Bispecific antibodies and uses thereof

Abstract

The invention 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 and a second antigen binding region, wherein the first antigen binding region has HLA-G binding activity; and a second antigen binding region, the 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 comprises a heavy chain complementarity determining region HCDR, and the HCDR comprises at least one selected from SEQ ID NO: 1-3 or an amino acid sequence in a conservative modification form of the at least one selected from SEQ ID NO: 1-3. The bispecific antibody can be combined with CD3 and HLA-G so as to promote T cell activation, proliferation and cytokine secretion and effectively promote PBMC to kill tumor cells, and the bispecific antibody has good anti-cancer activity; and the bispecific antibody has low binding activity with T cells and high binding activity with tumor cells, and has good clinical application value and drug development value.

Description

Technical Field

[0001] The present application belongs to the field of antibodies, specifically, 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 kits. Background Art

[0002] Cancer is a major disease that affects human survival and development. According to the latest data, approximately 19 million new cancer cases and 10 million cancer deaths occur worldwide each year, and both incidence and mortality rates are on the rise. Besides surgical resection, traditional cancer treatments such as chemotherapy and radiotherapy are associated with significant side effects and a high risk of recurrence. In recent years, immunotherapy, including tumor-targeted antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hotspot and a new hope in the fight against cancer. Immunotherapy, exemplified by PD-1 / L1, has demonstrated tremendous potential. However, even the PD-1 / L1 therapy, currently the most widely approved, has an overall response rate of only 30%, leaving many patients unable to benefit. One major reason for this is that immune checkpoint therapy is ineffective against "cold tumors." T cells recognize neoantigens (neoantigens)—antigens expressed by tumor gene mutations—through the T cell receptors (TCRs) on their surface. However, some tumors have a low frequency of genetic mutations and a limited number of neoantigens, making them "cold tumors." Current immune checkpoint therapies, such as PD-1 / L1 therapy, achieve anti-cancer goals by restoring the function of T cells themselves. However, in "cold tumors", T cells cannot effectively recognize tumors, resulting in the ineffectiveness of immune checkpoint therapy for "cold tumors".

[0003] CD3-based bispecific antibodies (hereinafter referred to as "CD3 bispecific antibodies") recruit T cells to reach local tumors, bridge T cells and tumors, promote T cell activation, and kill tumors. This type of bispecific antibody does 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, so they can also "ignore" the inhibitory signals of immune checkpoint molecules to a certain extent; however, CD3 bispecific antibodies also promote the production of a large number of pro-inflammatory cytokines, such as TNFα, IL-6, etc., triggering a strong cytokine storm and excessive immune response, causing damage to the body, and severe cases may be life-threatening. Therefore, CD3-based bispecific antibodies have good application prospects in clinical practice, but their safety needs to be further improved.

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

[0005] HLA-G is a type I transmembrane protein with three extracellular domains. 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, ovarian, and breast cancers, demonstrating its high tumor specificity.

[0006] Currently, there is a lack of highly safe bispecific antibodies targeting CD3 and HLA-G on the market. Therefore, the development of CD3×HLA-G bispecific antibodies is very valuable. 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 validation has shown that this bispecific antibody exhibits weak binding to T cells but strong binding to tumor cells, exhibits high cytotoxicity, exhibits enhanced anticancer activity, and is safer, demonstrating promising clinical application and drug development value.

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

[0009] In the first aspect of the present application, the present application proposes a bispecific antibody. According to an embodiment of the present application, the bispecific antibody comprises: a first antigen binding region, the first antigen binding region having HLA-G binding activity; and a second antigen binding region, the 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 comprises a heavy chain complementary determining region HCDR, and the HCDR comprises an amino acid sequence selected from at least one of SEQ ID NOs: 1 to 3 or a conservatively modified form thereof. In some examples of the present application, the bispecific antibody can bind to CD3 and HLA-G, thereby promoting T cell activation, proliferation and cytokine secretion, effectively promoting PBMC to kill tumor cells, and has good anti-cancer activity; and the bispecific antibody has low binding activity with T cells and high binding activity with tumor cells, and has good clinical application value and drug development value.

[0010] In the second aspect of the present application, the present application proposes a nucleic acid molecule. According to the embodiments of the present application, the nucleic acid molecule encodes the bispecific antibody described in the first aspect of the present application. In some examples of the present 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 to kill 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, and has good clinical application and drug development value.

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

[0012] In the fourth aspect of the present application, the present application proposes a method for preparing the bispecific antibody described in the first aspect. According to an embodiment of the present application, the method comprises: introducing the expression vector described in the third aspect of the present application into a cell; culturing the cell under conditions suitable for protein expression and secretion to obtain the bispecific antibody. In some examples of the present 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 secretion of cytokines, effectively promoting PBMC to kill tumor cells, and having good anti-cancer activity; and the bispecific antibody has low binding activity with T cells and high binding activity with tumor cells.

[0013] In a fifth aspect of the present application, a recombinant cell is provided. According to embodiments of the present application, the recombinant cell carries the bispecific antibody described in the first aspect of the present application, 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 the present application, the cell can efficiently express the bispecific antibody under appropriate conditions.

[0014] In the sixth aspect of the present application, the present application proposes a pharmaceutical composition. According to the embodiments of the present application, the pharmaceutical composition comprises: the bispecific antibody described in the first aspect of the present application, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect. In some examples of the present application, the bispecific antibody can effectively promote PBMC to kill tumor cells and has better anti-cancer activity; it has low T cell binding activity, higher safety, and good clinical application value and drug development value. Thus, the obtained drug can be further used to prevent and / or treat CD3 and / or HLA-G mediated related diseases.

[0015] In the seventh aspect of the present application, the present application proposes 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, wherein the drug is used to treat or prevent tumors. In some examples of the present application, the bispecific antibody can specifically bind to CD3 and HLA-G, thereby promoting T cell activation, proliferation and secretion of cytokines, effectively promoting PBMC to kill tumor cells. Drugs containing a series of substances such as the bispecific antibody also have significant effects in treating or preventing tumors.

[0016] In an eighth aspect of the present application, the present application provides for 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 preparing a kit for detecting CD3 and / or HLA-G. Those skilled in the art will appreciate that the features and advantages of the bispecific antibodies described above also apply to this use and are not further elaborated here.

[0017] In the ninth aspect of the present application, the present application proposes a kit. According to an embodiment of the present application, the kit comprises the bispecific antibody described in the first aspect. In some examples of the present application, the bispecific antibody can bind with high affinity. In some examples of the present application, the bispecific antibody can specifically bind to CD3 and HLA-G, thereby promoting T cell activation, proliferation and secretion of cytokines, and effectively promoting PBMC to kill tumor cells. Therefore, the kit comprising the bispecific antibody can be used to detect CD3 and / or HLA-G protein. 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 judge the status of an individual, such as after obtaining the CD3 and HLA-G protein levels of the individual, judging whether their CD3 and HLA-G protein levels are higher or lower than normal levels.

[0018] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 Schematic diagram of the structure of the CD3×HLA-G bispecific antibody provided according to the examples of the present application;

[0021] Figure 2 Schematic diagram of flow cytometry results of binding of the CD3×HLA-G bispecific antibody provided in the Examples of the present application to human peripheral blood CD8 T cells;

[0022] Figure 3 Schematic diagram of flow cytometry results of binding of the CD3×HLA-G bispecific antibody provided in the Examples of the present application to JEG3 tumor cells;

[0023] Figure 4 Schematic diagram of flow cytometry results of binding of the CD3×HLA-G bispecific antibody provided in the Examples of the present application to SKOV3-HLA-G tumor cells;

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

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

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

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

[0028] Figure 9 This is a schematic diagram of the results of the CD3×HLA-G bispecific antibody provided in the examples of the present application promoting the secretion of IL-2 and IFN-γ cytokines by PBMC;

[0029] Figure 10 This is a schematic diagram showing the results of the CD3×HLA-G bispecific antibody provided in the examples of the present application promoting PBMC to kill SKOV3-HLA-G tumor cells;

[0030] Figure 11 This is a schematic diagram of the results of the CD3×HLA-G bispecific antibody provided in the examples of the present application promoting PBMC to kill JEG3 tumor cells;

[0031] Figure 12Schematic diagram of the in vivo efficacy results of the CD3×HLA-G bispecific antibody provided in the examples of the present application in a mouse tumor model. DETAILED DESCRIPTION

[0032] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0033] In this application, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise specifically defined.

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

[0035] In this application, certain regions within the variable region have a higher degree of variation in amino acid composition and sequence, referred to as "hypervariable regions (HVRs)." Hypervariable regions are where antigen and antibody bind, and are therefore also referred to as complementarity-determining regions (CDRs). Both the heavy and light chain variable regions have three CDRs. For example, amino acid residues around 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and around 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 from a "hypervariable loop" (e.g., amino acid residues around 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and around 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 that can bind to CD3. Such an antibody is also referred to herein as an "antibody that binds to CD3." The term "anti-HLA-G antibody" refers to an antibody that can bind to HLA-G. Such an antibody is also referred to herein as an "antibody that binds to HLA-G."

[0037] As used herein, the term "antigen-binding fragment" is equivalent to "antibody fragment" or "antigen-binding antibody fragment" and may include a portion of an intact antibody, generally the 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 can increase half-life by chemical modification, such as the addition of poly(alkylene) glycols, such as polyethylene glycol ("PEGylation") (PEGylated fragments referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" is polyethylene glycol) or by incorporation into liposomes.

[0038] As used herein, the term "humanized antibody" refers to a recombinant antibody obtained by replacing the amino acid sequence of the non-CDR (Fv framework region (FR)) amino acids of the constant region and variable region of a monoclonal antibody from one species (such as a mouse) with the amino acid sequence of the non-CDR amino acids of the constant region and variable region of an antibody from another species (such as a human) using recombinant DNA technology. That is, when the constant region of an antibody is humanized, it is called a chimeric antibody, and when the amino acid sequence of the non-CDR amino acids of the constant region and variable region is fully humanized, it is called a humanized antibody. The method of humanization can be carried out with reference to conventional antibody engineering technology and will not be described in detail here.

[0039] As used herein, 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 amino acids of a monoclonal antibody from one species (such as mouse) with the constant region of an antibody from another species (such as human).

[0040] In this application, the amino acid sequences of the listed CDRs are shown in accordance with the IMGT definition rules. However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as the Kabat rules, the Chothia rules, etc. It should be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or a region thereof (such as a variable region) should be understood to cover the complementary determining regions defined by any of the above-mentioned known schemes described in the present invention. Although the scope of protection claimed in this application is based on the sequences shown in the IMGT definition rules, the amino acid sequences corresponding to the definition rules of other CDRs 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 and has the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0042] With respect to 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 sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical to the nucleotides at the corresponding positions in the second sequence] minus [the total number of nucleotides in the first sequence] and then multiplying by [100%], where each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence - relative to the first nucleotide 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 known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings. Some other techniques, computer algorithms and setups for determining the degree of sequence identity are described, for example, 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] With respect to polypeptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more polypeptides or designated sequences thereof when optimally aligned and compared (with appropriate insertions or deletions of nucleotides). The percent homology between two sequences varies with the number of identical positions shared by the sequences when the sequences are optimally aligned (i.e., % homology = number of identical positions / total number of positions x 100), where optimal alignment is determined by taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences and the length of each gap. Sequence comparison and percent identity determination between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting Examples below.

[0044] In the present application, under the premise of not substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art can replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present application to obtain variants of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The variant sequence described in the present application can have at least 80% identity (or homology) with the reference sequence, which means at least 80% with each reference sequence, 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%, 99.9% identity. The sequence identity described in the present application can be measured using sequence analysis software. For example, the computer program BLAST, in particular BLASTP or TBLASTN, with default parameters is used. The amino acid sequences described in the present invention are all shown in the manner of N-terminus to C-terminus.

[0045] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0046] As used herein, the term "pharmaceutical composition" generally refers to unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with liquid carriers, finely divided solid carriers, or both.

[0047] As used herein, the term "pharmaceutically acceptable" refers to substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic response), ie, substances with a reasonable benefit / risk ratio.

[0048] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except to the extent that any conventional excipient is incompatible with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated by the present invention.

[0049] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient by a suitable means. The antibody or antigen-binding fragment, recombinant protein, multispecific antibody, conjugate, or pharmaceutical composition of the present invention can be administered by any common route as long as it reaches the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, and the like, but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.

[0050] As used herein, the term "treatment" refers to any process used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0051] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0052] In this application, the amino acid sequences involved are shown in Table 1.

[0053] The present application proposes a bispecific antibody targeting CD3 and HLA-G, an expression vector, a method for preparing the bispecific antibody, a recombinant cell, a pharmaceutical composition, a pharmaceutical use, and a kit, which are described in detail below.

[0054] Bispecific antibodies

[0055] In a first aspect, the present application provides a bispecific antibody. According to an embodiment of the present 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 comprises a heavy chain complementarity determining region (HCDR), and the HCDR comprises an amino acid sequence selected from at least one of SEQ ID NOs: 1-3 or conservatively modified forms thereof.

[0056] The bispecific antibody of the present application can promote T cell activation, proliferation and cytokine secretion, effectively promote PBMC to kill 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, and has good clinical application value and drug development value.

[0057] In some examples of the present application, the HCDRs of the anti-HLA-G antibody include: HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 1, or having at least 80% homology to SEQ ID NO: 1, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 2, or having at least 80% homology to SEQ ID NO: 2, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 3, or having at least 80% homology to SEQ ID NO: 3. Anti-HLA-G antibodies based on the aforementioned HCDR sequences have high binding affinity and specificity for HLA-G.

[0058] In some preferred examples of the present application, the HCDRs include: HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively. Experimental verification has shown that anti-HLA-G antibodies having the amino acid sequences set forth in SEQ ID NOs: 1 to 3 have high binding affinity and specificity for HLA-G.

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

[0060] In some preferred examples of the present application, the LCDR comprises: LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 4 to 6, respectively. Experimental verification has shown that anti-HLA-G antibodies having amino acid sequences as shown in SEQ ID NOs: 4 to 6 have high binding affinity and specificity for HLA-G.

[0061] It should be noted that one or more amino acid residues in the HCDRs and / or LCDRs of the aforementioned anti-HLA-G antibodies may be replaced with other amino acid residues from the same side chain family, and the retained function of the altered antibodies can be tested using the functional assays described herein. Preferably, the number of conservative modifications does not exceed one or two.

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

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

[0064] In some examples of the present 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 a rabbit antibody, a mouse antibody, a sheep antibody, a goat antibody, a primate antibody, or mutants thereof.

[0065] In some preferred examples of the present application, the heavy chain variable region of the anti-HLA-G antibody comprises an amino acid sequence selected from SEQ ID NO: 7 or an amino acid sequence having at least 80% homology to SEQ ID NO: 7. In some more preferred examples of the present 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 examples of the present 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 an amino acid sequence having at least 80% homology to SEQ ID NO: 8. In some more preferred examples of the present 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] The bispecific antibody based on the above heavy chain variable region and light chain variable region sequences has high binding affinity and specificity for HLA-G.

[0068] In some examples of the present application, the second antigen-binding region includes an anti-CD3 antibody, which includes a heavy chain complementarity determining region (HCDR), and the HCDR of the anti-CD3 antibody comprises an amino acid sequence selected from at least one of SEQ ID NOs: 9 to 11 or a conservative modification thereof.

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

[0070] In some preferred examples of the present application, the HCDR comprises: HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 9 to 11, respectively. Experimental verification has shown that anti-CD3 antibodies having amino acid sequences as shown in SEQ ID NOs: 9 to 11 have high binding affinity and specificity to CD3.

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

[0072] In some preferred examples of the present application, the LCDR comprises: LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 12 to 14, respectively. Experimental verification has shown that anti-CD3 antibodies having amino acid sequences as shown in SEQ ID NOs: 12 to 14 have high binding affinity and specificity for CD3.

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

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

[0075] In some preferred examples of the present 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 a rabbit antibody, a mouse antibody, a sheep antibody, a goat antibody, a primate antibody or a mutant thereof.

[0076] In some preferred examples of the present application, the heavy chain variable region of the anti-CD3 antibody comprises an amino acid sequence selected from SEQ ID NO: 15 or an amino acid sequence having at least 80% homology to SEQ ID NO: 15. In some more preferred examples of the present 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 examples of the present application, the light chain variable region of the anti-CD3 antibody comprises an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 80% homology to SEQ ID NO: 16. In some more preferred examples of the present 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] The bispecific antibody based on the above heavy chain variable region and light chain variable region sequences has high binding affinity and specificity for CD3.

[0079] In some examples of the present application, the first antigen-binding region comprises a first anti-HLA-G antibody Fab fragment, which comprises an anti-HLA-G antibody heavy chain variable region, an HLA-G antibody light chain variable region, a CL fragment, and a CH1 fragment, wherein the CL fragment and the CH1 fragment are linked by a disulfide bond, 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 the present application, the first antigen binding region further includes a first Fc fragment.

[0081] In some examples of the present application, the C-terminus of the heavy chain variable region of the anti-HLA-G antibody is connected to the N-terminus of the CH1 fragment, the C-terminus of the light chain variable region of the anti-HLA-G antibody 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 the present application, the heavy chain constant region of the anti-HLA-G antibody comprises at least one selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; and / or the light chain constant region comprises a κ-type or λ-type light chain constant region. In some specific embodiments, the heavy chain constant region comprises a human IgG, IgA, IgM, IgE, or IgD, such as human IgG1.

[0083] In other specific embodiments, the heavy chain constant region comprises a protein selected from murine IgG, IgA, IgM, IgE or IgD, such as murine IgG1.

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

[0085] In some examples of the present application, the heavy chain variable region and the light chain variable region of the anti-CD3 antibody are connected via a connecting peptide. In some preferred examples of the present application, the anti-CD3 antibody is a single-chain antibody.

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

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

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

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

[0090] In some examples of the present application, the C-terminus of the heavy chain variable region of the anti-CD3 antibody is connected to the N-terminus of the connecting peptide, the C-terminus of the connecting 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 connecting peptide, the C-terminus of the connecting 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 the present application, the first antigen binding region and the second antigen binding region are connected via a Knob-in-hole structure. In a specific example of the present application, the aforementioned bispecific antibody structure is as follows: Figure 1 As shown in A.

[0092] In some examples of the present application, the second antigen-binding region further comprises a second anti-HLA-G antibody Fab fragment, which comprises an anti-HLA-G antibody heavy chain variable region, an HLA-G antibody light chain variable region, a CL fragment, and a CH1 fragment, wherein the CL fragment and the CH1 fragment are linked by a disulfide bond, 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 the present application, the second anti-HLA-G antibody Fab fragment, the anti-CD3 antibody and the second Fc fragment are connected. The C-terminus of the anti-HLA-G antibody Fab fragment is connected to the N-terminus of the anti-CD3 antibody, and the C-terminus of the anti-CD3 antibody is connected to the N-terminus of the second Fc fragment. In a specific example of the present application, the aforementioned bispecific antibody structure is as follows Figure 1 As shown in B.

[0094] In some examples of the present 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 the present 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: 18 and SEQ ID NO: 20.

[0096] The bispecific antibody 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 to kill tumor cells, has good anti-cancer activity, and higher safety, and has practical clinical application value and drug development value.

[0097] Nucleic acid molecules

[0098] In another aspect of the present application, the present application provides a nucleic acid molecule encoding the aforementioned bispecific antibody. In some examples of the present application, the aforementioned nucleic acid molecule can encode a bispecific antibody that can simultaneously target CD3 and HLA-G.

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

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

[0101] expression vector

[0102] In another aspect of the present application, an expression vector is provided. According to embodiments of the present application, the expression vector carries the aforementioned nucleic acid molecule. The aforementioned expression vector includes optional control amino acid sequences, such as one or more control amino acid sequences that are operably linked to the nucleic acid molecule. The control amino acid sequences can direct the expression of the nucleic acid molecule in a host. The resulting vector can effectively express the aforementioned bispecific antibody.

[0103] It should be noted that when the nucleic acid molecule is linked to a vector, the nucleic acid molecule and the control elements on the vector may be directly or indirectly linked, as long as these control elements are capable of controlling translation and expression of the nucleic acid molecule. These control elements may be derived directly from the vector itself or exogenously, i.e., not derived from the vector itself. Of course, it is sufficient that the nucleic acid molecule and the control elements are operably linked.

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

[0105] As used herein, the term "operably linked" refers to the attachment of an exogenous gene to a vector so that control elements within the vector, such as transcriptional control amino acid sequences such as and translational control amino acid sequences such as , can function as intended to regulate the transcription and translation of the exogenous gene. Commonly used vectors include viral vectors, plasmids, and bacteriophages. Expression vectors according to certain embodiments of the present invention, when introduced into appropriate recipient cells, can effectively express the aforementioned nucleic acid molecules under the mediation of a regulatory system, thereby enabling the in vitro production of large quantities of the protein encoded by the nucleic acid molecules.

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

[0107] In some examples of the present application, the vector comprises at least one selected from a plasmid vector, an adenoviral vector, a lentiviral vector, and an adeno-associated viral vector.

[0108] Method for preparing bispecific antibodies

[0109] In another aspect, the present application provides a method for preparing the aforementioned bispecific antibody, 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 aforementioned specific antibody to be expressed in large quantities in vitro.

[0110] In some examples of the present application, the cell is a eukaryotic cell.

[0111] recombinant cells

[0112] In another aspect of the present application, a recombinant cell is provided, which carries the aforementioned nucleic acid molecule or expression vector. In some examples of the present 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 appropriate conditions.

[0113] In some examples of the present application, the recombinant cell is a prokaryotic cell, a eukaryotic cell, or a bacteriophage. It should be noted that the eukaryotic cell does not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0114] In some examples of the present application, the prokaryotic cell is Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0115] In some examples of the present application, the eukaryotic cell is a fungus, an insect cell, a plant cell, or a mammalian cell.

[0116] In some examples of the present application, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Trichoderma.

[0117] In some examples of the present application, the insect cells are fall armyworm cells; in some examples of the present application, the plant cells are tobacco plant cells; in some examples of the present 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 the present application, the recombinant cell is a mammalian cell. When the cell is a mammalian cell, the expression efficiency of the antibody or antigen-binding fragment thereof is higher.

[0119] In some examples of the present application, the recombinant cell is a BHK cell, a CHO cell, a COS cell, or a NSO cell.

[0120] It should be noted that the "suitable conditions" described in this specification refer to conditions suitable for the expression of the bispecific antibodies of this application. Those skilled in the art will readily appreciate that conditions suitable for the expression of bispecific antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of bispecific antibodies based on the specific laboratory environment.

[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 cell. In some examples of this application, the pharmaceutical composition can effectively promote PBMCs to kill tumor cells, exhibiting anti-cancer activity; it can also produce less pro-inflammatory cytokines, offering improved safety, and possessing promising clinical application and drug development value. The resulting drug can be further used to prevent and / or treat diseases mediated by CD3 and / or HLA-G.

[0123] In some examples of the present application, a pharmaceutically acceptable excipient is further included.

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

[0125] In some examples of the present application, the pharmaceutical composition is an injection.

[0126] It should be noted that the pharmaceutical composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the components contained in the pharmaceutical composition can 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 can be administered to the subject simultaneously or sequentially.

[0127] The drug of the present application contains a safe and effective amount of the active ingredient (bispecific antibody) of the present application and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be compatible with the mode of administration. The dosage form of the drug of the present application is an injection, an oral preparation (tablet, capsule, oral solution), a transdermal agent, and a sustained-release agent. For example, it can be prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. The drug is preferably manufactured under sterile conditions.

[0128] The effective amount of the active ingredients described herein may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0129] The pharmaceutically acceptable excipients described herein 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 compatible with the mode of administration, as is well known to those skilled in the art.

[0130] Use in preparing medicines

[0131] In another aspect of this application, the present application proposes the use of the aforementioned bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells, or pharmaceutical compositions in the preparation of a medicament for treating or preventing tumors. 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 a medicament that can be clinically used to prevent or treat diseases mediated by CD3 and / or HLA-G.

[0132] In some examples of the present application, the aforementioned tumor includes 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, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0133] Use in the preparation of kits

[0134] In another aspect of the present application, the present application proposes the use of the aforementioned bispecific antibody, nucleic acid molecule, expression vector, recombinant cell or pharmaceutical composition in preparing a kit for detecting CD3 and / or HLA-G.

[0135] As previously described, the bispecific antibodies of the present invention are capable of specifically binding to CD3 and HLA-G. Therefore, these bispecific antibodies can be used to detect CD3 and / or HLA-G. Furthermore, they can be used to prepare CD3 and / or HLA-G-related kits for scientific research, such as for the qualitative or quantitative detection of CD3 and / or HLA-G protein molecules in biological samples. More specifically, they can be used in kits for immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of CD3 and / or HLA-G with antibodies. These kits may contain any one or more of the following: an antagonist, the bispecific antibody of the present invention, or a reference drug material; a protein purification column; an immunoglobulin affinity purification buffer; and a cell assay diluent. The bispecific antibodies of the present invention can be used in various diagnostic tests, for example, to detect various diseases or the presence of drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for CD3 and / or HLA-G-mediated diseases by testing serum or blood from a subject.

[0136] In another aspect of the present application, the present application provides a kit comprising the aforementioned bispecific antibody. In some examples of the present application, the aforementioned kit can accurately detect CD3 and HLA-G proteins in a sample.

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

[0138] Disease treatment methods

[0139] In another aspect of the present application, the present application provides a method for preventing and / or treating diseases mediated by CD3 and / or HLA-G. According to an embodiment of the present application, the method comprises administering to a subject a pharmaceutically acceptable amount of a bispecific antibody, nucleic acid molecule, vector or transformant, recombinant cell, or pharmaceutical composition.

[0140] It should be noted that the terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal being evaluated for treatment and / or being treated. In one embodiment, 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 infection, and the like. The subject can be a human, but also includes other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, and the like.

[0141] The effective amount of the antibody or antigen-binding fragment thereof, conjugate, nucleic acid, vector or transformant or pharmaceutical composition described herein may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

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

[0143] In some examples of the present 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, gastric 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 sequence 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] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not specified, they are carried out according to the technology or conditions described in the literature in this area or according to the product instructions.The reagents or instruments used are not specified by the manufacturer, and 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) using ExpiCHO Expression Medium (purchased from Thermo Fisher) and adjust the cell concentration to 6×10 6 / mL to obtain ExpiCHO cell solution;

[0155] 2. When the double antibody is of A configuration ( Figure 1 ), pcDNA3.4 vector containing CD3 antibody, HLA-G antibody heavy chain, and HLA-G antibody light chain (commissioned to Nanjing GenScript for synthesis) was added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a; or, when the bispecific antibody is of B configuration ( Figure 1), pcDNA3.4 vector containing HLA-G heavy chain-CD3 antibody, HLA-G antibody heavy chain, and HLA-G antibody light chain (commissioned to Nanjing GenScript for synthesis) was added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a;

[0156] 3. Add 160 μL of ExpiFectamine CHO 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 a transfection mixture, and add the entire transfection mixture to 50 mL of ExpiCHO cell solution within 5 minutes;

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

[0159] 6. Use Protein A purification column (purchased from Nanomicro) to affinity purify from the culture supernatant to obtain the target antibody.

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

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

[0162]

[0163] Note: For antibody configuration, see Figure 1 .

[0164] Example 2 Bispecific Antibody Flow Cytometry Binding Experiment

[0165] Flow cytometry experiments were 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 addition of the antibody was used to determine the binding properties of the antibody and cells.

[0166] (1) Dilute PBMC to 2×10 6 / ml, added to a 1.5ml EP tube at a volume of 100μl / tube, 10μl / tube of goat serum was added thereto, and the tube was blocked at 4℃ for 30min. Serial dilutions of the bispecific antibody 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, respectively), and control hIgG1LALA (purchased from Bio-Innovation) were added and incubated at 4°C for 30 min. Add 1 ml of PBS to the EP tube and centrifuge at 3500 rpm for 5 min at 4°C. Discard the supernatant and wash again with PBS. Discard the supernatant after centrifugation and resuspend the cells in 100 μl / tube of PBS. Add 1 μl / tube of Alexa-647-labeled goat anti-human IgG 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 and centrifuge and discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze by 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. The binding of CD3×HLA-G1:1 and CD3×HLA-G 2:1 to T cells is weaker than that of RG6353, and CD3×HLA-G 2:1 has the weakest binding to T cells.

[0168] (2) JEG3 and SKOV3-HLA-G tumor cells were diluted to 2×10 6 / ml, added to a volume of 100 μl / tube in a 1.5 ml EP tube, 10 μl / tube of goat serum was added, and the cells were blocked at 4°C for 30 min. Serial dilutions of 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 control hIgG1LALA (purchased from Bio-Bio) were added and incubated at 4°C for 30 min. Add 1 ml of PBS to the EP tube and centrifuge at 3500 rpm for 5 minutes at 4°C. Discard the supernatant and wash once more with PBS. Discard the supernatant after centrifugation. Resuspend the cells in 100 μl / tube of PBS and add 1 μl / tube of Alexa-647-labeled goat anti-human IgG secondary antibody (Jackson Labs). Incubate at 4°C in the dark for 30 minutes. Wash twice with PBS and centrifuge. Discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze by flow cytometry.

[0169] The results are as follows Figure 3 As shown, both CD3×HLA-G 1:1 and CD3×HLA-G 2:1 were able to bind to JEG3 cells with similar binding activities.

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

[0171] Example 3 Bispecific Antibodies Promote T Cell Expression and Activation

[0172] The bispecific antibody prepared in Example 1 was added to the PBMC and JEG3 tumor cell co-incubation system. After 48 h of culture, 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 properties of the bispecific antibody in inducing T cell activation.

[0173] (1) JEG3 cells were diluted to 1×10 5 / ml, added to 96-well plate;

[0174] (2) Using complete RPMI 1640 medium, the bispecific antibody 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 the control hIgG1LALA (purchased from Bio-Bio) were serially diluted and added to a 96-well plate at 20 μl / well;

[0175] (3) PBMC (purchased from Saili Biotechnology) were diluted to 1.25×10 6 / ml, added to 96-well plate, 80 μl / well;

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

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

[0178] (6) Wash twice with PBS and discard the supernatant after centrifugation.

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

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

[0181] The results are as follows Figure 6 As shown in the data, both CD3×HLA-G 1:1 and CD3×HLA-G 2:1 can promote the activation of CD8 T cells and the expression of CD69 and CD25, and the effective dose of 2:1 dual antibody is lower than that of 1:1 dual 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 PBMC and JEG3 tumor cell co-incubation system. After 72 h of culture, the CFSE fluorescence intensity in CD4 T cells and CD8 T cells was detected by flow cytometry to determine the T cell proliferation-inducing properties of the bispecific antibody.

[0184] (1) JEG3 cells were diluted to 1×10 5 / ml, added to 96-well plate;

[0185] (2) Using complete RPMI 1640 medium, the bispecific antibody 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 the control hIgG1LALA (purchased from Bio-Bio) were serially diluted and added to a 96-well plate at 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 using complete RPMI 1640 medium and added to a 96-well plate at 80 μl / well.

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

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

[0189] (6) Wash twice with PBS and discard the supernatant after centrifugation.

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

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

[0192] The results are as follows Figure 8 As shown in the data, both CD3×HLA-G 1:1 and CD3×HLA-G 2:1 can promote CD8 T cell proliferation. The two configurations of bispecific antibodies have similar intensities in promoting CD8 T cell proliferation, and the effective dose of the 2:1 configuration of bispecific antibodies is lower.

[0193] Example 5 Experiment on Bispecific Antibodies Promoting Cytokine Secretion by PBMC

[0194] The bispecific antibody prepared in Example 1 was added to the PBMC and JEG3 tumor cell co-incubation system. After 48 h of culture, 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 5 / ml, added to 96-well plates, and cultured in a 37°C, 5% CO2 incubator for 48 h;

[0196] (b) Using complete RPMI 1640 medium, the bispecific antibody 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 the control hIgG1LALA (purchased from Bio-Bio) were serially diluted and added to a 96-well plate at 20 μl / well.

[0197] (c) PBMC (purchased from Saili Biotechnology) were diluted to 1.25×10 6 / ml, added to 96-well plate, 80 μl / well;

[0198] (d) The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 48 h;

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

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

[0201] The results are as follows Figure 9As shown, both CD3×HLA-G 1:1 and CD3×HLA-G 2:1 can promote PBMC to secrete immune-activating cytokines IL-2 and IFN-γ, and the effective dose of the 2:1 configuration is lower;

[0202] Example 6 Bispecific Antibodies Promote PBMCs to Kill Tumor Cells

[0203] This example aims to detect the ability of the bispecific antibody prepared in Example 1 to promote PBMC to kill tumor cells.

[0204] (a) Complete RPMI-1640 medium was added to a 16-well RTCA plate at a volume of 50 μL / well and calibrated on the instrument.

[0205] (b) JEG-3 and SKOV3-HLA-G tumor cells were diluted to 2×10 5 / mL, and added to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then the cell coefficient was detected using the xCELLigence RTCA MP device at 37°C and 5% CO2 for 24 hours;

[0206] (c) 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), and control hIgG1LALA (purchased from Bio-Tech) to the RTCA plate obtained in step (2) using complete RPMI-1640 medium at a volume of 20 μl / well;

[0207] (4) PBMC (purchased from Saili Biotechnology) were diluted to 6.25×10 5 / ml or 1.25×10 6 pcs / ml, and added to the RTCA plate obtained in step (3) at a volume of 80 μl / well;

[0208] (5) The reaction system obtained in step (4) was incubated at 37° C. and 5% CO 2 for 24 h to detect the cell coefficient using an xCELLigence RTCA MP device.

[0209] The results are as follows Figure 10 As shown, both CD3×HLA-G 1:1 and CD3×HLA-G 2:1 can promote PBMC to kill SKOV3-HLA-G tumor cells, and the effective doses of the two dual antibodies are similar;

[0210] The results are as follows Figure 11 As shown in the figure, both CD3×HLA-G 1:1 and CD3×HLA-G 2:1 can promote PBMC to kill JEG3 tumor cells, and the killing activity of 2:1 dual antibody is stronger than that of 1:1 dual antibody.

[0211] Example 7 Verification of the anti-tumor effect of bispecific antibodies in mouse models

[0212] The in vivo efficacy experiment was used to detect the anti-cancer function of the bispecific antibody prepared in Example 1 of the present application in promoting immune reconstitution in mice.

[0213] (1) On day -14, NSG mice were subcutaneously injected with 5×10 6 SKOV3-HLA-G tumor cells;

[0214] (2) On day -7, human PBMC (purchased from Saili Biotechnology) were transfused into NSG mice (purchased from Southern Model) via the tail vein at an injection volume of 5×10 6 / Only;

[0215] (3) On day 0, mice were weighed and tumor volumes were measured. Mice were divided into groups based on the weighing results and tumor volumes.

[0216] (4) On days 0, 3, 7, and 10, mice were injected 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 injection of the above antibodies, the tumor volume was measured and the mice were weighed twice a week.

[0218] The results are as follows Figure 12 As shown, CD3×HLA-G 2:1 has a significant anti-cancer effect, and the tumor completely regressed after treatment.

[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 to kill tumor cells, and has 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, has good anti-cancer activity, and higher safety, and has good clinical application value and drug development value.

[0220] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as the contents disclosed by the present invention and fall within the scope of protection of the present invention.

[0221] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0222] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A bispecific antibody, characterized in that: include: a first antigen-binding region having HLA-G binding activity; and a second antigen-binding region having CD3 binding activity; The first antigen binding region comprises an anti-HLA-G antibody, the anti-HLA-G antibody comprises a heavy chain complementarity determining region (HCDR), and the HCDR comprises an amino acid sequence selected from at least one of SEQ ID NOs: 1 to 3 or a conservative modification thereof.

2. The bispecific antibody according to claim 1, characterized in that The HCDR of the anti-HLA-G antibody includes: HCDR1, HCDR2 and HCDR3, wherein: The HCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 1 or having at least 80% homology to SEQ ID NO: 1, The HCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 2 or having at least 80% homology to SEQ ID NO: 2, The HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 3 or a sequence having at least 80% homology to SEQ ID NO: 3; Preferably, the HCDR comprises: HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 1 to 3, respectively; Preferably, the anti-HLA-G antibody further comprises: a light chain complementary determining region LCDR, wherein the LCDR comprises: LCDR1, LCDR2 and LCDR3, wherein: The LCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having at least 80% homology to SEQ ID NO: 4, The LCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 5 or having at least 80% homology to SEQ ID NO: 5, The LCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 6 or at least 80% homologous to SEQ ID NO: 6; Preferably, the LCDR comprises: LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 4 to 6, respectively; Optionally, the anti-HLA-G antibody further comprises: a heavy chain framework region; Optionally, the anti-HLA-G antibody further comprises: a light chain framework region; Preferably, at least a portion of the heavy chain framework region and the light chain framework region are independently derived from at least one of a rabbit antibody, a mouse antibody, a sheep antibody, a goat antibody, a primate antibody, or a mutant thereof; Preferably, the heavy chain variable region of the anti-HLA-G antibody comprises an amino acid sequence selected from SEQ ID NO: 7 or an amino acid sequence having at least 80% homology to SEQ ID NO: 7; preferably, the amino acid sequence of the heavy chain variable region of the anti-HLA-G antibody is as shown in SEQ ID NO: 7; Preferably, the light chain variable region of the anti-HLA-G antibody comprises an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% homology to SEQ ID NO: 8; preferably, the amino acid sequence of the light chain variable region of the anti-HLA-G antibody is as shown in SEQ ID NO:

8.

3. The bispecific antibody according to claim 1, characterized in that The second antigen-binding region comprises an anti-CD3 antibody, wherein the anti-CD3 antibody comprises a heavy chain complementarity determining region (HCDR), wherein the HCDR comprises an amino acid sequence selected from at least one of SEQ ID NOs: 9 to 11 or a conservative modification thereof; Optionally, the HCDR comprises: HCDR1, HCDR2 and HCDR3, wherein The HCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% homology to SEQ ID NO: 9, The HCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 10 or having at least 80% homology to SEQ ID NO: 10, The HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 11 or a sequence having at least 80% homology to SEQ ID NO: 11; Preferably, the HCDR comprises: HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 9 to 11, respectively; Preferably, the anti-CD3 antibody further comprises: a light chain complementary determining region LCDR, wherein the LCDR comprises: LCDR1, LCDR2 and LCDR3, wherein: The LCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 12 or having at least 80% homology to SEQ ID NO: 12, The LCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 13 or having at least 80% homology to SEQ ID NO: 13, The LCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 14 or at least 80% homologous to SEQ ID NO: 14; Preferably, the LCDR comprises: LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 12 to 14, respectively; Optionally, the anti-CD3 antibody further comprises: a heavy chain framework region; Optionally, the anti-CD3 antibody further comprises: a light chain framework region; Preferably, at least a portion of the heavy chain framework region and the light chain framework region are independently derived from at least one of a rabbit antibody, a mouse antibody, a sheep antibody, a goat antibody, a primate antibody, or a mutant thereof; Preferably, the heavy chain variable region of the anti-CD3 antibody comprises an amino acid sequence selected from SEQ ID NO: 15 or an amino acid sequence having at least 80% homology to SEQ ID NO: 15; preferably, the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody is as shown in SEQ ID NO: 15; Preferably, the light chain variable region of the anti-CD3 antibody comprises an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 80% homology to SEQ ID NO: 16; preferably, the amino acid sequence of the light chain variable region of the anti-CD3 antibody is as shown in SEQ ID NO:

16.

4. The bispecific antibody according to claim 2, characterized in that The first antigen-binding region comprises a first anti-HLA-G antibody Fab fragment, which comprises an anti-HLA-G antibody heavy chain variable region, an HLA-G antibody light chain variable region, a CL fragment, and a CH1 fragment, wherein the CL fragment and the CH1 fragment are connected by a disulfide bond, the C-terminus of the anti-HLA-G antibody heavy chain variable region is connected to the N-terminus of the CH1 fragment, and the C-terminus of the anti-HLA-G antibody light chain variable region is connected to the N-terminus of the CL fragment; Optionally, the first antigen binding region further comprises a first Fc fragment; Optionally, the C-terminus of the heavy chain variable region of the anti-HLA-G antibody is connected to the N-terminus of the CH1 fragment, the C-terminus of the light chain variable region of the anti-HLA-G antibody 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.

5. The bispecific antibody according to claim 3, characterized in that The heavy chain variable region and the light chain variable region are connected via a connecting peptide; Optionally, the C-terminus of the heavy chain variable region of the anti-CD3 antibody is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the light chain variable region; or The C-terminus of the light chain variable region of the anti-CD3 antibody is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the heavy chain variable region; Optionally, the connecting peptide has the amino acid sequence shown in SEQ ID NO: 17; Optionally, the second antigen binding region further comprises a second Fc fragment; Optionally, the anti-CD3 antibody is linked to the second Fc fragment; Optionally, the C-terminus of the anti-CD3 antibody heavy chain variable region is connected to the N-terminus of the connecting peptide, the C-terminus of the connecting 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 connecting peptide, the C-terminus of the connecting 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.

6. The bispecific antibody according to claim 4 or 5, characterized in that The first antigen binding region and the second antigen binding region are connected via a Knob-in-hole structure.

7. The bispecific antibody according to claim 6, characterized in that The second antigen-binding region further comprises a second anti-HLA-G antibody Fab fragment, wherein the second anti-HLA-G antibody Fab fragment comprises an anti-HLA-G antibody heavy chain variable region, an HLA-G antibody light chain variable region, a CL fragment, and a CH1 fragment, wherein the CL fragment and the CH1 fragment are connected by a disulfide bond, the C-terminus of the anti-HLA-G antibody heavy chain variable region is connected to the N-terminus of the CH1 fragment, and the C-terminus of the anti-HLA-G antibody light chain variable region is connected to the N-terminus of the CL fragment; Optionally, the second anti-HLA-G antibody Fab fragment, the anti-CD3 antibody and the second Fc fragment are linked; Optionally, the C-terminus of the anti-HLA-G antibody Fab fragment is connected to the N-terminus of the anti-CD3 antibody, and the C-terminus of the anti-CD3 antibody is connected to the N-terminus of the second Fc fragment.

8. The bispecific antibody according to claim 1, wherein 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; Optionally, 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: 18 and SEQ ID NO:

20.

9. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the bispecific antibody according to any one of claims 1 to 8; Optionally, the nucleic acid molecule is selected from DNA.

10. An expression vector, characterized in that Carrying the nucleic acid molecule according to claim 9.

11. A method for preparing the bispecific antibody according to any one of claims 1 to 8, characterized in that: include: introducing the expression vector according to claim 10 into a cell; culturing the cells under conditions suitable for protein expression and secretion to obtain the bispecific antibody; Optionally, the cell is a eukaryotic cell.

12. A recombinant cell, characterized in that The recombinant cell carries the bispecific antibody according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, or the expression vector according to claim 10.

13. A pharmaceutical composition, characterized in that include: The bispecific antibody according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, the expression vector according to claim 10, or the recombinant cell according to claim 12; Optionally, further comprising a pharmaceutically acceptable excipient; Optionally, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers; Optionally, the pharmaceutical composition is an injection.

14. Use of the bispecific antibody according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, the expression vector according to claim 10, the recombinant cell according to claim 12, or the pharmaceutical composition according to claim 13 in the preparation of a medicament for treating or preventing a tumor.

15. The use according to claim 14, characterized in that The tumor includes 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.

16. Use of the bispecific antibody according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, the expression vector according to claim 10, the recombinant cell according to claim 12, or the pharmaceutical composition according to claim 13 in the preparation of a kit for detecting CD3 and / or HLA-G.

17. A kit, characterized in that The kit comprises the bispecific antibody according to any one of claims 1 to 8.

18. The kit according to claim 17, characterized in that The kit is used for detecting at least one of CD3 and HLA-G.

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