Antibodies binding to hla-a02 / mage-a4 complex and uses thereof

Abstract

The present application provides bispecific antibodies comprising a first antigen binding fragment that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex and a second antigen binding fragment that binds to an activating T cell antigen, single-domain antibodies that bind to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex, pharmaceutical compositions comprising the bispecific antibodies or the single-domain antibodies, and uses thereof.

Description

Invention Field

[0001] This invention generally relates to the fields of genetic engineering and biomedicine; specifically, this application relates to bispecific antibodies that bind to the HLA-AO2 / MAGE-A4 complex and / or the HLA-AO2 / MAGE-A8 complex, single-domain antibodies that bind to the HLA-AO2 / MAGE-A4 complex and / or the HLA-AO2 / MAGE-A8 complex, and their uses. Background of the Invention

[0002] Melanoma-associated antigen A (MAGE-A) belongs to the type I MAGE family and is an important member of the cancer-testis-associated antigen family. [1] The MAGE-A gene family is located in the q28 region of the X chromosome and has 12 members (MAGE-A1 to MAGE-A12). [2] All MAGE-A members share the same MAGE homology domain (MHD), a highly conserved domain of approximately 170 amino acids that exhibits high sequence homology with each other. [3] .

[0003] Melanoma-associated antigen 4 (MAGE-A4) is a member of the MAGE-A gene family. MAGE-A4 is located in the cytoplasm; some studies have also found MAGE-A4 in the cell nucleus.

[0004] The function of MAGE-A4 is not fully understood, but it may be involved in cell cycle progression, transcriptional control, cell survival, or apoptosis. In malignant tumors, overexpression of MAGE-A4 may be associated with tumor growth and metastasis, as well as poor patient prognosis.

[0005] MAGE-A4 protein exhibits the same expression pattern as MAGE-A family proteins, with restricted expression in localized testicular tissue, partial expression in ovarian and placental tissues, and abnormally high expression in cancer. [4,5] Clinical histological examination has shown that MAGE-A4 is positive in a wide range of cancers, including urothelial carcinoma, bladder cancer, lung cancer, ovarian cancer, esophageal squamous cell carcinoma, and oral squamous cell carcinoma. [6-11] .

[0006] As an intracellular protein, MAGE-A4 can be degraded in the proteasome and, after processing, presented on the cell surface via major histocompatibility complex I (MHC I). It then acts as a T-cell antigen, recognized by the T-cell receptor (TCR), and induces an immune response, such as the peptide MAGE-A4 derived from MAGE-A4. 230-239(GVYDGREHTV) in complex with HLA-A02 is presented on the cell surface, triggering T cell recognition and specific killing of tumor cells in a major histocompatibility complex (MHC)-dependent manner

[12] . Given the limited expression of MAGE-A4, it can be a potential molecular target for tumor diagnosis and immunotherapy [13,14] .

[0007] Bispecific antibodies (BsAb) that simultaneously bind to T cell surface CD3 and target cell (e.g., tumor cell) surface antigen establish an interaction between the target cell and the T cell, leading to activation of cytotoxic T cells, which lyse the target cell in a major histocompatibility complex (MHC)-independent manner. Such bispecific antibodies show promising prospects in the treatment of various tumors. T cell receptor-like antibodies (TCR-like antibodies), also known as TCR mimicking antibodies (TCRm), can effectively mimic TCRs to recognize MHC complexes of cell surface tumor-specific antigen peptides. TCRm that bind to intracellular proteins of the MAGE-A4 class can serve as the portion that binds to the target antigen in CD3 bispecific antibodies. Such bispecific antibodies can be more attractive due to the limitation of target antigen expression.

[0008] In 2020, there were 19.29 million new cancer cases worldwide, of which 4.57 million new cancer cases occurred in China, accounting for 23.7% of the world. In 2020, there were 9.96 million cancer deaths worldwide

[15] , 1.8 million lung cancer (18%), 920,000 colorectal cancer (9.2%), 830,000 liver cancer (8.3%), 770,000 gastric cancer (7.7%), 680,000 breast cancer (6.8%), 540,000 esophageal cancer (5.4%), 470,000 pancreatic cancer (4.7%), 380,000 prostate cancer (3.8%), 340,000 cervical cancer (3.4%), and 310,000 leukemia (3.1%). These ten cancers accounted for 70.4% of the total number of cancer deaths

[15] . Most of these cancers have no good treatment, so there is an urgent need for new treatment strategies and drugs. MAGE-A4, as an intracellular antigen, is expressed in various solid tumors and can be a new target for the treatment of solid tumors.

[0009] Based on the clinical needs, it is of great clinical significance to explore and develop bispecific antibodies targeting MAGE-A4-MHC. SUMMARY

[0010] In a first aspect, the present application provides a bispecific antibody comprising a first antigen binding fragment that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second antigen binding fragment that binds to an activating T cell antigen.

[0011] In some embodiments of the first aspect, the bispecific antibody is capable of mediating killing of HLA-A02 + / MAGE-A4 + tumor cells, and / or the bispecific antibody is capable of mediating killing of HLA-A02 + / MAGE-A4 + tumor cells.

[0012] In some embodiments of the first aspect, the first antigen binding fragment comprises a HCDR1 as set forth in SEQ ID NO: 32, a HCDR2 as set forth in SEQ ID NO: 33, and a HCDR3 as set forth in SEQ ID NO: 34; wherein the amino acid sequences of the HCDRs are according to the definition of Kabat.

[0013] In some embodiments of the first aspect, the first antigen binding fragment is in the form of a single domain antibody.

[0014] In some embodiments of the first aspect, the first antigen binding fragment comprises a monovalent or multivalent single domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex.

[0015] In some embodiments of the first aspect, the second antigen binding fragment comprises a HCDR1 as set forth in SEQ ID NO: 37, a HCDR2 as set forth in SEQ ID NO: 38, a HCDR3 as set forth in SEQ ID NO: 39, a LCDR1 as set forth in SEQ ID NO: 40, a LCDR2 as set forth in SEQ ID NO: 41, and a LCDR3 as set forth in SEQ ID NO: 42; wherein the amino acid sequences of the HCDRs are according to the definition of Kabat.

[0016] In some embodiments of the first aspect, the second antigen binding fragment is a single chain antibody (scFv) or a Fab fragment.

[0017] In some embodiments of the first aspect, the first antigen binding fragment comprises a bivalent single domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex; optionally, the first antigen binding fragment comprises two heavy chain variable regions of a monovalent single domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex.

[0018] In some embodiments of the first aspect, the first antigen binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 35, 36, 71 or 72; and / or

[0019] the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 44.

[0020] In some embodiments of the first aspect, the first antigen binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 35; and the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 44.

[0021] the first antigen binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 36; and the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 44.

[0022] the first antigen binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 71; and the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 44; or

[0023] the first antigen binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 72; and the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 44.

[0024] In some embodiments of the first aspect, the first antigen binding fragment and the second antigen binding fragment are connected by an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprises a first Fc fragment and a second Fc fragment, wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the first Fc fragment are C, S, A and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the second Fc fragment are C, S, A and V, respectively; wherein the antibody constant region amino acid positions are determined according to the EU numbering.

[0025] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are linked by an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment, wherein the amino acids at positions 234, 235 and 331 of the first Fc fragment and the second Fc fragment are F, E and S, respectively; wherein the amino acid positions of the antibody constant region are determined according to EU numbering.

[0026] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are linked by an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment including a first Fc fragment and a second Fc fragment, wherein one of the first Fc fragment and the second Fc fragment is linked to the first antigen-binding fragment, and the other of the first Fc fragment and the second Fc fragment is linked to the second antigen-binding fragment.

[0027] In some embodiments of the first aspect, the bispecific antibody comprises a first arm that binds to the HLA-AO2 / MAGE-A4 complex and / or the HLA-AO2 / MAGE-A8 complex and a second arm that binds to an activated T-cell antigen, wherein

[0028] The first arm comprises an amino acid sequence as shown in SEQ ID NO:45 or 74; and

[0029] The second arm contains an amino acid sequence as shown in SEQ ID NO:46.

[0030] Secondly, this application provides a single-domain antibody that binds the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex, comprising...

[0031] HCDR1, as shown in SEQ ID NO:32,

[0032] HCDR2, as shown in SEQ ID NO:33, and

[0033] HCDR3, as shown in SEQ ID NO:34;

[0034] The amino acid sequence of HCDR is defined according to Kabat.

[0035] In some embodiments of the second aspect, the single-domain antibody comprises an amino acid sequence as shown in SEQ ID NO:35, 36, 71 or 72.

[0036] Thirdly, this application provides a nucleic acid molecule that encodes the bispecific antibody described in the first aspect or the single-domain antibody described in the second aspect.

[0037] Fourthly, this application provides a pharmaceutical composition comprising the bispecific antibody described in the first aspect or the single-domain antibody described in the second aspect, as well as a pharmaceutically acceptable excipient, diluent, or carrier.

[0038] Fifthly, this application provides the use of the bispecific antibody described in the first aspect, the single-domain antibody described in the second aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a medicament for the prevention or treatment of HLA-AO2 / MAGE-A4 positive tumors and / or HLA-AO2 / MAGE-A8 positive tumors. Brief description of the attached diagram

[0039] Figure 1 The results of ELISA detection of the binding of the TCRm antibody N18D10-Fc against the HLA-A02 / MAGE-A4 complex to the recombinant protein MHC-P1 are shown.

[0040] Figure 2 The TCRm antibody N18D10-Fc against the HLA-A02 / MAGE-A4 complex and the antigenic peptide MAGE-A4 were displayed. 230-239 The result of pulsed T2 cell binding.

[0041] Figure 3 The TCRm antibody N18D10 against the HLA-A02 / MAGE-A4 complex was shown to be effective against MAGE-A4. 230-239 Results of T2 cell binding of (GVYDGREHTV) alanine scanning mutant peptide pulse.

[0042] Figure 4 The results of ELISA detection of the binding of TCRm antibody N18D10-h16-Fc to recombinant protein MHC-P1 are shown.

[0043] Figure 5 The results of ELISA detection of TCRm×CD3 bispecific antibody binding to both MHC-P1 and CD3 are shown.

[0044] Figure 6 The results show the activation of Jurkat-Dual cells by T2 cells mediated by TCRm×CD3 bispecific antibody-mediated antigen peptide pulses.

[0045] Figure 7 This demonstrates that the TCRm×CD3 bispecific antibody mediates HLA-A02. + / MAGE-A4 + The result of tumor cells activating Jurkat-Dual cells.

[0046] Figure 8Results showing TCRm x CD3 bispecific antibody mediated killing of antigen peptide pulsed T2 cells by PBMC cells.

[0047] Figure 9 Results showing TCRm x CD3 bispecific antibody mediated killing of A375 cells by PBMC cells.

[0048] Figure 10 Results showing TCRm x CD3 bispecific antibody mediated killing of multiple antigen positive tumor cells by PBMC cells.

[0049] Figure 11 Results showing TCRm x CD3 bispecific antibody mediated activation of Jurkat-Dual cells by MAGE-A4 230-239 (GVYDGREHTV) alanine scan peptide pulsed T2 cells.

[0050] Figure 12 Results showing TCRm x CD3 bispecific antibody mediated activation of Jurkat-Dual cells by similar peptide segment pulsed T2 cells.

[0051] Figure 13 Results showing TCRm x CD3 bispecific antibody does not mediate killing of antigen negative tumor cells by PBMC cells.

[0052] Figure 14 Results showing TCRm x CD3 bispecific antibody inhibits A375 tumor cell growth in hPBMC immune reconstituted mice.

[0053] SEQUENCE DESCRIPTION

[0054] SEQ ID NO: 1 shows the amino acid sequence of the antigen peptide MAGE-A4 230-239 from homo sapiens.

[0055] SEQ ID NO: 2 shows the amino acid sequence of MAGE-A4-S1 (MAGE-A4 230-239 similar peptide segment 1) from homo sapiens.

[0056] SEQ ID NO: 3 shows the amino acid sequence of MAGE-A4-S2 from homo sapiens.

[0057] SEQ ID NO: 4 shows the amino acid sequence of MAGE-A4-S3 from homo sapiens.

[0058] SEQ ID NO: 5 shows the amino acid sequence of MAGE-A4-S4 derived from homo sapiens.

[0059] SEQ ID NO: 6 shows the amino acid sequence of MAGE-A4-S5 derived from homo sapiens.

[0060] SEQ ID NO: 7 shows the amino acid sequence of MAGE-A4-S6 derived from homo sapiens.

[0061] SEQ ID NO: 8 shows the amino acid sequence of MAGE-A4-S7 derived from homo sapiens.

[0062] SEQ ID NO: 9 shows the amino acid sequence of MAGE-A4-S8 derived from homo sapiens.

[0063] SEQ ID NO: 10 shows the amino acid sequence of MAGE-A4-S9 derived from homo sapiens.

[0064] SEQ ID NO: 11 shows the amino acid sequence of MAGE-A4-S10 derived from homo sapiens.

[0065] SEQ ID NO: 12 shows the amino acid sequence of MAGE-A4-S11 derived from homo sapiens.

[0066] SEQ ID NO: 13 shows the amino acid sequence of MAGE-A4-S12 derived from homo sapiens.

[0067] SEQ ID NO: 14 shows the amino acid sequence of MAGE-A4-S13 derived from homo sapiens.

[0068] SEQ ID NO: 15 shows the amino acid sequence of MAGE-A4-S14 derived from homo sapiens.

[0069] SEQ ID NO: 16 shows the amino acid sequence of MAGE-A4-S15 derived from homo sapiens.

[0070] SEQ ID NO: 17 shows the amino acid sequence of MAGE-A4-S16 derived from homo sapiens.

[0071] SEQ ID NO: 18 shows the amino acid sequence of MAGE-A4-S17 derived from homo sapiens.

[0072] SEQ ID NO: 19 shows the amino acid sequence of MAGE-A4-S18 derived from homo sapiens.

[0073] SEQ ID NO: 20 shows the amino acid sequence of MAGE-A4-S19 derived from homo sapiens.

[0074] SEQ ID NO: 21 shows the amino acid sequence of MAGE-A4-S20 derived from homo sapiens.

[0075] SEQ ID NO: 22 shows the amino acid sequence of MAGE-A4-S21 derived from homo sapiens.

[0076] SEQ ID NO: 23 shows the amino acid sequence of MAGE-A4-S22 derived from homo sapiens.

[0077] SEQ ID NO: 24 shows the amino acid sequence of MAGE-A4-S23 derived from homo sapiens.

[0078] SEQ ID NO: 25 shows the amino acid sequence of MAGE-A4-S24 derived from homo sapiens.

[0079] SEQ ID NO: 26 shows the amino acid sequence of MAGE-A4-S25 derived from homo sapiens.

[0080] SEQ ID NO: 27 shows the amino acid sequence of MAGE-A4-S26 derived from homo sapiens.

[0081] SEQ ID NO: 28 shows the amino acid sequence of MAGE-A4-S27 derived from homo sapiens.

[0082] SEQ ID NO: 29 shows the amino acid sequence of MAGE-A4-S28 derived from homo sapiens.

[0083] SEQ ID NO: 30 shows the amino acid sequence of MAGE-A4-S29 derived from homo sapiens.

[0084] SEQ ID NO: 31 shows the amino acid sequence of MAGE-A4-S30 derived from homo sapiens.

[0085] SEQ ID NOs: 32-34 show the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the Camelus single domain antibody N18D10 and N18D10-h16, respectively.

[0086] SEQ ID NO: 35 shows the amino acid sequence of the heavy chain variable region of the Camelus single domain antibody N18D10.

[0087] SEQ ID NO: 36 shows the amino acid sequence of the heavy chain variable region of the humanized single domain antibody N18D10-h16.

[0088] SEQ ID NOs: 37-39 show the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the anti-human CD3 antibody IMCR, respectively.

[0089] SEQ ID NOs: 40-42 show the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region of the anti-human CD3 antibody IMCR, respectively.

[0090] SEQ ID NO: 43 shows the amino acid sequence of the heavy chain variable region of the anti-human CD3 antibody IMCR.

[0091] SEQ ID NO: 44 shows the amino acid sequence of the light chain variable region of the anti-human CD3 antibody IMCR.

[0092] SEQ ID NO: 45 shows the amino acid sequence of arm 2N18D10-h16-G1m3-FcHIn1 in the bispecific antibody 2N18D10-h16+IMCR.

[0093] SEQ ID NO: 46 shows the amino acid sequence of arm IMCR-anti-CD3-scFv-G1m3-FcKn1 in the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR.

[0094] SEQ ID NO: 47 shows the amino acid sequence of the HLA-A02 complex (MHC-P1) of GVYDGREHTV.

[0095] SEQ ID NO: 48 shows the amino acid sequence of MAGE-A4 of homo sapiens.

[0096] SEQ ID NO: 49 shows the amino acid sequence of a linker.

[0097] SEQ ID NO: 50 shows the nucleotide sequence of primer PCal-CH2R.

[0098] SEQ ID NO: 51 shows the amino acid sequence of the human (homo sapiens) CD3E extracellular region (hCD3E).

[0099] SEQ ID NO: 52 shows the amino acid sequence of the human (homo sapiens) CD3D extracellular region (hCD3D).

[0100] SEQ ID NO: 53 shows the amino acid sequence of a His tag.

[0101] SEQ ID NO: 54 shows the amino acid sequence of the Fc segment of a human (homo sapiens) IgGl antibody (hFc).

[0102] SEQ ID NO: 55 shows the amino acid sequence of the Fc segment of a mouse (mus musculus) IgG2a antibody (mFc).

[0103] SEQ ID NO: 56 shows the amino acid sequence of the human (homo sapiens) IgGl subtype heavy chain constant region.

[0104] SEQ ID NO: 57 shows the amino acid sequence of human IgGl subtype antibody heavy chain constant region mutant IgGl H.

[0105] SEQ ID NO: 58 shows the amino acid sequence of human IgGl subtype antibody heavy chain constant region mutant IgGl K.

[0106] SEQ ID NO: 59 shows the amino acid sequence of human IgGl subtype antibody heavy chain constant region mutant IgGl m3-H.

[0107] SEQ ID NO: 60 shows the amino acid sequence of human IgGl subtype antibody heavy chain constant region mutant IgGl m3-K.

[0108] SEQ ID NO: 61 shows the amino acid sequence of human IgGl subtype antibody heavy chain constant region mutant IgGl m3-H1n1.

[0109] SEQ ID NO: 62 shows the amino acid sequence of human IgGl subtype antibody heavy chain constant region mutant IgGl m3-Kn1.

[0110] SEQ ID NO:63 shows the amino acid sequence of the lambda subtype light chain constant region of homo sapiens.

[0111] SEQ ID NO:64 shows the amino acid sequence of the kappa subtype light chain constant region of homo sapiens

[0112] SEQ ID NO:65 shows the amino acid sequence of germline antibody DP47 heavy chain variable region (VH).

[0113] SEQ ID NO:66 shows the amino acid sequence of germline antibody DP47 light chain variable region (VK).

[0114] SEQ ID NO:67 shows the amino acid sequence of the alpha chain variable region (Va) of a control TCR that binds HLA-A02 / MAGE-4.

[0115] SEQ ID NO:68 shows the amino acid sequence of the beta chain variable region (Vp) of a control TCR that binds HLA-A02 / MAGE-4.

[0116] SEQ ID NO:69 shows the amino acid sequence of the first binding arm of the control IMC-C103C bifunctional molecule, TCRA-Des-G1m3-FcKn1.

[0117] SEQ ID NO:70 shows the amino acid sequence of TCRB-Des-G1m3-FcH1n1 in the control TCR.

[0118] SEQ ID NO:71 shows the amino acid sequence of the tandem heavy chain variable region of two camelid single domain antibodies, N18D10.

[0119] SEQ ID NO:72 shows the amino acid sequence of the tandem heavy chain variable region of two camelid single domain antibodies, N18D10-h16.

[0120] SEQ ID NO:73 shows the amino acid sequence of a single chain antibody of the anti-human (homo sapiens) CD3 antibody, IMCR.

[0121] SEQ ID NO:74 shows the amino acid sequence of 2N18D10-G1m3-FcHIn1 in the bispecific antibody 2N18D10+IMCR.

[0122] SEQ ID NO:75 shows the amino acid sequence of the second binding arm of the control IMC-C103C bifunctional molecule, IMCR-anti-CD3-scFv-TCRB-Des-G1m3-FcH1n1.

[0123] SEQ ID NO: 76 shows the heavy chain variable region (057G03VH) amino acid sequence of control antibody 057D03 that binds to the HLA-A02 / MAGE-A4 complex.

[0124] SEQ ID NO: 77 shows the light chain variable region (057G03VK) amino acid sequence of control antibody 057D03 that binds to the HLA-A02 / MAGE-A4 complex.

[0125] SEQ ID NO: 78 shows the amino acid sequence of the first component of the control anti-HLA-A02 / MAGE-A4 complex and CD3 bispecific antibody 057G03, 057G03-Fd-EE-IMCR-anti-CD3VK-G1m3-Kn1.

[0126] SEQ ID NO: 79 shows the amino acid sequence of the second component of the control anti-HLA-A02 / MAGE-A4 complex and CD3 bispecific antibody 057G03, 057G03-Fd-EE-G1m3-FcH1n1.

[0127] SEQ ID NO: 80 shows the amino acid sequence of the third component of the control anti-HLA-A02 / MAGE-A4 complex and CD3 bispecific antibody 057G03, 057G03VK+CK-RK.

[0128] SEQ ID NO: 81 shows the amino acid sequence of the fourth component of the control anti-HLA-A02 / MAGE-A4 complex and CD3 bispecific antibody 057G03, IMCR-anti-CD3VH-CK.

[0129] SEQ ID NO: 82 shows the amino acid sequence of the MAGE-A4 230-239 G1 position alanine scanning mutant peptide.

[0130] SEQ ID NO: 83 shows the amino acid sequence of the MAGE-A4 230-239 V2 position alanine scanning mutant peptide.

[0131] SEQ ID NO: 84 shows the amino acid sequence of the MAGE-A4 230-239 Y3 position alanine scanning mutant peptide.

[0132] SEQ ID NO: 85 shows the amino acid sequence of the MAGE-A4 230-239 D4 position alanine scanning mutant peptide.

[0133] SEQ ID NO: 86 shows the amino acid sequence of a MAGE-A4 230-239 The amino acid sequence of the alanine scanning mutant peptide segment at position G5.

[0134] SEQ ID NO: 87 shows the amino acid sequence of a MAGE-A4 230-239 The amino acid sequence of the alanine scanning mutant peptide segment at position R6.

[0135] SEQ ID NO: 88 shows the amino acid sequence of a MAGE-A4 230-239 The amino acid sequence of the alanine scanning mutant peptide segment at position E7.

[0136] SEQ ID NO: 89 shows the amino acid sequence of a MAGE-A4 230-239 The amino acid sequence of the alanine scanning mutant peptide segment at position H8.

[0137] SEQ ID NO: 90 shows the amino acid sequence of a MAGE-A4 230-239 The amino acid sequence of the alanine scanning mutant peptide segment at position T9.

[0138] SEQ ID NO: 91 shows the amino acid sequence of a MAGE-A4 230-239 The amino acid sequence of the alanine scanning mutant peptide segment at position V10.

[0139] SEQ ID NO: 92 shows the amino acid sequence of the human IgGl subtype antibody Fc segment mutant IgGl m3-FcHl n l.

[0140] SEQ ID NO: 93 shows the amino acid sequence of the human IgGl subtype antibody Fc segment mutant IgGl m3-FcKn l.

[0141] SEQ ID NO: 94 shows the amino acid sequence of human (homo sapiens) MAGE-A8. DETAILED DESCRIPTION

[0142] The inventors of the present application prepared a bispecific antibody (e.g., a TCRm bispecific antibody) by genetically engineering a first antigen binding fragment that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second antigen binding fragment that binds to an activating T cell antigen (e.g., a CD3 molecule) into a bispecific antibody (e.g., a TCRm bispecific antibody), and the first antigen binding fragment that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex binds to a complex of MAGE-A4 230-239 (GVYDGREHTV) and HLA-A02 and / or MAGE-A8 232-241The first antigen binding fragment binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex, and the second antigen binding fragment binds to an activating T cell antigen (e.g., CD3 molecule), thereby establishing an interaction between the target cell and the T cell, resulting in activation of the cytotoxic T cell to lyse the target cell (e.g., tumor cell) in a non-major histocompatibility complex (MHC)-dependent manner, to achieve the purpose of treating diseases (e.g., tumors). In various aspects of the present application, new bispecific antibodies comprising a first antigen binding fragment binding to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex and a second antigen binding fragment binding to an activating T cell antigen (e.g., CD3 molecule), single domain antibodies binding to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex, nucleic acid molecules encoding the bispecific antibodies or the single domain antibodies, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, methods for preparing and purifying the bispecific antibodies or the single domain antibodies, and medical and biological applications of the bispecific antibodies or the single domain antibodies are provided. According to the sequences of the bispecific antibodies or the single domain antibodies provided in the present application, the bispecific antibodies or the single domain antibodies binding to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex can be constructed as drugs for preventing or treating tumors in the clinic.

[0143] Unless otherwise indicated, the practice of the present application employs conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology.

[0144] Unless otherwise indicated, the terms used in the present application have the meanings commonly understood by those skilled in the art.

[0145] Definitions

[0146] The term "HLA-A02 / MAGE-A4 complex" as used herein refers to a complex of HLA-A02 molecule and MAGE-A4. In specific embodiments of the present application, "HLA-A02 / MAGE-A4 complex" refers to a complex of HLA-A02 molecule and MAGE-A4 of SEQ ID NO: 48 at positions 230-239 (MAGE-A4 230-239 ) of MAGE-A4. 230-239 complex.

[0147] The term "HLA-A02 / MAGE-A8 complex" as used herein refers to a complex of an HLA-A02 molecule with MAGE-A8. In particular embodiments of the application, "HLA-A02 / MAGE-A8 complex" refers to a complex of an HLA-A02 molecule with MAGE-A8 232-241 ) as shown in SEQ ID NO: 94. In particular embodiments of the application, "HLA-A02 / MAGE-A8 complex" refers to a complex of an HLA-A02 molecule with MAGE-A8 232-241 ) as shown in SEQ ID NO: 94.

[0148] The term "activating T cell antigen" as used herein refers to an antigenic determinant expressed on the surface of a T lymphocyte, in particular a cytotoxic T lymphocyte, which is capable of inducing T cell activation upon interaction with an antigen binding molecule. In particular, the interaction of an antigen binding molecule with an activating T cell antigen can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. In particular aspects, the activating T cell antigen is CD3.

[0149] The term "antibody" as used herein refers to an immunoglobulin molecule capable of specific binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like. "Antibody", as used herein, not only includes intact (i.e., full-length) antibodies, but also binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), variants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, bi-specific antibodies, linear antibodies, single-chain antibodies, single-domain antibodies, multi-specific antibodies (e.g., bi-specific antibodies), and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0150] Generally, intact or full-length antibodies comprise two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and a first, second, and third constant region (CHI, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). Full-length antibodies can be of any class, e.g., IgD, IgE, IgG, IgA, or IgM (or a subclass thereof), although the antibodies need not be of any particular class. An immunoglobulin can be assigned to a class based on the amino acid sequence of its constant regions. In general, immunoglobulins are of five major classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0151] The term "bispecific antibody" as used herein refers to an antibody that has the ability to bind to two antigen epitopes simultaneously. The two antigen epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have a variety of structural configurations. For example, a bispecific antibody can be composed of two Fc fragments and two binding moieties fused to each of them respectively (similar to a natural antibody, except that the two arms bind to different antigen targets or epitopes), the antigen binding moieties can be single domain antibodies, single chain antibodies (scFv) or Fab fragments. When directed against the epitopes of two given antigens, the two different binding moieties of a bispecific antibody are each bound to the N-terminus of one Fc fragment, the antigen binding moieties of the two arms can have four combinations: single domain antibody + Fab fragment, single domain antibody + scFv, scFv + single domain antibody and Fab fragment + single domain antibody. The Fc fragment can contain mutations that can ensure heterodimerization of the heavy chains, the KIH technology (knob-in-hole, KIH) is a strategy to solve the heterodimerization of heavy chains. Generally, the KIH technology refers to the formation of a structure that is conducive to the pairing of hetero-hybrid antibodies by modifying the amino acid sequence of the CH3 region, which can form a bispecific antibody while maintaining the structure of a normal antibody as much as possible. For guidance on KIH technology, see, for example, "An efficient route to human bispecific IgG", A. Margaret Merchant et al., Nature Biotechnology, Volume 16, 1998, which is incorporated herein by reference in its entirety.

[0152] The terms "binding moiety" or "binding fragment" as used herein are used interchangeably to refer to a portion or region of an intact antibody molecule that is responsible for binding to an antigen. An antigen binding domain can comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementarity determining regions, CDR1, CDR2, and CDR3.

[0153] It is well known to those skilled in the art that the complementarity determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that contribute most to the affinity and specificity of an antibody. There are two common ways of defining the CDR sequences of a VH or VL, the Kabat definition and the Chothia definition. (See, e.g., Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989)). For a given variable region sequence of an antibody, the CDR sequences can be determined according to either the Kabat definition or the Chothia definition. In embodiments of the present application, the CDR sequences are defined using the Kabat definition.

[0154] For a given variable region sequence of an antibody, the CDR sequences in the variable region sequence can be analyzed in a number of ways, for example, using the online software Abysis (http: / / www.abysis.org / ).

[0155] For a general antibody, examples of antigen binding fragments include, but are not limited to: (1) a Fab fragment, which can be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab')2 fragment, which can be a bivalent fragment having two Fab' fragments that are connected by a disulfide bridge at the hinge region (i.e., a dimer of Fab' fragments); (3) a Fv fragment having a single arm of a VL and VH domain of an antibody; (4) a single chain Fv (scFv), which can be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; (5) a (scFv)2, which can comprise two VH domains and two VL domains connected by a peptide linker, the two VL domains being combined with the two VH domains via disulfide bridges; and (6) a single domain antibody format.

[0156] In bispecific antibody construction, "binding moieties" include, but are not limited to, single domain antibody formats, Fab fragment formats, and / or single chain antibody (scFv) formats.

[0157] The term "single chain antibody (scFv)" as used herein refers to an antibody of a single chain structure, generally constructed using genetic engineering techniques, comprising one polypeptide chain of a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker is usually designed between the heavy chain variable region and the light chain variable region so that the heavy chain variable region and the light chain variable region can fold into a correct conformation capable of binding to an antigen.

[0158] The term "Fab (fragment antigen binding) fragment", "Fab portion" or similar terms as used herein refer to an antibody fragment capable of binding to an antigen produced after treating an intact antibody with papain, including an intact light chain (VL-CL), a heavy chain variable region and a CH1 fragment (VH-CH1).

[0159] The term "single domain antibody" as used herein refers to a heavy chain single variable domain antibody naturally lacking a light chain, such an antibody comprising a heavy chain variable region (VHH) and a conventional CH2 and CH3 region (e.g., one set or two sets (heavy chain variable region (VHH) and conventional CH2 and CH3 region)). A VHH structure cloned and expressed alone has a structural stability comparable to that of a conventional heavy chain antibody and a binding activity to an antigen, and is known as the smallest unit capable of binding to a target antigen. The single domain antibody is also referred to as a Nanobody (Nb).

[0160] The terms "Fc fragment", "Fc domain" and "Fc portion" as used herein are used interchangeably and refer to a portion of an antibody heavy chain constant region, including a hinge, a CH2 fragment and a CH3 fragment of the heavy chain constant region, and are determined with reference to the EU numbering of a human IgGl antibody.

[0161] The term "specifically binds" as used herein refers to a non-random binding reaction between two molecules, for example, an antibody to an epitope of an antigen.

[0162] The term "tumor" as used herein refers to a neoplasm or solid lesion formed by the growth of abnormal cells. A tumor can be benign, pre-malignant, or malignant.

[0163] The term "malignant tumor" as used herein refers to or describes a physiological condition of a mammal that is typically characterized by unregulated cell growth. Exemplary malignant tumors include carcinomas, solid tumors, melanomas, sarcomas, hematological tumors, germ cell tumors, and blast cell tumors. More specific examples of malignant tumors include esophageal carcinoma, urothelial carcinoma, oral cancer, sarcoma, head and neck cancer, bladder cancer, melanoma, ovarian cancer, colorectal cancer, breast cancer, renal cell carcinoma, lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma, cervical cancer, hepatic carcinoma, gastric cancer including gastrointestinal cancer, prostate cancer, pancreatic cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, liver cancer, urological cancer, hepatoma, endometrial or uterine cancer, salivary gland carcinoma, squamous cell carcinoma (e.g., squamous cell carcinoma of the head and neck), vulvar cancer, thyroid cancer, anal cancer, penile cancer, multiple myeloma and B-cell lymphoma, brain cancer, and associated metastases.

[0164] The term "hematological tumor" as used herein refers to a tumor that results from the uncontrolled growth proliferation of abnormal cells, which in most cases originate in the bone marrow, which is also where blood cells are produced. Exemplary hematological tumors include various types of leukemia, multiple myeloma, and malignant lymphoma. More specific examples of hematological tumors include acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia, large granular lymphocyte leukemia, juvenile myelomonocytic leukemia, B-cell prolymphocytic leukemia, Burkitt's leukemia, and adult T-cell leukemia, non-Hodgkin's lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, primary macroglobulinemia (macroglobulinemia), splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, B-cell chronic lymphocytic lymphoma, classical Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, adult T-cell lymphoma, extranodal nasal-type NK / T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Sezary syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disorder, primary cutaneous anaplastic large-cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma-not otherwise specified, and anaplastic large-cell lymphoma.

[0165] As used herein, the term "solid tumor" refers to a tangible mass that can be palpated by clinical examination such as X-ray, CT scan, B-ultrasound or palpation. Clinically diagnosed solid tumors are divided into two types, malignant and benign. Malignant solid tumors include: esophageal cancer (e.g., esophageal squamous cell carcinoma), urothelial carcinoma, oral cancer (e.g., oral squamous cell carcinoma), sarcoma (e.g., synovial sarcoma), head and neck cancer, liver cancer, lung cancer, bladder cancer, melanoma (metastatic melanoma), ovarian cancer, colorectal cancer, breast cancer, childhood Hodgkin's lymphoma: lymphocyte predominant type, nodular sclerosis type, mixed cell type, lymphocyte depletion type; childhood non-Hodgkin's lymphoma: prolymphoblastic lymphoma, small non-cleaved cell lymphoma (Burkitt's / non-Burkitt's lymphoma), diffuse large B-cell lymphoma, anaplastic large cell lymphoma, etc.; childhood kidney tumor: nephroblastoma (Wilms' tumor), renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, primitive neuroectodermal tumor of the kidney, etc.; childhood neuroblastoma: neuroblastoma, nodular neuroblastoma, nodular neuroblastoma; childhood extracranial germ cell tumor: mature teratoma, immature teratoma, endodermal sinus tumor (yolk sac tumor), seminoma, dysgerminoma, choriocarcinoma, embryonal carcinoma, etc.; osteosarcoma and chondrosarcoma; childhood rhabdomyosarcoma: embryonal type, alveolar type, pleomorphic type, etc.; soft tissue sarcoma: fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant schwannoma, alveolar soft part sarcoma, epithelioid sarcoma, clear cell sarcoma, malignant melanoma, synovial sarcoma, desmoplastic small round cell tumor, etc.; Ewing's family sarcoma: Ewing's sarcoma, primitive neuroectodermal tumor; childhood liver tumor: hepatoblastoma (embryonal type, fetal type, undifferentiated type), hepatocellular carcinoma; retinoblastoma; other tumors: posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, thymoma, pulmonary blastoma, pancreatic blastoma, islet cell tumor, ileocecal class carcinoma, mesothelioma, etc. Benign solid tumors include: lymphangioma, hemangioma, thyroglossal duct cyst, etc.

[0166] In a first aspect, the present application provides a bispecific antibody comprising a first antigen binding fragment that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second antigen binding fragment that binds to an activating T cell antigen.

[0167] In some embodiments of the first aspect, the bispecific antibody is capable of mediating killing of HLA-A02 + / MAGE-A4 + tumor cells by T cells, and / or the bispecific antibody is capable of mediating killing of HLA-A02 + / MAGE-A4 + tumor cells by PBMCs.

[0168] In some embodiments of the first aspect, the bispecific antibody is capable of mediating HLA-A02 + / MAGE-A4 + Tumor cells activated Jurkat-Dual cells.

[0169] In some embodiments of the first aspect, the binding epitope of the first antigen binding fragment to the HLA-A02 / MAGE-A4 complex comprises at least one of residues 232, 234, 235, 237, and 239 of MAGE-A4 set forth in SEQ ID NO: 48. In some particular embodiments, the binding epitope of the first antigen binding fragment to the HLA-A02 / MAGE-A4 complex comprises residues 232, 234, 235, 237, and 239 of MAGE-A4 set forth in SEQ ID NO: 48. In some embodiments, residues 232, 234, 235, 237, and 239 of MAGE-A4 set forth in SEQ ID NO: 48 correspond to amino acid residues 3, 5, 6, 8, and 10, respectively, of the MAGE-A4 (GVYDGREHTV) polypeptide set forth in SEQ ID NO: 1.

[0170] In some embodiments of the first aspect, the binding epitope of the first antigen binding fragment to the HLA-A02 / MAGE-A4 complex comprises at least one of residues 232, 234, 235, 237, and 239 of MAGE-A4 set forth in SEQ ID NO: 48. In some particular embodiments, the binding epitope of the first antigen binding fragment to the HLA-A02 / MAGE-A4 complex comprises residues 232, 234, 235, 237, and 239 of MAGE-A4 set forth in SEQ ID NO: 48. In some embodiments, residues 232, 234, 235, 237, and 239 of MAGE-A4 set forth in SEQ ID NO: 48 correspond to amino acid residues 3, 5, 6, 8, and 10, respectively, of the MAGE-A4 (GVYDGREHTV) polypeptide set forth in SEQ ID NO: 1. 230-239 In some embodiments of the first aspect, the binding epitope of the first antigen binding fragment to the HLA-A02 / MAGE-A4 complex comprises at least one of residues 232, 234, 235, 237, and 239 of MAGE-A4 set forth in SEQ ID NO: 48. In some particular embodiments, the binding epitope of the first antigen binding fragment to the HLA-A02 / MAGE-A4 complex comprises residues 232, 234, 235, 237, and 239 of MAGE-A4 set forth in SEQ ID NO: 48. In some embodiments, residues 232, 234, 235, 237, and 239 of MAGE-A4 set forth in SEQ ID NO: 48 correspond to amino acid residues 3, 5, 6, 8, and 10, respectively, of the MAGE-A4 (GVYDGREHTV) polypeptide set forth in SEQ ID NO: 1.

[0171] In some embodiments of the first aspect, the binding epitope of the first antigen binding fragment to the HLA-A02 / MAGE-A8 complex comprises positions 232-241 of MAGE-A8 set forth in SEQ ID NO: 94. In some embodiments, the amino acid sequence of positions 232-241 of MAGE-A8 set forth in SEQ ID NO: 94 is GLYDGREHSV (SEQ ID NO: 2).

[0172] In some embodiments of the first aspect, the activating T cell antigen is a CD3 molecule.

[0173] In some embodiments of the first aspect, the first antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:32, HCDR2 as shown in SEQ ID NO:33, and HCDR3 as shown in SEQ ID NO:34; wherein the amino acid sequence of HCDR is defined according to Kabat.

[0174] In some embodiments of the first aspect, the first antigen-binding fragment is in the form of a single-domain antibody.

[0175] In some embodiments of the first aspect, the first antigen-binding fragment comprises a monovalent or multivalent (e.g., 1, 2, 3, 4, 5, or 6-valent) single-domain antibody that binds to the HLA-A02 / MAGE-A4 complex.

[0176] In some specific embodiments of the first aspect, the first antigen-binding fragment comprises a bivalent single-domain antibody binding the HLA-AO2 / MAGE-A4 complex. In some embodiments, the first antigen-binding fragment comprises heavy chain variable regions of two monovalent single-domain antibodies binding the HLA-AO2 / MAGE-A4 complex. In some embodiments, the first antigen-binding fragment comprises heavy chain variable regions of two monovalent single-domain antibodies binding the HLA-AO2 / MAGE-A4 complex linked by a linker. In some embodiments, the linker is a linker comprising a GS flexible peptide, such as (GGGGS). n , where n is an integer ≥ 1. In some specific implementations, the connector is GGGGSGGGGSGGGGS (SEQ ID NO:49).

[0177] In some embodiments of the first aspect, the first antigen-binding fragment comprises a monovalent or multivalent (e.g., 1, 2, 3, 4, 5, or 6-valent) single-domain antibody that binds to the HLA-A02 / MAGE-A8 complex.

[0178] In some specific embodiments of the first aspect, the first antigen-binding fragment comprises a bivalent single-domain antibody binding the HLA-AO2 / MAGE-A8 complex. In some embodiments, the first antigen-binding fragment comprises heavy chain variable regions of two monovalent single-domain antibodies binding the HLA-AO2 / MAGE-A8 complex. In some embodiments, the first antigen-binding fragment comprises heavy chain variable regions of two monovalent single-domain antibodies binding the HLA-AO2 / MAGE-A8 complex linked by a linker. In some embodiments, the linker is a linker comprising a GS flexible peptide, such as (GGGGS). n , where n is an integer ≥ 1. In some specific implementations, the connector is GGGGSGGGGSGGGGS (SEQ ID NO:49).

[0179] In some embodiments of the first aspect, the first antigen binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 35.

[0180] In some embodiments of the first aspect, the first antigen binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 36.

[0181] In some embodiments of the first aspect, the first antigen binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 71.

[0182] In some embodiments of the first aspect, the first antigen binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 72.

[0183] In some embodiments of the first aspect, the amino acid sequence as set forth in SEQ ID NO: 71 comprises two amino acid sequences as set forth in SEQ ID NO: 35.

[0184] In some embodiments of the first aspect, the amino acid sequence as set forth in SEQ ID NO: 72 comprises two amino acid sequences as set forth in SEQ ID NO: 36.

[0185] In some embodiments of the first aspect, the second antigen binding fragment comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 43 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 44.

[0186] In some embodiments of the first aspect, the first antigen binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 35; and the second antigen binding fragment comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 43 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 44.

[0187] In some embodiments of the first aspect, the first antigen binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 36; and the second antigen binding fragment comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 43 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 44.

[0188] In some embodiments of the first aspect, the first antigen binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 71; and the second antigen binding fragment comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 43 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 44.

[0189] In some embodiments of the first aspect, the first antigen binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 72; and the second antigen binding fragment comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 43 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 44.

[0190] In some embodiments of the first aspect, the amino acid sequence of the first antigen binding fragment differs from the amino acid sequence as set forth in SEQ ID NO: 35, 36, 71, or 72 by a substitution, deletion, and / or addition of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0191] In some embodiments of the first aspect, the amino acid sequence of the first antigen binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence as set forth in SEQ ID NO: 35, 36, 71, or 72.

[0192] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence as set forth in SEQ ID NO: 35, 36, 71, or 72 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still retaining similar functionality of the first antigen binding fragment.

[0193] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added to the C-terminal or N-terminal region of the amino acid sequence as set forth in SEQ ID NO: 35, 36, 71, or 72, while the resulting amino acid sequence still retains similar functionality of the first antigen binding fragment.

[0194] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added or deleted from a region other than the C-terminal or N-terminal of the amino acid sequence as set forth in SEQ ID NO: 35, 36, 71, or 72, as long as the altered amino acid sequence substantially retains similar functionality of the first antigen binding fragment.

[0195] In some embodiments of the first aspect, wherein the amino acid sequence of the heavy chain variable region of the second antigen binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 43.

[0196] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen binding fragment differs from the amino acid sequence set forth in SEQ ID NO: 43 by a substitution, deletion, and / or addition of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0197] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 43.

[0198] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 43 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functionality of the heavy chain variable region of the second antigen binding fragment.

[0199] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 43, and the resulting amino acid sequence still retains similar functionality of the heavy chain variable region of the second antigen binding fragment.

[0200] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added or deleted from regions other than the C-terminal or N-terminal of the amino acid sequence set forth in SEQ ID NO: 43, as long as the altered amino acid sequence substantially retains similar functionality of the heavy chain variable region of the second antigen binding fragment.

[0201] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 44.

[0202] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen binding fragment differs from the amino acid sequence set forth in SEQ ID NO: 44 by a substitution, deletion, and / or addition of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0203] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 43.

[0204] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 43 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still retaining similar functionality of the heavy chain variable region of the second antigen binding fragment.

[0205] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 43, and the resulting amino acid sequence still retains similar functionality of the heavy chain variable region of the second antigen binding fragment.

[0206] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added or deleted from a region other than the C-terminal or N-terminal of the amino acid sequence set forth in SEQ ID NO: 43, as long as the altered amino acid sequence substantially retains similar functionality of the heavy chain variable region of the second antigen binding fragment.

[0207] In some embodiments of the first aspect, the first antigen binding fragment and the second antigen binding fragment are connected by an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprises a first Fc fragment and a second Fc fragment, wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the first Fc fragment are C, S, A and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the second Fc fragment are C, S, A and V, respectively; wherein the antibody constant region amino acid positions are determined according to EU numbering.

[0208] In some embodiments of the first aspect, the first antigen binding fragment and the second antigen binding fragment are connected via an antibody heavy chain constant region Fc fragment, which comprises a first Fc fragment and a second Fc fragment, wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, and the amino acids at positions 349, 366, 368 and 407 of the second Fc fragment are C, S, A and V, respectively; wherein the antibody constant region amino acid positions are determined according to EU numbering.

[0209] In some embodiments of the first aspect, the first antigen binding fragment and the second antigen binding fragment are connected via an antibody heavy chain constant region Fc fragment, which comprises a first Fc fragment and a second Fc fragment, wherein the amino acids at positions 234, 235 and 331 of the first Fc fragment and the second Fc fragment are F, E and S, respectively; wherein the antibody constant region amino acid positions are determined according to EU numbering.

[0210] In some embodiments of the first aspect, the first antigen binding fragment and the second antigen binding fragment are connected via an antibody heavy chain constant region Fc fragment, which comprises a first Fc fragment and a second Fc fragment, wherein one of the first Fc fragment and the second Fc fragment is connected to the first antigen binding fragment, and the other of the first Fc fragment and the second Fc fragment is connected to the second antigen binding fragment.

[0211] In some embodiments of the first aspect, the first antigen binding fragment is connected to the N-terminus of the first Fc fragment (e.g., an amino acid sequence such as SEQ ID NO: 92), e.g., at the C-terminus.

[0212] In some embodiments of the first aspect, the second antigen binding fragment is connected to the N-terminus of the second Fc fragment (e.g., an amino acid sequence such as SEQ ID NO: 93), e.g., at the C-terminus.

[0213] In some embodiments of the first aspect, the antibody heavy chain constant region Fc fragment is an Fc fragment of IgG1 subtype. In some embodiments, the first Fc fragment is an Fc fragment of IgG1 subtype; and / or the second Fc fragment is an Fc fragment of IgG1 subtype.

[0214] In some embodiments of the first aspect, the antibody heavy chain constant region Fc fragment is an Fc fragment of IgG1m3 subtype. In some embodiments, the first Fc fragment is an Fc fragment of IgG1m3 subtype; and / or the second Fc fragment is an Fc fragment of IgG1m3 subtype.

[0215] In some embodiments of the first aspect, the bispecific antibody comprises a first arm that binds to an HLA-A02 / MAGE-A4 complex and a second arm that binds to an activating T cell antigen, wherein

[0216] the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and

[0217] the second arm comprises an amino acid sequence as set forth in SEQ ID NO: 46.

[0218] In some embodiments of the first aspect, the bispecific antibody comprises a first arm that binds to an HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein

[0219] the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and

[0220] the second arm comprises an amino acid sequence as set forth in SEQ ID NO: 46.

[0221] In some embodiments of the first aspect, wherein the amino acid sequence of the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74.

[0222] In some embodiments of the first aspect, the amino acid sequence of the first arm differs from an amino acid sequence as set forth in SEQ ID NO: 45 or 74 by a substitution, deletion, and / or addition of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0223] In some embodiments of the first aspect, the amino acid sequence of the first arm has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to an amino acid sequence as set forth in SEQ ID NO: 45 or 74.

[0224] In some embodiments of the first aspect, a C-terminal or N-terminal region of the amino acid sequence as set forth in SEQ ID NO: 45 or 74 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functionality of the first arm.

[0225] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 45 or 74, and the resulting amino acid sequence still retains a similar function of the first arm.

[0226] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids can be added to or deleted from a region other than the C-terminal or N-terminal of the amino acid sequence set forth in SEQ ID NO: 45 or 74, as long as the altered amino acid sequence substantially retains a similar function of the first arm.

[0227] In some embodiments of the first aspect, the amino acid sequence of the second arm comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0228] In some embodiments of the first aspect, the amino acid sequence of the second arm differs from the amino acid sequence set forth in SEQ ID NO: 46 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, deletions, and / or additions of amino acids.

[0229] In some embodiments of the first aspect, the amino acid sequence of the second arm has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology to the amino acid sequence set forth in SEQ ID NO: 46.

[0230] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 46 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids, and the resulting amino acid sequence still retains a similar function of the second arm.

[0231] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 46, and the resulting amino acid sequence still retains a similar function of the second arm.

[0232] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids can be added or deleted outside the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 46, as long as the changed amino acid sequence substantially retains similar function of the second arm.

[0233] In a second aspect, the present application provides a single-domain antibody binding to HLA-A02 / MAGE-A4 complex and / or HLA-A02 / MAGE-A8 complex, comprising

[0234] HCDR1 as shown in SEQ ID NO: 32,

[0235] HCDR2 as shown in SEQ ID NO: 33, and

[0236] HCDR3 as shown in SEQ ID NO: 34.

[0237] wherein the amino acid sequences of the HCDRs are according to the definition of Kabat.

[0238] In some embodiments of the second aspect, the single-domain antibody comprises an amino acid sequence as shown in SEQ ID NO: 35.

[0239] In some embodiments of the second aspect, the single-domain antibody comprises an amino acid sequence as shown in SEQ ID NO: 36.

[0240] In some embodiments of the second aspect, the single-domain antibody comprises an amino acid sequence as shown in SEQ ID NO: 71.

[0241] In some embodiments of the second aspect, the single-domain antibody comprises an amino acid sequence as shown in SEQ ID NO: 72.

[0242] In some embodiments of the second aspect, the amino acid sequence of the single-domain antibody differs from the amino acid sequence shown in SEQ ID NO: 35, 36, 71, or 72 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, deletions, and / or additions of amino acids.

[0243] In some embodiments of the second aspect, the amino acid sequence of the single-domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology to SEQ ID NO: 35, 36, 71, or 72.

[0244] In some embodiments of the second aspect, the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 35, 36, 71 or 72 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still retaining similar functionality of the single-domain antibody.

[0245] In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 35, 36, 71 or 72, while the resulting amino acid sequence still retains similar functionality of the single-domain antibody.

[0246] In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added to or deleted from a region other than the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 35, 36, 71 or 72, as long as the altered amino acid sequence substantially retains similar functionality of the single-domain antibody.

[0247] In a third aspect, the present application provides nucleic acid molecules encoding the bispecific antibody of the first aspect or the single-domain antibody of the second aspect.

[0248] In some embodiments of the third aspect, the nucleic acid molecules can include DNA molecules and RNA molecules. The nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.

[0249] In some embodiments of the third aspect, the nucleic acid molecules can be operably linked to regulatory amino acid sequences that can be recognized by host cells transformed with the vectors.

[0250] In a fourth aspect, the present application provides pharmaceutical compositions comprising the bispecific antibody of the first aspect or the single-domain antibody of the second aspect and a pharmaceutically acceptable excipient, diluent or carrier.

[0251] In some embodiments of the fourth aspect, the pharmaceutical compositions are used for preventing or treating HLA-A02 / MAGE-A4 positive tumors.

[0252] In some embodiments of the fourth aspect, the HLA-A02 / MAGE-A4 positive tumor is selected from the group consisting of: esophageal cancer (e.g., esophageal squamous cell carcinoma), urothelial cancer, oral cancer (e.g., oral squamous cell carcinoma), sarcoma (e.g., synovial sarcoma), head and neck cancer, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer, and breast cancer.

[0253] In some embodiments of the fourth aspect, the pharmaceutical composition is used for preventing or treating HLA-A02 / MAGE-A8 positive tumor.

[0254] In some embodiments of the fourth aspect, the HLA-A02 / MAGE-A8 positive tumor is selected from the group consisting of: esophageal cancer (e.g., esophageal squamous cell carcinoma), urothelial cancer, oral cancer (e.g., oral squamous cell carcinoma), sarcoma (e.g., synovial sarcoma), head and neck cancer, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer, and breast cancer.

[0255] In some embodiments of the fourth aspect, the pharmaceutical composition can further comprise one or more of the following: lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives such as benzoic acid, sorbic acid, and calcium propionate; sweetening agents and / or flavoring agents, and the like.

[0256] In some embodiments of the fourth aspect, the pharmaceutical composition of the present application can be formulated into a tablet, a pill, a powder, a lozenge, an elixir, a suspension, an emulsion, a solution, a syrup, a suppository, a capsule, or the like.

[0257] In some embodiments of the fourth aspect, the pharmaceutical composition of the present application can be delivered by any physiologically acceptable mode of administration, including but not limited to oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.

[0258] In some embodiments of the fourth aspect, the pharmaceutical composition for therapeutic use can be stored in the form of a lyophilized preparation or an aqueous solution by mixing the reagents with the desired purity with, as appropriate, a pharmaceutically acceptable carrier, excipient, and the like.

[0259] In a fifth aspect, the present application provides use of the bispecific antibody of the first aspect, the single-domain antibody of the second aspect, or the pharmaceutical composition of the fourth aspect in the preparation of a medicament for preventing or treating HLA-A02 / MAGE-A4 positive tumor and / or HLA-A02 / MAGE-A8 positive tumor.

[0260] In some embodiments of the fifth aspect, the HLA-A02 / MAGE-A4 positive tumor is selected from the group consisting of: esophageal cancer (e.g., esophageal squamous cell carcinoma), urothelial cancer, oral cancer (e.g., oral squamous cell carcinoma), sarcoma (e.g., synovial sarcoma), head and neck cancer, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer, and breast cancer.

[0261] In some embodiments of the fifth aspect, the HLA-A02 / MAGE-A8 positive tumor is selected from the group consisting of: esophageal cancer (e.g., esophageal squamous cell carcinoma), urothelial cancer, oral cancer (e.g., oral squamous cell carcinoma), sarcoma (e.g., synovial sarcoma), head and neck cancer, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer, and breast cancer.

[0262] In some embodiments of the sixth aspect, the HLA-A02 / MAGE-A4 positive tumor is selected from the group consisting of: esophageal cancer (e.g., esophageal squamous cell carcinoma), urothelial cancer, oral cancer (e.g., oral squamous cell carcinoma), sarcoma (e.g., synovial sarcoma), head and neck cancer, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer, and breast cancer.

[0263] In some embodiments of the sixth aspect, the HLA-A02 / MAGE-A8 positive tumor is selected from the group consisting of: esophageal cancer (e.g., esophageal squamous cell carcinoma), urothelial cancer, oral cancer (e.g., oral squamous cell carcinoma), sarcoma (e.g., synovial sarcoma), head and neck cancer, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer, and breast cancer.

[0264] In some embodiments of the sixth aspect, the HLA-A02 / MAGE-A8 positive tumor is selected from the group consisting of: esophageal cancer (e.g., esophageal squamous cell carcinoma), urothelial cancer, oral cancer (e.g., oral squamous cell carcinoma), sarcoma (e.g., synovial sarcoma), head and neck cancer, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer, and breast cancer.

[0265] The present application also provides vectors comprising nucleic acid molecules encoding the bispecific antibodies of the first aspect, or the single-domain antibodies of the second aspect, and host cells comprising the nucleic acid molecules or vectors. In other aspects, the present application also provides methods of producing the bispecific antibodies of the first aspect, or the single-domain antibodies of the second aspect. In some embodiments, the methods of producing the bispecific antibodies of the first aspect, or the single-domain antibodies of the second aspect, comprise culturing the host cells to facilitate expression of the nucleic acid molecules. In some embodiments, the methods of producing the bispecific antibodies of the first aspect, or the single-domain antibodies of the second aspect, further comprise recovering the bispecific antibodies or the single-domain antibodies from the host cell culture medium.

[0266] It is to be understood that the foregoing detailed description has been presented for the purposes of clarity and that work in the art will be capable of practicing the present application without being limited to any particular embodiments described.

[0267] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present application in any way. Examples

[0268] Example 1: Preparation of recombinant proteins

[0269] References

[16] Preparation of antigen peptide MAGE-A4 230-239 disulfide trap single chain trimer (dtSCT) structure MHC I complex of GVYDGREHTV, i.e. HLA-A02 complex of GVYDGREHTV (MHC-P1, SEQ ID NO: 47); and human CD3E extracellular region (hCD3E, SEQ ID NO: 51), human CD3D extracellular region (hCD3D, SEQ ID NO: 52). These recombinant proteins all have a large number of post-translational modifications (such as glycosylation or disulfide bond, etc.), and thus it is more advantageous to use a mammalian cell expression system to maintain the structure and function of the recombinant protein. Adding a His tag (His, SEQ ID NO: 53) or Fc segment of human antibody IgG1 (Fc, SEQ ID NO: 54) or Fc segment of murine antibody IgG2a (mFc, SEQ ID NO: 55) at the C-terminus of the recombinant protein will be more advantageous for the purification of the recombinant protein and the identification of the function of the monoclonal antibody. In the preparation of the recombinant antibody, the antibody heavy chain constant region can be human IgG1 subtype (SEQ ID NO: 56) or various mutants of selected human IgG1 subtype, such as: IgG1H (SEQ ID NO: 57), IgG1K (SEQ ID NO: 58), IgG1m3-H (SEQ ID NO: 59), IgG1m3-K (SEQ ID NO: 60), IgG1m3-H1n1 (SEQ ID NO: 61) or IgG1m3-Kn1 (SEQ ID NO: 62), and the light chain constant region can be human lambda subtype (SEQ ID NO: 63) or human kappa subtype (SEQ ID NO: 64).

[0270] According to the amino acid sequences of various recombinant proteins of interest in the Uniprot database, the genes of the above-mentioned recombinant proteins (containing His tag, mFc or Fc coding genes) are designed and synthesized. The synthesized genes of various recombinant proteins are cloned into suitable eukaryotic expression vectors (such as pcDNA3.1 of Invitrogen Co.) by using conventional molecular biology techniques, and then the prepared recombinant protein expression plasmids are transfected into HEK293 cells (such as HEK293F cells of Invitrogen Co.) by using liposomes (such as 293fectin of Invitrogen Co.) or other cationic transfection reagents (such as PEI), and cultured in serum-free suspension culture conditions for 3-4 days. Then the culture supernatant is harvested by centrifugation and the like.

[0271] The recombinant proteins expressed by His tag fusion are subjected to one-step purification by using a metal chelate affinity chromatography column (such as HisTrap FF of GE Co.) for the recombinant proteins in the culture supernatant. The recombinant proteins expressed by mFc / Fc fusion are subjected to one-step purification by using a Protein A / G affinity chromatography column (such as Mabselect SURE of GE Co.). Then the recombinant proteins are replaced with PBS (pH 7.0) or other suitable buffers by using a desalting column (such as Hitrap desaulting of GE Co.). If necessary, the antibody sample can be filtered to remove bacteria, and then stored at -20°C after being divided and stored.

[0272] Example 2: Screening of antibodies binding to HAL-A02 / MAGE-A4 complex from camel immune library

[0273] 2.1 Construction of camel immune library

[0274] One healthy adult Bactrian camel was selected, and blood was taken before immunization to obtain background serum. For the first immunization, 1 mg of MHC-P1-His fusion protein was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple points; two weeks later, 1 mg of MHC-P1-His fusion protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple points for five times of booster immunization, and blood was taken before each immunization for antibody titer analysis; for the seventh immunization, 1 mg of MHC-P1-His fusion protein was used as an antigen without adjuvant, and the camel was subjected to impact immunization by subcutaneous injection at multiple points, and 200 ml of peripheral blood was collected 3 days later for lymphocyte separation.

[0275] Lymphocytes were isolated from 200 mL of camel peripheral blood using a camel peripheral blood lymphocyte isolation kit (Solarbio, CAT#P5750); total RNA was extracted from the lymphocytes using a total RNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd., CAT#DP430); and the camel single-domain antibody heavy chain variable region (VHH) was synthesized using the extracted total RNA as a template and a first-strand cDNA synthesis kit (Thermo Scientific, CAT#K1621). The reverse transcription primer was a gene-specific primer, and the primer pairing region was located in the antibody heavy chain constant region CH2 domain. The specific sequence was PCal-CH2R: TCCTTCCCCGTCAGCCAGTCCT (SEQ ID NO: 50). The synthesized cDNA was immediately stored at -70°C for standby use; then, the cDNA obtained by reverse transcription was used as a template, and a reference

[17] The primer was synthesized, and the camel single-domain antibody VHH gene was isolated by nested PCR amplification. Finally, the amplified VHH gene was cloned into the vector pADSCFV-S (see Chinese Patent Application No. 201510097117.0

[18] ), to construct a VHH library. The library capacity of this antibody library reached 1.5E+08, and the accuracy was 75%.

[0276] 2.2 Screening of camel immunization library

[0277] The recombinant MHC-P1-His prepared in Example 1 was used as an antigen, and a solid-phase screening strategy (experimental scheme, see Phage Display: A Laboratory Manual, Clackson, T., Lowman, H. B. Eds.; Ma Lan et al. Trans. Chemical Industry Press, 2008.5

[19] ) was used to screen the phage library displaying camel single-domain antibodies constructed in Example 2.1. Through binding, elution, neutralization, infection, and amplification, a total of three rounds of screening were performed, and finally the TCRm antibody N18D10 specifically binding to MHC-P1-His was obtained.

[0278] The nucleotide sequence encoding the heavy chain variable region of N18D10 (SEQ ID NO: 35) was cloned into a eukaryotic expression vector (such as pcDNA3.1 of Invitrogen Co.) fused with the nucleotide sequence encoding the Fc segment of human antibody IgG1 by using conventional molecular biology methods, to express the N18D10-Fc recombinant protein. At the same time, DP47 (germline gene antibody, see US Patent Application US 20160200833A1

[20] , the amino acid sequence of DP47VH is shown as SEQ ID NO: 65; the amino acid sequence of DP47VK is shown as SEQ ID NO: 66) monoclonal antibody DP47-IgG1 as a negative control. Refer to patent application WO 2017175006A1

[21] , the Vα (TCRA, SEQ ID NO: 67) and Vβ (TCRB, SEQ ID NO: 68) variable region genes of IMC-C103C were synthesized, respectively cloned into eukaryotic expression vectors containing Fc fragment nucleotide sequences fused with Cα containing Knob mutation and Fc fragment nucleotide sequences fused with Cβ containing Hole mutation, and the two chains of the TCR molecule were respectively TCRA-Des-G1m3-FcKn1 (SEQ ID NO: 69) and TCRB-Des-G1m3-FcH1n1 (SEQ ID NO: 70), co-expressed to bind MHC-P1 TCR control molecule, named P1-TCR.

[0279] Example 3: Identification of TCRm antibody binding to HLA-A02 / MAGE-A4 complex

[0280] 3.1 Affinity analysis of TCRm antibody against HLA-A02 / MAGE-A4 complex

[0281] The affinity of the anti-HLA-A02 / MAGE-A4 TCRm antibody N18D10 was determined by surface plasmon resonance using a Biacore T200. The relevant reagents and consumables, including the Amino Coupling Kit (BR-1000-50), Human Antibody Capture Kit (BR-1008-39), Series S CM5 Chips (14100530), and 10x HBS-EP, pH 7.4 (BR100669), were purchased from GE healthcare. The carboxylated CM5 chip surface was activated with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) according to the instructions in the kit, and the anti-human IgG (Fc) antibody (capture antibody) was diluted to 25 μg / mL with 10 mM sodium acetate, pH 5.0, and then injected at a flow rate of 10 μL / min to achieve a coupling amount of about 10,000 response units (RU). After injecting the capture antibody, 1 M ethanolamine was injected to block the unreacted groups. For the kinetics measurement, the TCRm antibody N18D10-Fc was diluted to 1 μg / mL, and injected at 10 μL / min to ensure that about 60 RU of antibody was captured by the anti-human Fc antibody. Then the MHC-P1-His was set at a series of concentration gradients (e.g., 1.23 nM, 3.74 nM, 11.1 nM, 33.3 nM, 100 nM), and injected at 30 μL / min from low concentration to high concentration at 25°C, with a binding time of 90 s and a dissociation time of 1200 s. The chip surface was regenerated by injecting a 3 M MgCl2 solution at 10 μL / min for 30 s. The Biacore T200 Evaluation Software version 3.2.1 was used to calculate the association rate (K a ) and dissociation rate (K d ) by fitting the binding and dissociation sensorgrams with a 1:1 binding model. The dissociation equilibrium constant (K d ) was calculated with the ratio K a / K D . The fitting results are shown in Table 1.

[0282] Table 1. Affinity of the anti-HLA-A02 / MAGE-A4 TCRm antibody N18D10-Fc binding to MHC-P1 of the HLA-A02 / MAGE-A4 complex

[0283] K a (M -1 s -1 )]]> K d (s -1 )]]> K D (M)]]> N18D10-Fc 1.851E+5 7.176E-5 3.878E-10

[0284] 3.2 Binding of the anti-HLA-A02 / MAGE-A4 TCRm antibody to the recombinant protein MHC-P1

[0285] TCRm antibody N18D10-Fc against HLA-A02 / MAGE-A4 complex was coated on 96-well ELISA plate, 5 μg / mL, 100 μL per well, coated overnight at 4°C. Blocked with blocking buffer PBS-0.1% Tween 20-3% skim milk for 1 hour at 37°C. Gradient dilution of recombinant protein MHC-P1-His with PBS, starting concentration of 30 μg / mL, 3-fold gradient dilution, a total of 10 concentration gradients, 100 μL per well into the blocked 96-well ELISA plate, incubated at 37°C for 1 hour. PBS-0.1% Tween 20 wash the ELISA plate, add HRP-labeled anti-His tag mouse monoclonal antibody (Beijing Kangwei Century Biotechnology Co., Ltd., CW0285M), incubate at 37°C for 1 hour. PBS-0.1% Tween 20 wash the ELISA plate, add OPD substrate color developing liquid, 5-10 minutes later, stop color development with 1M H2SO4, measure the optical density value at 492nm / 630nm dual wavelength with a microplate reader. Figure 1 The results show that N18D10-Fc specifically binds to MHC-P1.

[0286] 3.3 Binding of TCRm antibody against HLA-A02 / MAGE-A4 complex to T2 cells pulsed with antigen peptide

[0287] T2 cells (human lymphoma cells, HLA-A02 + , purchased from Shanghai Hongshun Biotechnology Co., Ltd.) in the logarithmic growth phase were collected, resuspended to 1×10 6 / mL with growth medium after centrifugation, and plated in 24-well plates at 1 mL per well. The peptide segment MAGE-A4 230-239 (GVYDGREHTV) was chemically synthesized (Jinsuirui Biotechnology Co., Ltd.), and added to the 24-well plate at a final concentration of 50 μg / mL, and pulsed overnight. After 16 hours of centrifugation, resuspend to 2×10 6 / mL with PBS buffer containing 1% BSA after centrifugation, and plate in 96-well V-bottom plates at 100 μL per well, and remove the supernatant after centrifugation. Prepare antibody N18D10-Fc, positive control sample P1-TCR and negative control DP47-IgG1 at a final concentration of 200 nM starting, 4-fold dilution of samples, 100 μL per well into the wells containing cells, incubate at 4°C for 1 hour. Then wash 3 times with 200 μL PBS, incubate goat anti-human IgG-FITC (Beijing Zhongshanjinqiao Biotechnology Co., Ltd., ZF-0308) at 100 μL per well, incubate at 4°C for 30 minutes in the dark. Then wash 3 times with 200 μL PBS, resuspend with 100 μL PBS, and detect the FITC channel with a flow cytometer (ACEA, Novocyte). Figure 2Results showed that N18D10-Fc specifically bound to the antigenic peptide MAGE-A4 230-239 Pulsed T2 cells, i.e. N18D10-Fc bound to cell surface HLA-A02 / MAGE-A4 complex.

[0288] 3.4 Anti-HLA-A02 / MAGE-A4 complex TCRm antibody binds to key amino acids of MAGE-A4

[0289] Chemically synthesized MAGE-A4 230-239 alanine scanning mutant peptides of MAGE-A4 230-239 alanine scanning mutant peptides of MAGE-A4 6 (GVYDGREHTV). T2 cells in logarithmic growth phase were collected, centrifuged and resuspended to 1 x 10 230-239 (GVYDGREHTV) and alanine scanning mutant peptides were added to the 24-well plate at a final concentration of 50 μg / mL for overnight pulsing. After 16 hours, centrifugation was performed and the cells were resuspended to 2 x 10 6 (GVYDGREHTV) and alanine scanning mutant peptides were added to the 24-well plate at a final concentration of 50 μg / mL for overnight pulsing. After 16 hours, centrifugation was performed and the cells were resuspended to 2 x 10 230-239 (GVYDGREHTV) and alanine scanning mutant peptides were added to the 24-well plate at a final concentration of 50 μg / mL for overnight pulsing. After 16 hours, centrifugation was performed and the cells were resuspended to 2 x 10 230-239 (GVYDGREHTV) and alanine scanning mutant peptides were added to the 24-well plate at a final concentration of 50 μg / mL for overnight pulsing. After 16 hours, centrifugation was performed and the cells were resuspended to 2 x 10 Figure 3 Results showed that MAGE-A4 230-239 (GVYDGREHTV) and alanine scanning mutant peptides were added to the 24-well plate at a final concentration of 50 μg / mL for overnight pulsing. After 16 hours, centrifugation was performed and the cells were resuspended to 2 x 10 230-239 (GVYDGREHTV) and alanine scanning mutant peptides were added to the 24-well plate at a final concentration of 50 μg / mL for overnight pulsing. After 16 hours, centrifugation was performed and the cells were resuspended to 2 x 10

[0290] Table 2. Binding of TCRm antibody N18D10 to T2 cells pulsed with alanine scanning mutant peptides of MAGE-A4 230-239 Binding of TCRm antibody N18D10 to T2 cells pulsed with alanine scanning mutant peptides of MAGE-A4

[0291] Binding of TCRm antibody N18D10 to T2 cells pulsed with alanine scanning mutant peptides of MAGE-A4 MAGE-A4 230-239 ]]> G1A V2A Y3A D4A G5A R6A E7A H8A T9A V10A N18D10 100.00％ 109.51％ 41.06％ -0.12％ 18.40％ -1.02％ -0.56％ 27.44％ -0.13％ 33.62％ 0.35％

[0292] Example 4: Humanization and identification of TCRm antibody against HLA-A02 / MAGE-A4 complex

[0293] 4.1 Humanization of TCRm antibody against HLA-A02 / MAGE-A4 complex

[0294] The TCRm antibody N18D10 was humanized to reduce its immunogenicity. The humanization scheme took the classic framework grafting strategy

[22] . The amino acid sequence of N18D10 was compared with the human antibody germline gene sequences in IMGT database, and appropriate germline gene sequences were selected to provide the framework regions 1 to 3 (FR1 + FR2 + FR3) of the antibody, and appropriate J region gene sequences were selected to provide the framework region 4 (FR4). This template can be selected according to various factors, such as: the relative total length of the antibody, the size of the CDR, the amino acid residues located at the junction between the framework region (FR) and the hypervariable region (CDR) of the antibody, the overall homology of the sequence, etc. The selected template can be a mixture of multiple sequences or can be a consensus template, with the purpose of maintaining the appropriate conformation of the parent complementarity determining region (CDR) as much as possible. At the same time, considering the solubility, stability and expression yield of the humanized antibody, the four hot spot amino acids 37F / 44E / 45R / 47F in FR2 were respectively back-mutated, and finally the humanized molecule N18D10-h16 was obtained.

[0295] Using conventional molecular biology methods, the nucleotide sequence encoding the variable region of N18D10-h16 (SEQ ID NO: 36) was cloned into a eukaryotic expression vector (such as pcDNA3.1 of Invitrogen Company) fused with the nucleotide sequence encoding the Fc fragment of human antibody IgG1, to express the N18D10-h16-Fc recombinant protein.

[0296] 4.2 Affinity analysis of humanized molecule of TCRm antibody against HLA-A02 / MAGE-A4 complex

[0297] Referring to Example 3.1, Biacore T200 was used to analyze the affinity of the recombinant TCRm antibody N18D10-h16-Fc against HLA-A02 / MAGE-A4 complex, and the results are shown in Table 3.

[0298] Table 3. Affinity of TCRm antibody N18D10-h16-Fc against HLA-A02 / MAGE-A4 complex to bind MHC-P1 complex

[0299] K a (M -1 s -1 )]]> K d (s -1 )]]> K D (M)]]> MHC-P1-His 1.631E+5 7.610E-5 4.665E-10

[0300] 4.3 Binding of TCRm antibody N18D10-h16-Fc against HLA-A02 / MAGE-A4 complex to MHC-P1

[0301] Referring to Example 3.2, the binding activity of the anti-HLA-A02 / MAGE-A4 complex antibody N18D10-h16-Fc to the recombinant protein MHC-P1-His was determined. Figure 4 The results showed that N18D10-h16-Fc specifically bound to the recombinant protein MHC-P1-His, i.e., N18D10-h16-Fc bound to the HLA-A02 / MAGE-A4 complex.

[0302] Example 5: Preparation and identification of TCRmxCD3 bispecific antibodies

[0303] 5.1 Preparation of TCRmxCD3 bispecific antibodies

[0304] The heavy chain variable region nucleotide sequence encoding the bivalent 2N18D10 or bivalent 2N18D10-h16 single-domain antibody against the HLA-A02 / MAGE-A4 complex and the nucleotide sequence encoding the single-chain antibody against CD3 (IMCR-anti-CD3-scFv, see anti-CD3 v9

[23] ) were cloned into suitable eukaryotic expression vectors, respectively, to co-express the bispecific antibodies against the HLA-A02 / MAGE-A4 complex and CD3. That is, the nucleotide sequence encoding 2N18D10 (SEQ ID NO: 71) or 2N18D10-h16 (SEQ ID NO: 72) was cloned into an eukaryotic expression vector fused with the nucleotide sequence encoding the Hole mutant Fc fragment IgG1m3-FcH1n1 (SEQ ID NO: 92), and the nucleotide sequence encoding IMCR-anti-CD3-ScFv (SEQ ID NO: 73) was cloned into an eukaryotic expression vector fused with the nucleotide sequence encoding the Knob mutant Fc fragment IgG1m3-FcKn1 (SEQ ID NO: 93). At the same time, the DP47 bispecific antibody based on the same structure was prepared as a negative control.

[0305] The constructed eukaryotic expression vectors expressing 2N18D10-G1m3-FcHIn1 (SEQ ID NO: 74) or 2N18D10-h16-G1m3-FcH1n1 (SEQ ID NO: 45) and the eukaryotic expression vector expressing IMCR-anti-CD3-ScFv-G1m3-FcKn1 (SEQ ID NO: 46) are co-transfected into HEK293F cells using liposomes, cultured for 3-5 days under serum-free suspension culture conditions, and then the culture supernatant is harvested by centrifugation and the like. The bispecific antibody in the culture supernatant is purified by Protein A affinity chromatography column (such as GE's Mabselect SURE, etc.), and then the desalting column (such as GE's Hitrap desaulting, etc.) is used to replace the recombinant protein storage buffer with PBS (pH 7.0) or other suitable buffers. The protein solution after desalting is purified by size exclusion chromatography (SEC) using Superdex 200 (GE) to obtain the target protein, which is named 2N18D10+IMCR and 2N18D10-h16+IMCR, respectively. If necessary, the antibody sample can be filtered to remove bacteria, and then stored at -20°C for standby.

[0306] Referring to patent application WO 2017175006A1, the single-chain antibody IMCR-anti-CD3-scFv of CD3 is fused at the N-terminus of the TCRB-Des-G1m3-FcH1n1 chain to prepare a bifunctional molecule IMC-C103C based on TCR and CD3, and the two chains of the TCR and CD3 bifunctional molecule are TCRB-Des-G1m3-FcKn1 (SEQ ID NO: 69) and IMCR-anti-CD3-scFv-TCRB-Des-G1m3-FcH1n1 (SEQ ID NO: 75), respectively.

[0307] Referring to patent application WO 2021122875A1

[24] , the variable region sequence of 057D03 was synthesized (the amino acid sequence of 057G03 VH is shown as SEQ ID NO: 76; the amino acid sequence of 057G03 VK is shown as SEQ ID NO: 77, and the anti-HLA-A02 / MAGE-A4 complex and CD3 bispecific antibody 057G03 based on the structure in the patent was prepared, and the four chains constituting the bispecific antibody were 057G03-Fd-EE-IMCR-anti-CD3 VK-G1m3-Kn1 (SEQ ID NO: 78), 057G03-Fd-EE-G1m3-FcH1n1 (SEQ ID NO: 79), 057G03 VK+CK-RK (SEQ ID NO: 80), and IMCR-anti-CD3 VH-CK (SEQ ID NO: 81), respectively.

[0308] The expression and purification of the above-mentioned IMC-C103C and 057G03 bispecific antibodies were performed according to the expression and purification of 2N18D10+IMCR and 2N18D10-h16+IMCR described above, and then the samples were filtered to remove bacteria, and then stored at -20°C for standby.

[0309] 5.2 Affinity analysis of TCRm x CD3 bispecific antibodies

[0310] According to Example 3.1, Biacore T200 was used to perform affinity analysis of recombinant TCRm x CD3 bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR, and the results are shown in Tables 4 and 5.

[0311] Table 4. TCRm x CD3 bispecific antibody binding MHC-P1 affinity

[0312] K a (M -1 s -1 )]]> K d (s -1 )]]> K D (M)]]> 2N18D10 + IMCR 1.961E+5 7.607E-5 3.880E-10 2N18D10-h16 + IMCR 1.820E+5 7.834E-5 4.304E-10 IMC-C103C 8.520E+4 4.594E-5 5.392E-10 057G03 5.071E+5 1.672E-2 3.297E-8

[0313] Table 5. TCRm x CD3 bispecific antibody binding CD3D-CD3E-mFc affinity

[0314] K a (M -1 s -1 )]]> K d (s -1 )]]> K D (M)]]> 2N18D10 + IMCR 2.037E+5 2.700E-4 1.325E-9 2N18D10-h16 + IMCR 1.946E+5 2.681E-4 1.378E-9 IMC-C103C 2.582E+5 2.797E-4 1.083E-9 057G03 1.178E+5 2.699E-4 2.291E-9

[0315] 5.3 Simultaneous recognition of antigen MHC-P1 and CD3 by TCRm x CD3 bispecific antibodies

[0316] The simultaneous binding of TCRm x CD3 bispecific antibodies to the double-antigen CD3 and MHC-P1 was detected by a conventional ELISA method.

[0317] CD3D-CD3E-mFc antigen coated 96-well ELISA plate (3 μg / mL, 100 μL per well) at 4°C overnight. Blocked with blocking buffer PBS-0.1% Tween 20-3% skim milk at 37°C for 1 hour. Gradient dilution of TCRm x CD3 bispecific antibodies with PBS, starting concentration 10 μg / mL, 3-fold gradient dilution, a total of 10 concentration gradients, 100 μL per well into the blocked 96-well ELISA plate, 37°C incubation for 1 hour. PBS-0.1% Tween 20 wash the ELISA plate, add MHC-P1-His antigen (10 μg / mL, 100 μL per well), 37°C incubation for 1 hour. PBS-0.1% Tween 20 wash the ELISA plate, then add HRP-labeled anti-His tag mouse monoclonal antibody (Beijing Kangwei Century Biotechnology Co., Ltd., cw0285M), 37°C incubation for 1 hour. PBS-0.1% Tween 20 wash the ELISA plate, add OPD substrate color developing liquid, 5-10 minutes after color development is terminated with 1M H2SO4, enzyme-labeled instrument 492 nm / 630 nm dual-wavelength determination of optical density value. ELISA analysis results as shown in Figure 5 Figure 6, TCRm x CD3 bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR can simultaneously recognize CD3 and MHC-P1 dual-direction antigens.

[0318] 5.4 TCRm x CD3 bispecific antibody-mediated Jurkat-Dual cell activation

[0319] 5.4.1 TCRm x CD3 bispecific antibody-mediated activation of antigen peptide-pulsed T2 cells Jurkat-Dual cells

[0320] Logarithmic growth phase T2 cells were collected, centrifuged and resuspended to 1 x 10 6 cells / mL with growth medium, 1 mL per well plated in a 24-well plate. Peptide MAGE-A4 230-239 (GVYDGREHTV) was added to the 24-well plate at a final concentration of 50 μg / mL, and pulsed overnight. After 16 hours, centrifugation, resuspension to 4 x 10 5 cells / mL with RPMI1640 medium, 50 μL per well plated in a cell plate. Logarithmic growth phase Jurkat-Dual cells (purchased from Invivogen) were collected, centrifuged and resuspended to 4 x 10 550 μL of the above-mentioned cells were added to each well of a cell plate seeded with T2 cells to obtain a final E:T ratio of 1:1. Then, bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR (initial concentration 7.5 nM, 4-fold serial dilution, 8 concentration points, 100 μL per well) were added. The negative control sample 2DP47+IMCR and the positive control samples 057G03 and IMC-C103C were used at the same concentrations as the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR. After incubation for 24 h, the supernatant was collected and processed according to the QUANTI-Luc standard. TM Instruction manual (QUANTI-Luc) TM The specific activation of Jurkat-Dual cells by T2 cells mediated by antigen peptide pulses induced by TCRm×CD3 bispecific antibodies was detected and analyzed using Invivogen and rep-qlc2. The results showed that the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR could specifically mediate the activation of Jurkat-Dual cells by T2 cells induced by antigen peptide pulses. Compared with 057G03 and IMC-C103C, the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR showed more pronounced activation of Jurkat-Dual cells. Figure 6 EC 50 The values ​​are shown in Table 6.

[0321] Table 6. T2 cell activation of Jurkat-Dual cells by exogenous pulsartan antigen peptide mediated by TCRm×CD3 bispecific antibody. 50 value

[0322]

[0323] 5.4.2 TCRm×CD3 bispecific antibody-mediated HLA-A02 + / MAGE-A4 + Tumor cells activate Jurkat-Dual cells

[0324] Collect A375 cells (human malignant melanoma cells, HLA-A02) in the logarithmic growth phase. + / MAGE-A4 + (Purchased from Nanjing Kebai Biotechnology Co., Ltd.). After digestion and centrifugation, the solution was resuspended in RPMI 1640 medium to a final volume of 4 × 10⁻⁶. 5 Jurkat-Dual cells were seeded at a density of 50 μL / mL in each well of a cell plate. Jurkat-Dual cells in the logarithmic growth phase were collected, centrifuged, and resuspended in RPMI 1640 medium to a final volume of 4 × 10⁻⁶. 5Individuals were selected according to the following criteria: age > 18 years; no HIV and HBV infection; normal blood routine test; non-pregnant or lactating women. Blood (50 mL each) was collected from normal volunteers, and all volunteers had signed informed consent. 5.5.1 Isolation of human peripheral blood mononuclear cells (PBMCs) TM The supernatant was collected after 48 hours of incubation, and the QUANTI-Luc TM , Invivogen, rep-qlc2) assay was used to detect and analyze the specific activation of Jurkat-Dual cells mediated by tumor cells under different conditions. The results showed that TCRm x CD3 bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR could mediate HLA-A02 + / MAGE-A4 + tumor cells A375 to activate Jurkat-Dual cells. Compared with 057G03 and IMC-C103C, TCRm x CD3 bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR had stronger activation activity on Jurkat-Dual cells Figure 7 ), and the EC 50 values are shown in Table 7. Table 7 TCRm x CD3 bispecific antibodies mediate HLA-A02 + / MAGE-A4 + tumor cells to activate Jurkat-Dual cells

[0325]

[0326] 5.5 TCRm x CD3 bispecific antibodies mediate PBMC specific killing of target cells

[0327] 5.5.1 Isolation of human peripheral blood mononuclear cells (PBMCs)

[0328] Blood (50 mL each) was collected from normal volunteers, and all volunteers had signed informed consent. The volunteers were selected according to the following criteria:

[0329] Age > 18 years;

[0330] No HIV and HBV infection;

[0331] Normal blood routine test;

[0332] Non-pregnant or lactating women.

[0333] PBMCs were isolated from whole blood of volunteers using Ficoll density gradient centrifugation and cultured in RPMI 1640 medium.

[0334] 5.5.2 TCRm×CD3 bispecific antibody-mediated PBMC killing of T2 cells induced by antigenic peptide pulses

[0335] Collect T2 cells in the logarithmic growth phase, centrifuge, and resuspend in growth medium to a concentration of 1×10⁻⁶. 6 Peptide MAGE-A4: 1 mL per well, seeded onto a 24-well plate. 230-239 (GVYDGREHTV) was added to a 24-well plate at a final concentration of 50 μg / mL and incubated overnight. After 16 hours, the plate was centrifuged and resuspended in RPMI 1640 medium to a final volume of 4 × 10⁻⁶. 5 50 μL of bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR were seeded per well in a 96-well plate. Then, bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR (starting at 30 nM, serially diluted 4-fold, 100 μL per well for 10 concentration points) were added. Negative control sample 2DP47+IMCR and positive control samples 057G03 and IMC-C103C were used at the same concentrations as the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR. PBMCs (effective cells) were resuspended to 4 × 10⁻⁶. 6 Add cells / mL to the above cell plate, 50 μL per well, for a final effector-to-target ratio of 10:1. Simultaneously, set up separate target cell controls (antigen peptide pulsed T2 cells), separate effector cell controls (PBMCs), and separate culture medium blank controls, and bring the volume of each well to 200 μL with culture medium. After incubation for 24 hours, collect the supernatant and refer to... Instructions for use of non-radioactive cytotoxicity test reagents ( The Non-Radioactive Cytotoxicity Assay (promega, G1780) was used to detect and analyze the killing rate of T2 cells induced by antigenic peptide pulses by PBMC cells mediated by TCRm×CD3 bispecific antibodies. The results showed that the TCRm×CD3 bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR could specifically mediate the killing of T2 cells induced by antigenic peptide pulses by PBMC cells; compared with 057G03 and IMC-C103C, the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR showed higher activity in PBMC cells killing T2 cells induced by antigenic peptide pulses (Table 8 and...). Figure 8 ).

[0336] Table 8. T2 cells mediated by TCRm×CD3 bispecific antibody to kill antigenic peptide pulses in PBMC cells

[0337]

[0338] 5.5.3 TCRm×CD3 bispecific antibody-mediated PBMC killing of HLA-A02 + / MAGE-A4 + Tumor cells

[0339] Collect different antigen double positives (HLA-A02) during the logarithmic growth phase. + / MAGE-A4 + The cells included A375 cells (human malignant melanoma cells), Hs695T cells (human malignant melanoma cells), U2-OS cells (human osteosarcoma cells), and negative control cells NIC-H520 cells (human lung squamous cell carcinoma cells, HLA-A02- / MAGE-A4). + ) and HepG2 (human liver cancer cells, HLA-A02) + / MAGE-A4 - After digestion and centrifugation, the solution was resuspended in RPMI 1640 medium to a final volume of 4 × 10⁻⁶. 5 Cells were seeded at a density of 50 μL / well in a cell plate. Then, bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR (starting at 30 nM, serially diluted 4-fold, 10 concentration spots, 100 μL per well) were added. Negative control sample 2DP47+IMCR and positive control samples 057G03 and IMC-C103C were used at the same concentrations as the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR. PBMCs (effective cells) were resuspended to 4 × 10⁻⁶. 6 Add cells / mL to the cell plate as described above, 50 μL per well, for a final effector-to-target ratio of 10:1. Simultaneously set up separate target cell controls, separate effector cell controls (PBMCs), and separate culture medium blank controls, and bring the volume of each well to 200 μL with culture medium. After incubation for 24-48 hours, collect the supernatant and refer to... Instructions for use of non-radioactive cytotoxicity test reagents ( The Non-Radioactive Cytotoxicity Assay (promega, G1780) was used to detect and analyze the killing rate of PBMCs against tumor cells mediated by the TCRm×CD3 bispecific antibody. The results showed that 2N18D10-h16+IMCR mediated the specific killing of tumor cells by PBMCs. Figure 10), 2N18D10+IMCR and 2N18D10-h16+IMCR showed stronger killing activity against A375 cells than 057G03 and IMC-C103C ( Figure 9 EC 50 (Values ​​are shown in Table 9).

[0340] Table 9. ECMO effect of TCRm×CD3 bispecific antibody-mediated killing of A375 cells by PBMCs 50 value

[0341]

[0342] Example 6: TCRm×CD3 bispecific antibody recognizes MAGE-A4 230-239 Key amino acid analysis of (GVYDGREHTV)

[0343] Chemical synthesis of MAGE-A4 230-239 Alanine-scanning peptide fragment of (GVYDGREHTV). Collect T2 cells in the logarithmic growth phase. After centrifugation, resuspend in growth medium to 1×10⁻⁶. 6 1 mL per well, plated onto a 24-well plate. MAGE-A4 230-239 (GVYDGREHTV) and the alanine-scanning mutant peptide were added to a 24-well plate at a final concentration of 50 μg / mL and incubated overnight. After 16 hours, the plate was centrifuged and resuspended in RPMI 1640 medium to a final volume of 4 × 10⁻⁶. 5 Jurkat-Dual cells were added at a concentration of 50 μL / well to each well of a 96-well cell plate. Jurkat-Dual cells in the logarithmic growth phase were collected, centrifuged, and resuspended in RPMI 1640 medium to a final volume of 4 × 10⁻⁶. 5 Cells / mL, 50 μL per well, were added to the preceding cell plate to obtain a final E:T ratio of 1:1. Then, 2N18D10-h16+IMCR bispecific antibody (starting concentration 30 nM, 4-fold serial dilution, 8 concentration points, 100 μL per well) was added. After incubation for 24 h, the supernatant was collected, and the culture was processed according to QUANTI-Luc... TM Instruction manual (QUANTI-Luc) TM The specific activation of Jurkat-Dual cells by T2 cells induced by different peptide pulses was detected and analyzed using Invivogen (rep-qlc2). The results showed that 2N18D10-h16+IMCR could not mediate the activation of Jurkat-Dual cells by T2 cells induced by alanine scan mutant peptide pulses at positions 3, 5, 6, 8, or 10. Figure 11 ), namely MAGE-A4 230-239 The amino acids at positions 3, 5, 6, 8, and 10 of (GVYDGREHTV) are the key amino acids for the 2N18D10-h16+IMCR binding.

[0344] Example 7: TCRm x CD3 bispecific antibody interaction analysis with similar peptides

[0345] Chemical synthesis of MAGE-A4 similar peptides in Table 10 230-239 of the similar peptide segment of MAGE-A4 (GVYDGREHTV).

[0346] Table 10. MAGE-A4 similar peptides information 230-239 of the similar peptide segment of MAGE-A4 (GVYDGREHTV).

[0347]

[0348]

[0349] T2 cells in log phase were collected. After centrifugation, resuspended to 1 x 10 6 cells / mL with growth medium, 1 mL per well plated in 24-well plates. MAGE-A4 230-239 similar peptide segments were added to the 24-well plates at a final concentration of 50 μg / mL, pulsed overnight. After 16 hours, centrifuged, resuspended to 4 x 10 5 cells / mL with RPMI1640 medium, 50 μL per well added to 96-well cell plates. Jurkat-Dual cells in log phase were collected, centrifuged, resuspended to 4 x 10 5 cells / mL with RPMI1640 medium, 50 μL per well added to the above cell plates to obtain a final E:T ratio of 1:1. Then, bispecific antibody 2N18D10-h16+IMCR was added (starting final concentration of 30 nM, 4-fold gradient dilution, 8 concentration points, 100 μL per well). Incubated for 24 h, supernatant was taken, and QUANTI-Luc TM assay (QUANTI-Luc TM , Invivogen, rep-qlc2) was used to detect and analyze the activation of Jurkat-Dual cells by different similar peptide pulsed T2 cells. Figure 12 The results of Example 7 show that 2N18D10-h16+IMCR can specifically mediate the activation of Jurkat-Dual cells by MAGE-A4 230-239T2 cells pulsed with MAGE-A4-M1 (i.e. MAGE-A4-S1) activated Jurkat-Dual cells, and also mediated activation of Jurkat-Dual cells by T2 cells pulsed with MAGE-A4-S1 (an antigenic peptide of MAGE-A8 presented by HLA-A02). MAGE-A8 is also a member of the MAGE family and has the same expression pattern as MAGE-A4. It is expressed in testis and placenta tissues in healthy people and in some tumor tissues in cancer patients.

[0350] Example 8: Analysis of the interaction of TCRm x CD3 bispecific antibodies with different antigen-negative tumor cells

[0351] PBMC were sorted according to Section 5.5.1.

[0352] Different tumor cells were collected, and the antigen information of the cells is shown in Table 11. According to Section 5.5.3, the killing experiment of PBMC mediated by 2N18D10-h16+IMCR bispecific antibodies on different antigen-negative tumor cells was performed. Figure 13 The results showed that 2N18D10-h16+IMCR bispecific antibodies only mediated the killing of PBMC on HLA-A02 + / MAGE-A4 + cells A375, but did not mediate the killing of PBMC on antigen-negative tumor cells.

[0353] Table 11. Antigen expression information of different tumor cells

[0354] Cells HLA-A02 MAGE-A4 Cells HLA-A02 MAGE-A4 A375 + + SW480 + - PANC-1 + - HT29 + - HepG2 + - NCI-H520 - + T2 + - PLC / PRF / 5 - -

[0355] Note: "+" represents positive, "-" represents negative.

[0356] Example 9: Tumor inhibition effect of TCRm x CD3 bispecific antibodies in hPBMC immune reconstituted mice

[0357] PBMC were sorted according to Section 5.5.1.

[0358] Forty 5-6 week old female NOG mice (purchased from Beijing Vantouda Biotechnology Co., Ltd.) were selected, and each mouse was intraperitoneally inoculated with 8x10 6 Personal PBMC, 1x10 7 A375 cells were inoculated subcutaneously on the right side of the NPG mice on the seventh day after the inoculation of human PBMC. When the average volume of the tumor was 70mm 3When the tumor size was determined, the animals were randomly grouped according to the tumor size, the grouping day was defined as day 0, and the administration was performed. The experiment was divided into 1 mg / kg group and 0.1 mg / kg group of the bispecific antibody 2N18D10-h16+IMCR, 2DP47+IMCR as the negative control group, and 057G03 as the positive control group, a total of 4 groups, 6 animals in each group, tail vein injection for administration, once a week, administration for three times, and continuous observation. The efficacy was evaluated according to the tumor inhibition rate TGI.

[0359] During the experiment, the animal's mental state was generally good. The test drug 2N18D10-h16+IMCR bispecific antibody had a significant inhibitory effect on tumor growth at a dose of 0.1 mg / kg (group 3), with a relative tumor inhibition rate TGI (%) of 99.09; and 3 animals had complete tumor regression, which had a very significant difference compared with the negative control group (group 1) (p<0.001). 2N18D10-h16+IMCR had a more significant inhibitory effect on tumor growth at a dose of 1 mg / kg (group 4), with a relative tumor inhibition rate TGI (%) of 104.2%; and 3 animals had complete tumor regression, which had a very significant difference compared with the negative control group (group 1) (p<0.001). The bispecific antibody 2N18D10-h16+IMCR effectively inhibited the growth of tumors, and was better than 057G03 (group 2). Figure 14 ) There was no animal death in each treatment group, and no obvious drug toxicity was observed.

[0360] All patents, patent application publications, and non-patent literature mentioned and / or listed in the present application are incorporated herein by reference in their entirety. The above describes exemplary embodiments of the present application, but those skilled in the art can modify or improve the exemplary embodiments described in the present application without departing from the spirit and scope of the present application, and the resulting modifications or equivalents also belong to the scope of the present application.

[0361] Sequence information

[0362] SEQ ID NO: 1

[0363] GVYDGREHTV

[0364] SEQ ID NO: 2

[0365] GLYDGREHSV

[0366] SEQ ID NO: 3

[0367] EVYDGREHSA

[0368] SEQ ID NO: 4

[0369] ISYSGVLHAV

[0370] SEQ ID NO: 5

[0371] GGYPGGIHEV

[0372] SEQ ID NO: 6

[0373] YLISQVEGHQV

[0374] SEQ ID NO: 7

[0375] DSYGGRHHRL

[0376] SEQ ID NO: 8

[0377] VGYPGRLHLW

[0378] SEQ ID NO: 9

[0379] FVYDEPGHAV

[0380] SEQ ID NO: 10

[0381] SQYSGQLHEV

[0382] SEQ ID NO: 11

[0383] ALYGRLEVV

[0384] SEQ ID NO: 12

[0385] NTYEGRPIYV

[0386] SEQ ID NO: 13

[0387] NTYEGRPIYV

[0388] SEQ ID NO: 14

[0389] KIYEGAYHV

[0390] SEQ ID NO: 15

[0391] RLYTGMHTV

[0392] SEQ ID NO: 16

[0393] GVYAGRPLSV

[0394] SEQ ID NO: 17

[0395] GVYQGRVSAV

[0396] SEQ ID NO: 18

[0397] AVLDGRELRV

[0398] SEQ ID NO: 19

[0399] RLYDGLFKV

[0400] SEQ ID NO: 20

[0401] DVYDGRFLV

[0402] SEQ ID NO: 21

[0403] SRYDGQIAV

[0404] SEQ ID NO: 22

[0405] NRYDGIYKV

[0406] SEQ ID NO: 23

[0407] NRYDGQVAV

[0408] SEQ ID NO: 24

[0409] LMYDGTKEV

[0410] SEQ ID NO: 25

[0411] HVYDGKFLARV

[0412] SEQ ID NO: 26

[0413] YIYDGELVSK

[0414] SEQ ID NO: 27

[0415] ATYDGQFMKL

[0416] SEQ ID NO: 28

[0417] FAYDGKDYL

[0418] SEQ ID NO: 29

[0419] KLYDGFQYL

[0420] SEQ ID NO: 30

[0421] SVYDGKLLI

[0422] SEQ ID NO: 31

[0423] YAYDGKDYI

[0424] SEQ ID NO: 32

[0425] DSDMG

[0426] SEQ ID NO: 33

[0427] TITTDGSTYYADSVKG

[0428] SEQ ID NO: 34

[0429] DLPILRPTPCGDSVLYNY

[0430] SEQ ID NO: 35

[0431] EVQLVESGGGSVQTGETLRLSCTVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSV

[0432] KGRFTISQDNAKTTAYLQMDSLKPEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTQVTVSS SEQ ID NO: 36

[0433] QVQLVESGGGLVKPGGSLRLSCAVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSV

[0434] KGRFTISQDNAKTSAYLQMNSLRAEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTTVTVSS SEQ ID NO: 37

[0435] GYTMN

[0436] SEQ ID NO: 38

[0437] LINPYKGVSTYNQKFKD

[0438] SEQ ID NO: 39

[0439] SGYYGDSDWYFDV

[0440] SEQ ID NO: 40

[0441] RASQDIRNYLN

[0442] SEQ ID NO: 41

[0443] YTSRLES

[0444] SEQ ID NO:42

[0445] QQGNTLPWT

[0446] SEQ ID NO:43

[0447] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ

[0448] KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS SEQ ID NO:44

[0449] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGS

[0450] GSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK

[0451] SEQ ID NO:45

[0452] QVQLVESGGGLVKPGGSLRLSCAVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSV

[0453] KGRFTISQDNAKTSAYLQMNSLRAEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTTVTVSS

[0454] GGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAVSGFTFDDSDMGWYRQAPGNECEL

[0455] VSTITTDGSTYYADSVKGRFTISQDNAKTSAYLQMNSLRAEDTAVYYCAADLPILRPTPCGDSV

[0456] LYNYWGQGTTVTVSSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0457] SQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

[0458] LPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYK

[0459] TTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0460] SEQ ID NO:46

[0461] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGS

[0462] GSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSG

[0463] GGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVST

[0464] YNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVS

[0465] SASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0466] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQP

[0467] REPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

[0468] SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0469] SEQ ID NO:47

[0470] GVYDGREHTVGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVD

[0471] LLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGG

[0472] GGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRM

[0473] EPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSD

[0474] WRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVE

[0475] WLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE

[0476] LVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE

[0477] SEQ ID NO:48

[0478] MSSEQKSQHCKPEEGVEAQEEALGLVGAQAPTTEEQEAAVSSSSPLVPGTLEEVPAAESAGPPQ

[0479] SPQGASALPTTISFTCWRQPNEGSSSQEEEGPSTSPDAESLFREALSNKVDELAHFLLRKYRAKE

[0480] LVTKAEMLERVIKNYKRCFPVIFGKASESLKMIFGIDVKEVDPASNTYTLVTCLGLSYDGLLGN

[0481] NQIFPKTGLLIIVLGTIAMEGDSASEEEIWEELGVMGVYDGREHTVYGEPRKLLTQDWVQENYL

[0482] EYRQVPGSNPARYEFLWGPRALAETSYVKVLEHVVRVNARVRIAYPSLREAALLEEEEGV

[0483] SEQ ID NO: 49

[0484] GGGGSGGGGSGGGGS

[0485] SEQ ID NO: 50

[0486] TCCTTCCCCGTCAGCCAGTCCT

[0487] SEQ ID NO: 51

[0488] QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKE

[0489] FSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

[0490] SEQ ID NO: 52

[0491] FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQ

[0492] VHYRMCQSCVELDPATVA

[0493] SEQ ID NO: 53

[0494] HHHHHH

[0495] SEQ ID NO: 54

[0496] ASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY

[0497] VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0498] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

[0499] LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0500] SEQ ID NO: 55

[0501] ASPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVN

[0502] NVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSV

[0503] RAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYF

[0504] MYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0505] SEQ ID NO: 56

[0506] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0507] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

[0508] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0509] HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF

[0510] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0511] YTQKSLSLSPGK

[0512] SEQ ID NO: 57

[0513] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0514] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

[0515] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0516] HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGF

[0517] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0518] YTQKSLSLSPGK

[0519] SEQ ID NO: 58

[0520] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0521] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

[0522] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0523] HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGF

[0524] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0525] YTQKSLSLSPGK

[0526] SEQ ID NO: 59

[0527] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0528] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKP

[0529] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0530] HQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGF

[0531] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0532] YTQKSLSLSPGK

[0533] SEQ ID NO: 60

[0534] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0535] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKP

[0536] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0537] HQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGF

[0538] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0539] YTQKSLSLSPGK

[0540] SEQ ID NO: 61

[0541] ASTKGPSVFPLAPSSKSTSEGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0542] SSVVTVPSSSLGTQTYICNVDHKPSNTKVDKTVERKSCVECPPCPAPEFEGGPSVFLFPPKPKDT

[0543] LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

[0544] WLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSD

[0545] IAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQK

[0546] SLSLSPGK

[0547] SEQ ID NO: 62

[0548] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0549] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVERKSCVECPPCPAPEFEGGPSVFLFPPKPKDT

[0550] LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

[0551] WLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPS

[0552] DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ

[0553] KSLSLSPGK

[0554] SEQ ID NO: 63

[0555] VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN

[0556] NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0557] SEQ ID NO: 64

[0558] VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST

[0559] YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0560] SEQ ID NO: 65

[0561] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS

[0562] SEQ ID NO: 66

[0563] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK

[0564] SEQ ID NO: 67

[0565] ANQVEQSPQSLIILEGKNVTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILDYAINTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVNRADGLYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFTCANAFNNSIIPEDTFFPSPESSCGGGGSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0566] SEQ ID NO:68

[0567] DVKVTQSSRYLVKRTGEKVFLECVQDAPLSKMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSSDQNSGDPYEQYFGPGTRLTVT

[0568] SEQ ID NO:69

[0569] ANQVEQSPQSLIILEGKNVTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILDYAINTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVNRADGLYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFTCANAFNNSIIPEDTFFPSPESSCGGGGSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0570] SEQ ID NO:70

[0571] DVKVTQSSRYLVKRTGEKVFLECVQDAPLSKMFWYRQDPGLGLRLIYFSY DVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSSDQNSGDP YEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSKAEISRTQKATLVCLATGFY PPHVELSWWVNGKEVHDGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQ DPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGGGGSASE RKSCVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVF SC S VMHEALHNHYTQKSLSLSPGK

[0572] SEQ ID NO: 71

[0573] EVQLVESGGGSVQTGETLRLSCTVSGFTFDDSDMGWYRQAPGNECELVSTITTDG STYYADSVKGRFTISQDNAKTTAYLQMDSLKPEDTAVYYCAADLPILRPTPCG DSVLYNYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGSVQTGETL RL S CT V S GFTFDDSDMGWYRQAPGNECELVSTITTDG STYYADSVKGRFT ISQDNAKTTAYLQMDSLKPEDTAVYYCAADLPILRPTPCGDSVLYNYWGQ GTQVTVSS

[0574] SEQ ID NO: 72

[0575] QVQLVESGGGLVKPGGSLRLSCAVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSVKGRFTISQDNAKTSAYLQMNSLRAEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTTVTVSSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSVKGRFTISQDNAKTSAYLQMNSLRAEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTTVTVSS

[0576] SEQ ID NO: 73

[0577] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS

[0578] SEQ ID NO: 74

[0579] EVQLVESGGGSVQTGETLRLSCTVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSVKGRFTISQDNAKTTAYLQMDSLKPEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQTGETLRLSCTVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSVKGRFTISQDNAKTTAYLQMDSLKPEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTQVTVSSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0580] SEQ ID NO: 75

[0581] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGS

[0582] GSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSG

[0583] GGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVST

[0584] YNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVS

[0585] SGGGGSDVKVTQSSRYLVKRTGEKVFLECVQDAPLSKMFWYRQDPGLGLRLIYFSYDVKMKE

[0586] KGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSSDQNSGDPYEQYFGPGTRLTVTEDLKN

[0587] VFPPEVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPAL

[0588] NDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC

[0589] GGGGSASERKSCVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN

[0590] WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISK

[0591] AKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0592] GSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0593] SEQ ID NO:76

[0594] AQLVESGGGLVQPGGSLRLSCAASAYFSFKAMSWVRQAPGKGLEWVGSISPSGGSTYYNDNVL

[0595] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDVGFFDEWGQGTLVTVSS

[0596] SEQ ID NO:77

[0597] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYDASIRDIGVPSRFSGSG

[0598] SGTDFTLTISSLQPEDFATYYCQQYSSYPYTFGQGTKLEIK

[0599] SEQ ID NO: 78

[0600] AQLVESGGGLVQPGGSLRLSCAASAYFSFKAMSWVRQAPGKGLEWVGSISPSGGSTYYNDNVL

[0601] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDVGFFDEWGQGTLVTVSSASTKGPSVFPLAP

[0602] SSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

[0603] QTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQD

[0604] IRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNT

[0605] LPWTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

[0606] VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVERKSCVECPPCPAPEF

[0607] EGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

[0608] STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTK

[0609] NQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF

[0610] SCSVMHEALHNHYTQKSLSLSPGK

[0611] SEQ ID NO: 79

[0612] AQLVESGGGLVQPGGSLRLSCAASAYFSFKAMSWVRQAPGKGLEWVGSISPSGGSTYYNDNVL

[0613] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDVGFFDEWGQGTLVTVSSASTKGPSVFPLAP

[0614] SSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

[0615] QTYICNVNHKPSNTKVDEKVERKSCVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVV

[0616] VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

[0617] NKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN

[0618] NYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0619] SEQ ID NO: 80

[0620] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYDASIRDIGVPSRFSGSG

[0621] SGTDFTLTISSLQPEDFATYYCQQYSSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVV

[0622] CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

[0623] VTHQGLSSPVTKSFNRGEC

[0624] SEQ ID NO: 81

[0625] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ

[0626] KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSAS

[0627] VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST

[0628] YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0629] SEQ ID NO: 82

[0630] AVYDGREHTV

[0631] SEQ ID NO: 83

[0632] GAYDGREHTV

[0633] SEQ ID NO: 84

[0634] GVADGREHTV

[0635] SEQ ID NO: 85

[0636] GVYAGREHTV

[0637] SEQ ID NO: 86

[0638] GVYDAREHTV

[0639] SEQ ID NO: 87

[0640] GVYDGAEHTV

[0641] SEQ ID NO: 88

[0642] GVYDGRAHTV

[0643] SEQ ID NO: 89

[0644] GVYDGREATV

[0645] SEQ ID NO: 90

[0646] GVYDGREHAV

[0647] SEQ ID NO: 91

[0648] GVYDGREHTA

[0649] SEQ ID NO: 92

[0650] ASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0651] SEQ ID NO: 93

[0652] ASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0653] SEQ ID NO: 94

[0654] MLLGQKSQRYKAEEGLQAQGEAPGLMDVQIPTAEEQKAASSSSTLIMGTLEEVTDSGSPSPPQSPEGASSSLTVTDSTLWSQSDEGSSSNEEEGPSTSPDPAHLESLFREALDEKVAELVRFLLRKYQIKEPVTKAEMLESVIKNYKNHFPDIFSKASECMQVIFGIDVKEVDPAGHSYILVTCLGLSYDGLLGDDQSTPKTGLLIIVLGMILMEGSRAPEEAIWEALSVMGLYDGREHSVYWKLRKLLTQEWVQENYLEYRQAPGSDPVRYEFLWGPRALAETSYVKVLEHVVRVNARVRISYPSLHEEALGEEKGV

[0655] [1] Plaen, E. D., Arden, K., Traversari et al., C, et al. (1994) Structure, chromosomal localization, and expression of 12 genes of the MAGE family. Immunogenetics 40(5), 360-369.

[0656] [2] Rogner, U. C, Wilke, K., Steck, E., et al. (1994) The melanoma antigen gene (MAGE) family is clustered in the chromosomal band Xq28. Genomics 29(3), 725-731.

[0657] [3] Hao, Y. H., Doyle, J. M., Ramanathan, S., et al. (2013) Regulation of WASH-dependent actin polymerization and protein trafficking by ubiquitination. Cell 152, 1051-1064.

[0658] [4]. Chomez, P., De, B. O., Bertrand, M., et al. (2001). An overview of the mage gene family with the identification of all human members of the family. Cancer Research 61(14), 5544-5551.

[0659] [5] Simpson, A., Caballero, O. L., Jungbluth, A., et al. (2005) Cancer / testis antigens, gametogenesis and cancer. Nature Reviews Cancer 5(8), 615.

[0660] [6] Sharma, P., Shen, Y., Wen, S., et al. (2006) Cancer-testis antigens: expression and correlation with survival in human urothelial carcinoma. Clinical Cancer Research an Official Journal of the American Association for Cancer Research 12(18), 5442-5447.

[0661] [7] Bergeron, A., Valérie, P., Hélène, L., et al. (2010) High frequency of mage-a4 and mage-a9 expression in high-risk bladder cancer. International Journal of Cancer 125(6), 1365-1371.

[0662] [8] Peikert, T., Specks, U., Farver, C., et al., (2006) Melanoma antigen A4 is expressed in non-small cell lung cancers and promotes apoptosis. Cancer Research 66(9), 4693-4700.

[0663] [9] Yakirevich, E., Sabo, E., Lavie, O., et al. (2003) Expression of the MAGE-A4 and NY-ESO-1 cancer-testis antigens in serous ovarian neoplasms. Clinical Cancer Research an Official Journal of the American Association for Cancer Research 9(17), 6453-6460.

[0664]

[10] Quillien, V., Raoul, J.L., Heresbach, D., et al. (1997) Expression of MAGE genes in esophageal squamous-cell carcinoma. Anticancer Research 17(1a), 387-391.

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[11] Ries, J., Schultze-Mosgau, S., Neukam, F., et al. (2005) Investigation of the expression of melanoma antigen-encoding genes (MAGE-A1 to-A6) in oral squamous cell carcinomas to determine potential targets for gene-based cancer immunotherapy. International Journal of Oncology 26(3), 817-824.

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[12] M.T. Duffour, P. Chaux, C. Lurquin, G., et al. (1999) A MAGE-A4 peptide presented by HLA-A02 is recognized by cytolytic T lymphocytes. European Journal of Immunology 29(10), 3329-3337.

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[13] . Sanderson, J. P., Crowley, D. J., Wiedermann, G. E., et al. (2019) Preclinical evaluation of an affinity-enhanced MAGE-A4-specific T-cell receptor for adop-tive T-cell therapy. Oncoimmunology 9, 1682381.

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[14] Ottaviani, S., Colau, D., Bruggen, P. V. D., et al. (2006) A new mage-4 antigenic peptide recognized by cytolytic lymphocytes on HLA-A024 carcinoma cells. Cancer Immunology Immunotherapy 55(7), 867-872.

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[16] Kotsiou, E., Brzostek, J., Lenart, I., et al. (2010) Dimerization of soluble disulfide trap single-chain major histocompatibility complex class I molecules dependent on peptide binding affinity. Antioxid Redox Signal 15(3):635-644.

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[21] WO 2017175006A1

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[24] WO 2021122875A1

Claims

1. A bispecific antibody comprising a first antigen-binding fragment that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second antigen-binding fragment that binds to an activating T cell antigen; wherein the first antigen-binding fragment comprises a HCDR1 as set forth in SEQ ID NO: 32, a HCDR2 as set forth in SEQ ID NO: 33, and a HCDR3 as set forth in SEQ ID NO: 34; wherein the second antigen-binding fragment comprises a HCDR1 as set forth in SEQ ID NO: 37, a HCDR2 as set forth in SEQ ID NO: 38, a HCDR3 as set forth in SEQ ID NO: 39, a LCDR1 as set forth in SEQ ID NO: 40, a LCDR2 as set forth in SEQ ID NO: 41, and a LCDR3 as set forth in SEQ ID NO: 42; and wherein the amino acid sequences of the HCDRs and LCDRs are defined according to Kabat.

2. The bispecific antibody of claim 1, wherein the binding epitope of the first antigen-binding fragment to the HLA-A02 / MAGE-A4 complex comprises positions 230-239 of MAGE-A4 set forth with reference to SEQ ID NO:

48.

3. The bispecific antibody of claim 2, wherein the binding epitope of the first antigen-binding fragment to the HLA-A02 / MAGE-A4 complex comprises at least one of residues 232, 234, 235, 237, and 239 of MAGE-A4 set forth with reference to SEQ ID NO:

48.

4. The bispecific antibody of claim 1, wherein the binding epitope of the first antigen-binding fragment to the HLA-A02 / MAGE-A8 complex comprises positions 232-241 of MAGE-A8 set forth with reference to SEQ ID NO:

94.

5. The bispecific antibody of claim 1, wherein the activating T cell antigen is a CD3 molecule.

6. The bispecific antibody of any one of claims 1-5, wherein the first antigen-binding fragment is in the form of a single domain antibody.

7. The bispecific antibody of claim 6, wherein the first antigen-binding fragment comprises a monovalent or multivalent single domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex.

8. The bispecific antibody of any one of claims 1-5, wherein the second antigen-binding fragment is a single chain antibody (scFv) or a Fab fragment.

9. The bispecific antibody of claim 8, wherein the second antigen-binding fragment is a scFv.

10. The bispecific antibody of any one of claims 1-5, 7, and 9, wherein the first antigen-binding fragment comprises a bivalent single domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex.

11. The bispecific antibody of claim 6, wherein the first antigen-binding fragment comprises a bivalent single-domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex.

12. The bispecific antibody of claim 8, wherein the first antigen-binding fragment comprises a bivalent single-domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex.

13. The bispecific antibody of claim 10, wherein the first antigen-binding fragment comprises two heavy chain variable regions of a monovalent single-domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex.

14. The bispecific antibody of claim 11 or 12, wherein the first antigen-binding fragment comprises two heavy chain variable regions of a monovalent single-domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex.

15. The bispecific antibody of claim 13, wherein the first antigen-binding fragment comprises two heavy chain variable regions of a monovalent single-domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex connected by a linker.

16. The bispecific antibody of claim 14, wherein the first antigen-binding fragment comprises two heavy chain variable regions of a monovalent single-domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex connected by a linker.

17. The bispecific antibody of claim 15 or 16, wherein the linker is (GGGGS) n wherein n is an integer > 1.

18. The bispecific antibody of claim 17, wherein the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 49).

19. The bispecific antibody of any one of claims 1-5, 7, 9, 11-13, 15, 16, and 18, wherein the first antigen-binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 35, 36, 71, or 72; and / or the second antigen-binding fragment comprises a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 43 and a light chain variable region of an amino acid sequence as set forth in SEQ ID NO:

44.

20. The bispecific antibody of claim 6, wherein the first antigen-binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 35, 36, 71, or 72; and / or the second antigen-binding fragment comprises a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 43 and a light chain variable region of an amino acid sequence as set forth in SEQ ID NO:

44.

21. The bispecific antibody of claim 8, wherein the first antigen-binding fragment comprises an amino acid sequence as set forth in SEQ ID NO: 35, 36, 71, or 72; and / or the second antigen-binding fragment comprises a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 43 and a light chain variable region of an amino acid sequence as set forth in SEQ ID NO:

44. ​ ​ ​ ​ ​ the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO:

44.

22. The bispecific antibody of claim 10, wherein the first antigen binding fragment comprises an amino acid sequence set forth in SEQ ID NO: 35, 36, 71, or 72; and / or the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO:

44.

23. The bispecific antibody of claim 14, wherein the first antigen binding fragment comprises an amino acid sequence set forth in SEQ ID NO: 35, 36, 71, or 72; and / or the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO:

44.

24. The bispecific antibody of claim 17, wherein the first antigen binding fragment comprises an amino acid sequence set forth in SEQ ID NO: 35, 36, 71, or 72; and / or the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO:

44.

25. The bispecific antibody of claim 19, wherein the first antigen binding fragment comprises an amino acid sequence set forth in SEQ ID NO: 35; and the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO:44; the first antigen binding fragment comprises an amino acid sequence set forth in SEQ ID NO: 36; and the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO:44; or the first antigen binding fragment comprises an amino acid sequence set forth in SEQ ID NO: 71; and the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO:44; or the first antigen binding fragment comprises an amino acid sequence set forth in SEQ ID NO: 72; and the second antigen binding fragment comprises a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:43 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO:

44.

26. The bispecific antibody of any one of claims 20-24, wherein the first antigen binding fragment comprises an amino acid sequence of SEQ ID NO: 35; and the second antigen binding fragment comprises a heavy chain variable region of an amino acid sequence of SEQ ID NO: 43 and a light chain variable region of an amino acid sequence of SEQ ID NO: 44; the first antigen binding fragment comprises an amino acid sequence of SEQ ID NO: 36; and the second antigen binding fragment comprises a heavy chain variable region of an amino acid sequence of SEQ ID NO: 43 and a light chain variable region of an amino acid sequence of SEQ ID NO: 44; or the first antigen binding fragment comprises an amino acid sequence of SEQ ID NO: 71; and the second antigen binding fragment comprises a heavy chain variable region of an amino acid sequence of SEQ ID NO: 43 and a light chain variable region of an amino acid sequence of SEQ ID NO: 44; or the first antigen binding fragment comprises an amino acid sequence of SEQ ID NO: 72; and the second antigen binding fragment comprises a heavy chain variable region of an amino acid sequence of SEQ ID NO: 43 and a light chain variable region of an amino acid sequence of SEQ ID NO:

44.

27. The bispecific antibody of any one of claims 1-5, 7, 9, 11-13, 15, 16, 18, and 20-25, wherein the first antigen binding fragment and the second antigen binding fragment are connected by an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment; wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the first Fc fragment are C, S, A, and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the second Fc fragment are C, S, A, and V, respectively; and / or the amino acids at positions 234, 235, and 331 of the first Fc fragment and the second Fc fragment are F, E, and S, respectively; and / or one of the first Fc fragment and the second Fc fragment is connected to the first antigen binding fragment, and the other of the first Fc fragment and the second Fc fragment is connected to the second antigen binding fragment; wherein, the antibody constant region amino acid positions are determined according to EU numbering.

28. The bispecific antibody of claim 6, wherein the first antigen binding fragment and the second antigen binding fragment are connected by an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment; wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the first Fc fragment are C, S, A, and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the second Fc fragment are C, S, A, and V, respectively; and / or the amino acids at positions 234, 235, and 331 of the first Fc fragment and the second Fc fragment are F, E, and S, respectively; and / or one of the first Fc fragment and the second Fc fragment is connected to the first antigen binding fragment, and the other of the first Fc fragment and the second Fc fragment is connected to the second antigen binding fragment; the antibody constant region amino acid positions are determined according to EU numbering. the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the first Fc fragment are C, S, A, and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the second Fc fragment are C, S, A, and V, respectively; and / or the amino acids at positions 234, 235, and 331 of the first Fc fragment and the second Fc fragment are F, E, and S, respectively; and / or one of the first Fc fragment and the second Fc fragment is linked to the first antigen binding fragment, and the other of the first Fc fragment and the second Fc fragment is linked to the second antigen binding fragment; wherein amino acid positions of antibody constant regions are determined according to EU numbering.

29. The bispecific antibody of claim 8, wherein the first antigen binding fragment and the second antigen binding fragment are linked by an antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment; wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the first Fc fragment are C, S, A, and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the second Fc fragment are C, S, A, and V, respectively; and / or the amino acids at positions 234, 235, and 331 of the first Fc fragment and the second Fc fragment are F, E, and S, respectively; and / or one of the first Fc fragment and the second Fc fragment is linked to the first antigen binding fragment, and the other of the first Fc fragment and the second Fc fragment is linked to the second antigen binding fragment; wherein amino acid positions of antibody constant regions are determined according to EU numbering.

30. The bispecific antibody of claim 10, wherein the first antigen binding fragment and the second antigen binding fragment are linked by an antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment; wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the first Fc fragment are C, S, A, and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the second Fc fragment are C, S, A, and V, respectively; and / or the amino acids at positions 234, 235, and 331 of the first Fc fragment and the second Fc fragment are F, E, and S, respectively; and / or one of the first Fc fragment and the second Fc fragment is linked to the first antigen binding fragment, and the other of the first Fc fragment and the second Fc fragment is linked to the second antigen binding fragment; amino acid positions of antibody constant regions are determined according to EU numbering. one of the first and second Fc fragments is connected to the first antigen binding fragment and the other of the first and second Fc fragments is connected to the second antigen binding fragment; wherein, the antibody constant region amino acid positions are determined according to EU numbering.

31. The bispecific antibody of claim 14, wherein the first and second antigen binding fragments are connected by an antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment; wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the first Fc fragment are C, S, A, and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the second Fc fragment are C, S, A, and V, respectively; and / or the amino acids at positions 234, 235, and 331 of the first and second Fc fragments are F, E, and S, respectively; and / or one of the first and second Fc fragments is connected to the first antigen binding fragment and the other of the first and second Fc fragments is connected to the second antigen binding fragment; wherein, the antibody constant region amino acid positions are determined according to EU numbering.

32. The bispecific antibody of claim 17, wherein the first and second antigen binding fragments are connected by an antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment; wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the first Fc fragment are C, S, A, and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the second Fc fragment are C, S, A, and V, respectively; and / or the amino acids at positions 234, 235, and 331 of the first and second Fc fragments are F, E, and S, respectively; and / or one of the first and second Fc fragments is connected to the first antigen binding fragment and the other of the first and second Fc fragments is connected to the second antigen binding fragment; wherein the antibody constant region amino acid positions are determined according to EU numbering.

33. The bispecific antibody of claim 19, wherein the first and second antigen binding fragments are connected by an antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment; wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the first Fc fragment are C, S, A, and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the second Fc fragment are C, S, A, and V, respectively; and / or the amino acids at positions 234, 235, and 331 of the first and second Fc fragments are F, E, and S, respectively; and / or one of the first and second Fc fragments is connected to the first antigen binding fragment and the other of the first and second Fc fragments is connected to the second antigen binding fragment; the antibody constant region amino acid positions are determined according to EU numbering. the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the first Fc fragment are C, S, A, and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the second Fc fragment are C, S, A, and V, respectively; and / or the amino acids at positions 234, 235, and 331 of the first Fc fragment and the second Fc fragment are F, E, and S, respectively; and / or one of the first Fc fragment and the second Fc fragment is linked to the first antigen binding fragment, and the other of the first Fc fragment and the second Fc fragment is linked to the second antigen binding fragment; wherein amino acid positions of antibody constant regions are determined according to EU numbering.

34. The bispecific antibody of claim 26, wherein the first antigen binding fragment and the second antigen binding fragment are linked by an antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment; wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the first Fc fragment are C, S, A, and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368, and 407 of the second Fc fragment are C, S, A, and V, respectively; and / or the amino acids at positions 234, 235, and 331 of the first Fc fragment and the second Fc fragment are F, E, and S, respectively; and / or one of the first Fc fragment and the second Fc fragment is linked to the first antigen binding fragment, and the other of the first Fc fragment and the second Fc fragment is linked to the second antigen binding fragment; wherein amino acid positions of antibody constant regions are determined according to EU numbering.

35. The bispecific antibody of claim 27, wherein the antibody heavy chain constant region Fc fragment is an Fc fragment of IgG1 subtype.

36. The bispecific antibody of any one of claims 28-34, wherein the antibody heavy chain constant region Fc fragment is an Fc fragment of IgG1 subtype.

37. The bispecific antibody of claim 35, wherein the antibody heavy chain constant region Fc fragment is an Fc fragment of IgG1 m3 subtype.

38. The bispecific antibody of claim 36, wherein the antibody heavy chain constant region Fc fragment is an Fc fragment of IgG1 m3 subtype.

39. The bispecific antibody of claim 27, wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, and the amino acids at positions 349, 366, 368, and 407 of the second Fc fragment are C, S, A, and V, respectively.

40. The bispecific antibody of any one of claims 28-34, wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, and the amino acids at positions 349, 366, 368, and 407 of the second Fc fragment are C, S, A, and V, respectively.

41. The bispecific antibody of any one of claims 1-5, 7, 9, 11-13, 15, 16, 18, 20-25, 28-35, and 37-39, comprising a first arm that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and the second arm comprises an amino acid sequence as set forth in SEQ ID NO:

46.

42. The bispecific antibody of claim 6, comprising a first arm that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and the second arm comprises an amino acid sequence as set forth in SEQ ID NO:

46.

43. The bispecific antibody of claim 8, comprising a first arm that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and the second arm comprises an amino acid sequence as set forth in SEQ ID NO:

46.

44. The bispecific antibody of claim 10, comprising a first arm that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and the second arm comprises an amino acid sequence as set forth in SEQ ID NO:

46.

45. The bispecific antibody of claim 14, comprising a first arm that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and the second arm comprises an amino acid sequence as set forth in SEQ ID NO:

46.

46. The bispecific antibody of claim 17, comprising a first arm that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and the second arm comprises an amino acid sequence as set forth in SEQ ID NO:

46. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and the second arm comprises an amino acid sequence as set forth in SEQ ID NO:

46.

47. The bispecific antibody of claim 19, comprising a first arm that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and the second arm comprises an amino acid sequence as set forth in SEQ ID NO:

46.

48. The bispecific antibody of claim 26, comprising a first arm that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and the second arm comprises an amino acid sequence as set forth in SEQ ID NO:

46.

49. The bispecific antibody of claim 27, comprising a first arm that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and the second arm comprises an amino acid sequence as set forth in SEQ ID NO:

46.

50. The bispecific antibody of claim 36, comprising a first arm that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and the second arm comprises an amino acid sequence as set forth in SEQ ID NO:

46.

51. The bispecific antibody of claim 40, comprising a first arm that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein the first arm comprises an amino acid sequence as set forth in SEQ ID NO: 45 or 74; and the second arm comprises an amino acid sequence as set forth in SEQ ID NO:

46.

52. A single domain antibody that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex, comprising a HCDR1 as set forth in SEQ ID NO: 32, a HCDR2 as set forth in SEQ ID NO: 33, and a HCDR3 as set forth in SEQ ID NO: 34; wherein the amino acid sequences of the HCDRs are according to the Kabat definition.

53. The single-domain antibody of claim 52, wherein the single-domain antibody comprises an amino acid sequence as set forth in SEQ ID NO: 35, 36, 71, or 72.

54. A nucleic acid molecule encoding the bispecific antibody of any one of claims 1-51 or the single-domain antibody of any one of claims 52-53.

55. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-51 or the single-domain antibody of any one of claims 52-53 and a pharmaceutically acceptable excipient, diluent, or carrier.

56. Use of the bispecific antibody of any one of claims 1-51, the single-domain antibody of any one of claims 52-53, or the pharmaceutical composition of claim 55 in the manufacture of a medicament for the prevention or treatment of an HLA-A02 / MAGE-A4 positive tumor and / or an HLA-A02 / MAGE-A8 positive tumor.

57. The use of claim 56, wherein the HLA-A02 / MAGE-A4 positive tumor and / or HLA-A02 / MAGE-A8 positive tumor is selected from the group consisting of esophageal cancer, urothelial cancer, oral cancer, sarcoma, head and neck cancer, lung cancer, liver cancer, bladder cancer, melanoma, ovarian cancer, colorectal cancer, and breast cancer.

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