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

Abstract

The present application provides a bispecific antibody comprising a first antigen binding fragment binding to an HLA-A02 / HBsAg complex and a second antigen binding fragment binding to an activated T cell antigen, a single domain antibody binding to an HLA-A02 / HBsAg 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-A02 / HBsAg complex, single domain antibodies binding to the HLA-A02 / HBsAg complex and their uses. Background of the Invention

[0003] Hepatitis B virus (HBV) is the pathogen that causes hepatitis B (abbreviated as HBV). Under an electron microscope, HBV can present three forms of particle structures: large spherical particles with a diameter of about 42nm, small spherical particles with a diameter of about 22nm, and tubular particles. [1,2] The large spherical particles (Dane particles) are complete virus particles, consisting of an envelope and a nucleocapsid. The envelope contains hepatitis B surface antigen (HBsAg), glycoproteins and cellular lipids, and the nucleocapsid contains core protein (HBcAg), circular double-stranded HBV-DNA and HBV-DNA polymerase. It is a complete form of the virus and is infectious. [3] .

[0004] There are at least 9 HBV genotypes, from A to I, and an uncertain J type. [4] The distribution of gene subtypes is somewhat regional. Type A is mainly distributed in Northern Europe, North America and Central Africa; Types B and C are mainly distributed in Asia; Type D is mainly distributed in the Mediterranean region, the Middle East and India; other types are distributed in South America and the African continent. [5-7] Among the people infected with HBV in my country, about 60% are infected with genotype B, 30% are infected with genotype C, and a small number are infected with genotype D and mixed infection of genotypes B and C. [8] .

[0005] According to the WHO, the HBsAg prevalence rate in the general population was 3.8% in 2019, with approximately 1.5 million new HBV infections, 296 million chronic infections, and 820,000 deaths from HBV-related diseases such as liver failure, cirrhosis, or hepatocellular carcinoma (HCC). [9] According to the Polaris International Epidemiology Collaboration, the HBsAg prevalence rate in the general population in my country was 6.1% in 2016, and the number of chronic HBV infections was 86 million. [8] Currently, there are two main types of drugs for the treatment of chronic HBV infection in clinical practice: nucleotide analogs (NA) and interferon. [8]. Based on different disease manifestations and treatment strategies, the current clinical cure rate can reach about 30%, but the HBV virus cannot be completely eliminated and a complete cure cannot be achieved. Based on the needs of disease control, a variety of new drugs are constantly being developed, such as: antisense RNA, siRNA, capsid inhibitors, monoclonal antibodies targeting HBsAg, therapeutic vaccines and immune detection targets, etc. Most single-drug effects are not ideal, and combination therapy is currently the main direction of clinical research.

[10] .

[0006] The T cell (antigen) receptor (T cell receptor, TCR) is a characteristic marker on the surface of all T cells. It binds to CD3 with a non-covalent bond to form a TCR-CD3 complex. The function of TCR is to recognize antigens, namely the peptide-MHC complex (p-MHC). The proteins in the cell are degraded and processed by the proteasome, and the resulting peptides are transported to the endoplasmic reticulum and assembled into a complex with MHC-I, which is then transported through the Golgi apparatus and finally presented on the cell surface to form antigens that can be recognized by TCR. Some of these proteins are proteins that are specifically expressed in tumors or diseases and can become specific targeted antigens in treatment. Through research, researchers have found that the preparation of TCR mimetic antibodies (TCRm) can effectively simulate TCR, recognize the MHC complex of tumor-specific antigens, and exert biological activity with the help of T cells. [11-13] Therefore, the development of TCRm×CD3 bispecific antibody drugs has become another important field of drug development. Hepatitis B surface antigen (HBsAg) is both a secretory protein and exists in cells. The degraded peptide fragments can also be presented to the cell surface.

[14] . It was identified that the HLA-A2 restricted peptide HBsAg 348-357 (GLSPTVWLSV) can be used as a targeting antigen peptide for drug development

[15] Due to the need for new treatment strategies and drugs, targeting HBsAg antigen peptides may bring new directions for the complete elimination of hepatitis B virus.

[0007] Based on clinical needs, exploring and developing CD3 bispecific antibodies targeting HBsAg-MHC has important biological and clinical significance. SUMMARY OF THE INVENTION

[0009] In a first aspect, the present application provides a bispecific antibody comprising a first antigen-binding fragment that binds to an HLA-A02 / HBsAg complex and a second antigen-binding fragment that binds to an activating T cell antigen.

[0010] In some embodiments of the first aspect, the bispecific antibody is capable of mediating HLA-A02 + / HBsAg +Tumor cells activate T cells, and / or the bispecific antibody can mediate PBMC killing of HLA-A02 + / HBsAg + Tumor cells.

[0011] In some embodiments of the first aspect, the first antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:32, HCDR2 as shown in SEQ ID NO:33, and HCDR3 as shown in SEQ ID NO:34; wherein the amino acid sequence of the HCDR is according to the Kabat definition.

[0012] In some embodiments of the first aspect, the first antigen-binding fragment is in the form of a single domain antibody.

[0013] In some embodiments of the first aspect, the first antigen-binding fragment comprises a monovalent or multivalent single domain antibody that binds to the HLA-A02 / HBsAg complex.

[0014] In some embodiments of the first aspect, the second antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:37, HCDR2 as shown in SEQ ID NO:38, HCDR3 as shown in SEQ ID NO:39, LCDR1 as shown in SEQ ID NO:40, LCDR2 as shown in SEQ ID NO:41 and LCDR3 as shown in SEQ ID NO:42; wherein the amino acid sequence of the HCDR is according to the Kabat definition.

[0015] 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.

[0016] In some embodiments of the first aspect, the first antigen-binding fragment comprises a bivalent single-domain antibody that binds to the HLA-A02 / HBsAg complex; optionally, the first antigen-binding fragment comprises two heavy chain variable regions of a monovalent single-domain antibody that binds to the HLA-A02 / HBsAg complex.

[0017] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 35, 36, 71 or 72; and / or

[0018] The second antigen-binding fragment comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:43 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:44.

[0019] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:35; and the second antigen-binding fragment comprises a heavy chain variable region with an amino acid sequence shown in SEQ ID NO:43 and a light chain variable region with an amino acid sequence shown in SEQ ID NO:44;

[0020] The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:36; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO:43 and the light chain variable region with the amino acid sequence shown in SEQ ID NO:44;

[0021] The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 71; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 43 and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 44; or

[0022] The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:72; and the second antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO:43 and the light chain variable region shown in SEQ ID NO:44.

[0023] 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.

[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 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.

[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, 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.

[0026] In some embodiments of the first aspect, the bispecific antibody comprises a first arm that binds to the HLA-A02 / HBsAg complex and a second arm that binds to an activating T cell antigen, wherein

[0027] The first arm comprises the amino acid sequence shown in SEQ ID NO: 45 or 46; and

[0028] The second arm comprises the amino acid sequence shown in SEQ ID NO:47.

[0029] In a second aspect, the present application provides a single domain antibody that binds to the HLA-A02 / HBsAg complex, comprising

[0030] HCDR1 as shown in SEQ ID NO:32,

[0031] HCDR2 as shown in SEQ ID NO:33, and

[0032] HCDR3 as shown in SEQ ID NO:34;

[0033] The amino acid sequence of HCDR is based on the Kabat definition.

[0034] In some embodiments of the second aspect, the binding epitope of the single domain antibody to the HLA-A02 / HBsAg complex includes one or more residues at positions 348-357 of HBsAg as shown in SEQ ID NO:51.

[0035] 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-A02 / HBsAg complex.

[0036] In some embodiments of the second aspect, the single domain antibody is in the form of a bivalent single domain antibody that binds to the HLA-A02 / HBsAg complex.

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

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

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

[0040] In a fifth aspect, the present application provides the use of the bispecific antibody described in the first aspect, the single domain antibody described in the second aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a medicament for preventing or treating diseases caused by HLA-A02 / HBsAg-positive HBV infection.

[0041] 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-A02 / HBsAg positive cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The results of ELISA detection of the binding of the anti-HLA-A02 / HBsAg complex TCRm antibody N1B5 to the recombinant protein MIP-H1-01 are shown.

[0044] Figure 2 The TCRm antibody N1B5 against the HLA-A02 / HBsAg complex and the antigen peptide HBsAg are shown 348-357 Results of pulsed T2 cell binding.

[0045] Figure 3 The TCRm antibody N1B5 against HLA-A02 / HBsAg complex and HBsAg 348-357 Results of T2 cell binding pulsed with alanine scanning mutant peptide (GLSPTVWLSV).

[0046] Figure 4 The results of ELISA detection of TCRm×CD3 bispecific antibody simultaneously recognizing MIP-H1-01 and CD3 are shown.

[0047] Figure 5 The results of TCRm×CD3 bispecific antibody-mediated activation of Jurkat-Dual cells by antigen peptide-pulsed T2 cells are shown.

[0048] Figure 6 TCRm×CD3 bispecific antibody mediated HLA-A02 + / HBsAg + Results of cell activation in Jurkat-Dual cells.

[0049] Figure 7 The results of TCRm×CD3 bispecific antibody-mediated PBMC cell killing of antigen peptide-pulsed T2 cells are shown.

[0050] Figure 8 The results showed that TCRm×CD3 bispecific antibody mediated the expression of PBMC cells in HLA-A02 + / HBsAg + Result of cell killing.

[0051] Fig. 9 The results showed that TCRm×CD3 bispecific antibody mediated HBsAg 348-357 (GLSPTVWLSV) Results of activation of Jurkat-Dual cells by T2 cells pulsed with alanine scanning mutant peptides.

[0052] Fig.10 The results of TCRm×CD3 bispecific antibody-mediated activation of Jurkat-Dual cells by similar peptide-pulsed T2 cells are shown.

[0053] Fig.11 It was shown that the TCRm×CD3 bispecific antibody did not mediate activation of Jurkat-Dual cells by antigen-negative target cells.

[0054] Fig.12 The results showing that TCRm×CD3 bispecific antibody inhibited the growth of HepG2-HBsAg tumor cells in hPBMC immune reconstitution mice.

[0055] Fig.13 A schematic diagram showing the hydrogen bonding and salt bridge interactions between the protein complex MIP-H1-01 and NIB5-h4 is shown, wherein the amino acid residues that form hydrogen bonding and salt bridge interactions are shown as stick models.

[0056] Sequence Description

[0057] SEQ ID NO: 1 shows an antigen peptide HBsAg derived from homo sapiens 348-357 The amino acid sequence of

[0058] SEQ ID NO: 2 shows the amino acid sequence of HBsAg-S1 derived from homo sapiens.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0088] SEQ ID NOs: 32-34 show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable regions of camel (Camelus) single domain antibodies N1B5 and N1B5-h4, respectively.

[0089] SEQ ID NO: 35 shows the amino acid sequence of the heavy chain variable region of the camel (Camelus) single domain antibody N1B5.

[0090] SEQ ID NO: 36 shows the amino acid sequence of the heavy chain variable region of the humanized single domain antibody N1B5-h4.

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

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

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

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

[0095] SEQ ID NO:45 shows the amino acid sequence of arm 2N1B5-G1m3-FcH1n1 in the bispecific antibody 2N1B5+IMCR.

[0096] SEQ ID NO:46 shows the amino acid sequence of arm 2N1B5-h4-G1m3-FcH1n1 in the bispecific antibody 2N1B5-h4+IMCR.

[0097] SEQ ID NO:47 shows the amino acid sequence of arm IMCR-anti-CD3-ScFv-G1m3-FcKn1 in 2N1B5-h4+IMCR.

[0098] SEQ ID NO:48 shows the amino acid sequence of the human (homo sapiens) CD3E extracellular region (hCD3E).

[0099] SEQ ID NO: 49 shows the amino acid sequence of the extracellular region of human (homo sapiens) CD3D (hCD3D).

[0100] SEQ ID NO:50 shows the amino acid sequence of the HLA-A02 complex (MIP-H1-01) of GLSPTVWLSV.

[0101] SEQ ID NO:51 shows the amino acid sequence of hepatitis B surface antigen (HBsAg).

[0102] SEQ ID NO:52 shows the amino acid sequence of the linker.

[0103] SEQ ID NO:53 shows the nucleotide sequence of primer PCal-CH2R.

[0104] SEQ ID NO:54 shows the amino acid sequence of the His tag.

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

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

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

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

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

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

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

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

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

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

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

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

[0117] SEQ ID NO:67 shows the amino acid sequence of the α chain variable region (Vα) of the control H1-TCR.

[0118] SEQ ID NO:68 shows the amino acid sequence of the variable region of the β chain (Vβ) of the control H1-TCR.

[0119] SEQ ID NO:69 shows the amino acid sequence of H1-TCRA-Des-G1m3-FcKn1 in the control H1-TCR.

[0120] SEQ ID NO:70 shows the amino acid sequence of H1-TCRB-Des-G1m3-FcH1n1 in the control H1-TCR.

[0121] SEQ ID NO:71 shows the amino acid sequence of the heavy chain variable regions of two camel single domain antibodies N1B5 in series.

[0122] SEQ ID NO:72 shows the amino acid sequence of the heavy chain variable regions of two camel single domain antibodies N1B5-h4 concatenated.

[0123] SEQ ID NO: 73 shows the amino acid sequence of the human IgG1 subtype antibody Fc region mutant IgG1m3-FcH1n1.

[0124] SEQ ID NO: 74 shows the amino acid sequence of the anti-human CD3E antibody IMCR single-chain antibody.

[0125] SEQ ID NO: 75 shows the amino acid sequence of the human IgG1 subtype antibody Fc region mutant IgG1m3-FcKn1.

[0126] SEQ ID NO: 76 shows the amino acid sequence of the second binding arm IMCR-anti-CD3-scFv-TCRB-Des-G1m3-FcH1n1 in the control IMC-I109V bifunctional molecule.

[0127] SEQ ID NO:77 shows HBsAg 348-357 Amino acid sequence of the G1 position alanine scanning mutant peptide.

[0128] SEQ ID NO:78 shows HBsAg 348-357 Amino acid sequence of the alanine scanning mutant peptide at position L2.

[0129] SEQ ID NO:79 shows HBsAg 348-357 Amino acid sequence of the alanine scanning mutant peptide at position S3.

[0130] SEQ ID NO:80 shows HBsAg 348-357 Amino acid sequence of the alanine scanning mutant peptide at position P4.

[0131] SEQ ID NO:81 shows HBsAg 348-357 Amino acid sequence of the alanine scanning mutant peptide at position T5.

[0132] SEQ ID NO:82 shows HBsAg 348-357 Amino acid sequence of the alanine scanning mutant peptide at position V6.

[0133] SEQ ID NO:83 shows HBsAg 348-357 Amino acid sequence of the alanine scanning mutant peptide at position W7.

[0134] SEQ ID NO:84 shows HBsAg 348-357 Amino acid sequence of the alanine scanning mutant peptide at position L8.

[0135] SEQ ID NO:85 shows HBsAg 348-357 Amino acid sequence of the alanine scanning mutant peptide at position S9.

[0136] SEQ ID NO:86 shows HBsAg 348-357 Amino acid sequence of the alanine scanning mutant peptide at position V10.

[0137] SEQ ID NO: 87 shows the amino acid sequence of an unrelated peptide.

[0138] SEQ ID NO: 88 shows the amino acid sequence of N1B5-h4-Twin-Strep-tag. DETAILED DESCRIPTION OF THE INVENTION

[0140] The inventors of the present application prepared a bispecific antibody (e.g., TCRm bispecific antibody) by genetic engineering means by using a first antigen-binding fragment that binds to the HLA-A02 / HBsAg complex and a second antigen-binding fragment that binds to an activated T cell antigen (e.g., CD3 molecule). The first antigen-binding fragment that binds to the HLA-A02 / HBsAg complex binds to HBsAg presented on the surface of a target cell (e.g., a cell infected with HBV). 348-357 (GLSPTVWLSV) and HLA-A02 complex, while the second antigen binding fragment binds to an activated T cell antigen (such as a CD3 molecule), thereby establishing an interaction between the target cell and the T cell, causing cytotoxic T cell activation, and lysing the target cell (such as an HBV-infected cell) in a non-histocompatibility complex (MHC)-dependent manner, thereby achieving the purpose of treating a disease (such as a disease caused by HBV infection). In various aspects of the present application, a new bispecific antibody comprising a first antigen binding fragment that binds to an HLA-A02 / HBsAg complex and a second antigen binding fragment that binds to an activated T cell antigen (such as a CD3 molecule), a single domain antibody that binds to an HLA-A02 / HBsAg 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-A02 / HBsAg complex can be constructed as drugs for clinical use in preventing or treating diseases caused by HLA-A02 / HBsAg-positive HBV infection.

[0141] 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.

[0142] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0143] definition

[0144] As used herein, the term "HLA-A02 / HBsAg complex" refers to a complex of an HLA-A02 molecule and HBsAg. In a specific embodiment of the present application, "HLA-A02 / HBsAg complex" refers to a complex of an HLA-A02 molecule and HBsAg at positions 348-357 (HBsAg 348-357 ) 348-357Complex.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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

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

[0149] 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.

[0150] 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)

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

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

[19] 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.

[0151] 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 / ).

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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).

[0156] 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).

[0157] 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.

[0158] 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.

[0159] As used herein, the term "tumor" refers to a neoplasm or solid lesion formed by abnormal cell growth. A tumor may be benign, pre-malignant, or malignant. In some embodiments of the present application, the tumor is liver cancer, such as liver cancer caused by HBV infection.

[0160] In a first aspect, the present application provides a bispecific antibody comprising a first antigen-binding fragment that binds to an HLA-A02 / HBsAg complex and a second antigen-binding fragment that binds to an activated T cell antigen.

[0161] In some embodiments of the first aspect, the bispecific antibody is capable of mediating HLA-A02 + / HBsAg + Tumor cells activate T cells, and / or the bispecific antibody can mediate PBMC killing of HLA-A02 + / HBsAg + Tumor cells.

[0162] In some embodiments of the first aspect, the bispecific antibody is capable of mediating HLA-A02 + / HBsAg +Tumor cells activate Jurkat-Dual cells.

[0163] In some embodiments of the first aspect, the binding epitope of the first antigen-binding fragment to the HLA-A02 / HBsAg complex includes one or more residues at positions 348-357 of HBsAg with reference to SEQ ID NO: 51. In some embodiments, the amino acid sequence at positions 348-357 of HBsAg with reference to SEQ ID NO: 51 is GLSPTVWLSV (SEQ ID NO: 1).

[0164] In some embodiments of the first aspect, the binding epitope of the first antigen binding fragment to the HLA-A02 / HBsAg complex includes at least one of residues 349, 351, 352, 353, 354, 355, 356 and 357 of HBsAg as shown in SEQ ID NO:51.

[0165] In some specific embodiments of the first aspect, the binding epitope of the first antigen-binding fragment to the HLA-A02 / HBsAg complex includes residues 349, 352, 353, 354, 355, 356, and 357 with reference to HBsAg as shown in SEQ ID NO: 51. In some embodiments, residues 349, 352, 353, 354, 355, 356, and 357 with reference to HBsAg as shown in SEQ ID NO: 51 correspond to residues 349, 352, 353, 354, 355, 356, and 357 with reference to HBsAg as shown in SEQ ID NO: 1. 348-357 The amino acid residues at positions 2, 5, 6, 7, 8, 9 and 10 of the (GLSPTVWLSV) polypeptide.

[0166] In some specific embodiments of the first aspect, the binding epitope of the first antigen-binding fragment to the HLA-A02 / HBsAg complex includes residues 351, 352, 353, 354, 355, 356, and 357 with reference to HBsAg as shown in SEQ ID NO: 51. In some embodiments, residues 351, 352, 353, 354, 355, 356, and 357 with reference to HBsAg as shown in SEQ ID NO: 51 correspond to residues 351, 352, 353, 354, 355, 356, and 357 with reference to HBsAg as shown in SEQ ID NO: 1. 348-357 The amino acid residues at positions 4, 5, 6, 7, 8, 9 and 10 of the (GLSPTVWLSV) polypeptide.

[0167] In some specific embodiments of the first aspect, the binding epitope of the first antigen-binding fragment to the HLA-A02 / HBsAg complex includes residues 349, 351, 352, 353, 354, 355, 356, and 357 with reference to HBsAg as shown in SEQ ID NO: 51. In some embodiments, residues 349, 351, 352, 353, 354, 355, 356, and 357 with reference to HBsAg as shown in SEQ ID NO: 51 correspond to residues 349, 351, 352, 353, 354, 355, 356, and 357 with reference to HBsAg as shown in SEQ ID NO: 1. 348-357 The amino acid residues at positions 2, 4, 5, 6, 7, 8, 9 and 10 of the (GLSPTVWLSV) polypeptide.

[0168] In some embodiments of the first aspect, the activating T cell antigen is a CD3 molecule.

[0169] In some embodiments of the first aspect, the first antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:32, HCDR2 as shown in SEQ ID NO:33, and HCDR3 as shown in SEQ ID NO:34; wherein the amino acid sequence of the HCDR is according to the Kabat definition.

[0170] In some embodiments of the first aspect, the first antigen-binding fragment is in the form of a single domain antibody.

[0171] In some embodiments of the first aspect, the first antigen-binding fragment comprises a monovalent or multivalent (eg, 1, 2, 3, 4, 5, or 6-valent) single domain antibody that binds to the HLA-A02 / HBsAg complex.

[0172] In some specific embodiments of the first aspect, the first antigen binding fragment comprises a bivalent single domain antibody that binds to the HLA-A02 / HBsAg complex. In some embodiments, the first antigen binding fragment comprises two heavy chain variable regions of monovalent single domain antibodies that bind to the HLA-A02 / HBsAg complex. In some embodiments, the first antigen binding fragment comprises two heavy chain variable regions of monovalent single domain antibodies that bind to the HLA-A02 / HBsAg complex that are directly fused. In some embodiments, the first antigen binding fragment comprises two heavy chain variable regions of monovalent single domain antibodies that bind to the HLA-A02 / HBsAg complex that are connected by a linker. In some embodiments, the linker is a linker comprising a GS-type flexible peptide, for example (GGGGS) n , wherein n is an integer ≥ 1. In some specific embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 52).

[0173] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:35.

[0174] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:36.

[0175] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:71.

[0176] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:72.

[0177] In some embodiments of the first aspect, the amino acid sequence shown in SEQ ID NO:71 comprises two amino acid sequences shown in SEQ ID NO:35.

[0178] In some embodiments of the first aspect, the amino acid sequence shown in SEQ ID NO:72 comprises two amino acid sequences shown in SEQ ID NO:36.

[0179] In some embodiments of the first aspect, the second antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:37, HCDR2 as shown in SEQ ID NO:38, HCDR3 as shown in SEQ ID NO:39, LCDR1 as shown in SEQ ID NO:40, LCDR2 as shown in SEQ ID NO:41 and LCDR3 as shown in SEQ ID NO:42; wherein the amino acid sequence of the HCDR is according to the Kabat definition.

[0180] 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.

[0181] In some embodiments of the first aspect, the second antigen-binding fragment is in the form of a scFv.

[0182] In some embodiments of the first aspect, the second antigen-binding fragment comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:43 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:44.

[0183] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:35; and the second antigen-binding fragment comprises the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:43 and the light chain variable region shown in the amino acid sequence of SEQ ID NO:44.

[0184] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:36; and the second antigen-binding fragment comprises the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:43 and the light chain variable region shown in the amino acid sequence of SEQ ID NO:44.

[0185] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:71; and the second antigen-binding fragment comprises the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:43 and the light chain variable region shown in the amino acid sequence of SEQ ID NO:44.

[0186] In some embodiments of the first aspect, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:72; and the second antigen-binding fragment comprises the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:43 and the light chain variable region shown in the amino acid sequence of SEQ ID NO:44.

[0187] In some embodiments of the first aspect, the amino acid sequence of the first antigen-binding fragment differs from the amino acid sequence shown in SEQ ID NO:35, 36, 71 or 72 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0188] In some embodiments of the first aspect, the amino acid sequence of the first antigen-binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology to the amino acid sequence shown in SEQ ID NO:35, 36, 71 or 72.

[0189] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:35, 36, 71 or 72 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar function of the first antigen-binding fragment.

[0190] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 35, 36, 71 or 72, and the resulting amino acid sequence still maintains similar function of the first antigen-binding fragment.

[0191] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 35, 36, 71 or 72, as long as the altered amino acid sequence substantially maintains the similar function of the first antigen-binding fragment.

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

[0193] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment differs from the amino acid sequence shown in SEQ ID NO:43 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0194] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the amino acid sequence shown in SEQ ID NO:43.

[0195] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:43 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar function of the heavy chain variable region of the second antigen-binding fragment.

[0196] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:43, and the resulting amino acid sequence still maintains similar function of the heavy chain variable region of the second antigen-binding fragment.

[0197] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO:43, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the similar second antigen-binding fragment.

[0198] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:44.

[0199] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen-binding fragment differs from the amino acid sequence shown in SEQ ID NO:44 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0200] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen-binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the amino acid sequence shown in SEQ ID NO:44.

[0201] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:44 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar function of the light chain variable region of the second antigen-binding fragment.

[0202] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:44, and the resulting amino acid sequence still maintains the similar function of the light chain variable region of the second antigen-binding fragment.

[0203] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO:44, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the similar second antigen-binding fragment.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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: 73).

[0209] 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: 75).

[0210] 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.

[0211] 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.

[0212] In some embodiments of the first aspect, the bispecific antibody comprises a first arm that binds to the HLA-A02 / HBsAg complex and a second arm that binds to an activating T cell antigen, wherein

[0213] The first arm comprises the amino acid sequence shown in SEQ ID NO: 45 or 46; and

[0214] The second arm comprises the amino acid sequence shown in SEQ ID NO:47.

[0215] In some embodiments of the first aspect, the amino acid sequence of the first arm comprises the amino acid sequence shown in SEQ ID NO: 45 or 46.

[0216] In some embodiments of the first aspect, the amino acid sequence of the first arm differs from the amino acid sequence shown in SEQ ID NO: 45 or 46 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0217] In some embodiments of the first aspect, the amino acid sequence of the first arm is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO:45 or 46.

[0218] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:45 or 46 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still maintaining similar function of the first arm.

[0219] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 45 or 46, and the resulting amino acid sequence still maintains similar function of the first arm.

[0220] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 45 or 46, as long as the altered amino acid sequence substantially maintains the function of the similar first arm.

[0221] 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:47.

[0222] 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:47 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0223] 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:47.

[0224] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:47 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.

[0225] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:47, and the resulting amino acid sequence still maintains a similar function of the second arm.

[0226] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids 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: 47, as long as the altered amino acid sequence substantially maintains similar function of the second arm.

[0227] In a second aspect, the present application provides a single domain antibody that binds to the HLA-A02 / HBsAg complex, comprising

[0228] HCDR1 as shown in SEQ ID NO:32,

[0229] HCDR2 as shown in SEQ ID NO:33, and

[0230] HCDR3 as shown in SEQ ID NO:34;

[0231] The amino acid sequence of HCDR is based on the Kabat definition.

[0232] In some embodiments of the second aspect, the binding epitope of the single domain antibody to the HLA-A02 / HBsAg complex includes one or more residues in positions 348-357 of HBsAg with reference to SEQ ID NO: 51. In some embodiments, the amino acid sequence of positions 348-357 of HBsAg with reference to SEQ ID NO: 51 is GLSPTVWLSV (SEQ ID NO: 1).

[0233] In some embodiments of the second aspect, the binding epitope of the single domain antibody to the HLA-A02 / HBsAg complex includes at least one of residues 349, 351, 352, 353, 354, 355, 356 and 357 of HBsAg as shown in SEQ ID NO:51.

[0234] In some specific embodiments of the second aspect, the binding epitope of the single domain antibody to the HLA-A02 / HBsAg complex includes residues 349, 352, 353, 354, 355, 356 and 357 of the HBsAg shown in SEQ ID NO: 51. In some embodiments, residues 349, 352, 353, 354, 355, 356 and 357 of the HBsAg shown in SEQ ID NO: 51 correspond to residues 349, 352, 353, 354, 355, 356 and 357 of the HBsAg shown in SEQ ID NO: 1. 348-357 The amino acid residues at positions 2, 5, 6, 7, 8, 9 and 10 of the (GLSPTVWLSV) polypeptide.

[0235] In some specific embodiments of the second aspect, the binding epitope of the single domain antibody to the HLA-A02 / HBsAg complex includes residues 351, 352, 353, 354, 355, 356 and 357 of HBsAg with reference to SEQ ID NO: 51. In some embodiments, residues 351, 352, 353, 354, 355, 356 and 357 of HBsAg with reference to SEQ ID NO: 51 correspond to residues 351, 352, 353, 354, 355, 356 and 357 of HBsAg with reference to SEQ ID NO: 1 348-357 The amino acid residues at positions 4, 5, 6, 7, 8, 9 and 10 of the (GLSPTVWLSV) polypeptide.

[0236] In some specific embodiments of the second aspect, the binding epitope of the single domain antibody to the HLA-A02 / HBsAg complex includes residues 349, 351, 352, 353, 354, 355, 356 and 357 of the HBsAg shown in SEQ ID NO: 51. In some embodiments, residues 349, 351, 352, 353, 354, 355, 356 and 357 of the HBsAg shown in SEQ ID NO: 51 correspond to residues 349, 351, 352, 353, 354, 355, 356 and 357 of the HBsAg shown in SEQ ID NO: 1. 348-357 The amino acid residues at positions 2, 4, 5, 6, 7, 8, 9 and 10 of the (GLSPTVWLSV) polypeptide.

[0237] 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-A02 / HBsAg complex.

[0238] In some embodiments of the second aspect, the single domain antibody is in the form of a monovalent single domain antibody that binds to the HLA-A02 / HBsAg complex.

[0239] In some embodiments of the second aspect, the single domain antibody is in the form of a bivalent single domain antibody that binds to the HLA-A02 / HBsAg complex.

[0240] 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-A02 / HBsAg complex in monovalent form.

[0241] In some specific embodiments of the second aspect, the single domain antibody comprises two heavy chain variable regions of a single domain antibody that binds to the HLA-A02 / HBsAg complex in monovalent form directly fused.

[0242] In some specific embodiments of the second aspect, the single-domain antibody comprises two monovalent heavy chain variable regions of a single-domain antibody that binds to the HLA-A02 / HBsAg complex connected by a linker. In some embodiments, the linker is a linker comprising a GS-type flexible peptide, such as (GGGGS)n, where n is an integer ≥ 1. In some specific embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 52).

[0243] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:35.

[0244] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:36.

[0245] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:71.

[0246] In some embodiments of the second aspect, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:72.

[0247] In some embodiments of the second aspect, the amino acid sequence of the single domain antibody differs from the amino acid sequence shown in SEQ ID NO:35, 36, 71 or 72 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0248] In some embodiments of the second aspect, the amino acid sequence of the single domain antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO:35, 36, 71 or 72.

[0249] In some embodiments of the second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:35, 36, 71 or 72 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar functions of the single domain antibody.

[0250] In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 35, 36, 71 or 72, and the resulting amino acid sequence still maintains similar functions of the single domain antibody.

[0251] In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 35, 36, 71 or 72, as long as the altered amino acid sequence substantially maintains similar function of the single domain antibody.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] In some embodiments of the fourth aspect, the pharmaceutical composition is used to prevent or treat diseases caused by HLA-A02 / HBsAg-positive HBV infection.

[0257] In some embodiments of the fourth aspect, the disease caused by the HLA-A02 / HBsAg-positive HBV infection is selected from the group consisting of: hepatitis B, cirrhosis, liver fibrosis and liver cancer.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] In a fifth aspect, the present application provides the use of the bispecific antibody described in the first aspect, the single domain antibody described in the second aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a medicament for preventing or treating diseases caused by HLA-A02 / HBsAg-positive HBV infection.

[0263] In some embodiments of the fifth aspect, the disease caused by HLA-A02 / HBsAg-positive HBV infection is selected from the group consisting of hepatitis B, cirrhosis, liver fibrosis and liver cancer.

[0264] In a sixth aspect, the present application provides a method for preventing or treating diseases caused by HLA-A02 / HBsAg-positive HBV infection, 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.

[0265] In some embodiments of the sixth aspect, the disease caused by HLA-A02 / HBsAg-positive HBV infection is selected from the group consisting of: hepatitis B, cirrhosis, liver fibrosis and liver cancer.

[0266] 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-A02 / HBsAg positive cells.

[0267] In some embodiments of the seventh aspect, the product is a kit, a test strip, a test card, or a microfluidic device.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] In some embodiments of the seventh aspect, the product may further include instructions for use of the product.

[0272] 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.

[0273] 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.

[0274] The following examples are for illustration purposes only and are not intended to limit the scope of this application. Example

[0275] Example 1: Preparation of recombinant protein

[0276] In the process of preparing bispecific antibodies based on HLA-A02 / HBsAg and CD3, a variety of different recombinant proteins are needed, including the extracellular region of human CD3E (hCD3E, SEQ ID NO: 48) and the extracellular region of human CD3D (hCD3D, SEQ ID NO: 49).

[20] Preparation of antigen peptide HBsAg 348-357The disulfide trap single chain trimer (dtSCT) structure MHCⅠ complex of (GLSPTVWLSV), namely the HLA-A02 complex of GLSPTVWLSV (MIP-H1-01, SEQ ID NO:50). These recombinant proteins have a large number of post-translational modifications (such as glycosylation or disulfide bonds, etc.), so the use of mammalian cell expression system will be more conducive to maintaining the structure and function of the recombinant protein. Adding His tag (His, SEQ ID NO:54) or the Fc segment of mouse antibody IgG2a (mFc, SEQ ID NO:55) to the C-terminus of these recombinant proteins will be more conducive to the purification of recombinant proteins and the identification of monoclonal antibody functions. When preparing recombinant antibodies, the antibody heavy chain constant region can be human IgG1 subtype (SEQ ID NO: 56) or various mutants of selected human IgG1 subtypes, such as: IgG1H (SEQ ID NO: 57), IgG1K (SEQ ID NO: 58), IgG1m3-H (SEQ ID NO: 59), IgG1m3-K (SEQID NO: 60), IgG1m3-H1n1 (SEQ ID NO: 61) or IgG1m3-Kn1 (SEQ ID NO: 62), and the light chain constant region can be human λ subtype (SEQ ID NO: 63) or human κ subtype (SEQ ID NO: 64).

[0277] 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 various synthesized recombinant protein genes are cloned into suitable eukaryotic expression vectors (such as pcDNA3.1 of Invitrogen, etc.) using conventional molecular biological techniques, and then the prepared recombinant protein expression plasmids are transfected into HEK293 cells (such as HEK293F cells of Invitrogen) using liposomes (such as 293fectin of Invitrogen, etc.) or other cationic transfection reagents (such as PEI, etc.), and cultured under serum-free suspension culture conditions for 3-4 days. The culture supernatant is then harvested by centrifugation or the like.

[0278] 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 antibody sample can be filtered and sterilized, and then stored in aliquots at -20°C.

[0279] Example 2: Screening of antibodies binding to the HLA-A02 / HBsAg complex from a camel immune library

[0280] 2.1 Construction of camel immune library

[0281] One healthy adult Bactrian camel was selected, and blood was collected before immunization to keep the background serum. For the first immunization, 1 mg of MIP-H1-01 fusion protein was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple points; for booster immunization at intervals of two weeks, 1 mg of MIP-H1-01 fusion protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple points, for a total of 5 booster immunizations, and blood was collected before each immunization for antibody titer analysis; for the seventh immunization, no adjuvant was added, 1 mg of MIP-H1-01 fusion protein was taken as an antigen, and multiple subcutaneous injections were injected for shock immunization, and 200 mL of peripheral blood was collected 3 days later for lymphocyte separation.

[0282] Lymphocytes were isolated from 200 mL of camel peripheral blood using a camel peripheral blood lymphocyte isolation kit (Solarbio, CAT#P5750); total RNA of lymphocytes was extracted using a cell total RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., CAT#DP430); the extracted total RNA was used as a template to synthesize camel single-domain antibody heavy chain variable region (VHH) cDNA 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 (SEQ ID NO:53). The synthesized cDNA was immediately stored at -70°C for future use; then the cDNA obtained by reverse transcription was used as a template, and references were used to synthesize the cDNA.

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

[22] ), constructing a VHH library with a library capacity of 1.41E+08 and an accuracy rate of 65.20%.

[0283] 2.2 Screening of camel immune library

[0284] The recombinant MIP-H1-01 prepared in Example 1 was used as an antigen and a solid phase screening strategy was used (the experimental scheme refers to Phage Display: A General Experimental Guide, edited by Clarkson, T. and Lowman, HB; translated by Ma Lan et al., Chemical Industry Press, May 2008).

[23] ) The phage library displaying camel single-domain antibodies constructed in Example 2.1 was screened, and three rounds of screening were performed by binding, elution, neutralization, infection, and amplification, and finally the TCRm (TCR simulation) antibody N1B5 that specifically binds to MIP-H1-01 was obtained.

[0285] Using conventional molecular biological methods, the nucleotide sequence encoding the N1B5 heavy chain variable region (SEQ ID NO: 35) was cloned into a eukaryotic expression vector (such as pcDNA3.1 from Invitrogen) fused with a nucleotide sequence encoding the Fc segment of human antibody IgG1 to express the N1B5-Fc recombinant protein. At the same time, DP47 (germline gene antibody, see US patent application US20160200833A1) was prepared.

[24] , the amino acid sequence of DP47VH is shown in SEQ ID NO: 65; the amino acid sequence of DP47VK is shown in SEQ ID NO: 66) monoclonal antibody DP47-IgG1 was used as a negative control. Refer to patent WO2020193745 A1

[25] , synthesized the Vα (TCRA, SEQ ID NO: 67) and Vβ (TCRB, SEQ ID NO: 68) variable region genes of the TCR of IMC-I109V, cloned them into the eukaryotic expression vectors of the Fc fragment nucleotide sequence containing the Knob mutation fused to Cα and the eukaryotic expression vectors of the Fc fragment nucleotide sequence containing the Hole mutation fused to Cβ, respectively, and co-transfected them to express the TCR control molecule binding to MIP-H1-01, named H1-TCR. The two chains of H1-TCR are H1-TCRA-Des-FcKn1 (SEQ ID NO: 69) and H1-TCRB-Des-G1m3-FcH1n1 (SEQ ID NO: 70).

[0286] Example 3: Identification of TCRm antibodies that bind to the HLA-A02 / HBsAg complex

[0287] 3.1 Affinity analysis of TCRm antibodies against HLA-A02 / HBsAg complex

[0288] The affinity of the anti-HLA-A2 / HBsAg TCRm antibody N1B5 was determined by surface plasmon resonance using Biacore T200. The amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), S series CM5 chip (14100530) and pH 7.4 10×HBS-EP (BR100669) and other related reagents and consumables were purchased from GE healthcare. According to the instructions in the kit, the carboxylated CM5 chip surface was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the anti-human IgG (Fc) antibody (capture antibody) was diluted to 25 μg / mL with 10 mM pH 5.0 sodium acetate and then injected at a flow rate of 10 μL / min to achieve a coupling amount of approximately 10,000 response units (RU). After the capture antibody was injected, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, the TCRm antibody N1B5- was diluted to 1 μg / mL and injected at 10 μL / min to ensure that about 70 RU of the antibody was captured by the anti-human Fc antibody. Then MIP-H1-01 was set to a series of concentration gradients (e.g., 1.23 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM) 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. A 3 M MgCl2 solution was injected at a flow rate of 10 μL / min for 30 s to regenerate the chip surface. The binding rate (K) was calculated by fitting the binding and dissociation sensorgrams with a 1:1 binding model using Biacore T200 Evaluation Software Version 3.2.1. a ) and dissociation rate (K d ). With the ratio K d / K a Calculate the dissociation equilibrium constant (K D ). The fitting results are shown in Table 1.

[0289] Table 1. Affinity of TCRm antibody N1B5 against HLA-A02 / HBsAg complex binding to MIP-H1-01

[0290] <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> N1B5 6.921E+5 9.696E-5 1.401E-10

[0291] 3.2 Binding of TCRm antibody against HLA-A02 / HBsAg complex and recombinant protein MIP-H1-01

[0292] The TCRm antibody N1B5 against the HLA-A02 / HBsAg complex was coated on a 96-well ELISA plate at 5 μg / mL, 100 μL per well, and coated overnight at 4°C. The plate was blocked at 37°C for 1 hour using a blocking solution PBS-0.1% Tween 20-3% skim milk. The recombinant protein MIP-H1-01 was diluted with PBS, with a starting concentration of 10 μg / mL, 3-fold gradient dilution, a total of 11 concentration gradients, 100 μL per well added to the blocked 96-well ELISA plate, and incubated at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20, and HRP-labeled anti-His tag mouse monoclonal antibody (Beijing Kangwei Century Biotechnology Co., Ltd., CW0285M) was added, and incubated at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20, and OPD substrate colorimetric solution was added. After 5-10 minutes, 1M H2SO4 was used to terminate the colorimetric development. The optical density value was measured by a 492nm / 630nm dual wavelength microplate reader. Figure 1 The results showed that N1B5 specifically binds to MIP-H1-01, that is, N1B5 specifically binds to the HLA-A02 / HBsAg complex.

[0293] 3.3 Binding of TCRm Antibody Against HLA-A02 / HBsAg Complex to Antigen Peptide-Pulsed T2 Cells

[0294] Collect T2 cells (human lymphoma cells, HLA-A02) in the logarithmic phase of growth + , purchased from Shanghai Hongshun Biotechnology Co., Ltd.), and resuspended in growth medium to 1×10 6 / mL, 1mL per well was plated in a 24-well plate. Chemically synthesized peptide HBsAg 348-357 (GLSPTVWLSV) (GenScript Biotech Co., Ltd.) was added to a 24-well plate at a final concentration of 50 μg / mL and pulsed overnight. After 16 hours, the cells were centrifuged and resuspended in PBS buffer containing 1% BSA to a concentration of 2 × 10 6 / mL, 100μL per well was spread in a 96-well V-bottom plate, and the supernatant was removed after centrifugation. Antibody N1B5, positive control sample H1-TCR and negative control DP47-IgG1 were prepared with PBS to a final concentration of 200nM, and the samples were diluted 4 times, for a total of 10 concentrations. 100μL per well was added to the wells containing cells and incubated at 4°C for 1 hour. Then wash 3 times with 200μL PBS, incubate with sheep anti-human IgG-FITC (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., ZF-0308), 100μL per well, incubate at 4°C in the dark for 30 minutes. Then wash 3 times with 200μL PBS, resuspend in 100μL PBS, and detect the FITC channel with a flow cytometer (ACEA, Novocyte). Figure 2The results showed that N1B5 could specifically bind to the antigen peptide HBsAg 348-357 Pulsed T2 cells, namely N1B5, bind to the HLA-A02 / HBsAg complex on the cell surface.

[0295] 3.4 Key amino acids for binding of TCRm antibody against HLA-A02 / HBsAg complex to HBsAg

[0296] Chemical synthesis of HBsAg 348-357 Alanine scanning mutant peptide (GLSPTVWLSV). Reference Example 3.3 Detection of N1B5 and HBsAg 348-357 Binding of N1B5 to T2 cells pulsed with alanine scanning mutant peptide (GLSPTVWLSV), wherein the amino acid sequence of the irrelevant peptide is shown in SEQ ID NO: 87. Binding of N1B5 to alanine scanning mutant peptides was calculated relative to the original peptide. Figure 3 The results showed that HBsAg 348-357 After positions 2, 4, 5, 6, 7, 8, 9, and 10 of (GLSPTVWLSV) were replaced by alanine, the binding ability of TCRm antibody N1B5 decreased by more than 70%, that is, HBsAg 348-357 The 2nd, 4th, 5th, 6th, 7th, 8th, 9th and 10th positions of (GLSPTVWLSV) are the key amino acids for N1B5 binding.

[0297] Example 4: Humanization and identification of TCRm antibodies against HLA-A02 / HBsAg complex

[0298] 4.1 Humanization of TCRm Antibody Against HLA-A02 / HBsAg Complex

[0299] The TCRm antibody N1B5 was humanized to reduce its immunogenicity. The humanization scheme adopted the classic framework transplantation strategy.

[26] . The amino acid sequence of N1B5 was compared with the human antibody germline gene sequence in the IMGT database, and the appropriate germline gene sequence was selected to provide the antibody framework regions 1 to 3 (FR1+FR2+FR3), and the appropriate J region gene sequence was selected to provide the framework region 4 (FR4). This template can be selected based on a variety of factors, such as: the relative total length of the antibody, the size of the CDR, the amino acid residues located at the junction between the antibody framework region (FR) and the hypervariable region (CDR), the homology of the entire sequence, etc. The selected template can be a mixture of multiple sequences or a common template, with the aim of maintaining the appropriate conformation of the parent complementarity determining region (CDR) as much as possible. At the same time, considering the solubility, stability and expression yield of the humanized antibody, the four hotspot amino acids 37F / 44E / 45R / 47F in its FR2 were reverse mutated, and the humanized molecule N1B5-h4 was finally obtained.

[0300] Using conventional molecular biological methods, the nucleotide sequence encoding the variable region of N1B5-h4 (SEQ ID NO: 36) was cloned into a eukaryotic expression vector (such as pcDNA3.1 from Invitrogen) fused with a nucleotide sequence encoding the Fc segment of human antibody IgG1 to express the N1B5-h4-Fc recombinant protein.

[0301] 4.2 Affinity analysis of humanized TCRm antibody against HLA-A02 / HBsAg complex

[0302] Referring to Example 3.1, the affinity analysis of the recombinant anti-HLA-A02 / HBsAg complex TCRm antibody N1B5-h4 was performed using Biacore T200, and the results are shown in Table 2. Table 2. Affinity of the anti-HLA-A02 / HBsAg complex TCRm antibody N1B5-h4 binding to MIP-H1-01

[0303] <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> N1B5-h4 6.556E+5 1.252E-4 1.910E-10

[0304] Example 5: Preparation and identification of TCRm×CD3 bispecific antibodies

[0305] 5.1 Preparation of TCRm×CD3 bispecific antibodies

[0306] The heavy chain variable region nucleotide sequence encoding the bivalent N1B5 or bivalent N1B5-h4 single domain antibody against HLA-A02 / HBsAg complex and the single chain antibody encoding anti-CD3 (IMCR-anti-CD3-scFv, see application No. US5821337A) were respectively

[27] The nucleotide sequence of anti-CD3 v9) was cloned into a suitable eukaryotic expression vector to co-express the bispecific antibody against HLA-A02 / HBsAg complex and CD3. The nucleotide sequence encoding 2N1B5 (SEQ ID NO: 71) or 2N1B5-h4 (SEQ ID NO: 72) was cloned into a eukaryotic expression vector fused with the nucleotide sequence of Fc fragment IgG1m3-FcH1n1 (SEQ ID NO: 73) encoding Hole mutation, and the nucleotide sequence encoding IMCR-anti-CD3-ScFv (SEQ ID NO: 74) was cloned into a eukaryotic expression vector fused with the nucleotide sequence of Fc fragment IgG1m3-FcKn1 (SEQ ID NO: 75) encoding Knob mutation. At the same time, the DP47 bispecific antibody 2DP47+IMCR based on the same structure was prepared as a negative control.

[0307] The constructed eukaryotic expression vector expressing 2N1B5-IgG1m3-FcH1n1 (SEQ ID NO: 45) or 2N1B5-h4-IgG1m3-FcH1n1 (SEQ ID NO: 46) and the eukaryotic expression vector expressing IMCR-anti-CD3-ScFv-IgG1m3-FcKn1 (SEQ ID NO: 47) 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 Company, etc.), and then the recombinant protein storage buffer was replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (such as Hitrapdesaulting of GE Company, etc.). The desalted protein solution was purified by size exclusion chromatography (SEC) using Superdex200 (GE) to obtain the target protein. If necessary, the antibody sample can be filtered and sterilized, then aliquoted and stored at -20°C for later use.

[0308] Prepare the bifunctional molecule IMC-I109V based on TCR and CD3. The single-chain antibody IMCR-anti-CD3-scFv of CD3 was fused to the N-terminus of the H1-TCRB-Des-G1m3-FcH1n1 chain. The two chains that make up IMC-I109V are H1-TCRA-Des-G1m3-FcKn1 (SEQ ID NO: 69) and IMCR-anti-CD3-scFv-TCRB-Des-G1m3-FcH1n1 (SEQ ID NO: 76). The expression and purification of the above IMC-I109V refer to the expression and purification of the bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR, after which the sample is filtered and sterilized, and then aliquoted and stored at -20°C for later use.

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

[0310] Referring to Example 3.1, affinity analysis of the recombinant TCRm×CD3 bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR was performed using Biacore T200. The results are shown in Tables 3 and 4.

[0311] Table 3. TCRm×CD3 bispecific antibody binding affinity to MIP-H1-01

[0312] <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> 2N1B5+IMCR 6.132E+5 1.452E-4 2.367E-10 2N1B5-h4+IMCR 2.629E+5 1.397E-4 5.314E-10 IMC-I109V 5.241E+4 8.693E-5 1.659E-9

[0313] Table 4. TCRm×CD3 bispecific antibody binding affinity to CD3D-CD3E-mFc

[0314] <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> 2N1B5+IMCR 5.918E+4 1.753E-4 2.962E-9 2N1B5-h4+IMCR 6.469E+4 1.32E-4 2.041E-9 IMC-I109V 7.019E+4 2.591E-4 3.692E-9

[0315] 5.3TCRm×CD3 bispecific antibody simultaneously recognizes antigens MIP-H1-01 and CD3

[0316] Conventional ELISA method was used to detect that TCRm×CD3 bispecific antibody simultaneously binds to the dual antigens CD3 and MIP-H1-01.

[0317] The CD3D-CD3E-mFc antigen was coated on a 96-well ELISA plate (3 μg / mL, 100 μL per well) and coated overnight at 4°C. The plate was blocked with a blocking solution of PBS-0.1% Tween 20-3% skim milk at 37°C for 1 hour. The TCRm×CD3 bispecific antibody was diluted to 10 μg / mL with PBS, and 100 μL per well was added to the blocked 96-well ELISA plate and incubated at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20, and the MIP-H1-01 antigen (10 μg / mL, 100 μL per well) was added and incubated at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20, and then the HRP-labeled anti-His tag mouse monoclonal antibody (Beijing Kangwei Century Biotechnology Co., Ltd., cw0285M) was added and incubated at 37°C for 1 hour. Wash the ELISA plate with PBS-0.1% Tween 20, add OPD substrate colorimetric solution, stop colorimetric development with 1M H2SO4 after 5-10 minutes, and measure the optical density with a dual wavelength of 492nm / 630nm using an ELISA reader. Figure 4 As shown: TCRm×CD3 bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR can simultaneously recognize CD3 and MIP-H1-01 dual antigens.

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

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

[0320] T2 cells in the logarithmic growth phase were collected, centrifuged and resuspended in growth medium to 1×10 6 pcs / mL, 1mL per well was plated in a 24-well plate. 348-357 (GLSPTVWLSV) was added to a 24-well plate at a final concentration of 50 μg / mL and pulsed overnight. After 16 hours, the cells were centrifuged and resuspended in RPMI1640 medium to 4 × 105 Jurkat-Dual cells (purchased from Invivogen) in the logarithmic growth phase were collected and resuspended in RPMI1640 medium to 4×10 5 / mL, 50μL per well was added to the cell plate inoculated with T2 cells to obtain a final E:T ratio of 1:1. Then add the bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR (starting concentration of 10nM, 3-fold gradient dilution, 11 concentration points, 100μL per well). The negative control sample 2DP47+IMCR and the positive control sample IMC-I109V were used at the same concentration as the bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR. Incubate for 24h, take the supernatant, and refer to QUANTI-Luc TM Instructions (QUANTI-Luc TM , Invivogen, rep-qlc2) detected and analyzed the specific activation of Jurkat-Dual cells by TCRm×CD3 bispecific antibody-mediated antigen peptide-pulsed T2 cells. Figure 5 The results showed that the bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR could specifically mediate the activation of Jurkat-Dual cells by antigen peptide-pulsed T2 cells; and compared with IMC-I109V, the activation of Jurkat-Dual cells by 2N1B5+IMCR and 2N1B5-h4+IMCR bispecific antibodies was more specific, and EC 50 See Table 5 for values.

[0321] Table 5. TCRm×CD3 bispecific antibody mediated activation of Jurkat-Dual cell EC by antigen peptide-pulsed T2 cells 50 value

[0322] 2N1B5+IMCR 2N1B5-h4+IMCR IMC-I109V <![CDATA[EC 50 (nM)]]> 0.002413 0.002758 0.06414

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

[0324] HepG2 (human hepatoma cells, purchased from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences). HepG2 is an HLA-A02 positive cell. Based on HepG2 cells, a cell line HepG2-HBsAg stably expressing HBsAg (hepatitis B surface antigen) was constructed. HepG2-HBsAg in the logarithmic growth phase was collected, digested and centrifuged, and then resuspended in RPMI1640 medium to 4×10 5 / mL, 50μL per well was plated in the cell plate. Jurkat-Dual cells in the logarithmic growth phase were collected, centrifuged and resuspended in RPMI1640 medium to 4×10 5 / mL, 50μL per well was added to the cell plate to obtain a final E:T ratio of 1:1. Then the bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR were added (starting concentration of 50nM, 4-fold gradient dilution, 10 concentration points, 100μL per well). The negative control sample 2DP47+IMCR and the positive control sample IMC-I109V were used at the same concentration as the bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR. After incubation for 48 hours, the supernatant was taken and the supernatant was referred to QUANTI-Luc TM Instructions (QUANTI-Luc TM , Invivogen, rep-qlc2) detection and analysis of HLA-A02 + / HBsAg + Cell-specific activation of Jurkat-Dual cells. Figure 6 The results showed that the TCRm×CD3 bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR could mediate HLA-A02 + / HBsAg + HepG2-HBsAg activated Jurkat-Dual cells; and compared with IMC-I109V, TCRm×CD3 bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR had stronger activation activity on Jurkat-Dual cells, EC 50 See Table 6 for values.

[0325] Table 6. TCRm×CD3 bispecific antibody mediated HLA-A02 + / HBsAg + Cell activation Jurkat-Dual cells

[0326] 2N1B5+IMCR 2N1B5-h4+IMCR IMC-I109V <![CDATA[EC 50 (nM)]]> 0.3894 0.4088 0.6211

[0327] 5.5 TCRm×CD3 bispecific antibody mediates PBMC-specific killing of target cells

[0328] 5.5.1 Isolation of human peripheral blood mononuclear cells (PBMC)

[0329] Blood was collected from normal volunteers (50 mL each), and all volunteers had signed informed consent. The volunteer selection criteria were:

[0330] Aged over 18 years old;

[0331] No HIV or HBV infection;

[0332] Routine blood test was normal;

[0333] Women who are not pregnant or breastfeeding;

[0334] PBMCs were isolated from whole blood of volunteers by Ficoll density gradient centrifugation and cultured in RPMI1640 medium.

[0335] 5.5.2 TCRm×CD3 bispecific antibody-mediated killing of PBMC by antigen peptide-pulsed T2 cells

[0336] T2 cells in the logarithmic growth phase were collected, centrifuged and resuspended in growth medium to 1×10 6 pcs / mL, 1mL per well was plated in a 24-well plate. 348-357 (GLSPTVWLSV) was added to a 24-well plate at a final concentration of 50 μg / mL and pulsed overnight. After 16 hours, the cells were centrifuged and resuspended in RPMI1640 medium to 4 × 10 5 / mL, 50μL per well was plated in a 96-well cell plate. Then the bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR were added (starting concentration was 2nM, 4-fold gradient dilution, 11 concentration points, 100μL per well). The negative control sample 2DP47+IMCR and the positive control sample IMC-I109V were used at the same concentration as the bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR. PBMC (effector cells) were resuspended to 4×10 6 / mL, add the above cell plate, 50μL per well, the final effector-target ratio is 10:1. At the same time, set up a single target cell control (antigen peptide pulsed T2 cells), a single effector cell control (PBMC), and a single culture medium blank control, and use culture medium to fill the volume of each well to 200μL. After incubation for 24 hours, take the supernatant and refer to Non-radioactive cytotoxicity assay reagents ( The results showed that the TCRm×CD3 bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR could specifically mediate the killing of PBMC cells against T2 cells pulsed with antigenic peptides; compared with IMC-I109V, the bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR had higher activity in mediating the killing of PBMC cells against T2 cells pulsed with antigenic peptides (Table 7 and Table 7). Figure 7 ).

[0337] Table 7. TCRm×CD3 bispecific antibody mediates PBMC cell killing of antigen peptide-pulsed T2 cells

[0338] 2N1B5+IMCR 2N1B5-h4+IMCR IMC-I109V <![CDATA[EC 50 (nM)]]> 0.000278 0.0002095 0.001455

[0339] 5.5.3 TCRm×CD3 bispecific antibody mediates PBMC killing of HLA-A02 + / HBsAg + cell

[0340] HepG2-HBsAg in the logarithmic growth phase were collected, digested and centrifuged, and then resuspended in RPMI1640 medium to 4 × 10 5 / mL, 50μL per well was plated in the cell plate. Then the bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR were added (starting concentration was 12.5nM, 4-fold gradient dilution, 9 concentration points, 100μL per well). The negative control sample 2DP47+IMCR and the positive control sample IMC-I109V were used at the same concentration as the bispecific antibodies 2N1B5+IMCR and 2N1B5-h4+IMCR. PBMC (effector cells) were resuspended to 4×10 6 / mL, add the above cell plate, 50μL per well, the final effector-target ratio is 10:1. At the same time, set up a single target cell control, a single effector cell control (PBMC), and a single culture medium blank control, and fill each well with culture medium to 200μL. After incubation for 48 hours, take the supernatant and refer to Non-radioactive cytotoxicity assay reagents ( Non-Radioactive Cytotoxicity Assay, Promega, G1780) Instructions for Detection and Analysis of TCRm×CD3 Bispecific Antibody-mediated PBMC Cells to HLA-A02 + / HBsAg + The results showed that 2N1B5+IMCR and 2N1B5-h4+IMCR specifically mediated the killing rate of PBMC cells to HLA-A02 + / HBsAg + The killing activity of cells is stronger than that of IMC-I109V ( Figure 8 , E.C. 50 Values ​​are shown in Table 8)

[0341] Table 8. ECs of TCRm×CD3 bispecific antibodies mediating PBMC cell killing of HepG2-HBsAg cells 50 value

[0342] 2N1B5+IMCR 2N1B5-h4+IMCR IMC-I109V <![CDATA[EC 50 (nM)]]> 0.008834 0.00952 0.1273

[0343] Example 6: TCRm×CD3 bispecific antibody recognizes HBsAg 348-357 Key amino acid analysis of (GLSPTVWLSV)

[0344] Chemical synthesis of HBsAg 348-357 Alanine scanning peptide (GLSPTVWLSV). Reference Example 5.4.1 detected and analyzed the specific activation of Jurkat-Dual cells by T2 cells pulsed with alanine scanning peptide. The results showed that 2N1B5-h4+IMCR could not effectively mediate the activation of Jurkat-Dual cells by T2 cells pulsed with alanine scanning mutant peptides at positions 2, 5, 6, 7, 8, 9 and 10 ( Fig. 9 ), namely HBsAg 348-357 The amino acids at positions 2, 5, 6, 7, 8, 9, and 10 of (GLSPTVWLSV) are the key amino acids for 2N1B5-h4+IMCR binding.

[0345] Example 7: Analysis of the interaction between TCRm×CD3 bispecific antibody and similar peptides

[0346] Chemical synthesis of HBsAg in Table 9 348-357 (GLSPTVWLSV) similar peptide.

[0347] Table 9. HBsAg 348-357 (GLSPTVWLSV) similar peptide information

[0348]

[0349]

[0350] Refer to Example 5.4.1 to detect and analyze the activation of Jurkat-Dual cells by T2 cells pulsed with different similar peptides. Fig.10 The results showed that 2N1B5-h4+IMCR only specifically mediated the antigen peptide HBsAg 348-357 (GLSPTVWLSV)-pulsed T2 cells activated Jurkat-Dual cells but did not mediate activation of Jurkat-Dual by other T2 cells loaded with similar peptides.

[0351] Example 8: Analysis of the interaction between TCRm×CD3 bispecific antibody and different antigen-negative tumor cells

[0352] Different tumor cells were collected, and the antigen information of the cells is shown in Table 10. Referring to 5.4.1 and 5.4.2, the specificity of the bispecific antibody 2N1B5-h4+IMCR in mediating tumor cell activation of Jurkat-Dual cells was evaluated. The results showed that 2N1B5-h4+IMCR only specifically mediated HLA-A02 + / HBsAg + Target cells activate Jurkat-Dual, but do not mediate activation of Jurkat-Dual by antigen-negative cells ( Fig.11 ).

[0353] Table 10. Antigen expression information of different cells

[0354]

[0355]

[0356] Note: “+” represents positive, “-” represents negative.

[0357] Example 9: Antitumor Effect of TCRm×CD3 Bispecific Antibody in hPBMC Immunoreconstitution Mice

[0358] PBMCs were obtained by sorting according to Example 5.5.1.

[0359] Thirty 5-6 week old female B-NDG mice (purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.) were selected and inoculated subcutaneously on the right back of the B-NDG mice with 1×10 7 Every other day, 8×10 HepG2-HBsAg cells were inoculated intraperitoneally in each mouse. 6 Individual PBMC. When the average tumor volume reaches 90 mm 3 The mice were randomly divided into groups according to the tumor size, and the day of grouping was defined as day 0, and the drug was administered. The experiment was divided into three groups: 2N1B5-h4+IMCR bispecific antibody group, 2DP47+IMCR negative control group, and IMC-I109V positive control group, with 7 mice in each group; the drug was administered by tail vein injection at a dose of 1 mg / kg, twice a week, for a total of six times, and continued to be observed. The efficacy was evaluated based on the tumor inhibition rate TGI.

[0360] The animals were generally in good mental state during the experiment. The bispecific antibody 2N1B5-h4+IMCR had a significant inhibitory effect on tumor growth at a dose of 1 mg / kg (Group 3). The relative tumor inhibition rate TGI (%) on day 11 was 76.01%, which was significantly different from the negative control group (Group 1) (p<0.0001). The bispecific antibody 2N1B5-h4+IMCR effectively inhibited HLA-A02 + / HBsAg+ positive tumor growth, and the effect was better than that of IMC-I109V (Group 2) ( Fig.12 There was no animal death in the treatment group, and no obvious drug toxicity was shown.

[0361] Example 10: Analysis of the binding site of TCRm antibody N1B5-h4 with HBsAg peptide 348-357 in the HLA-A02 / HBsAg complex

[0362] In order to study the binding site of TCRm antibody N1B5-h4 with HBsAg peptide 348-357 in HLA-A02 / HBsAg complex, recombinant protein N1B5-h4-Twin-Strep-tag (SEQ ID NO: 88) and recombinant protein MIP-H1-01 were prepared by HEK293 cells, and the two were co-incubated at a molecular ratio of 1:1 to form N1B5-h4 / MIP-H1-01 antigen-antibody complex. Complex crystals were formed under the conditions of 0.2 M calcium chloride + 0.1 M HEPES + 28% PEG400. X-ray diffraction experiments were performed on the complex crystals, and resolution was collected. The data were used and the crystal structure of the N1B5-h4 / MIP-H1-01 antigen-antibody complex was solved by the molecular replacement method.

[0363] The interaction area between N1B5-h4 and MIP-H1-01 is approximately The interaction modes include hydrogen bonds, salt bonds, van der Waals forces and hydrophobic interactions. The CDR3 of the heavy chain variable region of antibody N1B5-h4 is involved in the recognition of the 348-357th peptide of HBsAg in MIP-H1-01 (the 1st to 10th amino acid residues of MIP-H1-01). Fig.13 ). Among them, N1B5-h4 recognizes HBsAg 348-357 P4, T5, V6, W7, L8, S9 and V10 of (GLSPTVWLSV) (Table 11).

[0364] Table 11. Participation of N1B5-h4 / HBsAg 348-357 Amino acids that interact with peptides and how they work

[0365]

[0366] The key amino acids identified in Example 3.4 are 348-357 The key amino acids identified in Example 6 at positions 2, 4, 5, 6, 7, 8, 9 and 10 of (GLSPTVWLSV) are HBsAg 348-357(GLSPTVWLSV) at positions 2, 5, 6, 7, 8, 9 and 10. The methods used in Examples 3.4 and 6 cannot distinguish whether the decreased ability of the antibody to bind to the alanine scanning mutant peptide is due to the inability of the peptide to be properly loaded onto HLA (i.e., bind to HLA) after the amino acid residues are mutated to alanine, or whether it affects the binding of the mutated peptide to the antibody. HBsAg 348-357 The second amino acid residue is usually the site where the antigen peptide binds to HLA (i.e., the loading site). 348-357 After the leucine (L) at position 2 of the peptide is mutated to alanine (A), it may no longer bind to HLA. Therefore, although the activity mediated by the peptide detected in Examples 3.4 and 6 was significantly reduced, the leucine at position 2 may not be involved in the binding with the antibody.

[0367] The crystal structure in Example 10 shows that N1B5-h4 binds to HBsAg 348-357 The activity mediated by the 4th proline residue (4P) of N1B5-h4 was not significantly reduced after 4P was mutated to 4A in Example 6, indicating that N1B5-h4 and HBsAg in the complex 348-357 The binding at position 4 is not strict, and substitution of amino acids with similar properties has little effect on the interaction between the two, especially for molecules with bispecific antibody structures.

[0368] All patents, patent application disclosures, and non-patent documents mentioned and / or listed in this application are incorporated herein by reference in their entirety. The exemplary embodiments of the inventions of this application are described above, but without departing from the essence and scope of this application, those skilled in the art can modify or improve the exemplary embodiments described in this application, and the resulting variants or equivalents also belong to the scope of this application.

[0369] Sequence information

[0370] SEQ ID NO:32

[0371] TNCMG

[0372] SEQ ID NO:33

[0373] AIYTGGGRAVYADSVRG

[0374] SEQ ID NO:34

[0375] DWLGPDMTDIQVLGALPWFNY

[0376] SEQ ID NO:35

[0377] QVQLVESGGGSVQAGGSLRLSCAASDDTYMTNCMGWFRQAPGKEREGVAAIYTGGGRAVYADSVRGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADWLGPDMTDIQVLGALPWFNYWGQGTLVTVSS

[0378] SEQ ID NO:36

[0379] EVQLVESGGGLVQPGGSLRLSCAASDDTYMTNCMGWFRQAPGKERVGVAAIYTGGGRAVYADSVRGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADWLGPDMTDIQVLGALPWFNYWGQGTLVTVSS

[0380] SEQ ID NO:37

[0381] GYTMN

[0382] SEQ ID NO:38

[0383] LINPYKGVSTYNQKFKD

[0384] SEQ ID NO:39

[0385] SGYYGDSDWYFDV

[0386] SEQ ID NO:40

[0387] RASQDIRNYLN

[0388] SEQ ID NO:41

[0389] YTSRLES

[0390] SEQ ID NO:42

[0391] QQGNTLPWT

[0392] SEQ ID NO:43

[0393] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS

[0394] SEQ ID NO:44

[0395] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK

[0396] SEQ ID NO:45

[0397] QVQLVESGGGSVQAGGSLRLSCAASDDTYMTNCMGWFRQAPGKEREGVAAIYTGGGRAVYA

[0398] DSVRGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADWLGPDMTDIQVLGALPWFNYWGQ

[0399] GTLVTVSSGGGGSGGGGSGGGGSQVQLVESGGGSVQAGGSLRLSCAASDDTYMTNCMGWFR

[0400] QAPGKEREGVAAIYTGGGRAVYADSVRGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADW

[0401] LGPDMTDIQVLGALPWFNYWGQGTLVTVSSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDT

[0402] LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

[0403] WLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSD

[0404] IAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQK

[0405] SLSLSPGK

[0406] SEQ ID NO:46

[0407] EVQLVESGGGLVQPGGSLRLSCAASDDTYMTNCMGWFRQAPGKERVGVAAIYTGGGRAVYAD

[0408] SVRGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADWLGPDMTDIQVLGALPWFNYWGQG

[0409] TLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASDDTYMTNCMGWFRQA

[0410] PGKERVGVAAIYTGGGRAVYADSVRGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADWLG

[0411] PDMTDIQVLGALPWFNYWGQGTLVTVSSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMI

[0412] SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL

[0413] NGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAV

[0414] EWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS

[0415] LSPGK

[0416] SEQ ID NO:47

[0417] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGS

[0418] GSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSG

[0419] GGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVST

[0420] YNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVS

[0421] SASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0422] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQP

[0423] REPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

[0424] SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0425] SEQ ID NO:48

[0426] QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKE

[0427] FSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

[0428] SEQ ID NO:49

[0429] FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQ

[0430] VHYRMCQSCVELDPATVA

[0431] SEQ ID NO:50

[0432] GLSPTVWLSVGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDL

[0433] LKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGG

[0434] GSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRME

[0435] PRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDW

[0436] RFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWL

[0437] RRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELV

[0438] ETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE

[0439] SEQ ID NO:51

[0440] MGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDQWPEANQVGAGAFGPGFTPPHGG

[0441] LLGWSPQAQGILTTVPAAPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTTFHQALLDPRVRG

[0442] LYFPAGGSSSGTVNPVPTIVSPISSIFSRTGDPAPNMESTTSGFLGPLLVLQAGFFLLTRILTIPQSLD

[0443] SWWTSLNFLGEAPTCPGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDY

[0444] QGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWA

[0445] SVRFSWLSLLVPFVQWFAGLSPTVWLSVIWMMWYWGPSLYNILSPFLPLLPIFFCLWVYI

[0446] SEQ ID NO:52

[0447] GGGGSGGGGSGGGGS

[0448] SEQ ID NO:53

[0449] TCCTTCCCCGTCAGCCAGTCCT

[0450] SEQ ID NO:54

[0451] HHHHHH

[0452] SEQ ID NO:55

[0453] ASPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVN

[0454] NVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSV

[0455] RAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYF

[0456] MYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0457] SEQ ID NO:56

[0458] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0459] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

[0460] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0461] HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF

[0462] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0463] YTQKSLSLSPGK

[0464] SEQ ID NO:57

[0465] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0466] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

[0467] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0468] HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGF

[0469] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0470] YTQKSLSLSPGK

[0471] SEQ ID NO:58

[0472] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0473] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP

[0474] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0475] HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGF

[0476] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0477] YTQKSLSLSPGK

[0478] SEQ ID NO:59

[0479] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0480] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKP

[0481] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0482] HQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGF

[0483] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0484] YTQKSLSLSPGK

[0485] SEQ ID NO:60

[0486] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0487] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPKPKP

[0488] KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL

[0489] HQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGF

[0490] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0491] YTQKSLSLSPGK

[0492] SEQ ID NO:61

[0493] ASTKGPSVFPLAPSSKTSEGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0494] SSVVTVPSSSLGTQTYICNVDHKPSNTKVDKTVERSKVECPPPCPAPEFEGGPSVFLFPPKPKDT

[0495] LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

[0496] WLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSD

[0497] IAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQK

[0498] SLSLSPGK

[0499] SEQ ID NO:62

[0500] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

[0501] SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVERKSCVECPPCPAPEFEGGPSVFLFPPKPKDT

[0502] LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

[0503] WLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPS

[0504] DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ

[0505] KSLSLSPGK

[0506] SEQ ID NO:63

[0507] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNN

[0508] KYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0509] SEQ ID NO:64

[0510] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS

[0511] TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0512] SEQ ID NO:65

[0513] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADS

[0514] VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS

[0515] SEQ ID NO:66

[0516] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK

[0517] SEQ ID NO:67

[0518] AKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKGPELIMSIYSDGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAARNYKTDLLIFGTGTRLQVFP

[0519] SEQ ID NO:68

[0520] NAGVTQTPKFQVLKTGQSMTLQCAQDMSHGYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYATGGTGDLFFGEGSRLTVL

[0521] SEQ ID NO:69

[0522] AKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKGPELIMSIYSDGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAARNYKTDLLIFGTGTRLQVFPYIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFTCANAFNNSIIPEDTFFPSPESSCGGGGSASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0523] SEQ ID NO:70

[0524] NAGVTQTPKFQVLKTGQSMTLQCAQDMSHGYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYATGGTGDLFFGEGSRLTVLEDLKNVFPPEVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGGGGSASERKSCVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0525] SEQ ID NO:71

[0526] QVQLVESGGGSVQAGGSLRLSCAASDDTYMTNCMGWFRQAPGKEREGVAAIYTGGGRAVYADSVRGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADWLGPDMTDIQVLGALPWFNYWGQGTLVTVSSGGGGSGGGGSGGGGSQVQLVESGGGSVQAGGSLRLSCAASDDTYMTNCMGWFRQAPGKEREGVAAIYTGGGRAVYADSVRGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADWLGPDMTDIQVLGALPWFNYWGQGTLVTVSS

[0527] SEQ ID NO:72

[0528] EVQLVESGGGLVQPGGSLRLSCAASDDTYMTNCMGWFRQAPGKERVGVAAIYTGGGRAVYADSVRGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADWLGPDMTDIQVLGALPWFNYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASDDTYMTNCMGWFRQAPGKERVGVAAIYTGGGRAVYADSVRGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADWLGPDMTDIQVLGALPWFNYWGQGTLVTVSS

[0529] SEQ ID NO:73

[0530] ASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0531] SEQ ID NO:74

[0532] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS

[0533] SEQ ID NO:75

[0534] ASERKSSVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0535] SEQ ID NO:76

[0536] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVS

[0537] SGGGGSNAGVTQTPKFQVLKTGQSMTLQCAQDMSHGYMSWYRQDPGMGLRLIHYSVGAGIT

[0538] DQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYATGGTGDLFFGEGSRLTVLEDLKN

[0539] VFPPEVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVCTDPQPLKEQPAL

[0540] NDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC

[0541] GGGGSASERKSCVECPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN

[0542] WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISK

[0543] AKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0544] GSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0545] SEQ ID NO:77

[0546] ALSPTVWLSV

[0547] SEQ ID NO:78

[0548] GASPTVWLSV

[0549] SEQ ID NO:79

[0550] GLAPTVWLSV

[0551] SEQ ID NO:80

[0552] GLSATVWLSV

[0553] SEQ ID NO:81

[0554] GLSPAVWLSV

[0555] SEQ ID NO:82

[0556] GLSPVAWLSV

[0557] SEQ ID NO:83

[0558] GLSPTVALSV

[0559] SEQ ID NO:84

[0560] GLSPTVWASV

[0561] SEQ ID NO:85

[0562] GLSPTVWLAV

[0563] SEQ ID NO:86

[0564] GLSPTVWLSA

[0565] SEQ ID NO:87

[0566] FLLTRILTI

[0567] SEQ ID NO:88

[0568] EVQLVESGGGLVQPGGSLRLSCAASDDTYMTNCMGWFRQAPGKERVGVAAIYTGGGRAVYAD

[0569] SVRGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADWLGPDMTDIQVLGALPWFNYWGQG

[0570] TLVTVSSSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

[0571] References

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[0573] 2.Lee,H,M.,Banini,B,A.,(2019)Updates on chronic HBV:currentchallenges and future goals.Curr Treat Options Gastroenterol 17:271-291.

[0574] 3.Iannacone,M.,Guidotti,L,G.,(2022)Immunobiology and pathogenesis ofhepatitis B virus infection.Nature reviews.Immunology22(1):19-32.

[0575] 4.Mcnaughton,A,L.,V D’Arienzo,Ansari,M,A.,et al.(2018)Insights FromDeep Sequencing of the HBV Genome—Unique,Tiny,andMisunderstood.Gastroenterology 156(2):384-399.

[0576] 5.Stuyver,L.,De,G,S.,Van,G,C.,et al.,(2000)A new genotype ofhepatitis B virus:complete genome and phylogenetic relatedness.Journal ofGeneral Virology 81(1):67-74.

[0577] 6.Heléne.,Norder.,Berit.,et al.,(1993)Genetic relatedness ofhepatitis B viral strains of diverse geographical origin and naturalvariations in the primary structure of the surface antigen.Journal of GeneralVirology74:1341-1348.

[0578] 7.Lindh.,A,S.,Andersson.,et al.,(1997)Genotypes,nt 1858variants,andgeographic origin of hepatitis B virus--large-scale analysis using a newgenotyping method.The Journal of infectious diseases175:1285-1293.

[0579] 8. Chinese Society of Hepatology, Chinese Society of Infectious Diseases, (2022), Guidelines for the prevention and treatment of chronic hepatitis B, Chinese Journal of Hepatology, December, Vol. 30, No. 12.

[0580] 9.Global progress report on HIV,viral hepatitis and sexually transmitted infections,2021.Accountability for the global health sectorstrategies 2016-2021:actions for impact[EB / OL].https: / / apps.who.int / iris / bitstream / handle / 10665 / 342808 / 9789240030985-eng.pdf.

[0581] 10.Philips,C,A.,Ahamed,R.,Abduljaleel,J,K.,et al.,(2021)CriticalUpdates on Chronic Hepatitis B Virus Infection in 2021.Cureus 13(10):19152.

[0582] 11. Yixiang,

[0583] 12.Horig,H.,Lee,C,S.,Kaufman,H,L.(2002)Prostate-specific antigenvaccines for prostate cancer.Expert Opin Biol Ther2(4):395-408.

[0584] 13.Vlad,A,M.,Kettel,J,C.,Alajez,N,M.,et al.,(2004)MUC1 immunobiology:from discovery to clinical applications.Advances in Immunology 82:249-293.

[0585] 14.Blum,J,S.,Wearsch,P,A.,Cresswell,P.(2013)Pathways of antigenprocessing.Annu Rev Immunol 31:443–473.

[0586] 15.Nayersina,R.,Fowler,P.,Guilhot,S.,et al.,(1993)HLA A2 restrictedcytotoxic T lymphocyte responses to multiple hepatitis B surface antigenepitopes during hepatitis B virus infection.Journal of Immunology 150(10):4659-4671.

[0587] 16.A.Margaret Merchant et al.,An efficient route to human bispecificIgG,Nature Biotechnology,Volume 16,1998.

[0588] 17.Kabat,Sequences of Proteins of Immunological Interest,NationalInstitutes of Health,Bethesda,Md.(1991).

[0589] 18.A1-Lazikani et al., J. Mol. Biol. 273:927-948 (1997).

[0590] 19. Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989).

[0591] 20.Kotsiou,E.,Brzostek,J.,Lenart,I.,et al.,(2010)Dimerization of solubledisulfide trap single-chain major histocompatibility complex class Imolecules dependent on peptide binding affinity.Antioxid Redox Signal 15(3):635-644.

[0592] twenty one. 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.

[0593] 22.CN 201510097117.0

[0594] 23. Phage display: A general experimental guide / (US) Clarkson (Clackson, T.), (US) Lowman (Lowman, HB); (US) Translated by Ma Lan et al. Chemical Industry Press, 2008.5.

[0595] 24.US20160200833A1

[0596] 25.WO2020193745 A1

[0597] 26.Tan,P.,Mitchell,D,A.,Buss,T,N.,et al.(2022)"Superhumanized"antibodies:reduction of immunogenic potential by complementarity-determiningregion grafting with human germline sequences:application to an anti-CD28.JImmunol 169(2):1119-1125.

[0598] 27.US 5821337A。

Claims

1. A bispecific antibody comprising a first antigen-binding fragment that binds to an HLA-A02 / HBsAg complex and a second antigen-binding fragment that binds to an activated T cell antigen; Preferably, the binding epitope of the first antigen-binding fragment to the HLA-A02 / HBsAg complex includes one or more residues in positions 348-357 of HBsAg as shown in SEQ ID NO: 51; more preferably, the binding epitope of the first antigen-binding fragment to the HLA-A02 / HBsAg complex includes at least one of residues 349, 351, 352, 353, 354, 355, 356 and 357 of HBsAg as shown in SEQ ID NO: 51; and / or Preferably, the activating T cell antigen is a CD3 molecule.

2. The bispecific antibody according to claim 1, wherein The first antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:32, HCDR2 as shown in SEQ ID NO:33 and HCDR3 as shown in SEQ ID NO:34; preferably, the first antigen-binding fragment is in the form of a single-domain antibody; more preferably, the first antigen-binding fragment comprises a single-domain antibody that binds to the HLA-A02 / HBsAg complex monovalently or multivalently; and / or The second antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:37, HCDR2 as shown in SEQ ID NO:38, HCDR3 as shown in SEQ ID NO:39, LCDR1 as shown in SEQ ID NO:40, LCDR2 as shown in SEQ ID NO:41 and LCDR3 as shown in SEQ ID NO:42; preferably, the second antigen-binding fragment is 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-A02 / HBsAg complex; Optionally, the first antigen-binding fragment comprises two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A02 / HBsAg complex; preferably, the first antigen-binding fragment comprises two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A02 / HBsAg complex that are directly fused or two heavy chain variable regions of monovalent single-domain antibodies that bind to the HLA-A02 / HBsAg complex that are connected by a linker; more preferably, the linker is a linker comprising a GS-type flexible peptide, for example (GGGGS)n, wherein n is an integer ≥1.

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

44.

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: 35; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 43 and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 44; The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 36; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 43 and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 44; or The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 71; and the second antigen-binding fragment comprises the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 43 and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 44; or The first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:72; and the second antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO:43 and the light chain variable region shown in SEQ ID NO:

44.

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 according to any one of claims 1 to 6, comprising a first arm that binds to an HLA-A02 / HBsAg complex and a second arm that binds to an activating T cell antigen, wherein The first arm comprises the amino acid sequence shown in SEQ ID NO: 45 or 46; and The second arm comprises the amino acid sequence shown in SEQ ID NO:

47.

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 according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing or treating a disease caused by HLA-A02 / HBsAg-positive HBV infection; preferably, the disease caused by HLA-A02 / HBsAg-positive HBV infection is selected from: hepatitis B, cirrhosis, liver fibrosis and liver cancer.

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