Multiple antibodies that bind to HLA-A24 / PRAME complex and uses thereof

Abstract

The present application provides a bispecific antibody comprising a first antigen binding fragment that binds to an 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 an HLA-A24 / PRAME complex, a pharmaceutical composition comprising the bispecific antibody or the single domain antibody, and uses thereof.

Description

Field of the Invention

[0001] The present invention generally relates to the fields of genetic engineering and biomedicine; in particular, the present application relates to bispecific antibodies binding to the HLA-A24 / PRAME complex, single domain antibodies binding to the HLA-A24 / PRAME complex and their uses. Background of the Invention

[0003] Most proteins in the human proteome are intracellular, and some short peptides produced by the degradation of intracellular proteins in the proteasome can be 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 play a similar role as TCR in recognizing MHC / peptide complexes. These antibodies are called T cell receptor mimic molecules (TCRm) or TCR-like antibodies.

[0004] Preferentially Expressed Antigen in Melanoma (PRAME), a member of the cancer / testis antigen (CTA) gene family, is expressed at very low levels in normal adult tissues except testis, but is expressed 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] The PRAME gene has been reported to be hypermethylated in normal tissues; however, the gene is hypomethylated in most malignant cells. [3] .

[0005] PRAME is highly expressed in 88% of primary melanoma tissues and 95% of metastatic tissues [1] In addition to melanoma, PRAME is also widely expressed in many solid cancers, such as head and neck cancer, breast cancer, renal cell carcinoma, and non-small cell lung cancer. [4-7] PRAME is absent in normal hematopoietic tissues; however, some studies have found that PRAME is highly expressed in acute and chronic leukemias and Hodgkin's lymphoma. [8-10] Furthermore, the expression level of PRAME is a prognostic biomarker for poor clinical outcome in breast cancer and neuroblastoma [11,12] .

[0006] PRAME is degraded into small peptides in the proteasome, and some 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 immunotherapeutic intervention.

[0007] Bispecific antibodies (BsAb) are a type of artificial antibody that contains two different antigen binding sites. Bispecific antibodies are widely used in the field of biomedicine, especially in tumor immunotherapy. Bispecific antibodies targeting CD3, one arm can bind to CD3 in the TCR receptor complex on the surface of T cells, providing the first signal to activate T cells, and the other arm targets tumor antigens. Bispecific antibodies can bring tumor cells and T cells closer, activating T cells and directly killing tumor cells.

[0008] TCRm binding to PRAME-like intracellular proteins can be used as the target antigen-binding part of CD3 bispecific antibodies. Such bispecific antibodies may be more attractive due to the restricted expression of target antigens. Based on clinical needs, the exploration and development of bispecific antibodies targeting PRAME-MHC has important clinical significance. SUMMARY OF THE INVENTION

[0010] In a first aspect, the present application 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 activating T cell antigen.

[0011] In some embodiments of the first aspect, the bispecific antibody is capable of mediating HLA-A24 + / PRAME + Tumor cells activate T cells, and / or the bispecific antibody can mediate PBMC killing of HLA-A24 + / PRAME + Tumor cells.

[0012] In some embodiments of the first aspect, the first antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; wherein the amino acid sequence of the HCDR is according to the Kabat definition.

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

[0014] In some embodiments of the first aspect, the first antigen-binding fragment comprises a monovalent or multivalent single domain antibody that binds the HLA-A24 / PRAME complex.

[0015] In some embodiments of the first aspect, the second antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:6, HCDR2 as shown in SEQ ID NO:7, HCDR3 as shown in SEQ ID NO:8, LCDR1 as shown in SEQ ID NO:9, LCDR2 as shown in SEQ ID NO:10 and LCDR3 as shown in SEQ ID NO:11; wherein the amino acid sequence of HCDR is according to the Kabat definition.

[0016] 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.

[0017] In some embodiments of the first aspect, the first antigen-binding fragment comprises a bivalent single-domain antibody that binds to the HLA-A24 / PRAME complex; optionally, the first antigen-binding fragment comprises two heavy chain variable regions of a monovalent single-domain antibody that binds to the HLA-A24 / PRAME complex.

[0018] In some embodiments of the first aspect, the first antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 4, 5, 14 or 15; and / or

[0019] The second antigen-binding fragment comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:12 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:13.

[0020] In some embodiments of the first aspect, 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 with an amino acid sequence shown in SEQ ID NO:12 and a light chain variable region with an amino acid sequence shown in SEQ ID NO:13;

[0021] The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 5; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 12 and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 13; or

[0022] The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 14; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 12 and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 13; or

[0023] The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:15; and the second antigen-binding fragment comprises the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:12 and the light chain variable region shown in the amino acid sequence of SEQ ID NO:13.

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

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

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

[0027] 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 an activating T cell antigen, wherein

[0028] The first arm comprises the amino acid sequence shown in SEQ ID NO: 35 or 36; and

[0029] The second arm comprises the amino acid sequence shown in SEQ ID NO:37.

[0030] In a second aspect, the present application provides a single domain antibody that binds to the HLA-A24 / PRAME complex, comprising:

[0031] HCDR1 as shown in SEQ ID NO: 1,

[0032] HCDR2 as shown in SEQ ID NO: 2, and

[0033] HCDR3 as shown in SEQ ID NO:3;

[0034] The amino acid sequence of HCDR is based on the Kabat definition.

[0035] 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.

[0036] In some embodiments of the second aspect, the single domain antibody is in the form of a monovalent or multivalent single domain antibody that binds to the HLA-A24 / PRAME complex.

[0037] In some embodiments of the second aspect, the single domain antibody is in the form of a bivalent single domain antibody that binds the HLA-A24 / PRAME complex.

[0038] 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.

[0039] In a third aspect, the present application provides a nucleic acid molecule encoding the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect.

[0040] In a fourth aspect, the present application provides a pharmaceutical composition comprising the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect and a pharmaceutically acceptable excipient, diluent or carrier.

[0041] In a fifth aspect, the present application provides use of the bispecific antibody described in the first aspect, the single domain antibody described in the second aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a medicament for preventing or treating HLA-A24 / PRAME-positive tumors.

[0042] In a sixth aspect, the present application provides use of the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect in the preparation of a product for detecting HLA-A24 / PRAME positive cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1Antibodies against the HLA-A24 / PRAME complex were shown to bind to PRAME 301-309 Results of HT-29 cells pulsed with (LYVDSLFFL).

[0045] Figure 2 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 are shown.

[0046] Figure 3 The results of humanized bispecific antibody 2N83G3-h5×IMCR mediating PBMC killing of tumor cells are shown.

[0047] Figure 4 The humanized bispecific antibody 2N83G3-h5×IMCR mediated the activation of Jurkat-Dual cells by HT-29 cells pulsed with alanine-substituted peptide.

[0048] Figure 5 The results of humanized bispecific antibody 2N83G3-h5×IMCR-mediated PBMC killing of HT-29 cells transfected with PRAME or similar peptide fragments 8 / 11 / 12 / 16 genes are shown.

[0049] Figure 6 The results of humanized bispecific antibody 2N83G3-h5×IMCR mediating PBMC cell killing of 293T cells transfected with PRAME or similar peptide fragments 8 / 11 / 12 / 16 genes are shown.

[0050] Figure 7 The results of the anti-tumor activity of the humanized bispecific antibody 2N83G3-h5×IMCR in PBMC immune reconstitution model mice are shown.

[0051] Sequence Description

[0052] SEQ ID NOs: 1-3 show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable regions of llama single domain antibodies N83G3 and N83G3-h5, respectively.

[0053] SEQ ID NO: 4 shows the amino acid sequence of the heavy chain variable region of the llama (Llama) single domain antibody N83G3.

[0054] SEQ ID NO:5 shows the amino acid sequence of the heavy chain variable region of the humanized single domain antibody N83G3-h5.

[0055] SEQ ID NOs: 6-8 respectively show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the anti-human CD3 antibody IMCR.

[0056] SEQ ID NOs: 9-11 respectively show the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of the anti-human CD3 antibody IMCR.

[0057] SEQ ID NO: 12 shows the amino acid sequence of the heavy chain variable region of the anti-human CD3 antibody IMCR.

[0058] SEQ ID NO: 13 shows the amino acid sequence of the light chain variable region of the anti-human CD3 antibody IMCR.

[0059] SEQ ID NO: 14 shows the amino acid sequence of the heavy chain variable regions of two llama single domain antibodies N83G3 in tandem.

[0060] SEQ ID NO: 15 shows the amino acid sequence of the heavy chain variable regions of two llama single domain antibodies N83G3-h5 concatenated together.

[0061] SEQ ID NO: 16 shows the amino acid sequence of the single-chain antibody of the anti-human CD3 antibody IMCR.

[0062] SEQ ID NO: 17 shows the amino acid sequence of the extracellular region of human (homo sapiens) CD3E (hCD3E).

[0063] SEQ ID NO: 18 shows the amino acid sequence of the extracellular region of human (homo sapiens) CD3D (hCD3D).

[0064] SEQ ID NO: 19 shows the antigenic peptide PRAME derived from homo sapiens 301-309 The amino acid sequence of

[0065] SEQ ID NO: 20 shows the antigenic peptide PRAME 301-309 Amino acid sequence of the disulfide-trapped single-chain trimer complex with HLA-A24 (MIP-P3).

[0066] SEQ ID NO: 21 shows the amino acid sequence of the His tag.

[0067] SEQ ID NO: 22 shows the amino acid sequence of the Fc region (mFc) of mouse (mus musculus) IgG2a antibody.

[0068] SEQ ID NO: 23 shows the amino acid sequence of the heavy chain constant region of homo sapiens IgG1 subtype.

[0069] SEQ ID NO: 24 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1H.

[0070] SEQ ID NO: 25 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1K.

[0071] SEQ ID NO: 26 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-H.

[0072] SEQ ID NO: 27 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-K.

[0073] SEQ ID NO: 28 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-H1n1.

[0074] SEQ ID NO: 29 shows the amino acid sequence of the human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-Kn1.

[0075] SEQ ID NO: 30 shows the amino acid sequence of the human (homo sapiens) lambda subtype light chain constant region.

[0076] SEQ ID NO: 31 shows the amino acid sequence of the human (homo sapiens) kappa subtype light chain constant region

[0077] SEQ ID NO: 32 shows the amino acid sequence of the Fc region (hFc) of human (homo sapiens) IgG1 antibody.

[0078] SEQ ID NO: 33 shows the amino acid sequence of the human IgG1 subtype antibody Fc region mutant IgG1m3-FcH1n1.

[0079] SEQ ID NO: 34 shows the amino acid sequence of the human IgG1 subtype antibody Fc segment mutant IgG1m3-FcKn1

[0080] SEQ ID NO: 35 shows the amino acid sequence of 2N83G3-G1m3-FcH1n1 in the bispecific antibody 2N83G3×IMCR.

[0081] SEQ ID NO: 36 shows the amino acid sequence of 2N83G3-h5-G1m3-FcH1n1 in the bispecific antibody 2N83G3-h5×IMCR.

[0082] SEQ ID NO: 37 shows the amino acid sequence of the arm IMCR-scFv-G1m3-FcKn1 in the bispecific antibody 2N83G3×IMCR or 2N83G3-h5×IMCR.

[0083] SEQ ID NO: 38 shows the amino acid sequence of homo sapiens PRAME.

[0084] SEQ ID NO: 39 shows the amino acid sequence of XCR1 derived from homo sapiens.

[0085] SEQ ID NO: 40 shows the amino acid sequence of KIFBP derived from homo sapiens.

[0086] SEQ ID NO: 41 shows the amino acid sequence of CPVL derived from homo sapiens.

[0087] SEQ ID NO: 42 shows the amino acid sequence of PLD1 derived from homo sapiens.

[0088] SEQ ID NO:43 shows the amino acid sequence of HLA-A2402.

[0089] SEQ ID NO: 44 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 1.

[0090] SEQ ID NO: 45 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 2.

[0091] SEQ ID NO: 46 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 3.

[0092] SEQ ID NO: 47 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 4.

[0093] SEQ ID NO: 48 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 5.

[0094] SEQ ID NO: 49 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 6.

[0095] SEQ ID NO: 50 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 7.

[0096] SEQ ID NO:51 shows PRAME derived from homo sapiens 301-309 Amino acid sequence of similar peptide 8.

[0097] SEQ ID NO:52 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 9.

[0098] SEQ ID NO: 53 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 10.

[0099] SEQ ID NO: 54 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 11.

[0100] SEQ ID NO: 55 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 12.

[0101] SEQ ID NO: 56 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 13.

[0102] SEQ ID NO: 57 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 14.

[0103] SEQ ID NO: 58 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 15.

[0104] SEQ ID NO: 59 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 16.

[0105] SEQ ID NO:60 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 17.

[0106] SEQ ID NO:61 shows PRAME derived from homo sapiens 301-309 Amino acid sequence of similar peptide 18.

[0107] SEQ ID NO:62 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 19.

[0108] SEQ ID NO:63 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 20.

[0109] SEQ ID NO: 64 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 21.

[0110] SEQ ID NO:65 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 22.

[0111] SEQ ID NO: 66 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 23.

[0112] SEQ ID NO: 67 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 24.

[0113] SEQ ID NO:68 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 25.

[0114] SEQ ID NO: 69 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 26.

[0115] SEQ ID NO: 70 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 27.

[0116] SEQ ID NO: 71 shows PRAME derived from homo sapiens 301-309 Amino acid sequence of similar peptide 28.

[0117] SEQ ID NO: 72 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 29.

[0118] SEQ ID NO: 73 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 30.

[0119] SEQ ID NO: 74 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 31.

[0120] SEQ ID NO: 75 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 32.

[0121] SEQ ID NO: 76 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 33.

[0122] SEQ ID NO: 77 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 34.

[0123] SEQ ID NO: 78 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 35.

[0124] SEQ ID NO: 79 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 36.

[0125] SEQ ID NO: 80 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 37.

[0126] SEQ ID NO: 81 shows PRAME derived from homo sapiens 301-309 Amino acid sequence of similar peptide 38.

[0127] SEQ ID NO: 82 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 39.

[0128] SEQ ID NO: 83 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 40.

[0129] SEQ ID NO: 84 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 41.

[0130] SEQ ID NO: 85 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 42.

[0131] SEQ ID NO: 86 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 43.

[0132] SEQ ID NO: 87 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 44.

[0133] SEQ ID NO: 88 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 45.

[0134] SEQ ID NO: 89 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 46.

[0135] SEQ ID NO:90 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 47.

[0136] SEQ ID NO:91 shows PRAME derived from homo sapiens 301-309 Amino acid sequence of similar peptide 48.

[0137] SEQ ID NO:92 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 49.

[0138] SEQ ID NO:93 shows PRAME from homo sapiens 301-309 Amino acid sequence of similar peptide 50.

[0139] SEQ ID NO:94 shows the amino acid sequence of the linker.

[0140] SEQ ID NO:95 shows the nucleotide sequence of primer PCal-CH2R.

[0141] SEQ ID NO:96 shows the amino acid sequence of the negative control antibody DP47VH.

[0142] SEQ ID NO:97 shows the amino acid sequence of the negative control antibody DP47VK.

[0143] SEQ ID NO:98 shows the amino acid sequence of an unrelated peptide. DETAILED DESCRIPTION OF THE INVENTION

[0145] The inventors of the present application prepared a bispecific antibody (e.g., TCRm bispecific antibody) by genetic engineering means by combining 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., CD3 molecule). 301-309(LYVDSLFFL) and HLA-A24 complex, while the second antigen binding fragment binds to an activating T cell antigen (e.g., CD3 molecule), thereby establishing an interaction between the target cell and the T cell, causing cytotoxic T cell activation, and lysing the target cell (e.g., tumor cell) in a non-histocompatibility complex (MHC) dependent manner, thereby achieving the purpose of treating a disease (e.g., tumor). In various aspects of the present application, a new bispecific antibody comprising a first antigen binding fragment that binds to an HLA-A24 / PRAME complex and a second antigen binding fragment that binds to an activating T cell antigen (e.g., CD3 molecule), a single domain antibody that binds to an 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 the vector, a method for preparing and purifying the bispecific antibody or the single domain antibody, and medical and biological applications of the bispecific antibody or the single domain antibody are provided. According to the sequences of the bispecific antibodies or single-domain antibodies provided in the present application, bispecific antibodies or single-domain antibodies that bind to the HLA-A24 / PRAME complex can be constructed and used as drugs in clinical practice to prevent or treat tumors.

[0146] The practice of the present application employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology within the skill of the art.

[0147] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0148] definition

[0149] As used herein, the term "HLA-A24 / PRAME complex" refers to a complex of an HLA-A24 molecule and PRAME. In a specific embodiment of the present application, "HLA-A24 / PRAME complex" refers to a complex of an HLA-A24 molecule and PRAME at positions 301-309 (PRAME 301-309 ) 301-309 Complex.

[0150] As used herein, the term "activating T cell antigen" refers to an antigenic determinant expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, which can induce T cell activation when interacting with an antigen binding molecule. Specifically, the interaction of an antigen binding molecule with an activating T cell antigen can induce T cell activation by triggering the signal transduction cascade of the T cell receptor complex. In a specific aspect, the activating T cell antigen is a CD3 molecule.

[0151] The term "antibody" as used herein refers to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like. "Antibodies" as used herein include not only complete (i.e., full-length) antibodies, but also binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, bispecific antibodies, linear antibodies, single-chain antibodies, single-domain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0152] Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and the first, second and third constant regions (CH1, CH2 and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). A full-length antibody can be any type of antibody, such as IgD, IgE, IgG, IgA or IgM (or subclasses thereof), but the antibody need not belong to any particular class. Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the constant region of the heavy chain. Typically, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional structure of different classes of immunoglobulins are well known.

[0153] As used herein, the term "bispecific antibody" refers to an antibody that has the ability to bind to two antigen epitopes at the same time. The two antigen epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have a variety of structural configurations. For example, a bispecific antibody can be composed of two Fc fragments and two binding parts fused thereto respectively (similar to natural antibodies, the difference is that the two arms bind to different antigen targets or epitopes), and the antigen binding part can be a single domain antibody, a single chain antibody (scFv) or a Fab fragment. When targeting the epitopes of two given antigens, the two different binding parts of the bispecific antibody each bind to the N-terminus of an Fc fragment, and the antigen binding part configuration of the two arms can have four combinations: single domain antibody + Fab fragment, single domain antibody + scFv, scFv + single domain antibody and Fab fragment + single domain antibody. The Fc fragment can contain mutations that can ensure heavy chain heteropolymerization, and the KIH technology (knob-in-hole, KIH) is a strategy to solve heavy chain heteropolymerization. Generally, KIH technology refers to modifying the amino acid sequence of the CH3 region to form a structure that is conducive to the pairing of heterologous half antibodies, which can form a bispecific antibody while maintaining the structure of a normal antibody as much as possible. For guidance on KIH technology, see, for example, "An efficient route to human bispecific IgG", A. Margaret Merchant et al., Nature Biotechnology, Volume 16, 1998

[14] , which is incorporated herein by reference in its entirety.

[0154] As used herein, the terms "binding portion" or "binding fragment" are used interchangeably and refer to a portion or region of a complete antibody molecule that is responsible for binding to an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementary determining regions, CDR1, CDR2, and CDR3.

[0155] It is well known to those skilled in the art that the complementarity determining region (CDR, usually CDR1, CDR2 and CDR3) is the region in the variable region that has the greatest impact on the affinity and specificity of the antibody. There are two common definitions of the CDR sequence of VH or VL, namely the Kabat definition and the Chothia definition. (See, for example, Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991)

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

[16] ; and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989))

[17] For the variable region sequence of a given antibody, the CDR region sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In the embodiments of the present application, the Kabat definition of CDR sequences is used.

[0156] For a given antibody variable region sequence, the CDR region sequence in the variable region sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0157] For general antibodies, examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which can be monovalent fragments having a VL-CL chain and a VH-CH1 chain; (2) F(ab')2 fragments, which can be bivalent fragments having two Fab' fragments connected by a disulfide bridge in the hinge region (i.e., a dimer of Fab'); (3) Fv fragments having the VL and VH domains of a single arm of an antibody; (4) single-chain Fv (scFv), which can be a single polypeptide chain composed of a VH domain and a VL domain via a peptide connector; (5) (scFv)2, which can contain two VH domains connected by a peptide connector and two VL domains, wherein the two VL domains are combined with the two VH domains via a disulfide bridge; and (6) single-domain antibody formats.

[0158] In bispecific antibody constructions, the "binding moiety" includes, but is not limited to, a single domain antibody format, a Fab fragment format, and / or a single chain antibody (scFv) format.

[0159] As used herein, the term "single chain antibody (scFv, single chain fragment variable)" refers to an antibody with a single chain structure generally constructed using genetic engineering technology, comprising a polypeptide chain of a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker is usually designed between the heavy chain variable region and the light chain variable region so that the heavy chain variable region and the light chain variable region can fold into a correct conformation capable of binding to an antigen.

[0160] As used herein, the term "Fab (fragment antigen binding) fragment", "Fab portion" or similar terms refers to an antibody fragment capable of binding to an antigen produced after an intact antibody is treated with papain, including a complete light chain (VL-CL), a heavy chain variable region and a CH1 fragment (VH-CH1).

[0161] As used herein, the term "single domain antibody" refers to a heavy chain single variable domain antibody that naturally lacks a light chain, and such an antibody comprises a heavy chain variable region (VHH) and conventional CH2 and CH3 regions (e.g., one or two groups (heavy chain variable region (VHH) and conventional CH2 and CH3 regions)). The VHH structure cloned and expressed separately has a structural stability and antigen binding activity comparable to that of the original heavy chain antibody, and is the smallest unit known to bind to the target antigen. Single domain antibodies are also called nanobodies (Nanobody, Nb).

[0162] As used herein, the terms "Fc fragment", "Fc domain" and "Fc part" are used interchangeably and refer to a portion of the antibody heavy chain constant region, including the hinge region, the CH2 fragment and the CH3 fragment of the heavy chain constant region, and are determined with reference to the EU numbering of human IgG1 antibodies.

[0163] The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0164] As used herein, the term "tumor" refers to a neoplasm or solid lesion formed by abnormal cell growth. Tumors can be benign, pre-malignant or malignant.

[0165] As used herein, the term "malignancy" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. Exemplary malignancies include: carcinomas, solid tumors, melanomas, sarcomas, hematological tumors, germ cell tumors, and blastomas. More specific examples of malignancies include: melanomas, 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's lymphoma, neuroblastoma, endometrial or uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, multiple myeloma and B-cell lymphoma, brain cancer, and related metastatic lesions.

[0166] As used herein, the term "hematological tumor" refers to a tumor caused by uncontrolled growth and proliferation of abnormal cells. In most cases, the origin of these abnormal cells is the bone marrow, which is where blood cells are produced. Exemplary hematological tumors include various types of leukemia, multiple myeloma, and malignant lymphoma. More specific examples of hematological tumors include: acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute granulocytic leukemia, chronic myeloid leukemia, hairy cell leukemia (HCL), T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, juvenile myelomonocytic leukemia, B-cell prolymphocytic leukemia, Burkitt's leukemia and adult T-cell leukemia, non-Hodgkin's lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, primary macroglobulinemia ( macroglobulinemia), splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma, B-cell chronic lymphocytic lymphoma, classical Hodgkin lymphoma, Nodular lymphocyte-predominant Hodgkin lymphoma, adult T-cell lymphoma, extranodal nasal NK / T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Say-Schermer syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disease, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, not otherwise specified peripheral T-cell lymphoma, and anaplastic large cell lymphoma.

[0167] As used herein, the term "solid tumor" refers to a tangible mass that can be felt by clinical examination such as X-ray, CT scan, B-ultrasound or palpation. Solid tumors that have been clinically diagnosed and treated are divided into malignant and benign tumors. Malignant solid tumors include: 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, childhood Hodgkin lymphoma: lymphocyte-dominant type, nodular sclerosis type, mixed cell type, lymphocyte-depleted type; childhood non-Hodgkin lymphoma: prelymphoblastic lymphoma, small non-cleaved cell lymphoma (Burkitt / non-Burkitt lymphoma), diffuse large B-cell lymphoma, anaplastic large cell lymphoma, etc.; childhood kidney tumors: Wilms tumor (Wilms tumor), renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, renal primitive neuroectodermal tumor, etc.; childhood neuroblastoma: neuroblastoma, ganglioneuroblastoma, ganglioneuroma; childhood extracranial neoplasms: Germinoma: mature teratoma, immature teratoma, endodermal sinus tumor (yolk sac tumor), seminoma, dysgerminoma, choriocarcinoma, embryonal carcinoma, etc.; osteosarcoma and chondrosarcoma; childhood rhabdomyosarcoma: embryonal, alveolar, pleomorphic, etc.; soft tissue sarcoma: fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant neurilemoma, alveolar soft tissue sarcoma, epithelioid sarcoma, hyaline cell sarcoma, etc. Ewing's sarcoma, malignant melanoma, synovial sarcoma, desmoplastic small round cell tumor, etc.; Ewing's family sarcoma: Ewing's sarcoma, primitive neuroectodermal tumor; childhood liver tumors: hepatoblastoma (embryonal, fetal, undifferentiated), hepatocellular carcinoma; retinoblastoma; other tumors: posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, thymoma, pulmonary blastoma, pancreatic blastoma, islet cell tumor, ileocecal carcinoid, mesothelioma, etc. Benign solid tumors include: lymphangioma, hemangioma, thyroglossal duct cyst, etc.

[0168] In a first aspect, the present application 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 activating T cell antigen.

[0169] In some embodiments of the first aspect, the bispecific antibody is capable of mediating HLA-A24 + / PRAME + Tumor cells activate T cells, and / or the bispecific antibody can mediate PBMC killing of HLA-A24 + / PRAME + Tumor cells.

[0170] In some embodiments of the first aspect, the bispecific antibody is capable of mediating HLA-A24 + / PRAME + Tumor cells activate Jurkat-Dual cells.

[0171] In some embodiments of the first aspect, the binding epitope of the first antigen-binding fragment to the HLA-A24 / PRAME complex includes one or more residues at positions 301-309 of PRAME with reference to SEQ ID NO: 38. In some embodiments, the amino acid sequence at positions 301-309 of PRAME with reference to SEQ ID NO: 38 is LYVDSLFFL (SEQ ID NO: 19).

[0172] In some embodiments of the first aspect, the binding epitope of the first antigen-binding fragment to the HLA-A24 / PRAME complex includes at least one of residues 304, 305, 306, 307, 308 and 309 of PRAME as shown in SEQ ID NO:38.

[0173] In some specific embodiments of the first aspect, the binding epitope of the first antigen-binding fragment to the HLA-A24 / PRAME complex includes residues 304, 305, 306, 307, 308, and 309 of PRAME as shown in SEQ ID NO: 38. In some embodiments, residues 304, 305, 306, 307, 308, and 309 of PRAME as shown in SEQ ID NO: 38 correspond to residues 304, 305, 306, 307, 308, and 309 of PRAME as shown in SEQ ID NO: 19. 301-309 The amino acid residues at positions 4, 5, 6, 7, 8 and 9 of the (LYVDSLFFL) polypeptide.

[0174] In some embodiments of the first aspect, the activating T cell antigen is a CD3 molecule.

[0175] In some embodiments of the first aspect, the first antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; wherein the amino acid sequence of the HCDR is according to the Kabat definition.

[0176] In some embodiments of the first aspect, the first antigen-binding fragment is in the form of a single domain antibody.

[0177] In some embodiments of the first aspect, the first antigen-binding fragment comprises a monovalent or multivalent (eg, 1, 2, 3, 4, 5, or 6-valent) single domain antibody that binds to the HLA-A24 / PRAME complex.

[0178] In some specific embodiments of the first aspect, 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 two heavy chain variable regions of a monovalent single-domain antibody that binds to the HLA-A24 / PRAME complex.

[0179] In some embodiments of the first aspect, the first antigen-binding fragment comprises two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A24 / PRAME complex, directly fused.

[0180] In some embodiments of the first aspect, the first antigen-binding fragment comprises two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A24 / PRAME complex connected by 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, wherein n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or an integer or integer range between any two of the above values. In some embodiments, the linker is (G4S)n, (SG4)n or G4(SG4)n, wherein n is an integer from 2 to 4, such as 2, 3 or 4. In some specific embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 94).

[0181] In some embodiments of the first aspect, the second antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:6, HCDR2 as shown in SEQ ID NO:7, HCDR3 as shown in SEQ ID NO:8, LCDR1 as shown in SEQ ID NO:9, LCDR2 as shown in SEQ ID NO:10 and LCDR3 as shown in SEQ ID NO:11; wherein the amino acid sequence of HCDR is according to the Kabat definition.

[0182] 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.

[0183] In some specific embodiments of the first aspect, the second antigen-binding fragment is in the form of a scFv.

[0184] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:4.

[0185] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:5.

[0186] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:14.

[0187] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:15.

[0188] In some embodiments of the first aspect, the amino acid sequence shown in SEQ ID NO: 14 comprises two amino acid sequences shown in SEQ ID NO: 4.

[0189] In some embodiments of the first aspect, the amino acid sequence shown in SEQ ID NO: 15 comprises two amino acid sequences shown in SEQ ID NO: 5.

[0190] In some embodiments of the first aspect, the second antigen-binding fragment comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:12 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:13.

[0191] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:4; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO:12 and the light chain variable region with the amino acid sequence shown in SEQ ID NO:13.

[0192] In some embodiments of the first aspect, 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 with an amino acid sequence as shown in SEQ ID NO:12 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:13.

[0193] In some embodiments of the first aspect, 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 with an amino acid sequence as shown in SEQ ID NO:12 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:13.

[0194] In some embodiments of the first aspect, 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 with an amino acid sequence as shown in SEQ ID NO:12 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:13.

[0195] 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.

[0196] In some embodiments of the first aspect, the amino acid sequence of the first antigen-binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology to the amino acid sequence shown in SEQ ID NO:4, 5, 14 or 15.

[0197] 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 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar function of the first antigen-binding fragment.

[0198] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 4, 5, 14 or 15, and the resulting amino acid sequence still maintains similar function of the first antigen-binding fragment.

[0199] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 4, 5, 14 or 15, as long as the altered amino acid sequence substantially maintains the similar function of the first antigen-binding fragment.

[0200] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:12.

[0201] 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.

[0202] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the amino acid sequence shown in SEQ ID NO:12.

[0203] 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 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar function of the heavy chain variable region of the second antigen-binding fragment.

[0204] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can 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 similar function of the heavy chain variable region of the second antigen-binding fragment.

[0205] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 12, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the similar second antigen-binding fragment.

[0206] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:13.

[0207] 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.

[0208] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen-binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the amino acid sequence shown in SEQ ID NO:13.

[0209] 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 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar function of the light chain variable region of the second antigen-binding fragment.

[0210] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can 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 the similar function of the light chain variable region of the second antigen-binding fragment.

[0211] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 13, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the similar second antigen-binding fragment.

[0212] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, and the antibody heavy chain constant region Fc fragment includes a first Fc fragment and a second Fc fragment, wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the first Fc fragment are C, S, A and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the second Fc fragment are C, S, A and V, respectively; wherein the amino acid positions of the antibody constant region are determined according to EU numbering.

[0213] In some specific embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, and the antibody heavy chain constant region Fc fragment includes a first Fc fragment and a second Fc fragment, wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, and the amino acids at positions 349, 366, 368 and 407 of the second Fc fragment are C, S, A and V, respectively; wherein the amino acid positions of the antibody constant region are determined according to EU numbering.

[0214] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, and the antibody heavy chain constant region Fc fragment includes a first Fc fragment and a second Fc fragment, wherein the 234th, 235th and 331st amino acids of the first Fc fragment and the second Fc fragment are F, E and S respectively; wherein the amino acid positions of the antibody constant region are determined according to EU numbering.

[0215] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, which includes a first Fc fragment and a second Fc fragment, wherein one of the first Fc fragment and the second Fc fragment is connected to the first antigen-binding fragment, and the other of the first Fc fragment and the second Fc fragment is connected to the second antigen-binding fragment.

[0216] In some embodiments of the first aspect, the first antigen binding fragment (eg, at the C-terminus) is linked to the N-terminus of the first Fc fragment (eg, an amino acid sequence such as SEQ ID NO: 33).

[0217] In some embodiments of the first aspect, the second antigen binding fragment (eg, at the C-terminus) is linked to the N-terminus of the second Fc fragment (eg, an amino acid sequence such as SEQ ID NO: 34).

[0218] In some embodiments of the first aspect, the antibody heavy chain constant region Fc fragment is an Fc fragment of the IgG1 subtype. In some embodiments, the first Fc fragment is an Fc fragment of the IgG1 subtype; and / or the second Fc fragment is an Fc fragment of the IgG1 subtype.

[0219] In some embodiments of the first aspect, the antibody heavy chain constant region Fc fragment is an Fc fragment of the IgG1m3 subtype. In some embodiments, the first Fc fragment is an Fc fragment of the IgG1m3 subtype; and / or the second Fc fragment is an Fc fragment of the IgG1m3 subtype.

[0220] 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 an activating T cell antigen, wherein

[0221] The first arm comprises the amino acid sequence shown in SEQ ID NO: 35 or 36; and

[0222] The second arm comprises the amino acid sequence shown in SEQ ID NO:37.

[0223] In some embodiments of the first aspect, the amino acid sequence of the first arm comprises the amino acid sequence shown in SEQ ID NO: 35 or 36.

[0224] 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.

[0225] In some embodiments of the first aspect, the amino acid sequence of the first arm is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO:35 or 36.

[0226] 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 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still maintaining similar function of the first arm.

[0227] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 35 or 36, and the resulting amino acid sequence still maintains similar function of the first arm.

[0228] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 35 or 36, as long as the altered amino acid sequence substantially maintains the function of the similar first arm.

[0229] In some embodiments of the first aspect, the amino acid sequence of the second arm comprises the amino acid sequence shown in SEQ ID NO:37.

[0230] 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.

[0231] In some embodiments of the first aspect, the amino acid sequence of the second arm is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO:37.

[0232] 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 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still maintaining a similar function of the second arm.

[0233] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 37, and the resulting amino acid sequence still maintains a similar function of the second arm.

[0234] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 37, as long as the altered amino acid sequence substantially maintains similar function of the second arm.

[0235] In a second aspect, the present application provides a single domain antibody that binds to the HLA-A24 / PRAME complex, comprising:

[0236] HCDR1 as shown in SEQ ID NO: 1,

[0237] HCDR2 as shown in SEQ ID NO: 2, and

[0238] HCDR3 as shown in SEQ ID NO:3;

[0239] The amino acid sequence of HCDR is based on the Kabat definition.

[0240] 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 at positions 301-309 of PRAME with reference to SEQ ID NO: 38. In some embodiments, the amino acid sequence at positions 301-309 of PRAME with reference to SEQ ID NO: 38 is LYVDSLFFL (SEQ ID NO: 19).

[0241] 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 residues 304, 305, 306, 307, 308 and 309 of PRAME as shown in SEQ ID NO:38.

[0242] In some specific embodiments of the second aspect, the binding epitope of the single domain antibody to the HLA-A24 / PRAME complex includes residues 304, 305, 306, 307, 308 and 309 of PRAME as shown in SEQ ID NO: 38. In some embodiments, residues 304, 305, 306, 307, 308 and 309 of PRAME as shown in SEQ ID NO: 38 correspond to residues 304, 305, 306, 307, 308 and 309 of PRAME as shown in SEQ ID NO: 19. 301-309 The amino acid residues at positions 4, 5, 6, 7, 8 and 9 of the (LYVDSLFFL) polypeptide.

[0243] In some embodiments of the second aspect, the single domain antibody is in the form of a monovalent or multivalent single domain antibody that binds to the HLA-A24 / PRAME complex.

[0244] In some embodiments of the second aspect, the single domain antibody is in the form of a monovalent single domain antibody that binds the HLA-A24 / PRAME complex.

[0245] In some embodiments of the second aspect, the single domain antibody is in the form of a bivalent single domain antibody that binds the HLA-A24 / PRAME complex.

[0246] In some embodiments of the second aspect, the single domain antibody comprises two heavy chain variable regions of a single domain antibody that binds the HLA-A24 / PRAME complex in monovalent form.

[0247] In some specific embodiments of the second aspect, the single domain antibody comprises two monovalent forms of the heavy chain variable regions of the single domain antibody that binds the HLA-A24 / PRAME complex directly fused.

[0248] In some specific embodiments of the second aspect, the single-domain antibody comprises two monovalent forms of heavy chain variable regions of single-domain antibodies that bind to the HLA-A24 / PRAME complex connected by 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, wherein n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or an integer or integer range between any two of the above values. In some embodiments, the linker is (G4S)n, (SG4)n or G4(SG4)n, wherein n is an integer from 2 to 4, such as 2, 3 or 4. In some specific embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 94).

[0249] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:4.

[0250] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:5.

[0251] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:14.

[0252] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:15.

[0253] 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.

[0254] In some embodiments of the second aspect, the amino acid sequence of the single domain antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO:4, 5, 14 or 15.

[0255] 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 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar functions of the single domain antibody.

[0256] In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 4, 5, 14 or 15, and the resulting amino acid sequence still maintains similar functions of the single domain antibody.

[0257] In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 4, 5, 14 or 15, as long as the altered amino acid sequence substantially maintains similar function of the single domain antibody.

[0258] In a third aspect, the present application provides a nucleic acid molecule encoding the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect.

[0259] In some embodiments of the third aspect, the nucleic acid molecule may include a DNA molecule and an RNA molecule. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

[0260] In some embodiments of the third aspect, the nucleic acid molecule is operably linked to a regulatory nucleotide sequence that can be recognized by a host cell transformed with the vector.

[0261] In a fourth aspect, the present application provides a pharmaceutical composition comprising the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect and a pharmaceutically acceptable excipient, diluent or carrier.

[0262] In some embodiments of the fourth aspect, the pharmaceutical composition is used to prevent or treat HLA-A24 / PRAME positive tumors.

[0263] In some embodiments of the fourth aspect, the HLA-A24 / PRAME-positive tumor is selected from: 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's lymphoma, neuroblastoma, acute leukemia (e.g., acute myeloid leukemia or acute myeloid leukemia) and chronic leukemia (e.g., chronic myeloid leukemia or chronic myeloid leukemia).

[0264] In some embodiments of the fourth aspect, the pharmaceutical composition may further comprise one or more of the following substances: lubricants, such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives, such as benzoic acid, sorbic acid and calcium propionate; sweeteners and / or flavoring agents, etc.

[0265] In some embodiments of the fourth aspect, the pharmaceutical composition of the present application can be formulated into the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.

[0266] In some embodiments of the fourth aspect, the pharmaceutical composition of the present application can be delivered by any physiologically acceptable mode of administration, including but not limited to: oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.

[0267] In some embodiments of the fourth aspect, pharmaceutical compositions for therapeutic use can be formulated for storage in the form of lyophilized preparations or aqueous solutions by mixing reagents having the desired purity with pharmaceutically acceptable carriers, excipients, etc. as appropriate.

[0268] In a fifth aspect, the present application provides use of the bispecific antibody described in the first aspect, the single domain antibody described in the second aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a medicament for preventing or treating HLA-A24 / PRAME-positive tumors.

[0269] In some embodiments of the fifth aspect, the HLA-A24 / PRAME-positive tumor is selected from: 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's lymphoma, neuroblastoma, acute leukemia (e.g., acute myeloid leukemia or acute myeloid leukemia) and chronic leukemia (e.g., chronic myeloid leukemia or chronic myeloid leukemia).

[0270] In a sixth aspect, the present application provides a method for preventing or treating HLA-A24 / PRAME-positive tumors, comprising administering the bispecific antibody described in the first aspect, the single domain antibody described in the second aspect, or the pharmaceutical composition described in the fourth aspect to an individual in need.

[0271] In some embodiments of the sixth aspect, the HLA-A24 / PRAME-positive tumor is selected from: 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's lymphoma, neuroblastoma, acute leukemia (e.g., acute myeloid leukemia or acute myeloid leukemia) and chronic leukemia (e.g., chronic myeloid leukemia or chronic myeloid leukemia).

[0272] In a seventh aspect, the present application provides use of the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect in the preparation of a product for detecting HLA-A24 / PRAME positive cells.

[0273] In some embodiments of the seventh aspect, the product is a kit, a test strip, a test card, or a microfluidic device.

[0274] In some embodiments of the seventh aspect, the product may further comprise a detection label, such as colloidal gold, a chemiluminescent label, a fluorescent label, a nanoparticle label, and the like.

[0275] In some embodiments of the seventh aspect, the product may further include other reagents for detection, such as enzyme or colloidal gold-labeled antigens or antibodies, substrates, reference standards, diluents, washing solutions, and the like.

[0276] In some embodiments of the seventh aspect, the product may further include a reagent for processing a biological sample for detection, and the biological sample may be blood.

[0277] In some embodiments of the seventh aspect, the product may further include instructions for use of the product.

[0278] The present application also provides a vector comprising a nucleic acid molecule encoding the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect, and a host cell comprising the nucleic acid molecule or the vector. In other aspects, the present application also provides a method for producing the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect. In some embodiments, the method for producing the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect includes culturing a host cell to express the nucleic acid molecule. In some embodiments, the method for producing the bispecific antibody described in the first aspect or the single domain antibody described in the second aspect also includes recovering the bispecific antibody or the single domain antibody from the host cell culture medium.

[0279] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application, and is not intended to be limiting in any aspect. Those skilled in the art can make various modifications and changes to the embodiments.

[0280] The following examples are for illustration purposes only and are not intended to limit the scope of this application. Example

[0281] Example 1: Preparation of recombinant protein

[0282] In the process of preparing bispecific antibodies targeting HLA-A24 / PRAME and CD3, a variety of different recombinant proteins are needed, including the extracellular region of human CD3E (hCD3E, SEQ ID NO: 17) and the extracellular region of human CD3D (hCD3D, SEQ ID NO: 18).

[18] Prepare the PRAME antigen peptide LYVDSLFFL (SEQ ID NO: 19) and the dtSCT (disulfide trap single-chain trimer) structural complex of HLA-A24 (MIP-P3, SEQ ID NO: 20). These recombinant proteins have a large number of post-translational modifications (such as glycosylation or disulfide bonds, etc.), so the use of mammalian cell expression systems will be more conducive to maintaining the structure and function of the recombinant protein. Adding a His tag (His, SEQ ID NO: 21) or the Fc segment of mouse antibody IgG2a (mFc, SEQ ID NO: 22) to the C-terminus of the recombinant protein will be more conducive to the purification of the recombinant protein and the identification of the monoclonal antibody function. When preparing recombinant antibodies, the antibody heavy chain constant region can be human IgG1 subtype (SEQ ID NO: 23) or various mutants of selected human IgG1 subtypes, 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 can be human λ subtype (SEQ ID NO: 30) or human κ subtype (SEQID NO: 31).

[0283] According to the amino acid sequences of various target recombinant proteins in the Uniprot database, the genes of the above recombinant proteins (including His tags or mFc encoding genes) are designed and synthesized. The synthesized various recombinant protein genes are cloned into suitable eukaryotic expression vectors (such as pcDNA3.1 of Invitrogen, etc.) using conventional molecular biological techniques, and then the prepared recombinant protein expression plasmids are transfected into HEK293 cells (such as HEK293F of Invitrogen) using liposomes (such as 293fectin of Invitrogen, etc.) or other cationic transfection reagents (such as PEI, etc.), and cultured under serum-free suspension culture conditions for 3-4 days. The culture supernatant is then harvested by centrifugation or the like.

[0284] The recombinant protein expressed by His tag fusion is purified in one step by using a metal chelate affinity column (such as GE's HisTrap FF, etc.). The recombinant protein expressed by mFc fusion is purified in one step by using a ProteinA / G affinity column (such as GE's Mabselect SURE, etc.). Then, the recombinant protein storage buffer is replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (such as GE's Hitrapdesaulting, etc.). If necessary, the recombinant protein sample can be filtered and sterilized, and then stored in aliquots at -20°C.

[0285] Example 2: Screening and activity evaluation of anti-HLA-A24 / PRAME monoclonal antibodies

[0286] 2.1 Llama immunization and preparation of single domain antibody library

[0287] One healthy adult llama was selected, and blood was collected before immunization to keep the background serum. For the first immunization, 0.4 mg of MIP-P3-His fusion protein was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple points for immunization; for booster immunization at intervals of two weeks, 0.4 mg of MIP-P3-His fusion protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple points for immunization, for a total of 5 booster immunizations, and blood was collected before each immunization for antibody titer analysis; for the seventh immunization, no adjuvant was added, 0.4 mg of MIP-P3-His fusion protein was taken as an antigen, and multiple subcutaneous injections were injected for shock immunization, and 150 mL of peripheral blood was collected 3 days later for lymphocyte separation.

[0288] 150 mL of llama peripheral blood lymphocytes were isolated using a camel peripheral blood lymphocyte isolation kit (Solarbio, CAT#P5750); total RNA of lymphocytes was extracted using a cell total RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., CAT#DP430); the extracted total RNA was used as a template to synthesize the camel heavy chain variable region using a first-strand cDNA synthesis kit (Thermoscientific, CAT#K1621), and the reverse transcription primer used a gene-specific primer, and the primer pairing region was located in the CH2 domain of the antibody heavy chain constant region, with a specific sequence of PCal-CH2R: TCCTTCCCCGTCAGCCAGTCCT (SEQID NO:95). The synthesized cDNA was immediately stored at -70°C for future use; then the cDNA obtained by reverse transcription was used as a template, and references were used.

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

[20] ) to construct a VHH library. The library capacity of this antibody library reached 2.0E8, and the accuracy rate was 66.67%.

[0289] 2.2 Screening of camel immune library

[0290] References (see Chinese Patent Application No. 201510097117.0

[20] ), using the recombinant protein MIP-P3-mFc prepared in Example 1 as the antigen, using a solid phase screening strategy (the experimental protocol refers to Phage Display: A General Experimental Guide, edited by Clarkson, T., Lowman, HB; translated by Ma Lan et al., Chemical Industry Press, 2008.5

[21] ) The phage library displaying camel single domain antibodies constructed above was screened, and three rounds of screening were performed by binding, elution, neutralization, infection, and amplification. Finally, a TCRm antibody N83G3 (SEQ ID NO: 4) that specifically binds to MIP-P3-His was obtained.

[0291] Using conventional molecular biological methods, the nucleotide sequences of the N83G3 variable region were cloned into eukaryotic expression vectors (such as pcDNA3.1 from Invitrogen) fused with the nucleotide sequence encoding the Fc segment of human antibody IgG1 (Fc, SEQ ID NO: 32) to express the N83G3-Fc recombinant protein.

[0292] 2.3 Anti-HLA-A24 / PRAME antibody affinity analysis

[0293] The affinity of anti-HLA-A24 / PRAME monoclonal antibody was determined by surface plasmon resonance using Biacore T200. The amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), S series CM5 chip (14100530) and 10×HBS-EP at pH 7.4 (BR100669) and other related reagents and consumables were purchased from GE healthcare. According to the instructions in the kit, the carboxylated CM5 chip surface was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the anti-human IgG (Fc) antibody (capture antibody) was diluted to 25 μg / mL with 10 mM sodium acetate 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 (RU). After the injection of the capture antibody, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, the anti-HLA-A24 / PRAME monoclonal antibody was diluted to 1 μg / mL and injected at 10 μL / min to ensure that about 60 RU of the antibody was captured by the anti-human Fc antibody. Then, a series of concentration gradients (e.g., 0.37 nM, 1.11 nM, 3.33 nM, 10 nM, and 30 nM) of MIP-P3-His were set and injected from low to high concentrations at a flow rate of 30 μL / min, with an association time of 90 s and a dissociation time of 1200 s. The chip surface was regenerated by injecting 3 M MgCl2 at a flow rate of 10 μL / min for 30 s. The association rate (Ka) and dissociation rate (K) were calculated by fitting the association and dissociation sensorgrams with a 1:1 binding model using Biacore T200 Evaluation Software Version 3.2.1. d ). With the ratio K d / K a Calculate the dissociation equilibrium constant (K D ). The fitting results are shown in Table 1.

[0294] Table 1. Affinity constants of anti-HLA-A24 / PRAME antibodies binding to MIP-P3-His

[0295] <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> N83G3 6.963E+5 4.034E-5 5.793E-11

[0296] 2.4 Anti-HLA-A24 / PRAME antibody binds to PRAME 301-309 HT-29 cells pulsed with (LYVDSLFFL)

[0297] HT-29 (human colon cancer cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.) does not express PRAME protein and has two different HLA-A genes, HLA-A02 and HLA-A24. HT-29 cells in the logarithmic growth phase were digested and centrifuged and then resuspended in 1640 medium (Gibco, C11875500BT) to 1×10 7 pcs / mL, 1mL / well was plated in a 6-well cell culture plate. 301-309 The peptide (LYVDSLFFL, SEQ ID NO: 19, commissioned by GenScript Biotech Co., Ltd. to synthesize) was diluted to 114 nM with 1640 medium, and 1 mL / well was added to a 6-well cell culture plate containing cells. The cell plate was placed in a CO2 incubator at 37°C for 3 hours. After the pulse, the cells were washed once with PBS solution and resuspended in PBS to 2×10 6 pcs / mL, 100 μL / well was plated in a 96-well V-bottom plate. The test sample N83G3 and the negative control antibody DP47 (refer to US patent application US20160200833A1

[22] Preparation, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO: 96 and 97, respectively) were gradiently diluted, the starting concentration of the anti-HLA-A24 / PRAME antibody was 40nM, 4-fold gradient dilution, and a total of 8 concentration points. Take 100μL of anti-HLA-A24 / PRAME antibody and add it to the well containing cells and incubate at 4°C for 1 hour. Then wash 3 times with 200μL PBS solution, add goat anti-human IgG-FITC (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., ZF-0308), 100μL / well, incubate at 4°C in the dark for 30 minutes, wash 3 times with 200μL PBS solution, and resuspend with 100μL PBS solution and detect the FITC channel with a flow cytometer (ACEA, Novocyte). The results showed that the anti-HLA-A24 / PRAME antibody N83G3 can bind PRAME well. 301-309 Pulsed HT-29 cells ( Figure 1 ).

[0298] Example 3: Humanization and activity evaluation of anti-HLA-A24 / PRAME monoclonal antibodies

[0299] 3.1 Humanization of anti-HLA-A24 / PRAME monoclonal antibodies

[0300] Humanization studies were conducted on the TCRm antibody N83G3 to reduce its immunogenicity. The humanization scheme adopted the classic framework transplantation strategy

[23] . The amino acid sequence of N83G3 was compared with the human antibody germline gene sequence in the IMGT database, and the appropriate germline gene sequence was selected to provide the framework regions 1 to 3 (FR1+FR2+FR3) of the antibody, and the appropriate J region gene sequence was selected to provide the framework region 4 (FR4). This template can be selected based on a variety of factors, such as: the relative total length of the antibody, the size of the CDR, the amino acid residues located at the junction between the antibody framework region (FR) and the hypervariable region (CDR), the homology of the entire sequence, etc. The selected template can be a mixture of multiple sequences or a common template, the purpose of which is to maintain the appropriate conformation of the parent complementary determining region (CDR) as much as possible. At the same time, considering the solubility, stability and expression yield of the humanized antibody, the four hotspot amino acids 37F / 44E / 45R / 47F in its FR2 were reverse mutated, and the humanized mutant N83G3-h5 (SEQ ID NO: 5) was finally obtained.

[0301] 3.2 Affinity analysis of humanized antibodies

[0302] Referring to Example 2.3, affinity analysis of the anti-HLA-A24 / PRAME monoclonal antibody N83G3 humanized molecule N83G3-h5 was performed using Biacore T200. The results are shown in Table 2. Table 2. Affinity analysis of the anti-HLA-A24 / PRAME antibody N83G3 humanized molecule N83G3-h5

[0303] <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> N83G3 9.178E+5 4.67E-5 5.088E-11 N83G3-h5 7.368E+5 5.521E-5 7.474E-11

[0304] Example 4: Preparation and identification of TCRm×CD3 bispecific antibodies against HLA-A24 / PRAME complex

[0305] 4.1 Preparation of TCRm×CD3 bispecific antibodies

[0306] The nucleotide sequence encoding the variable region of bivalent N83G3 (2N83G3, SEQ ID NO: 14) and the nucleotide sequence encoding the anti-CD3 single-chain antibody (IMCR-ScFv, SEQ ID NO: 16, see anti-CD3 v9 of US Pat. No. US 5821337A) were used to construct the nucleotide sequence encoding the variable region of bivalent N83G3 (2N83G3, SEQ ID NO: 14) and the nucleotide sequence encoding the anti-CD3 single-chain antibody (IMCR-ScFv, SEQ ID NO: 16, see anti-CD3 v9 of US Pat. No. US 5821337A)

[24] ) were cloned into appropriate eukaryotic expression vectors, and co-expressed to construct a bispecific antibody against HLA-A24 / PRAME complex and CD3. That is, the nucleotide sequence encoding 2N83G3 was cloned into a eukaryotic expression vector fused with a nucleotide sequence encoding an Fc fragment IgG1m3-FcH1n1 (SEQ ID NO: 33) with a Hole mutation, and the nucleotide sequence encoding IMCR-anti-CD3-ScFv was cloned into a eukaryotic expression vector fused with a nucleotide sequence encoding an Fc fragment IgG1m3-FcKn1 (SEQ ID NO: 34) with a Knob mutation, to construct a bispecific antibody 2N83G3×IMCR.

[0307] The constructed eukaryotic expression vectors expressing 2N83G3-G1m3-FcH1n1 (SEQ ID NO: 35) and IMCR-ScFv-G1m3-FcKn1 (SEQ ID NO: 37) were co-transfected into HEK293F cells using liposomes, cultured for 3-5 days under serum-free suspension culture conditions, and then the culture supernatant was harvested by centrifugation or the like. The bispecific antibody in the culture supernatant was purified using a Protein A affinity chromatography column (such as Mabselect SURE of GE, etc.), and then the recombinant protein storage buffer was replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (such as Hitrap desaulting of GE, etc.). The desalted protein solution was purified by size exclusion chromatography (SEC) using Superdex200 (GE) to obtain the target protein. If necessary, the antibody sample can be filtered and sterilized, and then stored at -20°C for standby use.

[0308] 4.2 Construction of humanized TCRm×CD3 bispecific antibody against HLA-A24 / PRAME complex

[0309] 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 anti-CD3 single-chain antibody (IMCR-ScFv, SEQ ID NO: 16) were cloned into appropriate eukaryotic expression vectors, and co-expressed to construct a bispecific antibody against HLA-A24 / PRAME and CD3. That is, the nucleotide sequence encoding 2N83G3-h5 was cloned into a eukaryotic expression vector fused with a nucleotide sequence encoding the Hole mutation Fc fragment IgG1m3-FcH1n1 (SEQ ID NO: 33), and the nucleotide sequence encoding IMCR-ScFv was cloned into a eukaryotic expression vector fused with a nucleotide sequence encoding the Knob mutation Fc fragment IgG1m3-FcKn1 (SEQ ID NO: 34), to construct a humanized bispecific antibody 2N83G3-h5×IMCR.

[0310] 4.3 Affinity analysis of humanized bispecific antibodies

[0311] Referring to Example 2.3, affinity analysis of the humanized bispecific antibody was performed using Biacore T200. The results are shown in Tables 3 and 4.

[0312] Table 3. Affinity constants of humanized bispecific antibodies binding to MIP-P3-His

[0313] <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> 2N83G3×IMCR 1.406E+6 7.649E-5 5.442E-11 2N83G3-h5×IMCR 1.241E+6 8.064E-5 6.498E-11

[0314] Table 4. Affinity constants of humanized bispecific antibodies binding to CD3

[0315] <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> 2N83G3×IMCR 5.357E+4 2.446E-4 4.567E-9 2N83G3-h5×IMCR 5.046E+4 2.341E-4 4.64E-9

[0316] 4.4 Humanized bispecific antibodies mediate the activation of Jurkat-Dual cells by tumor cells

[0317] K562 human chronic myeloid leukemia cells (PRAME high expression, HLA-A24 negative, purchased from the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences). Our company transferred the HLA-A24 gene into K562 cells to construct the K562 / HLA-A24 cell line (PRAME + / HLA-A24 + ). K562 / HLA-A24 cells in the logarithmic growth phase were collected, centrifuged and resuspended in 1640 medium to 5×10 5 Jurkat-Dual cells (purchased from Invivogen) in the logarithmic growth phase were collected and resuspended in 1640 medium to 1×10 6 / mL, 50μL / well were plated in a 96-well cell plate to obtain a final E:T ratio of 1:1. Then, 2N83G3×IMCR bispecific antibody (2N83G3×IMCR BsAb, 50μL / well) or 2N83G3-h5×IMCR bispecific antibody (2N83G3-h5×IMCR BsAb, 50μL / well) or irrelevant antibody DP47×IMCR bispecific antibody (DP47×IMCR BsAb, 50μL / well) with a starting concentration of 80nM, 4-fold gradient dilution, and 10 concentration points were added. The cell plate was placed in a CO2 incubator at 37°C for 20 hours, and the supernatant was taken and compared with QUANTI-Luc TM Instructions (QUANTI-Luc TM , Invivogen, rep-qlc2) detection, analysis of HLA-A24 / PRAME×CD3 bispecific antibody-mediated activation of Jurkat-Dual cells by tumor cells. The results showed that both 2N83G3×IMCR BsAb and 2N83G3-h5×IMCR BsAb could mediate K562 / HLA-A24 tumor cells to activate Jurkat-Dual cells ( Figure 2 ).

[0318] 4.5 Humanized bispecific antibodies mediate PBMC killing of tumor cells

[0319] 4.5.1 Expression of PRAME in tumor cells and HLA-A locus typing

[0320] K562 (human chronic myeloid leukemia cells), HT-29 (human colon cancer cells), SK-Hep-1 (human liver cancer cells, purchased from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences), MEG-01 (human chronic myeloid 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.) were used to detect the intracellular PRAME expression level by Western blotting (commissioned by Beijing Liebaike Technology Co., Ltd.) and HLA-A locus typing (commissioned by Beijing Bio 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 express PRAME, but A375 and U-2OS cells are HLA-A24 negative. That is, only SK-Hep-1, MEG-01 cells and K562 / HLA-A24 cells have HLA-A24 on their surface + / PRAME + .

[0321] Table 5. Expression of PRAME in tumor cells and HLA-A locus typing

[0322] Cell Name PRAME expression (chemiluminescent signal value) HLA-A locus typing K562 4438571 HLA-A11:01 HLA-A31:01 SK-Hep-1 91150 HLA-A02:01 HLA-A24:02 MEG-01 118423 HLA-A24:02 HLA-A24:02 HT-29 4446 HLA-A02:01 HLA-A24:02 A375 323344 HLA-A01:01 HLA-A02:01 U-2OS 969099 HLA-A02:01 HLA-A32:01

[0323] 4.5.2 Isolation of human peripheral blood mononuclear cells (PBMC)

[0324] Blood (50 mL) was collected from normal volunteers, where the collected blood was provided by the inventor and his colleagues as volunteers, and all volunteers had signed informed consent. The inclusion criteria for volunteers were:

[0325] 1. Aged over 18;

[0326] 2. No HIV or HBV infection;

[0327] 3. Routine blood test results are normal;

[0328] 4. Non-pregnant or lactating women.

[0329] PBMCs were isolated from whole blood of volunteers by Ficoll density gradient centrifugation and cultured in 1640 medium.

[0330] 4.5.3 Humanized bispecific antibody 2N83G3-h5×IMCR mediates PBMC killing of tumor cells

[0331] K562 / HLA-A24 cells, SK-Hep-1 cells, MEG-01 cells, A375 cells, U-2OS cells, HT-29 cells and K562 cells were collected in the logarithmic growth phase. After centrifugation, they were resuspended in 1640 medium to 5×10 5 / mL, 100μL / well were plated in a 96-well cell plate. Then, a starting concentration of 80nM, 4-fold gradient dilution, 10 concentration points of 2N83G3-h5×IMCR bispecific antibody (50μL / well) or an irrelevant antibody DP47×IMCR bispecific antibody (50μL / well) was added. Finally, 50μL / well of PBMC (effector) was added to obtain a final E:T ratio of 5:1. At the same time, 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 culture medium blank control were set up, and the volume was filled to 200μL with culture medium. After incubating the cell plate in a CO2 incubator at 37°C for 20 hours, the supernatant was taken and referred to CytoTox Non-radioactive cytotoxicity assay reagent (CytoTox The humanized bispecific antibody 2N83G3-h5×IMCR only mediated the killing effect of PBMC on K562 / HLA-A24, SK-Hep-1 and MEG-01 tumor cells. 50 The values ​​are 0.01234, 0.2405 and 0.2599 respectively ( Figure 3 ).

[0332] 4.6 Activation of Jurkat-Dual cells by HT-29 cells pulsed with alanine-substituted peptide mediated by humanized bispecific antibodies

[0333] Referring to Example 2.4, PRAME 301-309 The peptide was substituted with alanine at positions 1, 2, 3, 4, 5, 6, 7, 8 and 9, and the corresponding peptide was synthesized and pulsed to HT-29 cells, wherein the amino acid sequence of the irrelevant peptide is shown in SEQ ID NO: 98. The results showed that the ability of the humanized bispecific antibody 2N83G3-h5×IMCR to activate Jurkat-Dual cells by 6, 7, 8, and 9 alanine-substituted peptide-pulsed cells was significantly reduced, and the ability to activate Jurkat-Dual cells after 4 and 5 substitutions was significantly reduced ( Figure 4 ).

[0334] Example 5: Specificity Verification

[0335] 5.1 Humanized bispecific antibody 2N83G3-h5×IMCR and PRAME 301-309 Specificity verification of peptide similarity

[0336] Cross-reactivity against homologous peptides derived from normal tissues (sharing key recognition residues) may cause severe and poorly predictable toxicity caused by TCR-like antibodies or TCR-based T cell therapies (see, e.g., Linette et al., Blood, (2013), 122:863-71, which reported fatal toxicity of MAGEA3-TCR therapy caused by off-target reactivity against proteins expressed by cardiac tissue

[25] ). Therefore, the specificity of these agents is crucial. In order to thoroughly define the specificity of the antibodies developed by our company, we 301-309The data of alanine mutants in the peptides that affect the active position of N83G3-h5 (see Example 4.6) were used to search for potential off-target peptides according to the definition formulas xYxxSLFFx, xYxDxLFFx, xYxxxLFFx, xYxxxxFFx, xxxxxLFFx, xYxxSLFxx, xxxDxLFFx, xxxxSLFFx, xxxxxxxFFx, and xYxxxxxFx through the databases (IEDB and HLA Ligand Atlas). A total of 50 peptides derived from different proteins were found, all of which had high affinity for HLA-A24, and these peptides were commissioned to be synthesized by GenScript Biotech Co., Ltd. Then, the cross-reactivity of the humanized bispecific antibody 2N83G3-h5×IMCR was verified by the activation method of Jurkat-Dual cells using peptide-pulsed HT-29 cells, as shown in Example 5.5. The results showed that the humanized bispecific antibody 2N83G3-h5×IMCR only mediated the activation of Jurkat-Dual cells by HT-29 cells pulsed with similar peptides 8 / 11 / 12 / 16, but not with PRAME 301-309 Compared with EC 50 The values ​​were increased by 337-fold / 93-fold / 482-fold / 97-fold, respectively; the humanized bispecific antibody 2N83G3-h5×IMCR did not mediate the activation of Jurkat-Dual cells by HT-29 cells pulsed with the other 46 similar peptides (Table 6).

[0337] Table 6. PRAME 301-309 Peptide similar to

[0338]

[0339]

[0340]

[0341] 5.2 Humanized bispecific antibody 2N83G3-h5×IMCR does not mediate PBMC killing of HLA-A24 and similar peptide double positive cells

[0342] 5.2.1 Construction of protein expression plasmids for PRAME and similar peptides 8 / 11 / 12 / 16

[0343] With reference to the UniProt database, the full-length genes of PRAME (SEQ ID NO: 38), XCR1 (similar peptide 8, SEQ ID NO: 39), KIFBP (similar peptide 11, SEQ ID NO: 40), CPVL (similar peptide 12, SEQ ID NO: 41) and PLD1 (similar peptide 16, SEQ ID NO: 42) were synthesized, and the various synthesized recombinant protein genes were cloned into appropriate eukaryotic expression vectors using conventional molecular biology techniques to construct full-length gene expression plasmids based on fusion GFP. Similarly, with reference to the UniProt database, the full-length gene of HLA-A2402 (SEQ ID NO: 43) was synthesized, and the synthesized gene was cloned into appropriate eukaryotic expression vectors using conventional molecular biology techniques to construct full-length gene expression plasmids.

[0344] 5.2.2 Humanized bispecific antibody 2N83G3-h5×IMCR mediates PBMC killing of HT-29 cells transfected with PRAME or similar peptides 8 / 11 / 12 / 16

[0345] HT-29 cells were plated one day before transfection, and the cells reached 50-90% confluence on the day of transfection. HT-29 cells in the logarithmic growth phase were digested and centrifuged, resuspended in 1640 medium and counted. 1.5×10 6 The cells were inoculated into a T25 cell culture flask and placed in a CO2 incubator at 37°C overnight. Transfection reagent ( DNA transfection reagent, Polyplus, 101000006) instructions, PRAME and similar peptide 8 / 11 / 12 / 16 gene expression plasmids were transfected into HT-29 cells respectively, and placed in a CO2 incubator at 37°C for 48 hours. After digestion and centrifugation of the transfected cells for 48 hours, the verification was performed with reference to Example 4.5. The results showed that the humanized bispecific antibody 2N83G3-h5×IMCR only mediated the killing of HT-29 cells transfected with the PRAME gene by PBMC, and almost could not mediate the killing of HT-29 cells transfected with similar peptide 8 / 11 / 12 / 16 genes ( Figure 5 ).

[0346] 5.2.3 Humanized bispecific antibody 2N83G3-h5×IMCR mediates PBMC killing of 293T cells transfected with PRAME or similar peptides 8 / 11 / 12 / 16

[0347] 293T (human embryonic kidney cells, PRAME-inexpressing, HLA-A24 negative, purchased from the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences). 293T cells were plated one day before transfection, and the cells reached 50-90% confluence on the day of transfection. 293T cells in the logarithmic growth phase were digested and centrifuged, resuspended in 1640 medium, and counted. 1.5×10 6 The cells were inoculated into a T25 cell culture flask and placed in a CO2 incubator at 37°C overnight. Transfection reagent ( DNA transfection reagent, Polyplus, 101000006) instructions, HLA-A2402 gene and PRAME or similar peptide 8 / 11 / 12 / 16 gene were co-transfected into 293T cells respectively, and placed in a CO2 incubator at 37°C for 24 hours. After digestion and centrifugation of the transfected cells for 24 hours, verification was performed with reference to Example 4.5. The results showed that the humanized bispecific antibody 2N83G3-h5×IMCR only mediated the killing of 293T cells co-transfected with HLA-A2402+PRAME gene by PBMC, and almost could not mediate the killing of 293T cells co-transfected with HLA-A2402+similar peptide 8 / 11 / 12 / 16 gene ( Figure 6 ).

[0348] Since the humanized bispecific antibody 2N83G3-h5×IMCR only mediated the activation of Jurkat-Dual cells by HT-29 cells pulsed with similar peptides 8 / 11 / 12 / 16, but not with PRAME 301-309 Compared with the HT-29 cells pulsed with peptides, the EC of HT-29 cells pulsed with similar peptides 8 / 11 / 12 / 16 50 The value was increased by about 90-500 times, and it was almost unable to mediate the killing of HT-29 cells transfected with similar peptide 8 / 11 / 12 / 16 genes and the killing of 293T cells co-transfected with HLA-A2402+similar peptide 8 / 11 / 12 / 16 genes. Therefore, the TCRm×CD3 bispecific antibody against the HLA-A24 / PRAME complex of the present application is only effective against HLA-A24. + / PRAME + Tumor-specific killing effect.

[0349] Example 6: Antitumor activity of humanized bispecific antibody 2N83G3-h5×IMCR in PBMC immune reconstitution model mice

[0350] Blood (50 mL each) was collected from normal volunteers, where the blood was provided by the inventor and his colleagues as volunteers, and all volunteers had signed informed consent. The inclusion criteria for volunteers were:

[0351] 1. Aged over 18;

[0352] 2. No HIV or HBV infection;

[0353] 3. Routine blood test results are normal;

[0354] 4. Non-pregnant or lactating women.

[0355] PBMCs were isolated from healthy human peripheral blood using Ficoll density gradient centrifugation. Forty 7-8 week old female NOG mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected and inoculated with 1×10 7 7 days after human PBMC inoculation, 1×10 7 K562 / HLA-A24 cells were inoculated, and the day of inoculation was defined as day 0. When the average tumor volume reached 110 mm 3 At the time of the experiment, the patients were randomly divided into 4 groups according to the tumor size: bispecific antibody 2N83G3-h5×IMCR (2N83G3-h5×IMCR BsAb) 1 mg / kg group (Group 2), bispecific antibody 2N83G3-h5×IMCR 0.1 mg / kg group (Group 3), bispecific antibody 2N83G3-h5×IMCR 0.01 mg / kg group (Group 4) and bispecific antibody unrelated antibody DP47×IMCR (DP47×IMCR BsAb) 1.5 mg / kg group (Group 1), with 8 mice in each group, and the drugs were injected into the tail vein for a total of three times at a frequency of once a week. The efficacy was evaluated according to the tumor growth volume.

[0356] The results showed that the humanized bispecific antibody 2N83G3-h5×IMCR had a significant inhibitory effect on tumor growth at a dose of 1 mg / kg and 0.1 mg / kg, with relative tumor inhibition rates (TGI) of 97.45% and 72.49%, respectively, P < 0.001 ( Figure 7 ).

[0357] Sequence information

[0358] SEQ ID NO:1

[0359] YV

[0360] SEQ ID NO:2

[0361] VITGSGNTNYADSVKG

[0362] SEQ ID NO:3

[0363] GRPSRYYRDSGTWADF

[0364] SEQ ID NO:4

[0365] QVQLVESGGGLVQAGGSLRLSCAASGASLDIYVMGWYRQATGKERDLVAVITGSGNTNYADSVKGRFTISRDDAKNTLYLQMSSLKPEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTQVTVSS

[0366] SEQ ID NO:5

[0367] EVQLVESGGGLVKPGGSLRLSCAASGASLDIYVMGWYRQAPGKGLELVAVITGSGNTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTTVTVSS

[0368] SEQ ID NO:6

[0369] GYTMN

[0370] SEQ ID NO:7

[0371] LINPYKGVSTYNQKFKD

[0372] SEQ ID NO:8

[0373] SGYYGDSDWYFDV

[0374] SEQ ID NO:9

[0375] RASQDIRNYLN

[0376] SEQ ID NO:10

[0377] YTSRLES

[0378] SEQ ID NO:11

[0379] QQGNTLPWT

[0380] SEQ ID NO:12

[0381] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS

[0382] SEQ ID NO:13

[0383] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK

[0384] SEQ ID NO:14

[0385] QVQLVESGGGLVQAGGSLRLSCAASGASLDIYVMGWYRQATGKERDLVAVITGSGNTNYADSVKGRFTISRDDAKNTLYLQMSSLKPEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVESGGGLVQAGGSLRLSCAASGASLDIYVMGWYRQATGKERDLVAVITGSGNTNYADSVKGRFTISRDDAKNTLYLQMSSLKPEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTQVTVSS

[0386] SEQ ID NO:15

[0387] EVQLVESGGGLVKPGGSLRLSCAASGASLDIYVMGWYRQAPGKGLELVAVITGSGNTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTTVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGASLDIYVMGWYRQAPGKGLELVAVITGSGNTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTTVTVSS

[0388] SEQ ID NO:16

[0389] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS

[0390] SEQ ID NO:17

[0391] QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

[0392] SEQ ID NO:18

[0393] FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVA

[0394] SEQ ID NO:20

[0395] LYVDSLFFLGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE

[0396] SEQ ID NO:21

[0397] HHHHHH

[0398] SEQ ID NO:22

[0399] ASPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0400] SEQ ID NO:23

[0401] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0402] SEQ ID NO:24

[0403] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0404] SEQ ID NO:25

[0405] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0406] SEQ ID NO:26

[0407] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPAEGEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0408] SEQ ID NO:27

[0409] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0410] SEQ ID NO:28

[0411] ASTKGPSVFPLAPSSKSTSEGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVDHKPSNTKVDKTVERKSCVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0412] SEQ ID NO:29

[0413] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVERKSCVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0414] SEQ ID NO:30

[0415] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0416] SEQ ID NO:31

[0417] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0418] SEQ ID NO:32

[0419] ASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0420] SEQ ID NO:33

[0421] ASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0422] SEQ ID NO:34

[0423] ASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0424] SEQ ID NO:35

[0425] QVQLVESGGGLVQAGGSLRLSCAASGASLDIYVMGWYRQATGKERDLVAVITGSGNTNYADSVKGRFTISRDDAKNTLYLQMSSLKPEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVESGGGLVQAGGSLRLSCAASGASLDIYVMGWYRQATGKERDLVAVITGSGNTNYADSVKGRFTISRDDAKNTLYLQMSSLKPEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTQVTVSSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0426] SEQ ID NO:36

[0427] EVQLVESGGGLVKPGGSLRLSCAASGASLDIYVMGWYRQAPGKGLELVAVITGSGNTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTTVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGASLDIYVMGWYRQAPGKGLELVAVITGSGNTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAGGRPSRYYRDSGTWADFWGQGTTVTVSSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0428] SEQ ID NO:37

[0429] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGSGGGSGGGSGGGSGGGSEGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYGDSDWYFDVWGQGTLVTSSASERKSVECPCPPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0430] SEQ ID NO:38

[0431] MERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN

[0432] SEQ ID NO:39

[0433] MESSGNPESTTFFYYDLQSQPCENQAWVFATLATTVLYCLVFLLSLVGNSLVLWVLVKYESLESLTNIFILNLCLSDLVFACLLPVWISPYHWGWVLGDFLCKLLNMIFSISLYSSIFFLTIMTIHRYLSVVSPLSTLRVPTLRCRVLVTMAVWVASILSSILDTIFHKVLSSGCDYSELTWYLTSVYQHNLFFLLSLGIILFCYVEILRTLFRSRSKRRHRTVKLIFAIVVAYFLSWGPYNFTLFLQTLFRTQIIRSCEAKQQLEYALLICRNLAFSHCCFNPVLYVFVGVKFRTHLKHVLRQFWFCRLQAPSPASIPHSPGAFAYEGASFY

[0434] SEQ ID NO:40

[0435] MANVPWAEVCEKFQAALALSRVELHKNPEKEPYKSKYSARALLEEVKALLGPAPEDEDERPEAEDGPGAGDHALGLPAEVVEPEGPVAQRAVRLAVIEFHLGVNHIDTEELSAGEEHLVKCLRLLRRYRLSHDCISLCIQAQNNLGILWSEREEIETAQAYLESSEALYNQYMKEVGSPPLDPTERFLPEEEKLTEQERSKRFEKVYTHNLYYLAQVYQHLEMFEKAAHYCHSTLKRQLEHNAYHPIEWAINAATLSQFYINKLCFMEARHCLSAANVIFGQTGKISATEDTPEAEGEVPELYHQRKGEIARCWIKYCLTLMQNAQLSMQDNIGELDLDKQSELRALRKKELDEEESIRKKAVQFGTGELCDAISAVEEKVSYLRPLDFEEARELFLLGQHYVFEAKEFFQIDGYVTDHIEVVQDHSALFKVLAFFETDMERRCKMHKRRIAMLEPLTVDLNPQYYLLVNRQIQFEIAHAYYDMMDLKVAIADRLRDPDSHIVKKINNLNKSALKYYQLFLDSLRDPNKVFPEHIGEDVLRPAMLAKFRVARLYGKIITADPKKELENLATSLEHYKFIVDYCEKHPEAAQEIEVELELSKEMVSLLPTKMERFRTKMALT

[0436] SEQ ID NO:41

[0437] MVGAMWKVIVSLVLLMPGPCDGLFRSLYRSVSMPPKGDSGQPLFLTPYIEAGKIQKGRELSLVGPFPGLNMKSYAGFLTVNKTYNSNLFFWFFPAQIQPEDAPVVLWLQGGPGGSSMFGLFVEHGPYVVTSNMTLRDRDFPWTTTLSMLYIDNPVGTGFSFTDDTHGYAVNEDDVARDLYSALIQFFQIFPEYKNNDFYVTGESYAGKYVPAIAHLIHSLNPVREVKINLNGIAIGDGYSDPESIIGGYAEFLYQIGLLDEKQKKYFQKQCHECIEHIRKQNWFEAFEILDKLLDGDLTSDPSYFQNVTGCSNYYNFLRCTEPEDQLYYVKFLSLPEVRQAIHVGNQTFNDGTIVEKYLREDTVQSVKPWLTEIMNNYKVLIYNGQLDIIVAAALTERSLMGMDWKGSQEYKKAEKKVWKIFKSDSEVAGYIRQVGDFHQVIIRGGGHILPYDQPLRAFDMINRFIYGKGWDPYVG

[0438] SEQ ID NO:42

[0439]

[0440] SEQ ID NO:43

[0441] HSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGLVLFGAVITGAVVAAVMWRRKSSDRKGGSYSQAASSDSAQGSDVSLTACKV

[0442] SEQ ID NO:94

[0443] GGGGSGGGGSGGGGS

[0444] SEQ ID NO:95

[0445] TCCTTCCCCGTCAGCCAGTCCT

[0446] SEQ ID NO:96

[0447] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS

[0448] SEQ ID NO:97

[0449] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK

[0450] SEQ ID NO:98

[0451] TYGPVFMCL

[0452] References

[0453] [1] Ikeda H, Lethé B, Lehmann F, et al., Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor. Immunity. 1997;6(2):199 - 208.

[0454] [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.

[0455] [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.

[0456] [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.

[0457] [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.

[0458] [6]Neumann E,Engelsberg A,Decker J,et al.,Heterogeneous expression ofthe tumor-associated antigens RAGE-1,PRAME,and glycoprotein 75in human renalcell carcinoma:candidates for T-cellbased immunotherapies?Cancer Res.1998;58(18):4090-4095.

[0459] [7]Thongprasert S,Yang PC,Lee JS,et al.,The prevalence of expressionof MAGE-A3 and PRAME tumor antigens in East and South East Asian non-smallcell lung cancer patients.Lung Cancer.2016;101:137-144.

[0460] [8]van Baren N,Chambost H,Ferrant A,et al.,PRAME,a gene encoding anantigen recognized on a human melanoma by cytolytic T cells,is expressed inacute leukaemia cells.Br J Haematol.1998;102(5):1376-1379.

[0461] [9]Radich JP,Dai H,Mao M,et al.,Gene expression changes associatedwith progression and response in chronic myeloid leukemia.Proc Natl Acad SciUSA.2006;103(8):2794-2799.

[0462]

[10] Willenbrock K,Kuppers R,Renne C,et al.,Common features anddifferences in the transcriptome of large cell anaplastic lymphoma andclassical Hodgkin's lymphoma.Haematologica.2006;91(5):596-604.

[0463]

[11] Doolan P,Clynes M,Kennedy S,Mehta JP,Crown J,O'DriscollL.Prevalence and prognostic and predictive relevance of PRAME in breastcancer.Breast Cancer Res Treat.2008;109(2):359-365.

[0464]

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

[0465]

[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)Characterizationof an antigen that is recognized on a melanoma showing partial HLA loss byCTL expressing an NK inhibitory receptor.Immunity,6,199–208.

[0466]

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

[0467]

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

[0468]

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

[0469]

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

[0470]

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

[0471]

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

[0472]

[20] CN 201510097117.0

[0473]

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

[0474]

[22] US20160200833A1

[0475]

[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. JImmunol 169(2):1119-1125.

[0476]

[24] US 5821337A

[0477]

[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 activating T cell antigen; Preferably, the binding epitope of the first antigen-binding fragment to the HLA-A24 / PRAME complex includes one or more residues 301-309 of PRAME as shown in SEQ ID NO: 38; more preferably, the binding epitope of the first antigen-binding fragment to the HLA-A24 / PRAME complex includes at least one of residues 304, 305, 306, 307, 308 and 309 of PRAME as shown in SEQ ID NO: 38; and / or Preferably, the activating T cell antigen is a CD3 molecule.

2. The bispecific antibody according to claim 1, wherein The first antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as 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 single-domain antibody that binds to the HLA-A24 / PRAME complex in a monovalent or multivalent manner; and / or The second antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:6, HCDR2 as shown in SEQ ID NO:7, HCDR3 as shown in SEQ ID NO:8, LCDR1 as shown in SEQ ID NO:9, LCDR2 as shown in SEQ ID NO:10 and LCDR3 as 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; more preferably, the second antigen-binding fragment is in the form of a scFv; in, The amino acid sequences of the HCDRs are according to the Kabat definition.

3. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding fragment comprises a bivalent single-domain antibody that binds to the HLA-A24 / PRAME complex; Optionally, the first antigen-binding fragment comprises two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A24 / PRAME complex; preferably, the first antigen-binding fragment comprises two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A24 / PRAME complex that are directly fused or two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A24 / PRAME complex that are connected by a linker; more preferably, the linker is a GS-type flexible peptide linker; most preferably, the linker is (G4S)n, (SG4)n or G4(SG4)n, wherein n is an integer from 1 to 10, such as an integer from 2 to 4.

4. The bispecific antibody according to any one of claims 1 to 3, wherein The first antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 4, 5, 14 or 15; and / or The second antigen-binding fragment comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:12 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

13.

5. The bispecific antibody of any one of claims 1 to 4, wherein the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 4; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 12 and the light chain variable region with the amino acid sequence 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 heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 12 and the light chain variable region with the amino acid sequence 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 heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 12 and the light chain variable region with the amino acid sequence 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 the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:12 and the light chain variable region shown in the amino acid sequence of SEQ ID NO:

13.

6. The bispecific antibody according to any one of claims 1 to 5, wherein The first antigen-binding fragment and the second antigen-binding fragment are connected through an antibody heavy chain constant region Fc fragment, and the antibody heavy chain constant region Fc fragment includes a first Fc fragment and a second Fc fragment; preferably, the antibody heavy chain constant region Fc fragment is an Fc fragment of the IgG1 subtype; more preferably, the antibody heavy chain constant region Fc fragment is an Fc fragment of the IgG1m3 subtype; wherein The amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the first Fc fragment are C, S, A and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the second Fc fragment are C, S, A and V, respectively; preferably, the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, and the amino acids at positions 349, 366, 368 and 407 of the second Fc fragment are C, S, A and V, respectively; and / or The amino acids at positions 234, 235 and 331 of the first Fc fragment and the second Fc fragment are F, E and S, respectively; and / or One of the first Fc fragment and the second Fc fragment is linked to the first antigen-binding fragment, and the other of the first Fc fragment and the second Fc fragment is linked to the second antigen-binding fragment; in, The amino acid positions of antibody constant regions are determined according to EU numbering.

7. The bispecific antibody of any one of claims 1 to 6, comprising a first arm that binds to the HLA-A24 / PRAME complex and a second arm that binds to an activating T cell antigen, wherein The first arm comprises the amino acid sequence shown in SEQ ID NO: 35 or 36; and The second arm comprises the amino acid sequence shown in SEQ ID NO:

37.

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

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

10. Use of the bispecific antibody of any one of claims 1 to 7 or the pharmaceutical composition of claim 9 in the preparation of a medicament for preventing or treating HLA-A24 / PRAME-positive tumors; preferably, the HLA-A24 / PRAME-positive tumors are selected from: 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's lymphoma, neuroblastoma, acute leukemia (e.g., acute myeloid leukemia or acute myeloid leukemia) and chronic leukemia (e.g., chronic myeloid leukemia or chronic myeloid leukemia).

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