Bispecific antibody and use thereof

Abstract

Disclosed in the present invention are a bispecific antibody targeting CD3 and PD-L1 and the use thereof. The bispecific antibody contains a first antigen binding region, a second antigen binding region and an Fc portion. The Fc portion contains a first Fc peptide fragment and a second Fc peptide fragment. The first antigen binding region contains an scFv fragment and a binding protein of a first molecule or a fragment thereof, the binding protein of the first molecule or the fragment thereof is linked to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is linked to the N-terminus of the first Fc peptide fragment. The second antigen binding region contains a binding protein of a second molecule or a fragment thereof, and the binding protein of the second molecule or the fragment thereof is linked to the N-terminus of the second Fc peptide fragment. The first molecule and the second molecule are both PD-L1, and the scFv fragment has a CD3 binding activity. The bispecific antibody of the present invention has the advantages of a good safety, a high anti-cancer activity, and a high value in clinical application and drug development.

Description

Bispecific antibodies and their applications Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to a bispecific antibody and its application. More specifically, the present invention relates to an isolated nucleic acid, an expression vector, a recombinant cell, a pharmaceutical composition and a kit and their application. Background Art

[0002] Cancer is a type of disease caused by the unlimited proliferation and spread of abnormal cells. It is a complex group of diseases that includes many types of cancer, such as breast cancer, lung cancer, colorectal cancer, etc. The development of cancer usually involves multiple gene mutations and genetic abnormalities, leading to uncontrolled proliferation and invasive growth of cells. Traditional cancer treatments include surgical resection, radiotherapy and chemotherapy. However, these treatments have many limitations, such as limited feasibility of surgical resection, toxic side effects and drug resistance. Therefore, researchers have been seeking new cancer treatment strategies, one of which is bispecific antibodies.

[0003] Bispecific antibodies are engineered antibodies that can simultaneously bind to two different antigens. They are typically composed of two monoclonal antibody domains and can simultaneously bind to antigens on the surface of tumor cells and antigens on the surface of immune cells (such as T cells). The structure of this bispecific antibody enables them to connect immune cells and cancer cells, thereby promoting the immune cells' attack and killing of cancer cells. The application of bispecific antibodies in cancer treatment has multiple mechanisms and advantages, including: Immune cell activation: Bispecific antibodies can tightly bind immune cells (such as T cells and natural killer cells) to cancer cells, activating the immune cells' killing function and enhancing their attack on cancer cells; Immune cell localization: Bispecific antibodies can localize immune cells to tumor tissues, increasing the local concentration of drugs and reducing toxic side effects on normal tissues; Inhibition of tumor growth signaling pathways: Some bispecific antibodies can inhibit the signaling pathways of tumor growth and metastasis by binding to antigens on the surface of tumor cells; Stimulation of immune response: Bispecific antibodies can activate the immune response, increase antigen presentation, T cell activation, and immune memory. As an emerging therapeutic strategy, bispecific antibodies have shown great potential in the field of cancer treatment. Therefore, there is an urgent need to develop a bispecific antibody with low toxicity and strong tumor killing ability. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0005] The present invention is accomplished based on the following findings of the inventors:

[0006] The bispecific antibody based on CD3 and PD-L1 recruits T cells to reach the local tumor, thereby bridging T cells and tumors, and then promoting T cell activation and killing tumors. This bispecific antibody does not require neoantigens and can guide T cells to kill "cold tumors". However, the bispecific antibody has a strong binding ability with T cells, which may trigger a strong cytokine storm and excessive immune response, thereby causing damage to the body, and severe cases may be life-threatening. The inventor unexpectedly discovered that the CD3 and PD-L1 bispecific antibody with the structure of the present invention can reduce its binding ability with T cells while, compared with the conventional configuration of the bispecific antibody (CD3×PDL1 1:1), it can also improve the killing activity against tumor cells, has the advantages of better anti-cancer activity and higher safety, and has good clinical application and drug development value.

[0007] Therefore, in a first aspect of the present invention, the present invention provides a bispecific antibody. According to an embodiment of the present invention, the bispecific antibody comprises: a first antigen-binding region, a second antigen-binding region, and an Fc portion;

[0008] The Fc portion comprises a first Fc peptide segment and a second Fc peptide segment;

[0009] The first antigen-binding region comprises an scFv fragment and a first molecule of a binding protein or a fragment thereof, wherein the first molecule of the binding protein or the fragment thereof is connected to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is connected to the N-terminus of the first Fc peptide segment; the second antigen-binding region comprises a second molecule of a binding protein or a fragment thereof, wherein the second molecule of the binding protein or the fragment thereof is connected to the N-terminus of the second Fc peptide segment; the first molecule and the second molecule are both PD-L1, and the scFv fragment has CD3 binding activity.

[0010] The bispecific antibodies according to the embodiments of the present invention can simultaneously bind to CD3 and PDL1, promoting T cell activation and cytokine secretion. They have strong binding ability to tumor cells and weak binding ability to T cells (potentially higher safety), thereby further facilitating the distribution of drugs bound to the bispecific antibodies into tumor tissues, increasing the effective concentration of drugs bound to the bispecific antibodies in tumor tissues, and reducing peripheral toxicity. Therefore, the bispecific antibodies according to the embodiments of the present invention have good clinical application and drug development value.

[0011] In a second aspect, the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid encodes the aforementioned bispecific antibody. According to an embodiment of the present invention, the isolated nucleic acid can encode a bispecific antibody that can simultaneously target CD3 and PDL1.

[0012] In a third aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the aforementioned isolated nucleic acid. The expression vector according to an embodiment of the present invention can effectively express the bispecific antibody described in the first aspect of the present invention.

[0013] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell carries the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the bispecific antibody described in the first aspect of the present invention. The recombinant cell according to an embodiment of the present invention is obtained by transfecting or transforming the expression vector described in the third aspect of the present invention. The recombinant cell can efficiently express the bispecific antibody described in the first aspect of the present invention under appropriate conditions.

[0014] In the fifth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the bispecific antibody described in the first aspect of the present invention, the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the recombinant cell described in the fourth aspect of the present invention. As mentioned above, the bispecific antibody of the first aspect can effectively promote T cells to kill tumor cells and has better anti-cancer activity; and the bispecific antibody of the first aspect can be prepared using the above-mentioned isolated nucleic acid, expression vector or recombinant cell. Therefore, the pharmaceutical composition containing the above-mentioned bispecific antibody can be further used to prevent and / or treat related diseases mediated by CD3 and / or PDL1.

[0015] In a sixth aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises the bispecific antibody described in the first aspect of the present invention, the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the recombinant cell described in the fourth aspect of the present invention. The kit according to embodiments of the present invention can effectively detect CD3 protein and / or PDL1 protein.

[0016] In the seventh aspect of the present invention, the invention proposes the use of the bispecific antibody described in the first aspect of the invention, the isolated nucleic acid described in the second aspect of the invention, the expression vector described in the third aspect of the invention, or the recombinant cell described in the fourth aspect of the invention in the preparation of a kit for detecting CD3 and / or PDL1 tumor targets.

[0017] In the eighth aspect of the present invention, the present invention proposes the use of the bispecific antibody described in the first aspect of the present invention, the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the recombinant cell described in the fourth aspect of the present invention, or the pharmaceutical composition described in the fifth aspect of the present invention in the preparation of a medicament for preventing and / or treating diseases mediated by CD3 and / or PDL1. As mentioned above, the bispecific antibody of the first aspect can effectively promote T cells to kill tumor cells and has better anti-cancer activity; and the bispecific antibody of the first aspect can be prepared using the above-mentioned isolated nucleic acid, expression vector or recombinant cell. Therefore, the medicament prepared using the above-mentioned bispecific antibody can be used to prevent or treat diseases mediated by CD3 and / or PDL1.

[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] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0020] FIG1 is a schematic diagram of the CD3×PDL1 bispecific antibody in Example 1 of the present invention.

[0021] FIG2 is a graph showing the ELISA results of the binding of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies to human CD3 protein in Example 2 of the present invention.

[0022] FIG3 is a graph showing the ELISA results of the binding of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies to monkey CD3 protein in Example 2 of the present invention.

[0023] FIG4 is a graph showing the ELISA results of the binding of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies to human PDL1 protein in Example 2 of the present invention.

[0024] FIG5 is a graph showing the ELISA results of the binding of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies to monkey PDL1 protein in Example 2 of the present invention.

[0025] FIG6 is a graph showing the ELISA results of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies bridging CD3 and PDL1 in Example 3 of the present invention.

[0026] FIG7 is a flow cytometry result showing the binding of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies to A-375 human melanoma cells in Example 4 of the present invention.

[0027] FIG8 is a flow cytometry result showing the binding of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies to A549 human lung cancer cells in Example 4 of the present invention.

[0028] FIG9 is a flow cytometry result showing the binding of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies to CD8 T cells in Example 4 of the present invention.

[0029] FIG10 is a graph showing the results of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies in Example 5 of the present invention promoting PBMC to kill PANC-1 human pancreatic cancer cells.

[0030] FIG11 is a graph showing the results of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies in Example 5 of the present invention promoting PBMC to kill A-375 human melanoma cells.

[0031] FIG12 is a graph showing the results of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies in Example 5 of the present invention promoting PBMC to kill HPAFII human pancreatic cancer cells.

[0032] FIG13 is a graph showing the results of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies in Example 5 of the present invention promoting PBMC to kill A549 human lung cancer cells.

[0033] FIG14 is a graph showing the results of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies in Example 5 of the present invention promoting PBMC to kill PC-3 human prostate cancer cells.

[0034] FIG15 is a graph showing the results of CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies in Example 5 of the present invention promoting PBMC to kill HCT-15 human colorectal cancer cells.

[0035] FIG16 is a graph showing the results of CD3×PDL1 2:1 antibody promoting IL-2 secretion by PBMC in Example 6 of the present invention.

[0036] FIG17 is a graph showing the results of CD3×PDL1 2:1 antibody promoting IFN-γ secretion by PBMC in Example 6 of the present invention.

[0037] FIG18 is a graph showing the in vivo efficacy of the CD3×PDL1 2:1 antibody in Example 7 of the present invention in an A-375 human melanoma subcutaneous tumor-bearing mouse model. DETAILED DESCRIPTION

[0038] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0039] It should be noted that 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 the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0040] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0041] Terms and Definitions

[0042] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0043] As used herein, 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, three-chain antibodies, 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.

[0044] Herein, certain regions within the variable region have a higher degree of variability 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.

[0045] As used herein, the term "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which is composed of the VH and CH1 of the heavy chain and a complete light chain, with the light chain and heavy chain connected by a disulfide bond.

[0046] As used herein, the term "F(ab')2 fragment" has two antigen-binding F(ab') portions linked together by a disulfide bond.

[0047] As used herein, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide.

[0048] As used herein, the term "bispecific antibody" is equivalent to "antibody fragment" or "antigen-binding antibody fragment" and may include an intact antibody or a portion thereof, generally the antigen-binding region or variable region. This includes, but is not limited to, Fv, scFv, Fab, Fab', F(ab')2, scFv-Fc fragments, 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.

[0049] In this article, the term "expression vector" generally refers to a carrier medium that carries a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression 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 expression vector also includes a vector with a variety of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0050] As used herein, the term "pharmaceutical composition" generally refers to a substance in unit dosage form, which can be prepared by methods commonly used in the pharmaceutical art. These methods include combining the active ingredient with one or more auxiliary ingredients that constitute a carrier. Typically, a pharmaceutical composition is prepared by thoroughly mixing the active bispecific antibody with a liquid carrier to ensure uniform mixing.

[0051] As used herein, the term "pharmaceutically acceptable" refers to any composition that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic response), ie, a substance with a reasonable benefit / risk ratio.

[0052] 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 for the scope of any conventional excipient being incompatible with the compound of the present invention, such as any adverse biological effect produced or any other component of the pharmaceutically acceptable composition interacting in a harmful manner, their use is also within the scope of the present invention.

[0053] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient via a suitable route. The bispecific antibodies or pharmaceutical compositions of the present invention can be administered via any common route, provided that they reach the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, and subcutaneous administration, but the present invention is not limited to these exemplified modes of administration. The pharmaceutical compositions of the present invention are administered via intravenous or subcutaneous injection.

[0054] As used herein, the term "treatment" is used to refer to obtaining 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 may be 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 an individual who is susceptible to the disease but has 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 to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug as described herein to an individual in need.

[0055] 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.

[0056] The present invention provides a bispecific antibody targeting CD3 and PDL1, an isolated nucleic acid, an expression vector, a recombinant cell, a pharmaceutical composition, a kit, and applications thereof, which are described in detail below.

[0057] Bispecific antibodies

[0058] In a first aspect of the present invention, a bispecific antibody is provided. According to an embodiment of the present invention, the bispecific antibody comprises: a first antigen-binding region, a second antigen-binding region, and an Fc portion;

[0059] The Fc portion comprises a first Fc peptide segment and a second Fc peptide segment;

[0060] The first antigen-binding region comprises an scFv fragment and a first molecule of a binding protein or fragment thereof, wherein the first molecule of the binding protein or fragment thereof is linked to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is linked to the N-terminus of the first Fc peptide segment; the second antigen-binding region comprises a second molecule of a binding protein or fragment thereof, wherein the second molecule of the binding protein or fragment thereof is linked to the N-terminus of the second Fc peptide segment; the first molecule and the second molecule are both PD-L1, and the scFv fragment has CD3 binding activity. The bispecific antibody according to the embodiment of the present invention can simultaneously bind to CD3 on the surface of T cells and to PDL1 on the surface of tumor cells, promoting T cell activation and cytokine secretion. It has strong binding ability to tumor cells and weak binding ability to T cells (helping to reduce nonspecific activation and potential toxic reactions in normal tissues and improve safety), thereby further facilitating the distribution of drugs (small molecule drugs, targeted drugs, radionuclides, cytotoxins, immunomodulators, etc.) bound to the bispecific antibody to tumor tissue, increasing the effective concentration of drugs bound to the bispecific antibody in tumor tissue, and reducing peripheral toxicity. Therefore, the bispecific antibodies according to the embodiments of the present invention have good clinical application and drug development value.

[0061] According to an embodiment of the present invention, the above-mentioned bispecific antibody may further include at least one of the following additional technical features:

[0062] According to an embodiment of the present invention, the binding protein or fragment thereof is selected from at least one of an antibody or an antigen-binding fragment thereof, a receptor, and a ligand.

[0063] According to an embodiment of the present invention, the binding protein or fragment thereof of the first molecule and / or the second molecule is independently selected from at least one of a Fab fragment or a F(ab')2 fragment.

[0064] According to an embodiment of the present invention, the binding protein or fragment thereof of the first molecule is a first Fab fragment, and / or the binding protein or fragment thereof of the second molecule is a second Fab fragment.

[0065] According to an embodiment of the present invention, the first Fab fragment and the second Fab fragment both contain: a first heavy chain variable region CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, a first heavy chain variable region CDR2 with an amino acid sequence as shown in SEQ ID NO: 2, a first heavy chain variable region CDR3 with an amino acid sequence as shown in SEQ ID NO: 3, a first light chain variable region CDR1 with an amino acid sequence as shown in SEQ ID NO: 4, a first light chain variable region CDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and a first light chain variable region CDR3 with an amino acid sequence as shown in SEQ ID NO: 6.

[0066] According to an embodiment of the present invention, the first Fab fragment and / or the second Fab fragment comprises a first heavy chain framework region and / or a first light chain framework region.

[0067] According to an embodiment of the present invention, at least a portion of the first heavy chain framework region and / or the first light chain framework region is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy bovine antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody or a mutant thereof, preferably at least one of a murine antibody, a human antibody and a primate antibody.

[0068] According to an embodiment of the present invention, the first heavy chain variable region of the first Fab fragment and the second Fab fragment are each independently selected from an amino acid sequence as shown in SEQ ID NO: 7 or having at least 90% identity therewith, and / or the first light chain variable region of the first Fab fragment and the second Fab fragment are each independently selected from an amino acid sequence as shown in SEQ ID NO: 8 or having at least 90% identity therewith.

[0069] According to an embodiment of the present invention, the first Fab fragment and / or the second Fab fragment further comprises a CH1 fragment and a CL fragment;

[0070] The C-terminus of the first heavy chain variable region is connected to the N-terminus of the CH1 fragment, the C-terminus of the first light chain variable region is connected to the N-terminus of the CL fragment, and the CH1 fragment and the CL fragment are connected via an interchain disulfide bond.

[0071] According to an embodiment of the present invention, the CH1 fragment has an amino acid sequence as shown in SEQ ID NO: 25, and / or the CL fragment has an amino acid sequence as shown in SEQ ID NO: 26.

[0072] According to an embodiment of the present invention, the C-terminus of the CH1 fragment of the first Fab fragment is connected to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is connected to the N-terminus of the first Fc peptide segment; and / or the C-terminus of the CH1 fragment of the second Fab fragment is connected to the N-terminus of the second Fc peptide segment.

[0073] According to an embodiment of the present invention, the first Fab fragment and the second Fab fragment both comprise:

[0074] A first peptide segment having an amino acid sequence as shown in SEQ ID NO: 27, and a second peptide segment having an amino acid sequence as shown in SEQ ID NO: 28.

[0075] According to an embodiment of the present invention, the scFv fragment comprises: a second heavy chain variable region CDR1 with an amino acid sequence as shown in SEQ ID NO: 9, a second heavy chain variable region CDR2 with an amino acid sequence as shown in SEQ ID NO: 10, a second heavy chain variable region CDR3 with an amino acid sequence as shown in SEQ ID NO: 11, a second light chain variable region CDR1 with an amino acid sequence as shown in SEQ ID NO: 12, a second light chain variable region CDR2 with an amino acid sequence as shown in SEQ ID NO: 13, and a second light chain variable region CDR3 with an amino acid sequence as shown in SEQ ID NO: 14. Thus, when the scFv fragment of the bispecific antibody of the present invention has the above-mentioned CDRs amino acid sequences, it can further reduce CD3 binding activity and enhance tumor cell binding ability, and can further enhance the activity of promoting T cells to kill tumor cells.

[0076] According to an embodiment of the present invention, the scFv fragment comprises a second heavy chain framework region and / or a second light chain framework region.

[0077] According to an embodiment of the present invention, at least a portion of the second heavy chain framework region and / or the second light chain framework region is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody or a mutant thereof, preferably at least one of a murine antibody, a human antibody and a primate antibody.

[0078] It should be noted that the immunoglobulins described herein can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules, including engineered subclasses with altered Fc portions that provide reduced or enhanced effector cell activity. The immunoglobulins can be derived from any species.

[0079] According to an embodiment of the present invention, the scFv fragment comprises:

[0080] A second heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 15, or at least 90% identical thereto, and a second light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 16, or at least 90% identical thereto.

[0081] According to an embodiment of the present invention, the scFv fragment further comprises: connecting peptide 1;

[0082] wherein the N-terminus of the connecting peptide 1 is connected to the C-terminus of the second heavy chain variable region, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the second light chain variable region; or

[0083] The N-terminus of the connecting peptide 1 is connected to the C-terminus of the second light chain variable region, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the second heavy chain variable region.

[0084] According to an embodiment of the present invention, the connecting peptide 1 has an amino acid sequence as shown in (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0085] According to an embodiment of the present invention, the scFv fragment has the amino acid sequence shown in SEQ ID NO:17.

[0086] According to an embodiment of the present invention, the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO:18.

[0087] According to an embodiment of the present invention, the first antigen binding region further includes a connecting peptide 2.

[0088] The C-terminus of CH1 of the first Fab fragment is connected to the N-terminus of the connecting peptide 2, and the C-terminus of the connecting peptide 2 is connected to the N-terminus of the scFv fragment.

[0089] According to an embodiment of the present invention, the connecting peptide 2 has an amino acid sequence as shown in (GGGGS)n, wherein n is an integer greater than or equal to 1.

[0090] According to an embodiment of the present invention, the connecting peptide 2 has an amino acid sequence as shown in (GGGGS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0091] According to an embodiment of the present invention, the connecting peptide 2 has an amino acid sequence as shown in SEQ ID NO:19.

[0092] According to an embodiment of the present invention, the first Fc peptide segment and the second Fc peptide segment are connected via a knob-into-hol structure.

[0093] According to an embodiment of the present invention, the first Fc peptide segment and the second Fc peptide segment are both human Fc peptide segments.

[0094] According to an embodiment of the present invention, the human Fc peptide segment is a human IgG1 Fc peptide segment.

[0095] According to an embodiment of the present invention, the first Fc peptide segment has an amino acid sequence as shown in SEQ ID NO: 20, and the second Fc peptide segment has an amino acid sequence as shown in SEQ ID NO: 21.

[0096] According to an embodiment of the present invention, the bispecific antibody comprises: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 22 or an amino acid sequence with at least 80% identity thereto; a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 23 or an amino acid sequence with at least 80% identity thereto; a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 24 or an amino acid sequence with at least 80% identity thereto; and a fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 24 or an amino acid sequence with at least 80% identity thereto.

[0097] It should be noted that, under the premise of not substantially affecting the CD3 and PD-L1 binding activity of the multispecific 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 amino acid sequence of the polypeptide chain contained in the multispecific antibody of the present invention to obtain variants of the amino acid sequence of the polypeptide chain of the bispecific antibody. 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 polypeptide chain. The amino acid sequence of the above-mentioned variant of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference amino acid sequence.

[0098] According to an embodiment of the present invention, the bispecific antibody comprises: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 22, a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 23, a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 24, and a fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 24. The bispecific antibody has high tumor cell binding ability and low T cell binding ability, has strong cancer cell killing ability and high safety. In particular, the inventors unexpectedly discovered that the bispecific antibody of the present invention, while reducing T cell binding ability, further improves tumor cell killing ability, has good anti-cancer activity and good clinical application and drug development value.

[0099] Isolated nucleic acids

[0100] In a second aspect, the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid encodes the aforementioned bispecific antibody. According to an embodiment of the present invention, the isolated nucleic acid can encode a bispecific antibody that can simultaneously target CD3 and PDL1.

[0101] It should be noted that, for nucleic acid molecules mentioned herein, 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 herein, the other strand complementary thereto is also disclosed. In addition, if the amino acid sequence of a molecule in the present invention includes either a DNA form or an RNA form, disclosure of one form implies disclosure of the other.

[0102] Those skilled in the art will appreciate that the aforementioned features and advantages of bispecific antibodies are also applicable to the isolated nucleic acid and will not be elaborated here.

[0103] expression vector

[0104] In a third aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the aforementioned isolated nucleic acid. The expression vector according to an embodiment of the present invention can effectively express the bispecific antibody described in the first aspect of the present invention.

[0105] When the isolated nucleic acid is linked to the expression vector, the isolated nucleic acid can be directly or indirectly linked to the control elements on the expression vector, as long as these control elements are capable of controlling translation and expression of the isolated nucleic acid. Of course, these control elements can be directly derived from the expression vector itself or exogenous, i.e., not derived from the expression vector itself. The isolated nucleic acid and the control elements can be operably linked.

[0106] According to an embodiment of the present invention, the isolated nucleic acid can be obtained by operably linking the isolated nucleic acid to a commercially available expression vector (such as a plasmid or viral vector). The expression vector in the present invention is not particularly limited, and commonly used plasmids such as pSeTag2, PEE14, pMH3, etc. can be used.

[0107] As used herein, the term "operably linked" refers to linking an exogenous gene to a vector so that control elements within the vector, such as amino acid sequences for transcriptional control and amino acid sequences for translational control, 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, upon introduction into appropriate recipient cells, can effectively express the aforementioned isolated nucleic acid under the mediation of a regulatory system, thereby enabling the in vitro production of large quantities of the protein encoded by the isolated nucleic acid.

[0108] According to an embodiment of the present invention, the above expression vector may further include at least one of the following additional technical features:

[0109] According to an embodiment of the present invention, the expression vector is a eukaryotic vector or a prokaryotic vector.

[0110] According to an embodiment of the present invention, the expression vector includes at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.

[0111] Those skilled in the art will appreciate that the aforementioned features and advantages of bispecific antibodies are also applicable to this expression vector and will not be elaborated here.

[0112] recombinant cells

[0113] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell carries the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the bispecific antibody described in the first aspect of the present invention. The recombinant cell according to an embodiment of the present invention is obtained by transfecting or transforming the expression vector described in the third aspect of the present invention. The recombinant cell can efficiently express the bispecific antibody described in the first aspect of the present invention under appropriate conditions.

[0114] According to an embodiment of the present invention, the recombinant cell may further include at least one of the following additional technical features:

[0115] According to an embodiment of the present invention, the recombinant cell is a prokaryotic cell, a eukaryotic cell or a bacteriophage.

[0116] According to an embodiment of the present invention, the prokaryotic cell is Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0117] According to an embodiment of the present invention, the eukaryotic cell is a fungus, an insect cell, a plant cell or a mammalian cell.

[0118] According to an embodiment of the present invention, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Trichoderma.

[0119] According to an embodiment of the present invention, the insect cell is a fall armyworm cell; according to an embodiment of the present invention, the plant cell is a tobacco plant cell; according to an embodiment of the present invention, the mammalian cell is a BHK cell, a CHO cell, a COS cell, a myeloma cell or a human embryonic kidney 293 cell; and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0120] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0121] According to an embodiment of the present invention, the recombinant cell is a BHK cell, a CHO cell, a COS cell or a NSO cell.

[0122] It should be noted that the "suitable conditions" described in this specification refer to conditions suitable for the expression of the bispecific antibodies described herein. 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 the bispecific antibodies based on the specific laboratory environment.

[0123] Those skilled in the art will appreciate that the aforementioned features and advantages of bispecific antibodies are also applicable to the recombinant cell and will not be elaborated here.

[0124] Pharmaceutical composition

[0125] In the fifth aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the bispecific antibody described in the first aspect of the present invention, the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the recombinant cell described in the fourth aspect of the present invention. According to an embodiment of the present invention, the bispecific antibody can effectively promote PBMC to kill tumor cells, has better anti-cancer activity, and has good clinical application value and drug development value. Therefore, the pharmaceutical composition containing the above-mentioned bispecific antibody can be further used to prevent and / or treat related diseases mediated by CD3 and / or PDL1.

[0126] According to an embodiment of the present invention, the pharmaceutical composition may further include at least one of the following additional technical features:

[0127] According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0128] According to an embodiment of the present invention, the pharmaceutical composition is an injection.

[0129] 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.

[0130] The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable excipient. Such excipients include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical composition formulation should be compatible with the mode of administration. The pharmaceutical composition of the present invention is in the form of an injection. For example, it can be prepared using conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0131] The effective amount of the bispecific antibody of the present invention 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 one of ordinary skill in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to, pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be proportionally reduced.

[0132] The pharmaceutically acceptable excipients of the present invention 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.

[0133] Those skilled in the art will appreciate that the aforementioned features and advantages of the bispecific antibody are also applicable to the pharmaceutical composition and will not be elaborated here.

[0134] Reagent test kit

[0135] In a sixth aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the bispecific antibody of the first aspect of the present invention, the isolated nucleic acid of the second aspect of the present invention, the expression vector of the third aspect of the present invention, or the recombinant cell of the fourth aspect of the present invention.

[0136] As described above, the bispecific antibodies of the embodiments of the present invention can specifically bind to CD3 and PDL1. The kits containing CD3 protein and / or PDL1 protein developed using this property can be used for research related to CD3 protein and / or PDL1 protein, such as for detecting and / or enriching and / or isolating and purifying CD3 protein and / or PDL1 protein from humans or other mammals.

[0137] The kit can effectively detect, enrich or separate and purify CD3 protein and / or PDL1 protein in biological samples, and further be used for scientific research, such as qualitative or quantitative detection of CD3 protein and / or PDL1 protein molecules in biological samples. More specifically, it can be used for immunoblotting, immunoprecipitation, and other kits that involve the use of CD3 protein and / or PDL1 protein and antibody specific binding properties for detection. These kits may contain any one or more of the following: antagonists, bispecific antibodies of the present invention or drug reference materials, protein purification columns, immunoglobulin affinity purification buffers, and cell assay diluents. The bispecific antibodies of the present invention can be used for different types of diagnostic tests, for example, to detect the presence of various diseases or drugs, toxins or other proteins in vitro or in vivo. For example, it can be used to test CD3 and / or PDL1-mediated related diseases by testing the serum or blood of the subject.

[0138] Those skilled in the art will appreciate that the aforementioned features and advantages of bispecific antibodies are also applicable to this kit and will not be described in detail here.

[0139] Use in the preparation of kits

[0140] In the seventh aspect of the present invention, the invention proposes the use of the bispecific antibody described in the first aspect of the invention, the isolated nucleic acid described in the second aspect of the invention, the expression vector described in the third aspect of the invention, or the recombinant cell described in the fourth aspect of the invention in the preparation of a kit for detecting CD3 and / or PDL1 tumor targets.

[0141] According to an embodiment of the present invention, the tumor target is PD-L1.

[0142] According to an embodiment of the present invention, the PD-L1-mediated diseases include cancer or transplant rejection, autoimmune diseases and infectious diseases.

[0143] According to an embodiment of the present invention, the cancer includes 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] As previously mentioned, the bispecific antibodies of the embodiments of the present invention are capable of specifically binding to CD3 and PDL1. Therefore, the bispecific antibodies can be used to detect CD3 and / or PDL1. Furthermore, they can be used to prepare CD3 and / or PDL1-related kits and used in scientific research, such as for the qualitative or quantitative detection of CD3 and / or PDL1 protein molecules in biological samples. More specifically, they can be used in kits such as immunoblotting and immunoprecipitation that utilize the specific binding properties of CD3 and / or PDL1 and antibodies for detection. These kits may contain any one or more of the following: antagonists, bispecific antibodies of the present invention or drug reference materials, protein purification columns, immunoglobulin affinity purification buffers, and cell assay diluents. The bispecific antibodies of the present invention can be used in different types of 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 detect CD3 and / or PDL1 by testing the serum or blood of a subject.

[0145] Those skilled in the art will appreciate that the aforementioned features and advantages of bispecific antibodies are also applicable to this use and will not be elaborated here.

[0146] Use in preparing medicines

[0147] In the eighth aspect of the present invention, the present invention proposes the use of the bispecific antibody described in the first aspect of the present invention, the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the recombinant cell described in the fourth aspect of the present invention, or the pharmaceutical composition described in the fifth aspect of the present invention in the preparation of a medicament for preventing and / or treating CD3 and / or PDL1-mediated related diseases.

[0148] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0149] According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0150] According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0151] Those skilled in the art will appreciate that the aforementioned features and advantages of bispecific antibodies are also applicable to this use and will not be elaborated here.

[0152] Disease treatment methods

[0153] In a ninth aspect, the present invention provides a method for preventing and / or treating diseases mediated by CD3 and / or PDL1. According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned bispecific antibody, the aforementioned isolated nucleic acid, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned pharmaceutical composition.

[0154] 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.

[0155] The effective amount of the bispecific antibody, isolated nucleic acid, expression vector, recombinant cell, or pharmaceutical composition of the present invention 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 one 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, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, and the like. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be proportionally reduced.

[0156] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0157] According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0158] According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0159] According to an embodiment of the present invention, the PDL1-mediated disease is cancer, a disease caused by transplant rejection, an autoimmune disease, or an infectious disease.

[0160] According to an embodiment of the present invention, 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.

[0161] Those skilled in the art will appreciate that the aforementioned features and advantages of bispecific antibodies are also applicable to this method and will not be elaborated here.

[0162] The amino acid sequences involved in the present invention are detailed in Table 1.

[0163] Table 1: Amino acid sequence description

[0164] The present invention will be described in detail below through examples. In the examples or test examples, if no specific conditions are specified, the experimental methods were carried out under conventional conditions.

[0165] 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 indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0166] Example 1: Production of antibodies

[0167] The specific experimental procedures for antibody production are as follows: (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×10 6 / mL to obtain ExpiCHO cell solution. (2) When the bispecific antibody is in A configuration (Figure 1), the pcDNA3.4 vector containing the three coding sequences of CD3 antibody, PD-L1 antibody heavy chain, and PD-L1 antibody light chain (commissioned by Nanjing GenScript for synthesis) is added to 2mL OptiSFM culture medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a; or, when the bispecific antibody is in B configuration (Figure 1), the pcDNA3.4 vector containing the three coding sequences of PD-L1 heavy chain-CD3 antibody, PD-L1 antibody heavy chain, and PD-L1 antibody light chain (commissioned by Nanjing GenScript for synthesis) is added to 2mL OptiSFM culture medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a. (3) 160μL ExpiCHO Expression Medium transfection reagent (purchased from Thermo Fisher) is added to 2mL OptiSFM culture medium (purchased from Thermo Fisher) to obtain solution b. (4) Solution a and solution b were then mixed to obtain a transfection mixture, and the entire transfection mixture was added to 50 mL of ExpiCHO cell solution within 5 minutes. (5) After culturing at 37°C, 5% CO2 for 1 day, 8 mL of feed and 300 μL of Enhancer (purchased from Thermo Fisher) were added, and the cells were transferred to 32°C, 5% CO2 for 9 days. The culture supernatant was harvested, with 8 mL of feed added on the 5th day. (6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from NanoMicro).

[0168] In this example, two CD3 and PD-L1 bispecific antibodies were prepared (one with a conventional CD3 × PD-L1 1:1 configuration) to investigate the various properties of the bispecific antibodies of the present invention. For descriptions of the configurations and amino acid sequences of each antibody, see Figure 1, Tables 1, and 2.

[0169] Table 2: Bispecific antibody structure and corresponding amino acid sequence in Example 1

[0170] Note: The configurations of class A and B antibodies are shown in Figure 1.

[0171] Example 2: ELISA binding experiment of the antibody of the present invention

[0172] ELISA is used to test the binding properties of bispecific antibodies. The antigen protein is coated onto a 96-well plate, and the strength of the signal after the antibody is added is used to determine the binding properties of the bispecific antibody and the antigen protein.

[0173] (1) Human PDL1-His protein, monkey PDL1 protein, human CD3E&D protein, and monkey CD3E&D protein (purchased from Kaixia) were diluted to 2 μg / mL with PBS buffer and added to a 96-well plate at a volume of 100 μL / well. The plate was incubated at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer. 200 μL / well of PBS / 10% BSA was added and the plate was incubated at 37°C for 2 h for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST. Then, 100 μL / well of the bispecific antibody to be tested, CD3×PDL1 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 29, the amino acid sequence of the PDL1 antibody heavy chain is shown in SEQ ID NO: 23, and the amino acid sequence of the PDL1 antibody light chain is shown in SEQ ID NO: 24), CD3×PDL1 2:1 (the amino acid sequence of the PDL1 antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 22, the amino acid sequence of the PDL1 antibody heavy chain is shown in SEQ ID NO: 23, and the amino acid sequence of the PDL1 antibody light chain is shown in SEQ ID NO: 24), and the control hIgG1LALA (purchased from Bio-Tech) were added in a gradient dilution with PBST / 0.05% BSA, and incubated at 37°C for 1 h. Remove the reaction mixture, wash the plate six times with PBST, and then add 100 μL / well of HRP (horseradish peroxidase)-labeled anti-human IgG secondary antibody (purchased from Jackson Lab) diluted in PBST / 0.05% BSA. Incubate at 37°C for 1 hour. Wash the plate six times with PBST, then add 80 μL / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and terminate the reaction by adding 80 μL / well of 4 M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0174] The results are shown in Figure 2 . Both CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies were able to bind to human CD3E&D protein with comparable binding abilities.

[0175] The results are shown in Figure 3 . Both CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies were able to bind to monkey CD3E&D protein with comparable binding abilities.

[0176] The results are shown in Figure 4. Both CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies were able to bind to human PDL1-His protein with comparable binding abilities.

[0177] The results are shown in Figure 5. Both CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies were able to bind to monkey PDL1 protein with comparable binding abilities.

[0178] Example 3: Antibody ELISA bridging experiment

[0179] ELISA assays were used to test the bridging binding properties of bispecific antibodies. CD3E&D antigen proteins were coated onto 96-well plates. After the antibodies were added, the cells were detected using biotinylated PDL1 protein. The signal strength was used to determine the binding properties of the bispecific antibody bridging CD3E&D and PDL1 proteins.

[0180] CD3 E&D protein (purchased from Acro) was diluted to 2 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL / well. The plate was incubated at 4°C overnight. The PBS buffer in the 96-well plate was aspirated and washed six times with PBST (PBS pH 7.2 containing 0.1% Tween 20). Then, 200 μL / well of PBS / 10% BSA was added and the plate was incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed six times with PBST. Then, 100 μL / well of a 1:1 dilution of the bispecific antibody to be tested (CD3×PDL1 1:1, CD3 single-chain antibody amino acid sequence shown in SEQ ID NO:29, PDL1 heavy chain amino acid sequence shown in SEQ ID NO:23, and PDL1 light chain amino acid sequence shown in SEQ ID NO:24) and CD3×PDL1 2:1 (PDL1 heavy chain-CD3 single-chain antibody amino acid sequence shown in SEQ ID NO:22, PDL1 heavy chain amino acid sequence shown in SEQ ID NO:23, and PDL1 light chain amino acid sequence shown in SEQ ID NO:24) were added, along with control hIgG1LALA (purchased from Bio-Bio). The reaction mixture was removed, and the plate was washed six times with PBST. PDL1-Biotin diluted to the appropriate concentration was added, and the plate was incubated at 37°C for 1 hour. Remove the reaction mixture, wash the plate six times with PBST, and then add 100 μL / well of HRP (horseradish peroxidase)-conjugated Streptavidin secondary antibody (purchased from Southern Biotech) diluted in PBST / 0.05% BSA. Incubate at 37°C for 1 hour. Wash the plate six times with PBST, then add 80 μL / well of TMB (tetramethylbenzidine). Incubate at room temperature for 3 minutes. Terminate the reaction by adding 80 μL / well of 4 M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0181] The results are shown in Figure 6 . CD3×PDL1 1:1 and CD3×PDL1 2:1 antibodies were able to bridge CD3E&D and PDL1 proteins, and the bridging activities of the two antibodies were comparable.

[0182] Example 4: Bispecific Antibody Flow Cytometry Binding Experiment

[0183] Flow cytometry experiments are used to detect the binding properties of bispecific antibodies. Antibodies are added to cells, and the strength of the signal after the addition of the antibodies is used to determine the binding properties of the antibodies and cells.

[0184] (1) A-375 human melanoma cells and A549 human lung cancer cells were diluted to 2×10 6 / mL, added to a volume of 100 μL / tube in a 1.5 mL EP tube, and 10 μL / tube of goat serum was added. The tube was blocked at 4°C for 30 min. A serial dilution of CD3×PDL1 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:29, the amino acid sequence of the PDL1 antibody heavy chain is shown in SEQ ID NO:23, and the amino acid sequence of the PDL1 antibody light chain is shown in SEQ ID NO:24), CD3×PDL1 2:1 (the amino acid sequence of the PDL1 antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO:22, the amino acid sequence of the PDL1 antibody heavy chain is shown in SEQ ID NO:23, and the amino acid sequence of the PDL1 antibody light chain is shown in SEQ ID NO:24), and the control hIgG1LALA (purchased from Bio-Bio) were added and incubated at 4°C for 30 min. 1 mL of PBS was added to the EP tube, and the tube was centrifuged at 3500 rpm for 5 min at 4°C. The supernatant was discarded and the tube was washed once with PBS. After centrifugation, discard the supernatant 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 incubate at 4°C in the dark for 30 min. Wash twice with PBS and centrifuge. Discard the supernatant. Resuspend the cells in 200 μL / tube of PBS and analyze using flow cytometry.

[0185] The results are shown in Figure 7 . Both CD3×PDL1 1:1 and CD3×PDL1 2:1 were able to bind to A-375 human melanoma cells, and the binding activities of the two antibodies were comparable.

[0186] The results are shown in Figure 8 . Both CD3×PDL1 1:1 and CD3×PDL1 2:1 were able to bind to A549 human lung cancer cells, and the binding activities of the two antibodies were comparable.

[0187] (2) 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, and the tube was blocked at 4°C for 30min. Serial dilutions of the bispecific antibody CD3×PDL1 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:29, the amino acid sequence of the PDL1 antibody heavy chain is shown in SEQ ID NO:23, and the amino acid sequence of the PDL1 antibody light chain is shown in SEQ ID NO:24), CD3×PDL1 2:1 (the amino acid sequence of the PDL1 antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO:22, the amino acid sequence of the PDL1 antibody heavy chain is shown in SEQ ID NO:23, and the amino acid sequence of the PDL1 antibody light chain is shown in SEQ ID NO:24), and the control hIgG1LALA (purchased from Bio-Bio) were added and incubated at 4°C for 30min. 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 using a flow cytometer.

[0188] The results are shown in Figure 9 . Both CD3×PDL1 1:1 and CD3×PDL1 2:1 were able to bind to T cells, and the CD3×PDL1 2:1 antibody bound to T cells weaker than the conventional CD3×PDL1 1:1 antibody.

[0189] Example 5: Experiment on Bispecific Antibodies Promoting PBMC to Kill Tumor Cells

[0190] Detect the ability of bispecific antibodies to promote PBMC to kill tumor cells.

[0191] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate.

[0192] (2) PANC-1 human pancreatic cancer cells, A-375 human melanoma cells, HPAF II human pancreatic cancer cells, A549 human lung cancer cells, PC-3 human prostate cancer cells, and HCT-15 human colorectal cancer 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 a ×CELLigence RTCAMP device at 37°C and 5% CO2 for 24 h;

[0193] (3) Add the gradient dilutions of the bispecific antibody CD3×PDL1 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 29, the amino acid sequence of the PDL1 antibody heavy chain is shown in SEQ ID NO: 23, and the amino acid sequence of the PDL1 antibody light chain is shown in SEQ ID NO: 24), CD3×PDL1 2:1 (the amino acid sequence of the PDL1 antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 22, the amino acid sequence of the PDL1 antibody heavy chain is shown in SEQ ID NO: 23, and the amino acid sequence of the PDL1 antibody light chain is shown in SEQ ID NO: 24), and the control hIgG1LALA (purchased from Bio-Tech) to the RTCA plate obtained in step (2) using complete RPMI-1640 medium, with an addition volume of 20 μL / well;

[0194] (4) PBMC (purchased from Saili Bio) were diluted to 1.25×10 6 pcs / mL, added to the RTCA plate obtained in step (3), with an addition volume of 80 μL / well;

[0195] (5) The reaction system obtained in step (4) was incubated at 37°C and 5% CO2 for 24 hours using a ×CELLigence RTCAMP device to detect the cell coefficient.

[0196] The results, as shown in Figures 10, 11, 12, 13, 14, and 15, show that both CD3×PDL1 1:1 and CD3×PDL1 2:1 can promote PBMCs to kill PANC-1 human pancreatic cancer cells, A-375 human melanoma cells, HPAF II human pancreatic cancer cells, A549 human lung cancer cells, PC-3 human prostate cancer cells, and HCT-15 human colorectal cancer cells. Furthermore, the inventors unexpectedly discovered that while the CD3×PDL1 2:1 antibody has a weakened ability to bind to T cells, its ability to promote T cell tumor killing is stronger than that of the conventional CD3×PDL1 1:1 antibody.

[0197] Example 6: Experiment on Bispecific Antibodies Promoting Cytokine Secretion by PBMC

[0198] Bispecific antibodies were added to the co-incubation system of PBMC and HCT-15 human colorectal cancer cells. After 48 hours of culture, the culture supernatant was collected and the cytokine content in the supernatant was detected to determine the characteristics of cytokine release triggered by the bispecific antibody.

[0199] (1) HCT-15 cells were diluted to 1×10 5 / mL, added to 96-well plates, and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0200] (2) Using complete RPMI 1640 medium, the bispecific antibody CD3×PDL1 2:1 (the amino acid sequence of the PDL1 antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 22, the amino acid sequence of the PDL1 antibody heavy chain is shown in SEQ ID NO: 23, and the amino acid sequence of the PDL1 antibody light chain is shown in SEQ ID NO: 24) was serially diluted and added to a 96-well plate at 20 μL / well;

[0201] (3) PBMC (purchased from Saili Biotechnology) were diluted to 1.25×10 6 / mL, added to 96-well plate, 80μL / well;

[0202] (4) The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 48 h;

[0203] (5) Centrifuge at 300 g for 10 min at room temperature and collect the cell culture supernatant;

[0204] (6) The cytokine content in the supernatant was detected using a CBA kit (purchased from BD).

[0205] The results are shown in Figures 16 and 17 . In the presence of target cells, CD3×PDL1 2:1 can activate PBMCs and promote their secretion of cytokines IL-2 and IFN-γ.

[0206] The above results show that the 2:1 configuration (configuration B) bispecific antibody of the present invention has the ability to promote PBMC to secrete immune-activating cytokines.

[0207] Example 7: Anticancer Experiment of Dual Antibodies in Mouse Models

[0208] In vivo efficacy experiments were used to detect the anti-cancer function of the bispecific antibody of the present invention in promoting immune reconstitution in mice.

[0209] (1) On day -8, NSG mice were subcutaneously injected with 5×10 6 A-375 human melanoma cells;

[0210] (2) On day -8, human PBMC (purchased from Saili Biotechnology) were transfused into NSG mice (purchased from Southern Model) through the tail vein at an injection volume of 5×10 6 / Only;

[0211] (3) On day 0, the mice were weighed and the tumor volume was measured. Based on the weighing results and tumor volume, the mice were divided into a control group and a treatment group;

[0212] (4) On days 0, 3, and 6, mice were injected with the CD3×PDL1 2:1 mixture (the amino acid sequence of the PDL1 antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 22, the amino acid sequence of the PDL1 antibody heavy chain is shown in SEQ ID NO: 23, and the amino acid sequence of the PDL1 antibody light chain is shown in SEQ ID NO: 24) and the solvent control PBS, 250 μL per mouse;

[0213] (5) After injection of the above antibodies, the tumor volume was measured and the mice were weighed twice a week.

[0214] As shown in FIG18 , the results showed that CD3×PDL1 2:1 administered at a dose of 0.1 mg / kg had significant anticancer activity.

[0215] The above experimental results show that the bispecific antibodies of the present invention can bind to CD3 and PDL1, thereby promoting T cell activation and cytokine secretion, effectively promoting PBMC to kill tumor cells, and have good anti-cancer activity; the bispecific antibodies of the present invention can achieve high-affinity binding to tumor cells and low-affinity binding to T cells, with higher anti-cancer activity and higher safety. In summary, the bispecific antibodies of the present invention can promote immune cells to fight cancer, have good anti-cancer activity, and higher safety, and have good clinical application value and drug development value.

[0216] The preferred embodiments of the present invention have been 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 disclosed in the present invention and fall within the scope of protection of the present invention.

[0217] 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.

[0218] 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: Comprising: a first antigen binding region, a second antigen binding region and an Fc portion; The Fc portion comprises a first Fc peptide segment and a second Fc peptide segment; The first antigen binding region comprises an scFv fragment and a first molecule of a binding protein or a fragment thereof, the first molecule of a binding protein or a fragment thereof is connected to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is connected to the N-terminus of the first Fc peptide segment; The second antigen binding region comprises a second molecule of binding protein or a fragment thereof, and the second molecule of binding protein or a fragment thereof is connected to the N-terminus of the second Fc peptide segment; The first molecule and the second molecule are both PD-L1, and the scFv fragment has CD3 binding activity.

2. The bispecific antibody according to claim 1, characterized in that The binding protein or fragment thereof is selected from at least one of an antibody or an antigen-binding fragment thereof, a receptor, and a ligand.

3. The bispecific antibody according to claim 1, characterized in that The binding protein or fragment thereof of the first molecule and / or the second molecule is independently selected from at least one of a Fab or a F(ab')2 fragment.

4. The bispecific antibody according to claim 1, characterized in that The binding protein or fragment thereof of the first molecule is a first Fab fragment, and / or the binding protein or fragment thereof of the second molecule is a second Fab fragment.

5. The bispecific antibody according to claim 4, characterized in that The first Fab fragment and the second Fab fragment both comprise: The amino acid sequence of the first heavy chain variable region CDR1 is shown in SEQ ID NO: 1, The amino acid sequence of the first heavy chain variable region CDR2 is shown in SEQ ID NO: 2, The amino acid sequence of the first heavy chain variable region CDR3 is shown in SEQ ID NO: 3, The amino acid sequence of the first light chain variable region CDR1 is shown in SEQ ID NO: 4, The amino acid sequence of the first light chain variable region CDR2 is shown in SEQ ID NO:5, The amino acid sequence of the first light chain variable region CDR3 is shown in SEQ ID NO:

6.

6. The bispecific antibody according to claim 4, characterized in that The first Fab fragment and / or the second Fab fragment comprises a first heavy chain framework region and / or a first light chain framework region.

7. The bispecific antibody according to claim 6, characterized in that At least a portion of the first heavy chain framework region and / or the first light chain framework region is derived from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody or a mutant thereof, preferably at least one of a mouse antibody, a human antibody and a primate antibody.

8. The bispecific antibody according to claim 5, characterized in that The first heavy chain variable regions of the first Fab fragment and the second Fab fragment are each independently selected from an amino acid sequence as shown in SEQ ID NO:7 or having at least 90% identity thereto, and / or the first light chain variable regions of the first Fab fragment and the second Fab fragment are each independently selected from an amino acid sequence as shown in SEQ ID NO:8 or having at least 90% identity thereto.

9. The bispecific antibody according to claim 5, characterized in that The first Fab fragment and / or the second Fab fragment further comprises a CH1 fragment and a CL fragment; The C-terminus of the first heavy chain variable region is connected to the N-terminus of the CH1 fragment, the C-terminus of the first light chain variable region is connected to the N-terminus of the CL fragment, and the CH1 fragment and the CL fragment are connected via an interchain disulfide bond.

10. The bispecific antibody according to claim 9, characterized in that The CH1 fragment has the amino acid sequence shown in SEQ ID NO:25, and / or the CL fragment has the amino acid sequence shown in SEQ ID NO:

26.

11. The bispecific antibody according to claim 4, characterized in that The first Fab fragment and the second Fab fragment both comprise: A first peptide segment having an amino acid sequence as shown in SEQ ID NO:27, and a second peptide segment having an amino acid sequence as shown in SEQ ID NO:

28.

12. The bispecific antibody according to claim 9, characterized in that The C-terminus of the CH1 fragment of the first Fab fragment is connected to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is connected to the N-terminus of the first Fc peptide segment; and / or The C-terminus of the CH1 fragment of the second Fab fragment is connected to the N-terminus of the second Fc peptide segment.

13. The bispecific antibody according to any one of claims 1 to 12, characterized in that: The scFv fragment comprises: The amino acid sequence of the second heavy chain variable region CDR1 is shown in SEQ ID NO:9, The second heavy chain variable region CDR2 has an amino acid sequence as shown in SEQ ID NO: 10, The second heavy chain variable region CDR3 having an amino acid sequence as shown in SEQ ID NO: 11, The amino acid sequence of the second light chain variable region CDR1 is shown in SEQ ID NO: 12, The amino acid sequence of the second light chain variable region CDR2 is shown in SEQ ID NO: 13, The amino acid sequence of the second light chain variable region CDR3 is shown in SEQ ID NO:

14.

14. The bispecific antibody according to any one of claims 1 to 12, characterized in that: The scFv fragment comprises a second heavy chain framework region and / or a second light chain framework region.

15. The bispecific antibody according to claim 14, characterized in that At least a portion of the second heavy chain framework region and / or the second light chain framework region is derived from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody or a mutant thereof, preferably at least one of a mouse antibody, a human antibody and a primate antibody.

16. The bispecific antibody according to any one of claims 1 to 12, characterized in that: The scFv fragment comprises: A second heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, or at least 90% identical thereto, and a second light chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, or at least 90% identical thereto.

17. The bispecific antibody according to claim 16, characterized in that The scFv fragment further comprises: a connecting peptide 1; wherein the N-terminus of the connecting peptide 1 is connected to the C-terminus of the second heavy chain variable region, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the second light chain variable region; or The N-terminus of the connecting peptide 1 is connected to the C-terminus of the second light chain variable region, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the second heavy chain variable region.

18. The bispecific antibody according to claim 17, characterized in that The connecting peptide 1 has an amino acid sequence as shown in (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

19. The bispecific antibody according to claim 17, characterized in that The scFv fragment has the amino acid sequence shown in SEQ ID NO:

17.

20. The bispecific antibody according to claim 17, characterized in that The connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO:

18.

21. The bispecific antibody according to claim 17, characterized in that: The first antigen binding region further comprises a connecting peptide 2; The C-terminus of CH1 of the first Fab fragment is connected to the N-terminus of the connecting peptide 2, and the C-terminus of the connecting peptide 2 is connected to the N-terminus of the scFv fragment.

22. The bispecific antibody according to claim 21, characterized in that The connecting peptide 2 has an amino acid sequence as shown in (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

23. The bispecific antibody according to claim 21, characterized in that The connecting peptide 2 has an amino acid sequence as shown in SEQ ID NO:

19.

24. The bispecific antibody according to any one of claims 1 to 4, characterized in that The first Fc peptide segment and the second Fc peptide segment are connected via a knob-into-hole structure.

25. The bispecific antibody according to any one of claims 1 to 4, characterized in that The first Fc peptide segment and the second Fc peptide segment are both human Fc peptide segments.

26. The bispecific antibody according to claim 25, characterized in that The human Fc peptide segment is a human IgG1 Fc peptide segment.

27. The bispecific antibody according to any one of claims 1 to 4, characterized in that The first Fc peptide segment has an amino acid sequence as shown in SEQ ID NO:20, and the second Fc peptide segment has an amino acid sequence as shown in SEQ ID NO:

21.

28. The bispecific antibody according to any one of claims 2 to 4, characterized in that: The bispecific antibody comprises: A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 22, or an amino acid sequence having at least 80% identity thereto; a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 23, or an amino acid sequence at least 80% identical thereto; a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO:24, or an amino acid sequence at least 80% identical thereto; A fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 24, or an amino acid sequence at least 80% identical thereto.

29. The bispecific antibody according to any one of claims 2 to 4, characterized in that The bispecific antibody comprises: A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO:22, a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO:23, a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO:24, and a fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO:

24.

30. An isolated nucleic acid, characterized in that Encoding the bispecific antibody according to any one of claims 1 to 29.

31. An expression vector, characterized in that Containing the isolated nucleic acid of claim 30.

32. The expression vector according to claim 31, characterized in that The expression vector is a eukaryotic vector or a prokaryotic vector.

33. The expression vector according to claim 31, characterized in that The expression vector comprises at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.

34. A recombinant cell, characterized in that Carrying the isolated nucleic acid of claim 30, the expression vector of any one of claims 31 to 33; or expressing the bispecific antibody of any one of claims 1 to 29.

35. The recombinant cell according to claim 34, characterized in that The recombinant cell is a prokaryotic cell, a eukaryotic cell or a bacteriophage.

36. A pharmaceutical composition, characterized in that The invention comprises: the bispecific antibody according to any one of claims 1 to 29, the isolated nucleic acid according to claim 30, or the expression vector according to any one of claims 31 to 33.

37. The pharmaceutical composition according to claim 36, characterized in that Further includes pharmaceutically acceptable excipients.

38. The pharmaceutical composition according to claim 36, characterized in that The pharmaceutical composition is an injection.

39. A kit, characterized in that The method comprises: the bispecific antibody according to any one of claims 1 to 29, the isolated nucleic acid according to claim 30, the expression vector according to any one of claims 31 to 33, or the recombinant cell according to claim 34 or 35.

40. Use of the bispecific antibody of any one of claims 1 to 29, the isolated nucleic acid of claim 30, the expression vector of any one of claims 31 to 33, or the recombinant cell of claim 34 or 35 in the preparation of a kit for detecting CD3 and / or PD-L1 tumor targets.

41. The use according to claim 40, characterized in that The tumor target is PD-L1.

42. The use according to claim 41, characterized in that The PD-L1-mediated diseases include cancer or transplant rejection, autoimmune diseases and infectious diseases.

43. The use according to claim 42, characterized in that The cancer includes at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

44. Use of the bispecific antibody of any one of claims 1 to 29, the isolated nucleic acid of claim 30, the expression vector of any one of claims 31 to 33, the recombinant cell of claim 34 or 35, or the pharmaceutical composition of claim 36 in the preparation of a drug for preventing and / or treating CD3 and / or PDL1-mediated related diseases.

45. The use according to claim 44, characterized in that The CD3-mediated related diseases include autoimmune diseases.

46. ​​The use according to claim 45, characterized in that The autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

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