Multiple antibodies that bind to the HLA-A24 / PRAME complex and their use

Abstract

The present invention provides a bispecific antibody comprising a first antigen-binding fragment that binds to the HLA-A24 / PRAME complex and a second antigen-binding fragment that binds to an activated T cell antigen, a single-domain antibody that binds to the HLA-A24 / PRAME complex, and a pharmaceutical composition comprising the bispecific antibody or the single-domain antibody, as well as the use thereof.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to Chinese Patent Application No. 202311429311.5, filed on 31 October 2023, the entirety of which is incorporated herein by reference.

[0002] This invention generally relates to the fields of genetic engineering and biopharmaceuticals, and more specifically to bispecific antibodies that bind to the HLA-A24 / PRAME complex, single-domain antibodies that bind to the HLA-A24 / PRAME complex, and their uses. [Background technology]

[0003] Most proteins in the human proteome reside within cells. These intracellular proteins are degraded by proteasomes, and some of the resulting short peptides are presented on the cell surface by MHC (major histocompatibility complex) molecules, making them potential targets for cancer immunotherapy. Antibodies targeting these MHC / peptide complexes are developed to mimic the function of TCRs in recognizing MHC / peptide complexes. These antibodies are called T cell receptor mimetic molecules (TCRm) or TCR-like antibodies.

[0004] The Preferentially Expressed Antigen in Melanoma (PRAME) belongs to the cancer / testicular antigen (CTA) gene family and is expressed at extremely low levels in normal adult tissues other than the testes, but at high levels in various cancer cells. [1] The PRAME gene is located on the reverse strand of chromosome 22 (22q11.22), is approximately 12 kilobases long, and contains a leucine-rich repeat domain. [2] While the PRAME gene is reported to be hypermethylated in normal tissues, it is hypomethylated in most malignant cells. [3] .

[0005] PRAME is highly expressed in 88% of primary melanoma tissue and 95% of metastatic melanoma tissue. [1] In addition to melanoma, it is widely expressed in many solid tumors, including head and neck cancer, breast cancer, renal cell carcinoma, and non-small cell lung cancer. [4-7] PRAME is not detected in normal hematopoietic tissue, but high expression has been reported in acute leukemia, chronic leukemia, and Hodgkin lymphoma. [8~10] Furthermore, PRAME expression levels are a prognostic biomarker for poor clinical outcomes in breast cancer and neuroblastoma. [11、12] .

[0006] PRAME is broken down into small polypeptides within the proteasome, and some of these peptides bind to MHC molecules to form complexes that are presented on the cell surface. LYVDSLFFL corresponds to amino acids 301-309 of the full-length PRAME protein and is present on the cell surface in complex with HLA-A24.

[13] This HLA-peptide complex provides a useful target for TCRm-based immunotherapy interventions.

[0007] Bispecific antibodies (BsAbs) are artificial antibodies that possess two different antigen-binding sites. Bispecific antibodies are widely used in the biopharmaceutical field, particularly in tumor immunotherapy. A CD3-targeting bispecific antibody has one arm that binds to CD3 in the TCR receptor complex on the surface of T cells, providing a primary signal to activate T cells, while the other arm targets tumor antigens. This brings tumor cells and T cells closer together, enabling T cell activation and simultaneous direct damage to tumor cells.

[0008] TCRm, which binds to PRAME-like intracellular proteins, can function as a component that binds to the target antigen in CD3 bispecific antibodies. This type of bispecific antibody may be a more attractive option due to its ability to restrict the expression of the target antigen. Based on clinical needs, the exploration and development of bispecific antibodies targeting PRAME-MHC has significant clinical importance. [Overview of the Initiative]

[0009] According to a first aspect, the present invention provides a bispecific antibody comprising a first antigen-binding fragment that binds to the HLA-A24 / PRAME complex and a second antigen-binding fragment that binds to an activated T cell antigen.

[0010] In some embodiments of the first aspect, the bispecific antibody is HLA-A24 + / PRAME + capable of mediating activation of T cells by tumor cells, and / or the bispecific antibody is capable of mediating killing of HLA-A24 + / PRAME + tumor cells by PBMCs (peripheral blood mononuclear cells).

[0011] In some embodiments of the first aspect, the first antigen-binding fragment comprises HCDR1 set forth in SEQ ID NO: 1, HCDR2 set forth in SEQ ID NO: 2, and HCDR3 set forth in SEQ ID NO: 3, wherein the amino acid sequences of the HCDRs are defined by Kabat. In some embodiments of the first aspect, the first antigen-binding fragment is in the form of a single domain antibody. 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-A24 / PRAME complex.

[0012] In some embodiments of the first aspect, the second antigen-binding fragment comprises HCDR1 set forth in SEQ ID NO: 6, HCDR2 set forth in SEQ ID NO: 7, HCDR3 set forth in SEQ ID NO: 8, LCDR1 set forth in SEQ ID NO: 9, LCDR2 set forth in SEQ ID NO: 10, and LCDR3 set forth in SEQ ID NO: 11, wherein the amino acid sequences of the HCDRs are defined by Kabat.

[0013] In some embodiments of the first aspect, the second antigen-binding fragment is in the form of a single-chain antibody (scFv) or a Fab fragment. In some embodiments of the first aspect, the first antigen-binding fragment comprises a bivalent single-domain antibody bound to the HLA-A24 / PRAME complex, and optionally, the first antigen-binding fragment comprises heavy chain variable regions of two monovalent single-domain antibodies bound to the HLA-A24 / PRAME complex.

[0014] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 4, 5, 14, or 15, and / or The second antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown as SEQ ID NO:12 and a light chain variable region whose amino acid sequence is shown as SEQ ID NO:13.

[0015] In some embodiments of the first aspect, the first antigen-binding fragment comprises an amino acid sequence shown in SEQ ID NO:4, and the second antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:12 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:13. The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:5, and the second antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:12 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:13, or The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:14, and the second antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:12 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:13, or The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:15, and the second antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:12 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:13.

[0016] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are linked via an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising the first Fc fragment and the 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, where the amino acid positions of the constant region of the antibody are determined according to EU numbering.

[0017] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are linked via an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising the first Fc fragment and the second Fc fragment, wherein the amino acids at positions 234, 235, and 331 of the first Fc fragment and the second Fc fragment are F, E, and S, respectively, where the amino acid positions of the constant region of the antibody are determined according to EU numbering.

[0018] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are linked via an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising the first Fc fragment and the second Fc fragment, wherein either the first Fc fragment or the second Fc fragment is linked to the first antigen-binding fragment, and the other of the first Fc fragment or the second Fc fragment is linked to the second antigen-binding fragment.

[0019] In some embodiments of the first aspect, the bispecific antibody comprises a first arm that binds to the HLA-A24 / PRAME complex and a second arm that binds to the activated T cell antigen, where, The first arm comprises the amino acid sequence shown in SEQ ID NO: 35 or 36, The second arm contains the amino acid sequence shown in SEQ ID NO:37.

[0020] According to a second aspect, the present invention provides a single-domain antibody that binds to the HLA-A24 / PRAME complex, which, HCDR1, indicated by SEQ ID NO:1 HCDR2, as shown in SEQ ID NO:2, and Contains HCDR3 as indicated by SEQ ID NO:3. Here, the amino acid sequence of HCDR is defined by Kabat.

[0021] In some embodiments of the second aspect, the binding epitope of the single-domain antibody to the HLA-A24 / PRAME complex includes one or more residues from positions 301-309 of PRAME, as shown in SEQ ID NO:38. In some embodiments of the second aspect, the single-domain antibody is in the form of a monovalent or polyvalent single-domain antibody that binds to the HLA-A24 / PRAME complex. In some embodiments of the second aspect, the single-domain antibody is in the form of a bivalent single-domain antibody that binds to the HLA-A24 / PRAME complex. In some embodiments of the second aspect, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO: 4, 5, 14, or 15.

[0022] According to a third aspect, the present invention provides a nucleic acid molecule encoding a bispecific antibody as described in the first aspect or a single-domain antibody as described in the second aspect.

[0023] According to a fourth aspect, the present invention provides a pharmaceutical composition comprising a bispecific antibody described in the first aspect or a single-domain antibody described in the second aspect, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0024] According to a fifth aspect, the present invention provides the use of the bispecific antibody described in the first aspect, the single-domain antibody described in the second aspect, or the pharmaceutical composition described in the fourth aspect in the manufacture of a pharmaceutical for the prevention or treatment of HLA-A24 / PRAME-positive tumors.

[0025] According to a sixth aspect, the present invention provides the use of the bispecific antibody described in the first aspect or the single-domain antibody described in the second aspect in the manufacture of a product for detecting HLA-A24 / PRAME-positive cells. [Brief explanation of the drawing]

[0026] [Figure 1] This figure shows the results of antibodies against the HLA-A24 / PRAME complex binding to HT-29 cells pulsed with PRAME301-309 (LYVDSLFFL). [Figure 2] This figure shows the results of the activation of Jurkat-Dual cells by tumor cells, mediated by the bispecific antibody 2N83G3×IMCR and the humanized bispecific antibody 2N83G3-h5×IMCR. [Figure 3]This figure shows the results of tumor cell damage by PBMCs mediated by the humanized bispecific antibody 2N83G3-h5×IMCR. [Figure 4] This figure shows the activation effect of HT-29 cells pulsed with alanine-substituted peptides on Jurkat-Dual cells, mediated by the humanized bispecific antibody 2N83G3-h5×IMCR. [Figure 5] This figure shows the results of injury to HT-29 cells transfected with PRAME or similar peptide segments 8 / 11 / 12 / 16 genes by PBMCs, mediated by the humanized bispecific antibody 2N83G3-h5×IMCR. [Figure 6] This figure shows the results of injury to 293T cells transfected with RAME or similar peptide segments 8 / 11 / 12 / 16 genes by PBMC cells, mediated by the humanized bispecific antibody 2N83G3-h5×IMCR. [Figure 7] This figure shows the results of the antitumor activity of the humanized bispecific antibody 2N83G3-h5×IMCR in a PBMC immune reconstitution model mouse.

[0027] [Explanation of the sequence] SEQ ID NO:1-3 represent the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable regions of the single-domain antibodies N83G3 and N83G3-h5 for Llama, respectively. SEQ ID NO:4 shows the amino acid sequence of the heavy chain variable region of the Llama single-domain antibody N83G3. SEQ ID NO:5 shows the amino acid sequence of the heavy chain variable region of the humanized single-domain antibody N83G3-h5. SEQ ID NO:6~8 shows the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the anti-human CD3 antibody IMCR, respectively.

[0028] SEQ ID NO:9~11 shows the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the anti-human CD3 antibody IMCR, respectively. SEQ ID NO:12 shows the amino acid sequence of the heavy chain variable region of the anti-human CD3 antibody IMCR. SEQ ID NO:13 shows the amino acid sequence of the light chain variable region of the anti-human CD3 antibody IMCR. SEQ ID NO:14 shows the amino acid sequence in which the heavy chain variable regions of two llama single-domain antibodies N83G3 are tandem-linked. SEQ ID NO:15 shows the amino acid sequence in which the heavy chain variable regions of two llama single-domain antibodies N83G3-h5 are tandem-linked. SEQ ID NO:16 shows the amino acid sequence of the single-chain antibody of the anti-human CD3 antibody IMCR.

[0029] SEQ ID NO:17 shows the amino acid sequence of the extracellular domain of human (homo sapiens) CD3E (hCD3E). SEQ ID NO:18 shows the amino acid sequence of the extracellular region of human (homo sapiens) CD3D (hCD3D). SEQ ID NO:19 is a human (homo sapiens) derived antigen peptide PRAME 301-309 The amino acid sequence is shown. SEQ ID NO:20 is the antigen peptide PRAME 301-309 The amino acid sequence of the disulfide-trapped single-strand trimer complex (MIP-P3) of HLA-A24 is shown. SEQ ID NO:21 indicates the amino acid sequence of the His tag. SEQ ID NO:22 shows the amino acid sequence of the Fc segment (mFc) of the mouse (mus musculus) IgG2a antibody.

[0030] SEQ ID NO:23 shows the amino acid sequence of the constant region of the heavy chain of human (homo sapiens) IgG1 subtype. SEQ ID NO:24 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1H. SEQ ID NO:25 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1K. SEQ ID NO:26 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-H. SEQ ID NO:27 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-K. SEQ ID NO:28 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-H1n1.

[0031] SEQ ID NO:29 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-Kn1. SEQ ID NO:30 shows the amino acid sequence of the constant region of the human (homo sapiens) λ isotype light chain. SEQ ID NO:31 represents the amino acid sequence of the constant region of the human (homo sapiens) κ isotype light chain. SEQ ID NO:32 shows the amino acid sequence of the Fc segment (hFc) of the human (homo sapiens) IgG1 antibody. SEQ ID NO:33 shows the amino acid sequence of the human IgG1 subtype antibody Fc segment variant IgG1m3-FcH1n1. SEQ ID NO:34 shows the amino acid sequence of the human IgG1 subtype antibody Fc segment variant IgG1m3-FcKn1. SEQ ID NO:35 shows the amino acid sequence of 2N83G3-G1m3-FcH1n1 in the bispecific antibody 2N83G3×IMCR.

[0032] SEQ ID NO:36 shows the amino acid sequence of 2N83G3-h5-G1m3-FcH1n1 in the bispecific antibody 2N83G3-h5×IMCR. SEQ ID NO:37 shows the amino acid sequence of arm IMCR-scFv-G1m3-FcKn1 in the bispecific antibody 2N83G3×IMCR or 2N83G3-h5×IMCR. SEQ ID NO:38 shows the amino acid sequence of human (homo sapiens) PRAME. SEQ ID NO:39 shows the amino acid sequence of XCR1 derived from human (homo sapiens). SEQ ID NO:40 shows the amino acid sequence of KIFBP derived from human (homo sapiens).

[0033] SEQ ID NO:41 shows the amino acid sequence of CPVL derived from human (homo sapiens). SEQ ID NO:42 shows the amino acid sequence of PLD1 derived from human (homo sapiens). SEQ ID NO:43 shows the amino acid sequence of HLA-A2402. SEQ ID NO:44 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 1 is shown. SEQ ID NO:45 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 2 is shown.

[0034] SEQ ID NO:46 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 3 is shown. SEQ ID NO:47 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 4 is shown. SEQ ID NO:48 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 5 is shown. SEQ ID NO:49 is derived from human (homo sapiens) PRAME 301-309The amino acid sequence of similar peptide segment 6 is shown. SEQ ID NO:50 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 7 is shown.

[0035] SEQ ID NO:51 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 8 is shown. SEQ ID NO:52 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 9 is shown. SEQ ID NO:53 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 10 is shown. SEQ ID NO:54 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 11 is shown. SEQ ID NO:55 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 12 is shown.

[0036] SEQ ID NO:56 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 13 is shown. SEQ ID NO:57 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 14 is shown. SEQ ID NO:58 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 15 is shown. SEQ ID NO:59 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 16 is shown. SEQ ID NO:60 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 17 is shown.

[0037] SEQ ID NO:61 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 18 is shown. SEQ ID NO:62 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 19 is shown. SEQ ID NO:63 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 20 is shown. SEQ ID NO:64 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 21 is shown. SEQ ID NO:65 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 22 is shown.

[0038] SEQ ID NO:66 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 23 is shown. SEQ ID NO:67 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 24 is shown. SEQ ID NO:68 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 25 is shown. SEQ ID NO:69 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 26 is shown. SEQ ID NO:70 is derived from human (homo sapiens) PRAME 301-309The amino acid sequence of similar peptide segment 27 is shown.

[0039] SEQ ID NO:71 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 28 is shown. SEQ ID NO:72 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 29 is shown. SEQ ID NO:73 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 30 is shown. SEQ ID NO:74 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 31 is shown. SEQ ID NO:75 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 32 is shown.

[0040] SEQ ID NO:76 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 33 is shown. SEQ ID NO:77 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 34 is shown. SEQ ID NO:78 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 35 is shown. SEQ ID NO:79 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 36 is shown. SEQ ID NO:80 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 37 is shown.

[0041] SEQ ID NO:81 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 38 is shown. SEQ ID NO:82 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 39 is shown. SEQ ID NO:83 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 40 is shown. SEQ ID NO:84 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 41 is shown. SEQ ID NO:85 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 42 is shown.

[0042] SEQ ID NO:86 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 43 is shown. SEQ ID NO:87 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 44 is shown. SEQ ID NO:88 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 45 is shown. SEQ ID NO:89 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 46 is shown. SEQ ID NO:90 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 47 is shown. SEQ ID NO:91 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 48 is shown.

[0043] SEQ ID NO:92 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequence of similar peptide segment 49 is shown. SEQ ID NO:93 is derived from human (homo sapiens) PRAME 301-309 The amino acid sequences of similar peptide segments 50 are shown. SEQ ID NO:94 indicates the amino acid sequence of the linker. SEQ ID NO:95 shows the nucleotide sequence of primer PCal-CH2R. SEQ ID NO:96 shows the amino acid sequence of the negative control antibody DP47VH. SEQ ID NO:97 shows the amino acid sequence of the negative control antibody DP47VK. SEQ ID NO:98 shows the amino acid sequence of an unrelated peptide. Detailed description of the invention

[0044] The inventors of this application prepared a bispecific antibody (e.g., a TCRm bispecific antibody) by combining a first antigen-binding fragment that binds to the HLA-A24 / PRAME complex with a second antigen-binding fragment that binds to activated T cell antigens (e.g., CD3 molecules) using genetic engineering techniques. The first antigen-binding fragment that binds to the HLA-A24 / PRAME complex is PRAME, which is presented on the surface of target cells (tumor cells). 301-309The first antigen-binding fragment binds to the HLA-A24 / PRAME complex, while the second antigen-binding fragment binds to an activated T cell antigen (e.g., the CD3 molecule), thereby establishing an interaction between target cells and T cells, inducing activation of cytotoxic T cells, lysing target cells (e.g., tumor cells) in a histocompatibility complex (MHC)-dependent manner, and achieving the objective of treating a disease (e.g., a tumor). Various aspects of this application provide a novel bispecific antibody comprising a first antigen-binding fragment that binds to the HLA-A24 / PRAME complex and a second antigen-binding fragment that binds to an activated T cell antigen (e.g., the CD3 molecule), a single-domain antibody that binds to the HLA-A24 / PRAME 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 vector, a method for producing and purifying the bispecific antibody or the single-domain antibody, and medical and biological applications of the bispecific antibody or the single-domain antibody. Based on the sequence of the bispecific antibody or single-domain antibody according to the present invention, a bispecific antibody or single-domain antibody that binds to the HLA-A24 / PRAME complex can be constructed and used clinically as a pharmaceutical to prevent or treat tumors.

[0045] Unless otherwise specified, the implementation of this invention utilizes conventional molecular biology, microbiology, cell biology, biochemistry, and immunology techniques in the art. Unless otherwise specified, terms used in this application have the meanings generally understood by those skilled in the art.

[0046] [Definition] As used herein, the term "HLA-A24 / PRAME complex" refers to a complex of the HLA-A24 molecule and PRAME. In the specific embodiments of this application, the "HLA-A24 / PRAME complex" refers to a complex of the HLA-A24 molecule and the PRAME molecule at positions 301-309 as shown in SEQIDNO:38 (PRAME 301-309 ) and HLA-A24 / PRAME 301-309 It refers to a complex.

[0047] As used herein, the term "activated 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 by interacting with antigen-binding molecules. Specifically, the interaction between antigen-binding molecules and activated T cell antigens can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. In certain embodiments, the activated T cell antigen is the CD3 molecule.

[0048] As used herein, the term “antibody” 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, etc. As used herein, “antibody” includes not only intact antibodies (i.e., full-length antibodies), but also their binding fragments (e.g., Fab, Fab', F(ab')2, Fv), their variants, fusion proteins containing the antibody moiety, 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 containing an antigen recognition site with the desired specificity, such as glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0049] Typically, intact antibodies or full-length antibodies contain two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and 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). Full-length antibodies may be antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or the subclasses listed above), but antibodies do not need to belong to any particular class. Immunoglobulins can be designated into 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. Some of these classes can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are publicly known.

[0050] As used herein, the term "bispecific antibody" refers to an antibody that has the ability to simultaneously bind to two different antigenic epitopes. The two antigenic epitopes may be located on different antigens or on the same antigen. A bispecific antibody may have multiple structural configurations. For example, a bispecific antibody may consist of two Fc fragments and two binding sites that fuse to each of them (similar to a native antibody except that the two arms bind to different antigenic targets or epitopes), and the antigen-binding sites may be single-domain antibodies, single-chain antibodies (scfv), or Fab fragments. When targeting epitopes of two specific antigens, the two different binding sites of the bispecific antibody each bind to the N-terminus of a single Fc fragment, and the antigen-binding sites of the two arms can be in four combinations: single-domain antibody + Fab fragment, single-domain antibody + scFv, scFv + single-domain antibody, and Fab fragment + single-domain antibody. Fc fragments can contain mutations that ensure heavy chain heteromerization, and KIH (knob-in-hole) technology is a strategy to address heavy chain heteropolymerization. Typically, KIH technology refers to a technique that modifies the amino acid sequence of the CH3 region to form a structure favorable to pairing heterologous incomplete antibodies, thereby maintaining the structure of the normal antibody as much as possible while constructing a bispecific antibody. 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.

[14] Please refer to the following, the full text of which is incorporated herein by reference.

[0051] As used herein, the terms “binding region” or “binding fragment” are used interchangeably and refer to a portion or region of an intact antibody molecule involved in binding to an antigen. The antigen-binding domain may include a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL typically contains three complementarity-determining regions: CDR1, CDR2, and CDR3.

[0052] Those skilled in the art know that the complementarity-determining region (CDR, usually consisting of CDR1, CDR2, and CDR3) is the region within the variable region that has the greatest influence on the affinity and specificity of the antibody. There are two common methods for defining VH or VL CDR sequences: the Kabat definition and the Chothia definition. (For example, "Kabat, “Sequences of Proteins of Immunological Interest”, National Institutes of Health, Bethesda, Md. (1991)")

[15] ”, “A1-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997)

[16] " and "Martin et al., Proc. Natl. Acad. Sci.USA86: 9268-9272 (1989)

[17] (See "). For the variable region sequence of a particular antibody, the CDR region sequence in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In embodiments of the present invention, Kabat is used to define the CDR sequence.

[0053] The variable region sequence of a specific antibody can be analyzed using various methods, such as the CDR sequence of the variable region, which can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0054] For typical antibodies, examples of antigen-binding fragments include, but are not limited to, (1) a Fab fragment which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab')2 fragment which may be a bivalent fragment having two Fab' fragments linked by a disulfide crosslink in the hinge region (i.e., a dimer of Fab'); (3) an Fv fragment having VL and VH domains of a single arm of the antibody; (4) a single-chain Fv(scFv) which may be a single polypeptide chain composed of a VH domain and a VL domain via a peptide linker; (5) a (scFv)2 which may include two VH domains linked by a peptide linker and two VL domains bound to the two VH domains via a disulfide crosslink; and (6) a single-domain antibody. In the construction of bispecific antibodies, the “binding site” includes, but is not limited to, the form of a single-domain antibody, a Fab fragment, and / or a single-chain antibody (scFv).

[0055] As used herein, the term "single-chain fragment variable (scFv)" refers to an antibody with a single-chain structure, typically constructed using genetic engineering techniques, consisting of a polypeptide chain containing a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker (a linking peptide) is usually designed between the heavy chain variable region and the light chain variable region to ensure that the two regions fold into the correct conformation for binding to an antigen.

[0056] As used herein, the terms “Fab (fragment antigen binding) fragment,” “Fab moiety,” or similar terms refer to an antigen-binding antibody fragment produced by treating an intact antibody with papain, which includes the intact light chain (VL-CL), heavy chain variable region, and CH1 fragment (VH-CH1).

[0057] As used herein, the term "single-domain antibody" refers to a single variable-domain antibody of a heavy chain that naturally lacks a light chain. Such antibodies contain a heavy chain variable region (VHH) and the usual CH2 and CH3 regions (e.g., one or two groups of heavy chain variable regions (VHH) and the usual CH2 and CH3 regions). A VHH structure cloned and expressed alone is the smallest known unit capable of binding to a target antigen, possessing structural stability and antigen-binding activity comparable to that of the original heavy chain antibody. Single-domain antibodies are also called nanobodies (Nb). As used herein, the terms “Fc fragment,” “Fc domain,” and “Fc portion” are used interchangeably and refer to a portion of the constant region of the antibody heavy chain, including the hinge region, the CH2 fragment of the heavy chain constant region, and the CH3 fragment, as determined according to the EU numbering of human IgG1 antibodies.

[0058] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope. As used herein, the term "tumor" refers to a neoplasm or solid lesion formed by abnormal cell proliferation. Tumors may be benign, precancerous, or malignant.

[0059] As used herein, the term “malignant tumor” refers to or describes a physiological condition in mammals typically characterized by uncontrolled cell proliferation. Exemplary malignant tumors include carcinoma, solid tumors, melanoma, sarcoma, hematological malignancies, germ cell tumors, and blastomas. More specific examples of malignant tumors include melanoma, non-small cell lung cancer, small cell lung cancer, breast cancer (e.g., triple-negative breast cancer), renal cell carcinoma, esophageal cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), cervical cancer, bladder cancer, hepatocellular carcinoma, gastric cancer, Hodgkin lymphoma, neuroblastoma, endometrial cancer, or uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, multiple myeloma, B-cell lymphoma, brain tumors, and associated metastases.

[0060] As used herein, the term “hematological malignancy” refers to a tumor caused by the uncontrolled growth and proliferation of abnormal cells. Often, these abnormal cells originate in the bone marrow, which is also where blood cells are produced. Exemplary hematological malignancies include various leukemias, multiple myeloma, and malignant lymphoma. More specific examples of hematological malignancies include acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute granulocytic leukemia, chronic granulocytic leukemia, pilocytic cell leukemia (HCL), pre-T-cell lymphocytic leukemia, macrogranulol lymphocytic leukemia, juvenile myelomonocytic leukemia, pre-B-cell lymphocytic leukemia, Burkitt's leukemia, and adult T-cell leukemia, non-Hodgkin lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoblastic lymphoma, lymphoplasmacytic lymphoma, and primary macroglobulinemia. JPEG2026536595000001.jpg10170, splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, intranodal 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 exudative lymphoma, Burkitt lymphoma, B-cell chronic lymphocytic lymphoma, classical Hodgkin's disease Examples include NK / T-cell lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, adult T-cell lymphoma, extranodal nasal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Celgene syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disorder, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, nonspecific peripheral T-cell lymphoma, and anaplastic large cell lymphoma.

[0061] As used herein, the term "solid tumor" refers to a tangible mass (lump) that can be detected (palpated) by clinical examinations such as X-ray, CT scan, B-ultrasound, and palpation. Clinically diagnosed and treated solid tumors are classified into two types: malignant and benign. Malignant solid tumors include melanoma (metastatic melanoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), esophageal cancer (e.g., esophageal squamous cell carcinoma), urothelial carcinoma, oral cancer (e.g., oral squamous cell carcinoma), sarcoma (e.g., synovial sarcoma), liver cancer, lung cancer, bladder cancer, ovarian cancer, colorectal cancer, breast cancer, pediatric Hodgkin lymphoma (lymphocyte-dominant type, nodular sclerosis type, mixed cell type, lymphopenic type), and pediatric non-Hodgkin lymphoma (prolymphoblastic lymphoma). Lymphoma, small non-incisional nuclear cell lymphoma (Burkitt lymphoma / non-Burkitt lymphoma), diffuse large B-cell lymphoma, anaplastic large cell lymphoma, etc., pediatric renal tumors (nephroblastoma (Wilms tumor), clear cell carcinoma, rhabdomyosarcomatoid tumor of the kidney, clear cell sarcoma of the kidney, primordial neuroectodermal tumor of the kidney, etc.), pediatric neuroblastoma (neuroblastoma, ganglioneuroblastoma, ganglioneuroma), pediatric extracranial germ cell tumors (mature malformations) Tumors, immature teratomas, endoderm sinus tumors (yolk sac tumors), seminomas (cytomas), dyserodermal tumors (asexual cell tumors), choriocarcinoma (choriocarcinoma), embryonic carcinoma, etc.), osteosarcoma and chondrosarcoma, pediatric rhabdomyosarcoma (embryonic, acinar, pleomorphic, etc.), soft tissue sarcomas (fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant schwannoma, alveolar soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma, malignant melanoma) These include synovial sarcoma, fibrous round cell tumors, etc., Ewing sarcoma family tumors (Ewing sarcoma, primitive neuroectodermal tumors), pediatric liver tumors (hepatoblastoma (embryonic, fetal, undifferentiated), hepatocellular carcinoma), retinoblastoma, and other tumors (posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, thymoma, pulmonary blastoma, pancreatic blastoma, islet cell tumor, ileocecal carcinoid tumor, mesothelioma, etc.). Benign solid tumors include lymphangiomas, hemangiomas, and thyroglossal duct cysts.

[0062] According to a first aspect, the present invention provides a bispecific antibody comprising a first antigen-binding fragment that binds to the HLA-A24 / PRAME complex and a second antigen-binding fragment that binds to an activated T cell antigen. In some embodiments of the first aspect, the bispecific antibody is HLA-A24 + / PRAME + It can mediate T cell activation by tumor cells, and / or the bispecific antibody can mediated HLA-A24 by PBMCs (peripheral blood mononuclear cells). + / PRAME + It can mediate damage to tumor cells. In some embodiments of the first aspect, the bispecific antibody is HLA-A24 + / PRAME + It can mediate the activation of Jurkat-Dual cells by tumor cells.

[0063] In some embodiments of the first aspect, the binding epitope between the first antigen-binding fragment and the HLA-A24 / PRAME complex includes one or more residues from positions 301-309 of PRAME, shown in SEQ ID NO:38. In some embodiments, the amino acid sequence from positions 301-309 of PRAME, shown in SEQ ID NO:38, is LYVDSLFFL (SEQ ID NO:19).

[0064] In some embodiments of the first aspect, the binding epitope between the first antigen-binding fragment and the HLA-A24 / PRAME complex includes at least one of the residues at positions 304, 305, 306, 307, 308, and 309 of PRAME, as shown in SEQ ID NO:38.

[0065] In some specific embodiments of the first embodiment, the binding epitope between the first antigen-binding fragment and the HLA-A24 / PRAME complex includes residues at positions 304, 305, 306, 307, 308, and 309 of PRAME, shown in SEQ ID NO:38. In some embodiments, the residues at positions 304, 305, 306, 307, 308, and 309 of PRAME, shown in SEQ ID NO:38, include residues at positions 304, 305, 306, 307, 308, and 309 of PRAME, shown in SEQ ID NO:19. 301-309This corresponds to the amino acid residues at positions 4, 5, 6, 7, 8, and 9 of the (LYVDSLFFL) polypeptide.

[0066] In some embodiments of the first aspect, the activated T cell antigen is a CD3 molecule. In some embodiments of the first aspect, the first antigen-binding fragment comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:3, where the amino acid sequences of HCDR are defined by Kabat. In some embodiments of the first aspect, the first antigen-binding fragment is in the form of a single-domain antibody.

[0067] In some embodiments of the first aspect, the first antigen-binding fragment comprises a monovalent or polyvalent (e.g., 1, 2, 3, 4, 5, or 6-valent) single-domain antibody bound to the HLA-A24 / PRAME complex. In some specific embodiments of the first embodiment, the first antigen-binding fragment comprises a bivalent single-domain antibody that binds to the HLA-A24 / PRAME complex. In some embodiments, the first antigen-binding fragment comprises heavy chain variable regions of two monovalent single-domain antibodies that bind to the HLA-A24 / PRAME complex. In some embodiments of the first aspect, the first antigen-binding fragment comprises the heavy chain variable regions of two monovalent single-domain antibodies that are directly fused to the HLA-A24 / PRAME complex.

[0068] In some embodiments of the first aspect, the first antigen-binding fragment comprises heavy chain variable regions of two monovalent single-domain antibodies linked via a linker to the HLA-A24 / PRAME complex. In some embodiments, the linker is a GS-type flexible peptide linker. In some embodiments, the linker is (G4S)n, (SG4)n, or G4(SG4)n, where n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), or an integer or range of integers between any two of the above numbers. In some embodiments, the linker is (G4S)n, (SG4)n, or G4(SG4)n, where n is an integer from 2 to 4, e.g., 2, 3, or 4. In some specific embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 94).

[0069] In some embodiments of the first aspect, the second antigen-binding fragment includes HCDR1 shown in SEQ ID NO:6, HCDR2 shown in SEQ ID NO:7, HCDR3 shown in SEQ ID NO:8, LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:10, and LCDR3 shown in SEQ ID NO:11, where the amino acid sequences of HCDR are defined by Kabat.

[0070] In some specific embodiments of the first aspect, the second antigen-binding fragment is in the form of a single-chain antibody (scFv) or a Fab fragment. In some embodiments of the first aspect, the second antigen-binding fragment is in the form of an scFv. In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:4.

[0071] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:5. In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:14. In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:15.

[0072] In some embodiments of the first aspect, the amino acid sequence shown in SEQ ID NO:14 includes the two amino acid sequences shown in SEQ ID NO:4.

[0073] In some embodiments of the first aspect, the amino acid sequence shown in SEQ ID NO:15 includes the two amino acid sequences shown in SEQ ID NO:5. In some embodiments of the first aspect, the second antigen-binding fragment includes a heavy chain variable region whose amino acid sequence is shown as SEQ ID NO:12 and a light chain variable region whose amino acid sequence is shown as SEQ ID NO:13. In some embodiments of the first aspect, the first antigen-binding fragment comprises an amino acid sequence shown in SEQ ID NO:4, and the second antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:12 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:13.

[0074] In some embodiments of the first aspect, the first antigen-binding fragment comprises an amino acid sequence shown in SEQ ID NO: 5, and the second antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 12 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO: 13. In some embodiments of the first aspect, the first antigen-binding fragment comprises an amino acid sequence shown in SEQ ID NO:14, and the second antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:12 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:13. In some embodiments of the first aspect, the first antigen-binding fragment comprises an amino acid sequence shown in SEQ ID NO:15, and the second antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:12 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:13.

[0075] 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: 4, 5, 14, or 15 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In some embodiments of the first aspect, the amino acid sequence of the first antigen-binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology with the amino acid sequence shown in SEQ ID NO: 4, 5, 14, or 15.

[0076] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 4, 5, 14, or 15 may be truncated by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the same function as the first antigen-binding fragment is still maintained.

[0077] In some embodiments of the first aspect, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, fifteen, twenty

[0078] In some embodiments of the first aspect, the modified amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids added or deleted in a region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 4, 5, 14, or 15, as long as the modified amino acid sequence substantially maintains the same function as the first antigen-binding fragment. In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment includes the amino acid sequence indicated by SEQ ID NO:12. 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:12 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions.

[0079] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology with the amino acid sequence shown in SEQ ID NO:12. In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:12 may be further truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the same function as the heavy chain variable region of the second antigen-binding fragment is still maintained.

[0080] 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 to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:12, and the resulting amino acid sequence still maintains a function similar to that of the heavy chain variable region of the second antigen-binding fragment. In some embodiments of the first aspect, the modified amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids added or deleted in a region other than the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:12, as long as the modified amino acid sequence maintains substantially the same function as the heavy chain variable region of the second antigen-binding fragment. In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen-binding fragment includes the amino acid sequence shown in SEQ ID NO:13.

[0081] 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:13 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions.

[0082] 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 more homology with the amino acid sequence shown in SEQ ID NO:13.

[0083] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:13 may be further cleaved by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and a function similar to that of the light chain variable region of the second antigen-binding fragment is still maintained.

[0084] In some embodiments of the first aspect, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, fifteen, twenty, twenty-five or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:13, and the resulting amino acid sequence still maintains a function similar to that of the light chain variable region of the second antigen-binding fragment.

[0085] In some embodiments of the first aspect, the modified amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids added or deleted in a region other than the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:13, as long as the modified amino acid sequence maintains substantially the same function as the light chain variable region of the second antigen-binding fragment.

[0086] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are linked via an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising the first Fc fragment and the 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, where the amino acid positions of the constant region of the antibody are determined according to EU numbering.

[0087] In some specific embodiments of the first embodiment, the first antigen-binding fragment and the second antigen-binding fragment are linked via an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising the first Fc fragment and the second Fc fragment, where 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, where the amino acid positions of the constant region of the antibody are determined according to EU numbering.

[0088] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are linked via an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising the first Fc fragment and the second Fc fragment, wherein the amino acids at positions 234, 235, and 331 of the first Fc fragment and the second Fc fragment are F, E, and S, respectively, where the amino acid positions of the constant region of the antibody are determined according to EU numbering.

[0089] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are linked via an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising the first Fc fragment and the second Fc fragment, wherein either the first Fc fragment or the second Fc fragment is linked to the first antigen-binding fragment, and the other of the first Fc fragment or the second Fc fragment is linked to the second antigen-binding fragment.

[0090] In some embodiments of the first aspect, the first antigen-binding fragment (e.g., the C-terminus) is ligated to the N-terminus of the first Fc fragment (e.g., the amino acid sequence shown in SEQ ID NO:33). In some embodiments of the first aspect, the second antigen-binding fragment (e.g., the C-terminus) is ligated to the N-terminus of the second Fc fragment (e.g., the amino acid sequence shown in SEQ ID NO:34).

[0091] In some embodiments of the first embodiment, the heavy chain constant region Fc fragment of the antibody 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.

[0092] In some embodiments of the first aspect, the heavy chain constant region Fc fragment of the antibody is an IgG1m3 allotype Fc fragment. In some embodiments, the first Fc fragment is an IgG1m3 allotype Fc fragment, and / or the second Fc fragment is an IgG1m3 allotype Fc fragment.

[0093] In some embodiments of the first aspect, the bispecific antibody comprises a first arm that binds to the HLA-A24 / PRAME complex and a second arm that binds to the activated T cell antigen, where, The first arm comprises the amino acid sequence shown in SEQ ID NO: 35 or 36, and The second arm contains the amino acid sequence shown in SEQ ID NO:37.

[0094] In some embodiments of the first aspect, the amino acid sequence of the first arm includes the amino acid sequence shown in SEQ ID NO: 35 or 36. 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: 35 or 36 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions.

[0095] In some embodiments of the first aspect, the amino acid sequence of the first arm has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology with the amino acid sequence shown in SEQ ID NO:35 or 36. 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 or 36 may be cleaved by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the same function as the first arm is still maintained.

[0096] In some embodiments of the first aspect, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteen, twelve, thirteen, fourteen, fifteen, twenty, twenty-five or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 35 or 36, and the resulting amino acid sequence still maintains the same function as the first arm.

[0097] In some embodiments of the first aspect, the modified amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids added or deleted in a region other than the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 35 or 36, as long as the modified amino acid sequence maintains substantially the same function as the first arm.

[0098] In some embodiments of the first aspect, the amino acid sequence of the second arm includes the amino acid sequence shown in SEQ ID NO:37. 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:37 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In some embodiments of the first aspect, the amino acid sequence of the second arm has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology with the amino acid sequence shown in SEQ ID NO:37.

[0099] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:37 may be cleaved by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the same function as the second arm is still maintained.

[0100] In some embodiments of the first aspect, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteen, twelve, thirteen, fourteen, fifteen, twenty, twenty-five or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:37, and the resulting amino acid sequence still maintains the same function as the second arm.

[0101] In some embodiments of the first aspect, the modified amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids added or deleted in a region other than the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:37, as long as the modified amino acid sequence maintains substantially the same function as the second arm.

[0102] According to a second aspect, the present invention provides a single-domain antibody that binds to the HLA-A24 / PRAME complex, which HCDR1, indicated by SEQ ID NO:1 HCDR2, as shown in SEQ ID NO:2, and Contains HCDR3 as indicated by SEQ ID NO:3. Here, the amino acid sequence of HCDR is defined by Kabat.

[0103] In some embodiments of the second aspect, the binding epitope of the single-domain antibody to the HLA-A24 / PRAME complex includes one or more residues from positions 301-309 of PRAME, shown in SEQ ID NO:38. In some embodiments, the amino acid sequence from positions 301-309 of PRAME, shown in SEQ ID NO:38, is LYVDSLFFL (SEQ ID NO:19).

[0104] In some embodiments of the second aspect, the binding epitope of the single-domain antibody to the HLA-A24 / PRAME complex includes at least one of the residues at positions 304, 305, 306, 307, 308, and 309 of PRAME, as shown in SEQ ID NO:38.

[0105] In some specific embodiments of the second aspect, the binding epitope of the single-domain antibody to the HLA-A24 / PRAME complex includes the residues at positions 304, 305, 306, 307, 308, and 309 of PRAME shown in SEQ ID NO:38. In some embodiments, the residues at positions 304, 305, 306, 307, 308, and 309 of PRAME shown in SEQ ID NO:38 are the same as those in PRAME shown in SEQ ID NO:19. 301-309 This corresponds to the amino acid residues at positions 4, 5, 6, 7, 8, and 9 of the (LYVDSLFFL) polypeptide.

[0106] In some embodiments of the second aspect, the single-domain antibody is in the form of a monovalent or polyvalent single-domain antibody that binds to the HLA-A24 / PRAME complex. In some embodiments of the second aspect, the single-domain antibody is a monovalent single-domain antibody that binds to the HLA-A24 / PRAME complex.

[0107] In some embodiments of the second aspect, the single-domain antibody is in the form of a bivalent single-domain antibody that binds to the HLA-A24 / PRAME complex. In some embodiments of the second aspect, the single-domain antibody comprises a heavy chain variable region of a single-domain antibody in two monovalent forms that bind to the HLA-A24 / PRAME complex. In some specific embodiments of the second aspect, the single-domain antibody comprises a heavy chain variable region of two monovalent forms of the single-domain antibody that are directly fused to the HLA-A24 / PRAME complex.

[0108] In some specific embodiments of the second embodiment, the single-domain antibody comprises a heavy chain variable region of two monovalent forms of the single-domain antibody that bind to the HLA-A24 / PRAME complex, linked via a linker. In some embodiments, the linker is a GS-type flexible peptide linker. In some embodiments, the linker is (G4S)n, (SG4)n, or G4(SG4)n, where n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), or an integer or range of integers between any two of the above numbers. In some embodiments, the linker is (G4S)n, (SG4)n, or G4(SG4)n, where n is an integer from 2 to 4, e.g., 2, 3, or 4. In some specific embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 94).

[0109] In some embodiments of the second aspect, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:4. In some embodiments of the second aspect, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:5. In some embodiments of the second aspect, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:14. In some embodiments of the second aspect, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:15.

[0110] 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: 4, 5, 14, or 15 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In some embodiments of the second aspect, the amino acid sequence of the single-domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology with the amino acid sequence shown in SEQ ID NO: 4, 5, 14, or 15.

[0111] In some embodiments of the second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 4, 5, 14, or 15 may be cleaved by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the same function as the single-domain antibody is still maintained. In some embodiments of the second aspect, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, fifteen, twenty

[0112] In some embodiments of the second aspect, the modified amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids added or deleted in a region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 4, 5, 14, or 15, as long as the modified amino acid sequence substantially maintains the same function as the single-domain antibody.

[0113] According to a third aspect, the present invention provides a nucleic acid molecule encoding a bispecific antibody as described in the first aspect or a single-domain antibody as described in the second aspect. In some embodiments of the third aspect, the nucleic acid molecule may include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, or it may be cDNA. In some embodiments of the third aspect, the nucleic acid molecule is operably linked to a regulatory nucleotide sequence, which can be identified by a host cell transformed with the vector.

[0114] According to a fourth aspect, the present invention provides a pharmaceutical composition comprising a bispecific antibody described in the first aspect or a single-domain antibody described in the second aspect, and a pharmaceutically acceptable excipient, diluent, or carrier. In some embodiments of the fourth aspect, the pharmaceutical composition is used to prevent or treat HLA-A24 / PRAME-positive tumors.

[0115] In some embodiments of the fourth aspect, the HLA-A24 / PRAME-positive tumor is selected from the group consisting of melanoma, non-small cell lung cancer, small cell lung cancer, breast cancer (e.g., triple-negative breast cancer), renal cell carcinoma, esophageal cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), cervical cancer, bladder cancer, hepatocellular carcinoma, gastric cancer, Hodgkin lymphoma, neuroblastoma, acute leukemia (e.g., acute granulocytic leukemia or acute myeloid leukemia), and chronic leukemia (e.g., chronic granulocytic leukemia or chronic myeloid leukemia).

[0116] In some embodiments of the fourth aspect, the pharmaceutical composition may further contain one or more substances such as lubricants (e.g., talc, magnesium stearate, and mineral oil), wetting agents, emulsifiers, suspending agents, preservatives (e.g., benzoic acid, sorbic acid, calcium propionate), sweeteners, and / or flavoring agents. In some embodiments of the fourth aspect, the pharmaceutical composition according to the present invention may be prepared in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules.

[0117] In some embodiments of the fourth aspect, the pharmaceutical composition according to the present invention can be delivered by any physiologically acceptable method 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.). In some embodiments of the fourth aspect, the therapeutic pharmaceutical composition can be formulated for storage in the form of a lyophilized preparation or an aqueous solution by mixing a reagent of the required purity with any pharmaceutically acceptable carrier, excipient, etc.

[0118] According to a fifth aspect, the present invention provides the use of the bispecific antibody described in the first aspect, the single-domain antibody described in the second aspect, or the pharmaceutical composition described in the fourth aspect in the manufacture of a pharmaceutical for the prevention or treatment of HLA-A24 / PRAME-positive tumors.

[0119] In some embodiments of the fifth aspect, the HLA-A24 / PRAME-positive tumor is selected from the group consisting of melanoma, non-small cell lung cancer, small cell lung cancer, breast cancer (e.g., triple-negative breast cancer), renal cell carcinoma, esophageal cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), cervical cancer, bladder cancer, hepatocellular carcinoma, gastric cancer, Hodgkin lymphoma, neuroblastoma, acute leukemia (e.g., acute granulocytic leukemia or acute myeloid leukemia), and chronic leukemia (e.g., chronic granulocytic leukemia or chronic myeloid leukemia).

[0120] According to a sixth aspect, the present invention provides a method for preventing or treating an HLA-A24 / PRAME-positive tumor, comprising administering a bispecific antibody according to the first aspect, a single-domain antibody according to the second aspect, or a pharmaceutical composition according to the fourth aspect to an individual in need thereof. In some embodiments of the sixth aspect, the HLA-A24 / PRAME-positive tumor is selected from the group consisting of melanoma, non-small cell lung cancer, small cell lung cancer, breast cancer (e.g., triple-negative breast cancer), renal cell carcinoma, esophageal cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), cervical cancer, bladder cancer, hepatocellular carcinoma, gastric cancer, Hodgkin lymphoma, neuroblastoma, acute leukemia (e.g., acute granulocytic leukemia or acute myeloid leukemia), and chronic leukemia (e.g., chronic granulocytic leukemia or chronic myeloid leukemia).

[0121] According to the seventh aspect, the present invention provides the use of the bispecific antibody described in the first aspect or the single-domain antibody described in the second aspect in the manufacture of a product for detecting HLA-A24 / PRAME-positive cells. In some embodiments of the seventh aspect, the product is a kit, a test strip, a detection card, or a microfluidic device. In some embodiments of the seventh aspect, the product may further include a detection label such as colloidal gold, a chemiluminescent label, a fluorescent label, or a nanoparticle label.

[0122] In some embodiments of the seventh aspect, the product may further include enzymes or other reagents used for detection, such as colloidal gold-labeled antigens / antibodies, substrates, reference standards, diluents, and washing solutions. In some embodiments of the seventh aspect, the product may further include a reagent for processing a biological sample for detection, the biological sample being blood. In some embodiments of the seventh aspect, the product may further include a product instruction manual.

[0123] The present invention further provides a vector comprising a nucleic acid molecule encoding a bispecific antibody according to a first embodiment or a single-domain antibody according to a second embodiment, and a host cell comprising the nucleic acid molecule or vector. In other embodiments, the present invention further provides a method for producing a bispecific antibody according to a first embodiment or a single-domain antibody according to a second embodiment. In some embodiments, the method for producing a bispecific antibody according to a first embodiment or a single-domain antibody according to a second embodiment comprises culturing host cells to facilitate the expression of the nucleic acid molecule. In some embodiments, the method for producing a bispecific antibody according to a first embodiment or a single-domain antibody according to a second embodiment further comprises recovering the bispecific antibody or the single-domain antibody from the host cell culture medium.

[0124] The above detailed description is intended solely to enable those skilled in the art to clearly understand the present invention, but it should be understood that it is not intended to limit any aspect. Those skilled in the art can make various modifications and changes to the above embodiments. The following examples illustrate, but do not limit, the scope of the present invention. [Examples]

[0125] [Example 1] Preparation of recombinant protein The process of preparing bispecific antibodies targeting HLA-A24 / PRAME and CD3 requires the use of various recombinant proteins, including the human CD3E extracellular domain (hCD3E, SEQ ID NO: 17) and the human CD3D extracellular domain (hCD3D, SEQ ID NO: 18).

[18] Referring to [reference], a dtSCT (disulfide trap single-chain trimer) structural complex (MIP-P3, SEQ ID NO:20) of the PRAME antigen peptide LYVDSLFFL (SEQ ID NO:19) and HLA-A24 was prepared. Because these recombinant proteins possess numerous post-translational modifications (e.g., glycosylation and disulfide bonds), utilizing mammalian cell expression systems offers advantages in preserving the structure and function of the recombinant proteins. Adding a His tag (His, SEQ ID NO:21) or the Fc segment of the mouse antibody IgG2a (mFc, SEQ ID NO:22) to the C-terminus of the recombinant protein offers advantages in purifying the recombinant protein and identifying the function of the monoclonal antibody. When preparing recombinant antibodies, the antibody heavy chain constant region may be a human IgG1 subtype (SEQ ID NO:23) or various variants of a selected human IgG1 subtype, such as IgG1H (SEQ ID NO:24), IgG1K (SEQ ID NO:25), IgG1m3-H (SEQ ID NO:26), IgG1m3-K (SEQ ID NO:27), IgG1m3-H1n1 (SEQ ID NO:28), or IgG1m3-Kn1 (SEQ ID NO:29), and the light chain constant region may be a human λ isotype (SEQ ID NO:30) or a human κ isotype (SEQ ID NO:31).

[0126] Based on the amino acid sequences of various target recombinant proteins in the Uniprot database, the genes for the above recombinant proteins (including His tag or mFc coding genes) were designed and synthesized. Using standard molecular biology techniques, the synthesized recombinant protein genes were cloned into appropriate eukaryotic expression vectors (e.g., Invitrogen's pcDNA3.1). Subsequently, the prepared recombinant protein expression plasmids were transfected into HEK293 cells (e.g., Invitrogen's HEK293F) using liposomes (e.g., Invitrogen's 293fectin) or other cationic transfection reagents (e.g., PEI), and cultured in serum-free suspension for 3-4 days. The culture supernatant was then collected by methods such as centrifugation.

[0127] Recombinant proteins expressed by His tag fusion were purified in a single step using a metal chelate affinity chromatography column (e.g., GE's HisTrap FF). Recombinant proteins expressed by mFc fusion were purified in a single step using a Protein A / G affinity chromatography column (e.g., GE's Mabselect SURE). Subsequently, the storage buffer for the recombinant proteins was replaced with PBS (pH 7.0) or another suitable buffer using a desalting column (e.g., GE's Hitrap desaulting). If necessary, the recombinant protein samples were filtered and sterilized, then aliquoted and stored at -20°C.

[0128] [Example 2] Screening and activity evaluation of anti-HLA-A24 / PRAME monoclonal antibodies 2.1 Preparation of llama immunization and single-domain antibody libraries One healthy adult camel was selected, and blood was collected before immunization to retain baseline serum. In the initial immunization, 0.4 mg of MIP-P3-His fusion protein was emulsified with Freund's complete adjuvant and administered by subcutaneous multi-point injection. Booster immunization was performed at two-week intervals, with 0.4 mg of MIP-P3-His fusion protein emulsified with Freund's incomplete adjuvant and administered by subcutaneous multi-point injection, for a total of five booster immunizations. Blood was collected before each immunization for antibody titer analysis. In the seventh immunization, no adjuvant was added, and a high-dose booster immunization was performed using 0.4 mg of MIP-P3-His fusion protein as the antigen by subcutaneous multi-point injection. Three days later, 150 mL of peripheral blood was collected and lymphocytes were isolated.

[0129] 150 mL of llama peripheral blood lymphocytes were isolated using a camel peripheral blood lymphocyte isolation kit (Solarbio, CAT#P5750). Total RNA from the lymphocytes was extracted using a total RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., CAT#DP430). Using the extracted total RNA as a template, the camel heavy chain variable region was synthesized using a first-strand cDNA synthesis kit (Thermo Scientific, CAT#K1621). Gene-specific primers were used for reverse transcription, and the primer pairing region was located in the CH2 domain of the antibody heavy chain constant region, with the specific sequence being PCal-CH2R:TCCTTCCCCGTCAGCCAGTCCT (SEQ ID NO:95). The synthesized cDNA was immediately stored at -70°C for later use. Next, using the cDNA obtained by reverse transcription as a template, the literature

[19] Primers were synthesized by referring to [reference], and the camel VHH gene was amplified and isolated using nested PCR. Finally, the amplified VHH gene was cloned into the vector pADSCFV-S (Chinese Patent Application No. 201510097117.0).

[20] (See reference) A VHH library was constructed. The library volume of this antibody library reached 2.0E8, and the accuracy rate was 66.67%.

[0130] 2.2 Screening of a Camel Immunology Library References (Chinese Patent Application No. 201510097117.0)

[20] Based on the above, the recombinant protein MIP-P3-mFc prepared in Example 1 was used as the antigen, and a solid-phase screening strategy (the experimental protocol is described in "Phage Display: General Experiment Manual / edited by Clackson, T. and Lowman, HB, translated by Ma Lan et al., Chemical Industry Press, May 2008") was used.

[21] Using (see reference), a phage library displaying the camel single-domain antibody constructed above was screened, and a total of three screenings were performed through the procedures of binding, elution, neutralization, infection, and amplification, ultimately yielding one strain of TCRm antibody N83G3 (SEQ ID NO:4) that specifically binds to MIP-P3-His.

[0131] Using conventional molecular biological methods, the nucleotide sequence of the N83G3 variable region was cloned into a eukaryotic expression vector (e.g., Invitrogen's pcDNA3.1) fused with the nucleotide sequence encoding the Fc segment (Fc, SEQ ID NO:32) of the human antibody IgG1, and the N83G3-Fc recombinant protein was expressed.

[0132] 2.3 Affinity analysis of anti-HLA-A24 / PRAME antibodies The affinity of an anti-HLA-A24 / PRAME monoclonal antibody was measured using surface plasmon resonance technology with a Biacore T200. All related reagents and consumables, including the amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), S-series CM5 tip (14100530), and pH 7.4 10×HBS-EP (BR100669), were purchased from GE Healthcare. Following the kit's instructions, the surface of the carboxylated CM5 chip was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Anti-human IgG (Fc) antibody (capture antibody) was diluted to 25 μg / mL with 10 mM sodium acetate at pH 5.0 and then injected at a flow rate of 10 μL / min to achieve a coupling amount of approximately 10,000 response units (RUs). After injection of the capture antibody, unreacted groups were blocked by injecting 1 M ethanolamine. In kinetic measurements, anti-HLA-A24 / PRAME monoclonal antibody was diluted to 1 μg / mL and injected at 10 μL / min, ensuring that approximately 60 RUs of antibody were captured by the anti-human Fc antibody. Subsequently, MIP-P3-His was injected at a flow rate of 30 μL / min with a series of concentration gradients (e.g., 0.37 nM, 1.11 nM, 3.33 nM, 10 nM, and 30 nM) from low to high concentration. The chip surface was then regenerated by injecting 3 M MgCl2 at a flow rate of 10 μL / min for 30 seconds with a binding time of 90 seconds and a dissociation time of 1200 seconds. The binding and dissociation sensor map was fitted using a 1:1 binding model with version 3.2.1 of the Biacore T200 evaluation software to determine the binding rate (K a ) and dissociation rate (K d The ratio K was calculated. d / K a The dissociation equilibrium constant (K D The value was calculated. The fitting results are shown in Table 1.

[0133] [Table 1]

[0134] 2.4 Anti-HLA-A24 / PRAME Antibody and PRAME 301-309 Binding to HT-29 cells pulsed with (LYVDSLFFL) HT-29 (human colon cancer cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.) does not express PRAME protein and carries two different HLA-A genes, HLA-A02 and HLA-A24. HT-29 cells in the logarithmic growth phase were collected, digested and centrifuged, then resuspended to 1×10 7 cells / mL using 1640 medium (Gibco, C11875500BT), and seeded into a 6-well cell culture plate at 1 mL / well. PRAME 301-309 peptide (LYVDSLFFL, SEQIDNO:19, synthesis entrusted to GenScript Biotechnology Co., Ltd.) was diluted to 114 nM with 1640 medium, added to the 6-well cell culture plate containing cells at 1 mL / well, and subjected to pulse treatment in a CO2 incubator at 37°C for 3 hours. After the pulse treatment was completed, the cells were washed once with PBS solution, and resuspended to 2×10 6 cells / mL with PBS, and seeded into a 96-well V-bottom plate at 100 μL / well. Using PBS, the test sample N83G3 and the negative control antibody DP47 (U.S. Patent Application US20160200833A1

[22] The antibody was prepared using the reference provided, and the amino acid sequences of the heavy chain variable region and light chain variable region are as shown in SEQIDNO:96 and 97, respectively. Dilution was performed with a concentration gradient to create a total of eight concentration points with an initial concentration of 40 nM of anti-HLA-A24 / PRAME antibody, using a 4-fold concentration gradient. 100 μL of anti-HLA-A24 / PRAME antibody was added to cell-containing wells and incubated at 4°C for 1 hour. Subsequently, the cells were washed three times with 200 μL of PBS solution, and 100 μL / well of goat anti-human IgG-FITC (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., ZF-0308) was added. After incubation in the dark at 4°C for 30 minutes, the cells were washed three times with 200 μL of PBS solution, resuspended in 100 μL of PBS solution, and the FITC channel was detected using a flow cytometer (ACEA, Novocyte). The results showed that anti-HLA-A24 / PRAME antibody N83G3 was found to be PRAME 301-309 It was shown to bind well to pulsed HT-29 cells (Figure 1).

[0135] [Example 3] Humanization and activity evaluation of anti-HLA-A24 / PRAME monoclonal antibody 3.1 Humanization of anti-HLA-A24 / PRAME monoclonal antibodies Humanization studies were conducted to reduce the immunogenicity of the TCRm antibody N83G3. The humanization scheme employed a classical framework transplantation strategy.

[23] . The amino acid sequence of N83G3 was compared with the human antibody germline gene sequences in the IMGT database, an appropriate germline gene sequence was selected to provide the framework regions 1 to 3 (FR1+FR2+FR3) of the antibody, and an appropriate J region gene sequence was selected to provide the framework region 4 (FR4). This template can be selected based on various factors, such as the relative full length of the antibody, the size of the CDRs, the amino acid residues located at the junctions between the antibody framework regions (FR) and the hypervariable regions (CDR), and the homology of the entire sequence. The selected template may be a mixture of multiple sequences or a consensus template, so as to maintain the proper conformation of the parent complementarity determining regions (CDRs) as much as possible. At the same time, considering the properties of the humanized antibody such as solubility, stability and expression yield, back mutations were respectively performed on the four hotspot amino acids 37F / 44E / 45R / 47F in FR2, and finally the humanized variant N83G3-h5 (SEQ ID NO: 5) was obtained.

[0136] 3.2 Affinity analysis of humanized antibody With reference to Example 2.3, the affinity analysis of the humanized molecule N83G3-h5 of the anti-HLA-A24 / PRAME monoclonal antibody N83G3 was performed using Biacore T200, and the results are shown in Table 2.

[0137] [Table 2]

[0138] [Example 4] Preparation and identification of TCRm×CD3 bispecific antibody against anti-HLA-A24 / PRAME complex 4.1 Production of TCRm×CD3 bispecific antibody The nucleotide sequence encoding the variable region of bivalent N83G3 (2N83G3, SEQ ID NO: 14), and the anti-CD3 single-chain antibody (IMCR-ScFv, SEQ ID NO: 16, anti-CD3 v9 of U.S. Patent No. US5821337A

[24] The nucleotide sequences encoding (see reference) were cloned into appropriate eukaryotic expression vectors and co-expressed to construct bispecific antibodies against the anti-HLA-A24 / PRAME complex and CD3. Specifically, the nucleotide sequence encoding 2N83G3 was cloned into a eukaryotic expression vector fused with the nucleotide sequence of the Fc fragment IgG1m3-FcH1n1 (SEQIDNO:33) encoding the Hole mutation, and the nucleotide sequence encoding IMCR-anti-CD3-ScFv was cloned into a eukaryotic expression vector fused with the nucleotide sequence of the Fc fragment IgG1m3-FcKn1 (SEQIDNO:34) encoding the Knob mutation, thereby constructing the bispecific antibody 2N83G3×IMCR.

[0139] Eukaryotic expression vectors expressing 2N83G3-G1m3-FcH1n1 (SEQIDNO:35) and IMCR-ScFv-G1m3-FcKn1 (SEQIDNO:37) were co-transfected into HEK293F cells using liposomes. After culturing under serum-free suspension conditions for 3-5 days, the culture supernatant was collected by centrifugation or other means. The bispecific antibodies in the culture supernatant were purified using a Protein A affinity chromatography column (e.g., GE's Mabselect SURE), and then the recombinant protein storage buffer was changed to PBS (pH 7.0) or other suitable buffer using a desalting column (e.g., GE's Hitrap desalting). The desalted protein solution was purified by size exclusion chromatography (SEC) using Superdex200 (GE) to obtain the target protein. If necessary, antibody samples were filtered and sterilized, then aliquoted and stored at -20°C.

[0140] 4.2 Construction of a TCRm × CD3 bispecific antibody of a humanized anti-HLA-A24 / PRAME complex Referring to Example 4.1, the nucleotide sequence encoding the variable region of bivalent N83G3-h5 (2N83G3-h5, SEQ ID NO:15) and the nucleotide sequence encoding the single-chain antibody against CD3 (IMCR-ScFv, SEQIDNO:16) were cloned into appropriate eukaryotic expression vectors and co-expressed to construct bispecific antibodies against HLA-A24 / PRAME and CD3. Specifically, the nucleotide sequence encoding 2N83G3-h5 was cloned into a eukaryotic expression vector fused with the nucleotide sequence of the Fc fragment IgG1m3-FcH1n1 (SEQIDNO:33) encoding the Hole mutation, and the nucleotide sequence encoding IMCR-ScFv was cloned into a eukaryotic expression vector fused with the nucleotide sequence of the Fc fragment IgG1m3-FcKn1 (SEQIDNO:34) encoding the Knob mutation, thereby constructing the humanized bispecific antibody 2N83G3-h5×IMCR.

[0141] 4.3 Affinity analysis of humanized bispecific antibodies Referring to Example 2.3, affinity analysis of humanized bispecific antibodies was performed using Biacore T200, and the results are shown in Tables 3 and 4.

[0142] [Table 3]

[0143] [Table 4]

[0144] 4.4 Activation of tumor cells against Jurkat-Dual cells mediated by humanized bispecific antibodies K562 human chronic myeloid leukemia cells (high PRAME expression, HLA-A24 negative, purchased from the Cell Resources Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences). Our company transfected K562 cells with the HLA-A24 gene to create the K562 / HLA-A24 cell line (PRAME). + / HLA-A24 +A culture medium was constructed. Logarithmic K562 / HLA-A24 cells were collected, centrifuged, and incubated in 1640 medium for 5 × 10⁶ days. 5 The cells were resuspended at 100 μL / mL and seeded into 96-well plates. Jurkat-Dual cells (purchased from Invivogen) in the logarithmic growth phase were collected, centrifuged, and incubated in 1640 medium for 1 × 10⁶ cells. 6 The cells were resuspended at 50 μL / well and seeded into 96-well plates to achieve a final E:T ratio of 1:1. Subsequently, 2N83G3×IMCR bispecific antibody (2N83G3×IMCRBsAb, 50 μL / well), 2N83G3-h5×IMCR bispecific antibody (2N83G3-h5×IMCRBsAb, 50 μL / well), or an unrelated antibody, DP47×IMCR bispecific antibody (DP47×IMCRBsAb, 50 μL / well), were added at an initial concentration of 80 nM, with 10 concentration points of 4-fold serial dilutions. The cell plates were placed in a CO2 incubator and incubated at 37°C for 20 hours. The supernatant was then collected and processed using QUANTI-Luc. TM Instruction manual (QUANTI-Luc TM Measurements were performed according to Invivogen (rep-qlc2), and the activation of Jurkat-Dual cells by tumor cells mediated by HLA-A24 / PRAME×CD3 bispecific antibodies was analyzed. The results showed that both 2N83G3×IMCR BsAb and 2N83G3-h5×IMCR BsAb could mediate the activation of Jurkat-Dual cells by K562 / HLA-A24 tumor cells (Figure 2).

[0145] 4.5 Tumor cell damage by PBMCs mediated by humanized bispecific antibodies 4.5.1 PRAME expression and HLA-A locus typing in tumor cells The intracellular PRAME expression levels were detected by protein blotting for six cell types: K562 (human chronic myeloid leukemia cells), HT-29 (human colon cancer cells), SK-Hep-1 (human liver cancer cells, purchased from the Cell Resources Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), MEG-01 (human chronic grain cell leukemia cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.), A375 (human malignant melanoma cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.), and U-2OS (human osteosarcoma cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.). Typing detection of the HLA-A gene locus was performed using JPEG2026536595000006.jpg778 (commissioned to Beijing Boao Jingdian Biotechnology Co., Ltd.). The results are shown in Table 5. Except for HT-29 cells, K562, SK-Hep-1, MEG-01, A375, and U-2OS cells all expressed PRAME, but A375 and U-2OS cells were HLA-A24 negative. In other words, only the surface of SK-Hep-1, MEG-01, and K562 / HLA-A24 cells showed HLA-A24. + / PRAME + That was the case.

[0146] [Table 5]

[0147] 4.5.2 Isolation of human peripheral blood mononuclear cells (PBMCs) Blood (50 mL) was collected from healthy volunteers. The collected blood was provided by the inventor and their colleagues on a volunteer basis, and all volunteers had signed informed consent forms. The volunteer selection criteria were as follows: 1. Must be over 18 years old. 2. Not infected with HIV or HBV. 3. The blood test results are normal. 4. Not being pregnant or breastfeeding. PBMCs were isolated from volunteer whole blood using Ficoll density gradient centrifugation and cultured in 1640 medium.

[0148] 4.5.3 Tumor cell injury by PBMCs mediated by the humanized bispecific antibody 2N83G3-h5×IMCR Logarithmic growth phase K562 / HLA-A24 cells, SK-Hep-1 cells, MEG-01 cells, A375 cells, U-2OS cells, HT-29 cells, and K562 cells were harvested, respectively. After centrifugation, they were incubated in 1640 medium for 5 × 10⁶ days. 5 The cells were resuspended at 100 μL / mL and seeded into 96-well cell plates. Next, 2N83G3-h5×IMCR bispecific antibody (50 μL / well) or the unrelated antibody DP47×IMCR bispecific antibody (50 μL / well) was added at an initial concentration of 80 nM, with 10 concentration points in a 4-fold dilution gradient. Finally, 50 μL / well of PBMC (effector) was added to obtain a final E:T ratio of 5:1. Simultaneously, a single target cell control (K562 / HLA-A24 cells, SK-Hep-1 cells, MEG-01 cells, A375 cells, U-2OS cells, HT-29 cells, or K562 cells), a single effector cell control (PBMC), and a single medium blank control were set up and refilled with medium to 200 μL in volume. Cell plates were placed in a CO2 incubator and incubated at 37°C for 20 hours. The supernatant was then collected, and the rate of T cell damage to tumor cells mediated by the bispecific antibody was detected and analyzed according to the instructions for the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780). The results showed that the humanized bispecific antibody 2N83G3-h5×IMCR mediated only the damaging effects of PBMCs on K562 / HLA-A24, SK-Hep-1, and MEG-01 tumor cells, while EC (European Cholesterol) was detected. 50 The values ​​were 0.01234, 0.2405, and 0.2599, respectively (Figure 3).

[0149] 4.6 Activation of Jurkat-Dual cells by humanized bispecific antibodies mediated by pulsed alanine-substituted peptides from HT-29 cells. Refer to Example 2.4, PRAME301-309 Peptides were each substituted with alanine at positions 1, 2, 3, 4, 5, 6, 7, 8, and 9, and the corresponding peptides were synthesized and pulsed on HT-29 cells. The amino acid sequence of the unrelated peptide is shown in SEQIDNO:98. As a result, the activation ability of cells pulsed with the alanine-substituted peptides at positions 6, 7, 8, and 9, mediated by the humanized bispecific antibody 2N83G3-h5×IMCR, towards Jurkat-Dual cells was significantly reduced, and the activation ability of Jurkat-Dual cells was also clearly reduced after substitutions at positions 4 and 5 (Figure 4).

[0150] [Example 5] Verification of specificity 5.1 Humanized bispecific antibody 2N83G3-h5×IMCR and PRAME 301-309 Verification of the specificity of peptides against similar peptides Cross-reactivity to homologous peptides derived from normal tissues (sharing key recognition residues) can lead to severe and unpredictable toxicity from TCR-like antibodies or TCR-based T-cell therapies (see, for example, Linette et al., Blood, (2013), 122:863-71, which reported lethal toxicity from MAGE A3-TCR therapy due to off-target reactivity to proteins expressed in cardiac tissue).

[25] Therefore, the specificity of these drugs is extremely important. To fully define the specificity of the antibodies we have developed, PRAME 301-309Based on alanine variant data affecting the N83G3-h5 active site in peptides (see Example 4.6), potential off-target peptides were searched for via databases (IEDB and HLA Ligand Atlas) according to their respective definition formulas: xYxxSLFFx, xYxDxLFFx, xYxxxLFFx, xYxxxxFFx, xxxxxLFFx, xYxxSLFxx, xxxDxLFFx, xxxxSLFFx, xxxxxxFFx, and xYxxxxxFx. Fifty peptides derived from different proteins were discovered, all showing high affinity for HLA-A24. These peptides were synthesized by Genscript Biotech Co., Ltd. Subsequently, cross-reactivity of the humanized bispecific antibody 2N83G3-h5×IMCR was verified using HT-29 cells pulsed with the peptides and activation methods against Jurkat-Dual cells (see Example 5.5 for details). As a result, the humanized bispecific antibody 2N83G3-h5×IMCR mediated only the activation of Jurkat-Dual cells by HT-29 cells pulsed with similar peptide segments 8, 11, 12, and 16, but PRAME 301-309 Compared to HT-29 cells pulsed with peptides, EC 50 The values ​​increased by 337-fold, 93-fold, 482-fold, and 97-fold, respectively, and the humanized bispecific antibody 2N83G3-h5×IMCR did not mediate Jurkat-Dual cell activation by HT-29 cells pulsed with the remaining 46 similar peptides (Table 6).

[0151] [Table 6] JPEG2026536595000009.jpg253163JPEG2026536595000010.jpg200155

[0152] 5.2 The humanized bispecific antibody 2N83G3-h5×IMCR does not mediate damage to cells positive for both HLA-A24 and similar peptide segments by PBMCs. 5.2.1 Construction of protein expression plasmids for PRAME and similar peptide segments 8 / 11 / 12 / 16 Referring to the UniProt database, full-length genes for PRAME (SEQ ID NO: 38), XCR1 (similar peptide segment 8, SEQ ID NO: 39), KIFBP (similar peptide segment 11, SEQ ID NO: 40), CPVL (similar peptide segment 12, SEQ ID NO: 41), and PLD1 (similar peptide segment 16, SEQ ID NO: 42) were synthesized. Using standard molecular biology techniques, the synthesized recombinant protein genes were cloned into appropriate eukaryotic expression vectors to construct GFP-fused full-length gene expression plasmids. Similarly, referring to the UniProt database, the full-length gene for HLA-A2402 (SEQ ID NO: 43) was synthesized, and using standard molecular biology techniques, the synthesized gene was cloned into an appropriate eukaryotic expression vector to construct a full-length gene expression plasmid.

[0153] 5.2.2 Damage to HT-29 cells transfected with PRAME or similar peptide segments 8 / 11 / 12 / 16 genes by PBMCs, mediated by the humanized bispecific antibody 2N83G3-h5×IMCR. HT-29 cells were seeded on plates the day before transfection and allowed to reach 50-90% confluence on the day of transfection. Specifically, HT-29 cells in the logarithmic growth phase were digested and centrifuged, then resuspended in 1640 medium and the cell count was measured, resulting in 1.5 × 10⁶ cells each. 6Individual cells were inoculated into T25 cell culture flasks and cultured overnight in a 37°C CO2 incubator. The following day, HT-29 cells were transfected with PRAME and the gene expression plasmids for similar peptide segments 8 / 11 / 12 / 16, respectively, according to the instructions for use of the jetOPTIMUS® transfection reagent (Polyplus, 101000006), and cultured for 48 hours in a 37°C CO2 incubator. After digestion and centrifugation of the cells 48 hours after transfection, verification was performed referring to Example 4.5. The results showed that the humanized bispecific antibody 2N83G3-h5×IMCR mediated only PBMC damage in HT-29 cells transfected with the PRAME gene, and hardly mediated damage in HT-29 cells transfected with similar peptide segments 8 / 11 / 12 / 16 genes (Figure 5).

[0154] 5.2.3 Damage to 293T cells transfected with PRAME or similar peptide segments 8 / 11 / 12 / 16 genes by PBMCs, mediated by the humanized bispecific antibody 2N83G3-h5×IMCR 293T cells (human embryonic kidney cells, PRAME non-expressing, HLA-A24 negative, purchased from the Cell Resources Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences). 293T cells were seeded on plates the day before transfection and allowed to reach 50-90% confluence on the day of transfection. Specifically, 293T cells in the logarithmic growth phase were digested and centrifuged, then resuspended in 1640 medium and counted, with each cell count being 1.5 × 10⁶. 6The cells were inoculated into a T25 cell culture flask and cultured overnight in a 37°C CO2 incubator. The following day, 293T cells were co-transfected with the HLA-A2402 gene and PRAME or similar peptide segments 8 / 11 / 12 / 16 according to the instructions for use of the jetOPTIMUS® DNA transfection reagent (Polyplus, 101000006), and cultured for 24 hours in a 37°C CO2 incubator. After digestion and centrifugation of the cells 24 hours after transfection, verification was performed according to Example 4.5. The results showed that the humanized bispecific antibody 2N83G3-h5×IMCR mediated only PBMC damage in 293T cells co-transfected with the HLA-A2402+PRAME gene, but hardly mediated damage in 293T cells co-transfected with the HLA-A2402+ similar peptide segments 8 / 11 / 12 / 16 (Figure 6).

[0155] The humanized bispecific antibody 2N83G3-h5×IMCR activates Jurkat-Dual cells only through pulsed HT-29 cells treated with similar peptide segments 8 / 11 / 12 / 16, but PRAME 301-309 Compared to HT-29 cells pulsed with peptides, HT-29 cells pulsed with similar peptide segments 8 / 11 / 12 / 16 showed improved EC (Emission Control). 50 The values ​​increased by approximately 90 to 500 times, and furthermore, it hardly mediated damage to HT-29 cells transfected with similar peptide segments 8 / 11 / 12 / 16 genes, nor to 293T cells transfected with HLA-A2402 + similar peptide segments 8 / 11 / 12 / 16 genes. Therefore, the TCRm × CD3 bispecific antibody against the anti-HLA-A24 / PRAME complex according to the present invention is effective against HLA-A24 + / PRAME + It exhibits a specific damaging effect only against tumors.

[0156] [Example 6] Tumor suppression activity of humanized bispecific antibody 2N83G3-h5×IMCR in PBMC immunoreconstituted mouse model. Blood samples (50 mL each) were collected from healthy volunteers. The collected blood was provided by the inventor and their colleagues on a volunteer basis, and all volunteers had signed informed consent forms. The volunteer selection criteria were as follows: 1. Must be over 18 years old. 2. Not infected with HIV or HBV. 3. The blood test results are normal. 4. Not being pregnant or breastfeeding.

[0157] PBMCs were isolated from the peripheral blood of healthy individuals using Ficoll density gradient centrifugation. Forty female NOG mice aged 7-8 weeks (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were selected, and 1 × 10⁶ septal molecules were extracted from the tail vein of each NOG mouse. 7 Human PBMCs were inoculated into each mouse. Seven days after human PBMC inoculation, 1 × 10¹⁶ K562 / HLA-A24 cells were found subcutaneously on the right side of each mouse. 7 Each dose was administered, and the day of administration was defined as day 0. The average tumor volume was 110 mm². 3 Upon reaching a certain stage, the animals were randomly divided into groups based on tumor size. The study was divided into four groups of eight animals each: a bispecific antibody 2N83G3-h5×IMCR (2N83G3-h5×IMCR BsAb) 1 mg / kg group (Group 2), a bispecific antibody 2N83G3-h5×IMCR 0.1 mg / kg group (Group 3), a bispecific antibody 2N83G3-h5×IMCR 0.01 mg / kg group (Group 4), and a bispecific antibody-unrelated antibody DP47×IMCR (DP47×IMCR BsAb) 1.5 mg / kg group (Group 1). The antibody was administered via tail vein injection once a week for a total of three times. The efficacy of the treatment was evaluated based on the tumor growth volume.

[0158] The results showed that the humanized bispecific antibody 2N83G3-h5×IMCR significantly inhibited tumor growth at doses of 1 mg / kg and 0.1 mg / kg, with relative tumor suppression rates (TGI%) of 97.45% and 72.49%, respectively, with P<0.001 (Figure 7).

[0159] [Sequence Information] SEQ ID NO: 1 IYVMG SEQ ID NO: 2 VITGSGNTNYADSVKG SEQ ID NO: 3 GRPSRYYRDSGTWADF SEQ ID NO: 4 QVQLVESGGGLVQAGGSLRLLSCAASGASLDIYVMGWYRQATGKERDLVAVITGSGNTNYADSVKGRFTISRDDAKNTLYLQMSSLKPEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTQVTVSS SEQ ID NO: 5 EVQLVESGGGLVKPGGSLRLSCAASGASLDIYVMGWYRQAPGKGLELVAVITGSGNTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTTVTVSS SEQ ID NO: 6 GYTMN SEQ ID NO: 7 LINPYKGVSTYNQKFKD SEQ ID NO: 8 SGYYGDSDWYFDV SEQ ID NO: 9 RASQDIRNYLN SEQ ID NO: 10 YTSRLES SEQ ID NO: 11 QQGNTLPWT SEQ ID NO: 12 EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS SEQ ID NO: 13 DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK SEQ ID NO: 14 QVQLVESGGGLVQAGGSLRLSCAASGASLDIYVMGWYRQATGKERDLVAVITGSGNTNYADSVKGRFTISRDDAKNTLYLQMSSLKPEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVESGGGLVQAGGSLRLSCAASGASLDIYVMGWYRQATGKERDLVAVITGSGNTNYADSVKGRFTISRDDAKNTLYLQMSSLKPEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTQVTVSS SEQ ID NO: 15 EVQLVESGGGLVKPGGSLRLSCAASGASLDIYVMGWYRQAPGKGLELVAVITGSGNTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTTVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGASLDIYVMGWYRQAPGKGLELVAVITGSGNTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTTVTVSS SEQ ID NO: 16 DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS SEQ ID NO: 17 QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD SEQ ID NO: 18 FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVA SEQ ID NO: 20 LYVDSLFFLGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE SEQ ID NO: 21 HHHHHH SEQ ID NO: 22 ASPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 23 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 24 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 25 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 26 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPAEGEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 27 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 28 ASTKGPSVFPLAPSSKSTSEGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVDHKPSNTKVDKTVERKSCVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 29 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVERKSCVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 30 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 31 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 32 ASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 33 ASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 34 ASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 35 QVQLVESGGGLVQAGGSLRLSCAASGASLDIYVMGWYRQATGKERDLVAVITGSGNTNYADSVKGRFTISRDDAKNTLYLQMSSLKPEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVESGGGLVQAGGSLRLSCAASGASLDIYVMGWYRQATGKERDLVAVITGSGNTNYADSVKGRFTISRDDAKNTLYLQMSSLKPEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTQVTVSSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 36 EVQLVESGGGLVKPGGSLRLSCAASGASLDIYVMGWYRQAPGKGLELVAVITGSGNTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTTVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGASLDIYVMGWYRQAPGKGLELVAVITGSGNTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTTVTVSSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 37 DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGSGGGSGGGSGGGSGGGSEGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYGDSDWYFDVWGQGTLVTSSASERKSVECPCPPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 38 MERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN SEQ ID NO: 39 MESSGNPESTTFFYYDLQSQPCENQAWVFATLATTVLYCLVFLLSLVGNSLVLWVLVKYESLESLTNIFILNLCLSDLVFACLLPVWISPYHWGWVLGDFLCKLLNMIFSISLYSSIFFLTIMTIHRYLSVVSPLSTLRVPTLRCRVLVTMAVWVASILSSILDTIFHKVLSSGCDYSELTWYLTSVYQHNLFFLLSLGIILFCYVEILRTLFRSRSKRRHRTVKLIFAIVVAYFLSWGPYNFTLFLQTLFRTQIIRSCEAKQQLEYALLICRNLAFSHCCFNPVLYVFVGVKFRTHLKHVLRQFWFCRLQAPSPASIPHSPGAFAYEGASFY SEQ ID NO: 40 MANVPWAEVCEKFQAALALSRVELHKNPEKEPYKSKYSARALLEEVKALLGPAPEDEDERPEAEDGPGAGDHALGLPAEVVEPEGPVAQRAVRLAVIEFHLGVNHIDTEELSAGEEHLVKCLRLLRRYRLSHDCISLCIQAQNNLGILWSEREEIETAQAYLESSEALYNQYMKEVGSPPLDPTERFLPEEEKLTEQERSKRFEKVYTHNLYYLAQVYQHLEMFEKAAHYCHSTLKRQLEHNAYHPIEWAINAATLSQFYINKLCFMEARHCLSAANVIFGQTGKISATEDTPEAEGEVPELYHQRKGEIARCWIKYCLTLMQNAQLSMQDNIGELDLDKQSELRALRKKELDEEESIRKKAVQFGTGELCDAISAVEEKVSYLRPLDFEEARELFLLGQHYVFEAKEFFQIDGYVTDHIEVVQDHSALFKVLAFFETDMERRCKMHKRRIAMLEPLTVDLNPQYYLLVNRQIQFEIAHAYYDMMDLKVAIADRLRDPDSHIVKKINNLNKSALKYYQLFLDSLRDPNKVFPEHIGEDVLRPAMLAKFRVARLYGKIITADPKKELENLATSLEHYKFIVDYCEKHPEAAQEIEVELELSKEMVSLLPTKMERFRTKMALT SEQ ID NO: 41 MVGAMWKVIVSLVLLMPGPCDGLFRSLYRSVSMPPKGDSGQPLFLTPYIEAGKIQKGRELSLVGPFPGLNMKSYAGFLTVNKTYNSNLFFWFFPAQIQPEDAPVVLWLQGGPGGSSMFGLFVEHGPYVVTSNMTLRDRDFPWTTTLSMLYIDNPVGTGFSFTDDTHGYAVNEDDVARDLYSALIQFFQIFPEYKNNDFYVTGESYAGKYVPAIAHLIHSLNPVREVKINLNGIAIGDGYSDPESIIGGYAEFLYQIGLLDEKQKKYFQKQCHECIEHIRKQNWFEAFEILDKLLDGDLTSDPSYFQNVTGCSNYYNFLRCTEPEDQLYYVKFLSLPEVRQAIHVGNQTFNDGTIVEKYLREDTVQSVKPWLTEIMNNYKVLIYNGQLDIIVAAALTERSLMGMDWKGSQEYKKAEKKVWKIFKSDSEVAGYIRQVGDFHQVIIRGGGHILPYDQPLRAFDMINRFIYGKGWDPYVG SEQ ID NO: 42 SEQ ID NO: 43 HSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGLVLFGAVITGAVVAAVMWRRKSSDRKGGSYSQAASSDSAQGSDVSLTACKV SEQ ID NO: 94 GGGGSGGGGSGGGGS SEQ ID NO: 95 TCCTTCCCCGTCAGCCAGTCCT SEQ ID NO: 96 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS SEQ ID NO: 97 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK SEQ ID NO: 98 TYGPVFMCL

[0160] References JPEG2026536595000011.jpg14170[2] Wadelin F, Fulton J, McEwan PA, Spriggs KA, Emsley J, Heery DM. Leucine-rich repeat protein PRAME: expression, potential functions and clinical implications for leukaemia. Mol Cancer. 2010; 9:226. [3] Goodison S, Urquidi V. The cancer testis antigen PRAME as a biomarker for solid tumor cancer management. Biomark Med. 2012;6(5):629-632. [4] Figueiredo DL, Mamede RC, Proto-Siqueira R, Neder L, Silva WA Jr, Zago MA. Expression of cancer testis antigens in head and neck squamous cell carcinomas. Head Neck. 2006;28(7):614-619. [5] van't Veer LJ, Dai H, van de Vijver MJ, et al., Gene expression profiling predicts clinical outcome of breast cancer. Nature. 2002;415(6871):530-536. [6] Neumann E, Engelsberg A, Decker J, et al., Heterogeneous expression of the tumor-associated antigens RAGE-1, PRAME, and glycoprotein 75 in human renal cell carcinoma: candidates for T-cellbased immunotherapies? Cancer Res. 1998; 58(18):4090-4095. [7] Thongprasert S, Yang PC, Lee JS, et al., The prevalence of expression of MAGE-A3 and PRAME tumor antigens in East and South East Asian non-small cell lung cancer patients. Lung Cancer. 2016; 101:137-144. [8] van Baren N, Chambost H, Ferrant A, et al., PRAME, a gene encoding an antigen recognized on a human melanoma by cytolytic T cells, is expressed in acute leukaemia cells. Br J Haematol. 1998; 102(5):1376-1379. [9] Radich JP, Dai H, Mao M, et al., Gene expression changes associated with progression and response in chronic myeloid leukemia. Proc Natl Acad Sci USA. 2006;103(8):2794-2799.

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[12] Oberthuer A, Hero B, Spitz R, Berthold F, Fischer M. The tumor-associated antigen PRAME is universally expressed in high-stage neuroblastoma and associated with poor outcome. Clin Cancer Res. 2004;10(13):4307-4313.

[13] Ikeda, H., Lethe, B., Lehmann, F., Van Baren, N., Baurain, J.F., De Smet, C., Chambost, H., Vitale, M., Moretta, A., Boon, T., Coulie, P.G. (1997) Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor. Immunity, 6, 199- 208.

[14] A. Margaret Merchant et al., An efficient route to human bispecific IgG, Nature Biotechnology, Volume 16, 1998.

[15] . Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991).

[16] . A1-Lazikani et al., J. Mol. Biol. 273:927-948 (1997).

[17] . Martin et al., Proc. Natl. Acad. Sci. USA86:9268-9272 (1989).

[18] . Kotsiou, E., Brzostek, J., Lenart, I., et al., (2010) Dimerization of soluble disulfide trap single-chain major histocompatibility complex class I molecules dependent on peptide binding affinity. Antioxid Redox Signal 15(3):635-644. JPEG2026536595000012.jpg13170

[20] CN 201510097117.0

[21] Phage Display: A General Experimental Manual / Edited by Clackson, T. and Lowman, HB., translated by Ma Lan et al., Chemical Press, May 2008.

[22] US20160200833A1

[23] 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. J Immunol 169(2):1119-1125.

[24] US 5821337A

[25] Linette et al., Blood, (2013), 122:863-71.

Claims

1. A bispecific antibody comprising a first antigen-binding fragment that binds to the HLA-A24 / PRAME complex and a second antigen-binding fragment that binds to an activated T cell antigen, Preferably, the binding epitope between the first antigen-binding fragment and the HLA-A24 / PRAME complex comprises one or more residues from positions 301 to 309 of the PRAME shown in SEQ ID NO: 38, more preferably, the binding epitope between the first antigen-binding fragment and the HLA-A24 / PRAME complex comprises at least one residue from positions 304, 305, 306, 307, 308, and 309 of the PRAME shown in SEQ ID NO: 38, and / or, Preferably, the activated T cell antigen is a CD3 molecule. Bispecific antibodies.

2. The first antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3, preferably the first antigen-binding fragment is in the form of a single-domain antibody, more preferably the first antigen-binding fragment comprises a monovalent or polyvalent single-domain antibody that binds to the HLA-A24 / PRAME complex, and / or The second antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 6, HCDR2 shown in SEQ ID NO: 7, HCDR3 shown in SEQ ID NO: 8, LCDR1 shown in SEQ ID NO: 9, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 11, preferably the second antigen-binding fragment is in the form of a single-chain antibody (scFv) or a Fab fragment, and more preferably the second antigen-binding fragment is in the form of an scFv. Here, the amino acid sequence of HCDR is defined by Kabat. The bispecific antibody according to claim 1.

3. The first antigen-binding fragment comprises a single-domain antibody that binds to a bivalent HLA-A24 / PRAME complex. Optionally, the first antigen-binding fragment comprises the heavy chain variable region of two monovalent single-domain antibodies bound to the HLA-A24 / PRAME complex, preferably the first antigen-binding fragment comprises either the directly fused heavy chain variable region of two monovalent single-domain antibodies bound to the HLA-A24 / PRAME complex, or the linker linked to the heavy chain variable region of two monovalent single-domain antibodies bound to the HLA-A24 / PRAME complex, more preferably the linker is a GS-type flexible peptide linker, most preferably the linker is (G4S)n, (SG4)n, or G4(SG4)n, where n is an integer from 1 to 10, for example, an integer from 2 to 4. A bispecific antibody according to claim 1 or 2.

4. The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 4, 5, 14, or 15, and / or The second antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown as SEQ ID NO: 12 and a light chain variable region whose amino acid sequence is shown as SEQ ID NO:

13. A bispecific antibody according to any one of claims 1 to 3.

5. The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 4, and the second antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 12 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:

13. The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 5, and the second antigen-binding fragment comprises the amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 12 and the amino acid sequence of a light chain variable region shown in SEQ ID NO: 13, or The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 14, and the second antigen-binding fragment comprises the amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 12 and the amino acid sequence of a light chain variable region shown in SEQ ID NO: 13, or The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 15, and the second antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 12 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:

13. A bispecific antibody according to any one of claims 1 to 4.

6. The first antigen-binding fragment and the second antigen-binding fragment are linked via an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising the first Fc fragment and the second Fc fragment, preferably the antibody heavy chain constant region Fc fragment being an IgG1 subtype Fc fragment, and more preferably the antibody heavy chain constant region Fc fragment being an IgG1m3 allotype Fc fragment, where, 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, 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 Either the first Fc fragment or the second Fc fragment is linked to the first antigen-binding fragment, and the other of the first Fc fragment or the second Fc fragment is linked to the second antigen-binding fragment. Here, the amino acid positions in the constant region of the antibody are determined according to EU numbering. A bispecific antibody according to any one of claims 1 to 5.

7. It comprises a first arm that binds to the HLA-A24 / PRAME complex and a second arm that binds to the activated T cell antigen, where, The first arm comprises the amino acid sequence shown in SEQ ID NO: 35 or 36, and The second arm comprises the amino acid sequence shown in SEQ ID NO: 37, A bispecific antibody according to any one of claims 1 to 6.

8. A nucleic acid molecule encoding a bispecific antibody according to any one of claims 1 to 7.

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

10. Used to prevent or treat HLA-A24 / PRAME-positive tumors, Preferably, the HLA-A24 / PRAME-positive tumor is selected from the group consisting of melanoma, non-small cell lung cancer, small cell lung cancer, breast cancer (e.g., triple-negative breast cancer), renal cell carcinoma, esophageal cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), cervical cancer, bladder cancer, hepatocellular carcinoma, gastric cancer, Hodgkin lymphoma, neuroblastoma, acute leukemia (e.g., acute granulocytic leukemia or acute myeloid leukemia), and chronic leukemia (e.g., chronic granulocytic leukemia or chronic myeloid leukemia). The pharmaceutical composition according to claim 9.

11. Use of a bispecific antibody according to any one of claims 1 to 7, or a pharmaceutical composition according to claim 9 or 10, in the manufacture of a pharmaceutical for the prevention or treatment of HLA-A24 / PRAME-positive tumors, Preferably, the HLA-A24 / PRAME-positive tumor is selected from the group consisting of melanoma, non-small cell lung cancer, small cell lung cancer, breast cancer (e.g., triple-negative breast cancer), renal cell carcinoma, esophageal cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), cervical cancer, bladder cancer, hepatocellular carcinoma, gastric cancer, Hodgkin lymphoma, neuroblastoma, acute leukemia (e.g., acute granulocytic leukemia or acute myeloid leukemia), and chronic leukemia (e.g., chronic granulocytic leukemia or chronic myeloid leukemia).

12. A method for preventing or treating HLA-A24 / PRAME-positive tumors, The process involves administering a bispecific antibody according to any one of claims 1 to 7, or a pharmaceutical composition according to claim 9 or 10, to an individual in need thereof. Preferably, the HLA-A24 / PRAME-positive tumor is selected from the group consisting of melanoma, non-small cell lung cancer, small cell lung cancer, breast cancer (e.g., triple-negative breast cancer), renal cell carcinoma, esophageal cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), cervical cancer, bladder cancer, hepatocellular carcinoma, gastric cancer, Hodgkin lymphoma, neuroblastoma, acute leukemia (e.g., acute granulocytic leukemia or acute myeloid leukemia), and chronic leukemia (e.g., chronic granulocytic leukemia or chronic myeloid leukemia).

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