Antibody binding to HLA-a02 / AFP complex and use thereof

Abstract

Provided are a bispecific antibody comprising a first antigen-binding fragment binding to an HLA-A02 / AFP complex and a second antigen-binding fragment binding to an activated T-cell antigen, a single-domain antibody binding to an HLA-A02 / AFP complex, a pharmaceutical composition comprising the bispecific antibody or the single-domain antibody, and use thereof.

Description

Antibodies that bind to the HLA-A02 / AFP complex and their applications

[0001] Related applications

[0002] This application claims priority and benefit to Chinese Patent Application No. CN 202411595443.X, filed on November 8, 2024, entitled “Antibody that binds to HLA-A02 / AFP complex and its use thereof”, which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] This application generally relates to the fields of genetic engineering and antibody pharmaceuticals; more specifically, this application relates to single-domain antibodies that bind to the HLA-AO2 / AFP complex, bispecific antibodies that bind to the HLA-AO2 / AFP complex and activated T-cell antigens, and their uses. Background Technology

[0004] AFP (alpha-fetoprotein), also known as alpha-fetoprotein, is expressed during fetal development and is a major component of fetal serum. During development, this protein is produced at high levels by the yolk sac and liver and is gradually suppressed after birth. [1] In healthy individuals, AFP remains at low levels throughout life, but it is abnormally expressed in hepatocellular carcinoma (HCC). [2、3] .

[0005] The 5-year survival rate for liver cancer is only about 15%. [4、5] Liver cancer is one of the malignant tumors with the lowest reported 5-year survival rates, with a total of 1.2 million new cases worldwide each year. According to cancer data released by the World Health Organization in 2020, China has more than 410,000 new cases of liver cancer and more than 390,000 deaths annually, accounting for 45% and 47% of the world's total, respectively. Nearly half of the world's new cases and deaths from liver cancer are in China, and this number may rise due to the prevalence of hepatitis B and hepatitis C. [6、7] Treatment typically includes surgery and chemotherapy; however, these are only beneficial in the early stages of the disease. Therefore, new treatment methods are needed.

[0006] T cell receptors (TCRs) are characteristic markers on the surface of all T cells. They bind nonvalently to CD3, forming the TCR-CD3 complex. The function of the TCR is to recognize antigens, specifically peptide-MHC complexes (p-MHC). Intracellular proteins are degraded by the proteasome, and the resulting peptides are transported to the endoplasmic reticulum to assemble with MHC into complexes. These complexes are then transported via the Golgi apparatus and finally presented on the cell surface, forming antigens that can be recognized by the TCR. Some of these proteins are specifically expressed in tumors or diseases and can serve as specific target antigens in treatment. Researchers have discovered that preparing TCR mimic antibodies (TCRm) can effectively mimic the TCR, recognizing MHC complexes of tumor-specific antigens and exerting biological activity with the help of T cells. [8、9、10] Therefore, the development of bispecific antibody drugs based on TCRm×anti-CD3 has become another important area of ​​drug development.

[0007] After being produced intracellularly, AFP is degraded into small polypeptide molecules and binds to MHC (master histocompatibility complex) molecules to form a complex, which is then presented to the cell surface. The HLA-A02-restricted AFP epitope FMNKFIYEI...

[0011] (SEQ ID NO:1) provides a suitable target for novel immunotherapeutic interventions; the antigenic peptide is naturally processed and eluted from HepG2 (HLA-A02 positive) liver cancer cell lines and detected by mass spectrometry, and has been shown to target AFP. 158-166 :FMNKFIYEI generates T cell clones

[0012] However, no TCRm antibodies that recognize this peptide have been reported yet.

[0008] Based on clinical needs, exploring and developing TCRm×anti-CD3 bispecific antibodies targeting AFP has important biological and clinical significance. Summary of the Invention

[0009] In one aspect, this application provides a bispecific antibody comprising a first antigen-binding fragment that binds to the HLA-A02 / AFP complex and a second antigen-binding fragment that binds to activated T-cell antigens.

[0010] In some embodiments of the first aspect, the bispecific antibody is capable of mediating HLA-A02. + / AFP + Tumor cells activate T cells, and / or the bispecific antibody can mediate the killing of HLA-A02 cells by human peripheral blood mononuclear cells (PBMCs). + / AFP + Tumor cells.

[0011] In some embodiments of the first aspect, the binding epitope of the first antigen-binding fragment to the HLA-A02 / AFP complex includes one or more residues from positions 158-166 (SEQ ID NO:1) of the AFP shown with reference to SEQ ID NO:58. In some specific embodiments, the binding epitope of the first antigen-binding fragment to the HLA-A02 / AFP complex includes at least one of residues from positions 159, 160, 162, 164, and 165 of the AFP shown with reference to SEQ ID NO:58.

[0012] In some embodiments of the first aspect, the first antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:18, HCDR2 as shown in SEQ ID NO:19, and HCDR3 as shown in SEQ ID NO:20, wherein the amino acid sequence of HCDR is defined according to Kabat.

[0013] In some embodiments of the first aspect, the first antigen-binding fragment is in the form of a single-domain antibody. For example, the first antigen-binding fragment comprises a monovalent or multivalent single-domain antibody that binds to the HLA-AO2 / AFP complex.

[0014] In some embodiments of the first aspect, the activating T-cell antigen is a CD3 molecule.

[0015] In some embodiments of the first aspect, the second antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:60, HCDR2 as shown in SEQ ID NO:61, HCDR3 as shown in SEQ ID NO:62, LCDR1 as shown in SEQ ID NO:64, LCDR2 as shown in SEQ ID NO:65, and LCDR3 as shown in SEQ ID NO:66, wherein the amino acid sequences of HCDR and LCDR are defined according to Kabat.

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

[0017] In some embodiments of the first aspect, the first antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:17 or 27, and / or the second antigen-binding fragment comprises a heavy chain variable region as shown in SEQ ID NO:59 and a light chain variable region as shown in SEQ ID NO:63.

[0018] For example, the first antigen-binding fragment may comprise the amino acid sequence shown in SEQ ID NO:17, and the second antigen-binding fragment may comprise a heavy chain variable region as shown in SEQ ID NO:59 and a light chain variable region as shown in SEQ ID NO:63. Alternatively, the first antigen-binding fragment may comprise the amino acid sequence shown in SEQ ID NO:27, and the second antigen-binding fragment may comprise a heavy chain variable region as shown in SEQ ID NO:59 and a light chain variable region as shown in SEQ ID NO:63.

[0019] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are linked by an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment. For example, the antibody heavy chain constant region Fc fragment may be an Fc fragment of the IgG1 subtype. In some specific embodiments, the antibody heavy chain constant region Fc fragment may be an Fc fragment of the IgG1m3 subtype.

[0020] In some embodiments of the first aspect, 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 in the antibody constant region are determined according to EU numbering.

[0021] In some embodiments of the first aspect, 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 in the antibody constant region are determined according to EU numbering.

[0022] In some embodiments of the first aspect, the amino acids at positions 234, 235, and 331 of the first Fc fragment and the second Fc fragment are F, E, and S, respectively, wherein the amino acid positions of the antibody constant region are determined according to EU numbering.

[0023] In some embodiments of the first aspect, 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.

[0024] In some embodiments of the first aspect, the bispecific antibody comprises a first arm that binds to the HLA-AO2 / AFP complex and a second arm that binds to an activated T-cell antigen, wherein

[0025] The first arm comprises an amino acid sequence as shown in SEQ ID NO:31 or 32; and

[0026] The second arm contains an amino acid sequence as shown in SEQ ID NO:33.

[0027] Secondly, this application provides a single-domain antibody that binds to the HLA-A02 / AFP complex, which contains

[0028] HCDR1, as shown in SEQ ID NO:18,

[0029] HCDR2, as shown in SEQ ID NO:19, and

[0030] HCDR3, as shown in SEQ ID NO:20;

[0031] The amino acid sequence of HCDR is defined according to Kabat.

[0032] In some embodiments of the second aspect, the binding epitope of the single-domain antibody to the HLA-A02 / AFP complex includes one or more residues from positions 158-166 (SEQ ID NO:1) of the AFP shown in SEQ ID NO:58. In some specific embodiments, the binding epitope of the first antigen-binding fragment to the HLA-A02 / AFP complex includes at least one of residues from positions 159, 160, 162, 164, and 165 of the AFP shown in SEQ ID NO:58.

[0033] 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 / AFP complex.

[0034] In some embodiments of the second aspect, the single-domain antibody comprises an amino acid sequence as shown in SEQ ID NO:17 or 27.

[0035] Thirdly, this application provides a nucleic acid molecule that encodes the bispecific antibody described in the first aspect or the single-domain antibody described in the second aspect.

[0036] Fourthly, this application provides a pharmaceutical composition comprising the bispecific antibody described in the first aspect or the single-domain antibody described in the second aspect, as well as a pharmaceutically acceptable excipient, diluent, or carrier.

[0037] Fifthly, this application provides the use of the bispecific antibody described in the first aspect, the single-domain antibody described in the second aspect, the nucleic acid molecule described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a medicament for the prevention or treatment of HLA-AO2 / AFP-positive tumors.

[0038] In some implementations of the fifth aspect, the HLA-A02 / AFP positive tumor is selected from hepatocellular carcinoma and gastric cancer. Attached Figure Description

[0039] Figure 1 shows the results of ELISA detection of the binding of TCRm antibody N9H11 against the HLA-A02 / AFP complex to recombinant protein MIP-A1-A0201.

[0040] Figure 2 shows the results of T2 cells detected by flow cytometry in which N9H11 binds to the antigenic peptide AFP158-166 pulse.

[0041] Figure 3 shows that the amino acids at positions 2, 3, 5, 7, and 8 of AFP158-166 (FMNKFIYEI) are the key amino acids for N9H11 binding.

[0042] Figure 4 shows the results of ELISA detection of the TCRm×anti-CD3 bispecific antibody simultaneously recognizing MIP-A1-A0201 and CD3.

[0043] Figure 5 shows the results of T2 cell activation of Jurkat-Dual cells by bispecific antibodies N9H11×anti-CD3 and N9H11-h4×anti-CD3 specifically mediating antigen peptide pulses.

[0044] Figure 6 shows that the bispecific antibodies N9H11×anti-CD3 and N9H11-h4×anti-CD3 can specifically mediate the PBMC response to HLA-A02. + / AFP + The result of HepG2 killing of cells.

[0045] Figure 7 shows that the amino acids at positions 2, 3, 5, 7, and 8 of AFP158-166 (FMNKFIYEI) are key amino acids for N9H11-h4×anti-CD3 binding.

[0046] Figure 8 shows the results of T2 cells loaded with an AFP158-166 (FMNKFIYEI) similar peptide not mediating Jurkat-Dual activation by the bispecific antibody N9H11-h4×anti-CD3.

[0047] Figure 9 shows that the bispecific antibody N9H11-h4×anti-CD3 specifically mediates the HLA-A02 response in PBMC cells. + / AFP + The result is the effective killing of target cells.

[0048] Figure 10 shows the results of the bispecific antibody N9H11-h4×anti-CD3 not mediating the effective killing of non-HLA-A02 allele tumor cells by PBMCs.

[0049] Figure 11 shows the inhibition of HLA-A02 by the bispecific antibody N9H11-h4×anti-CD3 in mice. + / AFP + The result of HepG2 tumor cell proliferation.

[0050] Sequence Description

[0051] SEQ ID NO:1 shows the amino acid sequence of the antigenic peptide at positions 158-166 of human alpha-fetoprotein (AFP).

[0052] SEQ ID NO:2 shows the amino acid sequence of the extracellular region (hCD3E) of human (homo sapiens) CD3E.

[0053] SEQ ID NO:3 shows the amino acid sequence of the extracellular region (hCD3D) of human (homo sapiens) CD3D.

[0054] SEQ ID NO:4 shows the antigenic peptide AFP. 158-166 The amino acid sequence of the complex (MIP-A1-A0201) of the disulfide bond-trapping single-chain trimer of HLA-A0201.

[0055] SEQ ID NO:5 shows the amino acid sequence of the His tag.

[0056] SEQ ID NO:6 shows the amino acid sequence of the Fc segment (mFc) of the mouse (mus musculus) IgG2a antibody.

[0057] SEQ ID NO:7 shows the amino acid sequence of the constant region of the heavy chain of the human (homo sapiens) IgG1 subtype.

[0058] SEQ ID NO:8 shows the amino acid sequence of the human (homo sapiens) IgG1 subtype antibody heavy chain constant region mutant IgG1H.

[0059] SEQ ID NO:9 shows the amino acid sequence of the human (homo sapiens) IgG1 subtype antibody heavy chain constant region mutant IgG1K.

[0060] SEQ ID NO:10 shows the amino acid sequence of the human (homo sapiens) IgG1 subtype antibody heavy chain constant region mutant IgG1m3-H.

[0061] SEQ ID NO:11 shows the amino acid sequence of the human (homo sapiens) IgG1 subtype antibody heavy chain constant region mutant IgG1m3-K.

[0062] SEQ ID NO:12 shows the amino acid sequence of the human (homo sapiens) IgG1 subtype antibody heavy chain constant region mutant IgG1m3-H1n1.

[0063] SEQ ID NO:13 shows the amino acid sequence of the human (homo sapiens) IgG1 subtype antibody heavy chain constant region mutant IgG1m3-Kn1.

[0064] SEQ ID NO:14 shows the amino acid sequence of the constant region of the light chain of the human (homo sapiens) λ subtype.

[0065] SEQ ID NO:15 shows the amino acid sequence of the constant region of the light chain of the human (homo sapiens) κ subtype.

[0066] SEQ ID NO:16 shows the primer sequence designed from the amino acid constant region CH2 of the heavy chain of the llama antibody.

[0067] SEQ ID NO:17 shows the amino acid sequence of the heavy chain variable region of the llama (Llama) single-domain antibody N9H11.

[0068] SEQ ID NO:18-20 show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of llama (Llama) single-domain antibodies N9H11 and N9H11-h4, respectively.

[0069] SEQ ID NO:21 shows the amino acid sequence of the variable region of the heavy chain of the negative control antibody DP47.

[0070] SEQ ID NO:22 shows the amino acid sequence of the variable region of the light chain of the negative control antibody DP47.

[0071] SEQ ID NO:23-26 show the FR1, FR2, FR3 and FR4 amino acid sequences of the humanized molecule N9H11-h4, a single-domain antibody against llamas.

[0072] SEQ ID NO:27 shows the amino acid sequence of the heavy chain variable region of the humanized single-domain antibody N9H11-h4.

[0073] SEQ ID NO:28 shows the amino acid sequence of a single-chain antibody (scFv) against human (homo sapiens) CD3 antibody.

[0074] SEQ ID NO:29 shows the amino acid sequence of the Fc fragment mutant IgG1m3-FcH1n1 of the human (homo sapiens) IgG1 subtype antibody.

[0075] SEQ ID NO:30 shows the amino acid sequence of the Fc fragment mutant IgG1m3-FcKn1 of the human (homo sapiens) IgG1 subtype antibody.

[0076] SEQ ID NO:31 shows the amino acid sequence of N9H11-G1m3-FcHIn1 in the bispecific antibody N9H11×anti-CD3.

[0077] SEQ ID NO:32 shows the amino acid sequence of N9H11-h4-G1m3-FcHIn1 in the bispecific antibody N9H11-h4×anti-CD3.

[0078] SEQ ID NO:33 shows the amino acid sequence of arm anti-CD3-scFv-G1m3-FcKn1 in the bispecific antibody N9H11×anti-CD3 or N9H11-h4×anti-CD3.

[0079] SEQ ID NO:34-42 show the scanning mutant peptide sequences of the first to ninth amino acids (alanine) of the antigenic peptide at positions 158-166 of human (homo sapiens) alpha-fetoprotein (AFP).

[0080] SEQ ID NO:43 shows the amino acid sequence of the irrelevant antigenic peptide used in the examples.

[0081] SEQ ID NO:44 shows an AFP sample from a human (homo sapiens). 158-166 The amino acid sequence of similar peptide 1.

[0082] SEQ ID NO:45 shows an AFP sample from a human (homo sapiens). 158-166 The amino acid sequence of similar peptide 2.

[0083] SEQ ID NO:46 shows an AFP sample from a human (homo sapiens). 158-166 The amino acid sequence of similar peptide 3.

[0084] SEQ ID NO:47 shows an AFP sample from a human (homo sapiens). 158-166 The amino acid sequence of similar peptide 4.

[0085] SEQ ID NO:48 shows an AFP sample from a human (homo sapiens). 158-166 The amino acid sequence of similar peptide 5.

[0086] SEQ ID NO:49 shows an antigen-like peptide 1 with amino acids that have N9H11 binding key site similarity properties.

[0087] SEQ ID NO:50 shows an antigen-like peptide 2 with similar amino acids that bind to the N9H11 key site.

[0088] SEQ ID NO:51 shows an antigen-like peptide 3 with similar amino acids that bind to the N9H11 key site.

[0089] SEQ ID NO:52 shows an antigen-like peptide 4 with similar amino acids that bind to the N9H11 key site.

[0090] SEQ ID NO:53 shows an antigen-like peptide 5 with amino acids that have N9H11 binding key site similarity properties.

[0091] SEQ ID NO:54 shows an antigen-like peptide 6 with amino acids that have N9H11 binding key site similarity properties.

[0092] SEQ ID NO:55 shows an antigen-like peptide 7 with amino acids that have N9H11 binding key site similarity properties.

[0093] SEQ ID NO:56 shows an antigen-like peptide 8 with amino acids that have N9H11 binding key site similarity properties.

[0094] SEQ ID NO:57 shows an antigen-like peptide 9 with similar amino acids that bind to the N9H11 key site.

[0095] SEQ ID NO:58 shows the amino acid sequence of human (homo sapiens) AFP.

[0096] SEQ ID NO:59 shows the amino acid sequence of the variable region of the heavy chain of the anti-human (homo sapiens) CD3 antibody.

[0097] SEQ ID NO:60-62 show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the anti-human (homo sapiens) CD3 antibody, respectively.

[0098] SEQ ID NO:63 shows the amino acid sequence of the variable region of the light chain of the anti-human (homo sapiens) CD3 antibody.

[0099] SEQ ID NO:64-66 show the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of the anti-human (homo sapiens) CD3 antibody, respectively. Detailed Implementation

[0100] The inventors of this application have prepared a bispecific antibody (e.g., TCRm bispecific antibody) by genetic engineering to combine a first antigen-binding fragment that binds to the HLA-A02 / AFP complex and a second antigen-binding fragment that binds to an activating T cell antigen (e.g., CD3 molecule). The first antigen-binding fragment of the HLA-A02 / AFP complex binds to the complex of AFP epitope peptide FMNKFIYEI (SEQ ID NO:1) and HLA-A02 presented on the surface of target cells (e.g., tumor cells), while the second antigen-binding fragment binds to an activating T cell antigen (e.g., CD3 molecule). This establishes an interaction between the target cell and the T cell, causing T cell activation and lysis of the target cell (e.g., tumor cell), thereby achieving the purpose of treating the disease (e.g., tumor). In various aspects of this application, bispecific antibodies comprising a first antigen-binding fragment binding to the HLA-A02 / AFP complex and a second antigen-binding fragment binding to an activating T-cell antigen (e.g., CD3 molecule), single-domain antibodies binding to the HLA-A02 / AFP complex, nucleic acid molecules encoding the bispecific antibody or the single-domain antibody, vectors containing the nucleic acid molecules, host cells containing the nucleic acid molecules or the vector, methods for preparing the bispecific antibody or the single-domain antibody, and medical and biological applications of the bispecific antibody or the single-domain antibody are provided. Based on the sequences of the bispecific antibody or single-domain antibody provided in this application, bispecific antibodies or single-domain antibodies binding to the HLA-A02 / AFP complex can be constructed as drugs for clinical use in the prevention or treatment of diseases (e.g., tumors).

[0101] Unless otherwise specified, this application is implemented using conventional molecular biology, microbiology, cell biology, biochemistry and immunology techniques in the art.

[0102] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0103] definition

[0104] As used herein, the term "HLA-AO2 / AFP complex" refers to a complex of the HLA-AO2 molecule and AFP. In some specific embodiments of this application, "HLA-AO2 / AFP complex" refers to the HLA-AO2 molecule and position 158-166 (AFP) of the AFP shown in SEQ ID NO:58. 158-166 The amino acid sequence is shown in SEQ ID NO:1, which is HLA-A02 / AFP of FMNKFIYEI. 158-166 Complex.

[0105] As used herein, the term "activating T-cell antigen" refers to antigenic determinants expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, which can induce T-cell activation upon interaction with antigen-binding molecules. Specifically, the interaction between antigen-binding molecules and activating T-cell antigens can induce T-cell activation by triggering a signaling cascade within the T-cell receptor complex. In a particular aspect, the activating T-cell antigen is the CD3 molecule.

[0106] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, peptides, etc. The term "antibody" as used herein includes not only complete (i.e., full-length) antibodies, but also their binding fragments (e.g., Fab, Fab', F(ab')2, Fv), their variants, fusion proteins containing 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 containing antigen recognition sites of desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0107] Typically, a full-length or complete antibody consists of two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). Full-length antibodies can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses mentioned above), but the antibody does not need to belong to any specific class. Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the constant region of the heavy chain. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

[0108] As used herein, the term "bispecific antibody" refers to an antibody that simultaneously binds to two antigenic epitopes. These two epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have various structural configurations. For example, a bispecific antibody can consist of two Fc fragments and two binding portions fused to them (similar to natural antibodies, except that the two arms bind to different antigenic targets or epitopes). The antigen-binding portions can be single-domain antibodies, single-chain antibodies (scFv), or Fab fragments. When targeting two given antigenic epitopes, the two different binding portions of the bispecific antibody each bind to the N-terminus of an Fc fragment. The antigen-binding portion 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 ensure heavy chain heteropolymerization; KIH (knob-in-hole) technology is one strategy to address heavy chain heteropolymerization. Typically, KIH (kidney-in-heap) technology refers to modifying the amino acid sequence of the CH3 region to create a structure that facilitates the pairing of heterologous half-antibodies, thus forming bispecific antibodies while preserving as much of the normal antibody structure 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.

[0013] The full text of that document is incorporated into this paper through citation.

[0109] The antigen-binding region may contain a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL typically contains three complementarity-determining regions, CDR1, CDR2, and CDR3.

[0110] It is well known to those skilled in the art that complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on antibody affinity and specificity. There are two common definitions for the CDR sequence of VH or VL: the Kabat definition and the Chothia definition. (See, for example, Kabat, “Sequences of Proteins of Immunological Interest”, National Institutes of Health, Bethesda, MD. (1991))

[0014] ;A1-Lazikani et al., J. Mol. Biol. 273:927-948 (1997)

[0015] ; and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989))

[0016] For a given antibody's variable region sequence, the CDR region sequence in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In the embodiments of this application, the Kabat definition of the CDR sequence is used.

[0111] For a given antibody's variable region sequence, the CDR region sequence can be analyzed in various ways, such as using the online software Abysis (http: / / www.abysis.org / ).

[0112] Examples of antigen-binding fragments for general antibodies include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having VL-CL chains and VH-CH1 chains; (2) F(ab')2 fragments, which may be divalent fragments having two Fab' fragments connected by disulfide bridges (i.e., Fab' dimers) in the hinge region; (3) Fv fragments having a single arm of antibody with VL and VH domains; (4) single-chain Fv(scFv), which may be a single polypeptide chain consisting of VH and VL domains connected by peptide linkers; (5) (scFv)2, which may contain two VH domains and two VL domains connected by peptide linkers, the two VL domains being combined with the two VH domains via disulfide bridges; and (6) single-domain antibody forms.

[0113] In the construction of bispecific antibodies, "antigen-binding fragments" include, but are not limited to, single-domain antibody forms, Fab fragment forms, and / or single-chain antibody (scFv) forms.

[0114] As used in this article, "single-chain antibody (scFv, single-chain fragment variable)" refers to an antibody with a single-chain structure, typically constructed using genetic engineering techniques. It is a polypeptide chain containing a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker is usually designed between the heavy and light chain variable regions so that they can fold into the correct conformation to bind the antigen.

[0115] As used herein, the terms “Fab (fragment antigen binding) fragment,” “Fab portion,” or similar terms refer to antibody fragments that bind to antigens produced by treating an intact antibody with papain, including the intact light chain (VL-CL), the heavy chain variable region, and the CH1 fragment (VH-CH1).

[0116] As used herein, the term "single-domain antibody" refers to a naturally occurring heavy chain monovariable domain antibody lacking the light chain. Such antibodies contain a heavy chain variable region (VHH) and conventional CH2 and CH3 regions (e.g., one or two groups of regions: the heavy chain variable region (VHH) and conventional CH2 and CH3 regions). The VHH structure, cloned and expressed independently, possesses structural stability and antigen-binding activity comparable to the original heavy chain antibody and is the smallest known unit capable of binding to a target antigen. Single-domain antibodies are also known as nanobodies (Nb).

[0117] As used herein, the terms “Fc fragment,” “Fc domain,” and “Fc portion” are used interchangeably to refer to a portion of the antibody heavy chain constant region, including the hinge region, the CH2 and CH3 fragments of the heavy chain constant region, and are determined with reference to the EU numbering of human IgG1 antibodies.

[0118] As used in this article, "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.

[0119] As used herein, the term "tumor" refers to a growth or solid lesion formed by abnormal cell growth. Tumors can be benign, pre-malignant, or malignant. In some embodiments of this application, the tumor is selected from hepatocellular carcinoma and gastric cancer. In some specific embodiments, the tumor is hepatocellular carcinoma. In some other specific embodiments, the tumor is gastric cancer.

[0120] As used herein, the term "therapeutic effective dose" or "effective dose" refers to a dose that has a therapeutic effect on a subject, such as: in subjects who have been given the dose, the symptoms or state of the disease are alleviated, reduced, or eliminated, or the development of the symptoms or state of the disease is delayed or suppressed compared to subjects who have not been given the dose.

[0121] In one aspect, this application provides a bispecific antibody comprising a first antigen-binding fragment that binds to the HLA-A02 / AFP complex and a second antigen-binding fragment that binds to activated T-cell antigens.

[0122] In some embodiments of the first aspect, the bispecific antibody is capable of mediating HLA-A02. + / AFP + Tumor cells activate T cells, and / or the bispecific antibody can mediate PBMC killing of HLA-A02. + / AFP + Tumor cells. For example, the bispecific antibody can mediate HLA-A02. + / AFP + Tumor cells activate Jurkat-Dual cells.

[0123] In some embodiments of the first aspect, the binding epitope of the first antigen-binding fragment to the HLA-A02 / AFP complex includes one or more residues from positions 158-166 (SEQ ID NO:1) of the AFP shown with reference to SEQ ID NO:58. In some specific embodiments, the binding epitope of the first antigen-binding fragment to the HLA-A02 / AFP complex includes at least one of residues from positions 159, 160, 162, 164, and 165 of the AFP shown with reference to SEQ ID NO:58.

[0124] In some embodiments of the first aspect, the first antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:18, HCDR2 as shown in SEQ ID NO:19, and HCDR3 as shown in SEQ ID NO:20, wherein the amino acid sequence of HCDR is defined according to Kabat.

[0125] In some embodiments of the first aspect, the first antigen-binding fragment is in the form of a single-domain antibody. For example, the first antigen-binding fragment comprises a monovalent or multivalent (e.g., 1, 2, 3, 4, 5, or 6-valent) single-domain antibody that binds the HLA-AO2 / AFP complex. In some specific embodiments, the first antigen-binding fragment comprises a monovalent single-domain antibody that binds the HLA-AO2 / AFP complex. In some specific embodiments, the first antigen-binding fragment comprises a bivalent single-domain antibody that binds the HLA-AO2 / AFP complex.

[0126] In some embodiments, the first antigen-binding fragment comprises heavy chain variable regions of two monovalent single-domain antibodies that bind to the HLA-A02 / AFP complex.

[0127] In some embodiments of the first aspect, the first antigen-binding fragment comprises the heavy chain variable regions of two directly fused monovalent single-domain antibodies that bind to the HLA-A02 / AFP complex.

[0128] In some embodiments of the first aspect, the first antigen-binding fragment comprises heavy chain variable regions of two monovalent single-domain antibodies binding the HLA-A02 / AFP complex, linked by a linker. In some embodiments, the linker is a GS-type flexible peptide linker. In some embodiments, the linker is (G4S)n, (SG4)n, or G4(SG4)n, where n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or an integer or range between any two of the above values. In some embodiments, the linker is (G4S)n, (SG4)n, or G4(SG4)n, where n is an integer from 2 to 4, such as 2, 3, or 4.

[0129] In some embodiments of the first aspect, the activating T-cell antigen is a CD3 molecule.

[0130] In some embodiments of the first aspect, the second antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:60, HCDR2 as shown in SEQ ID NO:61, HCDR3 as shown in SEQ ID NO:62, LCDR1 as shown in SEQ ID NO:64, LCDR2 as shown in SEQ ID NO:65, and LCDR3 as shown in SEQ ID NO:66, wherein the amino acid sequences of HCDR and LCDR are defined according to Kabat.

[0131] In some embodiments of the first aspect, the second antigen-binding fragment is in the form of a single-chain antibody (scFv) or a Fab fragment. In some specific embodiments, the second antigen-binding fragment is in the form of scFv.

[0132] In some embodiments of the first aspect, the first antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:17 or 27.

[0133] In some embodiments of the first aspect, the amino acid sequence of the first antigen-binding fragment may be substituted, deleted, and / or added by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids different from the amino acid sequence shown in SEQ ID NO: 17 or 27.

[0134] In some embodiments of the first aspect, the amino acid sequence of the first antigen-binding fragment may have 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:17 or 27.

[0135] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:17 or 27 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining the function of a similar first antigen-binding fragment.

[0136] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 17 or 27, and the resulting amino acid sequence still retains a similar function to the first antigen-binding fragment.

[0137] 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 regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 17 or 27, as long as the altered amino acid sequence substantially maintains the function of the first antigen-binding fragment.

[0138] 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:59 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:63.

[0139] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment may be substituted, deleted, and / or added by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids different from the amino acid sequence shown in SEQ ID NO:59.

[0140] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment may have 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:59.

[0141] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:59 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still retaining the function of the heavy chain variable region of the second antigen-binding fragment.

[0142] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:59, and the resulting amino acid sequence still retains the function of the heavy chain variable region of the second antigen-binding fragment.

[0143] 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 regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO:59, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the second antigen-binding fragment.

[0144] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen-binding fragment may be substituted, deleted, and / or added by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids different from the amino acid sequence shown in SEQ ID NO:63.

[0145] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the second antigen-binding fragment may have 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:63.

[0146] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:63 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still retaining the function of a similar light chain variable region of the second antigen-binding fragment.

[0147] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:63, and the resulting amino acid sequence still retains the function of the light chain variable region similar to that of the second antigen-binding fragment.

[0148] 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 regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO:63, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the second antigen-binding fragment.

[0149] For example, the first antigen-binding fragment may contain an amino acid sequence as shown in SEQ ID NO:17, and the second antigen-binding fragment may contain a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:59 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:63.

[0150] Alternatively, the first antigen-binding fragment may comprise an amino acid sequence as shown in SEQ ID NO:27, and the second antigen-binding fragment may comprise a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:59 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:63.

[0151] In some embodiments of the first aspect, the first antigen-binding fragment and the second antigen-binding fragment are linked by an antibody heavy chain constant region Fc fragment, the antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment.

[0152] In some embodiments, the antibody heavy chain constant region Fc fragment may be an Fc fragment of the IgG1 subtype (e.g., an Fc fragment of the human IgG1 subtype with an amino acid sequence as shown in SEQ ID NO:7).

[0153] In some embodiments, the Fc fragment of the antibody heavy chain constant region can be various mutants of the human IgG1 subtype, including but not limited to: IgG1H (SEQ ID NO:8), IgG1K (SEQ ID NO:9), IgG1m3-H (SEQ ID NO:10), IgG1m3-K (SEQ ID NO:11), IgG1m3-H1n1 (SEQ ID NO:12), or IgG1m3-Kn1 (SEQ ID NO:13). In some specific embodiments, the Fc fragment of the antibody heavy chain constant region can be an Fc fragment of the IgG1m3 subtype, for example, an Fc fragment of the IgG1m3 subtype with an amino acid sequence as shown in any one of SEQ ID NO:10 to SEQ ID NO:13.

[0154] In some embodiments of the first aspect, 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 in the antibody constant region are determined according to EU numbering.

[0155] In some embodiments of the first aspect, 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 in the antibody constant region are determined according to EU numbering.

[0156] In some embodiments of the first aspect, the amino acids at positions 234, 235, and 331 of the first Fc fragment and the second Fc fragment are F, E, and S, respectively, wherein the amino acid positions of the antibody constant region are determined according to EU numbering.

[0157] In some embodiments of the first aspect, 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 some specific embodiments, the first antigen-binding fragment (e.g., at its C-terminus) is linked to the N-terminus of the first Fc fragment. In some specific embodiments, the second antigen-binding fragment (e.g., at its C-terminus) is linked to the N-terminus of the second Fc fragment.

[0158] In some embodiments of the first aspect, the bispecific antibody comprises a first arm that binds to the HLA-AO2 / AFP complex and a second arm that binds to an activated T-cell antigen, wherein

[0159] The first arm comprises an amino acid sequence as shown in SEQ ID NO:31 or 32; and

[0160] The second arm contains an amino acid sequence as shown in SEQ ID NO:33.

[0161] Secondly, this application provides a single-domain antibody that binds to the HLA-A02 / AFP complex, which contains

[0162] HCDR1, as shown in SEQ ID NO:18,

[0163] HCDR2, as shown in SEQ ID NO:19, and

[0164] HCDR3, as shown in SEQ ID NO:20;

[0165] The amino acid sequence of HCDR is defined according to Kabat.

[0166] In some embodiments of the second aspect, the binding epitope of the single-domain antibody to the HLA-A02 / AFP complex includes one or more residues from positions 158-166 (SEQ ID NO:1) of the AFP shown in SEQ ID NO:58. In some specific embodiments, the binding epitope of the first antigen-binding fragment to the HLA-A02 / AFP complex includes at least one of residues from positions 159, 160, 162, 164, and 165 of the AFP shown in SEQ ID NO:58.

[0167] In some embodiments of the second aspect, the single-domain antibody is a monovalent or multivalent (e.g., 1, 2, 3, 4, 5, or 6-valent) single-domain antibody form that binds to the HLA-A02 / AFP complex.

[0168] In some specific embodiments of the second aspect, the single-domain antibody comprises the heavy chain variable region of two directly fused monovalent forms of single-domain antibodies binding the HLA-A02 / AFP complex.

[0169] In some specific embodiments of the second aspect, the single-domain antibody comprises the heavy chain variable region of two monovalent single-domain antibodies binding the HLA-A02 / AFP complex, linked by a linker. In some embodiments, the linker is a GS-type flexible peptide linker. In some embodiments, the linker is (G4S)n, (SG4)n, or G4(SG4)n, where n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or an integer or range of any two of the above values. In some embodiments, the linker is (G4S)n, (SG4)n, or G4(SG4)n, where n is an integer from 2 to 4, such as 2, 3, or 4.

[0170] In some embodiments of the second aspect, the single-domain antibody comprises an amino acid sequence as shown in SEQ ID NO:17 or 27.

[0171] In some embodiments of the second aspect, the amino acid sequence of the single-domain antibody may be substituted, deleted, and / or added by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids different from the amino acid sequence shown in SEQ ID NO:17 or 27.

[0172] In some embodiments of the second aspect, the amino acid sequence of the single-domain antibody may have 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:17 or 27.

[0173] In some embodiments of the second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:17 or 27 may 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 to the single-domain antibody.

[0174] 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 to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 17 or 27, and the resulting amino acid sequence still retains a similar function to the single-domain antibody.

[0175] 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 regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 17 or 27, as long as the altered amino acid sequence substantially maintains a similar function to the single-domain antibody.

[0176] Thirdly, this application provides a nucleic acid molecule that encodes the bispecific antibody described in the first aspect or the single-domain antibody described in the second aspect.

[0177] In some embodiments of the third aspect, the nucleic acid molecule may include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0178] In some embodiments of the third aspect, the nucleic acid molecule is operatively linked to a regulatory nucleotide sequence for expression in a host cell. In some embodiments, the nucleic acid molecule is operatively linked to a regulatory nucleotide sequence contained in a vector (e.g., a plasmid) for expression in a host cell. The host cell may be derived from mammals, such as mice, rats, dogs, cats, sheep, cattle, monkeys, camels, llamas, humans, etc. The host cell may be cultured in a culture medium to express the bispecific antibody or single-domain antibody and subsequently recovered to harvest the bispecific antibody or single-domain antibody.

[0179] Fourthly, this application provides a pharmaceutical composition comprising the bispecific antibody described in the first aspect or the single-domain antibody described in the second aspect, as well as a pharmaceutically acceptable excipient, diluent, or carrier.

[0180] In some embodiments of the fourth aspect, the pharmaceutical composition is used for the prevention or treatment of HLA-AO2 / AFP-positive tumors. The HLA-AO2 / AFP-positive tumors may be, for example, hepatocellular carcinoma or gastric cancer.

[0181] Fifthly, this application provides the use of the bispecific antibody described in the first aspect, the single-domain antibody described in the second aspect, the nucleic acid molecule described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a medicament for the prevention or treatment of HLA-AO2 / AFP-positive tumors.

[0182] In some implementations of the fifth aspect, the HLA-A02 / AFP positive tumor is selected from hepatocellular carcinoma and gastric cancer.

[0183] In some embodiments of the fourth and / or fifth aspects, the pharmaceutical composition and / or the pharmaceutical product 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, etc.

[0184] In some embodiments of the fourth and / or fifth aspects, the pharmaceutical composition and / or the drug may be formulated as tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules.

[0185] In some embodiments of the fourth and / or fifth aspects, the pharmaceutical composition and / or medicament of this application may be delivered using any physiologically acceptable mode of administration, including but not limited to: parenteral administration, intraperitoneal administration, intravascular injection (e.g., intravenous injection), subcutaneous administration, transdermal administration, etc.

[0186] In some embodiments of the fourth and / or fifth aspects, the pharmaceutical composition and / or the drug may be formulated for storage as a lyophilized formulation or an aqueous solution by mixing reagents having the desired purity with, as appropriate, pharmaceutically acceptable carriers, excipients, etc.

[0187] This article also provides methods for preventing or treating tumors (e.g., HLA-A02 / AFP-positive tumors), the methods comprising administering to an individual in need an effective amount of the bispecific antibody of the first aspect, the single-domain antibody of the second aspect, the nucleic acid molecule of the third aspect, and / or the pharmaceutical composition of the fourth aspect.

[0188] In some implementations, the HLA-A02 / AFP positive tumor is selected from hepatocellular carcinoma and gastric cancer.

[0189] Sixthly, this application provides the use of the bispecific antibody described in the first aspect or the single-domain antibody described in the second aspect in the preparation of products for detecting HLA-A02 / AFP positive cells.

[0190] In some embodiments of the sixth aspect, the product is a reagent kit, test strip, test card, or microfluidic device.

[0191] In some embodiments of the sixth aspect, the product may also include detection markers, such as colloidal gold, chemiluminescent markers, fluorescent markers, nanoparticle markers, etc.

[0192] In some embodiments of the sixth aspect, the product may also include other reagents for detection, such as enzymes or colloidal gold-labeled antigens or antibodies, substrates, reference standards, diluents, washing solutions, etc.

[0193] In some embodiments of the sixth aspect, the product may further include reagents for processing biological samples for detection, the biological samples being blood.

[0194] In some embodiments of the sixth aspect, the product may also include a product instruction manual.

[0195] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments.

[0196] The following examples are for illustrative purposes only and are not intended to limit the scope of this application.

[0197] Example

[0198] Example 1: Preparation of recombinant protein

[0199] In the preparation of bispecific antibodies targeting HLA-A02 / AFP and CD3, various recombinant proteins are required, including the extracellular domain of human CD3E (hCD3E, SEQ ID NO:2) and the extracellular domain of human CD3D (hCD3D, SEQ ID NO:3). Meanwhile, referring to the literature [Kotsiou E, Brzostek J, Lenart I, Antoniou AN, Dyson J, Gould KG. Dimerization of soluble disulfide trap single-chain major histocompatibility complex class I molecules dependent on peptide binding affinity. Antioxid Redox Signal. 2011 Aug 1; 15(3):635-44.]

[0017] A complex of AFP antigen peptide FMNKFIYEI (SEQ ID NO:1) and HLA-A0201 in the form of a dtSCT (disulfide trap single-chain trimer) structure (MIP-A1-A0201, SEQ ID NO:4) was prepared. These recombinant proteins exhibit numerous post-translational modifications (such as glycosylation or disulfide bonds), thus utilizing mammalian cell expression systems is more conducive to maintaining the structure and function of the recombinant proteins. Adding a His tag (His, SEQ ID NO:5) or the Fc fragment of mouse antibody IgG2a (mFc, SEQ ID NO:6) to the C-terminus of the recombinant protein will further facilitate the purification of the recombinant protein and the identification of monoclonal antibody function. When preparing recombinant antibodies, the constant region of the antibody heavy chain can be human IgG1 subtype (SEQ ID NO:7) or various mutants of selected human IgG1 subtypes, such as: IgG1H (SEQ ID NO:8), IgG1K (SEQ ID NO:9), IgG1m3-H (SEQ ID NO:10), IgG1m3-K (SEQ ID NO:11), IgG1m3-H1n1 (SEQ ID NO:12) or IgG1m3-Kn1 (SEQ ID NO:13), and the constant region of the light chain can be human λ subtype (SEQ ID NO:14) or human κ subtype (SEQ ID NO:15).

[0200] Based on the amino acid sequence of the target recombinant protein from the Uniprot database, genes for the aforementioned recombinant proteins (containing His tags or mFc encoding genes) were designed and synthesized. Using conventional molecular biology techniques, the synthesized recombinant protein genes were cloned into suitable eukaryotic expression vectors (such as Invitrogen's pcDNA3.1). Then, using liposomes (such as Invitrogen's 293fectin) or other cationic transfection reagents (such as PEI), the prepared recombinant protein expression plasmids were transfected into HEK293 cells (such as Invitrogen's HEK293F) and cultured in serum-free suspension for 3-4 days. The culture supernatant was then harvested by centrifugation or other methods.

[0201] Recombinant proteins expressed via His-tag fusion were purified in one step from the culture supernatant using a metal chelate affinity chromatography column (such as GE's HisTrap FF). Recombinant proteins expressed via mFc fusion were purified in one step using a Protein A / G affinity chromatography column (such as GE's Mabselect SURE). The recombinant protein storage buffer was then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (such as GE's Hitrap desaulting). If necessary, the recombinant protein samples can be filtered for sterilization and then aliquoted and stored at -20°C.

[0202] Example 2: Screening of antibodies binding to the HLA-AO2 / AFP complex from a camel immune bank

[0203] 2.1 Construction of the Camel Immunity Bank

[0204] One healthy adult llama was selected, and blood was collected to obtain baseline serum before immunization. For the first immunization, 0.4 mg of MIP-A1-A0201-His fusion protein was emulsified with Fischer's complete adjuvant and injected subcutaneously at multiple sites. Two weeks later, booster immunizations were performed, with 0.4 mg of MIP-P3-His fusion protein emulsified with Fischer's incomplete adjuvant and injected subcutaneously at multiple sites. A total of 5 booster immunizations were performed, with blood collected before each immunization for antibody titer analysis. For the seventh immunization, no adjuvant was added, and 0.4 mg of MIP-A1-A0201-His fusion protein was used as the antigen and injected subcutaneously at multiple sites for pulse immunization. Three days later, 150 ml of peripheral blood was collected for lymphocyte separation.

[0205] 150 ml of llamas were isolated from peripheral blood lymphocytes using a camel peripheral blood lymphocyte isolation kit (Solarbio, CAT#P5750). Total RNA was extracted from the lymphocytes using a total RNA extraction kit (Tiangen, CAT#DP430). Using the extracted total RNA as a template, the variable region of the camel heavy chain was synthesized using a first-strand cDNA synthesis kit (Thermo Scientific, CAT#K1621). Gene-specific primers were used for reverse transcription, with the primer pairing region located in the CH2 domain of the antibody heavy chain constant region, specifically the sequence PCal-CH2R: TCCTTCCCCGTCAGCCAGTCCT (SEQ ID NO:16). The synthesized cDNA was immediately stored at -70℃ for later use. Then, using the cDNA obtained from reverse transcription as a template, [reference missing]. E, Poignavent V, Vincke C, Ritzenthaler C, Muyldermans S, Monsion B. Construction of High-Quality Camel Immune Antibody Libraries. Methods Mol Biol. 2018; 1701:169-187.]

[0018] Primers were synthesized, and the camel VHH gene was isolated by nested PCR amplification. Finally, the amplified VHH gene was cloned into the vector pADSCFV-S (see Chinese Invention Patent Application 201510097117.0).

[0019] A VHH library was constructed. This antibody library has a capacity of 4.5E+08 and an accuracy of 63.3%.

[0206] 2.2 Screening of the camel immune bank

[0207] Using the recombinant MIP-A1-A0201-his prepared in Example 1 as the antigen, a solid-phase screening strategy was employed (experimental protocol referenced [Phage Display: A Universal Guide to Experimentation / Edited by (US) Clackson, T., and (US) Lowman, HB; translated by Ma Lan et al. Chemical Industry Press, 2008.5]).

[0020] The phage library displaying camel single-domain antibodies constructed in Example 2.1 was screened through three rounds of screening: binding, elution, neutralization, infection, and amplification. Finally, the TCRm (TCR mimic) antibody N9H11 (SEQ ID NO:17) that specifically binds to MIP-A1-A0201 was obtained.

[0208] Using conventional molecular biology techniques, the nucleotide sequence encoding the N9H11 heavy chain variable region (SEQ ID NO:17) was cloned into a eukaryotic expression vector (such as Invitrogen's pcDNA3.1) fused with the Fc segment encoding the human antibody IgG1, to express the N9H11-Fc recombinant protein. Simultaneously, a monoclonal antibody DP47-IgG1 (a germline gene antibody, referring to US patent application US20160200833A1, with the amino acid sequence of DP47VH as shown in SEQ ID NO:21; and the amino acid sequence of DP47VK as shown in SEQ ID NO:22) was prepared as a negative control.

[0209] Example 3: Identification of TCRm antibodies binding to the HLA-A02 / AFP complex

[0210] 3.1 Affinity analysis of the anti-HLA-A02 / AFP complex TCRm

[0211] The affinity of TCRm antibody N9H11 against the HLA-A02 / AFP complex was determined using surface plasmon resonance (SPR) technology with a Biacore T200. All reagents and consumables, including the amino-coupled assay kit (BR100050), human antibody capture kit (29234600), Series S sensor chip CM5 (29149603), and 10×HBS-EP (BR100669) at pH 7.4, were purchased from Cytiva. Following the kit instructions, the carboxylated CM5 chip surface was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The anti-human IgG (Fc) antibody (capture antibody) was diluted to 25 μg / mL with 10 mM pH 5.0 sodium acetate and injected at a flow rate of 10 μL / min to achieve a conjugation volume of approximately 10,000 response units (RU). After injection of the capture antibody, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, the TCRm antibody N9H11-Fc was diluted to 1 μg / mL and injected at a flow rate of 10 μL / min, ensuring that approximately 100 RU of antibody was captured by the anti-human Fc antibody. Then, a series of concentration gradients (e.g., 2.47 nM, 7.41 nM, 22.2 nM, 66.7 nM, 200 nM) of MIP-A1-A0201-his were set, and injected at 30 μL / min from low to high concentration at 25 °C. The binding time was 60 s, and the dissociation time was 1200 s. The chip surface was then regenerated by injecting 3M MgCl2 solution at 10 μL / min for 30 s. Using Biacore T200 evaluation software version 3.0, the binding rate (Ka) and dissociation rate (Kd) were calculated by fitting the binding and dissociation sensor maps using a 1:1 binding model. The dissociation equilibrium constant (KD) was calculated using the ratio Kd / Ka. The fitting results are shown in Table 1. Table 1. Affinity of TCRm antibody N9H11 against HLA-A02 / AFP complex to MIP-A1-A0201.

[0212] 3.2 Binding of the anti-HLA-A02 / AFP complex TCRm to the MIP-A1-A0201 recombinant protein

[0213] The prepared MIP-A1-A0201-his was coated onto 96-well plates (1 μg / ml, 100 μl / well) and incubated overnight at 4°C. Blocking was performed using PBS-0.1% Tween 20-3% skim milk blocking buffer at 37°C for 1 hour. The TCRm antibody against the HLA-A02 / AFP complex was serially diluted with PBS, starting at 10 μg / ml, with 11 4-fold serial dilutions, and 100 μl / well was added to each well of the blocked 96-well ELISA plate. The plates were incubated at 37°C for 1 hour. The ELISA plates were washed with PBS-0.1% Tween 20, and HRP-labeled mouse anti-human IgG monoclonal antibody (Beijing Bio-Sens Biotechnology Co., Ltd., bsm-0297M-HRP) was added, followed by incubation at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20, and OPD substrate chromogenic solution was added. After 5-10 minutes, the chromogenic reaction was terminated with 1M H2SO4. The optical density was measured at 492nm / 630nm using a microplate reader. The results showed (Figure 1) that N9H11 specifically binds to MIP-A1-A0201, that is, N9H11 specifically binds to the HLA-A02 / AFP complex.

[0214] 3.3 Binding of anti-HLA-A02 / AFP complex TCRm to antigenic peptide pulsed T2 cells

[0215] Collect T2 cells (human lymphoma cells, HLA-A02) in the logarithmic growth phase. + Purchased from Shanghai Hongshun Biotechnology Co., Ltd., the sample was centrifuged and resuspended in growth medium to a concentration of 1×10⁻⁶. 6 / mL, 1mL per well in a 24-well plate. Chemically synthesized peptide AFP 158-166 (FMNKFIYEI) (GenScript Biotech Inc.), and added to a final concentration of 50 μg / mL into a 24-well plate, incubated overnight. After 16 hours, centrifuged, and resuspended in PBS buffer containing 1% BSA to a final volume of 2 × 10⁻⁶. 6 100 μL of N9H11 antibody and DP47-IgG1 negative control were seeded into each well of a 96-well V plate and centrifuged, then the supernatant was discarded. Antibody N9H11 and negative control DP47-IgG1 were prepared to an initial concentration of 100 nM using PBS, with 4-fold dilutions, for a total of 10 concentrations. 100 μL of each sample was added to the well containing cells and incubated at 4°C for 1 hour. The cells were then washed three times with 200 μL of PBS and incubated with goat anti-human IgG-FITC (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., ZF-0308) at 100 μL per well, incubated at 4°C in the dark for 30 minutes. The cells were then washed three times with 200 μL of PBS, resuspended in 100 μL of PBS, and the FITC channels were detected by flow cytometry (ACEA, Novocyte). The results showed (Figure 2) that N9H11 specifically binds to the antigenic peptide AFP. 158-166Pulsed T2 cells, namely N9H11, bind to the HLA-A02 / AFP complex on the cell surface.

[0216] 3.4 Key amino acids of AFP bound to TCRm antibody against HLA-A02 / AFP complex

[0217] Chemical synthesis of AFP 158-166 (FMNKFIYEI) alanine scanning mutant peptide. Refer to Example 3.3 for the detection of N9H11 and AFP. 158-166 Binding of the alanine-scanning mutant peptide (FMNKFIYEI) to T2 cells. The binding of N9H11 to the alanine-scanning mutant peptide relative to the original peptide was calculated, and the results (Figure 3) show that AFP... 158-166 When alanine is substituted at positions 2, 3, 5, 7, and 8 of (FMNKFIYEI), the binding affinity of TCRm antibody N9H11 decreases by 80% or more, i.e., AFP... 158-166 The 2nd, 3rd, 5th, 7th, and 8th amino acids of (FMNKFIYEI) are key amino acids for N9H11 binding.

[0218] Example 4: Humanization and identification of TCRm antibody against HLA-A02 / AFP complex

[0219] 4.1 Humanization of TCRm antibodies against HLA-A02 / AFP complex

[0220] The TCRm antibody N9H11 was humanized to reduce its immunogenicity. The humanization protocol employed a classic framework transplantation strategy.

[0021] The amino acid sequence of N9H11 was compared with human antibody germline gene sequences in the IMGT database. Suitable germline gene sequences were selected to provide frame regions 1 to 3 (FR1+FR2+FR3) of the antibody, as shown in SEQ ID NO:23 / 24 / 25, and suitable J region gene sequences were selected to provide frame region 4 (FR4), as shown in SEQ ID NO:26. This template can be selected based on various factors, such as the relative total length of the antibody, the size of the CDR, the amino acid residues located at the junction between the antibody frame region (FR) and the hypervariable region (CDR), and the overall sequence homology. The selected template can be a mixture of multiple sequences or a shared template, with the aim of maintaining the appropriate conformation of the parental complementarity-determining region (CDR) as much as possible. Simultaneously, considering the solubility, stability, and expression yield of the humanized antibody, four hotspot amino acids 37F / 44E / 45R / 47F in FR2 were reverse-mutated, ultimately yielding the humanized molecule N9H11-h4 (SEQ ID NO:27).

[0221] Using conventional molecular biology techniques, the nucleotide sequence encoding the variable region of N9H11-h4 (SEQ ID NO: 27) was cloned into a eukaryotic expression vector (such as Invitrogen's pcDNA3.1) that fused with the Fc segment encoding human antibody IgG1, and the N9H11-h4-Fc recombinant protein was expressed.

[0222] 4.2 Affinity analysis of humanized TCRm antibodies against HLA-A02 / AFP complex

[0223] Referring to Example 3.1, the affinity of the recombinant anti-HLA-A02 / AFP complex TCRm antibody N9H11-h4 was analyzed using a Biacore T200, and the results are shown in Table 2.

[0224] Table 2. Affinity of TCRm antibody N9H11-h4 against HLA-A02 / AFP complex to MIP-H1-01

[0225] Example 5: Preparation and identification of TCRm×anti-CD3 bispecific antibody

[0226] 5.1 Preparation of TCRm×anti-CD3 bispecific antibody

[0227] The nucleotide sequences encoding the variable regions of N9H11 (N9H11, SEQ ID NO:17) and N9H11-h4 (N9H11-h4, SEQ ID NO:27) and the single-chain antibody encoding anti-CD3 (anti-CD3-ScFv, SEQ ID NO:28, see patent US005821337A) are compared with those encoding the single-chain antibody against CD3 (anti-CD3-ScFv, SEQ ID NO:28, see patent US005821337A).

[0022] The nucleotide sequences of anti-CD3 v9 were cloned into suitable eukaryotic expression vectors to co-express bispecific antibodies against HLA-A02 / AFP and CD3. Specifically, the nucleotide sequences encoding N9H11 or N9H11-h4 were cloned into eukaryotic expression vectors fused with the nucleotide sequence of the Fc fragment IgG1m3-FcH1n1 (SEQ ID NO:29) encoding the Hole mutation, and the nucleotide sequences encoding anti-CD3-ScFv were cloned into eukaryotic expression vectors fused with the nucleotide sequence of the Fc fragment IgG1m3-FcKn1 (SEQ ID NO:30) encoding the Knob mutation.

[0228] The constructed eukaryotic expression vectors expressing N9H11-G1m3-FcH1n1 (SEQ ID NO:31) or N9H11-h4-G1m3-FcH1n1 (SEQ ID NO:32) and anti-CD3-ScFv-G1m3-FcKn1 (SEQ ID NO:33) were co-transfected into HEK293F cells using liposomes. The cells were cultured in serum-free suspension for 3-5 days, and the culture supernatant was harvested by centrifugation. The bispecific antibodies in the culture supernatant were purified using a Protein A affinity chromatography column (e.g., GE's Mabselect SURE). The recombinant protein preservation buffer was then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (e.g., GE's Hitrap desaulting). The desalted protein solution was purified by size exclusion chromatography (SEC) using a Superdex 200 (GE) to obtain the target protein. If necessary, antibody samples can be filtered and sterilized, then aliquoted and stored at -20°C for later use.

[0229] 5.2 Affinity analysis of TCRm×anti-CD3 bispecific antibody

[0230] Referring to Example 3.1, the affinity analysis of the recombinant TCRm×anti-CD3 bispecific antibody was performed using Biacore T200, and the fitting results are shown in Tables 3 and 4.

[0231] Table 3: Affinity of TCRm×anti-CD3 bispecific antibody to MIP-A1-A0201

[0232] Table 4: Affinity of TCRm×anti-CD3 bispecific antibody to CD3D-CD3E-mFc

[0233] 5.3 The TCRm×anti-CD3 bispecific antibody simultaneously recognizes antigens MIP-A1-A0201 and CD3.

[0234] The TCRm×anti-CD3 bispecific antibody was detected using a conventional ELISA method, which simultaneously binds to both bidirectional antigens CD3 and MIP-A1-A0201.

[0235] Coat 96-well ELISA plates with CD3D-CD3E-mFc antigen (3 μg / mL, 100 μL per well) overnight at 4°C. Block with blocking buffer PBS-0.1% Tween 20-3% skim milk at 37°C for 1 hour. Dilute TCRm×anti-CD3 bispecific antibody to 10 μg / mL with PBS, add 100 μL per well to the blocked 96-well ELISA plates, and incubate at 37°C for 1 hour. Wash the ELISA plates with PBS-0.1% Tween 20, add MIP-A1-A0201 antigen (2 μg / mL, 100 μL per well), and incubate at 37°C for 1 hour. Wash the ELISA plates with PBS-0.1% Tween 20, then add HRP-labeled anti-his-tagged mouse monoclonal antibody (Beijing Kangwei Century Biotechnology Co., Ltd., cw0285M), and incubate at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20, and OPD substrate chromogenic solution was added. The chromogenic reaction was stopped with 1M H2SO4 after 5-10 minutes. The optical density was measured at 492nm / 630nm using a microplate reader. The ELISA results are shown in Figure 4. The TCRm×anti-CD3 bispecific antibodies N9H11×anti-CD3 and N9H11-h4×anti-CD3 can simultaneously recognize CD3 and MIP-A1-A0201.

[0236] 5.4 T2 cell activation of Jurkat-Dual cells mediated by TCRm×anti-CD3 bispecific antibody-mediated antigen peptide pulses

[0237] Collect T2 cells in the logarithmic growth phase, centrifuge, and resuspend in growth medium to a concentration of 1×10⁻⁶. 6 / mL, 1mL per well in a 24-well plate. Peptide AFP 158-166 (FMNKFIYEI) was added to a 24-well plate at a final concentration of 50 μg / mL and incubated overnight. After 16 hours, the plate was centrifuged and resuspended in RPMI 1640 medium to a final volume of 4 × 10⁻⁶. 5 50 μL of Jurkat-Dual cells (purchased from Invivogen) were seeded per well at a concentration of 10 mL / mL into cell plates. Jurkat-Dual cells in the logarithmic growth phase were collected, centrifuged, and resuspended in RPMI 1640 medium to a final volume of 4 × 10⁻⁶. 5 / mL, 50μL per well was added to the cell plate seeded with T2 cells to obtain a final E:T ratio of 1:1. Then, bispecific antibodies N9H11×anti-CD3 and N9H11-h4×anti-CD3 (starting concentration 30nM, 4-fold serial dilution, 10 concentration points, 100μL per well) were added. The negative control sample DP47×anti-CD3 was used at the same concentration as the bispecific antibodies. After incubation for 24h, the supernatant was collected, and the specific activation of Jurkat-Dual cells by T2 cells mediated by the TCRm×anti-CD3 bispecific antibody was detected and analyzed according to the QUANTI-Luc™ instructions (QUANTI-Luc™, Invivogen, rep-qlc2). The results showed that the bispecific antibodies N9H11×anti-CD3 and N9H11-h4×anti-CD3 could specifically mediate the activation of Jurkat-Dual cells by T2 cells mediated by the antigen peptide pulse (Figure 5), EC 50 The values ​​are shown in Table 5.

[0238] Table 5. T2 cell activation of Jurkat-Dual cell ECGs mediated by TCRm×anti-CD3 bispecific antibody-mediated antigen peptide pulses 50 value

[0239] 5.5 TCRm×anti-CD3 bispecific antibody mediates PBMC-specific killing of target cells

[0240] 5.5.1 Isolation of human peripheral blood mononuclear cells (PBMCs)

[0241] Blood was collected from healthy volunteers (50 mL each), and all volunteers had signed informed consent forms. Volunteer selection criteria were as follows:

[0242] Age over 18 years old;

[0243] No HIV or HBV infection;

[0244] Blood routine test results were normal;

[0245] Non-pregnant or breastfeeding women;

[0246] PBMCs were isolated from whole blood of volunteers using Ficoll density gradient centrifugation and cultured in RPMI 1640 medium.

[0247] 5.5.2 TCRm×anti-CD3 bispecific antibody-mediated PBMC killing of HLA-A02 + / AFP + cell

[0248] HepG2 cells (human liver cancer cells, purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were HLA-AO2 and AFP-positive. HepG2 cells in the logarithmic growth phase were collected, digested, centrifuged, and resuspended in RPMI 1640 medium to a concentration of 4 × 10⁻⁶. 5 / mL, 50μL per well, were seeded into cell plates. Then, bispecific antibodies N9H11×anti-CD3 and N9H11-h4×anti-CD3 (starting concentration 10nM, serially diluted 3-fold, 10 concentration spots, 100μL per well) were added. The negative control sample DP47×anti-CD3 was used at the same concentration as the bispecific antibodies. PBMCs (effective cells) were resuspended to 4×10⁶ m³ / well. 6 Add the above cell plate at a concentration of / mL, 50μL per well, for a final effector-to-target ratio of 10:1. Simultaneously set up separate target cell controls, separate effector cell controls (PBMCs), and separate culture medium blank controls, and bring the volume of each well to 200μL with culture medium. After incubation for 24 hours, collect the supernatant and refer to... Non-radioactive cytotoxicity detection reagents ( The Non-Radioactive Cytotoxicity Assay (Promega, G1780) describes the detection and analysis of TCRm×anti-CD3 bispecific antibody-mediated PBMC cell activity against HLA-A02. + / AFP + HepG2 cell killing. The results are shown in Figure 6 and Table 6: The bispecific antibodies N9H11×anti-CD3 and N9H11-h4×anti-CD3 can specifically mediate the killing of HLA-A02+ / AFP+ HepG2 cells by PBMCs.

[0249] Table 6. ECMO effect of TCRm×anti-CD3 bispecific antibody-mediated killing of HepG2 cells by PBMCs 50 value

[0250] Example 6: TCRm×anti-CD3 bispecific antibody recognizes AFP 158-166 Key amino acid analysis of (FMNKFIYEI)

[0251] Chemical synthesis of AFP 158-166The alanine-scanning peptide of (FMNKFIYEI), sequence information is shown in Table 7. Referring to Example 5.4, the specific activation of Jurkat-Dual cells by T2 cells pulsed with the alanine-scanning peptide was detected and analyzed. The results showed that N9H11-h4×anti-CD3 could not effectively mediate the activation of Jurkat-Dual cells by T2 cells pulsed with the alanine-scanning mutant peptide at positions 2, 3, 5, 7, and 8 (Figure 7), i.e., AFP... 158-166 The amino acids at positions 2, 3, 5, 7, and 8 of (FMNKFIYEI) are key amino acids for N9H11-h4×anti-CD3 binding.

[0252] Table 7: AFP 158-166 (FMNKFIYEI) alanine scanning peptide

[0253] Example 7: Interaction analysis between TCRm×anti-CD3 bispecific antibody and similar peptides

[0254] Chemical synthesis of AFP in Table 8 158-166 Similar peptides to (FMNKFIYEI), AFP-S1 / S2 / S3 / S4 / S5, were derived from searches for AFP-SFP-S1 / S2 / S3 / S4 / S5. 158-166 (FMNKFIYEI) contains antigenic peptides with 5 or more identical amino acids at the same position, while N9H11-S1~S9 are searched based on the binding of N9H11 to key amino acids. Antigenic peptides with N9H11 binding to key amino acids or binding to amino acids with similar properties at the key position are identified. Referring to Example 5.4, the activation of Jurkat-Dual cells by T2 cells with different similar peptide pulses was detected and analyzed. The results showed (Figure 8) that N9H11-h4×anti-CD3 specifically mediates the antigenic peptide AFP. 158-166 (FMNKFIYEI) pulses activate Jurkat-Dual cells in T2 cells without mediating other AFP-loaded cells. 158-166 T2 cells with (FMNKFIYEI)-like peptides activate Jurkat-Dual.

[0255] Table 8.AFP 158-166 (FMNKFIYEI) similar peptides

[0256] Example 8: Interaction analysis between TCRm×anti-CD3 bispecific antibody and antigen-negative tumor cells

[0257] Different tumor cells were collected, and their antigen information is shown in Table 9. Following Examples 5.5.1 and 5.5.2, the cytotoxic activity of PBMC cells mediated by the bispecific antibody N9H11-h4×anti-CD3 against tumor cells was evaluated. The results showed that N9H11-h4×anti-CD3 specifically mediated the cytotoxic activity of PBMC cells against HLA-A02. + / AFP + It effectively kills target cells without mediating the killing of antigen-negative cells by PBMCs (Figure 9).

[0258] Table 9. Information on the expression of different cell antigens Note: "+" represents positive, and "-" represents negative.

[0259] Example 9: Specificity analysis of TCRm×anti-CD3 bispecific antibody against 20 HLA-A alleles with high gene frequencies in Chinese populations

[0260] Based on the 20 HLA-A alleles with high gene frequencies in the Chinese population listed in Table 10, different tumor cell lines were collected. Following Examples 5.5.1 and 5.5.2, the specificity of the killing activity of PBMC cells mediated by the bispecific antibody N9H11-h4×anti-CD3 against tumor cells was evaluated. The results showed that N9H11-h4×anti-CD3 specifically mediated the killing activity of PBMC cells against HLA-A02. + / AFP + It effectively kills target cells without mediating PBMCs to effectively kill tumor cells that do not have the HLA-A02 allele (regardless of whether AFP is positive) (Figure 10).

[0261] Table 10. 20 HLA-A alleles with high gene frequency in Chinese populations Note: Cells containing the A*02:01 gene in the table do not express AFP.

[0262] Example 10: TCRm×anti-CD3 bispecific antibody inhibits tumor growth in mice

[0263] NOG mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) (female, 6-8 weeks old) were used as experimental subjects. Human peripheral blood lymphocytes were collected 14 days before the start of the experiment according to Example 5.5.1 and inoculated into NOG mice at a rate of 1*10^7 / mouse. Seven days before the start of the experiment, mice were subcutaneously injected unilaterally into the abdomen with a suspension of cultured and collected HepG2 tumor cells at a rate of 1*10^7 / mouse (injection volume 100ul) to establish a mouse model of PBMC immune reconstitution of human liver cancer cells xenograft.

[0264] On the first day of the experiment, the major diameter (a) and minor diameter (b) of the formed tumor of each mouse were measured with vernier calipers, and the tumor volume was calculated according to the following formula: V=a*b^2 / 2; the mice were randomly divided into groups according to the tumor volume, with 10 mice in each group; after the grouping was completed, the drugs were administered according to the protocol in Table 11.

[0265] From the start of the experiment, the tumor diameter and volume of mice were measured and calculated every 3 days using the method described above, continuing until the mice's movement was affected by the excessive size of the tumor or the tumor regressed. The data were then compiled and statistically analyzed for the tumor volume of each group of mice. The experimental results are shown in Figure 11. The low dose of N9H11-h4×anti-CD3 bispecific antibody (0.1 mg / kg) significantly inhibited HLA-A02 in vivo. + / AFP + Proliferation of HepG2 tumor cells.

[0266] Table 11: Evaluation scheme for the inhibition of tumor growth in mice by TCRm×anti-CD3 bispecific antibody

[0267] Sequence information

[0268] SEQ ID NO:1(AFP 158-166 )

[0269] SEQ ID NO:2(hCD3E)

[0270] SEQ ID NO:3(hCD3D)

[0271] SEQ ID NO:4(MIP-A1-A0201)

[0272] SEQ ID NO:5(His)

[0273] SEQ ID NO:6(mFc)

[0274] SEQ ID NO:7(CH-IgG1)

[0275] SEQ ID NO:8(CH-IgG1H)

[0276] SEQ ID NO:9(CH-IgG1K)

[0277] SEQ ID NO:10(CH-IgG1m3-H)

[0278] SEQ ID NO:11(CH-IgG1m3-K)

[0279] SEQ ID NO:12(CH-IgG1m3-H1n1)

[0280] SEQ ID NO:13(CH-IgG1m3-Kn1)

[0281] SEQ ID NO:14(Cλ)

[0282] SEQ ID NO:15(Cκ)

[0283] SEQ ID NO:16(PCal-CH2R)

[0284] SEQ ID NO:17(N9H11)

[0285] SEQ ID NO:18(N9H11-HCDR1)

[0286] SEQ ID NO:19(N9H11-HCDR2)

[0287] SEQ ID NO:20(N9H11-HCDR3)

[0288] SEQ ID NO:21(DP47VH)

[0289] SEQ ID NO:22(DP47VK)

[0290] SEQ ID NO:23(N9H11-h4-FR1)

[0291] SEQ ID NO:24(N9H11-h4-FR2)

[0292] SEQ ID NO:25(N9H11-h4-FR3)

[0293] SEQ ID NO:26(N9H11-h4-FR4)

[0294] SEQ ID NO:27(N9H11-h4)

[0295] SEQ ID NO:28(anti-CD3-scFv)

[0296] SEQ ID NO:29(IgG1m3-FcH1n1)

[0297] SEQ ID NO:30(IgG1m3-FcKn1)

[0298] SEQ ID NO:31(N9H11-G1m3-FcH1n1)

[0299] SEQ ID NO:32(N9H11-h4-G1m3-FcH1n1)

[0300] SEQ ID NO:33(anti-CD3-ScFv-G1m3-FcKn1)

[0301] SEQ ID NO:34(AFP-F1A)

[0302] SEQ ID NO:35(AFP-M2A)

[0303] SEQ ID NO:36(AFP-N3A)

[0304] SEQ ID NO:37(AFP-K4A)

[0305] SEQ ID NO:38(AFP-F5A)

[0306] SEQ ID NO:39(AFP-I6A)

[0307] SEQ ID NO:40(AFP-Y7A)

[0308] SEQ ID NO:41 (AFP-E8A)

[0309] SEQ ID NO:42 (AFP-I9A)

[0310] SEQ ID NO:43 (Irrelevant peptide)

[0311] SEQ ID NO:44 (AFP-S1)

[0312] SEQ ID NO:45 (AFP-S2)

[0313] SEQ ID NO:46 (AFP-S3)

[0314] SEQ ID NO:47 (AFP-S4)

[0315] SEQ ID NO:48 (AFP-S5)

[0316] SEQ ID NO:49 (N9H11-S1)

[0317] SEQ ID NO:50 (N9H11-S2)

[0318] SEQ ID NO:51 (N9H11-S3)<s

[0319] SEQ ID NO:52 (N9H11-S4)

[0320] SEQ ID NO:53 (N9H11-S5)

[0321] ] / >SEQ ID NO:54 (N9H11-S6)

[0322] SEQ ID NO:55 (N9H11-S7)

[0323] SEQ ID NO:56 (N9H11-S8)

[0324] Note: There seems to be a closing tag issue in the original text at line 52 where it says

[0321] ]> instead of

[0321] as in other lines. I've translated it as

[0321] ] / > in the English version to maintain the consistency with the original format. If this is an error in the original, it might need to be corrected before using the translation in a proper context.SEQ ID NO:57 (N9H11 - S9)

[0325] SEQ ID NO:58 (AFP)

[0326] SEQ ID NO:59 (anti - CD3 - VH)

[0327] SEQ ID NO:60 (anti - CD3 - HCDR1)

[0328] SEQ ID NO:61 (anti - CD3 - HCDR2)

[0329] SEQ ID NO:62 (anti - CD3 - HCDR3)

[0330] SEQ ID NO:63 (anti - CD3 - VK)

[0331] SEQ ID NO:64 (anti - CD3 - LCDR1)

[0332] SEQ ID NO:65 (anti - CD3 - LCDR2)

[0333] SEQ ID NO:66 (anti - CD3 - LCDR3)

[0334] References

[0335] 1. Masseyeff R, Gilli J, Krebs B, Calluaud A, Bonet C. Evolution of alpha - fetoprotein serum levels throughout life in humans and rats, and during pregnancy in the rat. Ann N Y Acad Sci 1975; 259: 17 - 28

[0336] 2.Nakabayashi H, Hashimoto T, Miyao Y, et al. A position-dependent silencer plays a major role in repressing alpha-fetoprotein expression in human hepatoma. Mol Cell Biol. 1991; 11:5885-93.

[0337] 3.Lazarevich NL.Molecular mechanisms of alpha-fetoprotein gene expression.Biochemistry(Mosc).2000;65:117-33.

[0338] 4. Tayob N, Kanwal F, Alsarraj A, Hernaez R, El-Serag HB. The Performance of AFP, AFP-3, DCP as Biomarkers for Detection of Hepatocellular Carcinoma(HCC): A Phase 3Biomarker Study in the United States. Clin Gastroenterol Hepatol. 2023 Feb; 21(2): 415-423.e4.

[0339] 5. Kim DY, Han KH. Epidemiology and surveillance of hepatocellular carcinoma. Liver Cancer. 2012; 1:2-14.

[0340] 6. Liu Zongchao, Li Zhexuan, Zhang Yang, et al. Interpretation of the 2020 Global Cancer Statistics Report [J]. Electronic Journal of Comprehensive Cancer Treatment, 2021, 7(2): 1-13.

[0341] 7.Sung H,Ferlay J,Siegel RL,Laversanne M,Soerjomataram I,Jemal A,Bray F.Global Cancer Statistics 2020:GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36Cancers in 185Countries.CA Cancer J Clin.2021May;71(3):209-249.

[0342] 8.Yixiang,X.,To'A,S,G.,Ningyan,Z.,et al.,(2019)T-cell receptor mimic(TCRm)antibody therapeutics against intracellular proteins.Antibody Therapeutics 2(1):22-32.

[0343] 9.Horig,H.,Lee,C,S.,Kaufman,H,L.(2002)Prostate-specific antigen vaccines for prostate cancer.Expert Opin Biol Ther2(4):395-408.

[0344] 10.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.

[0345] 11.Butterfield LH,Meng WS,Koh A,Vollmer CM,Ribas A,Dissette VB,Faull K,Glaspy JA,McBride WH,Economou JS.T cell responses to HLA-A*0201-restricted peptides derived from human alpha fetoprotein.J Immunol.2001Apr 15;166(8):5300-8.

[0346] 12.Pichard V,Royer PJ,Richou C,Cauchin E,Goebes K,Gaignerie A,Masliah C,Gournay J,Gregoire M,Ferry N.Detection,isolation,and characterization of alpha-fetoprotein-specific T cell populations and clones using MHC class I multimer magnetic sorting.J Immunother.2008 Apr;31(3):246-53.

[0347] 13.A.Margaret Merchant et al.An efficient route to human bispecific IgG.Nature Biotechnology,Volume 16,1998.

[0348] 14.Kabat.Sequences of Proteins of Immunological Interest.National Institutes of Health,Bethesda,Md.(1991).

[0349] 15.A1-Lazikani et al.J.Mol.Biol.273:927-948(1997).

[0350] 16.Martin et al.Proc.Natl.Acad.Sci.USA 86:9268-9272(1989).

[0351] 17.Kotsiou E,Brzostek J,Lenart I,Antoniou AN,Dyson J,Gould KG.Dimerization of soluble disulfide trap single-chain major histocompatibility complex class I molecules dependent on peptide binding affinity.Antioxid Redox Signal.2011Aug 1;15(3):635-44.

[0352] 18. E, Poignavent V, Vincke C, Ritzenthaler C, Muyldermans S, Monsion B. Construction of High-Quality Camel Immune Antibody Libraries. Methods Mol Biol. 2018; 1701:169-187.

[0353] 19. Chinese Invention Patent Application No. 201510097117.0

[0354] 20. Phage Display: A Universal Laboratory Guide / Edited by Clackson, T., and Lowman, HB.; Translated by Ma Lan et al. Chemical Industry Press, May 2008.

[0355] 21.Tan,P.,Mitchell,D,A.,Buss,T,N.,et al.(2022)"Superhumanized"antibodies:reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences:application to an anti-CD28.J Immunol 169(2):1119-1125.

[0356] 22.US005821337A.

[0357] All publications and patent documents referenced in this specification are incorporated herein by reference as if each publication or patent were expressly indicated to be incorporated herein by reference individually. Various changes and equivalent substitutions may be made to the embodiments disclosed in this application without departing from the true spirit and scope of the disclosure. Unless the context otherwise requires, any feature, step, or embodiment of the embodiments disclosed herein may be used in combination with any other feature, step, or embodiment.

Claims

1. A bispecific antibody comprising a first antigen-binding fragment that binds to the HLA-AO2 / AFP complex and a second antigen-binding fragment that binds to activated T-cell antigens; Preferably, the binding epitope of the first antigen-binding fragment to the HLA-AO2 / AFP complex includes one or more residues from positions 158-166 (SEQ ID NO:1) of the AFP shown in SEQ ID NO:58; more preferably, the binding epitope of the first antigen-binding fragment to the HLA-AO2 / AFP complex includes at least one of residues from positions 159, 160, 162, 164, and 165 of the AFP shown in SEQ ID NO:58; and / or Preferably, the activating T-cell antigen is a CD3 molecule.

2. The bispecific antibody as described in claim 1, wherein... The first antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:18, HCDR2 as shown in SEQ ID NO:19, and HCDR3 as shown in SEQ ID NO:20; preferably, the first antigen-binding fragment is in the form of a single-domain antibody; more preferably, the first antigen-binding fragment comprises a monovalent or multivalent single-domain antibody binding the HLA-AO2 / AFP complex; and / or The second antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:60, HCDR2 as shown in SEQ ID NO:61, HCDR3 as shown in SEQ ID NO:62, LCDR1 as shown in SEQ ID NO:64, LCDR2 as shown in SEQ ID NO:65, and LCDR3 as shown in SEQ ID NO:66; 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 scFv; in, The amino acid sequences of HCDR and LCDR are defined according to Kabat.

3. The bispecific antibody according to any one of claims 1-2, wherein The first antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:17 or 27; and / or The second antigen-binding fragment comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:59 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:63; Optionally, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:17, and the second antigen-binding fragment comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:59 and a light chain variable region with the amino acid sequence shown in SEQ ID NO:63; or Optionally, the first antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:27, and the second antigen-binding fragment comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:59 and a light chain variable region with the amino acid sequence shown in SEQ ID NO:

63.

4. The bispecific antibody according to any one of claims 1-3, wherein The first antigen-binding fragment and the second antigen-binding fragment are linked by an Fc fragment in the constant region of the antibody heavy chain, wherein the Fc fragment includes a first Fc fragment and a second Fc fragment; preferably, the Fc fragment in the constant region of the antibody heavy chain is an Fc fragment of the IgG1 subtype; more preferably, the Fc fragment in the constant region of the antibody heavy chain is an Fc fragment of the IgG1m3 subtype; wherein The first Fc fragment comprises amino acid C at position 354 and amino acid W at position 366, or the first Fc fragment comprises amino acid C at position 349, amino acid S at position 366, amino acid A at position 368, and amino acid V at position 407; and the second Fc fragment comprises amino acid C at position 354 and amino acid W at position 366, or the second Fc fragment comprises amino acid C at position 349, amino acid S at position 366, amino acid A at position 368, and amino acid V at position 407; preferably, the first Fc fragment comprises amino acid C at position 354 and amino acid W at position 366, and the second Fc fragment comprises amino acid C at position 349, amino acid S at position 366, amino acid A at position 368, and amino acid V at position 407; and / or The first Fc fragment and the second Fc fragment contain amino acid F at position 234, amino acid E at position 235, and amino acid S at position 331; 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 in the antibody constant region are determined according to EU numbering.

5. The bispecific antibody according to any one of claims 1-4, comprising a first arm binding to the HLA-AO2 / AFP complex and a second arm binding to an activated T-cell antigen, wherein... The first arm comprises an amino acid sequence as shown in SEQ ID NO:31 or 32; and The second arm contains an amino acid sequence as shown in SEQ ID NO:

33.

6. A single-domain antibody that binds to the HLA-A02 / AFP complex, which contains HCDR1, as shown in SEQ ID NO:18, HCDR2, as shown in SEQ ID NO:19, and HCDR3, as shown in SEQ ID NO:20; Preferably, the single-domain antibody comprises an amino acid sequence as shown in SEQ ID NO:17 or 27; in, The amino acid sequence of HCDR is defined according to Kabat.

7. A nucleic acid molecule encoding a bispecific antibody as described in any one of claims 1-5 or a single-domain antibody as described in claim 6.

8. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-5 or the single-domain antibody of claim 6, and a pharmaceutically acceptable excipient, diluent, or carrier.

9. Use of the bispecific antibody of any one of claims 1-5, the single-domain antibody of claim 6, the nucleic acid molecule of claim 7, or the pharmaceutical composition of claim 8 in the preparation of a medicament for the prevention or treatment of HLA-AO2 / AFP-positive tumors; preferably, the HLA-AO2 / AFP-positive tumors are selected from: hepatocellular carcinoma and gastric cancer.

10. The bispecific antibody of any one of claims 1-5, the single-domain antibody of claim 6, the nucleic acid molecule of claim 7, or the pharmaceutical composition of claim 8, for the prevention or treatment of HLA-AO2 / AFP positive tumors; Preferably, the HLA-A02 / AFP positive tumor is selected from hepatocellular carcinoma and gastric cancer.

11. A method for preventing or treating HLA-AO2 / AFP-positive tumors, the method comprising administering to an individual in need an effective amount of any one of the bispecific antibodies of claims 1-5, the single-domain antibody of claim 6, the nucleic acid molecule of claim 7, or the pharmaceutical composition of claim 8; Preferably, the HLA-A02 / AFP positive tumor is selected from hepatocellular carcinoma and gastric cancer.

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