Bispecific antibodies and their use

Abstract

The application belongs to the field of antibodies, and particularly relates to a bispecific antibody and application thereof. The bispecific antibody comprises: a first antigen binding region having HLA-G binding activity; and a second antigen binding region having CD3 binding activity; wherein the first antigen binding region comprises an anti-HLA-G antibody, and the anti-HLA-G antibody comprises a heavy chain complementarity determining region (HCDR) comprising an amino acid sequence selected from at least one of SEQ ID NO: 1-3 or a conservatively modified form thereof. The bispecific antibody can bind to CD3 and HLA-G, thereby promoting T cell activation, proliferation and secretion of cytokines, effectively promoting PBMC killing of tumor cells, and having good anticancer activity. Moreover, the bispecific antibody has low binding activity to T cells and high binding activity to tumor cells, and has good clinical application value and drug development value.

Description

Technical Field

[0001] This application belongs to the field of antibodies, specifically relating to bispecific antibodies and their applications, and more specifically to bispecific antibodies and their preparation methods, nucleic acid molecules, expression vectors, recombinant cells, pharmaceutical compositions, pharmaceutical uses, and reagent kits. Background Technology

[0002] Besides surgical resection, traditional cancer treatments such as chemotherapy and radiotherapy have significant side effects and a high recurrence rate. In recent years, immunotherapy, including tumor-targeting antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hotspot and a source of new hope in the fight against cancer. Immunotherapy, represented by PD-1 / L1, has shown great potential. However, even PD-1 / L1 therapy, which currently has the broadest approved indications, has an overall response rate of only 30%, meaning that many more patients cannot benefit from it. One major reason is that immune checkpoint therapy is ineffective against "cold tumors." T cells recognize neoantigens, which are antigens of tumor gene mutations, through their surface T cell receptors (TCRs). Some tumors have a low frequency of gene mutations and few types of neoantigens, and are called "cold tumors." Current immune checkpoint therapies, such as PD-1 / L1 therapy, achieve their anti-cancer purpose by restoring the function of T cells themselves. However, in "cold tumors," T cells cannot effectively recognize the tumor, resulting in the ineffectiveness of immune checkpoint therapy against "cold tumors."

[0003] CD3-based bispecific antibodies (hereinafter referred to as "CD3 bispecific antibodies") recruit T cells to reach the tumor site, bridging T cells and the tumor, promoting T cell activation and tumor killing. These bispecific antibodies do not require neoantigens and can guide T cells to kill "cold tumors." After binding to T cells and tumor cells, CD3 bispecific antibodies trigger strong activation signals, thus to some extent "ignoring" the inhibitory signals of immune checkpoint molecules. However, CD3 bispecific antibodies also promote the production of large amounts of pro-inflammatory cytokines, such as TNFα and IL-6, triggering a strong cytokine storm and excessive immune response, causing damage to the body, and in severe cases, potentially life-threatening. Therefore, CD3-based bispecific antibodies have promising clinical applications, but their safety needs further improvement.

[0004] One approach to addressing the safety concerns of CD3 bispecific antibodies is to enhance their affinity for tumor targets while simultaneously reducing their binding activity to T cells. This allows for greater local distribution of the CD3 bispecific antibody drug to the tumor, increasing local drug concentration, decreasing peripheral drug concentration, reducing off-target toxicity, lowering pro-inflammatory cytokine production levels, and reducing on-target toxicity. Therefore, there is an urgent clinical need to develop CD3 bispecific antibodies with improved safety and higher clinical application value.

[0005] HLA-G is a type I transmembrane protein with three domains in its extracellular domain. Together with β-microglobulin, it forms a non-classical MHC-I protein that presents antigenic peptides. HLA-G is not expressed in normal tissues but is highly expressed in various tumors, including renal cell carcinoma, ovarian cancer, and breast cancer, exhibiting high tumor specificity.

[0006] Currently, there is a lack of highly safe bispecific antibodies against CD3 and HLA-G on the market. Therefore, developing a CD3×HLA-G bispecific antibody is of great value. Summary of the Invention

[0007] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a highly safe bispecific antibody targeting CD3 and HLA-G. Experimental verification has shown that this bispecific antibody exhibits weak binding to T cells, strong binding to tumor cells, high cytotoxic activity, better anticancer activity, and higher safety, demonstrating good clinical application and drug development value.

[0008] Specifically, this application provides the following technical solution:

[0009] In a first aspect, this application proposes a bispecific antibody. According to embodiments of this application, the bispecific antibody comprises: a first antigen-binding region having HLA-G binding activity; and a second antigen-binding region having CD3 binding activity; wherein the first antigen-binding region comprises an anti-HLA-G antibody, the anti-HLA-G antibody comprising a heavy chain complementarity-determining region (HCDR), the HCDR containing an amino acid sequence selected from at least one of SEQ ID NO: 1-3 or its conserved modified form. In some examples of this application, the bispecific antibody can bind to CD3 and HLA-G, thereby promoting T cell activation, proliferation, and cytokine secretion, effectively promoting PBMC killing of tumor cells, and exhibiting good anti-cancer activity; moreover, this bispecific antibody has low binding activity with T cells and high binding activity with tumor cells, possessing good clinical application value and drug development value.

[0010] In a second aspect of this application, a nucleic acid molecule is proposed. According to embodiments of this application, the nucleic acid molecule encodes the bispecific antibody described in the first aspect of this application. In some examples of this application, the bispecific antibody encoded by the nucleic acid molecule can bind to CD3 and HLA-G with high affinity, thereby promoting T cell activation, proliferation, and cytokine secretion, effectively promoting PBMC killing of tumor cells, and exhibiting good anti-cancer activity; moreover, this bispecific antibody has low binding activity with T cells but high binding activity with tumor cells, possessing good clinical application and drug development value.

[0011] In a third aspect of this application, an expression vector is provided. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the second aspect of this application. In some examples of this application, the expression vector can efficiently express the bispecific antibody in suitable host cells.

[0012] In a fourth aspect of this application, a method for preparing the bispecific antibody described in the first aspect is provided. According to embodiments of this application, the method includes: introducing the expression vector described in the third aspect of this application into cells; culturing the cells under conditions suitable for protein expression and secretion to obtain the bispecific antibody. In some examples of this application, the bispecific antibody prepared by this method can bind to CD3 and HLA-G with high affinity, thereby promoting T cell activation, proliferation, and cytokine secretion, effectively promoting PBMC killing of tumor cells, and exhibiting good anti-cancer activity; moreover, this bispecific antibody has low binding activity with T cells but high binding activity with tumor cells.

[0013] In a fifth aspect of this application, a recombinant cell is provided. According to embodiments of this application, the recombinant cell carries the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, or the expression vector described in the third aspect. The recombinant cell is obtained by transfection or transformation of the expression vector. In some examples of this application, the cell can efficiently express the aforementioned bispecific antibody under suitable conditions.

[0014] In a sixth aspect of this application, a pharmaceutical composition is proposed. According to embodiments of this application, the pharmaceutical composition comprises: the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fifth aspect. In some examples of this application, the bispecific antibody can effectively promote PBMC killing of tumor cells, exhibiting better anti-cancer activity; it has low binding activity to T cells, resulting in higher safety and good clinical application and drug development value. Therefore, the obtained drug can be further used for the prevention and / or treatment of CD3 and / or HLA-G mediated diseases.

[0015] In a seventh aspect of this application, the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a drug for the treatment or prevention of tumors. In some examples of this application, the bispecific antibody can specifically bind to CD3 and HLA-G, thereby promoting T cell activation, proliferation, and cytokine secretion, effectively promoting PBMC killing of tumor cells. Drugs containing the bispecific antibody and a series of other substances also have significant therapeutic or preventative effects against tumors.

[0016] In an eighth aspect of this application, the application discloses the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cells described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a kit for detecting CD3 and / or HLA-G. Those skilled in the art will understand that the features and advantages of the bispecific antibody described above also apply to this use, and will not be repeated here.

[0017] In a ninth aspect of this application, a kit is provided. According to embodiments of this application, the kit comprises the bispecific antibody described in the first aspect. In some examples of this application, the bispecific antibody can bind with high affinity to CD3 and HLA-G, thereby promoting T cell activation, proliferation, and cytokine secretion, effectively promoting PBMC killing of tumor cells. Therefore, a kit containing the bispecific antibody can be used to detect CD3 and / or HLA-G proteins. The kit can be used for scientific research, such as qualitative or quantitative detection of CD3 and HLA-G proteins in biological samples, and can also be used to assess individual status, such as determining whether an individual's CD3 and HLA-G protein levels are higher or lower than normal levels after obtaining the individual's CD3 and HLA-G protein levels.

[0018] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1This is a schematic diagram of the CD3×HLA-G bispecific antibody structure provided according to an embodiment of this application;

[0021] Figure 2 This is a schematic diagram of the flow cytometry results of the binding of CD3×HLA-G bispecific antibody to human peripheral blood CD8 T cells according to the embodiments of this application;

[0022] Figure 3 This is a schematic diagram of the flow cytometry results of the binding of CD3×HLA-G bispecific antibody to JEG3 tumor cells according to the embodiments of this application;

[0023] Figure 4 This is a schematic diagram of the flow cytometry results of the binding of CD3×HLA-G bispecific antibody to SKOV3-HLA-G tumor cells according to the embodiments of this application;

[0024] Figure 5 This is a schematic diagram showing the results of CD3×HLA-G bispecific antibody promoting CD4 T cell activation and expression of CD69 and CD25 according to the embodiments of this application;

[0025] Figure 6 This is a schematic diagram showing the results of CD3×HLA-G bispecific antibody promoting CD8 T cell activation and expression of CD69 and CD25 according to the embodiments of this application;

[0026] Figure 7 This is a schematic diagram illustrating the results of CD3×HLA-G bispecific antibody promoting CD4 T cell proliferation according to the embodiments of this application;

[0027] Figure 8 This is a schematic diagram showing the results of CD3×HLA-G bispecific antibody promoting CD8 T cell proliferation according to the embodiments of this application;

[0028] Figure 9 This is a schematic diagram showing the results of CD3×HLA-G bispecific antibody promoting PBMC secretion of IL-2 and IFN-γ cytokines according to the embodiments of this application;

[0029] Figure 10 This is a schematic diagram showing the results of CD3×HLA-G bispecific antibody promoting PBMC killing SKOV3-HLA-G tumor cells according to the embodiments of this application;

[0030] Figure 11 This is a schematic diagram showing the results of CD3×HLA-G bispecific antibody promoting PBMC killing JEG3 tumor cells according to the embodiments of this application;

[0031] Figure 12This is a schematic diagram showing the efficacy of the CD3×HLA-G bispecific antibody provided in the embodiments of this application in a mouse tumor model. Detailed Implementation

[0032] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0033] In this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0034] In this application, the term "antibody" generally refers to an antibody that recognizes one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, triple-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854 4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy-chain, full-length light-chain, or intact immunoglobulin molecules; or the immunologically active portion of any of these polypeptides, i.e., a molecule or portion thereof containing an antigen-binding site that specifically binds to a target antigen of interest, such targets including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases.

[0035] In this application, certain regions within the variable region exhibit a higher degree of variation in amino acid composition and sequence, termed "hypervariable region (HVR)." The hypervariable region is the location where antigens and antibodies bind, and is therefore also called the complementarity-determining region (CDR). Both the heavy chain and light chain variable regions contain three CDR regions. For example, these typically include amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., amino acid residues near 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)).

[0036] In this application, the term "anti-CD3 antibody" refers to an antibody capable of binding to CD3. Such an antibody is also referred to herein as a "CD3-binding antibody." The term "anti-HLA-G antibody" refers to an antibody capable of binding to HLA-G. Such an antibody is also referred to herein as an "HLA-G-binding antibody."

[0037] In this document, the term "antigen-binding fragment" is equivalent to "antibody fragment" or "antigen-binding antibody fragment," and can include a portion of a complete antibody, generally an antigen-binding region or variable region. This includes, but is not limited to: Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, scFv-Fc fragments, or bispecific antibodies (BsAbs), linear antibodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol ("PEGylated," "PEGylated") (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol).

[0038] In this article, the term "humanized antibody" refers to a recombinant antibody obtained by replacing all non-CDR (Fv backbone (FR)) amino acid sequences in the constant and variable regions of a monoclonal antibody from one species (e.g., mouse) with the non-CDR amino acid sequences in the constant and variable regions of an antibody from another species (e.g., human). That is, when the constant region of an antibody is humanized, it is called a chimeric antibody, and when all non-CDR amino acid sequences in both the constant and variable regions are humanized, it is called a humanized antibody. The humanization method can be performed using conventional antibody engineering techniques, and will not be elaborated upon here.

[0039] In this article, the term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of the constant region of a monoclonal antibody from one species (such as a mouse) with the constant region of an antibody from another species (such as a human).

[0040] In this application, the amino acid sequences of the listed CDRs are all as shown in the IMGT definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as the Kabat rule, the Chothia rule, etc. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described in any of the known schemes described above. Although the scope of protection claimed in this application is based on the sequences shown in the IMGT definition rules, amino acid sequences corresponding to other CDR definition rules should also be included in the scope of protection of this application.

[0041] In this application, the term "amino acid" is represented by a single-letter or three-letter code, with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Proline (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Valine (valine).

[0042] For nucleotides, the terms "homology," "identity," or "similarity" are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of "sequence homology" between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings and known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0 967 284, EP 1 085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0043] For peptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0044] In this application, without substantially affecting antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain sequences of the antibody or its functional fragments. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences described in this application can have at least 80% identity (or homology) with the reference sequences, meaning at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with each reference sequence. The sequence consistency described in this application can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this invention are shown from the N-terminus to the C-terminus.

[0045] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0046] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0047] In this article, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0048] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0049] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, conjugates, or pharmaceutical compositions of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0050] In this document, the term "treatment" means used to refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0051] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0052] The amino acid sequences involved in this application are shown in Table 1.

[0053] This application discloses a bispecific antibody targeting CD3 and HLA-G, an expression vector, a method for preparing the bispecific antibody, recombinant cells, a pharmaceutical composition, pharmaceutical uses, and a kit, which will be described in detail below.

[0054] Bispecific antibodies

[0055] In a first aspect, this application provides a bispecific antibody. According to an embodiment of this application, the bispecific antibody comprises: a first antigen-binding region having HLA-G binding activity; and a second antigen-binding region having CD3 binding activity; wherein the first antigen-binding region comprises an anti-HLA-G antibody, the anti-HLA-G antibody comprising a heavy chain complementarity-determining region (HCDR), the HCDR containing an amino acid sequence selected from at least one of SEQ ID NO: 1 to 3 or a conserved modified form thereof.

[0056] The bispecific antibody of this application can promote T cell activation, proliferation and secretion of cytokines, effectively promote PBMC killing of tumor cells and has good anti-cancer activity; moreover, the bispecific antibody has low binding activity with T cells and high binding activity with tumor cells, which has good clinical application value and drug development value.

[0057] In some examples of this application, the HCDR of the anti-HLA-G antibody includes: HCDR1, HCDR2, and HCDR3, wherein HCDR1 contains an amino acid sequence as shown in SEQ ID NO:1 or having at least 80% homology with SEQ ID NO:1, HCDR2 contains an amino acid sequence as shown in SEQ ID NO:2 or having at least 80% homology with SEQ ID NO:2, and HCDR3 contains an amino acid sequence as shown in SEQ ID NO:3 or having at least 80% homology with SEQ ID NO:3. Anti-HLA-G antibodies based on the aforementioned HCDR sequences exhibit high binding affinity and specificity to HLA-G.

[0058] In some preferred embodiments of this application, the HCDR includes HCDR1, HCDR2, and HCDR3, respectively, with amino acid sequences as shown in SEQ ID NO:1–3. Experimental verification has shown that anti-HLA-G antibodies having amino acid sequences as shown in SEQ ID NO:1–3 exhibit high binding affinity and specificity to HLA-G.

[0059] In some examples of this application, the anti-HLA-G antibody further comprises a light chain complementarity-determining region (LCDR), wherein the LCDR comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 contains an amino acid sequence as shown in SEQ ID NO:4 or having at least 80% homology with SEQ ID NO:4, LCDR2 contains an amino acid sequence as shown in SEQ ID NO:5 or having at least 80% homology with SEQ ID NO:5, and LCDR3 contains an amino acid sequence as shown in SEQ ID NO:6 or having at least 80% homology with SEQ ID NO:6. Anti-HLA-G antibodies based on the aforementioned LCDR sequences exhibit high binding affinity and specificity to HLA-G.

[0060] In some preferred embodiments of this application, the LCDR comprises LCDR1, LCDR2, and LCDR3, respectively, having amino acid sequences as shown in SEQ ID NO:4–6. Experimental verification has shown that anti-HLA-G antibodies having amino acid sequences as shown in SEQ ID NO:4–6 exhibit high binding affinity and specificity to HLA-G.

[0061] It should be noted that one or more amino acid residues in the aforementioned anti-HLA-G antibody HCDR and / or LCDR can be replaced by other amino acid residues from the same side chain family, and the retention function of the modified antibody can be tested using the functional assay methods described herein. Preferably, the number of conserved modifications does not exceed one or two.

[0062] In some examples of this application, the anti-HLA-G antibody further includes a heavy chain framework region.

[0063] In some examples of this application, the anti-HLA-G antibody further includes a light chain framework region.

[0064] In some examples of this application, at least a portion of the aforementioned heavy chain framework region and light chain framework region are independently derived from at least one of rabbit-derived antibodies, mouse-derived antibodies, sheep-derived antibodies, goat-derived antibodies, primate-derived antibodies, or mutants thereof.

[0065] In some preferred embodiments of this application, the heavy chain variable region of the anti-HLA-G antibody comprises an amino acid sequence selected from or having at least 80% homology with SEQ ID NO:7. In some more preferred embodiments of this application, the amino acid sequence of the heavy chain variable region of the anti-HLA-G antibody is as shown in SEQ ID NO:7.

[0066] In some preferred embodiments of this application, the light chain variable region of the anti-HLA-G antibody comprises an amino acid sequence as shown in SEQ ID NO:8 or having at least 80% homology with SEQ ID NO:8. In some more preferred embodiments of this application, the amino acid sequence of the light chain variable region of the anti-HLA-G antibody is as shown in SEQ ID NO:8.

[0067] Bispecific antibodies based on the aforementioned heavy chain variable region and light chain variable region sequences exhibit high affinity and specificity for HLA-G binding.

[0068] In some examples of this application, the second antigen-binding region includes an anti-CD3 antibody, the anti-CD3 antibody including a heavy chain complementarity-determining region (HCDR), and the HCDR of the anti-CD3 antibody containing an amino acid sequence selected from at least one of SEQ ID NO: 9 to 11 or a conserved modified form thereof.

[0069] In some examples of this application, the HCDR includes: HCDR1, HCDR2, and HCDR3, wherein: HCDR1 contains an amino acid sequence as shown in SEQ ID NO:9 or having at least 80% homology with SEQ ID NO:9; HCDR2 contains an amino acid sequence as shown in SEQ ID NO:10 or having at least 80% homology with SEQ ID NO:10; and HCDR3 contains an amino acid sequence as shown in SEQ ID NO:11 or having at least 80% homology with SEQ ID NO:11. Anti-CD3 antibodies based on the aforementioned HCDR sequences exhibit high binding affinity and specificity to CD3.

[0070] In some preferred embodiments of this application, the HCDR includes HCDR1, HCDR2, and HCDR3, respectively, with amino acid sequences as shown in SEQ ID NO:9-11. Experimental verification has shown that anti-CD3 antibodies having amino acid sequences as shown in SEQ ID NO:9-11 exhibit high binding affinity and specificity to CD3.

[0071] In some examples of this application, the anti-CD3 antibody further comprises a light chain complementarity-determining region (LCDR), wherein the LCDR comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 contains an amino acid sequence as shown in SEQ ID NO:12 or having at least 80% homology with SEQ ID NO:12, LCDR2 contains an amino acid sequence as shown in SEQ ID NO:13 or having at least 80% homology with SEQ ID NO:13, and LCDR3 contains an amino acid sequence as shown in SEQ ID NO:14 or having at least 80% homology with SEQ ID NO:14. Anti-CD3 antibodies based on the aforementioned LCDR sequences exhibit high binding affinity and specificity to CD3.

[0072] In some preferred embodiments of this application, the LCDR comprises LCDR1, LCDR2, and LCDR3, respectively, having amino acid sequences as shown in SEQ ID NO:12-14. Experimental verification has shown that anti-CD3 antibodies having amino acid sequences as shown in SEQ ID NO:12-14 exhibit high binding affinity and specificity to CD3.

[0073] In some examples of this application, the anti-CD3 antibody further includes a heavy chain framework region.

[0074] In some examples of this application, the anti-CD3 antibody further includes a light chain framework region.

[0075] In some preferred embodiments of this application, at least a portion of the heavy chain framework region and the light chain framework region are independently derived from at least one of rabbit-derived antibodies, mouse-derived antibodies, sheep-derived antibodies, goat-derived antibodies, primate-derived antibodies, or mutants thereof.

[0076] In some preferred embodiments of this application, the heavy chain variable region of the anti-CD3 antibody comprises an amino acid sequence selected from or having at least 80% homology with SEQ ID NO:15. In some more preferred embodiments of this application, the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody is as shown in SEQ ID NO:15.

[0077] In some preferred embodiments of this application, the light chain variable region of the anti-CD3 antibody comprises an amino acid sequence as shown in SEQ ID NO:16 or having at least 80% homology with SEQ ID NO:16. In some more preferred embodiments of this application, the amino acid sequence of the light chain variable region of the anti-CD3 antibody is as shown in SEQ ID NO:16.

[0078] Bispecific antibodies based on the above heavy chain variable region and light chain variable region sequences exhibit high binding affinity and specificity for CD3.

[0079] In some examples of this application, the first antigen-binding region includes a first anti-HLA-G antibody Fab fragment, which includes an anti-HLA-G antibody heavy chain variable region, an HLA-G antibody light chain variable region, a CL fragment, and a CH1 fragment. The CL fragment and the CH1 fragment are linked by disulfide bonds. The C-terminus of the anti-HLA-G antibody heavy chain variable region is linked to the N-terminus of the CH1 fragment, and the C-terminus of the anti-HLA-G antibody light chain variable region is linked to the N-terminus of the CL fragment.

[0080] In some examples of this application, the first antigen-binding region further includes a first Fc fragment.

[0081] In some examples of this application, the C-terminus of the variable region of the anti-HLA-G antibody heavy chain is connected to the N-terminus of the CH1 fragment, the C-terminus of the variable region of the anti-HLA-G antibody light chain is connected to the N-terminus of the CL fragment, and the C-terminus of the CH1 fragment is connected to the N-terminus of the first Fc fragment.

[0082] In some examples of this application, the heavy chain constant region of the anti-HLA-G antibody includes at least one selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; and / or, the light chain constant region includes a light chain constant region selected from κ-type or λ-type. In some specific embodiments, the heavy chain constant region includes human IgG, IgA, IgM, IgE, or IgD, such as human IgG1.

[0083] In other specific embodiments, the heavy chain constant region includes a selection from mouse IgG, IgA, IgM, IgE, or IgD, such as mouse IgG1.

[0084] It should be noted that, in order to further improve the bioacceptability of the antibody, the antibody can also be humanized, that is, the antibody is a chimeric antibody or a humanized antibody.

[0085] In some examples of this application, the heavy chain variable region and the light chain variable region of the anti-CD3 antibody are linked by a linker peptide. In some preferred examples of this application, the anti-CD3 antibody is a single-chain antibody.

[0086] In some examples of this application, the C-terminus of the anti-CD3 antibody heavy chain variable region is connected to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is connected to the N-terminus of the light chain variable region; or the C-terminus of the anti-CD3 antibody light chain variable region is connected to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is connected to the N-terminus of the heavy chain variable region.

[0087] In some examples of this application, the linker peptide has an amino acid sequence as shown in SEQ ID NO:17.

[0088] In some examples of this application, the second antigen-binding region further includes a second Fc fragment.

[0089] In some examples of this application, the anti-CD3 antibody is linked to the second Fc fragment.

[0090] In some examples of this application, the C-terminus of the heavy chain variable region of the anti-CD3 antibody is connected to the N-terminus of the linker peptide, the C-terminus of the linker peptide is connected to the N-terminus of the light chain variable region, and the C-terminus of the light chain variable region is connected to the N-terminus of the second Fc region; or the C-terminus of the light chain variable region of the anti-CD3 antibody is connected to the N-terminus of the linker peptide, the C-terminus of the linker peptide is connected to the N-terminus of the heavy chain variable region, and the C-terminus of the heavy chain variable region is connected to the N-terminus of the second Fc region.

[0091] In some examples of this application, the first antigen-binding region and the second antigen-binding region are connected by a knob-in-hole structure. In a specific example of this application, the aforementioned bispecific antibody structure is as follows: Figure 1 As shown in Figure A.

[0092] In some examples of this application, the second antigen-binding region further includes a second anti-HLA-G antibody Fab fragment, which includes an anti-HLA-G antibody heavy chain variable region, an HLA-G antibody light chain variable region, a CL fragment, and a CH1 fragment. The CL fragment and the CH1 fragment are linked by disulfide bonds. The C-terminus of the anti-HLA-G antibody heavy chain variable region is linked to the N-terminus of the CH1 fragment, and the C-terminus of the anti-HLA-G antibody light chain variable region is linked to the N-terminus of the CL fragment.

[0093] In some examples of this application, the aforementioned second anti-HLA-G antibody Fab fragment, the anti-CD3 antibody, and the second Fc fragment are linked. Specifically, the C-terminus of the anti-HLA-G antibody Fab fragment is linked to the N-terminus of the anti-CD3 antibody, and the C-terminus of the anti-CD3 antibody is linked to the N-terminus of the second Fc fragment. In a specific example of this application, the aforementioned bispecific antibody structure is as follows... Figure 1 As shown in B.

[0094] In some examples of this application, the first antigen-binding region of the bispecific antibody has the amino acid sequence shown in SEQ ID NO:19 and SEQ ID NO:20, and the second antigen-binding region of the bispecific antibody has the amino acid sequence shown in SEQ ID NO:21.

[0095] In some examples of this application, the first antigen-binding region of the bispecific antibody has the amino acid sequences shown in SEQ ID NO:19 and SEQ ID NO:20, and the second antigen-binding region of the bispecific antibody has the amino acid sequences shown in SEQ ID NO:18 and SEQ ID NO:20.

[0096] The bispecific antibodies obtained in any of the above embodiments can bind to CD3 and HLA-G, thereby promoting T cell activation, proliferation, and cytokine secretion, effectively promoting PBMC killing of tumor cells, exhibiting good anti-cancer activity and higher safety, and possessing practical clinical application value and drug development value.

[0097] Nucleic acid molecules

[0098] In another aspect of this application, a nucleic acid molecule is proposed that encodes the aforementioned bispecific antibody. In some examples of this application, the aforementioned nucleic acid molecule may encode a bispecific antibody that simultaneously targets CD3 and HLA-G.

[0099] In some examples of this application, the nucleic acid molecule is DNA.

[0100] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned in this specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed as well. In addition, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0101] expression carrier

[0102] In another aspect of this application, an expression vector is proposed. According to an embodiment of this application, the expression vector carries the aforementioned nucleic acid molecule. The aforementioned expression vector includes an optional controlled amino acid sequence, such that the controlled amino acid sequence is operatively linked to the nucleic acid molecule. The controlled amino acid sequence can direct the expression of one or more controlled amino acid sequences of the nucleic acid molecule in a host. The vector thus constructed can effectively express the aforementioned bispecific antibody.

[0103] It should be noted that when linking the aforementioned nucleic acid molecules to the vector, the nucleic acid molecules can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. These control elements can originate directly from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the connection between the nucleic acid molecules and the control elements must be operably established.

[0104] According to embodiments of the present invention, the vector may refer to a cloning vector or an expression vector, and can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in this invention is not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0105] In this document, the term "operably ligated" refers to ligating a foreign gene to a vector such that the control elements within the vector, such as amino acid sequences controlling transcription and amino acid sequences controlling translation, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include viral vectors, plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into suitable recipient cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of the proteins encoded by the nucleic acid molecules.

[0106] In some examples of this application, the vector is a eukaryotic vector or a prokaryotic vector.

[0107] In some examples of this application, the vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

[0108] Bispecific antibody preparation method

[0109] In another aspect of this application, a method for preparing the aforementioned bispecific antibody is proposed, comprising: introducing the aforementioned expression vector into cells; and culturing the cells under conditions suitable for protein expression and secretion to obtain the bispecific antibody. This method enables the large-scale expression of the aforementioned specific antibody in vitro.

[0110] In some examples of this application, the cells are eukaryotic cells.

[0111] Recombinant cells

[0112] In another aspect of this application, a recombinant cell carrying the aforementioned nucleic acid molecule or expression vector is proposed. In some examples of this application, the recombinant cell is obtained by transfecting or transforming the expression vector, and the recombinant cell can efficiently express the aforementioned bispecific antibody under suitable conditions.

[0113] In some examples of this application, the recombinant cells are prokaryotic cells, eukaryotic cells, or bacteriophages. It should be noted that the eukaryotic cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0114] In some examples of this application, the prokaryotic cells are Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0115] In some examples of this application, the eukaryotic cells are fungal, insect, plant, or mammalian cells.

[0116] In some examples of this application, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomyces cerevisiae, or Trichoderma.

[0117] In some examples of this application, the insect cells are grass armyworm cells; in some examples of this application, the plant cells are tobacco plant cells; in some examples of this application, the mammalian cells are BHK cells, CHO cells, COS cells, myeloma cells, or human embryonic kidney 293 cells; and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0118] In some examples of this application, the recombinant cells are mammalian cells. When the cells are mammalian cells, the expression efficiency of the antibody or its antigen-binding fragment is higher.

[0119] In some examples of this application, the recombinant cells are BHK cells, CHO cells, COS cells, or NSO cells.

[0120] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the bispecific antibody of this application. Those skilled in the art will readily understand that suitable conditions for bispecific antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for bispecific antibody expression based on the specific environment of their laboratory.

[0121] Pharmaceutical Composition

[0122] In another aspect of this application, a pharmaceutical composition is proposed, comprising: the aforementioned bispecific antibody, nucleic acid molecule, expression vector, or recombinant cells. In some examples of this application, the aforementioned pharmaceutical composition can effectively promote PBMC killing of tumor cells, exhibiting anticancer activity; it can produce fewer pro-inflammatory cytokines, resulting in higher safety and good clinical application and drug development value. Therefore, the obtained drug can be further used for the prevention and / or treatment of CD3 and / or HLA-G mediated diseases.

[0123] In some examples of this application, pharmaceutically acceptable excipients are further included.

[0124] In some examples of this application, the excipients include one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers.

[0125] In some examples of this application, the pharmaceutical composition is an injectable formulation.

[0126] It should be noted that the pharmaceutical composition includes combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the present invention. For example, the components contained in the pharmaceutical composition may be administered to the subject as a whole or separately. When the components contained in the pharmaceutical composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0127] The drug of this application contains a safe and effective amount of the active ingredient (bispecific antibody) of this application, as well as pharmaceutically acceptable excipients. These excipients include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage form of the drug of this application is an injection, an oral formulation (tablet, capsule, oral liquid), a transdermal formulation, or a sustained-release formulation. For example, it is prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The drug is preferably manufactured under aseptic conditions.

[0128] The effective amount of the active ingredient described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0129] Pharmaceutically acceptable excipients described in this application include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0130] Uses in drug preparation

[0131] In another aspect of this application, the use of the aforementioned bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells, or pharmaceutical compositions in the preparation of medicaments for the treatment or prevention of tumors is proposed. In some examples of this application, the bispecific antibodies and corresponding nucleic acid molecules, vectors, recombinant cells, or pharmaceutical compositions of this application can be further prepared into medicaments that can be clinically used for the prevention or treatment of CD3 and / or HLA-G mediated diseases.

[0132] In some examples of this application, the aforementioned tumors include at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0133] Uses in the preparation kit

[0134] In another aspect of this application, the application proposes the use of the aforementioned bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells or pharmaceutical compositions in the preparation of a kit for the detection of CD3 and / or HLA-G.

[0135] As previously described, the bispecific antibody of this application can specifically bind to CD3 and HLA-G, therefore, the bispecific antibody can be used to detect CD3 and / or HLA-G. Furthermore, it can be used to prepare CD3 and / or HLA-G related kits for scientific research, such as qualitative or quantitative detection of CD3 and / or HLA-G protein molecules in biological samples. More specifically, it can be used in kits involving the specific binding properties of CD3 and / or HLA-G and antibodies for detection, such as immunoblotting and immunoprecipitation. These kits may contain any one or more of the following: antagonists, the bispecific antibody of this invention, or pharmaceutical reference materials; protein purification columns; immunoglobulin affinity purification buffers; and cell assay diluents. The bispecific antibody of this application can be used for different types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, it can be used to test for CD3 and / or HLA-G-mediated diseases by detecting the serum or blood of a subject.

[0136] In another aspect of this application, a kit is provided comprising the aforementioned bispecific antibody. In some examples of this application, the aforementioned kit is capable of accurately detecting CD3 and HLA-G proteins in a sample.

[0137] In some examples of this application, the kit is used to detect at least one of CD3 and HLA-G.

[0138] Disease treatment methods

[0139] In another aspect of this application, a method for preventing and / or treating CD3 and / or HLA-G mediated diseases is provided. According to embodiments of this application, the method includes administering to a subject a pharmaceutically acceptable amount of a bispecific antibody, nucleic acid molecule, vector or transformant, recombinant cells, or pharmaceutical composition.

[0140] It should be noted that the terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one implementation, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects can be humans, but also include other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0141] The effective amount of the antibody or its antigen-binding fragment, conjugate, nucleic acid, carrier, transformant, or pharmaceutical composition described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0142] In some examples of this application, the HLA-G mediated disease is a tumor.

[0143] In some examples of this application, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0144] Table 1

[0145]

[0146]

[0147]

[0148]

[0149] Note: Figure 1 A bispecific antibody configuration consists of a first antigen-binding region (amino acid sequences shown in SEQ ID NO:19 and SEQ ID NO:20) and a second antigen-binding region (amino acid sequence shown in SEQ ID NO:21);

[0150] Figure 1 The B bispecific antibody configuration consists of a first antigen-binding region (amino acid sequences shown in SEQ ID NO:19 and SEQ ID NO:20) and a second antigen-binding region (amino acid sequences shown in SEQ ID NO:18 and SEQ ID NO:20).

[0151] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0152] Example 1 Antibody Preparation

[0153] The specific experimental procedures for antibody preparation are as follows:

[0154] 1. Culture ExpiCHO cells (purchased from Thermo Fisher) in ExpiCHO Expression Medium (purchased from Thermo Fisher) and adjust the cell concentration to 6 × 10⁶. 6 / mL, to obtain ExpiCHO cell solution;

[0155] 2. When the dual antibodies are in configuration A ( Figure 1 ), add the pcDNA3.4 vector (synthesized by Nanjing GenScript) containing CD3 antibody, HLA-G antibody heavy chain, and HLA-G antibody light chain to 2 mL of OptiSFM medium (purchased from Thermo Fisher) in a 1:1:1 ratio to obtain solution a; or, when the bispecific antibody is in configuration B ( Figure 1), add pcDNA3.4 vector (synthesized by Nanjing GenScript) containing HLA-G heavy chain-CD3 antibody, HLA-G antibody heavy chain and HLA-G antibody light chain to 2 mL OptiSFM medium (purchased from Thermo Fisher) in a ratio of 1:1:1 to obtain solution a;

[0156] 3. Add 160 μL of ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) to 2 mL of OptiSFM medium (purchased from Thermo Fisher) to obtain solution b;

[0157] 4. Then mix solution a and solution b to obtain the transfection mixture, and add the entire transfection mixture to 50 mL of ExpiCHO cell solution within 5 minutes;

[0158] 5. After culturing at 37℃ and 5% CO2 for 1 day, add 8mL Feed and 300μL Enhancer (purchased from Thermo Fisher), and then transfer to 32℃ and 5% CO2 for 9 days. Harvest the culture supernatant, adding 8mL Feed on the 5th day.

[0159] 6. The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from Nanomicro).

[0160] The amino acid sequences corresponding to different antibody configurations are shown in Tables 1 and 2.

[0161] Table 2. Structure and corresponding amino acid sequence of the bispecific antibody in Example 1.

[0162]

[0163] Note: See antibody configuration. Figure 1 .

[0164] Example 2: Bispecific antibody flow cytometry combined with experiment

[0165] Flow cytometry was used to detect the binding properties of the bispecific antibody prepared in Example 1. The antibody was added to the cells, and the strength of the signal after the antibody was added was used to determine the binding properties between the antibody and the cells.

[0166] (1) Dilute PBMC with PBS to 2×10 6 / ml, add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of goat serum, and block at 4℃ for 30min. Add serially diluted bispecific antibodies CD3×HLA-G 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:21, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20), CD3×HLA-G 2:1 (the amino acid sequence of the HLA-G heavy chain-CD3 single-chain antibody is shown in SEQ ID NO:18, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20), RG6353 (a CD3×HLA-G bispecific antibody developed by Roche, consisting of four chains, with sequences of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25), and control hIgG1LALA (purchased from Bio-Tech), and incubate at 4°C for 30 min. Add 1 ml of PBS to an EP tube, centrifuge at 3500 rpm for 5 min at 4°C, discard the supernatant, and wash once more with PBS. After centrifugation, discard the supernatant again, resuspend the cells in 100 μl / tube of PBS, add 1 μl / tube of Alexa-647-labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson Lab) and 0.5 μl / tube of PerCP-Cy5.5-labeled anti-human CD8 antibody, and incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze using flow cytometry.

[0167] The results are as follows Figure 2 As shown, CD3×HLA-G 1:1, CD3×HLA-G 2:1 and RG6353 can all bind to T cells, with CD3×HLA-G 2:1 having the weakest binding to T cells.

[0168] (2) Dilute JEG3 and SKOV3-HLA-G tumor cells to 2×10⁻⁶ with PBS. 6 / ml, add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of goat serum, and block at 4℃ for 30min. Add serially diluted CD3×HLA-G 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:21, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20), CD3×HLA-G 2:1 (the amino acid sequence of the HLA-G heavy chain-CD3 single-chain antibody is shown in SEQ ID NO:18, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20), RG6353 (a CD3×HLA-G bispecific antibody developed by Roche, consisting of four chains, with sequences of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25), and control hIgG1LALA (purchased from Bio-Tech), and incubate at 4°C for 30 min. Add 1 ml of PBS to an EP tube, centrifuge at 3500 rpm for 5 min at 4°C, discard the supernatant, and wash once more with PBS. After centrifugation, discard the supernatant again, resuspend the cells in 100 μl / tube of PBS, add 1 μl / tube of Alexa-647-labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson Lab), and incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze using flow cytometry.

[0169] The results are as follows Figure 3 As shown, CD3×HLA-G 1:1, CD3×HLA-G 2:1, and RG6353 can all bind to JEG3 cells, and their binding activities are similar.

[0170] The results are as follows Figure 4 As shown, both CD3×HLA-G 2:1 and RG6353 can bind to SKOV3-HLA-G cells, and their binding activities are similar.

[0171] Example 3: Bispecific antibody promotes T cell expression and activation

[0172] The bispecific antibody prepared in Example 1 was added to the co-incubation system of PBMC and JEG3 tumor cells. After culturing for 48 hours, the expression of CD25 and CD69 on the surface of CD4 T cells and CD8 T cells was detected by flow cytometry to determine the characteristics of T cell activation induced by the bispecific antibody.

[0173] (1) JEG3 cells were diluted to 1×10⁻⁶ using complete RPMI 1640 medium.5 Add / ml to a 96-well plate;

[0174] (2) Using complete RPMI 1640 medium, the following bispecific antibodies were prepared: CD3×HLA-G 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:21, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20), CD3×HLA-G 2:1 (the amino acid sequence of the HLA-G heavy chain-CD3 single-chain antibody is shown in SEQ ID NO:18, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20), and RG6353 (a CD3×HLA-G bispecific antibody developed by Roche, consisting of four chains, with sequences SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25). NO:25), control hIgG1LALA (purchased from Baiying Biotechnology) was serially diluted and added to a 96-well plate, 20 μl / well;

[0175] (3) PBMC (purchased from Selene Biotechnology) was diluted to 1.25 × 10⁻⁶ using complete RPMI 1640 medium. 6 Add 80 μl / well to a 96-well plate;

[0176] (4) Incubate the 96-well plate at 37°C in a 5% CO2 incubator for 72 hours;

[0177] (5) Add PerCP-Cy5.5 labeled CD8 antibody, BV605 labeled CD4 antibody, PE labeled CD25 antibody, and BV421 labeled CD69 antibody, and incubate at 4°C in the dark for 30 min.

[0178] (6) Wash twice with PBS, centrifuge and discard the supernatant.

[0179] (7) Resuspend the cells in 200 μl / tube of PBS and perform the analysis using a flow cytometer.

[0180] The results are as follows Figure 5 As shown, CD3×HLA-G 1:1, CD3×HLA-G 2:1, and RG6353 can all promote CD4 T cell activation and expression of CD69 and CD25.

[0181] The results are as follows Figure 6As shown, CD3×HLA-G 1:1, CD3×HLA-G 2:1, and RG6353 can all promote CD8 T cell activation and expression of CD69 and CD25, and the effective dose of the 2:1 bispecific antibody is lower than that of the 1:1 bispecific antibody.

[0182] Example 4: Bispecific antibodies promote T cell proliferation

[0183] PBMC cells were labeled with CFSE fluorescein, and the bispecific antibody prepared in Example 1 was added to the co-incubation system of PBMC and JEG3 tumor cells. After culturing for 72 hours, the CFSE fluorescence intensity of CD4 T cells and CD8 T cells was detected by flow cytometry to determine the characteristics of T cell proliferation induced by the bispecific antibody.

[0184] (1) JEG3 cells were diluted to 1×10⁻⁶ using complete RPMI 1640 medium. 5 Add / ml to a 96-well plate;

[0185] (2) Using complete RPMI 1640 medium, the following bispecific antibodies were prepared: CD3×HLA-G 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:21, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20), CD3×HLA-G 2:1 (the amino acid sequence of the HLA-G heavy chain-CD3 single-chain antibody is shown in SEQ ID NO:18, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20), and RG6353 (a CD3×HLA-G bispecific antibody developed by Roche, consisting of four chains, with sequences SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25). NO:25), control hIgG1LALA (purchased from Baiying Biotechnology) was serially diluted and added to a 96-well plate, 20 μl / well;

[0186] (3) PBMCs were labeled with 5 μM CFSE. After labeling, PBMCs (purchased from Saili Biotechnology) were diluted to 1.25 × 10⁶ / ml with complete RPMI 1640 medium and added to 96-well plates, 80 μl / well.

[0187] (4) Incubate the 96-well plate at 37°C in a 5% CO2 incubator for 72 hours;

[0188] (5) Add PerCP-Cy5.5 labeled CD8 antibody and BV605 labeled CD4 antibody, and incubate at 4°C in the dark for 30 min.

[0189] (6) Wash twice with PBS, centrifuge and discard the supernatant.

[0190] (7) Resuspend the cells in 200 μl / tube of PBS and perform the analysis using a flow cytometer.

[0191] The results are as follows Figure 7 As shown, CD3×HLA-G 1:1, CD3×HLA-G 2:1, and RG6353 can all promote CD4 T cell proliferation, and the 1:1 configuration promotes CD4 T cell proliferation more strongly than the 2:1 configuration. RG6353 promotes CD4 T cell proliferation most strongly.

[0192] The results are as follows Figure 8 As shown, CD3×HLA-G 1:1, CD3×HLA-G 2:1, and RG6353 can all promote CD8 T cell proliferation, but the effective dose of CD3×HLA-G 2:1 and RG6353 to promote CD8 T cell proliferation is lower than that of CD3×HLA-G 1:1.

[0193] Example 5: Bispecific antibody-induced PBMC cytokine secretion experiment

[0194] The bispecific antibody prepared in Example 1 was added to the co-incubation system of PBMC and JEG3 tumor cells. After culturing for 48 hours, the culture supernatant was collected, and the cytokine content in the supernatant was detected to determine the characteristics of the bispecific antibody in inducing cytokine release.

[0195] (a) JEG3 cells were diluted to 1×10⁻⁶ using complete RPMI 1640 medium. 5 Add / ml to a 96-well plate and incubate at 37°C in a 5% CO2 incubator for 48 hours;

[0196] (b) Using complete RPMI 1640 medium, the following bispecific antibodies were prepared: CD3×HLA-G 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:21, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20), CD3×HLA-G 2:1 (the amino acid sequence of the HLA-G heavy chain-CD3 single-chain antibody is shown in SEQ ID NO:18, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20), and RG6353 (a CD3×HLA-G bispecific antibody developed by Roche, consisting of four chains, with sequences SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25). NO:25), control hIgG1LALA (purchased from Baiying Biotechnology) was serially diluted and added to a 96-well plate, 20 μl / well;

[0197] (c) Dilute PBMC (purchased from Selene Biotechnology) to 1.25 × 10⁻⁶ using complete RPMI 1640 medium. 6 Add 80 μl / well to a 96-well plate;

[0198] (d) Incubate the 96-well plate at 37°C in a 5% CO2 incubator for 48 h;

[0199] (e) Centrifuge at 300g for 10 min at room temperature and collect the cell culture supernatant;

[0200] (f) The cytokine content in the supernatant was detected using the CBA kit (purchased from BD).

[0201] The results are as follows Figure 9 As shown, CD3×HLA-G 1:1, CD3×HLA-G 2:1, and RG6353 can all promote the secretion of immune-activating cytokines IL-2 and IFN-γ by PBMCs, and the effective dose of the 2:1 configuration is lower than that of the 1:1 configuration.

[0202] Example 6: Bispecific antibody promotes PBMC killing of tumor cells

[0203] This embodiment aims to test the ability of the bispecific antibody prepared in Example 1 to promote the killing of tumor cells by PBMCs.

[0204] (a) Add 50 μL of complete RPMI-1640 medium to a 16-well RTCA plate and calibrate it.

[0205] (b) Dilute JEG-3 and SKOV3-HLA-G tumor cells to 2 × 10⁻⁶ using complete RPMI-1640 medium. 5 / mL, at a volume of 50μL / well, were individually added to the RTCA plate obtained in step (1), and then the cell coefficient was detected for 24h at 37°C and 5% CO2 using an xCELLigence RTCAMP device;

[0206] (c) Using complete RPMI-1640 medium, serially diluted bispecific antibodies CD3×HLA-G 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:21, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20), CD3×HLA-G 2:1 (the amino acid sequence of the HLA-G heavy chain-CD3 single-chain antibody is shown in SEQ ID NO:18, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20), and RG6353 (a CD3×HLA-G bispecific antibody developed by Roche, consisting of four chains, with sequences of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25), were prepared. NO:25), control hIgG1LALA (purchased from Baiying Biotechnology), added to the RTCA plate obtained in step (2), with an addition volume of 20 μl / well;

[0207] (4) Dilute PBMC (purchased from Selene Biotechnology) to 6.25 × 10⁻⁶ using complete RPMI-1640 medium. 5 1.25 × 10⁻⁶ cells / ml or 1.25 × 10⁻⁶ cells / ml 6 Add 80 μl / well to the RTCA plate obtained in step (3);

[0208] (5) The reaction system obtained in step (4) was subjected to 37°C and 5% CO2 for 24 h using the xCELLigence RTCAMP device to detect the cell coefficient.

[0209] The results are as follows Figure 10 As shown, CD3×HLA-G 1:1, CD3×HLA-G 2:1, and RG6353 can all promote PBMC killing of SKOV3-HLA-G tumor cells, and the 2:1 conformation antibody is superior to the 1:1 conformation bispecific antibody.

[0210] The results are as follows Figure 11As shown, both CD3×HLA-G 2:1 and RG6353 can promote the killing of JEG3 tumor cells by PBMCs, and the two bispecific antibodies have similar killing activities.

[0211] Example 7: Validation of the antitumor effect of bispecific antibodies in a mouse model

[0212] In vivo efficacy experiments were used to detect the anticancer function of the bispecific antibody of this invention in promoting immune reconstitution in mice.

[0213] (1) On day -21, NSG mice were implanted with tumors subcutaneously on the right ventral side, and each mouse was injected with 5×10 6 One SKOV3-HLA-G tumor cell;

[0214] (2) On day -7, human PBMCs (purchased from Selvi Biotech) were transfused into NSG mice (purchased from Southern Model) via the tail vein at a dose of 5 × 10⁻⁶. 6 / Only;

[0215] (3) On day 0, the mice were weighed and the tumor volume was measured. Based on the weighing results and tumor volume, the mice were grouped.

[0216] (4) On days 0, 5, and 9, mice were injected via tail vein with the CD3×HLA-G 2:1 (the amino acid sequence of the HLA-G heavy chain-CD3 single chain antibody is shown in SEQ ID NO:18, the amino acid sequence of the HLA-G antibody heavy chain is shown in SEQ ID NO:19, and the amino acid sequence of the HLA-G antibody light chain is shown in SEQ ID NO:20) and the solvent control PBS, 250 μl per mouse;

[0217] (4) After the above-mentioned antibodies were injected, the tumor volume was measured twice a week and the mice were weighed.

[0218] The results are as follows Figure 12 As shown, CD3×HLA-G 2:1 has a significant anti-cancer effect, with tumor volume at the start of treatment being around 300 mmHg. 3 In conclusion, dual antibodies can still exert significant anti-cancer effects.

[0219] The above experimental results show that the bispecific antibody obtained in this application can bind to CD3 and HLA-G, thereby promoting T cell activation, proliferation, and cytokine secretion, effectively promoting PBMC killing of tumor cells, and exhibiting good anti-cancer activity. The bispecific antibody of this application has high tumor-binding activity, low T-cell-binding activity, higher anti-cancer activity, and higher safety. In summary, the bispecific antibody of this application can promote immune cell anti-cancer activity, has good anti-cancer activity, and higher safety, possessing good clinical application value and drug development value.

[0220] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

[0221] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0222] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A bispecific antibody, characterized in that, include: The first antigen-binding region has HLA-G binding activity; and The second antigen-binding region has CD3 binding activity; The first antigen-binding region includes an anti-HLA-G antibody Fab fragment and a first Fc fragment. The anti-HLA-G antibody Fab fragment includes an anti-HLA-G antibody heavy chain variable region, an HLA-G antibody light chain variable region, a CL fragment, and a CH1 fragment. The CL fragment and the CH1 fragment are linked by disulfide bonds. The C-terminus of the anti-HLA-G antibody heavy chain variable region is connected to the N-terminus of the CH1 fragment. The C-terminus of the anti-HLA-G antibody light chain variable region is connected to the N-terminus of the CL fragment. The C-terminus of the CH1 fragment is connected to the N-terminus of the first Fc fragment. The second antigen-binding region includes an anti-CD3 single-chain antibody and a second Fc fragment. The heavy chain variable region of the anti-CD3 single-chain antibody is linked to the light chain variable region of the anti-CD3 single-chain antibody via a linker peptide. The C-terminus of the heavy chain variable region of the anti-CD3 single-chain antibody is linked to the N-terminus of the linker peptide, the C-terminus of the linker peptide is linked to the N-terminus of the light chain variable region, and the C-terminus of the light chain variable region is linked to the N-terminus of the second Fc fragment; or The C-terminus of the light chain variable region of the anti-CD3 single-chain antibody is connected to the N-terminus of the linker peptide, the C-terminus of the linker peptide is connected to the N-terminus of the heavy chain variable region, and the C-terminus of the heavy chain variable region is connected to the N-terminus of the second Fc fragment. The first Fc segment and the second Fc segment are connected by a Knob-in-hole structure; The amino acid sequences corresponding to the heavy chain complementarity-determining regions HCDR1-3 of the Fab fragment of the anti-HLA-G antibody are shown in SEQ ID NO: 1-3, the light chain complementarity-determining regions are LCDR1-3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is DTS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

6. The amino acid sequences corresponding to the heavy chain complementarity-determining regions HCDR1-3 of the anti-CD3 single-chain antibody are shown in SEQ ID NO: 9-11, the light chain complementarity-determining regions are LCDR1-3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is GTN, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

14.

2. A bispecific antibody, characterized in that, include: The first antigen-binding region has HLA-G binding activity; and The second antigen-binding region has CD3 binding activity; The first antigen-binding region includes an anti-HLA-G antibody Fab fragment and a first Fc fragment. The anti-HLA-G antibody Fab fragment includes an anti-HLA-G antibody heavy chain variable region, an HLA-G antibody light chain variable region, a CL fragment, and a CH1 fragment. The CL fragment and the CH1 fragment are linked by disulfide bonds. The C-terminus of the anti-HLA-G antibody heavy chain variable region is connected to the N-terminus of the CH1 fragment. The C-terminus of the anti-HLA-G antibody light chain variable region is connected to the N-terminus of the CL fragment. The C-terminus of the CH1 fragment is connected to the N-terminus of the first Fc fragment. The second antigen-binding region includes an anti-HLA-G antibody Fab fragment, an anti-CD3 single-chain antibody, and a second Fc fragment. The C-terminus of the anti-HLA-G antibody Fab fragment is connected to the N-terminus of the anti-CD3 single-chain antibody, and the C-terminus of the anti-CD3 single-chain antibody is connected to the N-terminus of the second Fc fragment. The first Fc fragment and the second Fc fragment are connected through a Knob-in-hole structure. The amino acid sequences corresponding to the heavy chain complementarity-determining regions HCDR1-3 of the Fab fragment of the anti-HLA-G antibody are shown in SEQ ID NO: 1-3, the light chain complementarity-determining regions are LCDR1-3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is DTS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

6. The amino acid sequences corresponding to the heavy chain complementarity-determining regions HCDR1-3 of the anti-CD3 single-chain antibody are shown in SEQ ID NO: 9-11, the light chain complementarity-determining regions are LCDR1-3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is GTN, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

14.

3. The bispecific antibody according to claim 1 or 2, characterized in that, The anti-HLA-G antibody further includes a heavy chain framework region.

4. The bispecific antibody according to claim 3, characterized in that, The anti-HLA-G antibody further includes a light chain framework region.

5. The bispecific antibody according to claim 4, characterized in that, At least a portion of the heavy chain framework region and the light chain framework region of the anti-HLA-G antibody are independently derived from at least one of rabbit-derived antibodies, mouse-derived antibodies, sheep-derived antibodies, goat-derived antibodies, or primate-derived antibodies.

6. The bispecific antibody according to claim 5, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-HLA-G antibody is shown in SEQ ID NO:

7.

7. The bispecific antibody according to claim 5, characterized in that, The amino acid sequence of the light chain variable region of the anti-HLA-G antibody is shown in SEQ ID NO:

8.

8. The bispecific antibody according to claim 1 or 2, characterized in that, The anti-CD3 single-chain antibody further includes a heavy chain framework region.

9. The bispecific antibody according to claim 8, characterized in that, The anti-CD3 single-chain antibody further includes a light chain framework region.

10. The bispecific antibody according to claim 9, characterized in that, At least a portion of the heavy chain framework region and the light chain framework region of the anti-CD3 single-chain antibody are independently derived from at least one of rabbit-derived antibodies, mouse-derived antibodies, sheep-derived antibodies, goat-derived antibodies, or primate-derived antibodies.

11. The bispecific antibody according to claim 10, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO:

15.

12. The bispecific antibody according to claim 11, characterized in that, The amino acid sequence of the light chain variable region of the anti-CD3 single-chain antibody is shown in SEQ ID NO:

16.

13. The bispecific antibody according to claim 1, characterized in that, The linker peptide has an amino acid sequence as shown in SEQ ID NO:

17.

14. The bispecific antibody according to claim 1, characterized in that, The first antigen-binding region of the bispecific antibody has the amino acid sequence shown in SEQ ID NO: 19 and SEQ ID NO: 20, and the second antigen-binding region of the bispecific antibody has the amino acid sequence shown in SEQ ID NO:

21.

15. The bispecific antibody according to claim 2, characterized in that, The first antigen-binding region of the bispecific antibody has the amino acid sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20, and the second antigen-binding region of the bispecific antibody has the amino acid sequences shown in SEQ ID NO: 18 and SEQ ID NO:

20.

16. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bispecific antibody as described in any one of claims 1 to 15.

17. The nucleic acid molecule according to claim 16, characterized in that, The nucleic acid molecules are selected from DNA.

18. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 16 or 17.

19. A method for preparing the bispecific antibody according to any one of claims 1 to 15, characterized in that, include: The expression vector of claim 18 is introduced into cells; The cells are cultured under conditions suitable for protein expression and secretion in order to obtain the bispecific antibody.

20. The method according to claim 19, characterized in that, The cells in question are eukaryotic cells.

21. A recombinant cell, characterized in that, The recombinant cells carry the bispecific antibody as described in any one of claims 1 to 15, the nucleic acid molecule as described in claim 16 or 17, or the expression vector as described in claim 18.

22. A pharmaceutical composition, characterized in that, include: The bispecific antibody according to any one of claims 1 to 15, the nucleic acid molecule according to claim 16 or 17, the expression vector according to claim 18, or the recombinant cell according to claim 21.

23. The pharmaceutical composition according to claim 22, characterized in that, The pharmaceutical composition further includes pharmaceutically acceptable excipients.

24. The pharmaceutical composition according to claim 23, characterized in that, The excipients include one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

25. The pharmaceutical composition according to any one of claims 22-24, characterized in that, The pharmaceutical composition is an injectable preparation.

26. Use of the bispecific antibody according to any one of claims 1 to 15, the nucleic acid molecule according to claim 16 or 17, the expression vector according to claim 18, the recombinant cell according to claim 21, or the pharmaceutical composition according to any one of claims 22 to 25 in the preparation of a medicament for treating a tumor, wherein the tumor is ovarian cancer.

27. Use of the bispecific antibody of any one of claims 1 to 15, the nucleic acid molecule of claim 16 or 17, the expression vector of claim 18, the recombinant cell of claim 21, or the pharmaceutical composition of any one of claims 22 to 25 in the preparation of a kit for detecting CD3 and / or HLA-G.

28. A reagent kit, characterized in that, The kit contains the bispecific antibody as described in any one of claims 1 to 15.

29. The reagent kit according to claim 28, characterized in that, The kit is used to detect at least one of CD3 and HLA-G.

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