B2+, HLA-A2+), H522 (MAGE-B2+, HLA-A2+), H1299-A2 (MAGE-B2+, HLA-A2-expressing), H1299 (MAGE-B2+, HLA-A2-), H1355 (MAGE-B2+, HLA-A2+), H1755 (MAGE-B2+, HLA-A2+), and DFC-1032 (MAGE-B2+, HLA-A2+) at an E:T ratio of 10:1. After overnight incubation, markers of TCR downstream activation, CD137, CD69, IFN-γ, and TNF-α, were detected by ICS assay. M26 peptide-pulsed T2, HSAEC2-KT, and H1299 served as negative controls. After co-culture with MB2-231 peptide-pulsed T2, H2023, H1395, H1299-A2, and H1755, the levels of CD137, CD69, IFN-γ, and TNF-α were significantly enhanced compared to the negative controls. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above. [Figure 10-6] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0032] Melanoma-associated antigen B2 (MAGE-B2), also known as cancer / testis antigen 3.2 (UniProt No. O15479) (CT3.2), is encoded by a gene located on the X chromosome. As measured at the protein and RNA levels, MAGE-B2 is expressed in the testis but not in other normal tissues. MAGE-B2 is overexpressed in several cancers, including lung cancer, liver cancer, head and neck cancer, gastric cancer, glioblastoma, and colorectal cancer. One HLA-A2 (e.g., HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, or HLA-A*0205)-restricted peptide (GVYDGEEHSV, SEQ ID NO: 1) was eluted from ovarian cancer cells and identified (Barnea et al., 2002). Epitopes were also identified from peptidome analysis of glioblastoma multiforme cells T98G and U-87 ( Shraibman et al., 2016 ).

[0033] In this study, antigen-specific CTLs that recognize antigens endogenously presented on HLA-matched allogeneic tumor cell lines using the MAGE-B2 peptide epitope were generated from patient peripheral blood mononuclear cells (PBMCs). These antigen-specific CTLs stimulated by antigen-presenting cells displaying this HLA-A2-restricted MAGE-B2 peptide were shown to be selectively cytotoxic to lung cancer cells.

[0034] Thus, in certain embodiments, the present disclosure provides a TCR that recognizes and specifically binds to the MAGE-B2 HLA-A2-restricted epitope GVYDGEEHSV (SEQ ID NO: 1). The present disclosure also provides a nucleotide sequence encoding this TCR, and an expression vector containing this nucleotide sequence that can be used to modify naive T cells and generate MAGE-B2-specific T cells. The present disclosure further provides the use of MAGE-B2-specific T cells for treatment, such as adoptive cell therapy, for cancer patients, such as HLA-A2-positive cancer patients whose malignant cells express the MAGE-B2 antigen. The antigen-specific T cells, such as CTLs, provided herein can be used to target solid tumors.

[0035] I. Definition As used in this specification and the appended claims, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells, and a reference to a "peptide" includes a reference to one or more peptides and equivalents thereof (e.g., polypeptides) known to those skilled in the art.

[0036] As used in this specification and the appended claims, the term "or" means "and / or" unless expressly stated to refer to alternatives only or unless the alternatives are mutually exclusive.

[0037] The term "another," as used in this specification and the appended claims, may mean at least a second or more.

[0038] As used herein, the term "about" indicates that a particular value or measurement includes the inherent variation associated with the device being used to obtain the measurement or calculate the value, or the natural variation that exists among study subjects.

[0039] The term "essentially free," as used herein with respect to a component of a solution (e.g., a preparation of one or more proteins, polymers, or small molecules), means that the preparation was not formulated to include that component, or that such component is present only in trace amounts (e.g., as a contaminant). In certain embodiments, a preparation of a molecule of interest is essentially free of a particular component if the preparation contains less than 0.05% (w / w) of that component. In certain embodiments, a preparation of a molecule of interest is essentially free of a particular component if the preparation contains less than 0.01% (w / w) of that component. In certain embodiments, a preparation of a molecule of interest is essentially free of a particular component if the amount of that component in the preparation cannot be detected using standard analytical methods (e.g., UV spectrophotometry, mass spectrometry, nuclear magnetic resonance spectroscopy, etc.).

[0040] The term "enriched" as used herein with respect to a component of a solution or suspension (e.g., a preparation of one or more cell types, proteins, polymers, or small molecules) means that the preparation has been formulated to contain that component in a higher than normal concentration or in greater than normal numbers (e.g., a suspension of lymphocytes may be enriched for effector T lymphocytes).

[0041] As used herein, the terms "treat," "treatment," "treating," and the like refer to the process of improving, alleviating, or otherwise alleviating the symptoms of a disease or condition in a subject, for example, by administering a therapeutic agent to the subject or by performing a surgical, clinical, or other medical procedure on the subject.

[0042] As used herein, the terms "subject" or "patient" are used interchangeably herein to refer to an individual, e.g., a human, or a non-human organism such as a primate, mammal, or vertebrate.

[0043] As used herein, terms such as "therapeutically effective" or "therapeutically beneficial" refer to a therapeutic agent or surgical, clinical, or other medical procedure that improves, alleviates, or otherwise relieves one or more symptoms of a disease, disorder, or condition, thereby improving the health of a subject with a disease, disorder, or condition, for example, by reducing the frequency or severity of the signs or symptoms of the disease, disorder, or condition. Thus, a therapeutically effective or therapeutically beneficial cancer treatment may, for example, reduce tumor size, reduce the rate of tumor growth, and reduce the likelihood of tumor dissemination or metastasis.

[0044] As used herein, the terms "pharmaceutically acceptable" or "pharmacologically acceptable" refer to a pharmaceutical preparation of a therapeutic agent that does not produce an adverse, allergic, or other undesired reaction when administered to a mammalian or vertebrate subject. Such preparations should be formulated in accordance with Good Manufacturing Practice (GMP) standards for sterility, pyrogenicity, purity, and any other relevant standards required by the FDA Office of Biological Standards.

[0045] As used herein, the term "pharmaceutically acceptable carrier" refers to any chemical compound or solvent known to those skilled in the art used to formulate a therapeutic agent for delivery to a mammalian or vertebrate subject, such as aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solution, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, fluid and nutrient replenishers, and any combination thereof.

[0046] As used herein, the terms "unit dose," "dose," or "dosage" refer to a formulation of a therapeutic agent suitable for administration to a mammalian or vertebrate subject, containing a predetermined amount of agent expected to be therapeutically effective in the subject when administered by an appropriate route and according to a desired treatment regimen. The actual dosage of a particular therapeutic agent to be administered to a subject can be empirically determined by a healthcare provider in light of various physical and physiological parameters, including, for example, the subject's weight, age, health, and sex, the type of disease being treated, the degree of disease progression, previous or concurrent therapeutic interventions, the route of administration, and the efficacy, stability, and toxicity of the particular therapeutic substance.

[0047] II. MAGE-B2 TCR Methods and Compositions In certain embodiments, the present disclosure provides a MAGE-B2 peptide epitope comprising the sequence GVYDGEEHSV (SEQ ID NO: 1). The MAGE-B2 peptide epitope can be contacted with or used to stimulate a population of T cells to induce proliferation of T cells that recognize or bind the MAGE-B2 peptide epitope. The MAGE-B2 peptide epitope can be administered to a subject, such as a human patient, to enhance the subject's immune response to cancer. The MAGE-B2 peptide epitope can be included in active immunotherapy (e.g., cancer vaccines) or passive immunotherapy (e.g., adoptive cell therapy). Active immunotherapy involves immunizing a subject with purified tumor antigens or MAGE-B2 peptide epitopes (natural or engineered). Alternatively, antigen-presenting cells pulsed with the MAGE-B2 peptide epitope (or transfected with a gene encoding the tumor antigen) can be administered to the subject. The MAGE-B2 peptide epitope may be modified or may include one or more mutations, such as, for example, substitution mutations. Adoptive cell therapy may involve administering cells to a subject, where the cells (e.g., cytotoxic T cells) have been sensitized in vitro to the MAGE-B2 peptide epitope.

[0048] In particular, T cells can be activated and expanded ex vivo for adoptive cell therapy within a short period of time, such as 6-8 weeks. T cells can be isolated from peripheral blood (e.g., CD4 T cells) using, for example, tetramer-induced sorting and rapid expansion protocols (REP). + T cells, CD8 +The peptide or corresponding polynucleotide (e.g., full-length MAGE-B2 or a MAGE-B2 peptide epitope) can then be loaded into HLA-A2-positive dendritic cells, lymphoblastoid cell lines (LCLs), PBMCs, or artificial antigen-presenting cells (aAPCs), which can then be co-cultured with T cells for several rounds of stimulation to generate antigen-specific CTL cell lines or clones. Furthermore, manipulation of immunoregulatory parameters can enhance the effector function and long-term in vivo persistence of these expanded antigen-specific T cells. These CTLs can be used in adoptive cell therapy for MAGE-B2 and HLA-A2-positive cancer patients. Other MAGE-B2-specific cells that can be generated from this disclosure include NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs), and induced pluripotent stem (iPS) cells. These cells can be isolated from blood or umbilical cord. The antigen-specific cells of the present disclosure can be autologous or allogeneic.

[0049] In another method, antigen-specific cells can be generated by using the MAGE-B2 TCRs provided herein (e.g., SEQ ID NOS: 2-5 or 18-22). In this method, the TCR sequences are inserted into T cells (e.g., CD4 + T cells, CD8 + They can be inserted into vectors (e.g., retroviral or lentiviral vectors) that are introduced into host cells, such as T cells, γδ T cells, and Tregs), NK cells, invariant NK cells, NKT cells, MSCs, or iPS cells, to generate antigen-specific cells that can be used in adoptive cell therapy for cancer patients.

[0050] The MAGE-B2 peptide epitopes and TCR sequences are provided below. [ka] [ka]

[0051] The MAGE-B2-231 C5 TCR sequence is provided below, with the signal peptide underlined and the variable regions in italics. [ka] [ka]

[0052] A. MAGE-B2 peptide In some embodiments, the present disclosure includes a MAGE-B2 peptide epitope. The MAGE-B2 peptide epitope may have the amino acid sequence of the HLA-A2-restricted MAGE-B2 peptide GVYDGEEHSV, SEQ ID NO: 1. The MAGE-B2 peptide epitope may have an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, 99, or 100 percent sequence identity to the peptide sequence of SEQ ID NO: 1.

[0053] A MAGE-B2 peptide epitope can comprise or consist of 7 to 35 amino acids, preferably 8 to 35 amino acid residues, even more preferably 8 to 25 amino acids, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length, or any range derivable therein. For example, a MAGE-B2 peptide epitope of the present disclosure can, in some embodiments, comprise or consist of the MAGE-B2 peptide epitope of SEQ ID NO: 1. An antigenic peptide can comprise an immunoreactive MAGE-B2 peptide epitope and can include additional sequences. The additional sequences can be derived from the native antigen or can be heterologous, and such sequences can be, but need not be, immunogenic. In some embodiments, the MAGE-B2 peptide epitope is capable of selectively binding to HLA-A2, in particular HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, or HLA-A*0205.

[0054] As will be appreciated by those skilled in the art, MHC molecules can bind peptides of various sizes, but typically cannot bind full-length proteins. While MHC class I molecules have traditionally been described as binding to peptides 8-11 amino acids long, it has been shown that 15-amino acid peptides can bind to MHC class I molecules by bulging in the center of the binding site or extending from the MHC class I binding groove. As will be readily appreciated by those skilled in the art, naturally occurring full-length tumor antigens, such as MAGE-B2, selectively bind class II MHC and may therefore be endocytosed and not useful for inducing T cell proliferation. Generally, naturally occurring full-length tumor antigen proteins do not exhibit these properties and are therefore not useful for these immunotherapeutic purposes.

[0055] In certain embodiments, the MAGE-B2 peptide epitopes are immunogenic or antigenic. As shown in the Examples below, the MAGE-B2 peptide epitopes of the present disclosure can promote T cell proliferation.

[0056] The MAGE-B2 peptide epitope can be a recombinant peptide, a synthetic peptide, a purified peptide, an immobilized peptide, a detectably labeled peptide, an encapsulated peptide, or a vector-expressed peptide (e.g., a peptide encoded by a nucleic acid in a vector comprising a heterologous promoter operably linked to the nucleic acid). In some embodiments, a synthetic MAGE-B2 peptide epitope can be administered to a subject, such as a human patient, to elicit an immune response in the subject. Synthetic peptides may exhibit certain advantages over recombinantly expressed peptides, such as a reduced risk of bacterial contamination. MAGE-B2 peptides can also be included in pharmaceutical compositions, such as vaccine compositions, formulated for administration to mammalian or human subjects.

[0057] 1. Cell-penetrating peptides In some embodiments, immunotherapy may utilize a MAGE-B2 peptide epitope of the present disclosure associated with a cell-penetrating agent, such as a liposome or cell-penetrating peptide (CPP). Antigen-presenting cells (such as dendritic cells) pulsed with the peptide may be used to enhance anti-tumor immunity. In some embodiments, immunotherapy may utilize a nucleic acid encoding a MAGE-B2 peptide epitope of the present disclosure, the nucleic acid being delivered, for example, in a viral or non-viral vector.

[0058] Cell-penetrating peptides that can be covalently linked to tumor antigen-specific peptides (e.g., MAGE-B2 peptides) include, for example, HIV Tat, herpesvirus VP22, Drosophila antennapedia homeobox gene product, signal sequences, fusion sequences, or protegrin I. Covalently linking a peptide to a CPP can prolong presentation of the peptide by dendritic cells and thus enhance anti-tumor immunity. In some embodiments, a MAGE-B2 peptide of the present disclosure (e.g., contained within a peptide or polyepitope string) can be covalently linked (e.g., via a peptide bond) to a CPP to generate a fusion protein. In other embodiments, a MAGE-B2 peptide epitope or a nucleic acid encoding a peptide epitope can be encapsulated within or associated with a liposome, exocytosis vesicle, or exosome, such as a multilamellar, vesicular, or multivesicular liposome.

[0059] In some embodiments, cellular uptake is promoted by attaching lipids, such as stearate or myristate, to polypeptides. Lipidation has been shown to enhance the passage of peptides into cells. Attaching lipid moieties is another way in which the present disclosure increases cellular uptake of polypeptides. Cellular uptake is further discussed below.

[0060] The MAGE-B2 peptide epitopes of the present disclosure may be included in a liposomal vaccine composition. For example, the liposomal composition may be or include a proteoliposomal composition.

[0061] In some embodiments, the MAGE-B2 peptide epitope may associate with a nanoparticle to form a nanoparticle-polypeptide complex. In some embodiments, the nanoparticle is a liposome or other lipid-based nanoparticle, such as a lipid-based vesicle (e.g., DOTAP:cholesterol vesicle). In other embodiments, the nanoparticle is an iron oxide-based superparamagnetic nanoparticle. In some embodiments, the nanoparticle is a semiconductor nanocrystal or semiconductor quantum dot, both of which can be used in optical imaging. In further embodiments, the nanoparticle may be a nanoshell comprising a gold layer on a silica core.

[0062] 2. Biological functional equivalent The MAGE-B2 peptide epitopes of the present disclosure can be modified to contain amino acid substitutions, insertions, and / or deletions that do not alter their respective interactions with HLA class protein binding regions, such as HLA-A2. As a non-limiting example, certain amino acids in the MAGE-B2 peptides disclosed herein can be substituted for other amino acids without appreciable loss of HLA binding, as demonstrated by undetectably altered peptide binding to HLA-A2. Thus, MAGE-B2 peptides disclosed herein (or nucleic acids encoding such peptides) that have altered sequence and / or structure but not altered biological utility or activity are contemplated to remain within the scope of the compositions and methods disclosed herein.

[0063] It is also well understood by those skilled in the art that the concept of having a limited number of changes that can be made within a defined portion of a molecule while still maintaining an acceptable level of equivalent biological activity is inherent in the definition of a biologically functional equivalent peptide.A biologically functional equivalent peptide is therefore defined herein as a peptide in which most or not all of a particular amino acid can be substituted.Of course, multiple distinct peptides with different substitutions can be easily made and used in accordance with the present disclosure.

[0064] Those skilled in the art also recognize that if certain residues, e.g., residues in a particular epitope, are shown to be particularly important to the biological or structural properties of a peptide, such residues generally cannot be replaced. This may be the case in the present disclosure, as mutations in the MAGE-B2 peptides disclosed herein may result in a loss of species specificity, thereby reducing the usefulness of the resulting peptide for use in the methods of the present disclosure. Thus, peptides that are antigenic (e.g., specifically bind HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, or HLA-A*0205) and contain conservative amino acid substitutions are understood to be included in the present disclosure. Conservative substitutions are least likely to dramatically alter the activity of a protein. A "conservative amino acid substitution" refers to the replacement of an amino acid with a chemically similar amino acid, i.e., replacing a nonpolar amino acid with another nonpolar amino acid, replacing a polar amino acid with another polar amino acid, replacing an acidic residue with another acidic amino acid, etc.

[0065] Amino acid substitutions that may be used in modifying the MAGE-B2 peptides disclosed herein are generally based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues, that alanine, glycine, and serine are all similar in size, and that phenylalanine, tryptophan, and tyrosine all have similar overall shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine are defined herein as biologically functional equivalents. In some embodiments, mutations may enhance TCR-pMHC interaction and / or peptide-MHC binding.

[0066] The present disclosure also contemplates isoforms of the MAGE-B2 peptides disclosed herein. Isoforms contain the same number and types of amino acids as the peptides of the present disclosure, but the isoforms have different molecular structures. Isoforms contemplated by the present disclosure have the same properties as the peptides of the present disclosure described herein.

[0067] Non-standard amino acids can be incorporated into proteins by chemical modification of existing amino acids or by de novo synthesis of the peptides disclosed herein. Non-standard amino acids refer to amino acids whose chemical structure differs from the 20 standard amino acids encoded by the genetic code.

[0068] In some embodiments, the present disclosure contemplates chemical derivatives of the MAGE-B2 peptides disclosed herein. A "chemical derivative" refers to a peptide having one or more residues that have been chemically derivatized by reaction of a side chain functional group, while retaining biological activity and utility. Such derivatized peptides include, for example, those in which free amino groups have been derivatized to form certain salts or by alkylation and / or acylation, including p-toluenesulfonyl, carbobenzoxy, t-butylocycarbonyl, chloroacetyl, formyl, or acetyl groups, among others. Free carboxyl groups may be derivatized to form organic or inorganic salts, methyl and ethyl esters or other types of esters, or hydrazides, and preferably amides (primary or secondary). Chemical derivatives may include peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline may be substituted for serine, and ornithine may be substituted for lysine.

[0069] It should be noted that all amino acid residue sequences herein are represented by formulas whose left-right orientation is in the conventional direction from amino to carboxy terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues. The amino acids described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional properties described herein are retained by the protein.

[0070] The preferred MAGE-B2 peptides or analogs thereof preferably specifically or preferentially bind HLA-A2. Whether or not a particular tumor antigen-specific peptide or labeled peptide, or analog thereof, can bind HLA-A2, or the degree of binding, can be determined, and this determination can be assessed using in vitro assays, such as enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescence assay (FA), turbidimetry, flow cytometry assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, mass spectrometry assay, particle-based assay, inhibition assay, and / or avidity assay.

[0071] B. Engineered MAGE-B2-specific cells In some embodiments, the present disclosure provides MAGE-B2-specific TCRs. The TCRs may comprise an α chain CDR of SEQ ID NOs: 6-12 and / or a β chain CDR of SEQ ID NOs: 13-17. The TCRs may comprise an α chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or similarity to SEQ ID NOs: 2-3, and / or a β chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or similarity to SEQ ID NOs: 4-5. Polypeptides and polynucleotides encoding the α chain and / or β chain of the MAGE-B2 TCRs provided herein are also provided herein. Further provided herein are cells, such as T cells, NK cells, invariant NK cells, NKT cells, MSCs, or iPS cells, engineered to express the MAGE-B2-specific TCRs provided herein. These non-T cell effector immune cells may express the TCR together with a CD3 molecule or other signaling domain linked to the TCR, which initiates signal transduction in these cells.

[0072] The engineered immune cells can be constructed using any of many well-established gene transfer methods known to those skilled in the art. In certain embodiments, the engineered cells are constructed using a viral vector-based gene transfer method to introduce a nucleic acid encoding a MAGE-B2-specific TCR. The viral vector-based gene transfer method can include a lentiviral vector, a retroviral vector, an adenovirus, or an adeno-associated virus vector. In certain embodiments, the engineered cells are constructed using a non-viral vector-based gene transfer method to introduce a nucleic acid encoding a MAGE-B2-specific TCR. The TCR vector can include an α chain polypeptide and a β chain polypeptide, which can be linked by a linker domain or an IRES sequence. The linker domain can include one or more cleavage sites, such as a furin cleavage site and / or a P2A cleavage site, which can be separated by a spacer, such as SGSG or GSG. In certain embodiments, the non-viral vector-based gene transfer method comprises a gene editing method selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) nucleases. In certain embodiments, the non-viral vector-based gene editing method comprises a transfection or transformation method selected from the group consisting of lipofection, nucleofection, virosomes, liposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced DNA uptake.

[0073] C. Soluble TCR and BiTE Furthermore, the present disclosure provides soluble TCRs that can be used to directly treat HLA-A2-positive cancer patients. Soluble bispecific T cell-engaging molecules (BiTEs) can be generated by linking MAGE-B2 TCRs to CD3-specific Fab fragments. These bispecific molecules can bind to tumor cell surfaces via MAGE-B2 TCR binding to peptide / HLA complexes, and the CD3-specific Fab fragments crosslink TCRs on target T cells, etc. This leads to cell activation and target cell elimination. Therefore, these soluble bispecific TCR constructs can be used directly to treat cancer patients.

[0074] Finally, soluble TCR can be used as a probe for the diagnostic evaluation of peptide / MHC in tumor cells, or to direct therapeutic molecules to tumor site.This soluble TCR molecule can also be labeled with a tracer such as fluorescent probe or radioactive probe, and then used for the diagnostic evaluation of the presentation of peptide / MHC in tumor cells.In addition, this soluble TCR molecule can be linked with therapeutic molecules such as toxins, and then these therapeutic molecules can be directed to tumor site for the treatment of cancer patients.

[0075] In some embodiments, the present disclosure provides soluble TCRs, such as the MAGE-B2-specific TCRs provided herein. Soluble TCRs can be used to investigate specific TCR-pMHC interactions, as diagnostic tools for detecting infections, or to detect autoimmune disease markers. Soluble TCRs can also be used in staining, for example, to stain cells for the presence of specific peptide antigens presented in the context of MHC. Similarly, soluble TCRs can be used to deliver therapeutic agents, such as cytotoxic or immunostimulatory compounds, to cells presenting specific antigens. Soluble TCRs may also be used to inhibit T cells, for example, T cells that respond to autoimmune peptide antigens. In some aspects, the TCR is linked to another molecule that delivers cells to the vicinity of a tumor. In further aspects, the TCR delivers toxins, cytokines, costimulatory ligands, or inhibitory ligands, directing molecules, cells, or compounds to target cells expressing peptide-MHC.

[0076] In some aspects, the present disclosure provides a soluble T cell receptor (sTCR) comprising (i) all or a portion of the TCR alpha chain (e.g., SEQ ID NO: 2 or 3), excluding its transmembrane domain, and (ii) all or a portion of the TCR beta chain (e.g., SEQ ID NO: 4 or 5), excluding its transmembrane domain, wherein (i) and (ii) comprise at least a portion of the functional variable and constant domains of the TCR chains, respectively, and are linked by disulfide bonds between constant domain residues that are not present in the native TCR.

[0077] In some embodiments, the soluble TCR comprises a TCR α or γ chain extracellular domain dimerized to a TCR β or δ chain extracellular domain, respectively, by a pair of C-terminal dimerization peptides, such as leucine zippers.

[0078] The soluble TCRs of the present disclosure may be provided in a substantially pure form or as a purified or isolated preparation, for example, substantially free of other proteins.

[0079] The plurality of soluble TCRs of the present disclosure may be provided in a multivalent complex. Thus, in one aspect, the present disclosure provides a multivalent TCR complex comprising a plurality of soluble TCRs described herein. Each of the plurality of soluble TCRs is preferably identical.

[0080] In its simplest form, a multivalent TCR complex according to the present disclosure comprises a multimer of two, three, four, or more T cell receptor molecules associated with each other (e.g., covalently or otherwise linked), preferably via a linker molecule. Suitable linker molecules include, but are not limited to, multivalent attachment molecules such as avidin, streptavidin, neutravidin, and extravidin, each of which has four binding sites for biotin. Thus, biotinylated TCR molecules can be formed into multimers of TCRs with multiple TCR binding sites. The number of TCR molecules in a multimer depends on the amount of TCR relative to the amount of linker molecules used to create the multimer, and also on the presence or absence of any other biotinylated molecules. Preferred multimers are dimeric, trimeric, or tetrameric TCR complexes.

[0081] Suitable structures for use in the methods of the present invention include membrane structures such as liposomes, and solid structures, preferably particles such as beads, e.g., latex beads. Other structures that can be coated on the outside with T cell receptor molecules are also suitable. Preferably, the structures are coated with T cell receptor multimers rather than individual T cell receptor molecules.

[0082] In the case of liposomes, the T cell receptor molecule or multimer thereof may be attached to or otherwise associated with the membrane, techniques for this being well known to those skilled in the art.

[0083] A label or other moiety, such as a toxic or therapeutic moiety, may be included in the multivalent TCR complex of the present disclosure. For example, the label or other moiety may be included in a mixed molecule multimer. An example of such a multimeric molecule is a tetramer containing three TCR molecules and one peroxidase molecule. This can be achieved by mixing the TCR and enzyme in a 3:1 molar ratio to produce a tetrameric complex and isolating the desired complex from any complexes that do not contain the correct ratio of molecules. These mixed molecules can contain any combination of molecules as long as steric hindrance does not impair or significantly impair the desired function of the molecules. The configuration of the binding sites on the streptavidin molecule is favorable for mixed tetramers because steric hindrance is unlikely to occur.

[0084] Alternatively or additionally, the TCRs (or multivalent complexes thereof) of the present disclosure may be associated (e.g., covalently or otherwise linked) with a therapeutic agent, which may be, for example, a toxic moiety for use in cell killing, or an immunostimulatory agent such as an interleukin or cytokine. Multivalent TCR complexes of the present disclosure may have enhanced binding ability to TCR ligands compared to non-multimeric T cell receptor heterodimers. Thus, multivalent TCR complexes according to the present disclosure are particularly useful for tracking or targeting cells presenting specific antigens in vitro or in vivo, and are also useful as intermediates for the production of additional multivalent TCR complexes with such uses. Accordingly, the TCRs or multivalent TCR complexes may be provided in pharmaceutically acceptable formulations for use in vivo.

[0085] The present disclosure also provides a method for delivering a therapeutic agent to a target cell, comprising contacting a potential target cell with a TCR or multivalent TCR complex according to the present disclosure under conditions that allow attachment of the TCR or multivalent TCR complex to the target cell, wherein the TCR or multivalent TCR complex is specific for a TCR ligand and has a therapeutic agent associated therewith.

[0086] In particular, soluble TCR or multivalent TCR complexes can be used to deliver therapeutic agents to the location of cells that present specific antigens.This can be useful in many situations, especially for tumors.The therapeutic agent can be delivered so that it exerts its effect locally, not just in the cells to which it binds.Therefore, one particular strategy envisions anti-tumor molecules linked to T cell receptors or multivalent TCR complexes that are specific for tumor antigens.

[0087] Many therapeutic agents can be used for this purpose, such as radioactive compounds, enzymes (e.g., perforin), or chemotherapeutic agents (e.g., cisplatin). To ensure that the toxic effect is exerted at the desired location, the toxin can be placed inside a liposome linked to streptavidin, resulting in a slow release of the compound. This prevents damaging effects during transport within the body and ensures that the toxin has its maximum effect after binding to the relevant antigen-presenting cell by the TCR.

[0088] The soluble TCR of the present disclosure can be used to modulate T cell activation by binding to specific TCR ligands, thereby inhibiting T cell activation. Autoimmune diseases involving T cell-mediated inflammation and / or tissue damage, such as type I diabetes, may be suitable for this approach. This use requires knowledge of the specific peptide epitope presented by the relevant pMHC.

[0089] The use of the soluble TCRs and / or multivalent TCR complexes of the present disclosure in the preparation of compositions for the treatment of cancer or autoimmune disease is also envisaged.

[0090] Also provided is a method for treating cancer or an autoimmune disease comprising administering to a patient in need thereof an effective amount of a soluble TCR and / or multivalent TCR complex of the present disclosure.

[0091] As is common in anti-cancer and autoimmune therapies, the soluble TCRs of the present disclosure may be used in combination with other agents for the treatment of cancer and autoimmune diseases, as well as other related conditions seen in similar patient populations.

[0092] III.How to use In another aspect, provided herein is a method for treating cancer in a subject, comprising administering to a subject a therapeutically effective amount of MAGE-B2 cells, such as T cells, NK cells, invariant NK cells, NKT cells, MSCs, or iPS cells produced by any of the methods provided herein. A method comprising administering to a subject a therapeutically effective amount of a population of TCR-specific cells. The cells from which TILs can be cultured in vitro or from which tumor antigen-specific CTLs can be generated in vitro can be adoptively transferred into a subject with cancer.

[0093] Provided herein is a method for treating cancer or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of MAGE-B2-specific T cell therapy. Also provided herein is adoptive T cell therapy using genetically engineered TCR-transduced T cells (in which the TCR is conjugated to other bioreactive proteins, such as anti-CD3). In a further embodiment, a method for treating cancer is provided, comprising immunizing a subject with a purified tumor antigen or an immunodominant tumor antigen-specific peptide.

[0094] The MAGE-B2 peptides provided herein can be used to develop cancer vaccines or immunogens. These peptide-specific vaccines or immunogens can be used to directly immunize cancer patients to induce anti-tumor immune responses in vivo, or to expand antigen-specific T cells in vitro by stimulating APCs loaded with the peptide or encoding polynucleotide. These large numbers of T cells can be adoptively transferred into patients to induce tumor regression.

[0095] The tumors for which this treatment method is useful include any malignant cell type that expresses MAGE-B2, such as those found in solid tumors or blood tumors.Exemplary solid tumors include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary blood tumors include bone marrow tumors, malignant diseases of T or B cells, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric or stomach cancer (including digestive cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0096] Cancers, especially those of the following histological types: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumor; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma adenocarcinoma); papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinic cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomus angiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma melanoma); acral lentiginous melanoma; nodular melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mixed Müllerian tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor, malignant Phyllodes tumor, malignant;synovial sarcoma;mesothelioma, malignant;dysgerminoma;embryonal carcinoma;teratoma, malignant;ovarial goiter, malignant;choriocarcinoma;mesonephroma, malignant;angiosarcoma;hemangioendothelioma, malignant;Kaposi's sarcoma;hemangiopericytoma, malignant;lymphangiosarcoma;osteosarcoma;parosteal osteosarcoma;chondrosarcoma;chondroblastoma, malignant;mesenchymal chondrosarcoma;giant cell tumor of bone;Ewing's sarcoma;odontogenic tumor, malignant;ameloblastic odontosarcoma;Ameloblastoma, malignant; Ameloblastoma fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrillary astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal; Cerebellar sarcoma; Ganglioneuroma; Neuroblastoma; Retinoblastoma; Olfactory nerve tumor; Meningioma, malignant; Neurofibrosarcoma; Neurofibroschuma, malignant; Granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; lateral granuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate The disease may be, but is not limited to, aggressive diffuse NHL, aggressive immunoblastic NHL, aggressive lymphoblastic NHL, aggressive small non-cleaved cell NHL, giant mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloblastic leukemia.

[0097] In certain embodiments, the method further comprises performing lymphodepletion prior to administering a therapeutically effective amount of the population of MAGE-B2 TCR cells. In certain embodiments, the lymphodepletion comprises non-myeloablative lymphodepleting chemotherapy. In certain embodiments, the non-myeloablative lymphodepleting chemotherapy comprises administering cyclophosphamide and fludarabine.

[0098] In certain embodiments, the method further comprises administering to the subject, either concomitantly with or subsequent to the autologous T cells, a T cell growth factor that promotes the growth and activation of the autologous T cells. In certain embodiments, the T cell growth factor comprises any suitable growth factor that promotes the growth and activation of the autologous T cells. In certain embodiments, the T cell growth factor is selected from the group consisting of interleukin (IL)-2, IL-7, IL-15, and IL-12, and combinations thereof (e.g., IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2).

[0099] In certain embodiments, a therapeutically effective amount of a population of MAGE-B2 TCR-specific cells produced by any of the methods provided herein is administered intravenously, intratumorally, or intraperitoneally to a subject. The appropriate dosage of cell therapy can be determined based on the type of cancer being treated, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0100] A. Combination Therapy In certain embodiments, the methods provided herein further comprise administering to the subject at least one additional therapeutic agent. All additional therapeutic agents disclosed herein are administered to the subject in accordance with good clinical practice for each particular composition or treatment, taking into account any potential toxicity, possible side effects, and any other relevant factors.

[0101] In certain embodiments, the additional therapy may be immunotherapy, radiation therapy, surgery (e.g., surgical removal of tumor), chemotherapy, bone marrow transplant, or a combination of the foregoing. The additional therapy may be targeted therapy. In certain embodiments, the additional therapy is administered before the primary treatment (i.e., as adjuvant therapy). In certain embodiments, the additional therapy is administered after the primary treatment (i.e., as neoadjuvant therapy).

[0102] In certain embodiments, the additional therapy comprises immunotherapy. In certain embodiments, the immunotherapy comprises an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor inhibits an immune checkpoint protein selected from the group consisting of programmed cell death pathway 1 (PD-1 / CD279) and its ligands (PD-L1 / CD274 and PD-L2 / CD273), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4 / CD152), lymphocyte-activation gene 3 (LAG-3 / CD223), B- and T-lymphocyte attenuator (BTLA), T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT), T-cell immunoglobulin domain and mucin domain 3 (TIM-3 / HAVcr2), killer immunoglobulin-like receptor (KIR / CD158), V-domain immunoglobulin suppressor of T-cell activation (VISTA), and adenosine A2a receptor (A2aR).

[0103] In certain embodiments, the immune checkpoint inhibitor is a PD-1 binding antagonist. In certain embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In certain embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1-binding portion of PDL1 or PDL2 fused to an immunoglobulin constant region (e.g., an Fc region of an immunoglobulin sequence)).

[0104] In certain embodiments, the immune checkpoint inhibitor is a CTLA-4 binding antagonist. In certain embodiments, the CTLA-4 binding antagonist is an anti-CTLA-4 antibody. In certain embodiments, the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab and tremelimumab.

[0105] In certain embodiments, the additional therapeutic agent comprises radiation therapy treatment. In certain embodiments, the radiation therapy is selected from the group consisting of gamma rays (γ-rays), X-rays, microwaves, proton beam radiation, ultraviolet radiation, and directed delivery of a radioisotope to the tumor. In certain embodiments, the radiation therapy comprises X-ray treatment. In certain embodiments, the X-rays are administered in daily doses of 50-200 roentgens over a period of 3-4 weeks. In certain embodiments, the X-rays are administered in a single dose of 2000-6000 roentgens. In certain embodiments, the radiation therapy comprises directed delivery of a radioisotope to the tumor. Radiation dose ranges for radioisotopes vary widely, depending on the half-life of the isotope, the strength and type of radiation emitted, and the extent of uptake by tumor cells; however, determination of an appropriate therapeutically effective dose is within the level of one skilled in the art.

[0106] In certain embodiments, the additional therapeutic agent comprises administration of an agent for the treatment of side effects associated with the primary treatment (e.g., nausea, cachexia, etc.). In certain embodiments, the additional therapy comprises immunotherapy. In certain embodiments, the additional therapy comprises radiation therapy. In some embodiments, radiation therapy comprises gamma irradiation. In certain embodiments, the additional therapy comprises surgery. In certain embodiments, the additional therapy comprises a combination of radiation therapy and surgery. In certain embodiments, the additional therapy comprises treatment with a class of chemotherapeutic agent selected from the group consisting of alkylating agents, anthracyclines, cytoskeletal disruptors, epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, nucleotide analogs and nucleotide precursor analogs, peptide antibiotics, platinum-based compounds, retinoids, vinca alkaloids, and derivatives thereof.

[0107] The additional therapeutic methods contemplated herein can be administered before, after, or simultaneously with the administration of the compositions provided herein.In certain embodiments, the additional therapeutic methods are administered before the administration of the compositions provided herein.In certain embodiments, the additional therapeutic methods are administered after the administration of the compositions provided herein.In certain embodiments, the additional therapeutic methods are administered one or more intervals before or after the administration of the compositions provided herein.It is within the level of those skilled in the art to determine the appropriate interval for the administration of the additional therapeutic methods so that the subject being treated can benefit from the combined therapy.

[0108] B. Pharmaceutical Compositions In another aspect, provided herein are pharmaceutical compositions and formulations comprising MAGE-B2 TCR-specific cells and a pharmaceutically acceptable carrier.

[0109] The pharmaceutical compositions and formulations described herein can be prepared by mixing the active ingredient (such as an antibody or polypeptide) having the desired degree of purity in the form of an aqueous solution, e.g., saline (e.g., 0.9%) and human serum albumin (e.g., 10%), with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22 nd edition, Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers, e.g., phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins , such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG). [Example]

[0110] IV. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and can therefore be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of this disclosure, appreciate that many changes can be made in the specific embodiments disclosed and still obtain like or similar results, without departing from the spirit and scope of the invention.

[0111] Example 1 Generation and Characterization of MAGE-B2-Specific T Cells MAGE-B2 expression was analyzed in lung cancer cell lines and immortalized normal human small airway epithelial cells (HSAEC1-KT and HSAEC2-KT) (Fig. 1). MAGE-B2 protein was found to be strongly expressed in most lung cancer cell lines, but was hardly expressed in normal lung (ling) cell lines.

[0112] To generate MAGE-B2-specific CD8+ CTLs, dendritic cells were cultured in a 2000-well plate containing 1000 ng / ml MAGE-B2-specific CD8+ CTLs. T cells were then stimulated with RNA encoding an HLA-A2-restricted epitope. + Tetramers were detected by flow cytometry. T cells were then sorted, cloned, and expanded by random expansion protocol (REP). T cells were then characterized by functional screening before cloning of functional MAGE-B2-specific TCRs.

[0113] Thus, the MAGE-B2 HLA-A2-restricted epitope was used to generate MAGE-B2-specific cytotoxic T lymphocytes (CTLs). Naive T cells were derived from healthy HLA-A2 donors and stimulated with autologous mature dendritic cells (mDCs) pulsed with full-length MAGE-B2 RNA. After two rounds of stimulation, a tetramer bearing the HLA-A2-restricted MAGE-B2 epitope (GVYDGEEHSV; SEQ ID NO: 1) was used to detect the T cell population that recognized the epitope. CD8 + tetramer + The population was selected and expanded using the Rapid Expansion Protocol (REP) to generate CTL cell lines. Correlative CTL clones were generated using limiting dilution. Over 99% of the cells were CD8 + and tetramer + It was observed that (Figure 2).

[0114] Next, we tested the functional avidity of MAGE-B2-specific T cells. In a peptide titration assay, T2 cells were pulsed with different concentrations of MAGE-B2 peptide (10 pg / ml to 10 μg / ml) (Figure 3A). T2 cells were used as target cells and co-cultured with isolated MAGE-B2 CTL clones (E:T = 20:1). The cytotoxic activity of the CTL clones against the lung cancer cell line H2023 (HLA-A*0201) and the normal lung cell line HSAEC2-KT (HLA-A*0201) was measured (Figure 3B). Target cells were co-cultured with MAGE-B2 CTL clones at different E:T ratios. Cytotoxic activity was measured using standard 51 The MAGE-B2 CTL clone was detected by Cr release assay. It was observed that the MAGE-B2 CTL clone was cytotoxic against lung cancer cell lines, but not against normal lung cell lines (Figure 3B). Furthermore, HLA-A2 + Lung cancer cell lines H522, H1355, H1755, and DFC-1032 were used as target cells and cocultured with the MAGE-B2 CTL clone at different E:T ratios, and cytotoxicity was measured. The MAGE-B2 CTL clone was observed to be cytotoxic against the lung cancer cell lines DFC-1032 and H1755 (Figure 3C). Finally, we evaluated the cytotoxicity of the CTL clone against the parental lung cancer cell lines PC-9 and H1573, as well as both cell lines with forced HLA-A2 expression. Greater cytotoxic activity was observed against cell lines with forced HLA-A2 expression compared to the parental PC-9 and H1573 cells (Figure 3D).

[0115] To generate MAGE-B2 TCR-engineered T cells (TCR-T), the TCR from a MAGE-B2 CTL clone was cloned and inserted into the retroviral vector pMSGV1. A linker fragment containing a furin cleavage site, an SGSG linker, and a P2A cleavage site was inserted between the TCR-β and TCR-α chains to ensure equal expression of both chains under the MSCV promoter. Recombinant retrovirus was generated by cotransfection of the retroviral vector and the envelope vector RD114 into the packaging cell line GP2-293. Two to three days after transfection, the retrovirus-containing supernatant was used to infect allogeneic PBMCs that had been activated for two days with 50 ng / mg OKT3 and 300 U / ml IL-2. Another infection was performed one day after the first infection. Five days later, clear CD8 + tetramer + The TCR-T cell line was detected by flow cytometry (Figure 4). + tetramer + Populations were generated by selection and expansion using a rapid expansion protocol.

[0116] A peptide titration assay was performed using T2 cells as target cells pulsed with different concentrations of MAGE-B2 peptide (10 pg / ml to 10 μg / ml). T2 cells were co-cultured with a MAGE-B2 TCR-T cell line (E:T = 20:1). Cytotoxicity was assessed using standard 51 The cytotoxicity of MAGE-B2 TCR-T cells against the lung cancer cell line H2023 (HLA-A*0201) and the normal lung cell line HSAEC2-KT (HLA-A*0201) was also evaluated (Figure 5B). The lung cancer cell line H2023 and the normal lung cell line HSAEC2-KT were co-cultured with MAGE-B2 TCR-T cells at different E:T ratios. The killing activity was measured using a standard 51 Cr release assay was used to detect the MAGE-B2 TCR-T cell line, which was observed to be specifically cytotoxic against lung cancer cell lines.

[0117] Finally, we functionally characterized MAGE-B2 TCR-T cells by intracellular cytokine staining (ICS). MAGE-B2 TCR-T cell lines were cocultured with the lung cancer cell line H2023, the normal lung cell line HSAEC2-KT, T2 cells pulsed with MAGE-B2 peptide, and T2 cells pulsed with the MART-1 peptide M26. IFN-γ, TNF-α, IL-2, and antigen-specific response markers CD137 and CD69 were detected by ICS assay. Coculture of TCR-T cells with the lung cancer cell line H2023 or T2 cells pulsed with MAGE-B2 peptide significantly enhanced IFN-γ, TNF-α, IL-2, CD137, and CD69 levels in the MAGE-B2 TCR-T cell lines compared with coculture with the normal lung cell line HSAEC2-KT or T2 cells pulsed with the control peptide M26 (Figure 6).

[0118] Therefore, MAGE-B2 TCR-T cells can be used to treat HLA-A2 (e.g., HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, or HLA-A*0205)-positive patients with advanced or recurrent cancer by, for example, generating and expanding TCR gene-modified CTLs using allogeneic PBMCs. After functional detection (e.g., phenotype and cytotoxicity), the TCR-modified T cells are infused back into the patient.

[0119] Example 2 Materials and Methods To generate T cell clones: Full-length MAGE-B2 RNA was transfected into mature dendritic cells (DCs) from healthy HLA-A2 donors. The RNA-transfected DCs were co-cultured with naive T cells at a DC:T ratio of 1:10 in the presence of IL-21. One week later, the RNA-transfected DCs were used to restimulate the T cells. After two rounds of stimulation, CD8 and tetramer double-positive T cell populations were selected and expanded using the rapid expansion protocol (REP). T cell clones were generated by limiting dilution. Highly active CTL clones were screened via cytotoxicity assays against cancer cells.

[0120] Cloning of T cell receptors (TCRs) and construction of retroviral expression vectors: TCRs (including α and β chains) were cloned using the 5'-RACE method according to the manufacturer's instructions. TCR V-alpha and TCR V-beta usage were identified using the IMGT / V-QUEST annotation tool. For construction of TCR-expressing retroviral vectors, forward primers were designed according to the TCR V-alpha or beta usage. Reverse primers were designed according to the sequence of the TCR alpha or beta constant region. Expression cassettes containing the alpha and beta TCR chains separated by furin and P2A linker peptides were generated, and the full-length PCR products were cloned into the retroviral vector pMSGV1. The cloned DNA sequences were verified by sequencing.

[0121] Retrovirus generation and infection of human peripheral blood lymphocytes (PBLs): The pMSGV1 vector containing the TCR and the envelope vector RD114 were co-transfected into the packaging cell line GP2-293. After 6–8 h of transfection, the medium was refreshed. After 24 h, the supernatant was collected and added to a 6-well plate coated with 20 μg / mL RetroNectin, followed by centrifugation (2000 × g) at 32 °C for 2 h. The supernatant was then removed, and PBLs activated for 2 days with 50 ng / mL OKT3 and 300 U / mL IL-2 were added to the retrovirus-loaded plate, followed by centrifugation (1000 × g) at 32 °C for 10 min. The cells were then incubated overnight at 32 °C, and the procedure was repeated the next day (a total of two transductions). The cells were then expanded at 37 °C in a 5% CO2 incubator and split as needed.

[0122] Generation of TCR-engineered T cell clones: post-infection, CD8 + and tetramer + T cell populations were selected and expanded using a rapid expansion protocol (REP).

[0123] 51Cr release assay: The killing capacity of TCR-engineered T cells or CTL clones to lyse HLA-A2 tumor targets was assessed using standard 51 Cr release assay was used. Tumor or normal cells were treated with 200 μCi of 51 Cr for 2 hours at 37°C. The labeled target cells were washed and then incubated with effector cells at different ratios in 0.2 ml of complete medium for 4 hours at 37°C. The collected supernatants were counted using an automated gamma counter. Maximum and spontaneous 51 Cr release was determined by incubating labeled target cells in either trypan lysis buffer or medium for 4 hours at 37°C. Each data point was determined as the average of quadruplicate wells. The percent of specific lysis was calculated as follows: % killing = ((specific release - spontaneous release) / (total release - spontaneous release)) × 100.

[0124] Intracellular cytokine staining (ICS) assay: T cells were incubated with target cells at a 10:1 ratio in the presence of brefeldin A (BFA) overnight at 37°C. After co-culture, T cells were harvested and washed. First, cells were stained with flow antibodies against surface markers. Then, cells were washed, fixed with Fix Buffer, and then permeabilized using Permeabilizing Solution. The permeabilized cells were then stained with intracellular cytokine flow antibodies. Finally, the levels of cytokines produced within the cells were analyzed using FACS.

[0125] Example 3: Generation of MAGE-B2 HLA-A2-restricted peptide (MB2-231)-specific TCR-T An additional MAGE-B2-specific T cell population was generated using dendritic cells pulsed with the MAGE-B2 peptide (GVYDGEEHSV; SEQ ID NO: 1) to stimulate PBMCs from the same healthy donor (Figure 7). After two rounds of stimulation, CD8 T cells were detected in three wells of a single 48-well plate. + / tetramer +A small population of 100 positive wells was observed. Three positive wells were separately selected using the tetramer-induced selection technique and expanded once or twice by REP. CD8 and tetramer staining of the final product is shown in Figure 7.

[0126] The functional avidity of the three MAGE-B2-specific CTL cell lines was demonstrated by lysis of the T2 cell line pulsed with various concentrations of the MAGE-B2 peptide (GVYDGEEHSV; SEQ ID NO: 1) at an effector-to-target (E:T) ratio of 20:1. Cytotoxicity was detected by a standard 51Cr release assay (Figure 8A). The lung cancer cell line H2023 (HLA-A*0201 + , MAGE-B2 + ) and normal lung cell line HSAEC2-KT (HLA-A*0201 + , MAGE-B2 - The cytotoxicity of the three MAGE-B2-specific CTL cell lines against the lung cancer cell line H2023 (HLA-A*0201-, MAGE-B2+), H1299-A2 (HLA-A*0201-, MAGE-B2+), and H1299-A2 (HLA-A*0201-, MAGE-B2+) was also evaluated (Figure 8B). The lung cancer cell line H2023 and the normal lung cell line HSAEC2-KT were co-cultured with MAGE-B2 TCR-T cells at different E:T ratios. Killing activity was detected by a standard 51Cr release assay. All three MAGE-B2-specific CTL cell lines were observed to be specifically cytotoxic against the lung cancer cell line H2023 (Figure 8B). Other lung cancer cell lines were H1299 (HLA-A*0201-, MAGE-B2+), H1299-A2 (HLA-A*0201-, MAGE-B2+), and H1299-A2 (HLA-A*0201-, MAGE-B2+). + We further evaluated the cytotoxicity of the three MAGE-B2-specific CTL cell lines against H1395 (HLA-A*0201+, MAGE-B2+), H522 (HLA-A*0201+, MAGE-B2-), H1355 (HLA-A*0201+, MAGE-B2-), H1755 (HLA-A*0201+, MAGE-B2+), and DFC-1032 (HLA-A*0201+, MAGE-B2-) (Figures 8C, 8D, and 8E).

[0127] To generate MAGE-B2 TCR-engineered T cells (TCR-T), the TCR from the MAGE-B2 CTL cell line C5 was cloned and inserted into the retroviral vector pMSGV1. A linker fragment containing a furin cleavage site, an SGSG linker, and a P2A cleavage site was inserted between the TCR-β and TCR-α chains to ensure equal expression of both chains under the MSCV promoter. Recombinant retrovirus was generated by co-transfection of the retroviral vector and the envelope vector RD114 into the packaging cell line Phoenix-GP. Two to three days after transfection, the supernatant containing the retrovirus was used to infect PBMCs from an allogeneic HLA-A*0201+ healthy donor that had been activated for two days with 50 ng / mg OKT3 and 300 U / ml IL-2. Five days later, a distinct CD8+ tetramer+ population was detected by flow cytometry (Figure 9A). The CD8+ tetramer+ population was selected using tetramer-guided sorting technology and expanded by REP. CD8 and tetramer staining of the final product is shown in Figure 9A. The functional avidity of MAGE-B2 TCR-T was demonstrated by lysis of T2 cell lines pulsed with various concentrations of MAGE-B2 peptide (GVYDGEEHSV; SEQ ID NO: 1) at an effector-to-target (E:T) ratio of 20:1. Cytotoxicity was detected by a standard 51Cr release assay (Figure 9B). The cytotoxicity of MAGE-B2 TCR-T against the lung cancer cell line H2023 (HLA-A*0201+, MAGE-B2+) and the normal lung cell line HSAEC2-KT (HLA-A*0201+, MAGE-B2-) was also evaluated (Figure 9C). We further evaluated the cytotoxicity of MAGE-B2 TCR-T against other lung cancer cell lines: H1299 (HLA-A*0201-, MAGE-B2+), H1299-A2 (HLA-A*0201-expressing, MAGE-B2+), H1395 (HLA-A*0201+, MAGE-B2+), H522 (HLA-A*0201+, MAGE-B2-), H1355 (HLA-A*0201+, MAGE-B2-), H1755 (HLA-A*0201+, MAGE-B2+), and DFC-1032 (HLA-A*0201+, MAGE-B2-) (Figures 9D and 9E).

[0128] Finally, MAGE-B2 TCR-T cells were functionally characterized by intracellular cytokine staining (ICS). MAGE-B2 TCR-T cell lines were co-cultured with T2 cells pulsed with the MAGE-B2 peptide (GVYDGEEHSV; SEQ ID NO: 1) and with T2 cells pulsed with the MART-1 peptide M26 (as a control) (Figure 10A). The response of MAGE-B2-specific TCR-T cells to the lung cancer cell line H2023 and the normal lung cell line HSAEC2-KT (as a control) was also evaluated (Figure 10A). Furthermore, other lung cancer cell lines, H1395, H522, H1299, H1299-A2, DFC-1032, H1355, and H1755, were also used as targets to evaluate the function and specificity of MAGE-B2 TCR-T cells (Figures 10B and 10C). Cytokine release of IFN-γ, TNF-α, and upregulation of antigen-specific response markers CD137 and CD69 in MAGE-B2 TCR-T cell lines were detected by ICS.

[0129] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods and steps or the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Barnea et al., Eur J Immunol, 32(1):213-22, 2002. Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press, 2012. Shraibman et al., Mol Cell Proteomics, 15(9):3058-70, 2016.

Claims

1. 1. A polynucleotide encoding a polypeptide comprising a TCR alpha polypeptide comprising the sequences CDR1 (SEQ ID NO:7), CDR2 (SEQ ID NO:9), and CDR3 (SEQ ID NO:11), and a TCR beta polypeptide comprising the sequences CDR1 (SEQ ID NO:13), CDR2 (SEQ ID NO:15), and CDR3 (SEQ ID NO:17), wherein a T cell receptor (TCR) comprising said TCR alpha polypeptide and said TCR beta polypeptide is capable of binding to an antigenic peptide derived from melanoma-associated antigen B2 (MAGE-B2).

2. The polynucleotide of claim 1, wherein the polypeptide comprises a TCR alpha polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:3 and a TCR beta polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:

5.

3. The polynucleotide of claim 1, wherein the polypeptide comprises a TCR alpha polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:3 and a TCR beta polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:

5.

4. The polynucleotide of claim 1 , wherein the polypeptide comprises a TCR alpha polypeptide of SEQ ID NO: 3 and a TCR beta polypeptide of SEQ ID NO:

5.

5. The polynucleotide of claim 1 , wherein the polypeptide comprises a TCR alpha polypeptide of SEQ ID NO:

3.

6. 2. The polynucleotide of claim 1, wherein the polypeptide comprises a TCR beta polypeptide of SEQ ID NO:

5.

7. 1. A polynucleotide encoding a polypeptide comprising a TCR alpha polypeptide comprising the sequences CDR1 (SEQ ID NO:23), CDR2 (SEQ ID NO:25), and CDR3 (SEQ ID NO:27), and a TCR beta polypeptide comprising the sequences CDR1 (SEQ ID NO:29), CDR2 (SEQ ID NO:31), and CDR3 (SEQ ID NO:33), wherein a T cell receptor (TCR) comprising said TCR alpha polypeptide and said TCR beta polypeptide is capable of binding to an antigenic peptide derived from melanoma-associated antigen B2 (MAGE-B2).

8. The polynucleotide of claim 7, wherein the polypeptide comprises a TCR alpha polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 19 and a TCR beta polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:

21.

9. The polynucleotide of claim 7, wherein the polypeptide comprises a TCR alpha polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO: 19 and a TCR beta polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:

21.

10. 8. The polynucleotide of claim 7, wherein the polypeptide comprises a TCR alpha polypeptide of SEQ ID NO: 19 and a TCR beta polypeptide of SEQ ID NO:

21.

11. The polynucleotide of claim 7, wherein the polypeptide comprises a TCR alpha polypeptide of SEQ ID NO:

19.

12. 8. The polynucleotide of claim 7, wherein the polypeptide comprises a TCR beta polypeptide of SEQ ID NO:

21.

13. 1. A host cell engineered to express an isolated T cell receptor (TCR) capable of binding to an antigenic peptide derived from melanoma-associated antigen B2 (MAGE-B2), wherein the TCR comprises a TCR alpha polypeptide of SEQ ID NO:3 and a TCR beta polypeptide of SEQ ID NO:5, or a TCR alpha polypeptide of SEQ ID NO:19 and a TCR beta polypeptide of SEQ ID NO:

21.

14. The host cell of claim 13, wherein the antigenic peptide is HLA-A2 restricted.

15. The host cell of claim 13, wherein the antigenic peptide is HLA-A*0201 restricted.

16. The host cell of claim 13, wherein the TCR is a soluble TCR lacking a transmembrane domain.

17. The host cell of claim 16 , wherein the TCR further comprises a detectable label.

18. 18. The host cell of claim 16 or claim 17, wherein the host cell comprises a TCR complex, the TCR complex comprising the TCR and a therapeutic agent.

19. The host cell of any one of claims 13 to 18, wherein the host cell is an immune cell.

20. The host cell according to any one of claims 13 to 18, wherein the host cell is a NK cell, an invariant NK cell, a NKT cell, a mesenchymal stem cell (MSC), or an induced pluripotent stem (iPS) cell.

21. 19. The host cell of any of claims 13 to 18, wherein the host cell is isolated from umbilical cord or blood.

22. 20. The host cell of claim 19, wherein the immune cell is a T cell or a peripheral blood lymphocyte.

23. 23. The host cell of claim 22, wherein the T cell is a CD8+ T cell, a CD4+ T cell, or a γδ T cell.

24. 23. The host cell of claim 22, wherein the host cell is allogeneic or autologous.

25. 25. A pharmaceutical composition comprising a population of host cells according to any one of claims 13 to 24.

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