Anti-CD3 antibody and use thereof

Abstract

Provided are an anti-CD3 antibody or an antigen-binding fragment thereof, and a use thereof. The anti-CD3 antibody comprises: heavy chain variable region CDRs and light chain variable region CDRs, the heavy chain variable region CDRs having amino acid sequences as shown in any one of SEQ ID NOs: 1-3, 7-8 and 74-86 or a conservatively modified form thereof, and / or the light chain variable region CDRs having amino acid sequences as shown in any one of SEQ ID NOs: 4-6, 9 and 87-92 or a conservatively modified form thereof. The provided anti-CD3 antibody or antigen-binding fragment thereof can bind to human CD3 protein and reduce the production of pro-inflammatory cytokines, and a bispecific antibody constructed using same has high PBMC tumor-killing activity and weak promotion of the secretion of pro-inflammatory cytokines. A pharmaceutical composition prepared using the anti-CD3 antibody or the antigen-binding fragment thereof or a multi-specific antibody thereof has better safety, and has value for clinical applications and drug development.

Description

Anti-CD3 antibodies and their applications Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an anti-CD3 antibody or an antigen-binding fragment thereof and applications thereof. Background Art

[0002] Cancer is a major disease that affects human survival and development. According to the latest data, approximately 19 million new cancer cases and 10 million cancer deaths occur worldwide each year, and both incidence and mortality rates are on the rise. Besides surgical resection, traditional cancer treatments such as chemotherapy and radiotherapy are associated with significant side effects and high recurrence rates. In recent years, immunotherapy, including tumor-targeted antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hotspot and a new hope in the fight against cancer. Immunotherapy drugs, including monoclonal antibodies and bispecific antibodies, primarily work by promoting the function of autoimmune cells to achieve their anti-cancer effects, with minimal side effects and prolonged survival for patients who benefit. Approved immune checkpoint antibodies include PD1-1 / L1, CTLA-4, and LAG-3, while bispecific anti-cancer antibodies include blinatumomab (CD3×CD19), Kimtrak (CD3×GP100), Mosunetuzumab (CD3×CD20), and Tecvayli (CD3×BCMA). Bispecific antibodies have made great progress in the treatment of hematological tumors. They target CD19, CD20, and BCMA, and can encourage T cells to eliminate tumor cells, benefiting patients.

[0003] CD3-based bispecific antibodies (hereinafter referred to as "CD3 bispecific antibodies") recruit T cells to reach local tumors, bridge T cells and tumors, promote T cell activation, and kill tumors. This type of bispecific antibody does not require neoantigens and can guide T cells to kill "cold tumors." After binding to T cells and tumor cells, CD3 bispecific antibodies trigger strong activation signals, so they can also "ignore" the inhibitory signals of immune checkpoint molecules to a certain extent; however, CD3 bispecific antibodies also promote the production of a large number of pro-inflammatory cytokines, such as TNFα, IL-6, etc., triggering a strong cytokine storm and excessive immune response, causing damage to the body, and severe cases may be life-threatening. Therefore, CD3-based bispecific antibodies have good application prospects in clinical practice, but their safety needs to be further improved.

[0004] One approach to addressing the safety concerns of CD3 bispecific antibodies is to increase their affinity for tumor targets while simultaneously reducing their affinity for CD3. This allows for greater local distribution of the CD3 bispecific antibody drug to the tumor, increasing local tumor drug concentration, reducing peripheral drug concentration, and reducing off-target toxicity, pro-inflammatory cytokine production, and on-target toxicity. Therefore, there is an urgent need to develop anti-CD3 antibodies that can bind to the human CD3 protein with low affinity, thereby obtaining CD3 antibody drugs with improved safety, high clinical application, and high drug development value.

[0005] Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, one object of the present invention is to provide an anti-CD3 antibody or an antigen-binding fragment thereof. Another object of the present invention is to provide a multispecific antibody constructed using the anti-CD3 antibody or its antigen-binding fragment. The anti-CD3 antibody or its antigen-binding fragment of the present invention can bind to the human CD3 protein and can reduce the production of pro-inflammatory cytokines. The bispecific antibody constructed using it has high PBMC tumor-killing activity and weak promotion of pro-inflammatory cytokine secretion performance. Therefore, the pharmaceutical composition prepared using the anti-CD3 antibody or its antigen-binding fragment or its multispecific antibody has better safety and high clinical application and drug development value.

[0007] The present invention is based on the inventor's discovery and understanding of the following facts and problems:

[0008] Currently, the most widely used CD3 antibodies in bispecific antibodies are TR66 and UCHT1. On the one hand, neither of these two antibodies binds to monkey CD3, which increases the difficulty of non-clinical evaluation of drugs; on the other hand, these two antibodies have very strong binding affinity to T cells, which increases the safety risk of CD3 bispecific antibody drugs constructed based on them. The inventors have modified the affinity of Cross3, an anti-CD3 antibody with high CD3 affinity (KD=0.165nM) and human-monkey cross. Through a large number of screenings, they obtained a series of anti-CD3 antibodies that can bind to human CD3 protein with low affinity. Furthermore, based on the series of anti-CD3 antibodies, CD3 bispecific antibodies were constructed and 19 anti-CD3 antibodies were screened and obtained. Compared with Cross3, the specific recognition and binding of bispecific antibodies to tumor antigens are not affected by the CD3 antigen binding region. Further experimental results demonstrated that: 1) the CD3 bispecific antibody of the present invention binds to CD3E&D proteins and specifically binds to tumor antigens (such as PDL1 and Trop2), and its tumor antigen binding activity is substantially equivalent to that of a bispecific antibody constructed based on Cross3; 2) cellular assay results showed that the CD3 bispecific antibody of the present invention can bind to T cells and specifically bind to tumor cells (such as A-375-Trop2 cells, A-375 melanoma cells, HCT-15 colorectal cancer cells, and A549 lung cancer cells), and its tumor cell binding ability is substantially equivalent to that of a bispecific antibody constructed based on Cross3; 3) assay results for promoting cytokine secretion in PBMCs showed that the CD3 bispecific antibody of the present invention has reduced ability to promote the secretion of pro-inflammatory cytokines.

[0009] The inventors further verified the tumor cell killing activity of the above-mentioned CD3 bispecific antibodies through PBMC tumor cell killing experiments. The results showed that the bispecific antibodies constructed based on the anti-CD3 antibodies Cross313, Cross316, and Cross325 of the present invention (such as Cross313×Trop2, Cross316×Trop2, Cross325×Trop2, Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1) can achieve the highest killing effect or can promote 100% tumor cell killing by PBMC, and have comparable tumor cell killing effects to the bispecific antibodies constructed based on the anti-CD3 antibody Cross3 (such as Cross3×Trop2 and Cross3×PDL1).

[0010] The above experimental results show that the CD3 bispecific antibody developed based on the anti-CD3 antibody or antigen-binding fragment thereof of the present invention has high PBMC tumor-killing activity and weak promotion of pro-inflammatory cytokine secretion performance, better safety, and high value for clinical application and drug development.

[0011] Therefore, in a first aspect, the present invention provides an anti-CD3 antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region CDRs and a light chain variable region CDRs, wherein the heavy chain variable region CDRs have an amino acid sequence as shown in any one of SEQ ID NOs: 1-3, 7-8, and 74-86, or a conservative modification thereof; and / or the light chain variable region CDRs have an amino acid sequence as shown in any one of SEQ ID NOs: 4-6, 9, and 87-92, or a conservative modification thereof.

[0012] The anti-CD3 antibody or its antigen-binding fragment according to the embodiment of the present invention can bind to the human CD3 protein and reduce the production of pro-inflammatory cytokines. The CD3 bispecific antibody developed based on the CD3 antibody or its antigen-binding fragment has high PBMC tumor-killing activity and weak pro-inflammatory cytokine secretion performance; after being prepared into a drug, on the one hand, it can be distributed more to the local tumor, increasing the local drug concentration of the tumor and reducing the peripheral drug concentration, thereby reducing the risk of off-target toxicity; on the other hand, the bispecific antibody drug can reduce the production of pro-inflammatory cytokines and reduce the risk of on-target toxicity. Furthermore, the drug prepared using the anti-CD3 antibody or its antigen-binding fragment can be used to prevent and / or treat CD3-related diseases; the CD3 bispecific antibody drug developed based on the anti-CD3 antibody or its antigen-binding fragment has higher safety and can be used to prevent and / or treat cancer, tumor, infection or autoimmune disease. Therefore, the anti-CD3 antibody or its antigen-binding fragment of the present invention has good clinical application value and drug development value.

[0013] In a second aspect of the present invention, the present invention provides a multispecific antibody. According to an embodiment of the present invention, the multispecific antibody comprises:

[0014] a first antigen-binding region, wherein the first antigen-binding region comprises the anti-CD3 antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0015] The second antigen-binding region has a biomolecule-binding activity, and the biomolecule is not CD3.

[0016] As previously mentioned, multispecific antibodies (such as bispecific antibodies) developed based on the anti-CD3 antibodies or antigen-binding fragments thereof described in the first aspect of the present invention can, when formulated as drugs, be distributed more locally to the tumor, increasing local tumor drug concentration and reducing peripheral drug concentration, thereby reducing the risk of off-target toxicity. Furthermore, they can reduce the production of proinflammatory cytokines, thereby reducing the risk of on-target toxicity of multispecific antibodies (such as bispecific antibodies). Therefore, multispecific antibodies based on the anti-CD3 antibodies or antigen-binding fragments thereof described in the first aspect of the present invention have improved safety and high clinical application and drug development value.

[0017] In a third aspect, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises: the antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the multispecific antibody according to the second aspect of the present invention.

[0018] As mentioned above, the anti-CD3 antibodies or antigen-binding fragments thereof of the embodiments of the present invention can be used to prepare bispecific antibody drugs. On the one hand, the prepared bispecific antibody drugs can be distributed more locally to the tumor, increase the local drug concentration in the tumor, reduce the peripheral drug concentration, and thus reduce the risk of off-target toxicity; on the other hand, the bispecific antibodies can reduce the production of pro-inflammatory cytokines, reducing the risk of on-target toxicity of multispecific antibodies (such as bispecific antibodies). Furthermore, drugs prepared using the aforementioned anti-CD3 antibodies or antigen-binding fragments thereof can be used to prevent and / or treat CD3-related diseases; CD3 bispecific antibody drugs developed based on the anti-CD3 antibodies or antigen-binding fragments thereof are safer and can be used to prevent and / or treat cancer, tumors, infections, or autoimmune diseases. Therefore, the conjugate comprising the anti-CD3 antibody or antigen-binding fragment thereof of the first aspect of the present invention and the multispecific antibody of the second aspect of the present invention has good clinical application value and drug development value.

[0019] In a fourth aspect, the present invention provides a nucleic acid. According to embodiments of the present invention, the nucleic acid encodes the anti-CD3 antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or the multispecific antibody described in the second aspect of the present invention. According to embodiments of the present invention, the antibody or antigen-binding fragment thereof of the first aspect of the present invention, or the multispecific antibody of the second aspect of the present invention, encoded by the nucleic acid, is suitable for developing safer monoclonal antibody or bispecific antibody drugs. Furthermore, the protein encoded by the nucleic acid can be used to prevent and / or treat CD3-related diseases, or to prevent and / or treat cancer, tumors, infections, or autoimmune diseases.

[0020] In a fifth aspect, the present invention provides a vector or transformant. According to embodiments of the present invention, the vector or transformant contains the nucleic acid described in the fourth aspect of the present invention. Thus, the constructed vector or transformant can be used to effectively express the antibody or antigen-binding fragment thereof of the first aspect of the present invention, or the multispecific antibody of the second aspect of the present invention, and to obtain the conjugate of the third aspect of the present invention.

[0021] In a sixth aspect, the present invention provides a cell. According to embodiments of the present invention, the cell carries the nucleic acid described in the fourth aspect of the present invention, or the vector or transformant described in the fifth aspect of the present invention, or expresses the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or the multispecific antibody described in the second aspect of the present invention. According to embodiments of the present invention, the cell can efficiently express the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or the multispecific antibody described in the second aspect of the present invention under appropriate conditions, and further, obtains an antibody or antigen-binding fragment thereof, or a multispecific antibody thereof, with improved binding safety to the human CD3 protein.

[0022] In its seventh aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the multispecific antibody of the second aspect of the present invention, the conjugate of the third aspect of the present invention, the nucleic acid of the fourth aspect of the present invention, or the vector or transformant of the fifth aspect of the present invention. The resulting medicament can be further used to prevent and / or treat CD3-related diseases, or further used to prevent and / or treat CD3-related diseases and / or cancers, tumors, infections, or autoimmune diseases.

[0023] In an eighth aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the multispecific antibody according to the second aspect of the present invention. Thus, the resulting kit can be used for CD3-related research.

[0024] In a ninth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the multispecific antibody according to the second aspect of the present invention, in preparing a kit for detecting CD3. Those skilled in the art will appreciate that the aforementioned features and advantages of the antibody or antigen-binding fragment thereof also apply to this use and are not further elaborated here.

[0025] In its tenth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the multispecific antibody of the second aspect of the present invention, the conjugate of the third aspect of the present invention, the nucleic acid of the fourth aspect of the present invention, the vector or transformant of the fifth aspect of the present invention, or the pharmaceutical composition of the seventh aspect of the present invention in the preparation of a medicament for preventing and / or treating CD3-related diseases and / or cancer, tumors, infections, or autoimmune diseases. The antibody or antigen-binding fragment thereof, multispecific antibody and conjugate, nucleic acid, vector or transformant, or pharmaceutical composition of the present invention can be further prepared into a medicament, which can be clinically used to prevent or treat diseases.

[0026] Those skilled in the art will appreciate that the features and advantages described above for antibodies or antigen-binding fragments thereof, conjugates, nucleic acid molecules, vectors or transformants, and pharmaceutical compositions are also applicable to this use and will not be elaborated here. Beneficial effects:

[0027] (1) The anti-CD3 antibodies Cross303, Cross307, Cross308, Cross309, Cross310, Cross311, Cross312, Cross313, Cross314, Cross316, Cross317, Cross318, Cross323, Cross325, Cross326, Cross3ZH07, Cross3ZH09, Cross3ZH16, and Cross3ZH23 obtained by the present invention can reduce the production of pro-inflammatory cytokines caused by the CD3 antigen-binding portion compared to the parent antibody Cross3.

[0028] (2) The bispecific antibodies constructed from the antibodies Cross313, Cross316, and Cross325 obtained in the present invention have weaker T cell binding ability than the bispecific antibodies constructed from the parent antibody Cross3.

[0029] (3) The bispecific antibodies constructed from the antibodies Cross313, Cross316, and Cross325 obtained in the present invention can also promote PBMC to kill tumor cells to the greatest extent compared with the bispecific antibodies constructed from the parent antibody Cross3, and have weaker performance in promoting the production of pro-inflammatory cytokines and better safety.

[0030] 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

[0031] 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:

[0032] FIG1 is a comparison of amino acid mutations of an affinity-reduced antibody according to an embodiment of the present invention compared with the parent antibody Cross3;

[0033] FIG2 is a graph showing the SPR results of affinity determination between the parent antibody Cross3 and the affinity-reduced antibody and CD3E&D protein according to an embodiment of the present invention;

[0034] FIG3 is a graph showing the ELISA results of the binding of the parent antibody Cross3 and the affinity-reduced antibody to the CD3E&D protein according to an embodiment of the present invention;

[0035] FIG4 is a flow cytometry result diagram of the binding of the parent antibody Cross3 and the affinity-reduced antibody to CD8 T cells according to an embodiment of the present invention;

[0036] FIG5 is a schematic diagram of the configuration of the CD3×Trop2 and CD3×PDL1 bispecific antibodies composed of the parent antibody Cross3 and the affinity-reduced antibody according to an embodiment of the present invention;

[0037] FIG6 is a graph showing the ELISA results of the binding of the CD3×Trop2 bispecific antibody composed of the parent antibody Cross3 and the affinity-reduced antibody to the CD3E&D protein according to an embodiment of the present invention;

[0038] FIG7 is a graph showing the ELISA results of the binding of the CD3×Trop2 bispecific antibody composed of the parent antibody Cross3 and the affinity-reduced antibody to the Trop2 protein according to an embodiment of the present invention;

[0039] FIG8 is a graph showing the ELISA results of a CD3×Trop2 bispecific antibody composed of the parent antibody Cross3 and an affinity-reduced antibody bridging CD3E&D and Trop2 proteins according to an embodiment of the present invention;

[0040] FIG9 is a flow cytometry result showing the binding of a CD3×Trop2 bispecific antibody composed of the parent antibody Cross3 and an affinity-reduced antibody to CD8 T cells according to an embodiment of the present invention;

[0041] FIG10 is a flow cytometry result showing the binding of a CD3×Trop2 bispecific antibody composed of the parent antibody Cross3 and an affinity-reduced antibody according to an embodiment of the present invention to A-375-Trop2 melanoma cells;

[0042] FIG11 is a graph showing the results of CD3×Trop2 bispecific antibodies composed of the parent antibody Cross3 and affinity-reduced antibodies according to an embodiment of the present invention promoting PBMC to kill A-375-Trop2 melanoma cells;

[0043] FIG12 is a graph showing the results of Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 bispecific antibodies promoting PBMC to kill A-375-Trop2 melanoma cells according to an embodiment of the present invention;

[0044] FIG13 is a graph showing the results of Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 bispecific antibodies according to an embodiment of the present invention promoting the secretion of pro-inflammatory cytokines by PBMC;

[0045] FIG14 is a graph showing the ELISA results of binding of the CD3×PDL1 bispecific antibody composed of the parent antibody Cross3 and the affinity-reduced antibody to the CD3E&D protein according to an embodiment of the present invention;

[0046] FIG15 is a graph showing the ELISA results of the binding of the CD3×PDL1 bispecific antibody composed of the parent antibody Cross3 and the affinity-reduced antibody to the PDL1 protein according to an embodiment of the present invention;

[0047] FIG16 is a flow cytometry result showing the binding of a CD3×PDL1 bispecific antibody composed of the parent antibody Cross3 and an affinity-reduced antibody to CD8 T cells according to an embodiment of the present invention;

[0048] FIG17 is a flow cytometry result showing the binding of a CD3×PDL1 bispecific antibody composed of the parent antibody Cross3 and an affinity-reduced antibody to A-375 melanoma cells according to an embodiment of the present invention;

[0049] FIG18 is a flow cytometry result showing the binding of a CD3×PDL1 bispecific antibody composed of the parent antibody Cross3 and an affinity-reduced antibody to HCT-15 colorectal cancer cells according to an embodiment of the present invention;

[0050] FIG19 is a flow cytometry result showing the binding of a CD3×PDL1 bispecific antibody composed of the parent antibody Cross3 and an affinity-reduced antibody to A-549 lung cancer cells according to an embodiment of the present invention;

[0051] FIG20 is a graph showing the results of promoting PBMC to kill A-375 melanoma cells by the CD3×PDL1 bispecific antibody composed of the parent antibody Cross3 and the affinity-reduced antibody according to an embodiment of the present invention;

[0052] FIG21 is a graph showing the results of Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 promoting PBMC to kill A-375 melanoma cells according to an embodiment of the present invention;

[0053] FIG22 is a graph showing the results of promoting PBMC to kill HCT-15 colorectal cancer cells by the CD3×PDL1 bispecific antibody composed of the parent antibody Cross3 and the affinity-reduced antibody according to an embodiment of the present invention;

[0054] FIG23 is a graph showing the results of the CD3×PDL1 bispecific antibody composed of the parent antibody Cross3 and the affinity-reduced antibody according to an embodiment of the present invention promoting the secretion of pro-inflammatory cytokines by PBMC after co-incubation with A-375;

[0055] FIG24 is a graph showing the results of the CD3×PDL1 bispecific antibody composed of the parent antibody Cross3 and the affinity-reduced antibody according to an embodiment of the present invention promoting the secretion of pro-inflammatory cytokines by PBMC after co-incubation with HCT-15. DETAILED DESCRIPTION

[0056] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0057] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0058] 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.

[0059] Terms and Definitions

[0060] 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.

[0061] 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. 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 an immunologically active portion of any of these polypeptides, i.e., a molecule or portion thereof that contains 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.

[0062] 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. For example, it generally includes: amino acid residues around 23-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable region, and around 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region; and / or from a "hypervariable loop" (e.g., amino acid residues around 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable region, and around 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable region.

[0063] Herein, the term "anti-CD3 antibody" refers to an antibody that can bind to CD3. Such an antibody is also referred to herein as an "antibody that binds to CD3."

[0064] As used herein, the term "Fab-Linker" refers to a polypeptide segment formed by linking the Fab VH-CH1 and Fab VL-CL segments of an antibody Fab fragment via a linker peptide, including but not limited to scFab and scFabΔ (the linker peptide does not contain a disulfide bond).

[0065] As used herein, the term "antigen-binding fragment" is equivalent to "antibody fragment" or "antigen-binding antibody fragment" and may include a portion of an intact antibody, generally the binding region or variable region. This includes, but is not limited to, Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, scFv-Fc fragments or bispecific antibodies (BsAbs), linear antibodies, or any fragment that can increase half-life by chemical modification, such as the addition of poly(alkylene) glycols, such as polyethylene glycol ("PEGylation") (PEGylated fragments referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" is polyethylene glycol) or by incorporation into liposomes.

[0066] Herein, the term "chimeric antibody" refers to a recombinant antibody obtained by replacing the amino acid sequence of the constant region of a monoclonal antibody from one species (such as mouse) with the constant region of an antibody from another species (such as human) using recombinant DNA technology.

[0067] As used herein, the term "humanized antibody" refers to a recombinant antibody obtained by replacing all non-CDR (Fv framework region (FR)) amino acid sequences of the constant and variable regions of a monoclonal antibody from one species (e.g., mouse) with non-CDR amino acid sequences of the constant and variable regions of an antibody from another species (e.g., human) using recombinant DNA technology. That is, when the constant region of an antibody is humanized, it is called a chimeric antibody, while when the non-CDR amino acid sequences of the constant and variable regions are all humanized, it is called a humanized antibody. The humanization method can be performed with reference to conventional antibody engineering techniques and will not be described in detail here.

[0068] With respect to nucleotides, the terms "homology," "identity," or "similarity" are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of "sequence homology" between a first sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical to the nucleotides at the corresponding positions in the second sequence] minus [the total number of nucleotides in the first sequence] and then multiplying by [100%], where each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence - relative to the first nucleotide sequence - is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings. Some other techniques, computer algorithms and setups for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768-A.

[0069] With respect to polypeptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more polypeptides or designated sequences thereof when optimally aligned and compared (with appropriate insertions or deletions of nucleotides). The percent homology between two sequences varies with the number of identical positions shared by the sequences when the sequences are optimally aligned (i.e., % homology = number of identical positions / total number of positions x 100), where optimal alignment is determined by taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences and the length of each gap. Sequence comparison and percent identity determination between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting Examples below.

[0070] As used herein, the term "conservatively modified amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0071] As used herein, the term "conjugate" refers to an antibody or antigen-binding fragment thereof conjugated to a coupling moiety such as a carrier substance, drug, toxin, cytokine, protein tag, modifier, therapeutic agent, chemotherapeutic agent, etc., using any covalent or non-covalent bioconjugation strategy.

[0072] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0073] As used herein, the term "pharmaceutical composition" generally refers to unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with liquid carriers, finely divided solid carriers, or both.

[0074] As used herein, the term "pharmaceutically acceptable" refers to substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic response), ie, substances with a reasonable benefit / risk ratio.

[0075] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except to the extent that any conventional excipient is incompatible with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated by the present invention.

[0076] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient by a suitable means. The antibody or antigen-binding fragment, recombinant protein, multispecific antibody, conjugate, or pharmaceutical composition of the present invention can be administered by any common route as long as it reaches the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, and the like, but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.

[0077] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0078] 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.

[0079] The present invention provides an anti-CD3 antibody or antigen-binding fragment thereof, a multispecific antibody, a conjugate, a nucleic acid molecule, a vector or transformant, a cell, a pharmaceutical composition, a kit and uses thereof, which are described in detail below.

[0080] Anti-CD3 antibodies or antigen-binding fragments thereof

[0081] The present invention provides an anti-CD3 antibody or antigen-binding fragment thereof. The anti-CD3 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region CDRs and a light chain variable region CDRs, wherein the heavy chain variable region CDRs have an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 3, 7 to 8, and 74 to 86, or a conservative modification thereof; and / or the light chain variable region CDRs have an amino acid sequence as shown in any one of SEQ ID NOs: 4 to 6, 9, and 87 to 92, or a conservative modification thereof.

[0082] The anti-CD3 antibody or its antigen-binding fragment of the present invention can bind to the human CD3 protein and reduce the production of pro-inflammatory cytokines. The CD3 bispecific antibody developed based on the CD3 antibody or its antigen-binding fragment has high PBMC tumor-killing activity and weak pro-inflammatory cytokine secretion performance; after being prepared into a drug, on the one hand, it can be distributed more to the local tumor, increasing the local drug concentration of the tumor and reducing the peripheral drug concentration, thereby reducing the risk of off-target toxicity; on the other hand, the bispecific antibody drug can reduce the production of pro-inflammatory cytokines and reduce the risk of on-target toxicity. Furthermore, the drug prepared using the anti-CD3 antibody or its antigen-binding fragment can be used to prevent and / or treat CD3-related diseases; the CD3 bispecific antibody drug developed based on the anti-CD3 antibody or its antigen-binding fragment has higher safety and can be used to prevent and / or treat cancer, tumor, infection or autoimmune disease. Therefore, the anti-CD3 antibody or its antigen-binding fragment of the present invention has good clinical application value and drug development value.

[0083] It should be noted that one or more amino acid residues in the CDR region of the antibodies or antigen-binding fragments thereof of the present invention may be replaced by other amino acid residues from the same side chain family, and the retained function of the altered antibodies can be tested using the functional assays described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0084] According to an embodiment of the present invention, the heavy chain variable region CDRs have an amino acid sequence as shown in SEQ ID NOs: 1 to 2 or a conservative modification thereof, and an amino acid sequence as shown in one of SEQ ID NOs: 3, 7 to 8 and 74 to 86 or a conservative modification thereof; the light chain variable region CDRs have an amino acid sequence as shown in SEQ ID NOs: 4 to 5 or a conservative modification thereof, and an amino acid sequence as shown in one of SEQ ID NOs: 6, 9 and 87 to 92 or a conservative modification thereof.

[0085] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a heavy chain variable region CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a heavy chain variable region CDR3 having an amino acid sequence as shown in one of SEQ ID NO: 3, SEQ ID NOs: 7 to 8, and SEQ ID NOs: 74 to 86; a light chain variable region CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a light chain variable region CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a light chain variable region CDR3 having an amino acid sequence as shown in one of SEQ ID NOs: 6, 9, and 87 to 92.

[0086] According to an embodiment of the present invention, the heavy chain variable region CDR3 has an amino acid sequence as shown in one of SEQ ID NO: 3 and 7 to 8; and / or the light chain variable region CDR3 has an amino acid sequence as shown in SEQ ID NO: 6 or 9.

[0087] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises any one of the following groups:

[0088] 1) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 74, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0089] 2) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 75, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0090] 3) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 76, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0091] 4) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 77, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0092] 5) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 78, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0093] 6) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 79, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0094] 7) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 80, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0095] 8) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 81, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0096] 9) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 82, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0097] 10) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 83, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0098] 11) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0099] 12) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 7, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 6, respectively;

[0100] 13) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 8, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 87, respectively;

[0101] 14) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 8, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 88, respectively;

[0102] 15) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 8, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 9, respectively;

[0103] 16) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 84, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 89, respectively;

[0104] 17) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 85, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 90, respectively;

[0105] 18) having heavy chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 1, 2, and 86, respectively, and light chain variable region CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 4, 5, and 92, respectively;

[0106] 19) have heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 8, respectively, and have light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 91, respectively.

[0107] It should be noted that the antigen-binding fragment will be composed of a partial sequence of the heavy chain variable region or light chain variable region of the antibody from which it is derived, or contain a partial sequence of the light or heavy chain variable region, and the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived.

[0108] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region and / or a light chain framework region.

[0109] According to an embodiment of the present invention, at least a portion of the heavy chain framework region and / or the 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 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 mouse antibody, a human antibody, and a primate antibody.

[0110] It should be noted that, in order to further improve the bioacceptability of the antibody, the antibody may be humanized, that is, the antibody is a chimeric antibody or a humanized antibody.

[0111] As previously mentioned, one or more amino acid residues in the heavy or light chain variable region of the antibodies or antigen-binding fragments thereof of the present invention can be replaced by other amino acid residues from the same side chain family, and the retained function of the altered antibodies can be tested using the functional assays described herein.

[0112] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having an amino acid sequence as shown in one of SEQ ID NOs: 10, 12-13, 15-24, 27 and 31, or an amino acid sequence of a conservative modification thereof; and / or a light chain variable region having an amino acid sequence as shown in one of SEQ ID NOs: 11, 14, 25-26, 28 and 30, or an amino acid sequence of a conservative modification thereof.

[0113] It should be noted that, under the premise of not substantially affecting the CD3 binding activity of the antibody or its antigen-binding fragment (retaining at least 95% 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 heavy chain or light chain variable region of the antibody or its antigen-binding fragment of the present invention to obtain variants of the sequence of the antibody or its antigen-binding fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the heavy chain or light chain variable region. The sequence of the above variant of the present invention may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequence.

[0114] It should be noted that the sequence identity described in the present invention can be measured using sequence analysis software, such as the computer program BLAST using default parameters, particularly BLASTP or TBLASTN.

[0115] According to an embodiment of the present invention, the heavy chain variable region has an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in one of SEQ ID NOs: 10, 12-13, 15-24, 27 and 31; and / or, the light chain variable region has an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in one of SEQ ID NOs: 11, 14, 25-26, 28 and 30.

[0116] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises an amino acid sequence represented by any of the following combinations of heavy chain variable regions and light chain variable regions, or an amino acid sequence having at least 90% sequence identity thereto:

[0117] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof further comprises a constant region; wherein the constant region includes at least one of a heavy chain constant region and a light chain constant region.

[0118] According to an embodiment of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant 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 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.

[0119] 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.

[0120] According to an embodiment of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and / or, the light chain constant region includes a light chain constant region selected from κ type or λ type.

[0121] In some specific embodiments, the heavy chain constant region comprises a heavy chain constant region selected from human IgG, IgA, IgM, IgE or IgD, such as human IgG1, human IgG2, human IgG3, human IgG4, human IgA, human IgM, human IgE or human IgD.

[0122] In some specific embodiments, the heavy chain constant region comprises a heavy chain constant region selected from murine IgG, IgA, IgM, IgE or IgD, such as murine IgG1, murine IgG2a, murine IgG2b, murine IgG2c, murine IgG3, murine IgA, murine IgM, murine IgE or murine IgD.

[0123] According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from a murine antibody or a mutant thereof, or a human antibody or a mutant thereof.

[0124] It should be noted that the amino acid sequences described in the present invention are all shown from N-terminus to C-terminus.

[0125] According to an embodiment of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or, the N-terminus of the light chain constant region is connected to the N-terminus of the light chain variable region.

[0126] As mentioned above, under the premise of not substantially affecting the CD3 binding activity of the antibody or its antigen-binding fragment (retaining at least 95% 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 heavy chain or light chain constant region of the antibody or its antigen-binding fragment of the present invention to obtain variants of the sequence of the antibody or its antigen-binding fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the heavy chain or light chain constant region. The 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 sequence.

[0127] According to an embodiment of the present invention, the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO: 93 or an amino acid sequence that is at least 80% identical thereto; and / or, the light chain constant region has an amino acid sequence as shown in SEQ ID NO: 94 or an amino acid sequence that is at least 80% identical thereto.

[0128] According to an embodiment of the present invention, the heavy chain constant region and the light chain constant region have the amino acid sequences shown in SEQ ID NO: 93 and SEQ ID NO: 94, respectively.

[0129] According to an embodiment of the present invention, the C-terminus of the heavy chain variable region is connected to the N-terminus of the light chain variable region, or the N-terminus of the heavy chain variable region is connected to the C-terminus of the light chain variable region.

[0130] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof further comprises a first connecting peptide, the C-terminus of the heavy chain variable region is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the light chain variable region, or the C-terminus of the light chain variable region is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the heavy chain variable region.

[0131] In some specific embodiments, the first connecting peptide 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.

[0132] As previously described, the antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies) or comprise only an antigen-binding portion (e.g., Fab, Fab', Fab'-SH, F(ab')2, or Fv, scFv fragments), or may be modified to affect function. The present invention includes anti-CD3 antibodies with modified glycosylation patterns. In some embodiments, modification to remove undesirable glycosylation sites may be useful, or antibodies lacking fucose moieties on oligosaccharide chains to, for example, enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other embodiments, galactosylation modification may be performed to alter complement-dependent cytotoxicity (CDC).

[0133] According to an embodiment of the present invention, the antibody includes at least one of a full-length monoclonal antibody, a chimeric antibody, a humanized antibody, Fv, scFv, Fab, Fab', Fab'-SH and F(ab')2; the antigen-binding fragment of the antibody includes at least one of a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein and a minimum recognition unit.

[0134] As mentioned above, under the premise of not substantially affecting the CD3 binding activity of the antibody or its antigen-binding fragment (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 heavy chain or light chain, scFv fragment, Fab fragment or scFab fragment of the antibody or its antigen-binding fragment of the present invention to obtain variants of the sequence of the antibody or its antigen-binding fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the heavy chain or light chain. The 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 sequence.

[0135] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain having an amino acid sequence as shown in one of SEQ ID NOs: 32, 34, 35 and 37 to 46, or an amino acid sequence having at least 80% identity thereto; and / or a light chain having an amino acid sequence as shown in one of SEQ ID NOs: 33, 36 and 47 to 48, or an amino acid sequence having at least 80% identity thereto.

[0136] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof has an amino acid sequence shown in any one of the following heavy chain and light chain combinations or an amino acid sequence having at least 80% sequence identity thereto:

[0137] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof has an amino acid sequence shown in any one of SEQ ID NOs: 99 to 117 or an amino acid sequence having at least 80% sequence identity therewith.

[0138] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof has an amino acid sequence shown in any one of the following groups of Fab VL-CL and Fab VH-CH1 combinations or an amino acid sequence having at least 80% sequence identity thereto:

[0139] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof has an amino acid sequence as shown in any one of SEQ ID NOs: 142 to 160 or an amino acid sequence having at least 80% sequence identity therewith.

[0140] Multispecific antibodies

[0141] The present invention provides a multispecific antibody comprising: a first antigen-binding region comprising the aforementioned anti-CD3 antibody or antigen-binding fragment thereof; and a second antigen-binding region having biomolecule binding activity, wherein the biomolecule is not CD3.

[0142] As previously mentioned, multispecific antibodies (such as bispecific antibodies) developed based on the anti-CD3 antibodies or antigen-binding fragments thereof described in the first aspect of the present invention can, when formulated as drugs, be distributed more locally to the tumor, increasing local tumor drug concentration and reducing peripheral drug concentration, thereby reducing the risk of off-target toxicity. Furthermore, they can reduce the production of proinflammatory cytokines, thereby reducing the risk of on-target toxicity of multispecific antibodies (such as bispecific antibodies). Therefore, multispecific antibodies based on the anti-CD3 antibodies or antigen-binding fragments thereof described in the first aspect of the present invention have improved safety and high clinical application and drug development value.

[0143] According to an embodiment of the present invention, the multispecific antibody is selected from one of a bispecific antibody, a trispecific antibody and a tetraspecific antibody.

[0144] Bispecific antibodies are preferred.

[0145] According to an embodiment of the present invention, the bispecific antibody includes a symmetric bispecific antibody or an asymmetric bispecific antibody.

[0146] According to an embodiment of the present invention, the second antigen binding region is a binding protein of the biomolecule or a fragment thereof.

[0147] According to an embodiment of the present invention, the biological molecule is selected from at least one of the following: PDL1, CD47, TIGIT, CD73, CD33, CEACAM1, CEACAM5, CEACAM6, STING, WNT, Beta catenin, B7H3, VISITA, CD19, BCMA, CD22, CD20, CD123, CD38, CEA, CD25, CD46, CD138, PSCA, PSMA, PSA, MUC1, MUC16, NY-ESO-1, GD2, WT1, Mesothelin, MAGE-A3, GPC3, PRAME, Globo H, AFP, Trop2, FOLR1, SP, Sca-1, CD133, EPCAM.

[0148] 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.

[0149] According to an embodiment of the present invention, the bispecific antibody is an asymmetric bispecific antibody.

[0150] According to an embodiment of the present invention, the first antigen binding region comprises a first scFv fragment, a first Fv fragment, a first Fab fragment or a first Fab-Linker fragment.

[0151] According to an embodiment of the present invention, the first antigen binding region further comprises a first Fc peptide segment.

[0152] According to an embodiment of the present invention, the N-terminus of the first Fc peptide segment is connected to the C-terminus of the scFv fragment or the Fv fragment; or, the N-terminus of the first Fc peptide segment is connected to the C-terminus of CH1 of the Fab fragment or the Fab-Linker fragment.

[0153] According to an embodiment of the present invention, the first antigen binding region further comprises a second connecting peptide, the N-terminus of the first Fc peptide segment is connected to the C-terminus of the second connecting peptide, and the N-terminus of the second connecting peptide is connected to the C-terminus of the scFv fragment or the Fv fragment; or, the N-terminus of the first Fc peptide segment is connected to the C-terminus of the second connecting peptide, and the N-terminus of the second connecting peptide is connected to the C-terminus of CH1 of the Fab fragment or the Fab-Linker fragment.

[0154] In some specific embodiments, the second connecting peptide 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.

[0155] According to an embodiment of the present invention, the first Fc peptide segment is selected from a human Fc peptide segment, preferably a human IgG1 Fc peptide segment.

[0156] According to an embodiment of the present invention, the first Fc peptide segment has the amino acid sequence shown in SEQ ID NO: 118; or the first Fc peptide segment has the amino acid sequence shown in SEQ ID NO: 166.

[0157] According to an embodiment of the present invention, the first scFv fragment has an amino acid sequence shown in any one of SEQ ID NOs: 95 to 117.

[0158] According to an embodiment of the present invention, the first Fab fragment has an amino acid sequence shown in any one of the following combinations of Fab VL-CL and Fab VH-CH1:

[0159] According to an embodiment of the present invention, the first Fab-Linker has an amino acid sequence as shown in any one of SEQ ID NOs: 142 to 160.

[0160] According to an embodiment of the present invention, the first antigen binding region has an amino acid sequence as shown in any one of SEQ ID NOs: 49 to 52, 54 to 68.

[0161] According to an embodiment of the present invention, the biomolecule is Trop2 or PDL1.

[0162] According to an embodiment of the present invention, the second antigen binding region comprises a first anti-Trop2 antibody or an antigen-binding fragment thereof or a first anti-PDL1 antibody or an antigen-binding fragment thereof.

[0163] According to an embodiment of the present invention, the first anti-Trop2 antibody or antigen-binding fragment thereof is the second scFab fragment or the second Fab fragment of anti-Trop2; or, the first anti-PDL1 antibody or antigen-binding fragment thereof is the third scFab fragment or the third Fab fragment of anti-PDL1.

[0164] According to an embodiment of the present invention, the second scFab fragment has the amino acid sequence shown in SEQ ID NO: 161; or, the second Fab fragment has the amino acid sequence shown in SEQ ID NO: 162 and 70; or, the third scFab fragment has the amino acid sequence shown in SEQ ID NO: 163; or, the third Fab fragment has the amino acid sequence shown in SEQ ID NO: 164 and 72.

[0165] According to an embodiment of the present invention, the second antigen binding region further includes a second Fc fragment; wherein the N-terminus of the second Fc fragment is connected to the C-terminus of CH1 of the second scFab fragment, the second Fab fragment, the third scFab fragment or the third Fab fragment.

[0166] According to an embodiment of the present invention, the second Fc peptide segment is selected from a human Fc peptide segment, preferably a human IgG1 Fc peptide segment.

[0167] According to an embodiment of the present invention, the second Fc peptide segment has an amino acid sequence as shown in SEQ ID NO: 165.

[0168] 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-hole structure. According to an embodiment of the present invention, the second antigen-binding region has an amino acid sequence as shown in SEQ ID NO: 161 or 163; or, the second antigen-binding region has an amino acid sequence as shown in SEQ ID NO: 162 and 70; or, the second antigen-binding region has an amino acid sequence as shown in SEQ ID NO: 164 and 72.

[0169] In some optional embodiments of the present invention, the multispecific antibody of the present invention has the structure shown in FIG5 .

[0170] In some optional embodiments of the present invention, as shown in Figure 5A, the multispecific antibody of the present invention includes: a first peptide chain having an amino acid sequence as shown in any one of SEQ ID NOs: 49 to 52, 54 to 68, a second peptide chain having an amino acid sequence as shown in SEQ ID NO: 69, and a third peptide chain having an amino acid sequence as shown in SEQ ID NO: 70; or, a second peptide chain having an amino acid sequence as shown in SEQ ID NO: 71, and a third peptide chain having an amino acid sequence as shown in SEQ ID NO: 72.

[0171] In some optional embodiments of the present invention, as shown in Figure 5B, the multispecific antibody of the present invention comprises: a first peptide chain having an amino acid sequence as shown in any one of SEQ ID NOs: 49 to 52, 54 to 68, and a second peptide chain having an amino acid sequence as shown in SEQ ID NO: 161 or 163.

[0172] In some optional embodiments of the present invention, as shown in Figure 5C, the multispecific antibody of the present invention comprises: a first peptide chain having an amino acid sequence as shown in any one of SEQ ID NOs: 142 to 160, and a second peptide chain having an amino acid sequence as shown in SEQ ID NO: 69, and a third peptide chain having an amino acid sequence as shown in SEQ ID NO: 70; or, a second peptide chain having an amino acid sequence as shown in SEQ ID NO: 71, and a third peptide chain having an amino acid sequence as shown in SEQ ID NO: 72.

[0173] In some optional embodiments of the present invention, as shown in Figure 5D, the multispecific antibody of the present invention comprises: a first peptide chain having an amino acid sequence as shown in any one of SEQ ID NOs: 142 to 160, and a second peptide chain having an amino acid sequence as shown in SEQ ID NO: 161 or 163.

[0174] In some optional embodiments of the present invention, as shown in Figure 5E, the multispecific antibody of the present invention includes: a first peptide chain having an amino acid sequence as shown in SEQ ID NO:70, a third peptide chain having an amino acid sequence as shown in SEQ ID NO:69, and a fourth peptide chain having an amino acid sequence as shown in SEQ ID NO:70; or, a first peptide chain having an amino acid sequence as shown in SEQ ID NO:72, a third peptide chain having an amino acid sequence as shown in SEQ ID NO:71, and a fourth peptide chain having an amino acid sequence as shown in SEQ ID NO:72; and a second peptide chain, wherein the second peptide chain comprises a Fab VH-CH1 having an amino acid sequence as shown in SEQ ID NO:162 or 164 and a scFv having an amino acid sequence as shown in SEQ ID NOs:99 to 117, and the C-terminus of the Fab VH-CH1 is connected to the N-terminus of the scFv.

[0175] Conjugate

[0176] The present invention provides a conjugate comprising: the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned multispecific antibody;

[0177] A conjugate moiety is linked to the antibody or antigen-binding fragment thereof, or the multispecific antibody.

[0178] As previously mentioned, the anti-CD3 antibodies or antigen-binding fragments thereof of the embodiments of the present invention can be used to prepare multispecific antibodies, such as bispecific antibody drugs. On the one hand, the prepared bispecific antibody drugs can be distributed more locally to the tumor, increasing the local drug concentration in the tumor and reducing the peripheral drug concentration, thereby reducing the risk of off-target toxicity; on the other hand, the bispecific antibodies can reduce the production of pro-inflammatory cytokines, reducing the risk of on-target toxicity of the bispecific antibodies. Furthermore, drugs prepared using the anti-CD3 antibodies or antigen-binding fragments thereof can be used to prevent and / or treat CD3-related diseases; CD3 bispecific antibody drugs developed based on the anti-CD3 antibodies or antigen-binding fragments thereof have higher safety and can be used to prevent and / or treat cancer, tumors, infections, or autoimmune diseases. Therefore, the conjugate comprising the anti-CD3 antibody or antigen-binding fragment thereof of the first aspect of the present invention and the multispecific antibody of the second aspect of the present invention has good clinical application value and drug development value.

[0179] According to an embodiment of the present invention, the coupling portion includes at least one selected from a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier, a therapeutic agent and a chemotherapeutic agent.

[0180] Nucleic Acids

[0181] The present invention provides a nucleic acid encoding the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned multispecific antibody.

[0182] According to embodiments of the present invention, the antibody or antigen-binding fragment thereof of the first aspect of the present invention or the multispecific antibody of the second aspect of the present invention encoded by the nucleic acid is suitable for the development of safer monoclonal antibody or bispecific antibody drugs. Furthermore, the protein encoded by the nucleic acid can be used to prevent and / or treat CD3-related diseases or prevent and / or treat cancer, tumors, infections, or autoimmune diseases.

[0183] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one 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, the molecular sequences in the present invention include DNA or RNA forms, and disclosure of one of them implies that the other is also disclosed.

[0184] Vector or transformant

[0185] The present invention provides a vector or transformant. The vector or transformant contains the aforementioned nucleic acid. The vector or transformant may include optional control sequences operably linked to the nucleic acid molecule. The control sequences are one or more control sequences that direct the expression of the nucleic acid molecule in a host. The vector or transformant thus constructed can effectively express the antibody or antigen-binding fragment thereof of the first aspect of the present invention or the multispecific antibody of the second aspect of the present invention, as well as obtain the conjugate of the third aspect of the present invention.

[0186] When the nucleic acid molecule is linked to a vector or transformant, such as an expression vector, the nucleic acid molecule 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 nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself or exogenous, i.e., not derived from the vector itself. The nucleic acid molecule and the control elements can be operably linked.

[0187] According to an embodiment of the present invention, the vector may refer to a cloning vector or an expression vector, which can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0188] As used herein, the term "operably linked" refers to the attachment of an exogenous gene to a vector so that control elements within the vector, such as transcriptional and translational control sequences, 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 nucleic acid molecules under the mediation of a regulatory system, thereby enabling the in vitro production of large quantities of the protein encoded by the nucleic acid molecules.

[0189] According to an embodiment of the present invention, the vector is a eukaryotic vector or a prokaryotic vector.

[0190] According to an embodiment of the present invention, the vector comprises at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.

[0191] cell

[0192] The present invention provides a cell. The cell carries the aforementioned nucleic acid, vector, or transformant, or expresses the aforementioned antibody, antigen-binding fragment thereof, or multispecific antibody. Thus, according to embodiments of the present invention, the cell can, under appropriate conditions, efficiently express the antibody, antigen-binding fragment thereof, or multispecific antibody of the first aspect of the present invention, and further, can produce an antibody, antigen-binding fragment thereof, or multispecific antibody that binds to human CD3 protein and has improved safety.

[0193] According to an embodiment of the present invention, the cell is a prokaryotic cell, a eukaryotic cell or a bacteriophage.

[0194] According to an embodiment of the present invention, the prokaryotic cell is Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0195] According to an embodiment of the present invention, the eukaryotic cell is a fungus, an insect cell, a plant cell or a mammalian cell.

[0196] According to an embodiment of the present invention, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Trichoderma.

[0197] 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.

[0198] According to an embodiment of the present invention, the cell is a mammalian cell.

[0199] According to an embodiment of the present invention, the cell is a BHK cell, a CHO cell, a COS cell or a NSO cell.

[0200] It should be noted that the "suitable conditions" described in the present specification refer to conditions suitable for the expression of the antibodies, antigen-binding fragments thereof, or multispecific antibodies of the present invention. Those skilled in the art will readily appreciate that conditions suitable for the expression of antibodies, antigen-binding fragments thereof, or multispecific 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, a suitable cell culture environment, and a suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for expressing the recombinant antibodies based on the specific laboratory environment.

[0201] Pharmaceutical composition

[0202] The present invention provides a pharmaceutical composition. The pharmaceutical composition comprises: the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antibody, the aforementioned conjugate, the aforementioned nucleic acid, or the aforementioned vector or transformant. The resulting medicament can be further used to prevent and / or treat CD3-related diseases, or further used to prevent and / or treat CD3-related diseases and / or cancers, tumors, infections, or autoimmune diseases.

[0203] According to an embodiment of the present invention, the composition further comprises a pharmaceutically acceptable excipient.

[0204] According to an embodiment of the present invention, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0205] According to an embodiment of the present invention, the pharmaceutical composition is an injection.

[0206] 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 composition can be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components can be administered to the subject simultaneously or sequentially.

[0207] The medicament of the present invention contains a safe and effective amount of the active ingredient of the present invention and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be compatible with the mode of administration. The dosage form of the medicament of the present invention is an injection, oral preparation (tablets, capsules, oral liquid), transdermal preparation, or sustained-release preparation. For example, it can be prepared using conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. The medicament is preferably manufactured under sterile conditions.

[0208] The effective amount of the active ingredient 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, the 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 exigencies of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0209] 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.

[0210] Reagent test kit

[0211] The present invention provides a kit. The kit comprises the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned multispecific antibody. As previously described, the antibodies or antigen-binding fragments thereof according to the embodiments of the present invention can effectively and specifically bind to CD3. A CD3-related kit developed utilizing this property can be used in CD3-related research.

[0212] The kit can effectively detect, enrich, or separate and purify CD3 in biological samples for further scientific research, such as qualitative or quantitative detection of CD3 protein molecules in biological samples. More specifically, it can be used in kits for immunoblotting, immunoprecipitation, and other methods that utilize the specific binding properties of CD3 and antibodies. These kits may contain any one or more of the following: antagonists, CD3 antibodies, or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; and cell assay diluents. CD3 antibodies can be used in various diagnostic tests, such as in vitro or in vivo detection of various diseases or the presence of drugs, toxins, or other proteins. For example, they can be used to test for CD3-related diseases by testing the serum or blood of a subject.

[0213] Use in the preparation of kits

[0214] The present invention provides use of the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned multispecific antibody in preparing a kit for detecting CD3.

[0215] As previously mentioned, the antibodies or antigen-binding fragments thereof according to the embodiments of the present invention are capable of specifically binding to CD3. Therefore, the antibodies or antigen-binding fragments thereof can be used to detect CD3. Furthermore, they can be used to prepare CD3-related kits for scientific research, such as for the qualitative or quantitative detection of CD3 protein molecules in biological samples. More specifically, they can be used in kits for immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of CD3 and antibodies or their binding fragments. These kits may contain any one or more of the following: antagonists, CD3 antibodies, or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; and cell assay diluents. CD3 antibodies or antigen fragments can be used in various diagnostic tests, for example, to detect various diseases or the presence of drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for CD3-related diseases by testing serum or blood from a subject.

[0216] Use in preparing medicines

[0217] The present invention provides use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antibody, the aforementioned conjugate, the aforementioned nucleic acid, the aforementioned vector or transformant, or the aforementioned pharmaceutical composition in the preparation of a medicament for modulating an immune response and / or inhibiting tumor growth, or for preventing and / or treating CD3-related diseases and / or cancers or tumors or infections or autoimmune diseases.

[0218] According to an embodiment of the present invention, the cancer, tumor, infection or autoimmune disease is a Trop2 or PDL1 related disease.

[0219] According to an embodiment of the present invention, the cancer or tumor is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, rectal cancer, breast cancer, pancreatic cancer, prostate cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma or head and neck cancer.

[0220] According to an embodiment of the present invention, the cancer or tumor is at least one of non-small cell lung cancer, malignant melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, colorectal cancer, liver cancer or classical Hodgkin's lymphoma.

[0221] Disease treatment methods

[0222] The present invention provides a method for regulating immune response and / or inhibiting tumor growth or preventing and / or treating CD3-related diseases and / or cancer or tumor or infection or autoimmune disease.

[0223] According to an embodiment of the present invention, the method comprises: administering to a subject a pharmaceutically acceptable amount of the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned multispecific antibody, the above-mentioned conjugate, the above-mentioned nucleic acid, the above-mentioned vector or transformant, or the above-mentioned pharmaceutical composition.

[0224] 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.

[0225] The effective amount of the antibody or antigen-binding fragment thereof, multispecific antibody conjugate, nucleic acid, vector or transformant 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 selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0226] According to an embodiment of the present invention, the cancer, tumor, infection or autoimmune disease is a Trop2 or PDL1 related disease.

[0227] According to an embodiment of the present invention, the cancer or tumor is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, rectal cancer, breast cancer, pancreatic cancer, prostate cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma or head and neck cancer.

[0228] According to an embodiment of the present invention, the cancer or tumor is at least one of non-small cell lung cancer, malignant melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, colorectal cancer, liver cancer or classical Hodgkin's lymphoma.

[0229] The sequences involved in the present invention are detailed in Table 1.

[0230] Table 1 Amino acid sequence description

[0231] 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.

[0232] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not specified, they are carried out according to the technology or conditions described in the literature in this area or according to the product instructions.The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased commercially.

[0233] Example 1: Obtaining and producing affinity-reduced antibodies

[0234] During affinity maturation of natural antibodies, somatic high-frequency mutations are primarily concentrated in the CDR regions. Typically, single-point saturation mutagenesis is performed at each site in the CDR region in vitro to achieve sufficient mutational diversity without disrupting the protein structure. This approach allows for in vitro reproduction that is most similar to the somatic high-frequency mutations of natural antibodies in vivo. This method is generally used for affinity maturation (improvement) of antibodies. The present invention, in contrast, reduces the affinity of CD3 antibodies, using this approach as a reference.

[0235] Single-point saturation mutagenesis was performed on every amino acid site in the CDR region to construct an unbiased single-point saturation mutagenesis plasmid library of the parent antibody. Mutation hotspots that weakened antigen-specific binding were screened, and these hotspots were then combined to obtain candidate antibody mutation sequences.

[0236] Through this technical route and after a large number of screening tests, the affinity of the parent antibody Cross3 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16) was reduced to obtain the following low-affinity CD3 monoclonal antibodies: Cross303, Cross307, Cross308, Cross309, Cross310, Cross311, Cross312, Cross313, Cross314, Cross316, Cross317, Cross318, Cross323, Cross325, Cross326, Cross3ZH07, Cross3ZH09, Cross3ZH16, and Cross3ZH23. The amino acid sequences of the CDR region and the heavy and light chain amino acid sequences of the above-mentioned low-affinity CD3 monoclonal antibody are shown in Table 1 . The amino acid mutations in the CDR region compared with the parent antibody Cross3 are shown in Figure 1 .

[0237] The specific experimental procedures for the production of CD3 monoclonal antibodies 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) The pcDNA3.4 vector containing the heavy chain and light chain of the CD3 antibody (Cross3 heavy chain amino acid sequence is shown in SEQ ID NO: 35 and the light chain amino acid sequence is shown in SEQ ID NO: 33; Cross303 heavy chain amino acid sequence is shown in SEQ ID NO: 37 and the light chain amino acid sequence is shown in SEQ ID NO: 33; Cross307 heavy chain amino acid sequence is shown in SEQ ID NO: 38 and the light chain amino acid sequence is shown in SEQ ID NO: 33; Cross308 heavy chain amino acid sequence is shown in SEQ ID NO: 39 and the light chain amino acid sequence is shown in SEQ ID NO: 33; Cross309 heavy chain amino acid sequence is shown in SEQ ID NO: 40 and the light chain amino acid sequence is shown in SEQ ID NO: 33; Cross310 heavy chain amino acid sequence is shown in SEQ ID NO: 41 and the light chain amino acid sequence is shown in SEQ ID NO: 33; Cross311 heavy chain amino acid sequence is shown in SEQ ID NO: 42 and the light chain amino acid sequence is shown in SEQ ID NO: 43) was inserted into the vector. NO:33; the Cross312 heavy chain amino acid sequence is set forth in SEQ ID NO:43 and the light chain amino acid sequence is set forth in SEQ ID NO:33; the Cross313 heavy chain amino acid sequence is set forth in SEQ ID NO:32 and the light chain amino acid sequence is set forth in SEQ ID NO:33; the Cross314 heavy chain amino acid sequence is set forth in SEQ ID NO:44 and the light chain amino acid sequence is set forth in SEQ ID NO:33; the Cross316 heavy chain amino acid sequence is set forth in SEQ ID NO:34 and the light chain amino acid sequence is set forth in SEQ ID NO:33; the Cross317 heavy chain amino acid sequence is set forth in SEQ ID NO:45 and the light chain amino acid sequence is set forth in SEQ ID NO:33; the Cross318 heavy chain amino acid sequence is set forth in SEQ ID NO:46 and the light chain amino acid sequence is set forth in SEQ ID NO:33; the Cross323 heavy chain amino acid sequence is set forth in SEQ ID NO:45 and the light chain amino acid sequence is set forth in SEQ ID NO:47; the Cross325 heavy chain amino acid sequence is set forth in SEQ ID NO: NO:45 and the light chain amino acid sequence as shown in SEQ ID NO:36; Cross326 heavy chain amino acid sequence as shown in SEQ ID NO:46 and the light chain amino acid sequence as shown in SEQ ID NO:48) (commissioned to Nanjing GenScript for synthesis) was added to 2 mL of OptiSFM culture medium (purchased from Thermo Fisher) at a ratio of 1:1 to obtain solution A.(3) Add 160 μL of ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) to 2 mL of OptiSFM medium (purchased from Thermo Fisher) to obtain solution b. (4) Then mix solution a and solution b to obtain a transfection mixture, and add the entire transfection mixture to 50 mL of ExpiCHO cell solution within 5 minutes. (5) After culturing at 37°C and 5% CO2 for 1 day, add 8 mL of feed and 300 μL of Enhancer (purchased from Thermo Fisher), and transfer to 32°C and 5% CO2 for 9 days. The culture supernatant is harvested, with 8 mL of feed added on the 5th day. (6) Affinity purification is performed from the culture supernatant using a Protein A purification column (purchased from NanoMicro) to obtain the target antibody.

[0238] The specific experimental procedures for the production of bispecific antibodies 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) The pcDNA3.4 vector containing CD3 antibody, Trop2 antibody heavy chain and Trop2 antibody light chain (Cross3×Trop2 contains CD3 antibody shown in SEQ ID NO: 52 and Trop2 antibody heavy chain amino acid sequence shown in SEQ ID NO: 69 and light chain amino acid sequence shown in SEQ ID NO: 70; Cross303×Trop2 contains CD3 antibody shown in SEQ ID NO: 53 and Trop2 antibody heavy chain amino acid sequence shown in SEQ ID NO: 69 and light chain amino acid sequence shown in SEQ ID NO: 70; Cross307×Trop2 contains CD3 antibody shown in SEQ ID NO: 54 and Trop2 antibody heavy chain amino acid sequence shown in SEQ ID NO: 69 and light chain amino acid sequence shown in SEQ ID NO: 70; Cross308×Trop2 contains CD3 antibody shown in SEQ ID NO: 55 and Trop2 antibody heavy chain amino acid sequence shown in SEQ ID NO: 69 and light chain amino acid sequence shown in SEQ ID NO: 70) was added. NO:70; Cross309×Trop2 comprises the CD3 antibody shown in SEQ ID NO:56, the heavy chain amino acid sequence of the Trop2 antibody shown in SEQ ID NO:69, and the light chain amino acid sequence shown in SEQ ID NO:70; Cross310×Trop2 comprises the CD3 antibody shown in SEQ ID NO:57, the heavy chain amino acid sequence of the Trop2 antibody shown in SEQ ID NO:69, and the light chain amino acid sequence shown in SEQ ID NO:70; Cross311×Trop2 comprises the CD3 antibody shown in SEQ ID NO:58, the heavy chain amino acid sequence of the Trop2 antibody shown in SEQ ID NO:69, and the light chain amino acid sequence shown in SEQ ID NO:70; Cross312×Trop2 comprises the CD3 antibody shown in SEQ ID NO:59, the heavy chain amino acid sequence of the Trop2 antibody shown in SEQ ID NO:69, and the light chain amino acid sequence shown in SEQ ID NO:70; Cross313×Trop2 comprises the CD3 antibody shown in SEQ ID NO:49, and the heavy chain amino acid sequence of the Trop2 antibody shown in SEQ ID NO: ID NO:69 and a light chain amino acid sequence as shown in SEQ ID NO:70; Cross314×Trop2 comprises the CD3 antibody as shown in SEQ ID NO:60 and the Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and a light chain amino acid sequence as shown in SEQ ID NO:70;Cross316×Trop2 comprises the CD3 antibody set forth in SEQ ID NO:50, the heavy chain amino acid sequence of the Trop2 antibody set forth in SEQ ID NO:69, and the light chain amino acid sequence set forth in SEQ ID NO:70; Cross317×Trop2 comprises the CD3 antibody set forth in SEQ ID NO:61, the heavy chain amino acid sequence of the Trop2 antibody set forth in SEQ ID NO:69, and the light chain amino acid sequence set forth in SEQ ID NO:70; Cross318×Trop2 comprises the CD3 antibody set forth in SEQ ID NO:62, the heavy chain amino acid sequence of the Trop2 antibody set forth in SEQ ID NO:69, and the light chain amino acid sequence set forth in SEQ ID NO:70; Cross323×Trop2 comprises the CD3 antibody set forth in SEQ ID NO:63, the heavy chain amino acid sequence of the Trop2 antibody set forth in SEQ ID NO:69, and the light chain amino acid sequence set forth in SEQ ID NO:70; Cross325×Trop2 comprises the CD3 antibody set forth in SEQ ID NO:51, and the heavy chain amino acid sequence of the Trop2 antibody set forth in SEQ ID NO: NO:69 and the light chain amino acid sequence as shown in SEQ ID NO:70; Cross326×Trop2 comprising the CD3 antibody shown in SEQ ID NO:64 and the Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and the light chain amino acid sequence as shown in SEQ ID NO:70) (commissioned to Nanjing GenScript for synthesis), or, a pcDNA3.4 vector containing the CD3 antibody, the PDL1 antibody heavy chain and the PDL1 antibody light chain (Cross3×PDL1 comprising the CD3 antibody shown in SEQ ID NO:52 and the PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and the light chain amino acid sequence as shown in SEQ ID NO:72; Cross303×PDL1 comprising the CD3 antibody shown in SEQ ID NO:53 and the PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and the light chain amino acid sequence as shown in SEQ ID NO:72; Cross307×PDL1 comprising the CD3 antibody shown in SEQ ID NO:54 and the PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO: NO:71 and the light chain amino acid sequence as shown in SEQ ID NO:72; Cross308×PDL1 comprises the CD3 antibody as shown in SEQ ID NO:55 and the PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and the light chain amino acid sequence as shown in SEQ ID NO:72;Cross309×PDL1 comprises the CD3 antibody set forth in SEQ ID NO:56, the heavy chain amino acid sequence of the PDL1 antibody set forth in SEQ ID NO:71, and the light chain amino acid sequence set forth in SEQ ID NO:72; Cross310×PDL1 comprises the CD3 antibody set forth in SEQ ID NO:57, the heavy chain amino acid sequence of the PDL1 antibody set forth in SEQ ID NO:71, and the light chain amino acid sequence set forth in SEQ ID NO:72; Cross311×PDL1 comprises the CD3 antibody set forth in SEQ ID NO:58, the heavy chain amino acid sequence of the PDL1 antibody set forth in SEQ ID NO:71, and the light chain amino acid sequence set forth in SEQ ID NO:72; Cross312×PDL1 comprises the CD3 antibody set forth in SEQ ID NO:59, the heavy chain amino acid sequence of the PDL1 antibody set forth in SEQ ID NO:71, and the light chain amino acid sequence set forth in SEQ ID NO:72; Cross313×PDL1 comprises the CD3 antibody set forth in SEQ ID NO:49, and the heavy chain amino acid sequence of the PDL1 antibody set forth in SEQ ID NO: NO:71 and the light chain amino acid sequence is shown in SEQ ID NO:72; Cross314×PDL1 comprises the CD3 antibody shown in SEQ ID NO:60 and the PDL1 antibody heavy chain amino acid sequence is shown in SEQ ID NO:71 and the light chain amino acid sequence is shown in SEQ ID NO:72; Cross316×PDL1 comprises the CD3 antibody shown in SEQ ID NO:50 and the PDL1 antibody heavy chain amino acid sequence is shown in SEQ ID NO:71 and the light chain amino acid sequence is shown in SEQ ID NO:72; Cross317×PDL1 comprises the CD3 antibody shown in SEQ ID NO:61 and the PDL1 antibody heavy chain amino acid sequence is shown in SEQ ID NO:71 and the light chain amino acid sequence is shown in SEQ ID NO:72; Cross318×PDL1 comprises the CD3 antibody shown in SEQ ID NO:62 and the PDL1 antibody heavy chain amino acid sequence is shown in SEQ ID NO:71 and the light chain amino acid sequence is shown in SEQ ID NO:72; Cross323×PDL1 comprises the CD3 antibody shown in SEQ ID NO: NO:63 and the PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and the light chain amino acid sequence as shown in SEQ ID NO:72; Cross325×PDL1 comprises the CD3 antibody as shown in SEQ ID NO:51 and the PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and the light chain amino acid sequence as shown in SEQ ID NO:72;Cross326×PDL1 comprises the CD3 antibody shown in SEQ ID NO:64, the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO:71, and the light chain amino acid sequence shown in SEQ ID NO:72; Cross3ZH07×PDL1 comprises the CD3 antibody shown in SEQ ID NO:65, the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO:71, and the light chain amino acid sequence shown in SEQ ID NO:72; Cross3ZH09×PDL1 comprises the CD3 antibody shown in SEQ ID NO:66, the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO:71, and the light chain amino acid sequence shown in SEQ ID NO:72; Cross3ZH16×PDL1 comprises the CD3 antibody shown in SEQ ID NO:67, the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO:71, and the light chain amino acid sequence shown in SEQ ID NO:72; Cross3ZH23×PDL1 comprises the CD3 antibody shown in SEQ ID NO:68, and the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO: ID NO:71 and the light chain amino acid sequence is shown in SEQ ID NO:72) (commissioned to Nanjing GenScript for synthesis) were added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution A. (3) 160 μL of ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) was added to 2 mL of OptiSFM 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 and 5% CO2 for 1 day, 8 mL of Feed and 300 μL of Enhancer (purchased from Thermo Fisher) were added, and the culture was transferred to 32°C and 5% CO2 for 9 days. The culture supernatant was harvested, and 8 mL of Feed was added on the 5th day. (6) Protein A purification columns (purchased from Nanomicro) were used to affinity purify the culture supernatant to obtain the target CD3×Trop2 or CD3×PDL1 antibody. The antibody configuration is shown in FIG5 .

[0239] Example 2: Antibody affinity detection

[0240] Biacore is a method for analyzing biomolecular interactions based on the principle of optical surface plasmon resonance (SPR). It can not only detect the specific binding between antigens and antibodies, but also obtain intermolecular association rate constant (ka), dissociation rate constant (kd), equilibrium dissociation constant (KD) and other very important data in drug development, thereby calculating the affinity of the antibody.

[0241] In a Biacore 8K (Cytiva) system, the antibody was diluted to 10 μg / mL in running buffer (HBS-EP) and coupled to a CM5 chip (Cytiva) at a flow rate of 10 μL / min. The binding kinetics and affinity data of the low-affinity CD3 monoclonal antibody of Example 1 of the present invention were tested for CD3 E&D antigens at a flow rate of 30 μL / min, with an association time of 120 s and a dissociation time of 800 s. The results of the antibody affinity study are shown in Figure 2.

[0242] Example 3: CD3 monoclonal antibody ELISA binding experiment

[0243] In this example, ELISA was used to test the binding properties of the parental antibody Cross3 and a CD3-affinity-reducing antibody (production methods and heavy and light chain amino acid sequences are described in Example 1). The inventors coated the CD3 E&D proteins onto 96-well plates. The signal strength after antibody addition was used to determine the binding properties of the antibody to CD3 E&D.

[0244] 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 was aspirated from the 96-well plate, and the plate was washed six times with PBST (pH 7.2 PBS 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 serially diluted PBST / 0.05% BSA solution of the Cross3 antibody to be tested, Cross303, Cross307, Cross308, Cross309, Cross310, Cross311, Cross312, Cross313, Cross314, Cross316, Cross317, Cross318, Cross323, Cross325, Cross326, and control IgG1 (purchased from Bio-Bio) were added and incubated at 37°C for 1 h. The reaction mixture was removed, and the plate was washed six times with PBST. Then, 100 μL / well of an HRP-conjugated anti-human secondary antibody (Fab specific) (purchased from Sigma) diluted in PBST / 0.05% BSA was added and incubated at 37°C for 1 h. After washing the plate six times with PBST, add 80 μL / well of TMB (tetramethylbenzidine) and incubate at room temperature for 3 min. Then, add 80 μL / well of 4 M sulfuric acid to terminate the reaction. The absorbance is read at 450 nm using a microplate reader.

[0245] The results are shown in FIG3 , indicating that all CD3 antibodies of the present invention can bind to CD3 E&D. However, due to the limited resolution of the ELISA experiment, the ELISA results cannot reflect the affinity differences of the various CD3 monoclonal antibodies.

[0246] Example 4: CD3 monoclonal antibody flow cytometry binding experiment

[0247] PBMCs 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 the test antibodies Cross3, Cross303, Cross307, Cross308, Cross309, Cross310, Cross311, Cross312, Cross313, Cross314, Cross316, Cross317, Cross318, Cross323, Cross325, and Cross326 (production methods for Cross3 and antibodies with reduced CD3 affinity, as well as the amino acid sequences of their heavy and light chains, are provided in Example 1) and control IgG1 (purchased from Bio-Bio) were added, and the tube was 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 secondary antibody (purchased from Jackson Lab) and 0.5 μL / tube of PerCP-Cy5.5-labeled anti-human CD8 antibody. Incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μL / tube of PBS and analyze by flow cytometry.

[0248] The results are shown in Figure 4, which show that the CD3 antibodies Cross309, Cross310, Cross311, Cross312, Cross313, Cross314, Cross316, Cross317, Cross318, Cross323, and Cross325 of the present invention can bind to CD8 T cells with reduced CD3 affinity, and the CD3 antibody binding ability is weaker than that of the parent antibody Cross3.

[0249] Example 5: CD3×Trop2, CD3×PDL1 bispecific antibody ELISA binding experiment

[0250] This example uses ELISA to test the binding properties of CD3×Trop2 and CD3×PDL1 bispecific antibodies (production methods and heavy and light chain amino acid sequences are described in Example 1). The inventors coated CD3E&D, Trop2, or PDL1 proteins onto 96-well plates. The signal strength after antibody addition was used to determine the binding properties of the antibodies and the corresponding proteins.

[0251] (1) Dilute CD3E&D protein (purchased from Acro) to 2 μg / ml with PBS buffer, add 100 μL / well to a 96-well plate, and place at 4°C overnight. Aspirate the PBS buffer in the 96-well plate, wash the plate 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, add 200 μL / well PBS / 10% BSA, and incubate at 37°C for 2 hours for blocking. Remove the blocking solution, wash the plate 6 times with PBST, and add 100 μL / well PBST / 0.05% BSA gradient dilution of the tested bispecific antibodies Cross3×Trop2, Cross303×Trop2, Cross307×Trop2, Cross308×Trop2, Cross309×Trop2, Cross310×Trop2, Cross311×Trop2, Cross312×Trop2, Cross313×Trop2, Cross314×Trop2, Cross316×Trop2, Cross317×Trop2, Cross318×Trop2, Cross323×Trop2, Cross325×Trop2, Cross326×Trop 2. Cross3×PDL1, Cross303×PDL1, Cross307×PDL1, Cross308×PDL1, Cross309×PDL1, Cross310×PDL1, Cross311×PDL1, Cross312×PDL1, Cross313×PDL1, Cross314×PDL1, Cross316×PDL1, Cross317×PDL1, Cross318×PDL1, Cross323×PDL1, Cross325×PDL1, Cross326×PDL1, and control IgG1LALA (purchased from Bio-Tech), 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-conjugated anti-human secondary antibody (Fab specific) (purchased from Sigma) 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.

[0252] As shown in Figure 6, the CD3×Trop2 antibody of the present invention can bind to CD3E&D. Compared with the parent Cross3×Trop2 bispecific antibody, the affinity of the CD3 bispecific antibody is reduced and the binding of the CD3 bispecific antibody to CD3E&D protein is weakened, which is consistent with expectations.

[0253] As shown in Figure 14, the CD3×PDL1 antibody of the present invention can bind to CD3E&D. Compared with the parent Cross3×PDL1 bispecific antibody, the affinity of the CD3 bispecific antibody is reduced and the binding of the CD3 bispecific antibody to the CD3E&D protein is weakened, which is consistent with expectations.

[0254] (2) Dilute Trop2 protein (purchased from Acro) to 2 μg / ml in PBS buffer and add 100 μL / well to a 96-well plate. Incubate at 4°C overnight. Aspirate the PBS buffer from the 96-well plate, wash the plate six times with PBST (pH 7.2 PBS containing 0.1% Tween 20), and then add 200 μL / well of PBS / 10% BSA. Incubate 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 antibodies to be tested, Cross3×Trop2, Cross303×Trop2, Cross307×Trop2, Cross308×Trop2, Cross309×Trop2, Cross310×Trop2, Cross311×Trop2, Cross312×Trop2, Cross313×Trop2, Cross314×Trop2, Cross316×Trop2, Cross317×Trop2, Cross318×Trop2, Cross323×Trop2, Cross325×Trop2, Cross326×Trop2, and control IgG1LALA (purchased from Bio-Tech) diluted in PBST / 0.05% BSA were added 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-conjugated anti-human secondary antibody (Fab specific) (purchased from Sigma) 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.

[0255] As shown in FIG7 , it was shown that the CD3×Trop2 antibody of the present invention can bind to the Trop2 protein, and all bispecific antibodies bind to the Trop2 protein similarly.

[0256] (3) PDL1 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 the plate was washed six times with PBST (pH 7.2 PBS 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 h for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST. Then, 100 μL / well of the bispecific antibodies to be tested, Cross3×PDL1, Cross303×PDL1, Cross307×PDL1, Cross308×PDL1, Cross309×PDL1, Cross310×PDL1, Cross311×PDL1, Cross312×PDL1, Cross313×PDL1, Cross314×PDL1, Cross316×PDL1, Cross317×PDL1, Cross318×PDL1, Cross323×PDL1, Cross325×PDL1, Cross326×PDL1, and control IgG1LALA (purchased from Bio-Tech) diluted in PBST / 0.05% BSA were added 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-conjugated anti-human secondary antibody (Fab specific) (purchased from Sigma) 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.

[0257] As shown in FIG15 , it was demonstrated that the CD3×PDL1 antibody of the present invention can bind to the PDL1 protein.

[0258] Example 6: CD3×Trop2 Antibody ELISA Bridging Experiment

[0259] An ELISA assay was used to test the bridging binding properties of the CD3×Trop2 bispecific antibody (production methods and heavy and light chain amino acid sequences are described in Example 1). CD3E&D antigen proteins were coated onto a 96-well plate. After the antibody was added, the cells were detected using biotin-labeled Trop2 protein. The signal strength was used to determine the binding properties of the bispecific antibody bridging CD3E&D and Trop2 proteins.

[0260] 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 was aspirated from the 96-well plate, and the plate was washed six times with PBST (pH 7.2 PBS 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 h for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST. Then, 100 μl / well of the bispecific antibodies to be tested, Cross3×Trop2, Cross303×Trop2, Cross307×Trop2, Cross308×Trop2, Cross309×Trop2, Cross310×Trop2, Cross311×Trop2, Cross312×Trop2, Cross313×Trop2, Cross314×Trop2, Cross316×Trop2, Cross317×Trop2, Cross318×Trop2, Cross323×Trop2, Cross325×Trop2, Cross326×Trop2, and control hIgG1LALA (purchased from Bio-Tech), diluted serially with PBST / 0.05% BSA were added and incubated at 37°C for 1 h. Remove the reaction mixture, wash the plate six times with PBST, add Trop2-Biotin protein diluted to the appropriate concentration, and incubate at 37°C for 1 hour. Remove the reaction mixture, wash the plate six times with PBST, and add 100 μl / well of HRP (horseradish peroxidase)-labeled 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, add 80 μl / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and terminate the reaction by adding 80 μl / well of 4M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0261] The results are shown in FIG8 , indicating that the bispecific antibody of the present invention can bridge CD3E&D and Trop2 proteins, and the bridging ability of the parent Cross3×Trop2 antibody is stronger than that of the affinity-reduced CD3 bispecific antibody, which is consistent with expectations.

[0262] Example 7: Bispecific Antibody Flow Cytometry Binding Experiment

[0263] Flow cytometry experiments were used to detect the binding properties of bispecific antibodies. Bispecific antibodies (production methods and amino acid sequences of heavy and light chains are shown in Example 1) were added to cells, and the strength of the signal after the addition of the antibody was measured.

[0264] (1) Dilute PBMC to 2×10 6 / ml, added to a 1.5ml EP tube at a volume of 100μl / tube, 10μl / tube of goat serum was added, and the cells were blocked at 4℃ for 30min. Serial dilutions of bispecific antibodies Cross3×Trop2, Cross303×Trop2, Cross307×Trop2, Cross308×Trop2, Cross309×Trop2, Cross310×Trop2, Cross311×Trop2, Cross312×Trop2, Cross313×Trop2, Cross314×Trop2, Cross315×Trop2, Cross316×Trop2, Cross317×Trop2, Cross318×Trop2, Cross319×Trop2, Cross320×Trop2, Cross321×Trop2, Cross322×Trop2, Cross323×Trop2, Cross324×Trop2, Cross325×Trop2, Cross326×Trop2, Cross327×Trop2, Cross328×Trop2, Cross329×Trop2, Cross330×Trop2, Cross331 6×Trop2, Cross317×Trop2, Cross318×Trop2, Cross323×Trop2, Cross325×Trop2, Cross326×Trop2, Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, Cross325×PDL1, and control hIgG1LALA (purchased from Bio-Bio) were 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 cells were washed once with PBS. After centrifugation, the supernatant was discarded and the cells were resuspended in 100 μl / tube of PBS. 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 were added and incubated at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge and discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze by flow cytometry.

[0265] The results are shown in FIG9 , which show that the CD3×Trop2 antibody of the present invention can bind to CD8 T cells, and compared with the parent CD3 antibody Cross3×Trop2, the CD3 affinity is reduced and the ability of the bispecific antibody to bind to T cells is weakened, which is consistent with expectations.

[0266] The results are shown in Figure 16, which shows that the Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 antibodies of the present invention can bind to CD8 T cells, and the ability of Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 to bind to T cells is weaker than that of Cross3×PDL1, which is consistent with expectations.

[0267] (2) Dilute the tumor cells to 2×10 6 / ml, add 100μl / tube to 1.5ml EP tube, add 10μl / tube goat serum, block at 4℃ for 30min. Add serial dilutions of bispecific antibodies Cross3×Trop2, Cross303×Trop2, Cross307×Trop2, Cross308×Trop2, Cross309×Trop2, Cross310×Trop2, Cross311×Trop2, Cross312×Trop2, Cross313×Trop2, Cross314×Trop2, Cross316×Trop2, Cross317×Trop2, Cross318×Trop2, Cross323×Trop2, Cross325×Trop2, Cross326×Trop2, Cross3×PDL1, Cross303×PDL1, Cross307×P DL1, Cross308×PDL1, Cross309×PDL1, Cross310×PDL1, Cross311×PDL1, Cross312×PDL1, Cross313×PDL1, Cross314×PDL1, Cross316×PDL1, Cross317×PDL1, Cross318×PDL1, Cross323×PDL1, Cross325×PDL1, Cross326×PDL1, and control hIgG1LALA (purchased from Bio-Bio) were 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-conjugated 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 by flow cytometry.

[0268] The results are shown in FIG10 , which show that the CD3×Trop2 antibodies of the present invention can bind to A-375-Trop2 melanoma cells. Judging by the mean fluorescence intensity, all bispecific antibodies have similar binding abilities to A-375-Trop2 cells.

[0269] The results are shown in Figures 17, 18, and 19, indicating that the CD3xPDL1 antibody of the present invention can bind to A-375 melanoma cells, HCT-15 colorectal cancer cells, and A549 lung cancer cells.

[0270] Example 8: Experiment on Bispecific Antibodies Promoting PBMC to Kill Tumor Cells

[0271] The ability of bispecific antibodies to promote PBMC to kill A-375 melanoma cells and HCT-15 colorectal cancer cells was tested.

[0272] (a) Complete RPMI-1450 medium was added to a 16-well RTCA plate at a volume of 50 μL / well and calibrated on the instrument.

[0273] (b) Tumor cells were diluted to 2×10 5 / mL, and added to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then the cell coefficient was detected using the xCELLigence RTCA MP device at 37°C and 5% CO2 for 24 hours;

[0274] (c) Serial dilutions of the bispecific antibodies Cross3×Trop2, Cross303×Trop2, Cross307×Trop2, Cross308×Trop2, Cross309×Trop2, Cross310×Trop2, Cross311×Trop2, Cross312×Trop2, Cross313×Trop2, Cross314×Trop2, Cross316×Trop2, Cross317×Trop2, Cross318×Trop2, Cross323×Trop2, Cross325×Trop2, Cross326×Trop2, Cross3×PDL1, Cross303×PDL1, Cross307×Trop2, Cross319×Trop2, Cross328×Trop2, Cross330×Trop2, Cross331×Trop2, Cross332×Trop2, Cross333×Trop2, Cross334×Trop2, Cross335×Trop2, Cross336×Trop2, Cross337×Trop2, Cross338×Trop2, Cross339×Trop2, Cross340×Trop2, Cross341×Trop2, Cross342×Trop2, Cross343×Trop2, Cross344×Trop2 ×PDL1, Cross308×PDL1, Cross309×PDL1, Cross310×PDL1, Cross311×PDL1, Cross312×PDL1, Cross313×PDL1, Cross314×PDL1, Cross316×PDL1, Cross317×PDL1, Cross318×PDL1, Cross323×PDL1, Cross325×PDL1, Cross326×PDL1, Cross3ZH07×PDL1, Cross3ZH09×PDL1, Cross3ZH16×PDL1, Cross3ZH23×PDL1, and control hIgG1LALA (purchased from Bio-Tech) were added to the RTCA plate obtained in step (2) at a volume of 20 μl / well;

[0275] (4) PBMC (purchased from Miaoshun Biotechnology) were diluted to 1.25×106 pcs / ml, and added to the RTCA plate obtained in step (3) at a volume of 80 μl / well;

[0276] (5) The reaction system obtained in step (4) was incubated at 37° C. and 5% CO 2 for 24 h to detect the cell coefficient using an xCELLigence RTCA MP device.

[0277] As shown in Figure 11, the CD3×Trop2 antibody of the present invention promotes PBMC to kill A-375-Trop2 melanoma cells. The onset dose of the Cross3×Trop2 bispecific antibody is lower than that of the affinity-reduced bispecific antibody. Although the onset doses of Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 are high, the maximum killing ratio is consistent with that of Cross3×Trop2, and all can achieve the highest killing.

[0278] As shown in Figure 12 , Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 promoted PBMC to kill A-375-Trop2 melanoma cells. Although the effective doses of Cross313×Trop2 and Cross316×Trop2 were high, the highest killing ratio was consistent with that of Cross3×Trop2, and both could achieve the highest killing.

[0279] As shown in FIG20 , CD3×PDL1 of the present invention promotes PBMC to kill A-375 melanoma cells, and Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 have the highest killing effect, which is better than other bispecific antibodies.

[0280] As shown in FIG21 , Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 of the present invention promoted PBMC to kill A-375 melanoma cells, and Cross316×PDL1 had the highest killing effect, which was superior to Cross313×PDL1 and Cross325×PDL1.

[0281] As shown in FIG22 , CD3×PDL1 of the present invention promoted PBMC to kill HCT-15 colorectal cancer cells, and Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 were able to promote PBMC to kill HCT-15 colorectal cancer cells 100%.

[0282] Example 9: Experiment on Bispecific Antibodies Promoting Cytokine Secretion by PBMC

[0283] A bispecific antibody (production method and amino acid sequences of heavy and light chains are shown in Example 1) was added to the PBMC and tumor cell co-incubation system. After 48 h of incubation, the culture supernatant was collected and the cytokine content in the supernatant was detected to determine the characteristics of the bispecific antibody in inducing cytokine release.

[0284] (1) Dilute the tumor to 1×10 5 / ml, added to 96-well plates, and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0285] (2) The bispecific antibodies Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, Cross325×Trop2, Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, Cross325×PDL1, and control hIgG1LALA (purchased from Bio-Bio) were serially diluted in complete RPMI 1450 medium and added to a 96-well plate at 20 μl / well.

[0286] (3) PBMC (purchased from Saili Biotechnology) were diluted to 1.25×10 6 / ml, added to 96-well plate, 80 μl / well;

[0287] (4) Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 48 h;

[0288] (5) Centrifuge at 300 g for 10 min at room temperature and collect the cell culture supernatant;

[0289] (6) The cytokine content in the supernatant was detected using a CBA kit (purchased from BD).

[0290] As shown in Figure 13, Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 of the present invention promoted PBMC to secrete proinflammatory cytokines TNF-α, IL-6, IFN-γ, and IL-2, and the secretion of inflammatory factors promoted by Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 antibodies was weaker than that promoted by Cross3×Trop2.

[0291] As shown in Figures 23 and 24, Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 of the present invention promote the secretion of proinflammatory cytokines TNF-α and IFN-γ by PBMC, and Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 antibodies promote the secretion of inflammatory factors weaker than Cross3×PDL1.

[0292] 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.

[0293] 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. An anti-CD3 antibody or an antigen-binding fragment thereof, characterized in that: Contains: heavy chain variable region CDRs and light chain variable region CDRs; Wherein, the heavy chain variable region CDRs have an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 3, 7 to 8 and 74 to 86 or a conservatively modified form thereof; and / or the light chain variable region CDRs have an amino acid sequence as shown in any one of SEQ ID NOs: 4 to 6, 9 and 87 to 92 or a conservatively modified form thereof.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain variable region CDRs have an amino acid sequence as shown in SEQ ID NOs: 1 to 2 or a conservative modification thereof, and an amino acid sequence as shown in one of SEQ ID NOs: 3, 7 to 8, and 74 to 86 or a conservative modification thereof; The light chain variable region CDRs have an amino acid sequence as shown in SEQ ID NOs: 4 to 5 or a conservatively modified form thereof, and an amino acid sequence as shown in one of SEQ ID NOs: 6, 9 and 87 to 92 or a conservatively modified form thereof.

3. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The antibody or antigen-binding fragment thereof comprises: The heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 1, The heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 2, The heavy chain variable region CDR3 has an amino acid sequence as shown in one of SEQ ID NO: 3, SEQ ID NO: 7-8, and SEQ ID NO: 74-86; The light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO:4, The light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 5, The light chain variable region CDR3 has an amino acid sequence as shown in one of SEQ ID NOs: 6, 9 and 87-92.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The antibody or antigen-binding fragment thereof comprises any one of the following groups: 1) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 74, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; 2) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 75, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; 3) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 76, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; 4) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 77, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; 5) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 78, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; 6) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 79, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; 7) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 80, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; 8) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 81, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; 9) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 82, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; 10) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 83, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; 11) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; 12) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 7, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; 13) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 8, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 87, respectively; 14) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 8, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 88, respectively; 15) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 8, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 9, respectively; 16) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 84, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 89, respectively; 17) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 85, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 90, respectively; 18) having heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 86, respectively, and light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 92, respectively; 19) have heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 8, respectively, and have light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 91, respectively.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain framework region and / or a light chain framework region.

6. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: At least a portion of the heavy chain framework region and / or the 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.

7. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The antibody or antigen-binding fragment thereof comprises: A heavy chain variable region having an amino acid sequence as shown in one of SEQ ID NOs: 10, 12-13, 15-24, 27 and 31 or an amino acid sequence of a conservatively modified form thereof; and / or, A light chain variable region having an amino acid sequence as shown in one of SEQ ID NOs: 11, 14, 25-26, 28 and 30 or an amino acid sequence of a conservatively modified form thereof.

8. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The heavy chain variable region has an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in one of SEQ ID NOs: 10, 12-13, 15-24, 27 and 31; and / or, The light chain variable region has an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in one of SEQ ID NOs: 11, 14, 25-26, 28 and 30.

9. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that: The antibody or antigen-binding fragment thereof comprises an amino acid sequence shown in any of the following combinations of heavy chain variable regions and light chain variable regions or an amino acid sequence having at least 90% sequence identity thereto:

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region; Wherein, the constant region includes at least one of a heavy chain constant region and a light chain constant region.

11. The antibody or antigen-binding fragment thereof according to claim 10, characterized in that: At least a portion of at least one of the heavy chain constant region and the light chain constant 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.

12. The antibody or antigen-binding fragment thereof according to claim 10, characterized in that: The heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and / or, The light chain constant region comprises a light chain constant region selected from a kappa-type or a lambda-type.

13. The antibody or antigen-binding fragment thereof according to claim 11, characterized in that: The light chain constant region and the heavy chain constant region are both derived from a murine antibody or a mutant thereof, or a human antibody or a mutant thereof.

14. The antibody or antigen-binding fragment thereof according to claim 10, characterized in that: The N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or, The N-terminus of the light chain constant region is linked to the N-terminus of the light chain variable region.

15. The antibody or antigen-binding fragment thereof according to claim 10, characterized in that: The heavy chain constant region has an amino acid sequence as shown in SEQ ID NO: 93 or an amino acid sequence having at least 80% identity thereto; and / or, The light chain constant region has an amino acid sequence as shown in SEQ ID NO: 94 or an amino acid sequence having at least 80% identity thereto.

16. The antibody or antigen-binding fragment thereof according to claim 10, characterized in that: The C-terminus of the heavy chain variable region is connected to the N-terminus of the light chain variable region, or the N-terminus of the heavy chain variable region is connected to the C-terminus of the light chain variable region.

17. The antibody or antigen-binding fragment thereof according to claim 16, characterized in that: The antibody or antigen-binding fragment thereof further comprises a first connecting peptide, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the light chain variable region, or the C-terminus of the light chain variable region is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the heavy chain variable region.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody comprises at least one of a full-length monoclonal antibody, a chimeric antibody, a humanized antibody, Fv, scFv, Fab, Fab', Fab'-SH and F(ab')2; The antigen-binding fragment of the antibody includes at least one of a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein and a minimum recognition unit.

19. The antibody or antigen-binding fragment thereof according to claim 18, characterized in that: The antibody or antigen-binding fragment comprises: A heavy chain having an amino acid sequence as shown in one of SEQ ID NOs: 32, 34, 35 and 37-46, or an amino acid sequence having at least 80% identity thereto; and / or, A light chain having an amino acid sequence as shown in one of SEQ ID NOs: 33, 36 and 47-48, or an amino acid sequence having at least 80% identity thereto.

20. The antibody or antigen-binding fragment thereof according to claim 18, characterized in that: The antibody or antigen-binding fragment thereof has an amino acid sequence shown in any of the following heavy chain and light chain combinations or an amino acid sequence having at least 80% sequence identity thereto:

21. The antibody or antigen-binding fragment thereof according to claim 18, characterized in that: The antibody or antigen-binding fragment thereof has an amino acid sequence shown in any one of SEQ ID NOs: 99 to 117 or an amino acid sequence having at least 80% sequence identity therewith.

22. The antibody or antigen-binding fragment thereof according to claim 18, characterized in that: The antibody or antigen-binding fragment thereof has an amino acid sequence shown in any of the following groups of Fab VL-CL and Fab VH-CH1 combinations or an amino acid sequence having at least 80% sequence identity thereto:

23. The antibody or antigen-binding fragment thereof according to claim 18, characterized in that: The antibody or antigen-binding fragment thereof has an amino acid sequence as shown in any one of SEQ ID NOs: 142 to 160 or an amino acid sequence having at least 80% sequence identity thereto.

24. A multispecific antibody, characterized in that Include: A first antigen binding region, wherein the first antigen binding region comprises the anti-CD3 antibody or antigen binding fragment thereof according to any one of claims 1 to 23; The second antigen binding region has a biomolecule binding activity, and the biomolecule is not CD3.

25. The multispecific antibody according to claim 24, characterized in that The multispecific antibody is selected from a bispecific antibody, a trispecific antibody and a tetraspecific antibody, preferably a bispecific antibody.

26. The multispecific antibody according to claim 25, characterized in that The bispecific antibody includes a symmetric bispecific antibody or an asymmetric bispecific antibody.

27. The multispecific antibody according to claim 24, characterized in that The second antigen binding region is a binding protein of the biomolecule or a fragment thereof.

28. The multispecific antibody according to claim 24, characterized in that The biomolecule is selected from at least one of the following: PDL1, CD47, TIGIT, CD73, CD33, CEACAM1, CEACAM5, CEACAM6, STING, WNT, Beta catenin, B7H3, VISITA, CD19, BCMA, CD22, CD20, CD123, CD38, CEA, CD25, CD46, CD138, PSCA, PSMA, PSA, MUC1, MUC16, NY-ESO-1, GD2, WT1, Mesothelin, MAGE-A3, GPC3, PRAME, Globo H, AFP, Trop2, FOLR1, SP, Sca-1, CD133, EPCAM.

29. The multispecific antibody according to claim 27, 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.

30. The multispecific antibody according to claim 29, characterized in that The bispecific antibody is an asymmetric bispecific antibody.

31. The multispecific antibody according to claim 24, characterized in that The first antigen binding region comprises a first scFv fragment, a first Fv fragment, a first Fab fragment or a first Fab-Linker fragment.

32. The multispecific antibody according to claim 31, characterized in that The first antigen binding region further comprises a first Fc peptide segment.

33. The multispecific antibody according to claim 32, characterized in that The N-terminus of the first Fc peptide segment is connected to the C-terminus of the scFv fragment or the Fv fragment; or, The N-terminus of the first Fc peptide segment is connected to the C-terminus of CH1 of the Fab fragment or the Fab-Linker fragment.

34. The multispecific antibody according to claim 32, characterized in that The first antigen binding region further comprises a second connecting peptide, the N-terminus of the first Fc peptide segment is connected to the C-terminus of the second connecting peptide, and the N-terminus of the second connecting peptide is connected to the C-terminus of the scFv fragment or the Fv fragment; or, The N-terminus of the first Fc peptide segment is connected to the C-terminus of the second connecting peptide, and the N-terminus of the second connecting peptide is connected to the C-terminus of CH1 of the Fab fragment or Fab-Linker fragment.

35. The multispecific antibody according to claim 32, characterized in that The first Fc peptide segment is selected from human Fc peptide segments.

36. The multispecific antibody according to claim 35, characterized in that The first Fc peptide segment is a human IgG1 Fc peptide segment.

37. The multispecific antibody according to claim 32, characterized in that The first Fc peptide segment has the amino acid sequence shown in SEQ ID NO:118; or the first Fc peptide segment has the amino acid sequence shown in SEQ ID NO:

166.

38. The multispecific antibody according to claim 31, characterized in that The first scFv fragment has an amino acid sequence shown in any one of SEQ ID NOs: 95-117.

39. The multispecific antibody according to claim 31, characterized in that The first Fab fragment has an amino acid sequence shown in any of the following combinations of Fab VL-CL and Fab VH-CH1:

40. The multispecific antibody according to claim 31, characterized in that The first Fab-Linker has an amino acid sequence as shown in any one of SEQ ID NOs: 142-160.

41. The multispecific antibody according to claim 24, characterized in that The first antigen binding region has an amino acid sequence as shown in any one of SEQ ID NOs: 49-52, 54-68.

42. The multispecific antibody according to claim 28, characterized in that The biomolecule is Trop2 or PDL1.

43. The multispecific antibody according to claim 42, characterized in that The second antigen-binding region comprises a first anti-Trop2 antibody or an antigen-binding fragment thereof or a first anti-PDL1 antibody or an antigen-binding fragment thereof.

44. The multispecific antibody according to claim 43, characterized in that The first anti-Trop2 antibody or antigen-binding fragment thereof is a second scFab fragment or a second Fab fragment of anti-Trop2; or, The first anti-PDL1 antibody or antigen-binding fragment thereof is the third scFab fragment or the third Fab fragment of anti-PDL1.

45. The multispecific antibody according to claim 44, characterized in that The second scFab fragment has the amino acid sequence shown in SEQ ID NO: 161; or, The second Fab fragment has the amino acid sequence shown in SEQ ID NO: 162 and 70; or, The third scFab fragment has an amino acid sequence as shown in SEQ ID NO: 163; or, The third Fab fragment has the amino acid sequence shown in SEQ ID NOs: 164 and 72.

46. ​​The multispecific antibody according to claim 44, characterized in that The second antigen binding region further comprises a second Fc fragment; The N-terminus of the second Fc fragment is connected to the C-terminus of CH1 of the second scFab fragment, the second Fab fragment, the third scFab fragment or the third Fab fragment.

47. The multispecific antibody according to claim 46, characterized in that The second Fc peptide segment is selected from human Fc peptide segments.

48. The multispecific antibody according to claim 47, characterized in that The second Fc peptide segment is a human IgG1 Fc peptide segment.

49. The multispecific antibody according to claim 46, characterized in that The second Fc peptide segment has an amino acid sequence as shown in SEQ ID NO:

165.

50. The multispecific antibody according to claim 46, characterized in that The first Fc peptide segment and the second Fc peptide segment are connected via a knob-into-hole structure.

51. The multispecific antibody according to claim 43, characterized in that The second antigen binding region has an amino acid sequence as shown in SEQ ID NO: 161 or 163; or, The second antigen binding region has an amino acid sequence as shown in SEQ ID NO: 162 and 70; or, The second antigen binding region has the amino acid sequence shown in SEQ ID NO: 164 and 72.

52. A conjugate, characterized in that include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 or the multispecific antibody according to any one of claims 24 to 51; A conjugate moiety is linked to the antibody or antigen-binding fragment thereof, or the multispecific antibody.

53. The conjugate according to claim 52, characterized in that The coupling part includes at least one selected from a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier, a therapeutic agent and a chemotherapeutic agent.

54. A nucleic acid, characterized in that Encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 or the multispecific antibody according to any one of claims 24 to 51.

55. A vector or transformant, characterized in that: Containing the nucleic acid according to claim 54.

56. The vector or transformant according to claim 55, characterized in that The vector is a eukaryotic vector or a prokaryotic vector.

57. The vector or transformant according to claim 55, characterized in that The vector comprises at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.

58. A cell, characterized in that Carrying the nucleic acid of claim 54, the vector or transformant of any one of claims 55 to 57; or expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 23 or the multispecific antibody of any one of claims 24 to 51.

59. The cell according to claim 58, characterized in that The cell is a prokaryotic cell, a eukaryotic cell or a bacteriophage.

60. A pharmaceutical composition, characterized in that Comprising: the antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, the multispecific antibody according to any one of claims 24 to 51, the conjugate according to any one of claims 52 to 53, the nucleic acid according to claim 54, or the vector or transformant according to any one of claims 55 to 57.

61. The pharmaceutical composition according to claim 60, characterized in that Further includes pharmaceutically acceptable excipients.

62. The pharmaceutical composition according to claim 61, characterized in that The auxiliary materials include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

63. The pharmaceutical composition according to claim 60, characterized in that The pharmaceutical composition is an injection.

64. A kit, characterized in that The method comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 or the multispecific antibody according to any one of claims 24 to 51.

65. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 or the multispecific antibody according to any one of claims 24 to 51 in the preparation of a kit for detecting CD3.

66. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 23, the multispecific antibody of any one of claims 24 to 51, the conjugate of any one of claims 52 to 53, the nucleic acid of claim 54, the vector or transformant of any one of claims 55 to 57, or the pharmaceutical composition of any one of claims 60 to 63 in the preparation of a medicament for regulating an immune response and / or inhibiting tumor growth, or for preventing and / or treating a CD3-related disease and / or cancer or tumor or infection or autoimmune disease.

67. The use according to claim 66, characterized in that The cancer, tumor, infection or autoimmune disease is a Trop2 or PDL1 related disease.

68. The use according to claim 67, characterized in that The cancer or tumor is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, rectal cancer, breast cancer, pancreatic cancer, prostate cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma or head and neck cancer; and / or The cancer or tumor is at least one of non-small cell lung cancer, malignant melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, colorectal cancer, liver cancer or classical Hodgkin's lymphoma.

69. A method for regulating immune response and / or inhibiting tumor growth or preventing and / or treating CD3-related diseases and / or cancer or tumors or infections or autoimmune diseases, characterized in that: The method comprises: administering to a subject a pharmaceutically acceptable amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 23, the multispecific antibody of any one of claims 24 to 51, the conjugate of any one of claims 52 to 53, the nucleic acid of claim 54, the vector or transformant of any one of claims 55 to 57, or the pharmaceutical composition of any one of claims 60 to 63.

70. The method according to claim 69, characterized in that The cancer, tumor, infection or autoimmune disease is a Trop2 or PDL1 related disease.

71. The method according to claim 70, characterized in that The cancer or tumor is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, rectal cancer, breast cancer, pancreatic cancer, prostate cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma or head and neck cancer; and / or The cancer or tumor is at least one of non-small cell lung cancer, malignant melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, colorectal cancer, liver cancer or classical Hodgkin's lymphoma.

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