Multiple antibodies that bind to HLA-A02 / MAGE-A4 complex and uses thereof

Abstract

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 activated T cell antigen, a single domain antibody that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex, and a method for preparing the same. The invention also relates to a pharmaceutical composition containing the bispecific antibody or the single-domain antibody and application of the bispecific antibody or the single-domain antibody.

Description

Field of the Invention

[0001] The present invention generally relates to the fields of genetic engineering and biomedicine; in particular, the present application relates to bispecific antibodies that bind to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex, single domain antibodies that bind to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex, and their uses. Background of the Invention

[0003] Melanoma-associated antigen A (MAGE-A), which belongs to the type I MAGE family, is an important member of the cancer-testis-associated antigen [1] The MAGE-A gene family is located in the q28 region of chromosome X and has 12 members (MAGE-A1 to MAGE-A12). [2] All MAGE-A members have the same MAGE homology domain (MHD), which is a highly conserved domain consisting of approximately 170 amino acids with high sequence homology among them. [3] .

[0004] Melanoma-associated antigen 4 protein (MAGE-A4) is a member of the MAGE-A gene family. MAGE-A4 is localized in the cytoplasm; some studies have found that MAGE-A4 is also detected in the nucleus.

[0005] The function of MAGE-A4 is still unclear, but MAGE-A4 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 prognosis of patients.

[0006] The expression pattern of MAGE-A4 protein is consistent with that of MAGE-A family proteins, with restricted expression in local testicular tissue, partial expression in ovarian and placental tissues, and abnormally high expression in cancer [4,5] Through clinical histological identification, MAGE-A4 was detected in many cancers, including urothelial carcinoma, bladder cancer, lung cancer, ovarian cancer, esophageal squamous cell carcinoma, oral squamous cell carcinoma, etc. [6-11] .

[0007] As an intracellular protein, MAGE-A4 can be degraded in the proteasome and, after processing, presented on the cell surface through the major histocompatibility complex I (MHC I), and then recognized by the T cell receptor (TCR) as a T cell antigen and induces an immune response, such as the MAGE-A4-derived peptide MAGE-A4 230-239(GVYDGREHTV) and HLA-A02 are presented on the cell surface in the form of a complex, triggering T cell recognition and specific killing of tumor cells in a manner dependent on the major histocompatibility complex (MHC).

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

[0008] Bispecific antibodies (BsAb) that simultaneously bind to CD3 on the surface of T cells and antigens on the surface of target cells (e.g., tumor cells) establish an interaction between target cells and T cells, causing cytotoxic T cell activation and lysing target cells in a non-histocompatibility complex (MHC)-dependent manner. This type of bispecific antibody presents 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 simulate TCR and recognize the MHC complex of tumor-specific antigen peptides on the cell surface. TCRm that binds to intracellular proteins of the MAGE-A4 class can be used as the part of CD3 bispecific antibodies that bind to target antigens. This type of bispecific antibody may be more attractive due to the restriction of target antigen expression.

[0009] In 2020, there were 19.29 million new cancer cases worldwide, of which 4.57 million were in China, accounting for 23.7% of the world's total. In 2020, there were 9.96 million cancer deaths worldwide.

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

[15] Most of these cancers do not have good treatments, so new treatment strategies and drugs are urgently needed. MAGE-A4 is expressed as an intracellular antigen in a variety of solid tumors and may become a new target for solid tumor treatment.

[0010] Based on clinical needs, exploring and developing bispecific antibodies targeting MAGE-A4-MHC has important clinical significance. SUMMARY OF THE INVENTION

[0012] 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.

[0013] In some embodiments of the first aspect, the bispecific antibody is capable of mediating HLA-A02 + / MAGE-A4 + Tumor cells activate T cells, and / or the bispecific antibody can mediate PBMC killing of HLA-A02 + / MAGE-A4 + Tumor cells.

[0014] 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 the HCDR is according to the Kabat definition.

[0015] In some embodiments of the first aspect, the first antigen-binding fragment is in the form of a single domain antibody.

[0016] 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.

[0017] In some embodiments of the first aspect, the second antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:37, HCDR2 as shown in SEQ ID NO:38, HCDR3 as shown in SEQ ID NO:39, LCDR1 as shown in SEQ ID NO:40, LCDR2 as shown in SEQ ID NO:41, and LCDR3 as shown in SEQ ID NO:42; wherein the amino acid sequence of the HCDR is according to the Kabat definition.

[0018] In some embodiments of the first aspect, the second antigen-binding fragment is a single-chain antibody (scFv) or a Fab fragment.

[0019] 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.

[0020] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 35, 36, 71 or 72; and / or

[0021] The second antigen-binding fragment comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:43 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:44.

[0022] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:35; and the second antigen-binding fragment comprises a heavy chain variable region with an amino acid sequence shown in SEQ ID NO:43 and a light chain variable region with an amino acid sequence shown in SEQ ID NO:44;

[0023] The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:36; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO:43 and the light chain variable region with the amino acid sequence shown in SEQ ID NO:44;

[0024] The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 71; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 43 and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 44; or

[0025] The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:72; and the second antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO:43 and the light chain variable region shown in SEQ ID NO:44.

[0026] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, and the antibody heavy chain constant region Fc fragment includes 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 amino acid positions of the antibody constant region are determined according to EU numbering.

[0027] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, and the antibody heavy chain constant region Fc fragment includes a first Fc fragment and a second Fc fragment, wherein the 234th, 235th and 331st amino acids 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.

[0028] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, which includes 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.

[0029] In some embodiments of the first aspect, the bispecific antibody comprises 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

[0030] The first arm comprises the amino acid sequence shown in SEQ ID NO: 45 or 74; and

[0031] The second arm comprises the amino acid sequence shown in SEQ ID NO:46.

[0032] In a second aspect, the present application provides a single domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex, comprising

[0033] HCDR1 as shown in SEQ ID NO:32,

[0034] HCDR2 as shown in SEQ ID NO:33, and

[0035] HCDR3 as shown in SEQ ID NO:34;

[0036] The amino acid sequence of HCDR is based on the Kabat definition.

[0037] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO: 35, 36, 71 or 72.

[0038] In a third aspect, the present application provides a nucleic acid molecule encoding the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect.

[0039] In a fourth aspect, the present application provides a pharmaceutical composition comprising the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect and a pharmaceutically acceptable excipient, diluent or carrier.

[0040] In a fifth aspect, the present application provides 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 preventing or treating HLA-A02 / MAGE-A4-positive tumors and / or HLA-A02 / MAGE-A8-positive tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The results of ELISA detection of the binding of the anti-HLA-A02 / MAGE-A4 complex TCRm antibody N18D10-Fc to the recombinant protein MHC-P1 are shown.

[0043] Figure 2 The TCRm antibody N18D10-Fc against the HLA-A02 / MAGE-A4 complex and the antigen peptide MAGE-A4 are shown 230-239 Results of pulsed T2 cell binding.

[0044] Figure 3 The TCRm antibody N18D10 against the HLA-A02 / MAGE-A4 complex and MAGE-A4 230-239 Results of T2 cell binding pulsed with alanine scanning mutant peptide (GVYDGREHTV).

[0045] Figure 4 The results of ELISA detection of TCRm antibody N18D10-h16-Fc binding to recombinant protein MHC-P1 are shown.

[0046] Figure 5 The results of ELISA detection of TCRm×CD3 bispecific antibody binding to MHC-P1 and CD3 simultaneously are shown.

[0047] Figure 6 The results of TCRm×CD3 bispecific antibody-mediated activation of Jurkat-Dual cells by antigen peptide-pulsed T2 cells are shown.

[0048] Figure 7 TCRm×CD3 bispecific antibody mediated HLA-A02 + / MAGE-A4 + The result of tumor cells activating Jurkat-Dual cells.

[0049] Figure 8The results of TCRm×CD3 bispecific antibody-mediated PBMC cell killing of antigen peptide-pulsed T2 cells are shown.

[0050] Fig. 9 The results of TCRm×CD3 bispecific antibody-mediated PBMC cell killing of A375 cells are shown.

[0051] Fig.10 The results of TCRm×CD3 bispecific antibody-mediated PBMC cell killing of multiple antigen-positive tumor cells are shown.

[0052] Fig.11 TCRm×CD3 bispecific antibody mediated MAGE-A4 230-239 (GVYDGREHTV) Results of activation of Jurkat-Dual cells by T2 cells pulsed with alanine scanning peptide.

[0053] Fig.12 The results of TCRm×CD3 bispecific antibody-mediated activation of Jurkat-Dual cells by T2 cells pulsed with similar peptides are shown.

[0054] Fig.13 The results showed that TCRm×CD3 bispecific antibody did not mediate PBMC killing of antigen-negative tumor cells.

[0055] Fig.14 The results showing that TCRm×CD3 bispecific antibody inhibited the growth of A375 tumor cells in hPBMC immune reconstituted mice.

[0056] Sequence Description

[0057] SEQ ID NO: 1 shows an antigen peptide MAGE-A4 derived from homo sapiens 230-239 The amino acid sequence of

[0058] SEQ ID NO: 2 shows MAGE-A4-S1 (MAGE-A4 230-239 Amino acid sequence of similar peptide 1).

[0059] SEQ ID NO: 3 shows the amino acid sequence of MAGE-A4-S2 derived from homo sapiens.

[0060] SEQ ID NO: 4 shows the amino acid sequence of MAGE-A4-S3 derived from homo sapiens.

[0061] SEQ ID NO: 5 shows the amino acid sequence of MAGE-A4-S4 derived from homo sapiens.

[0062] SEQ ID NO: 6 shows the amino acid sequence of MAGE-A4-S5 derived from homo sapiens.

[0063] SEQ ID NO: 7 shows the amino acid sequence of MAGE-A4-S6 derived from homo sapiens.

[0064] SEQ ID NO: 8 shows the amino acid sequence of MAGE-A4-S7 derived from homo sapiens.

[0065] SEQ ID NO: 9 shows the amino acid sequence of MAGE-A4-S8 derived from homo sapiens.

[0066] SEQ ID NO: 10 shows the amino acid sequence of MAGE-A4-S9 derived from homo sapiens.

[0067] SEQ ID NO: 11 shows the amino acid sequence of MAGE-A4-S10 derived from homo sapiens.

[0068] SEQ ID NO: 12 shows the amino acid sequence of MAGE-A4-S11 derived from homo sapiens.

[0069] SEQ ID NO: 13 shows the amino acid sequence of MAGE-A4-S12 derived from homo sapiens.

[0070] SEQ ID NO: 14 shows the amino acid sequence of MAGE-A4-S13 derived from homo sapiens.

[0071] SEQ ID NO: 15 shows the amino acid sequence of MAGE-A4-S14 derived from homo sapiens.

[0072] SEQ ID NO: 16 shows the amino acid sequence of MAGE-A4-S15 derived from homo sapiens.

[0073] SEQ ID NO: 17 shows the amino acid sequence of MAGE-A4-S16 derived from homo sapiens.

[0074] SEQ ID NO: 18 shows the amino acid sequence of MAGE-A4-S17 derived from homo sapiens.

[0075] SEQ ID NO: 19 shows the amino acid sequence of MAGE-A4-S18 derived from homo sapiens.

[0076] SEQ ID NO: 20 shows the amino acid sequence of MAGE-A4-S19 derived from homo sapiens.

[0077] SEQ ID NO: 21 shows the amino acid sequence of MAGE-A4-S20 derived from homo sapiens.

[0078] SEQ ID NO: 22 shows the amino acid sequence of MAGE-A4-S21 derived from homo sapiens.

[0079] SEQ ID NO: 23 shows the amino acid sequence of MAGE-A4-S22 derived from homo sapiens.

[0080] SEQ ID NO: 24 shows the amino acid sequence of MAGE-A4-S23 derived from homo sapiens.

[0081] SEQ ID NO: 25 shows the amino acid sequence of MAGE-A4-S24 derived from homo sapiens.

[0082] SEQ ID NO: 26 shows the amino acid sequence of MAGE-A4-S25 derived from homo sapiens.

[0083] SEQ ID NO: 27 shows the amino acid sequence of MAGE-A4-S26 derived from homo sapiens.

[0084] SEQ ID NO: 28 shows the amino acid sequence of MAGE-A4-S27 derived from homo sapiens.

[0085] SEQ ID NO: 29 shows the amino acid sequence of MAGE-A4-S28 derived from homo sapiens.

[0086] SEQ ID NO: 30 shows the amino acid sequence of MAGE-A4-S29 derived from homo sapiens.

[0087] SEQ ID NO: 31 shows the amino acid sequence of MAGE-A4-S30 derived from homo sapiens.

[0088] SEQ ID NOs: 32-34 respectively show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable regions of camel (Camelus) single domain antibodies N18D10 and N18D10-h16.

[0089] SEQ ID NO: 35 shows the amino acid sequence of the heavy chain variable region of the camel (Camelus) single domain antibody N18D10.

[0090] SEQ ID NO: 36 shows the amino acid sequence of the heavy chain variable region of the humanized single domain antibody N18D10-h16.

[0091] SEQ ID NOs: 37-39 respectively show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the anti-human CD3 antibody IMCR.

[0092] SEQ ID NOs: 40-42 respectively show the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of the anti-human CD3 antibody IMCR.

[0093] SEQ ID NO: 43 shows the amino acid sequence of the heavy chain variable region of the anti-human CD3 antibody IMCR.

[0094] SEQ ID NO: 44 shows the amino acid sequence of the light chain variable region of the anti-human CD3 antibody IMCR.

[0095] SEQ ID NO: 45 shows the amino acid sequence of arm 2N18D10-h16-G1m3-FcHIn1 in the bispecific antibody 2N18D10-h16+IMCR.

[0096] SEQ ID NO: 46 shows the amino acid sequence of the arm IMCR-anti-CD3-scFv-G1m3-FcKn1 in the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR.

[0097] SEQ ID NO: 47 shows the amino acid sequence of the HLA-A02 complex (MHC-P1) of GVYDGREHTV.

[0098] SEQ ID NO: 48 shows the amino acid sequence of human (homo sapiens) MAGE-A4.

[0099] SEQ ID NO:49 shows the amino acid sequence of the linker.

[0100] SEQ ID NO:50 shows the nucleotide sequence of primer PCal-CH2R.

[0101] SEQ ID NO:51 shows the amino acid sequence of the human (homo sapiens) CD3E extracellular region (hCD3E).

[0102] SEQ ID NO: 52 shows the amino acid sequence of the extracellular region of human (homo sapiens) CD3D (hCD3D).

[0103] SEQ ID NO:53 shows the amino acid sequence of the His tag.

[0104] SEQ ID NO: 54 shows the amino acid sequence of the Fc region (hFc) of human (homo sapiens) IgG1 antibody.

[0105] SEQ ID NO:55 shows the amino acid sequence of the Fc region (mFc) of mouse (mus musculus) IgG2a antibody.

[0106] SEQ ID NO: 56 shows the amino acid sequence of the human (homo sapiens) IgG1 subtype heavy chain constant region.

[0107] SEQ ID NO: 57 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1H.

[0108] SEQ ID NO: 58 shows the amino acid sequence of human IgG1 subtype antibody heavy chain constant region mutant IgG1K.

[0109] SEQ ID NO: 59 shows the amino acid sequence of human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-H.

[0110] SEQ ID NO: 60 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-K.

[0111] SEQ ID NO: 61 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-H1n1.

[0112] SEQ ID NO: 62 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-Kn1.

[0113] SEQ ID NO: 63 shows the amino acid sequence of the human (homo sapiens) lambda subtype light chain constant region.

[0114] SEQ ID NO: 64 shows the amino acid sequence of the human (homo sapiens) kappa subtype light chain constant region

[0115] SEQ ID NO: 65 shows the amino acid sequence of the heavy chain variable region (VH) of the germline gene antibody DP47.

[0116] SEQ ID NO: 66 shows the amino acid sequence of the light chain variable region (VK) of the germline gene antibody DP47.

[0117] SEQ ID NO:67 shows the amino acid sequence of the α chain variable region (Vα) of a control TCR that binds to HLA-A02 / MAGE-4.

[0118] SEQ ID NO:68 shows the amino acid sequence of the variable region of the beta chain (Vβ) of a control TCR that binds to HLA-A02 / MAGE-4.

[0119] SEQ ID NO: 69 shows the amino acid sequence of the first binding arm TCRA-Des-G1m3-FcKn1 of the control IMC-C103C bifunctional molecule.

[0120] SEQ ID NO:70 shows the amino acid sequence of TCRB-Des-G1m3-FcH1n1 in the control TCR.

[0121] SEQ ID NO:71 shows the amino acid sequence of the heavy chain variable regions of two camel single domain antibodies N18D10 concatenated.

[0122] SEQ ID NO:72 shows the amino acid sequence of the heavy chain variable regions of two camel single domain antibodies N18D10-h16 in series.

[0123] SEQ ID NO: 73 shows the amino acid sequence of the single-chain antibody of the anti-human (homo sapiens) CD3 antibody IMCR.

[0124] SEQ ID NO:74 shows the amino acid sequence of 2N18D10-G1m3-FcHIn1 in the bispecific antibody 2N18D10+IMCR.

[0125] 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.

[0126] SEQ ID NO: 76 shows the amino acid sequence of the heavy chain variable region (057G03VH) of the control antibody 057D03 that binds to the HLA-A02 / MAGE-A4 complex.

[0127] SEQ ID NO: 77 shows the amino acid sequence of the light chain variable region (057G03VK) of the control antibody 057D03 that binds to the HLA-A02 / MAGE-A4 complex.

[0128] SEQ ID NO: 78 shows the amino acid sequence of the control anti-HLA-A02 / MAGE-A4 complex and the first component of the CD3 bispecific antibody 057G03, 057G03-Fd-EE-IMCR-anti-CD3VK-G1m3-Kn1.

[0129] SEQ ID NO: 79 shows the amino acid sequence of the second component 057G03-Fd-EE-G1m3-FcH1n1 of the control anti-HLA-A02 / MAGE-A4 complex and CD3 bispecific antibody 057G03.

[0130] SEQ ID NO: 80 shows the amino acid sequence of the third component 057G03VK+CK-RK of the control anti-HLA-A02 / MAGE-A4 complex and CD3 bispecific antibody 057G03.

[0131] SEQ ID NO: 81 shows the amino acid sequence of the control anti-HLA-A02 / MAGE-A4 complex and the fourth component IMCR-anti-CD3 VH-CK of the CD3 bispecific antibody 057G03.

[0132] SEQ ID NO:82 shows MAGE-A4 230-239 Amino acid sequence of the G1 position alanine scanning mutant peptide.

[0133] SEQ ID NO:83 shows MAGE-A4 230-239 Amino acid sequence of the alanine scanning mutant peptide at position V2.

[0134] SEQ ID NO:84 shows MAGE-A4 230-239 Amino acid sequence of the alanine scanning mutation peptide at position Y3.

[0135] SEQ ID NO:85 shows MAGE-A4 230-239 Amino acid sequence of the alanine scanning mutant peptide at position D4.

[0136] SEQ ID NO:86 shows MAGE-A4 230-239 Amino acid sequence of the G5 position alanine scanning mutant peptide.

[0137] SEQ ID NO:87 shows MAGE-A4 230-239 Amino acid sequence of the alanine scanning mutation peptide at position R6.

[0138] SEQ ID NO:88 shows MAGE-A4 230-239 Amino acid sequence of the alanine scanning mutant peptide at position E7.

[0139] SEQ ID NO:89 shows MAGE-A4 230-239 Amino acid sequence of the alanine scanning mutant peptide at position H8.

[0140] SEQ ID NO:90 shows MAGE-A4 230-239 Amino acid sequence of the alanine scanning mutant peptide at position T9.

[0141] SEQ ID NO:91 shows MAGE-A4 230-239 Amino acid sequence of the alanine scanning mutant peptide at position V10.

[0142] SEQ ID NO:92 shows the amino acid sequence of the human IgG1 subtype antibody Fc region mutant IgG1m3-FcH1n1.

[0143] SEQ ID NO:93 shows the amino acid sequence of the human IgG1 subtype antibody Fc region mutant IgG1m3-FcKn1.

[0144] SEQ ID NO:94 shows the amino acid sequence of human (homo sapiens) MAGE-A8. DETAILED DESCRIPTION OF THE INVENTION

[0146] The inventors of the present application prepared a bispecific antibody (e.g., TCRm bispecific antibody) by genetic engineering means by using 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 activated T cell antigen (e.g., CD3 molecule). The first antigen-binding fragment that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex binds to MAGE-A4 presented on the surface of a target cell (tumor cell). 230-239 (GVYDGREHTV) in complex with HLA-A02 and / or MAGE-A8 232-241The second antigen binding fragment binds to the activated T cell antigen (such as CD3 molecule), thereby establishing an interaction between the target cell and the T cell, causing the activation of cytotoxic T cells, and lysing the target cell (such as tumor cell) in a non-histocompatibility complex (MHC) dependent manner, thereby achieving the purpose of treating the disease (such as tumor). In various aspects of the present application, a novel 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 activated T cell antigen (e.g., a CD3 molecule), a single-domain antibody that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex, a nucleic acid molecule encoding the bispecific antibody or the single-domain antibody, a vector comprising the nucleic acid molecule, a host cell comprising the nucleic acid molecule or the vector, a method for preparing and purifying the bispecific antibody or the single-domain antibody, and medical and biological applications of the bispecific antibody or the single-domain antibody are provided. According to the sequence of the bispecific antibody or the single-domain antibody provided in the present application, a bispecific antibody or the single-domain antibody that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex can be constructed as a drug for clinical use in preventing or treating tumors.

[0147] The practice of the present application employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology within the skill of the art.

[0148] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0149] definition

[0150] As used herein, the term "HLA-A02 / MAGE-A4 complex" refers to a complex of an HLA-A02 molecule and MAGE-A4. In a specific embodiment of the present application, "HLA-A02 / MAGE-A4 complex" refers to a complex of an HLA-A02 molecule and positions 230-239 (MAGE-A4) of MAGE-A4 as shown in SEQ ID NO: 48. 230-239 ) complex HLA-A02 / MAGE-A4 230-239 Complex.

[0151] As used herein, the term "HLA-A02 / MAGE-A8 complex" refers to a complex of an HLA-A02 molecule and MAGE-A8. In a specific embodiment of the present application, "HLA-A02 / MAGE-A8 complex" refers to a complex of an HLA-A02 molecule and MAGE-A84 shown in SEQ ID NO: 94 at positions 232-241 (MAGE-A8 232-241 ) complex HLA-A02 / MAGE-A8 232-241 Complex.

[0152] As used herein, the term "activating T cell antigen" refers to an antigenic determinant expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, which can induce T cell activation when interacting with an antigen binding molecule. Specifically, the interaction of an antigen binding molecule with an activating T cell antigen can induce T cell activation by triggering the signal transduction cascade of the T cell receptor complex. In a specific aspect, the activating T cell antigen is CD3.

[0153] The term "antibody" as used herein refers to an immunoglobulin molecule that can specifically bind 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. "Antibodies" as used herein include not only complete (i.e., full-length) antibodies, but also binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, bispecific antibodies, linear antibodies, single-chain antibodies, single-domain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0154] Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and the first, second and third constant regions (CH1, CH2 and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). A full-length antibody can be any type of antibody, such as IgD, IgE, IgG, IgA or IgM (or subclasses thereof), but the antibody need not belong to any particular class. Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the constant region of the heavy chain. Typically, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional structure of different classes of immunoglobulins are well known.

[0155] As used herein, the term "bispecific antibody" refers to an antibody that has the ability to bind to two antigen epitopes at the same time. 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 parts fused thereto respectively (similar to natural antibodies, the difference is that the two arms bind to different antigen targets or epitopes), and the antigen binding part can be a single domain antibody, a single chain antibody (scFv) or a Fab fragment. When targeting the epitopes of two given antigens, the two different binding parts of the bispecific antibody each bind to the N-terminus of an Fc fragment, and the antigen binding part configuration 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 heavy chain heteropolymerization, and the KIH technology (knob-in-hole, KIH) is a strategy to solve heavy chain heteropolymerization. Generally, KIH technology refers to the formation of a structure that is conducive to the pairing of heterologous half 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.

[0156] As used herein, the terms "binding portion" or "binding fragment" are used interchangeably and refer to a portion or region of a complete antibody molecule that is responsible for binding to an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementary determining regions, CDR1, CDR2, and CDR3.

[0157] It is well known to those skilled in the art that the complementarity determining region (CDR, usually CDR1, CDR2 and CDR3) is the region in the variable region that has the greatest impact on the affinity and specificity of the antibody. There are two common ways to define the CDR sequence of VH or VL, namely the Kabat definition and the Chothia definition. (See, for example, Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); A1-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989)). For the variable region sequence of a given antibody, the CDR region sequence in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In the embodiments of the present application, the Kabat definition of CDR sequences is used.

[0158] For a given antibody variable region sequence, the CDR region sequence in the variable region sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0159] For general antibodies, examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which can be monovalent fragments having a VL-CL chain and a VH-CH1 chain; (2) F(ab')2 fragments, which can be bivalent fragments having two Fab' fragments connected by a disulfide bridge in the hinge region (i.e., a dimer of Fab'); (3) Fv fragments having the VL and VH domains of a single arm of an antibody; (4) single-chain Fv (scFv), which can be a single polypeptide chain composed of a VH domain and a VL domain via a peptide connector; (5) (scFv)2, which can contain two VH domains connected by a peptide connector and two VL domains, wherein the two VL domains are combined with the two VH domains via a disulfide bridge; and (6) single-domain antibody formats.

[0160] In bispecific antibody constructions, the "binding moiety" includes, but is not limited to, a single domain antibody format, a Fab fragment format, and / or a single chain antibody (scFv) format.

[0161] As used herein, the term "single chain antibody (scFv, single chain fragment variable)" refers to an antibody with a single chain structure generally constructed using genetic engineering technology, comprising a 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.

[0162] As used herein, the term "Fab (fragment antigen binding) fragment", "Fab portion" or similar terms refers to an antibody fragment capable of binding to an antigen produced after an intact antibody is treated with papain, including a complete light chain (VL-CL), a heavy chain variable region and a CH1 fragment (VH-CH1).

[0163] As used herein, the term "single domain antibody" refers to a heavy chain single variable domain antibody that naturally lacks a light chain, and such an antibody comprises a heavy chain variable region (VHH) and conventional CH2 and CH3 regions (e.g., one or two groups (heavy chain variable region (VHH) and conventional CH2 and CH3 regions)). The VHH structure cloned and expressed separately has a structural stability and antigen binding activity comparable to that of the original heavy chain antibody, and is the smallest unit known to bind to the target antigen. Single domain antibodies are also called nanobodies (Nanobody, Nb).

[0164] As used herein, the terms "Fc fragment", "Fc domain" and "Fc part" are used interchangeably and refer to a portion of the antibody heavy chain constant region, including the hinge region, the CH2 fragment and the CH3 fragment of the heavy chain constant region, and are determined with reference to the EU numbering of human IgG1 antibodies.

[0165] The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0166] As used herein, the term "tumor" refers to a neoplasm or solid lesion formed by abnormal cell growth. Tumors can be benign, pre-malignant or malignant.

[0167] As used herein, the term "malignancy" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. Exemplary malignancies include: carcinomas, solid tumors, melanomas, sarcomas, hematological tumors, germ cell tumors, and blastomas. More specific examples of malignancies include: esophageal cancer, urothelial carcinoma, oral cancer, sarcomas, 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 cell carcinoma, cervical cancer, hepatic carcinoma, gastric cancer including gastrointestinal cancer, prostate cancer, pancreatic cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, liver cancer, urinary tract cancer, hepatoma, endometrial or uterine cancer, salivary gland cancer, squamous cell carcinoma (e.g., squamous epithelial cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, multiple myeloma and B cell lymphoma, brain cancer and related metastatic lesions.

[0168] As used herein, the term "hematological tumor" refers to a tumor caused by uncontrolled growth and proliferation of abnormal cells. In most cases, the origin of these abnormal cells is the bone marrow, which is 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 myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia, large granular lymphocytic 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 lymphoma, B-cell chronic lymphocytic lymphoma, classical Hodgkin lymphoma, Nodular lymphocyte-predominant Hodgkin lymphoma, adult T-cell lymphoma, extranodal nasal NK / T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Say-Schermer syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disease, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, not otherwise specified peripheral T-cell lymphoma, and anaplastic large cell lymphoma.

[0169] As used herein, the term "solid tumor" refers to a tangible mass that can be felt by clinical examination such as X-ray, CT scan, B-ultrasound or palpation. Solid tumors that have been clinically diagnosed and treated are divided into malignant and benign tumors. 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 lymphoma: lymphocyte-dominant type, nodular sclerosis type, mixed cell type, lymphocyte-depleted type; childhood non-Hodgkin lymphoma: prelymphoblastic lymphoma, small non-cleaved cell lymphoma (Burkitt / non-Burkitt lymphoma), diffuse large B-cell lymphoma, anaplastic large cell lymphoma, etc.; childhood kidney tumors: Wilms tumor (Wilms tumor), renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, renal primitive neuroectodermal tumor, etc.; childhood neuroblastoma: neuroblastoma, ganglioneuroblastoma, ganglioneuroma; childhood extracranial germ cell tumor: adult Mature teratoma, immature teratoma, endodermal sinus tumor (yolk sac tumor), seminoma, dysgerminoma, choriocarcinoma, embryonal carcinoma, etc.; osteosarcoma and chondrosarcoma; childhood rhabdomyosarcoma: embryonal, alveolar, pleomorphic, etc.; soft tissue sarcoma: fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant neurilemmoma, alveolar soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma , malignant melanoma, synovial sarcoma, desmoplastic small round cell tumor, etc.; Ewing family sarcoma: Ewing sarcoma, primitive neuroectodermal tumor; childhood liver tumors: hepatoblastoma (embryonal, fetal, undifferentiated), hepatocellular carcinoma; retinoblastoma; other tumors: posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, thymoma, pulmonary blastoma, pancreatic blastoma, islet cell tumor, ileocecal carcinoid, mesothelioma, etc. Benign solid tumors include: lymphangioma, hemangioma, thyroglossal duct cyst, etc.

[0170] 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.

[0171] In some embodiments of the first aspect, the bispecific antibody is capable of mediating HLA-A02 + / MAGE-A4 + Tumor cells activate T cells, and / or the bispecific antibody can mediate PBMC killing of HLA-A02 + / MAGE-A4 + Tumor cells.

[0172] In some embodiments of the first aspect, the bispecific antibody is capable of mediating HLA-A02 + / MAGE-A4 + Tumor cells activate Jurkat-Dual cells.

[0173] In some embodiments of the first aspect, the binding epitope of the first antigen-binding fragment to the HLA-A02 / MAGE-A4 complex includes positions 230-239 with reference to MAGE-A4 as shown in SEQ ID NO: 48. In some embodiments, the amino acid sequence with reference to positions 230-239 of MAGE-A4 as shown in SEQ ID NO: 48 is GVYDGREHTV (SEQ ID NO: 1).

[0174] 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 with reference to MAGE-A4 as set forth in SEQ ID NO: 48. In some specific 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 with reference to MAGE-A4 as set forth in SEQ ID NO: 48. In some 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 with reference to MAGE-A4 as set forth in SEQ ID NO: 48 corresponds to residues 232, 234, 235, 237, and 239 with reference to MAGE-A4 as set forth in SEQ ID NO: 1. 230-239 The amino acid residues at positions 3, 5, 6, 8 and 10 of the (GVYDGREHTV) polypeptide.

[0175] In some embodiments of the first aspect, the binding epitope of the first antigen-binding fragment to the HLA-A02 / MAGE-A8 complex includes positions 232-241 of MAGE-A8 with reference to SEQ ID NO: 94. In some embodiments, the amino acid sequence with reference to positions 232-241 of MAGE-A8 with reference to SEQ ID NO: 94 is GLYDGREHSV (SEQ ID NO: 2).

[0176] In some embodiments of the first aspect, the activating T cell antigen is a CD3 molecule.

[0177] 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 the HCDR is according to the Kabat definition.

[0178] In some embodiments of the first aspect, the first antigen-binding fragment is in the form of a single domain antibody.

[0179] In some embodiments of the first aspect, the first antigen-binding fragment comprises a monovalent or multivalent (eg, 1, 2, 3, 4, 5, or 6-valent) single domain antibody that binds to the HLA-A02 / MAGE-A4 complex.

[0180] In some specific 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. In some embodiments, the first antigen-binding fragment comprises two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A02 / MAGE-A4 complex. In some embodiments, the first antigen-binding fragment comprises two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A02 / MAGE-A4 complex connected by a linker. In some embodiments, the linker is a linker comprising a GS flexible peptide, for example (GGGGS) n , wherein n is an integer ≥ 1. In some specific embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 49).

[0181] In some embodiments of the first aspect, the first antigen-binding fragment comprises a monovalent or multivalent (eg, 1, 2, 3, 4, 5, or 6-valent) single domain antibody that binds to the HLA-A02 / MAGE-A8 complex.

[0182] In some specific embodiments of the first aspect, the first antigen-binding fragment comprises a bivalent single-domain antibody that binds to the HLA-A02 / MAGE-A8 complex. In some embodiments, the first antigen-binding fragment comprises two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A02 / MAGE-A8 complex. In some embodiments, the first antigen-binding fragment comprises two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A02 / MAGE-A8 complex connected by a linker. In some embodiments, the linker is a linker comprising a GS flexible peptide, for example (GGGGS) n , wherein n is an integer ≥ 1. In some specific embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 49).

[0183] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:35.

[0184] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:36.

[0185] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:71.

[0186] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:72.

[0187] In some embodiments of the first aspect, the amino acid sequence shown in SEQ ID NO:71 comprises two amino acid sequences shown in SEQ ID NO:35.

[0188] In some embodiments of the first aspect, the amino acid sequence shown in SEQ ID NO:72 comprises two amino acid sequences shown in SEQ ID NO:36.

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

[0190] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:35; and the second antigen-binding fragment comprises the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:43 and the light chain variable region shown in the amino acid sequence of SEQ ID NO:44.

[0191] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:36; and the second antigen-binding fragment comprises the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:43 and the light chain variable region shown in the amino acid sequence of SEQ ID NO:44.

[0192] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:71; and the second antigen-binding fragment comprises the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:43 and the light chain variable region shown in the amino acid sequence of SEQ ID NO:44.

[0193] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:72; and the second antigen-binding fragment comprises the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:43 and the light chain variable region shown in the amino acid sequence of SEQ ID NO:44.

[0194] In some embodiments of the first aspect, the amino acid sequence of the first antigen-binding fragment 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 amino acid substitutions, deletions and / or additions.

[0195] 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 higher homology to the amino acid sequence shown in SEQ ID NO:35, 36, 71 or 72.

[0196] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown 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 maintaining similar function of the first antigen-binding fragment.

[0197] 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 shown in SEQ ID NO: 35, 36, 71 or 72, and the resulting amino acid sequence still maintains similar function of the first antigen-binding fragment.

[0198] 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 may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 35, 36, 71 or 72, as long as the altered amino acid sequence substantially maintains the similar function of the first antigen-binding fragment.

[0199] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:43.

[0200] 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 shown in SEQ ID NO:43 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0201] 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 higher homology with the amino acid sequence shown in SEQ ID NO:43.

[0202] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown 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 maintaining similar function of the heavy chain variable region of the second antigen-binding fragment.

[0203] 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 shown in SEQ ID NO:43, and the resulting amino acid sequence still maintains similar function of the heavy chain variable region of the second antigen-binding fragment.

[0204] 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 may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO:43, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the similar second antigen-binding fragment.

[0205] 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 shown in SEQ ID NO:44.

[0206] 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 shown in SEQ ID NO:44 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0207] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen-binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the amino acid sequence shown in SEQ ID NO:44.

[0208] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:44 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 maintaining similar function of the light chain variable region of the second antigen-binding fragment.

[0209] 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 shown in SEQ ID NO:44, and the resulting amino acid sequence still maintains the similar function of the light chain variable region of the second antigen-binding fragment.

[0210] 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 may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO:44, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the similar second antigen-binding fragment.

[0211] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, and the antibody heavy chain constant region Fc fragment includes 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 amino acid positions of the antibody constant region are determined according to EU numbering.

[0212] In some specific embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, and the antibody heavy chain constant region Fc fragment includes 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 amino acid positions of the antibody constant region are determined according to EU numbering.

[0213] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, and the antibody heavy chain constant region Fc fragment includes a first Fc fragment and a second Fc fragment, wherein the 234th, 235th and 331st amino acids 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.

[0214] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, which includes 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.

[0215] In some embodiments of the first aspect, the first antigen binding fragment (eg, at the C-terminus) is linked to the N-terminus of the first Fc fragment (eg, an amino acid sequence such as SEQ ID NO: 92).

[0216] In some embodiments of the first aspect, the second antigen binding fragment (eg, at the C-terminus) is linked to the N-terminus of the second Fc fragment (eg, an amino acid sequence such as SEQ ID NO: 93).

[0217] In some embodiments of the first aspect, the antibody heavy chain constant region Fc fragment is an Fc fragment of the IgG1 subtype. In some embodiments, the first Fc fragment is an Fc fragment of the IgG1 subtype; and / or the second Fc fragment is an Fc fragment of the IgG1 subtype.

[0218] In some embodiments of the first aspect, the antibody heavy chain constant region Fc fragment is an Fc fragment of the IgG1m3 subtype. In some embodiments, the first Fc fragment is an Fc fragment of the IgG1m3 subtype; and / or the second Fc fragment is an Fc fragment of the IgG1m3 subtype.

[0219] In some embodiments of the first aspect, the bispecific antibody comprises a first arm that binds to the HLA-A02 / MAGE-A4 complex and a second arm that binds to an activating T cell antigen, wherein

[0220] The first arm comprises the amino acid sequence shown in SEQ ID NO: 45 or 74; and

[0221] The second arm comprises the amino acid sequence shown in SEQ ID NO:46.

[0222] In some embodiments of the first aspect, the bispecific antibody comprises a first arm that binds to the HLA-A02 / MAGE-A8 complex and a second arm that binds to an activating T cell antigen, wherein

[0223] The first arm comprises the amino acid sequence shown in SEQ ID NO: 45 or 74; and

[0224] The second arm comprises the amino acid sequence shown in SEQ ID NO:46.

[0225] In some embodiments of the first aspect, the amino acid sequence of the first arm comprises the amino acid sequence shown in SEQ ID NO:45 or 74.

[0226] In some embodiments of the first aspect, the amino acid sequence of the first arm differs from the amino acid sequence shown in SEQ ID NO: 45 or 74 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0227] In some embodiments of the first aspect, the amino acid sequence of the first arm is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO:45 or 74.

[0228] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown 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 maintaining similar function of the first arm.

[0229] 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 shown in SEQ ID NO: 45 or 74, and the resulting amino acid sequence still maintains similar function of the first arm.

[0230] 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 may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 45 or 74, as long as the altered amino acid sequence substantially maintains the function of the similar first arm.

[0231] In some embodiments of the first aspect, the amino acid sequence of the second arm comprises the amino acid sequence shown in SEQ ID NO:46.

[0232] In some embodiments of the first aspect, the amino acid sequence of the second arm differs from the amino acid sequence shown in SEQ ID NO:46 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0233] In some embodiments of the first aspect, the amino acid sequence of the second arm is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO:46.

[0234] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown 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 while still maintaining a similar function of the second arm.

[0235] 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 shown in SEQ ID NO:46, and the resulting amino acid sequence still maintains a similar function of the second arm.

[0236] 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 may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 46, as long as the altered amino acid sequence substantially maintains similar function of the second arm.

[0237] In a second aspect, the present application provides a single domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex, comprising

[0238] HCDR1 as shown in SEQ ID NO:32,

[0239] HCDR2 as shown in SEQ ID NO:33, and

[0240] HCDR3 as shown in SEQ ID NO:34;

[0241] The amino acid sequence of HCDR is based on the Kabat definition.

[0242] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:35.

[0243] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:36.

[0244] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:71.

[0245] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:72.

[0246] 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 amino acid substitutions, deletions and / or additions.

[0247] In some embodiments of the second aspect, the amino acid sequence of the single domain antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO:35, 36, 71 or 72.

[0248] In some embodiments of the second aspect, the C-terminal or N-terminal region of the amino acid sequence shown 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 maintaining similar functions of the single domain antibody.

[0249] 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 be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 35, 36, 71 or 72, and the resulting amino acid sequence still maintains similar functions of the single domain antibody.

[0250] 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 may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 35, 36, 71 or 72, as long as the altered amino acid sequence substantially maintains similar function of the single domain antibody.

[0251] In a third aspect, the present application provides a nucleic acid molecule encoding the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect.

[0252] In some embodiments of the third aspect, the nucleic acid molecule may include a DNA molecule and an RNA molecule. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

[0253] In some embodiments of the third aspect, the nucleic acid molecule is operably linked to a regulatory amino acid sequence that can be recognized by a host cell transformed with the vector.

[0254] In a fourth aspect, the present application provides a pharmaceutical composition comprising the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect and a pharmaceutically acceptable excipient, diluent or carrier.

[0255] In some embodiments of the fourth aspect, the pharmaceutical composition is used to prevent or treat HLA-A02 / MAGE-A4 positive tumors.

[0256] In some embodiments of the fourth aspect, the HLA-A02 / MAGE-A4 positive tumor is selected from: 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, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer and breast cancer.

[0257] In some embodiments of the fourth aspect, the pharmaceutical composition is used to prevent or treat HLA-A02 / MAGE-A8 positive tumors.

[0258] In some embodiments of the fourth aspect, the HLA-A02 / MAGE-A8 positive tumor is selected from: 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, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer and breast cancer.

[0259] In some embodiments of the fourth aspect, the pharmaceutical composition may further comprise one or more of the following substances: lubricants, such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives, such as benzoic acid, sorbic acid and calcium propionate; sweeteners and / or flavoring agents, etc.

[0260] In some embodiments of the fourth aspect, the pharmaceutical composition of the present application can be formulated into the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.

[0261] 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, etc.

[0262] In some embodiments of the fourth aspect, pharmaceutical compositions for therapeutic use can be formulated for storage in the form of lyophilized preparations or aqueous solutions by mixing reagents having the desired purity with pharmaceutically acceptable carriers, excipients, etc. as appropriate.

[0263] In a fifth aspect, the present application provides 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 preventing or treating HLA-A02 / MAGE-A4-positive tumors and / or HLA-A02 / MAGE-A8-positive tumors.

[0264] In some embodiments of the fifth aspect, the HLA-A02 / MAGE-A4 positive tumor is selected from: 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, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer and breast cancer.

[0265] In some embodiments of the fifth aspect, the HLA-A02 / MAGE-A8 positive tumor is selected from: 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, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer and breast cancer.

[0266] In a sixth aspect, the present application provides a method for preventing or treating HLA-A02 / MAGE-A4-positive tumors and / or HLA-A02 / MAGE-A8-positive tumors, comprising administering 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 to an individual in need.

[0267] In some embodiments of the sixth aspect, the HLA-A02 / MAGE-A4 positive tumor is selected from: 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, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer and breast cancer.

[0268] In some embodiments of the sixth aspect, the HLA-A02 / MAGE-A8 positive tumor is selected from: 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, lung cancer, liver cancer, bladder cancer, melanoma (e.g., metastatic melanoma), ovarian cancer, colorectal cancer and breast cancer.

[0269] The present application also provides a vector comprising a nucleic acid molecule encoding the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect, and a host cell comprising the nucleic acid molecule or the vector. In other aspects, the present application also provides a method for producing the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect. In some embodiments, the method for producing the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect includes culturing a host cell to express the nucleic acid molecule. In some embodiments, the method for producing the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect also includes recovering the bispecific antibody or the single domain antibody from the host cell culture medium.

[0270] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application, and is not intended to be limiting in any aspect. Those skilled in the art can make various modifications and changes to the embodiments.

[0271] The following examples are for illustration purposes only and are not intended to limit the scope of this application. Example

[0272] Example 1: Preparation of recombinant protein

[0273] References

[16] Preparation of antigen peptide MAGE-A4 230-239 (GVYDGREHTV) disulfide trap single chain trimer (dtSCT) structure MHCⅠ complex, 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 have a large number of post-translational modifications (such as glycosylation or disulfide bonds, etc.), so the use of mammalian cell expression system will be more conducive to maintaining the structure and function of the recombinant protein. Adding a His tag (His, SEQ ID NO: 53) or the Fc segment of human antibody IgG1 (Fc, SEQ ID NO: 54) or the Fc segment of mouse antibody IgG2a (mFc, SEQ ID NO: 55) to the C-terminus of the recombinant protein will be more conducive to the purification of the recombinant protein and the identification of the function of the monoclonal antibody. When preparing recombinant antibodies, the antibody heavy chain constant region can be human IgG1 subtype (SEQ ID NO: 56) or various mutants of selected human IgG1 subtypes, 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 λ subtype (SEQ ID NO: 63) or human κ subtype (SEQ ID NO: 64).

[0274] According to the amino acid sequences of various target recombinant proteins in the Uniprot database, the genes of the above recombinant proteins (including His tags, mFc or Fc encoding genes) are designed and synthesized. The synthesized various recombinant protein genes are cloned into suitable eukaryotic expression vectors (such as pcDNA3.1 of Invitrogen, etc.) using conventional molecular biological techniques, and then the prepared recombinant protein expression plasmids are transfected into HEK293 cells (such as HEK293F cells of Invitrogen) using liposomes (such as 293fectin of Invitrogen, etc.) or other cationic transfection reagents (such as PEI, etc.), and cultured under serum-free suspension culture conditions for 3-4 days. The culture supernatant is then harvested by centrifugation or the like.

[0275] The recombinant protein expressed by His tag fusion is purified in one step by metal chelate affinity chromatography column (such as GE's HisTrap FF, etc.). The recombinant protein expressed by mFc / Fc fusion is purified in one step by ProteinA / G affinity chromatography column (such as GE's Mabselect SURE, etc.). Then, the recombinant protein storage buffer is replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (such as GE's Hitrap desaulting, etc.). If necessary, the antibody sample can be filtered and sterilized, and then stored in aliquots at -20°C.

[0276] Example 2: Screening of antibodies binding to the HAL-A02 / MAGE-A4 complex from a camel immune library

[0277] 2.1 Construction of camel immune library

[0278] One healthy adult Bactrian camel was selected, and blood was collected before immunization to keep the 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; for booster immunization at intervals of two weeks, 1 mg of MHC-P1-His fusion protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple points, for a total of 5 booster immunizations, and blood was collected before each immunization for antibody titer analysis; for the seventh immunization, no adjuvant was added, 1 mg of MHC-P1-His fusion protein was taken as an antigen, and multiple subcutaneous injections were performed for shock immunization, and 200 ml of peripheral blood was collected 3 days later for lymphocyte separation.

[0279] Lymphocytes were isolated from 200 mL of camel peripheral blood using a camel peripheral blood lymphocyte isolation kit (Solarbio, CAT#P5750); total RNA of lymphocytes was extracted using a cell total RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., CAT#DP430); the extracted total RNA was used as a template and the first-strand cDNA synthesis kit (Thermoscientific, CAT#K1621) was used to synthesize the camel single-domain antibody heavy chain variable region (VHH), and the reverse transcription primers were gene-specific primers, and the primer pairing region was located in the CH2 domain of the antibody heavy chain constant region, and the specific sequence was PCal-CH2R: TCCTTCCCCGTCAGCCAGTCCT (SEQ ID NO: 50). The synthesized cDNA was immediately stored at -70°C for future use; then the cDNA obtained by reverse transcription was used as a template, and references were used to synthesize the camel single-domain antibody heavy chain variable region (VHH).

[17] Primers were 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%.

[0280] 2.2 Screening of camel immune library

[0281] The recombinant MHC-P1-His prepared in Example 1 was used as an antigen and a solid phase screening strategy was used (the experimental scheme refers to Phage Display: General Experimental Guide / Clackson, T., Lowman, HB, ed.; Ma Lan et al., Chemical Industry Press, May 2008).

[19] ) The phage library displaying camel single-domain antibodies constructed in Example 2.1 was screened, and three rounds of screening were performed by binding, elution, neutralization, infection, and amplification, and finally the TCR mimic (TCRm) antibody N18D10 that specifically binds to MHC-P1-His was obtained.

[0282] Using conventional molecular biological methods, the nucleotide sequence encoding the N18D10 heavy chain variable region (SEQ ID NO: 35) was cloned into a eukaryotic expression vector (such as Invitrogen's pcDNA3.1, etc.) fused with a nucleotide sequence encoding the Fc segment of human antibody IgG1 to express the N18D10-Fc recombinant protein. At the same time, DP47 (germline gene antibody, refer to US patent application US 20160200833A1) was prepared.

[20] , the amino acid sequence of DP47VH is shown in SEQ ID NO: 65; the amino acid sequence of DP47VK is shown in SEQ ID NO: 66) monoclonal antibody DP47-IgG1 was used as a negative control. Reference 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 and cloned into a eukaryotic expression vector fused with a nucleotide sequence of an Fc fragment containing a Knob mutation of Cα and a eukaryotic expression vector fused with a nucleotide sequence of an Fc fragment containing a Hole mutation of Cβ, respectively. The two chains constituting the TCR molecule were TCRA-Des-G1m3-FcKn1 (SEQ ID NO: 69) and TCRB-Des-G1m3-FcH1n1 (SEQ ID NO: 70), respectively. A TCR control molecule binding to MHC-P1 was co-expressed and named P1-TCR.

[0283] Example 3: Identification of TCRm antibodies that bind to the HLA-A02 / MAGE-A4 complex

[0284] 3.1 Affinity analysis of TCRm antibodies against HLA-A02 / MAGE-A4 complex

[0285] The affinity of the anti-HLA-A02 / MAGE-A4 TCRm antibody N18D10 was determined by surface plasmon resonance using Biacore T200. The amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), S series CM5 chip (14100530) and pH 7.4 10×HBS-EP (BR100669) and other related reagents and consumables were purchased from GE healthcare. According to the instructions in the kit, the carboxylated CM5 chip surface was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the anti-human IgG (Fc) antibody (capture antibody) was diluted to 25 μg / mL with 10 mM pH 5.0 sodium acetate and then injected at a flow rate of 10 μL / min to achieve a coupling amount of approximately 10,000 response units (RU). After the capture antibody was injected, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, the TCRm antibody N18D10-Fc was diluted to 1 μg / mL and injected at 10 μL / min to ensure that about 60 RU of the antibody was captured by the anti-human Fc antibody. Then, a series of concentration gradients (e.g., 1.23 nM, 3.74 nM, 11.1 nM, 33.3 nM, 100 nM) of MHC-P1-His were set, and injection was performed from low concentration to high concentration at 30 μL / min at 25°C, with a binding time of 90 s and a dissociation time of 1200 s. A 3 M MgCl2 solution was injected at 10 μL / min for 30 s to regenerate the chip surface. The binding rate (K) was calculated by fitting the binding and dissociation sensorgrams with a 1:1 binding model using Biacore T200 evaluation software version 3.2.1. a ) and dissociation rate (K d ). With the ratio K d / K a Calculate the dissociation equilibrium constant (K D ). The fitting results are shown in Table 1.

[0286] Table 1. Affinity of TCRm antibody N18D10-Fc against HLA-A02 / MAGE-A4 complex binding to MHC-P1

[0287] <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> N18D10-Fc 1.851E+5 7.176E-5 3.878E-10

[0288] 3.2 Binding of TCRm Antibody Against HLA-A02 / MAGE-A4 Complex to Recombinant Protein MHC-P1

[0289] The TCRm antibody N18D10-Fc against the HLA-A02 / MAGE-A4 complex was coated on a 96-well ELISA plate at 5 μg / mL, 100 μL per well, and coated overnight at 4°C. The plate was blocked with a blocking solution of PBS-0.1% Tween 20-3% skim milk at 37°C for 1 hour. The recombinant protein MHC-P1-His was gradiently diluted with PBS, with a starting concentration of 30 μg / mL, 3-fold gradient dilution, a total of 10 concentration gradients, 100 μL per well was added to the blocked 96-well ELISA plate, and incubated at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20, and HRP-labeled anti-His tag mouse monoclonal antibody (Beijing Kangwei Century Biotechnology Co., Ltd., CW0285M) was added, and incubated at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20, and OPD substrate colorimetric solution was added. After 5-10 minutes, 1M H2SO4 was used to terminate the colorimetric development. The optical density value was measured by a 492nm / 630nm dual wavelength microplate reader. Figure 1 The results showed that N18D10-Fc specifically bound to MHC-P1.

[0290] 3.3 Binding of TCRm Antibody Against HLA-A02 / MAGE-A4 Complex to Antigen Peptide-Pulsed T2 Cells

[0291] Collect T2 cells (human lymphoma cells, HLA-A02) in the logarithmic phase of growth + , purchased from Shanghai Hongshun Biotechnology Co., Ltd.), and resuspended in growth medium to 1×10 6 / mL, 1mL per well was plated in a 24-well plate. Chemically synthesized peptide MAGE-A4 230-239 (GVYDGREHTV) (GenScript Biotech Co., Ltd.) was added to a 24-well plate at a final concentration of 50 μg / mL and pulsed overnight. After 16 hours, the cells were centrifuged and resuspended in PBS buffer containing 1% BSA to a concentration of 2×10 6 / mL, 100μL per well was plated in a 96-well V-bottom plate, and the supernatant was removed after centrifugation. Antibody N18D10-Fc, positive control sample P1-TCR and negative control DP47-IgG1 were prepared with PBS to a final concentration of 200nM, and 4-fold diluted samples were added to the wells containing cells at 100μL per well and incubated at 4°C for 1 hour. Then wash 3 times with 200μL PBS, incubate with sheep anti-human IgG-FITC (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., ZF-0308), 100μL per well, incubate at 4°C in the dark for 30 minutes. Then wash 3 times with 200μL PBS, resuspend in 100μL PBS, and detect the FITC channel with a flow cytometer (ACEA, Novocyte). Figure 2The results showed that N18D10-Fc specifically binds to the antigen peptide MAGE-A4 230-239 Pulsed T2 cells, namely N18D10-Fc binds to the HLA-A02 / MAGE-A4 complex on the cell surface.

[0292] 3.4 Key amino acids of TCRm antibody against HLA-A02 / MAGE-A4 complex binding to MAGE-A4

[0293] Chemical synthesis of MAGE-A4 230-239 Alanine scanning mutant peptide (GVYDGREHTV). Reference Example 3.3 Detection of N18D10 and MAGE-A4 230-239 T2 cells pulsed with alanine scanning mutant peptide (GVYDGREHTV) were collected in the logarithmic phase of growth, centrifuged and resuspended in growth medium to 1×10 6 1 mL / well was plated in a 24-well plate. 230-239 (GVYDGREHTV) and alanine scanning mutant peptides were added to a 24-well plate at a final concentration of 50 μg / mL and pulsed overnight. After 16 hours, the cells were centrifuged and resuspended in PBS buffer containing 1% BSA to 2×10 6 / mL, 100μL per well was plated in a 96-well V-bottom plate, and the supernatant was removed after centrifugation. The antibody N18D10-Fc was prepared with PBS to a final concentration of 10nM, 100μL per well was added to the cell plate, and incubated at 4°C for 1 hour. Then wash three times with 200μL PBS, incubate with sheep anti-human IgG-FITC (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., ZF-0308), 100μL per well, incubate at 4°C in the dark for 30 minutes. Then wash three times with 200μL PBS, resuspend in 100μL PBS, and detect the FITC channel with a flow cytometer (ACEA, Novocyte). The binding values ​​of N18D10 to the alanine scanning peptide relative to the original peptide were calculated, as shown in Tables 2 and 3. Figure 3 The results showed that MAGE-A4 230-239 When the 3rd, 5th, 6th, 8th or 10th position of (GVYDGREHTV) was replaced by alanine, the binding ability of TCRm antibody N18D10 decreased by more than 90%, that is, MAGE-A4 230-239 The 3rd, 5th, 6th, 8th and 10th positions of (GVYDGREHTV) are the key amino acids for N18D10 binding.

[0294] Table 2. TCRm antibody N18D10 and MAGE-A4 230-239 Binding of T2 cells pulsed with alanine scanning mutant peptide (GVYDGREHTV)

[0295] <![CDATA[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％

[0296] Example 4: Humanization and identification of TCRm antibodies against HLA-A02 / MAGE-A4 complex

[0297] 4.1 Humanization of TCRm Antibody Against HLA-A02 / MAGE-A4 Complex

[0298] The TCRm antibody N18D10 was humanized to reduce its immunogenicity. The humanization scheme adopted the classic framework transplantation strategy.

[22] . The amino acid sequence of N18D10 was compared with the human antibody germline gene sequence in the IMGT database, and the appropriate germline gene sequence was selected to provide the antibody framework regions 1 to 3 (FR1+FR2+FR3), and the appropriate J region gene sequence was selected to provide the framework region 4 (FR4). This template can be selected based on a variety of 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 antibody framework region (FR) and the hypervariable region (CDR), the homology of the entire sequence, etc. The selected template can be a mixture of multiple sequences or a common template, with the aim 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 hotspot amino acids 37F / 44E / 45R / 47F in its FR2 were reverse mutated, and the humanized molecule N18D10-h16 was finally obtained.

[0299] Using conventional molecular biological methods, the nucleotide sequence encoding the variable region of N18D10-h16 (SEQ ID NO: 36) was cloned into a eukaryotic expression vector (such as Invitrogen's pcDNA3.1, etc.) fused with a nucleotide sequence encoding the Fc segment of human antibody IgG1 to express the N18D10-h16-Fc recombinant protein.

[0300] 4.2 Affinity analysis of humanized TCRm antibodies against HLA-A02 / MAGE-A4 complex

[0301] Referring to Example 3.1, affinity analysis of the recombinant anti-HLA-A02 / MAGE-A4 complex TCRm antibody N18D10-h16-Fc was performed using Biacore T200. The results are shown in Table 3.

[0302] Table 3. Affinity of TCRm antibody N18D10-h16-Fc binding to MHC-P1 complex against HLA-A02 / MAGE-A4 complex

[0303] <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> MHC-P1-His 1.631E+5 7.610E-5 4.665E-10

[0304] 4.3 Binding of TCRm antibody N18D10-h16-Fc against HLA-A02 / MAGE-A4 complex to MHC-P1

[0305] Referring to Example 3.2, the binding activity of the anti-HLA-A02 / MAGE-A4 complex antibody N18D10-h16-Fc and 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, that is, N18D10-h16-Fc bound to the HLA-A02 / MAGE-A4 complex.

[0306] Example 5: Preparation and identification of TCRm×CD3 bispecific antibodies

[0307] 5.1 Preparation of TCRm×CD3 bispecific antibodies

[0308] The heavy chain variable region nucleotide sequence encoding the bivalent 2N18D10 or bivalent 2N18D10-h16 single domain antibody against HLA-A02 / MAGE-A4 complex and the single chain antibody encoding anti-CD3 (IMCR-anti-CD3-scFv, see anti-CD3 v9 of U.S. Patent Application No. US 5821337A) were respectively

[23] ) was cloned into a suitable eukaryotic expression vector to co-express a bispecific antibody 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 a eukaryotic expression vector fused with a nucleotide sequence encoding a Hole mutation Fc fragment IgG1m3-FcH1n1 (SEQ ID NO: 92), and the nucleotide sequence encoding IMCR-anti-CD3-ScFv (SEQ ID NO: 73) was cloned into a eukaryotic expression vector fused with a nucleotide sequence encoding a Knob mutation Fc fragment IgG1m3-FcKn1 (SEQ ID NO: 93). At the same time, a DP47 bispecific antibody based on the same structure was prepared as a negative control.

[0309] The constructed eukaryotic expression vector 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) were co-transfected into HEK293F cells using liposomes, cultured for 3-5 days under serum-free suspension culture conditions, and then the culture supernatant was harvested by centrifugation, etc. The bispecific antibody in the culture supernatant was purified using a Protein A affinity chromatography column (such as Mabselect SURE of GE Company, etc.), and then the recombinant protein storage buffer was replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (such as Hitrapdesaulting of GE Company, etc.). The desalted protein solution was purified by size exclusion chromatography (SEC) using Superdex200 (GE) to obtain the target proteins, which were named 2N18D10+IMCR and 2N18D10-h16+IMCR. If necessary, the antibody sample can be filtered and sterilized, and then stored in aliquots at -20°C for later use.

[0310] Referring to patent application WO 2017175006A1, the single-chain antibody IMCR-anti-CD3-scFv of CD3 was fused to the N-terminus of the TCRB-Des-G1m3-FcH1n1 chain to prepare the bifunctional molecule IMC-C103C based on TCR and CD3. The two chains constituting the TCR and CD3 bifunctional molecule were TCRA-Des-G1m3-FcKn1 (SEQ ID NO: 69) and IMCR-anti-CD3-scFv-TCRB-Des-G1m3-FcH1n1 (SEQ ID NO: 75).

[0311] Refer to patent application WO 2021122875A1

[24] , synthesized the variable region sequence of 057D03 (the amino acid sequence of 057G03VH is shown in SEQ ID NO:76; the amino acid sequence of 057G03VK is shown in SEQ ID NO:77), and prepared the anti-HLA-A02 / MAGE-A4 complex and CD3 bispecific antibody 057G03 based on the structure in the patent. The four chains constituting the bispecific antibody were 057G03-Fd-EE-IMCR-anti-CD3VK-G1m3-Kn1 (SEQ ID NO:78), 057G03-Fd-EE-G1m3-FcH1n1 (SEQ ID NO:79), 057G03VK+CK-RK (SEQ ID NO:80) and IMCR-anti-CD3VH-CK (SEQ ID NO:81).

[0312] The expression and purification of the above-mentioned IMC-C103C and 057G03 bispecific antibodies refer to the expression and purification of the above-mentioned 2N18D10+IMCR and 2N18D10-h16+IMCR, after which the samples are filtered and sterilized, and then packaged and stored at -20°C for future use.

[0313] 5.2 Affinity analysis of TCRm×CD3 bispecific antibodies

[0314] Referring to Example 3.1, affinity analysis of the recombinant TCRm×CD3 bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR was performed using Biacore T200. The results are shown in Tables 4 and 5.

[0315] Table 4. TCRm×CD3 bispecific antibody binding affinity to MHC-P1

[0316] <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[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

[0317] Table 5. TCRm×CD3 bispecific antibody binding CD3D-CD3E-mFc affinity

[0318] <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[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

[0319] 5.3 TCRm×CD3 bispecific antibody recognizes antigens MHC-P1 and CD3 simultaneously

[0320] Conventional ELISA method was used to detect the TCRm×CD3 bispecific antibody that simultaneously binds to the dual antigens CD3 and MHC-P1.

[0321] The CD3D-CD3E-mFc antigen was coated on a 96-well ELISA plate (3 μg / mL, 100 μL per well) and coated overnight at 4°C. The plate was blocked with a blocking solution of PBS-0.1% Tween 20-3% skim milk at 37°C for 1 hour. The TCRm×CD3 bispecific antibody was gradiently diluted with PBS, with a starting concentration of 10 μg / mL, 3-fold gradient dilution, a total of 10 concentration gradients, and 100 μL per well was added to the blocked 96-well ELISA plate and incubated at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20, and the MHC-P1-His antigen (10 μg / mL, 100 μL per well) was added and incubated at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20, and then the HRP-labeled anti-His tag mouse monoclonal antibody (Beijing Kangwei Century Biotechnology Co., Ltd., cw0285M) was added and incubated at 37°C for 1 hour. Wash the ELISA plate with PBS-0.1% Tween 20, add OPD substrate colorimetric solution, stop colorimetric development with 1M H2SO4 after 5-10 minutes, and measure the optical density with a dual wavelength of 492nm / 630nm using an ELISA reader. Figure 5 As shown, the TCRm×CD3 bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR can simultaneously recognize CD3 and MHC-P1 dual antigens.

[0322] 5.4 TCRm×CD3 bispecific antibody mediated Jurkat-Dual cell activation

[0323] 5.4.1 TCRm×CD3 bispecific antibody-mediated activation of Jurkat-Dual cells by antigen peptide-pulsed T2 cells

[0324] T2 cells in the logarithmic growth phase were collected, centrifuged and resuspended in growth medium to 1×10 6 pcs / mL, 1mL per well was plated in a 24-well plate. 230-239 (GVYDGREHTV) was added to a 24-well plate at a final concentration of 50 μg / mL and pulsed overnight. After 16 hours, the cells were centrifuged and resuspended in RPMI1640 medium to 4 × 10 5 Jurkat-Dual cells (purchased from Invivogen) in the logarithmic growth phase were collected and resuspended in RPMI1640 medium to 4×10 5 / mL, 50μL per well was added to the cell plate inoculated with T2 cells to obtain a final E:T ratio of 1:1. Then add the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR (starting and ending concentrations were 7.5nM, 4-fold gradient dilutions, 8 concentration points, 100μL per well). The negative control sample 2DP47+IMCR, the positive control samples 057G03 and IMC-C103C, all used the same concentrations as the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR. Incubate for 24h, take the supernatant, and refer to QUANTI-Luc TM Instructions (QUANTI-Luc TM , Invivogen, rep-qlc2) detected and analyzed the specific activation of Jurkat-Dual cells by TCRm×CD3 bispecific antibodies mediated by antigen peptide-pulsed T2 cells. The results showed that the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR could specifically mediate the activation of Jurkat-Dual cells by antigen peptide-pulsed T2 cells. Compared with 057G03 and IMC-C103C, the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR activated Jurkat-Dual cells more clearly ( Figure 6 ), E.C. 50 See Table 6 for values.

[0325] Table 6. EC of Jurkat-Dual cells activated by T2 cells mediated by exogenous pulsed antigen peptides by TCRm×CD3 bispecific antibodies 50 value

[0326]

[0327] 5.4.2 TCRm×CD3 bispecific antibody mediated HLA-A02 + / MAGE-A4 + Tumor cells activate Jurkat-Dual cells

[0328] A375 cells (human malignant melanoma cells, HLA-A02) were collected in the logarithmic phase of growth. + / MAGE-A4 + , purchased from Nanjing Kebai Biotechnology Co., Ltd.). After digestion and centrifugation, it was resuspended in RPMI1640 medium to 4×10 5 / mL, 50μL per well was plated in the cell plate. Jurkat-Dual cells in the logarithmic growth phase were collected, centrifuged and resuspended in RPMI1640 medium to 4×10 5 / mL, 50μL per well was added to the cell plate to obtain a final E:T ratio of 1:1. Then the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR were added (starting concentration was 30nM, 4-fold gradient dilution, 10 concentration points, 100μL per well). The negative control sample 2DP47+IMCR, the positive control samples 057G03 and IMC-C103C, the concentrations used were the same as the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR. After incubation for 48 hours, the supernatant was taken and the QUANTI-Luc TM Instructions (QUANTI-Luc TM , Invivogen, rep-qlc2) to detect and analyze the specific activation of Jurkat-Dual cells by tumor cells under different conditions. The results showed that TCRm×CD3 bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR could mediate HLA-A02 + / MAGE-A4 + Tumor cell A375 activates Jurkat-Dual cells. Compared with 057G03 and IMC-C103C, TCRm×CD3 bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR have stronger activation activity on Jurkat-Dual cells ( Figure 7 ), E.C. 50 The values ​​are shown in Table 7. Table 7 TCRm×CD3 bispecific antibody mediated HLA-A02 + / MAGE-A4 + Tumor cells activate Jurkat-Dual cells

[0329]

[0330] 5.5TCRm×CD3 bispecific antibody mediates PBMC-specific killing of target cells

[0331] 5.5.1 Isolation of human peripheral blood mononuclear cells (PBMC)

[0332] Blood was collected from normal volunteers (50 mL each), and all volunteers had signed informed consent. The volunteer selection criteria were:

[0333] Aged over 18 years old;

[0334] No HIV and HBV infection;

[0335] Routine blood test results were normal;

[0336] Not pregnant or breastfeeding women.

[0337] PBMCs were isolated from whole blood of volunteers by Ficoll density gradient centrifugation and cultured in RPMI1640 medium.

[0338] 5.5.2 TCRm×CD3 bispecific antibody mediates PBMC killing of antigen peptide-pulsed T2 cells

[0339] T2 cells in the logarithmic growth phase were collected, centrifuged and resuspended in growth medium to 1×10 6 pcs / mL, 1mL per well was plated in a 24-well plate. 230-239 (GVYDGREHTV) was added to a 24-well plate at a final concentration of 50 μg / mL and pulsed overnight. After 16 hours, the cells were centrifuged and resuspended in RPMI1640 medium to 4 × 10 5 / mL, 50μL per well was plated in a 96-well cell plate. Then the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR were added (starting concentration was 30nM, 4-fold gradient dilution, 10 concentration points, 100μL per well). The negative control sample 2DP47+IMCR, the positive control samples 057G03 and IMC-C103C, the concentrations used were the same as the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR. PBMC (effector cells) were resuspended to 4×10 6 / mL, add the above cell plate, 50μL per well, the final effector-target ratio is 10:1. At the same time, set up a single target cell control (antigen peptide pulsed T2 cells), a single effector cell control (PBMC), and a single culture medium blank control, and use culture medium to fill the volume of each well to 200μL. After incubation for 24 hours, take the supernatant and refer to Non-radioactive cytotoxicity assay reagent instructions ( Non-Radioactive Cytotoxicity Assay, promega, G1780) was used to detect and analyze the killing rate of PBMC cells against antigen peptide-pulsed T2 cells mediated by TCRm×CD3 bispecific antibodies. The results showed that TCRm×CD3 bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR could specifically mediate the killing of PBMC cells against antigen peptide-pulsed T2 cells; compared with 057G03 and IMC-C103C, 2N18D10+IMCR and 2N18D10-h16+IMCR bispecific antibodies mediated PBMC cells to kill antigen peptide-pulsed T2 cells with higher activity (Table 8 and Figure 8 ).

[0340] Table 8. TCRm×CD3 bispecific antibody mediates PBMC cell killing of antigen peptide-pulsed T2 cells

[0341]

[0342] 5.5.3 TCRm×CD3 bispecific antibody mediates PBMC killing of HLA-A02 + / MAGE-A4 + Tumor cells

[0343] Collect different antigen double positive (HLA-A02 + / MAGE-A4 + ) cells A375 cells (human malignant melanoma cells), Hs695T (human malignant melanoma cells), U2-OS (human osteosarcoma cells) and negative control cells NIC-H520 (human lung squamous cell carcinoma cells, HLA-A02- / MAGE-A4 + ) and HepG2 (human hepatoma cells, HLA-A02 + / MAGE-A4 - ). After digestion and centrifugation, resuspend in RPMI1640 medium to 4×10 5 / mL, 50μL per well was plated in the cell plate. Then the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR were added (starting concentration was 30nM, 4-fold gradient dilution, 10 concentration points, 100μL per well). The negative control sample 2DP47+IMCR, the positive control samples 057G03 and IMC-C103C, were used at the same concentration as the bispecific antibodies 2N18D10+IMCR and 2N18D10-h16+IMCR. PBMC (effector cells) were resuspended to 4×10 6 / mL, add the above cell plate, 50μL per well, the final effector-target ratio is 10:1. At the same time, set up a single target cell control, a single effector cell control (PBMC), and a single culture medium blank control, and use culture medium to fill the volume of each well to 200μL. After incubation for 24-48 hours, take the supernatant and refer to Non-radioactive cytotoxicity assay reagent instructions ( Non-Radioactive Cytotoxicity Assay, promega, G1780) was used to detect and analyze the killing rate of PBMC cells against tumor cells mediated by TCRm×CD3 bispecific antibody. The results showed that 2N18D10-h16+IMCR mediated the specific killing of PBMC against tumor cells ( Fig.10), 2N18D10+IMCR and 2N18D10-h16+IMCR had stronger killing activity against A375 cells than 057G03 and IMC-C103C ( Fig. 9 , E.C. 50 The values ​​are shown in Table 9).

[0344] Table 9. ECs of TCRm×CD3 bispecific antibodies mediating PBMC cell killing of A375 cells 50 value

[0345]

[0346] Example 6: TCRm×CD3 bispecific antibody recognizes MAGE-A4 230-239 Key amino acid analysis of (GVYDGREHTV)

[0347] Chemical synthesis of MAGE-A4 230-239 Alanine scanning peptides of (GVYDGREHTV). T2 cells in the logarithmic growth phase were collected. After centrifugation, they were resuspended in growth medium to 1×10 6 1 mL / well was plated in a 24-well plate. 230-239 (GVYDGREHTV) and alanine scanning mutant peptides were added to a 24-well plate at a final concentration of 50 μg / mL and pulsed overnight. After 16 hours, the cells were centrifuged and resuspended in RPMI1640 medium to 4×10 5 / mL, 50μL was added to each well of a 96-well cell plate. Jurkat-Dual cells in the logarithmic growth phase were collected, centrifuged and resuspended in RPMI1640 medium to 4×10 5 / mL, 50μL per well was added to the previous cell plate to obtain a final E:T ratio of 1:1. Then 2N18D10-h16+IMCR bispecific antibody was added (starting concentration was 30nM, 4-fold gradient dilution, 8 concentration points, 100μL per well). Incubate for 24h, take the supernatant, and refer to QUANTI-Luc TM Instructions (QUANTI-Luc TM , Invivogen, rep-qlc2) were used to detect and analyze the specific activation of Jurkat-Dual cells by T2 cells pulsed with different peptides. The results showed that 2N18D10-h16+IMCR could not mediate the activation of Jurkat-Dual cells by T2 cells pulsed with alanine scanning mutant peptides at positions 3, 5, 6, 8 or 10 ( Fig.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 2N18D10-h16+IMCR binding.

[0348] Example 7: Analysis of the interaction between TCRm×CD3 bispecific antibody and similar peptides

[0349] Chemical synthesis of MAGE-A4 in Table 10 230-239 (GVYDGREHTV) similar peptide.

[0350] Table 10. MAGE-A4 230-239 (GVYDGREHTV) similar peptide information

[0351]

[0352]

[0353] Collect T2 cells in the logarithmic growth phase. Centrifuge and resuspend in growth medium to 1×10 6 1 mL / well was plated in a 24-well plate. 230-239 (GVYDGREHTV) and similar peptides were added to a 24-well plate at a final concentration of 50 μg / mL and pulsed overnight. After 16 hours, the cells were centrifuged and resuspended in RPMI1640 medium to 4 × 10 5 / mL, 50μL was added to each well of a 96-well cell plate. Jurkat-Dual cells in the logarithmic growth phase were collected, centrifuged and resuspended in RPMI1640 medium to 4×10 5 / mL, 50μL per well was added to the above cell plate to obtain a final E:T ratio of 1:1. Then the bispecific antibody 2N18D10-h16+IMCR was added (starting and ending concentration was 30nM, 4-fold gradient dilution, 8 concentration points, 100μL per well). Incubate for 24h, take the supernatant, and refer to QUANTI-Luc TM Instructions (QUANTI-Luc TM , Invivogen, rep-qlc2) were used to detect and analyze the activation of Jurkat-Dual cells by T2 cells pulsed with different similar peptides. Fig.12 The results showed that 2N18D10-h16+IMCR could specifically mediate MAGE-A4 230-239T2 cells pulsed with MAGE-A4-M1 activate Jurkat-Dual cells, and also mediate the activation of Jurkat-Dual by cells pulsed with the same family sequence MAGE-A4-S1 (antigenic peptide of MAGE-A8 presented by HLA-A02); it does not mediate the activation of Jurkat-Dual by other T2 cells loaded with similar peptides. MAGE-A8 also belongs to the MAGE family and has the same expression pattern as MAGE-A4. It is restrictedly expressed in testicular and placental tissues in healthy people, and specifically expressed in some tumor tissues in cancer patients.

[0354] Example 8: Analysis of the interaction between TCRm×CD3 bispecific antibody and different antigen-negative tumor cells

[0355] PBMCs were isolated by referring to Section 5.5.1.

[0356] Different tumor cells were collected, and the antigen information of the cells is shown in Table 11. Referring to Section 5.5.3, the killing experiment of PBMC mediated by 2N18D10-h16+IMCR bispecific antibody against different antigen-negative tumor cells was carried out. Fig.13 The results showed that 2N18D10-h16+IMCR only mediated PBMC to HLA-A02 + / MAGE-A4 + The cytotoxicity of A375 cells does not mediate the killing of antigen-negative tumor cells by PBMCs.

[0357] Table 11. Antigen expression information of different tumor cells

[0358] cell HLA-A02 MAGE-A4 cell HLA-A02 MAGE-A4 A375 + + SW480 + - PANC-1 + - HT29 + - HepG2 + - NCI-H520 - + T2 + - PLC / PRF / 5 - -

[0359] Note: “+” represents positive, and “-” represents negative.

[0360] Example 9: Antitumor effect of TCRm×CD3 bispecific antibody in hPBMC immune reconstitution mice

[0361] PBMCs were isolated by referring to Section 5.5.1.

[0362] Forty 5-6 week old female NOG mice (purchased from Beijing Weitongda Biotechnology Co., Ltd.) were selected and each mouse was intraperitoneally inoculated with 8×10 6 On the seventh day of inoculation of human PBMC, 1 × 10 7 A375 cells. The average tumor volume is 70mm 3The patients were randomly divided into groups according to the tumor size, and the day of grouping was defined as day 0, and the drugs were administered. The experiment was divided into 1 mg / kg group and 0.1 mg / kg group of bispecific antibody 2N18D10-h16+IMCR, 2DP47+IMCR as negative control group, and 057G03 as positive control group, with a total of 4 groups, 6 mice in each group, and the drugs were administered by tail vein injection once a week for three times, and the observation was continued. The efficacy was evaluated according to the tumor inhibition rate TGI.

[0363] The animals were generally in good mental state during the experiment. 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 the tumors of 3 animals completely regressed, which was significantly different from 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 the tumors of 3 animals completely regressed, which was significantly different from the negative control group (Group 1) (p<0.001). The bispecific antibody 2N18D10-h16+IMCR effectively inhibited tumor growth and was better than 057G03 (Group 2) ( Fig.14 There was no animal death in each treatment group, and no obvious drug toxicity was shown.

[0364] All patents, patent application disclosures, and non-patent documents mentioned and / or listed in this application are incorporated herein by reference in their entirety. The exemplary embodiments of the inventions of this application are described above, but without departing from the essence and scope of this application, those skilled in the art can modify or improve the exemplary embodiments described in this application, and the resulting variants or equivalents also belong to the scope of this application.

[0365] Sequence information

[0366] SEQ ID NO:1

[0367] GVYDGREHTV

[0368] SEQ ID NO:2

[0369] GLYDGREHSV

[0370] SEQ ID NO:3

[0371] EVYDGREHSA

[0372] SEQ ID NO:4

[0373] ISYSGVLHAV

[0374] SEQ ID NO:5

[0375] GGYPGGIHEV

[0376] SEQ ID NO:6

[0377] YLISQVEGHQV

[0378] SEQ ID NO:7

[0379] DSYGGRHHRL

[0380] SEQ ID NO:8

[0381] VGYPGRLHLW

[0382] SEQ ID NO:9

[0383] FVYDEPGHAV

[0384] SEQ ID NO:10

[0385] SQYSGQLHEV

[0386] SEQ ID NO:11

[0387] ALYGRLEVV

[0388] SEQ ID NO:12

[0389] NTYEGRPIYV

[0390] SEQ ID NO:13

[0391] NTYEGRPIYV

[0392] SEQ ID NO:14

[0393] KIYEGAYHV

[0394] SEQ ID NO:15

[0395] RLYTGMHTV

[0396] SEQ ID NO:16

[0397] GVYAGRPLSV

[0398] SEQ ID NO:17

[0399] GVYQGRVSAV

[0400] SEQ ID NO:18

[0401] AVLDGRELRV

[0402] SEQ ID NO:19

[0403] RLYDGLFKV

[0404] SEQ ID NO:20

[0405] DVYDGRFLV

[0406] SEQ ID NO:21

[0407] SRYDGQIAV

[0408] SEQ ID NO:22

[0409] NRYDGIYKV

[0410] SEQ ID NO:23

[0411] NRYDGQVAV

[0412] SEQ ID NO:24

[0413] LMYDGTKEV

[0414] SEQ ID NO:25

[0415] HVYDGKFLARV

[0416] SEQ ID NO:26

[0417] YIYDGELVSK

[0418] SEQ ID NO:27

[0419] ATYDGQFMKL

[0420] SEQ ID NO:28

[0421] FAYDGKDYL

[0422] SEQ ID NO:29

[0423] KLYDGFQYL

[0424] SEQ ID NO:30

[0425] SVYDGKLLI

[0426] SEQ ID NO:31

[0427] YAYDGKDYI

[0428] SEQ ID NO:32

[0429] DSDMG

[0430] SEQ ID NO:33

[0431] TITTDGSTYYADSVKG

[0432] SEQ ID NO:34

[0433] DLPILRPTPCGDSVLYNY

[0434] SEQ ID NO:35

[0435] EVQLVESGGGSVQTGETLRLSCTVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSV

[0436] KGRFTISQDNAKTTAYLQMDSLKPEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTQVTVSSSEQ IDNO:36

[0437] QVQLVESGGGLVKPGGSLRLSCAVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSV

[0438] KGRFTISQDNAKTSAYLQMNSLRAEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTTVTVSSSEQ IDNO:37

[0439] GYTMN

[0440] SEQ ID NO:38

[0441] LINPYKGVSTYNQKFKD

[0442] SEQ ID NO:39

[0443] SGYYGDSDWYFDV

[0444] SEQ ID NO:40

[0445] RASQDIRNYLN

[0446] SEQ ID NO:41

[0447] YTSRLES

[0448] SEQ ID NO:42

[0449] QQGNTLPWT

[0450] SEQ ID NO:43

[0451] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ

[0452] KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSSEQ IDNO:44

[0453] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGS

[0454] GSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK

[0455] SEQ ID NO:45

[0456] QVQLVESGGGLVKPGGSLRLSCAVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSV

[0457] KGRFTISQDNAKTSAYLQMNSLRAEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTTVTVSS

[0458] GGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAVSGFTFDDSDMGWYRQAPGNECEL

[0459] VSTITTDGSTYYADSVKGRFTISQDNAKTSAYLQMNSLRAEDTAVYYCAADLPILRPTPCGDSV

[0460] LYNYWGQGTTVTVSSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0461] SQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

[0462] LPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYK

[0463] TTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0464] SEQ ID NO:46

[0465] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGS

[0466] GSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSG

[0467] GGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVST

[0468] YNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYCARSGYYGDSDWYFDVWGQGTLVTVS

[0469] SASERKSSVECPPCPAPEFEGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0470] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQP

[0471] REPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

[0472] SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0473] SEQ ID NO:47

[0474] GVYDGREHTVGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVD

[0475] LLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGG

[0476] GGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRM

[0477] EPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSD

[0478] WRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVE

[0479] WLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE

[0480] LVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE

[0481] SEQ ID NO:48

[0482] MSSEQKSQHCKPEEGVEAQEEALGLVGAQAPTTEEQEAAVSSSSPLVPGTLEEVPAAESAGPPQ

[0483] SPQGASALPTTISFTCWRQPNEGSSSQEEEGPSTSPDAESLFREALSNKVDELAHFLLRKYRAKE

[0484] LVTKAEMLERVIKNYKRCFPVIFGKASESLKMIFGIDVKEVDPASNTYTLVTCLGLSYDGLLGN

[0485] NQIFPKTGLLIIVLGTIAMEGDSASEEEIWEELGVMGVYDGREHTVYGEPRKLLTQDWVQENYL

[0486] EYRQVPGSNPARYEFLWGPRALAETSYVKVLEHVVRVNARVRIAYPSLREAALLEEEEGV

[0487] SEQ ID NO:49

[0488] GGGGSGGGGSGGGGS

[0489] SEQ ID NO:50

[0490] TCCTTCCCCGTCAGCCAGTCCT

[0491] SEQ ID NO:51

[0492] QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKE

[0493] FSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

[0494] SEQ ID NO:52

[0495] FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQ

[0496] VHYRMCQSCVELDPATVA

[0497] SEQ ID NO:53

[0498] HHHHHH

[0499] SEQ ID NO:54

[0500] ASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY

[0501] VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0502] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

[0503] LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0504] SEQ ID NO:55

[0505] ASPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVN

[0506] NVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSV

[0507] RAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYF

[0508] MYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0509] SEQ ID NO:56

[0510] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0511] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

[0512] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0513] HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF

[0514] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0515] YTQKSLSLSPGK

[0516] SEQ ID NO:57

[0517] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0518] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

[0519] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0520] HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGF

[0521] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0522] YTQKSLSLSPGK

[0523] SEQ ID NO:58

[0524] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0525] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

[0526] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0527] HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGF

[0528] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0529] YTQKSLSLSPGK

[0530] SEQ ID NO:59

[0531] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0532] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKP

[0533] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0534] HQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGF

[0535] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0536] YTQKSLSLSPGK

[0537] SEQ ID NO:60

[0538] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0539] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKP

[0540] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0541] HQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGF

[0542] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0543] YTQKSLSLSPGK

[0544] SEQ ID NO:61

[0545] ASTKGPSVFPLAPSSKTSEGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0546] SSVVTVPSSSLGTQTYICNVDHKPSNTKVDKTVERSKVECPPPCPAPEFEGGPSVFLFPPKPKDT

[0547] LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

[0548] WLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSD

[0549] IAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQK

[0550] SLSLSPGK

[0551] SEQ ID NO:62

[0552] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0553] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVERKSCVECPPCPAPEFEGGPSVFLFPPKPKDT

[0554] LMISRTPEVTVCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

[0555] WLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPS

[0556] DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ

[0557] KSLSLSPGK

[0558] SEQ ID NO:63

[0559] VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN

[0560] NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0561] SEQ ID NO:64

[0562] VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST

[0563] YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0564] SEQ ID NO:65

[0565] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS

[0566] SEQ ID NO:66

[0567] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK

[0568] SEQ ID NO:67

[0569] ANQVEQSPQSLIILEGKNVTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILDYAINTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVNRADGLYIPTFGRGTSLIVHP

[0570] SEQ ID NO:68

[0571] DVKVTQSSRYLVKRTGEKVFLECVQDAPLSKMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSSDQNSGDPYEQYFGPGTRLTVT

[0572] SEQ ID NO:69

[0573] ANQVEQSPQSLIILEGKNVTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILDYAINTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVNRADGLYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFTCANAFNNSIIPEDTFFPSPESSCGGGGSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0574] SEQ ID NO:70

[0575] DVKVTQSSRYLVKRTGEKVFLECVQDAPLSKMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSSDQNSGDPYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGGGGSASERKSCVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0576] SEQ ID NO:71

[0577] EVQLVESGGGSVQTGETLRLSCTVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSVKGRFTISQDNAKTTAYLQMDSLKPEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGSVQTGETLRLSCTVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSVKGRFTISQDNAKTTAYLQMDSLKPEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTQVTVSS

[0578] SEQ ID NO:72

[0579] QVQLVESGGGLVKPGGSLRLSCAVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSVKGRFTISQDNAKTSAYLQMNSLRAEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTTVTVSSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSVKGRFTISQDNAKTSAYLQMNSLRAEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTTVTVSS

[0580] SEQ ID NO:73

[0581] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS

[0582] SEQ ID NO:74

[0583] EVQLVESGGGSVQTGETLRLSCTVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSVKGRFTISQDNAKTTAYLQMDSLKPEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGSVQTGETLRLSCTVSGFTFDDSDMGWYRQAPGNECELVSTITTDGSTYYADSVKGRFTISQDNAKTTAYLQMDSLKPEDTAVYYCAADLPILRPTPCGDSVLYNYWGQGTQVTVSSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0584] SEQ ID NO:75

[0585] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGS

[0586] GSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSG

[0587] GGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVST

[0588] YNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVS

[0589] SGGGGSDVKVTQSSRYLVKRTGEKVFLECVQDAPLSKMFWYRQDPGLGLRLIYFSYDVKMKE

[0590] KGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSSDQNSGDPYEQYFGPGTRLTVTEDLKN

[0591] VFPPEVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPAL

[0592] NDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC

[0593] GGGGSASERKSCVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN

[0594] WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISK

[0595] AKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0596] GSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0597] SEQ ID NO:76

[0598] AQLVESGGGLVQPGGSLRLSCAASAYFSFKAMSWVRQAPGKGLEWVGSISPSGGSTYYNDNVL

[0599] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDVGFFDEWGQGTLVTVSS

[0600] SEQ ID NO:77

[0601] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYDASIRDIGVPSRFSGSG

[0602] SGTDFTLTISSLQPEDFATYYCQQYSSYPYTFGQGTKLEIK

[0603] SEQ ID NO:78

[0604] AQLVESGGGLVQPGGSLRLSCAASAYFSFKAMSWVRQAPGKGLEWVGSISPSGGSTYYNDNVL

[0605] GRFTISRDNSKNTLYLQMNSLRAEDTAVYCAKDVGFFDEWGQGTLVTVSSASTKGPSVFPLAP

[0606] SSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

[0607] QTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGSDIQMTQSPSSLSASVGDRVTITCRASQD

[0608] IRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNT

[0609] LPWTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

[0610] VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVERKSCVECPPCPAPEF

[0611] EGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

[0612] STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTK

[0613] NQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF

[0614] SCSVMHEALHNHYTQKSLSLSPGK

[0615] SEQ ID NO:79

[0616] AQLVESGGGLVQPGGSLRLSCAASAYFSFKAMSWVRQAPGKGLEWVGSISPSGGSTYYNDNVL

[0617] GRFTISRDNSKNTLYLQMNSLRAEDTAVYCAKDVGFFDEWGQGTLVTVSSASTKGPSVFPLAP

[0618] SSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

[0619] QTYICNVNHKPSNTKVDEKVERKSCVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVV

[0620] VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

[0621] NKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN

[0622] NYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0623] SEQ ID NO:80

[0624] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPCLLIYDASRIGVPSRFSGSG

[0625] SGTDFTLTISSLQPEDFATYYCQQYSSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVV

[0626] CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

[0627] VTHQGLSSPVTKSFNRGEC

[0628] SEQ ID NO:81

[0629] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ

[0630] KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSAS

[0631] VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST

[0632] YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0633] SEQ ID NO:82

[0634] AVYDGREHTV

[0635] SEQ ID NO:83

[0636] GAYDGREHTV

[0637] SEQ ID NO:84

[0638] GVADGREHTV

[0639] SEQ ID NO:85

[0640] GVYAGREHTV

[0641] SEQ ID NO:86

[0642] GVYDAREHTV

[0643] SEQ ID NO:87

[0644] GVYDGAEHTV

[0645] SEQ ID NO:88

[0646] GVYDGRAHTV

[0647] SEQ ID NO:89

[0648] GVYDGREATV

[0649] SEQ ID NO:90

[0650] GVYDGREHAV

[0651] SEQ ID NO:91

[0652] GVYDGREHTA

[0653] SEQ ID NO:92

[0654] ASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0655] SEQ ID NO:93

[0656] ASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0657] SEQ ID NO:94

[0658] MLLGQKSQRYKAEEGLQAQGEAPGLMDVQIPTAEEQKAASSSSTLIMGTLEEVTDSGSPSPPQSPEGASSSLTVTDSTLWSQSDEGSSSNEEEGPSTSPDPAHLESLFREALDEKVAELVRFLLRKYQIKEPVTKAEMLESVIKNYKNHFPDIFSKASECMQVIFGIDVKEVDPAGHSYILVTCLGLSYDGLLGDDQSTPKTGLLIIVLGMILMEGSRAPEEAIWEALSVMGLYDGREHSVYWKLRKLLTQEWVQENYLEYRQAPGSDPVRYEFLWGPRALAETSYVKVLEHVVRVNARVRISYPSLHEEALGEEKGV References

[0659] [1] Plaen, E.D., Arden, K., Traversarietal, C., et al. (1994) Structure, chromosomal localization, and expression of 12 genes of the MAGE family. Immunogenetics 40(5), 360–369.

[0660] [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.

[0661] [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.

[0662] [4].Chomez,P.,De,B.O.,Bertrand,M.,et al.(2001).An overview of themage gene family with the identification of all human members of thefamily.Cancer Research 61(14),5544-5551.

[0663] [5]Simpson,A.,Caballero,O.L.,Jungbluth,A.,et al.(2005)Cancer / testisantigens,gametogenesis and cancer.Nature Reviews Cancer 5(8),615.

[0664] [6]Sharma,P.,Shen,Y.,Wen,S.,et al.(2006)Cancer-testis antigens:expression and correlation with survival in human urothelialcarcinoma.Clinical Cancer Research an Official Journal of the AmericanAssociation for Cancer Research 12(18),5442-5447.

[0665] [7]Bergeron,A.,Valérie,P.,Hélène,L.,et al.(2010)High frequency ofmage-a4 and mage-a9expression in high-risk bladder cancer.InternationalJournal of Cancer 125(6),1365-1371.

[0666] [8]Peikert,T.,Specks,U.,Farver,C.,et al.,(2006)Melanoma antigen A4 isexpressed in non-small celllung cancers and promotes apoptosis.CancerResearch 66(9),4693–4700.

[0667] [9]Yakirevich,E.,Sabo,E.,Lavie,O.,et al.(2003)Expression of the MAGE-A4 and NY-ESO-1cancer-testis antigens in serous ovarian neoplasms.ClinicalCancer Research an Official Journal of the American Association for CancerResearch 9(17),6453-6460.

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[10] Quillien,V.,Raoul,J.L.,Heresbach,D.,et al.(1997)Expression ofMAGE 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 ofthe expression of melanoma antigen-encoding genes(MAGE-A1 to-A6)in oralsquamous cell carcinomas to determine potential targets for gene-based cancerimmunotherapy.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 peptidepresented by HLA-A02is recognized by cytolytic T lymphocytes.European Journalof 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-cellreceptor 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-4antigenic peptide recognized by cytolytict lymphocytes on HLA-A024 carcinomacells.Cancer Immunology Immunotherapy 55(7),867-872.

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[15] Sung,H.,Ferlay,J.,Siegel,R.L.,et al.(2021)Global CancerStatistics 2020:GLOBOCAN Estimates of Incidence and Mortality Worldwide for36Cancers in 185Countries.CA:a cancer journal for clinicians71(3),209-249.

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

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[17] E., Poignavent, V., Vincke, C., et al. (2018) Ritzenthaler C, Muyldermans S, Monsion B. Construction of High-Quality Camel Immune AntibodyLibraries. Methods Mol Biol 1701:169-187.

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[18] CN 201510097117.0

[0677]

[19] Phage display: A general experimental guide / (US) Clarkson, T., (US) Lowman, HB; (US) Translated by Ma Lan et al. Chemical Industry Press, 2008.5.

[0679]

[20] US20160200833A1

[0680]

[21] WO 2017175006A1

[0681]

[22] Tan, P., Mitchell, D, A., Buss, T, N., et al. (2022) "Superhumanized" antibodies: reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences: application to an anti-CD28. JImmunol 169(2):1119-1125.

[0682]

[23] US 5821337A

[0683]

[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; Preferably, the binding epitope of the first antigen-binding fragment to the HLA-A02 / MAGE-A4 complex includes residues 230-239 of MAGE-A4 as shown in SEQ ID NO:48; more preferably, the binding epitope of the first antigen-binding fragment to the HLA-A02 / MAGE-A4 complex includes at least one of residues 232, 234, 235, 237 and 239 of MAGE-A4 as shown in SEQ ID NO:48; and / or The binding epitope of the first antigen-binding fragment to the HLA-A02 / MAGE-A8 complex includes positions 232-241 of MAGE-A8 shown in SEQ ID NO: 94; and / or Preferably, the activating T cell antigen is a CD3 molecule.

2. The bispecific antibody according to claim 1, wherein 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; preferably, the first antigen-binding fragment is in the form of a single-domain antibody; more preferably, the first antigen-binding fragment comprises a single-domain antibody that binds to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex in a monovalent or multivalent manner; and / or The second antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:37, HCDR2 as shown in SEQ ID NO:38, HCDR3 as shown in SEQ ID NO:39, LCDR1 as shown in SEQ ID NO:40, LCDR2 as shown in SEQ ID NO:41, and LCDR3 as shown in SEQ ID NO:42; preferably, the second antigen-binding fragment is a single-chain antibody (scFv) or a Fab fragment; more preferably, the second antigen-binding fragment is a scFv; in, The amino acid sequences of the HCDRs are according to the Kabat definition.

3. The bispecific antibody of claim 1 or 2, 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; Optionally, the first antigen-binding fragment comprises two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex; preferably, the first antigen-binding fragment comprises two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A02 / MAGE-A4 complex and / or the HLA-A02 / MAGE-A8 complex connected by a linker; more preferably, the linker is (GGGGS) n , wherein n is an integer ≥1, for example GGGGSGGGGSGGGGS (SEQ ID NO: 49).

4. The bispecific antibody according to any one of claims 1 to 3, wherein The first antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 35, 36, 71 or 72; and / or The second antigen-binding fragment comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:43 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:44; Preferably, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:35; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO:43 and the light chain variable region with the amino acid sequence shown in SEQ ID NO:44; The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 36; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 43 and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 44; or The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 71; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 43 and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 44; or The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:72; and the second antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO:43 and the light chain variable region shown in SEQ ID NO:

44.

5. The bispecific antibody according to any one of claims 1 to 4, wherein The first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, and the antibody heavy chain constant region Fc fragment includes a first Fc fragment and a second Fc fragment; preferably, the antibody heavy chain constant region Fc fragment is an Fc fragment of the IgG1 subtype; more preferably, the antibody heavy chain constant region Fc fragment is an Fc fragment of the IgG1m3 subtype; 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; preferably, 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; 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; in, The amino acid positions of antibody constant regions are determined according to EU numbering.

6. The bispecific antibody according to any one of claims 1 to 5, 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 the amino acid sequence shown in SEQ ID NO: 45 or 74; and The second arm comprises the amino acid sequence shown in SEQ ID NO:

46.

7. A single domain antibody that binds to an HLA-A02 / MAGE-A4 complex and / or an HLA-A02 / MAGE-A8 complex, comprising 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; Preferably, the single-domain antibody comprises an amino acid sequence as shown in SEQ ID NO: 35, 36, 71 or 72; in, The amino acid sequences of the HCDRs are according to the Kabat definition.

8. A nucleic acid molecule encoding the bispecific antibody of any one of claims 1 to 6 or the single domain antibody of claim 7.

9. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 6 or the single domain antibody according to claim 7 and a pharmaceutically acceptable excipient, diluent or carrier.

10. Use of the bispecific antibody of any one of claims 1 to 6, the single domain antibody of claim 7 or the pharmaceutical composition of claim 9 in the preparation of a medicament for preventing or treating HLA-A02 / MAGE-A4-positive tumors and / or HLA-A02 / MAGE-A8-positive tumors; preferably, the HLA-A02 / MAGE-A4-positive tumors and / or HLA-A02 / MAGE-A8-positive tumors are selected from: esophageal cancer, urothelial carcinoma, 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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