Anti-CD3 antibodies and their applications

Abstract

This invention proposes an anti-CD3 antibody or its antigen-binding fragment and its applications. It comprises: 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 NO: 1-3, 7-8, and 74-86 or a conserved modified form thereof; and / or the light chain variable region CDRs have an amino acid sequence as shown in any one of SEQ ID NO: 4-6, 9, and 87-92 or a conserved modified form thereof. The anti-CD3 antibody or its antigen-binding fragment of this invention can bind to human CD3 protein and can reduce the production of pro-inflammatory cytokines. Bispecific antibodies constructed using this antibody exhibit high PBMC-based tumor-killing activity and weak pro-inflammatory cytokine secretion-promoting properties. Therefore, pharmaceutical compositions prepared using this anti-CD3 antibody or its antigen-binding fragment or its multispecific antibody have better safety and high clinical application and drug development value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically, this invention relates to an anti-CD3 antibody or its antigen-binding fragment and its applications. Background Technology

[0002] Cancer is a major disease affecting human survival and development. According to the latest data, there are approximately 19 million new cancer cases and 10 million cancer deaths worldwide each year, and both incidence and mortality rates are on the rise. Besides surgical resection, traditional cancer treatments such as chemotherapy and radiotherapy have significant side effects and are prone to recurrence. In recent years, immunotherapy, including tumor-targeting antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hotspot and a source of new hope in the fight against cancer. Immunotherapy drugs, including monoclonal antibodies and bispecific antibodies, mainly work by promoting the function of the body's own immune cells to achieve anti-cancer effects, characterized by fewer side effects and longer survival times for beneficiaries. 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 hematologic malignancies. Targeting CD19, CD20, and BCMA, they can promote T cells to eliminate tumor cells and benefit patients.

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

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

[0005] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, one object of the invention is to provide an anti-CD3 antibody or its antigen-binding fragment. Another object of the invention is to provide a multispecific antibody constructed using the said anti-CD3 antibody or its antigen-binding fragment. The anti-CD3 antibody or its antigen-binding fragment of the present invention can bind to human CD3 protein and can reduce the production of pro-inflammatory cytokines. The bispecific antibody constructed using it has high PBMC-killing tumor-killing activity and weak pro-inflammatory cytokine secretion-promoting properties. Therefore, pharmaceutical compositions prepared using this anti-CD3 antibody or its antigen-binding fragment or its multispecific antibody have better safety and higher clinical application and drug development value.

[0006] This invention is based on the inventor's discoveries and understanding of the following facts and problems:

[0007] Currently, the most commonly used bispecific CD3 antibodies are TR66 and UCHT1. On the one hand, neither of these antibodies binds to monkey CD3, increasing the difficulty of non-clinical drug evaluation; on the other hand, these two antibodies have very strong binding affinity to T cells, increasing the safety risks of CD3 bispecific antibody drugs constructed based on them. The inventors modified the affinity of Cross3, a high CD3 affinity (KD = 0.165 nM) anti-CD3 antibody with human-monkey cross-linking. Through extensive screening, they obtained a series of anti-CD3 antibodies that can bind to human CD3 protein with low affinity. Furthermore, based on this series of anti-CD3 antibodies, they constructed and screened 19 bispecific CD3 antibodies. Compared to Cross3, the specific recognition and binding of these bispecific antibodies to tumor antigens are not affected by the CD3 antigen-binding region. Further experimental results demonstrate 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 essentially equivalent to that of bispecific antibodies constructed based on Cross3; 2) Cell-level experimental results show 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 essentially equivalent to that of bispecific antibodies constructed based on Cross3 antibodies; 3) Experimental results on promoting PBMC cytokine secretion show that the CD3 bispecific antibody of the present invention has reduced pro-inflammatory cytokine secretion performance.

[0008] The inventors further verified the killing activity of the above-mentioned CD3 bispecific antibodies against tumor cells 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 promote 100% tumor cell killing by PBMCs, and have a comparable tumor cell killing effect to the bispecific antibodies constructed based on the anti-CD3 antibody Cross3 (such as Cross3×Trop2 and Cross3×PDL1).

[0009] The above experimental results show that the CD3 bispecific antibody developed based on the anti-CD3 antibody or its antigen-binding fragment of the present invention has high PBMC tumor-killing activity and weak ability to promote the secretion of pro-inflammatory cytokines, with better safety and high value for clinical application and drug development.

[0010] Therefore, in a first aspect, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof. According to embodiments of the present invention, the anti-CD3 antibody or its antigen-binding fragment comprises: heavy chain variable regions (CDRs) and light chain variable regions (CDRs), wherein the heavy chain variable regions (CDRs) have an amino acid sequence as shown in any one of SEQ ID NO: 1–3, 7–8, and 74–86 or a conserved modified form thereof; and / or the light chain variable regions (CDRs) have an amino acid sequence as shown in any one of SEQ ID NO: 4–6, 9, and 87–92 or a conserved modified form thereof.

[0011] The anti-CD3 antibody or its antigen-binding fragment according to embodiments of the present invention can bind to human CD3 protein and reduce the production of pro-inflammatory cytokines. The CD3 bispecific antibody developed based on this CD3 antibody or its antigen-binding fragment exhibits high PBMC-killing tumor-killing activity and weak pro-inflammatory cytokine secretion-promoting properties. When prepared as a drug, it can, on the one hand, distribute more extensively to the tumor site, increasing the local drug concentration and reducing the peripheral drug concentration, thereby reducing the risk of off-target toxicity; on the other hand, this bispecific antibody drug can reduce the production of pro-inflammatory cytokines, reducing the risk of on-target toxicity. Furthermore, the drug prepared using the aforementioned anti-CD3 antibody or its antigen-binding fragment can be used for the prevention and / or treatment of CD3-related diseases; the CD3 bispecific antibody drug developed based on the aforementioned anti-CD3 antibody or its antigen-binding fragment has higher safety and can be used for the prevention and / or treatment of cancer, tumors, infections, or autoimmune diseases. Therefore, the anti-CD3 antibody or its antigen-binding fragment of the present invention has good clinical application value and drug development value.

[0012] In a second aspect, the present invention provides a multispecific antibody. According to an embodiment of the present invention, the multispecific antibody comprises:

[0013] A first antigen-binding region, wherein the first antigen-binding region comprises the anti-CD3 antibody or its antigen-binding fragment as described in the first aspect of the present invention;

[0014] The second antigen-binding region has biomolecule binding activity, wherein the biomolecule is not CD3.

[0015] As previously described, multispecific antibodies (such as bispecific antibodies) developed based on the anti-CD3 antibody or its antigen-binding fragment described in the first aspect of this invention, when prepared as drugs, can be more widely distributed to the tumor site, increasing the local drug concentration and reducing the peripheral drug concentration, thereby reducing the risk of off-target toxicity. Simultaneously, they can reduce the production of pro-inflammatory cytokines, reducing the target toxicity risk of multispecific antibodies (such as bispecific antibodies). Therefore, multispecific antibodies based on the anti-CD3 antibody or its antigen-binding fragment described in the first aspect of this invention have better safety and higher clinical application and drug development value.

[0016] In a third aspect, the present invention provides a conjugate. According to embodiments of the present invention, the conjugate comprises: the antibody or its antigen-binding fragment described in the first aspect of the present invention, or the multispecific antibody described in the second aspect of the present invention.

[0017] As described above, the anti-CD3 antibody or its antigen-binding fragment of the present invention can be used to prepare bispecific antibody drugs. On the one hand, the prepared bispecific antibody drug can be distributed more extensively to the tumor site, increasing the local drug concentration and reducing the peripheral drug concentration, thereby reducing the risk of off-target toxicity. On the other hand, the bispecific antibody can reduce the production of pro-inflammatory cytokines, reducing the target toxicity risk of multispecific antibodies (such as bispecific antibodies). Furthermore, drugs prepared using the aforementioned 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, tumors, infections, or autoimmune diseases. Therefore, conjugates including the anti-CD3 antibody or its antigen-binding fragment of the first aspect of the present invention and the multispecific antibody of the second aspect of the present invention have good clinical application value and drug development value.

[0018] In a fourth aspect of the invention, a nucleic acid is provided. According to embodiments of the invention, the nucleic acid encodes the anti-CD3 antibody or its antigen-binding fragment as described in the first aspect of the invention, or the multispecific antibody as described in the second aspect of the invention. According to embodiments of the invention, the nucleic acid encoding the antibody or its antigen-binding fragment as described in the first aspect of the invention, or the multispecific antibody as described in the second aspect of the invention, is suitable for developing monoclonal or bispecific antibody drugs with higher safety. Further, the protein encoded by the nucleic acid can be used for the prevention and / or treatment of CD3-related diseases, or for the prevention and / or treatment of cancer, tumors, infections, or autoimmune diseases.

[0019] 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, using the constructed vector or transformant, the antibody of the first aspect of the present invention or its antigen-binding fragment, or the multispecific antibody of the second aspect of the present invention, can be effectively expressed, as well as the conjugate of the third aspect of the present invention can be obtained.

[0020] 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 described in the first aspect of the present invention or its antigen-binding fragment, or the multispecific antibody of the second aspect of the present invention. According to embodiments of the present invention, the cell can efficiently express the antibody of the first aspect of the present invention or its antigen-binding fragment, or the multispecific antibody of the second aspect of the present invention under suitable conditions, and further, obtains an antibody or its antigen-binding fragment, or its multispecific antibody, with improved safety in binding to human CD3 protein.

[0021] In a seventh aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: an antibody or antigen-binding fragment thereof according to a first aspect of the present invention, a multispecific antibody according to a second aspect of the present invention, a conjugate according to a third aspect of the present invention, a nucleic acid according to a fourth aspect of the present invention, or a carrier or transformant according to a fifth aspect of the present invention. Thus, the obtained pharmaceutical composition can be further used for the prevention and / or treatment of CD3-related diseases, or further used for the prevention and / or treatment of CD3-related diseases and / or cancer or tumors or infections or autoimmune diseases.

[0022] In an eighth aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises an antibody or antigen-binding fragment thereof from the first aspect of the present invention, or a multispecific antibody from the second aspect of the present invention. Thus, the obtained kit can be used for CD3-related research.

[0023] In a ninth aspect of the invention, the present invention provides for the use of the antibody or antigen-binding fragment thereof of the first aspect of the invention or the multispecific antibody of the second aspect of the invention in the preparation of a kit for detecting CD3. Those skilled in the art will understand that the features and advantages of the antibody or antigen-binding fragment thereof described above also apply to this use, and will not be repeated here.

[0024] In a tenth aspect of the invention, the invention provides for the use of the antibody or antigen-binding fragment thereof of the first aspect of the invention, the multispecific antibody of the second aspect of the invention, the conjugate of the third aspect of the invention, the nucleic acid of the fourth aspect of the invention, the carrier or transformant of the fifth aspect of the invention, or the pharmaceutical composition of the seventh aspect of the invention in the preparation of a medicament for the prevention and / or treatment of CD3-related diseases and / or cancer or tumors or infections or autoimmune diseases. The antibody or antigen-binding fragment thereof, the multispecific antibody and conjugate, the nucleic acid, the carrier or transformant, or the pharmaceutical composition of the invention can be further prepared into a medicament that can be used clinically for the prevention or treatment of diseases.

[0025] Those skilled in the art will understand that the features and advantages described above for antibodies or their antigen-binding fragments, conjugates, nucleic acid molecules, vectors or transformants and pharmaceutical compositions also apply to this use and will not be repeated here.

[0026] 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 in this invention can reduce the production of pro-inflammatory cytokines caused by the CD3 antigen binding site compared to the parent antibody Cross3.

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

[0029] (3) The bispecific antibodies constructed from the antibodies Cross313, Cross316, and Cross325 obtained in this invention can also promote the maximum killing of tumor cells by PBMCs compared with the bispecific antibodies constructed from the parent antibody Cross3. Moreover, the bispecific antibodies have a weaker ability to promote the production of pro-inflammatory cytokines and are safer.

[0030] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0031] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0032] Figure 1 This is a comparison chart of amino acid mutations between the affinity-reducing antibody and the maternal antibody Cross3 according to embodiments of the present invention;

[0033] Figure 2 This is a graph showing the SPR results of the affinity assay between the maternal antibody Cross3 and the affinity-reducing antibody and CD3E&D protein according to an embodiment of the present invention.

[0034] Figure 3 This is an ELISA result diagram of the binding of the maternal antibody Cross3 and the affinity-reducing antibody to CD3E&D protein according to an embodiment of the present invention.

[0035] Figure 4 This is a flow cytometry result of the binding of the maternal antibody Cross3 and the affinity-reducing antibody to CD8 T cells according to an embodiment of the present invention.

[0036] Figure 5 This is a schematic diagram of the configuration of the CD3×Trop2 and CD3×PDL1 bispecific antibodies composed of the maternal antibody Cross3 and the affinity-reducing antibody according to an embodiment of the present invention.

[0037] Figure 6 The image shows the ELISA results of the binding of the CD3×Trop2 bispecific antibody, composed of the maternal antibody Cross3 and the affinity-reducing antibody, to the CD3E&D protein according to an embodiment of the present invention.

[0038] Figure 7 The image shows the ELISA results of the binding of the CD3×Trop2 bispecific antibody, composed of the maternal antibody Cross3 and the affinity-reducing antibody, to the Trop2 protein according to an embodiment of the present invention.

[0039] Figure 8 The figure shows the ELISA results of CD3×Trop2 bispecific antibody bridging CD3E&D and Trop2 protein, which is composed of the maternal antibody Cross3 and the affinity-reducing antibody according to an embodiment of the present invention.

[0040] Figure 9 The image shows the flow cytometry results of CD8 T cells binding to CD3×Trop2 bispecific antibodies composed of the maternal antibody Cross3 and the affinity-reducing antibody according to an embodiment of the present invention.

[0041] Figure 10The image shows the flow cytometry results of A-375-Trop2 melanoma cells bound to the CD3×Trop2 bispecific antibody composed of the maternal antibody Cross3 and the affinity-reducing antibody according to an embodiment of the present invention.

[0042] Figure 11 This diagram illustrates the results of CD3×Trop2 bispecific antibody, composed of the maternal antibody Cross3 and an affinity-reducing antibody according to an embodiment of the present invention, promoting the killing of A-375-Trop2 melanoma cells by PBMCs.

[0043] Figure 12 The figure shows the results of Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 bispecific antibodies promoting PBMC killing of A-375-Trop2 melanoma cells according to embodiments of the present invention.

[0044] Figure 13 The diagram shows the results of the Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 bispecific antibodies promoting the secretion of pro-inflammatory cytokines by PBMCs according to embodiments of the present invention.

[0045] Figure 14 The image shows the ELISA results of the binding of the CD3×PDL1 bispecific antibody, composed of the maternal antibody Cross3 and the affinity-reducing antibody, to the CD3E&D protein according to an embodiment of the present invention.

[0046] Figure 15 The image shows the ELISA results of the binding of the CD3×PDL1 bispecific antibody, composed of the maternal antibody Cross3 and the affinity-reducing antibody, to the PDL1 protein according to an embodiment of the present invention.

[0047] Figure 16 The image shows the flow cytometry results of CD3×PDL1 bispecific antibody, composed of the maternal antibody Cross3 and the affinity-reducing antibody according to an embodiment of the present invention, binding to CD8 T cells.

[0048] Figure 17 The image shows the flow cytometry results of A-375 melanoma cells bound to the CD3×PDL1 bispecific antibody composed of the maternal antibody Cross3 and the affinity-reducing antibody according to an embodiment of the present invention.

[0049] Figure 18 This is a flow cytometry result of HCT-15 colorectal cancer cells, showing the binding of the CD3×PDL1 bispecific antibody, composed of the maternal antibody Cross3 and the affinity-reducing antibody according to an embodiment of the present invention, to HCT-15 colorectal cancer cells.

[0050] Figure 19 This is a flow cytometry result of A-549 lung cancer cells bound to the CD3×PDL1 bispecific antibody composed of the maternal antibody Cross3 and the affinity-reducing antibody according to an embodiment of the present invention.

[0051] Figure 20 This diagram shows the results of CD3×PDL1 bispecific antibody, composed of the maternal antibody Cross3 and the affinity-reducing antibody according to an embodiment of the present invention, promoting the killing of A-375 melanoma cells by PBMCs.

[0052] Figure 21 The diagram shows the results of Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 promoting PBMC killing of A-375 melanoma cells according to embodiments of the present invention.

[0053] Figure 22 This is a diagram showing the results of CD3×PDL1 bispecific antibody, composed of the maternal antibody Cross3 and the affinity-reducing antibody according to an embodiment of the present invention, promoting the killing of HCT-15 colorectal cancer cells by PBMCs.

[0054] Figure 23 The diagram shows the results of promoting the secretion of pro-inflammatory cytokines by PBMCs after co-incubation with A-375 using a CD3×PDL1 bispecific antibody composed of the maternal antibody Cross3 and an affinity-reducing antibody, according to an embodiment of the present invention.

[0055] Figure 24 The diagram shows the results of promoting the secretion of pro-inflammatory cytokines by PBMCs after co-incubation with HCT-15 using a CD3×PDL1 bispecific antibody composed of the maternal antibody Cross3 and an affinity-reducing antibody, according to an embodiment of the present invention. Detailed Implementation

[0056] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[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 technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0058] The endpoints and any values ​​of the ranges 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 endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0059] Terms and Definitions

[0060] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly 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 pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0061] In this article, the term "antibody" generally refers to an antibody that recognizes one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, triple-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854 4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy-chain, full-length light-chain, or intact immunoglobulin molecules; or the immunologically active portion of any of these polypeptides, i.e., molecules or portions thereof containing an antigen-binding site that specifically binds to a target antigen of interest, such targets including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases.

[0062] In this paper, certain regions within the variable region exhibit a higher degree of variation in amino acid composition and sequence, termed "hypervariable region (HVR)." The hypervariable region is the location where antigens and antibodies bind, and is therefore also called the complementarity-determining region (CDR). Both the heavy chain and light chain variable regions contain three CDR regions. For example, these typically include amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., amino acid residues near 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)).

[0063] In this article, the term "anti-CD3 antibody" refers to an antibody that can bind to CD3. This antibody is also referred to as "CD3-binding antibody" in this article.

[0064] In this article, the term "Fab-Linker" refers to a polypeptide fragment obtained by linking Fab VH-CH1 and Fab VL-CL of an antibody Fab fragment using a linker peptide. This includes, but is not limited to, scFab and scFab△ (the linker peptide does not contain disulfide bonds).

[0065] In this document, the term "antigen-binding fragment" is equivalent to "antibody fragment" or "antigen-binding antibody fragment," and can include a portion of a complete antibody, generally a binding region or variable region. This includes, but is not limited to: Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, scFv-Fc fragments, or bispecific antibodies (BsAbs), linear antibodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol ("PEGylated," "PEGylated") (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol).

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

[0067] In this article, the term "humanized antibody" refers to a recombinant antibody obtained by replacing all non-CDR (Fv backbone region (FR)) amino acid sequences in the constant and variable regions of a monoclonal antibody from one species (e.g., mouse) with non-CDR amino acid sequences in the constant and variable regions of an antibody from another species (e.g., human). That is, when the constant region of an antibody is humanized, it is called a chimeric antibody, while when all non-CDR amino acid sequences in both the constant and variable regions are humanized, it is called a humanized antibody. The humanization methods can be performed using conventional antibody engineering techniques and will not be elaborated upon here.

[0068] For nucleotides, the terms "homology," "identity," or "similarity" are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of "sequence homology" between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings and known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0 967 284, EP 1 085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0069] For peptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0070] In this document, the term "conservatively modified amino acid sequence" refers to an amino acid modification that does not significantly affect or alter the binding properties of an antibody containing that amino acid sequence. Such modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this invention using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is the replacement of an amino acid residue 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, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine).

[0071] In this article, the term "conjugate" is understood in context to refer to an antibody or its antigen-binding fragment that is conjugated to a coupling part such as a carrier substance, drug, toxin, cytokine, protein tag, modifier, therapeutic agent, or chemotherapeutic agent using any covalent or non-covalent biological conjugation strategy.

[0072] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0073] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0074] In this article, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0075] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0076] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, conjugates, or pharmaceutical compositions of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0077] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in an individual who is susceptible but has not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0078] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0079] This invention proposes an anti-CD3 antibody or its antigen-binding fragment, a multispecific antibody, a conjugate, a nucleic acid molecule, a vector or transformant, a cell, a pharmaceutical composition, a kit, and their applications, which will be described in detail below.

[0080] Anti-CD3 antibody or its antigen-binding fragment

[0081] This invention provides an anti-CD3 antibody or its antigen-binding fragment. The anti-CD3 antibody or its antigen-binding fragment comprises: heavy chain variable regions (CDRs) and light chain variable regions (CDRs), wherein the heavy chain variable regions (CDRs) have an amino acid sequence as shown in any one of SEQ ID NO: 1–3, 7–8, and 74–86 or a conserved modified form thereof; and / or the light chain variable regions (CDRs) have an amino acid sequence as shown in any one of SEQ ID NO: 4–6, 9, and 87–92 or a conserved modified form thereof.

[0082] The anti-CD3 antibody or its antigen-binding fragment of the present invention can bind to human CD3 protein and reduce the production of pro-inflammatory cytokines. The CD3 bispecific antibody developed based on this CD3 antibody or its antigen-binding fragment exhibits high PBMC-killing tumor-killing activity and weak pro-inflammatory cytokine secretion-promoting properties. When prepared as a drug, it can, on the one hand, distribute more extensively to the tumor site, increasing the local drug concentration and reducing the peripheral drug concentration, thereby reducing the risk of off-target toxicity; on the other hand, this bispecific antibody drug can reduce the production of pro-inflammatory cytokines, reducing the risk of on-target toxicity. Furthermore, the drug prepared using the aforementioned anti-CD3 antibody or its antigen-binding fragment can be used for the prevention and / or treatment of CD3-related diseases; the CD3 bispecific antibody drug developed based on the aforementioned anti-CD3 antibody or its antigen-binding fragment has higher safety and can be used for the prevention and / or treatment of cancer, tumors, infections, or autoimmune diseases. 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 antibody or its antigen-binding fragment of the present invention may be replaced by other amino acid residues from the same side chain family, and the retention function of the modified antibody can be tested using the functional assay methods described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0084] According to embodiments of the present invention, the heavy chain variable regions CDRs have amino acid sequences as shown in SEQ ID NO:1-2 or their conserved modified forms, and amino acid sequences as shown in one of SEQ ID NO:3, 7-8 and 74-86 or their conserved modified forms; the light chain variable regions CDRs have amino acid sequences as shown in SEQ ID NO:4-5 or their conserved modified forms, and amino acid sequences as shown in one of SEQ ID NO:6, 9 and 87-92 or their conserved modified forms.

[0085] According to embodiments of the present invention, the antibody or its antigen-binding fragment 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 any one of SEQ ID NO:3, SEQ ID NO:7-8, and SEQ ID NO:74-86; 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 any one of SEQ ID NO:6, 9, and 87-92.

[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 7-8; 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 embodiments of the present invention, the antibody or its antigen-binding fragment comprises any one of the following groups:

[0088] 1) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 74 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 6 respectively;

[0089] 2) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 75 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 6 respectively;

[0090] 3) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 76 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 6 respectively;

[0091] 4) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 77 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 6 respectively;

[0092] 5) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 78 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 6 respectively;

[0093] 6) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 79 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 6 respectively;

[0094] 7) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 80 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 6 respectively;

[0095] 8) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 81 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 6 respectively;

[0096] 9) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 82 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 6 respectively;

[0097] 10) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 83 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 6 respectively;

[0098] 11) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 3 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 6 respectively;

[0099] 12) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 7 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 6 respectively;

[0100] 13) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 8 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 87 respectively;

[0101] 14) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 8 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 88 respectively;

[0102] 15) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 8 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 9 respectively;

[0103] 16) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 84 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 89 respectively;

[0104] 17) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 85 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 90 respectively;

[0105] 18) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 86 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 92 respectively;

[0106] 19) having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2 and 8 respectively, and light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, 5 and 91 respectively.

[0107] It should be noted that the antigen-binding fragment will consist of a partial sequence of the heavy chain variable region or the light chain variable region of its source antibody, or contain partial sequences of the light and heavy chain variable regions, wherein the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as its source antibody.

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

[0109] According to embodiments of the present invention, at least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof; preferably, at least one of mouse antibodies, human antibodies, and primate antibodies.

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

[0111] As previously described, one or more amino acid residues in the heavy or light chain variable region of the antibody or its antigen-binding fragment of the present invention may be replaced by other amino acid residues from the same side chain family, and the retained function of the modified antibody may be tested using the functional assay methods described herein.

[0112] According to embodiments of the present invention, the antibody or its antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in one of SEQ ID NO: 10, 12-13, 15-24, 27 and 31 or an amino acid sequence in a conserved modified form thereof; and / or, a light chain variable region having an amino acid sequence as shown in one of SEQ ID NO: 11, 14, 25-26, 28 and 30 or an amino acid sequence in a conserved modified form thereof.

[0113] It should be noted that, without 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 substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the heavy or light chain variable region of the antibody or its antigen-binding fragment to obtain sequences of the antibody or its antigen-binding fragment. These variations are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the heavy or light chain variable region. The sequences of the above-mentioned variations of this invention can 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 this invention can be measured using sequence analysis software. For example, the computer program BLAST with default parameters, especially BLASTP or TBLASTN.

[0115] According to an embodiment of the present invention, 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 NO: 10, 12-13, 15-24, 27 and 31; and / or, the light 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 NO: 11, 14, 25-26, 28 and 30.

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

[0117] Antibody or its antigen-binding fragment group number Heavy chain variable region Light chain variable region 1 SEQ ID NO:15 SEQ ID NO:11 2 SEQ ID NO:16 SEQ ID NO:11 3 SEQ ID NO:17 SEQ ID NO:11 4 SEQ ID NO:17 SEQ ID NO:11 5 SEQ ID NO:19 SEQ ID NO:11 6 SEQ ID NO:20 SEQ ID NO:11 7 SEQ ID NO:21 SEQ ID NO:11 8 SEQ ID NO:22 SEQ ID NO:11 9 SEQ ID NO:23 SEQ ID NO:11 10 SEQ ID NO:24 SEQ ID NO:11 11 SEQ ID NO:10 SEQ ID NO:11 12 SEQ ID NO:12 SEQ ID NO:11 13 SEQ ID NO:13 SEQ ID NO:25 14 SEQ ID NO:13 SEQ ID NO:26 15 SEQ ID NO:13 SEQ ID NO:14 16 SEQ ID NO:27 SEQ ID NO:28 17 SEQ ID NO:29 SEQ ID NO:28 18 SEQ ID NO:13 SEQ ID NO:30 19 SEQ ID NO:31 SEQ ID NO:26

[0118] According to an embodiment of the present invention, the antibody or its antigen-binding fragment further comprises a constant region; wherein the constant region comprises at least one of a heavy chain constant region and a light chain constant region.

[0119] According to embodiments 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 mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

[0120] It should be noted that the immunoglobulins discussed 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 moieties that provide reduced or enhanced effector cell activity. Immunoglobulins can be derived from any species.

[0121] According to embodiments 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.

[0122] In some specific implementations, the heavy chain constant region includes 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.

[0123] In some specific implementations, the heavy chain constant region includes a heavy chain constant region selected from mouse IgG, IgA, IgM, IgE or IgD, such as mouse IgG1, mouse IgG2a, mouse IgG2b, mouse IgG2c, mouse IgG3, mouse IgA, mouse IgM, mouse IgE or mouse IgD.

[0124] According to an embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from mouse antibodies or their mutants, or human antibodies or their mutants.

[0125] It should be noted that the amino acid sequences mentioned in this invention are shown from the N-terminus to the C-terminus.

[0126] According to an embodiment of the present invention, the N end of the heavy chain constant region is connected to the C end of the heavy chain variable region; and / or, the N end of the light chain constant region is connected to the N end of the light chain variable region.

[0127] As previously stated, without 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 substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the constant region of the heavy or light chain of the antibody or its antigen-binding fragment to obtain sequences of the antibody or its antigen-binding fragment. These variations are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties may be substituted in the constant region of the heavy or light chain. The sequences of the above-described variations of this invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence.

[0128] According to embodiments 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 having at least 80% identity with it; 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 with it.

[0129] According to an embodiment of the present invention, the heavy chain constant region and the light chain constant region have amino acid sequences as shown in SEQ ID NO:93 and SEQ ID NO:94, respectively.

[0130] According to an embodiment of the present invention, the C-end of the heavy chain variable region is connected to the N-end of the light chain variable region, or the N-end of the heavy chain variable region is connected to the C-end of the light chain variable region.

[0131] According to an embodiment of the present invention, the antibody or its antigen-binding fragment further comprises a first linker peptide, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the first linker peptide, and the C-terminus of the first linker 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 linker peptide, and the C-terminus of the first linker peptide is connected to the N-terminus of the heavy chain variable region.

[0132] In some specific embodiments, the first linker peptide has an amino acid sequence as shown in (GGGGS)n, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0133] As previously described, the antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies) or contain only the 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 to antibodies lacking a fucose moiety on the oligosaccharide chain to, for example, enhance antibody-dependent cytotoxicity (ADCC) function. In other embodiments, galactosylation modification may be performed to alter complement-dependent cytotoxicity (CDC).

[0134] According to embodiments of the present invention, 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 comprises at least one of an F(ab')2 fragment, a Fab' fragment, a Fab fragment, an F(ab)2 fragment, an Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, and a minimum recognition unit.

[0135] As previously stated, without 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 substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the heavy or light chain, scFv fragment, Fab fragment, or scFab fragment of the antibody or its antigen-binding fragment to obtain variations of the sequence of the antibody or its antigen-binding fragment. These variations are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties may be substituted in the heavy or light chain. The sequences of the above-described variations of this invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence.

[0136] According to embodiments 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 NO:32, 34, 35 and 37-46 or an amino acid sequence having at least 80% identity with it; and / or a light chain having an amino acid sequence as shown in one of SEQ ID NO:33, 36 and 47-48 or an amino acid sequence having at least 80% identity with it.

[0137] According to embodiments of the present invention, the antibody or its antigen-binding fragment has an amino acid sequence as shown in any of the following combinations of heavy and light chains, or an amino acid sequence having at least 80% sequence identity with the following:

[0138] Antibody or its antigen-binding fragment group number Heavy chain Light chain 1 SEQ ID NO:37 SEQ ID NO:33 2 SEQ ID NO:38 SEQ ID NO:33 3 SEQ ID NO:39 SEQ ID NO:33 4 SEQ ID NO:40 SEQ ID NO:33 5 SEQ ID NO:41 SEQ ID NO:33 6 SEQ ID NO:42 SEQ ID NO:33 7 SEQ ID NO:43 SEQ ID NO:33 8 SEQ ID NO:44 SEQ ID NO:33 9 SEQ ID NO:45 SEQ ID NO:33 10 SEQ ID NO:46 SEQ ID NO:33 11 SEQ ID NO:32 SEQ ID NO:33 12 SEQ ID NO:34 SEQ ID NO:33 13 SEQ ID NO:35 SEQ ID NO:47 14 SEQ ID NO:35 SEQ ID NO:48 15 SEQ ID NO:35 SEQ ID NO:36 16 SEQ ID NO:95 SEQ ID NO:119 17 SEQ ID NO:96 SEQ ID NO:120 18 SEQ ID NO:97 SEQ ID NO:121 19 SEQ ID NO:98 SEQ ID NO:122

[0139] According to embodiments of the present invention, the antibody or its antigen-binding fragment has an amino acid sequence shown in any one of SEQ ID NO: 99 to 117 or an amino acid sequence having at least 80% sequence identity with it.

[0140] According to embodiments of the present invention, the antibody or its antigen-binding fragment has an amino acid sequence represented by any one of the following combinations of Fab VL-CL and Fab VH-CH1, or an amino acid sequence having at least 80% sequence identity with it:

[0141]

[0142]

[0143] According to embodiments of the present invention, the antibody or its antigen-binding fragment has an amino acid sequence as shown in any one of SEQ ID NO:142 to 160 or an amino acid sequence having at least 80% sequence identity with it.

[0144] Multispecific antibodies

[0145] This invention provides a multispecific antibody. The multispecific antibody includes: a first antigen-binding region, the first antigen-binding region comprising the above-mentioned anti-CD3 antibody or its antigen-binding fragment; and a second antigen-binding region, the second antigen-binding region having biomolecule binding activity, wherein the biomolecule is not CD3.

[0146] As previously described, multispecific antibodies (such as bispecific antibodies) developed based on the anti-CD3 antibody or its antigen-binding fragment described in the first aspect of this invention, when prepared as drugs, can be more widely distributed to the tumor site, increasing the local drug concentration and reducing the peripheral drug concentration, thereby reducing the risk of off-target toxicity. Simultaneously, they can reduce the production of pro-inflammatory cytokines, reducing the target toxicity risk of multispecific antibodies (such as bispecific antibodies). Therefore, multispecific antibodies based on the anti-CD3 antibody or its antigen-binding fragment described in the first aspect of this invention have better safety and higher clinical application and drug development value.

[0147] According to an embodiment of the present invention, the multispecific antibody is selected from one of bispecific antibodies, trispecific antibodies, and tetraspecific antibodies.

[0148] Bispecific antibodies are preferred.

[0149] According to embodiments of the present invention, the bispecific antibody includes a symmetrical bispecific antibody or an asymmetrical bispecific antibody.

[0150] According to an embodiment of the present invention, the second antigen-binding region is a binding protein of the biomolecule or a fragment thereof.

[0151] According to embodiments of the present invention, the biomolecules are 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.

[0152] According to embodiments of the present invention, the binding protein or a fragment thereof is selected from at least one of an antibody or its antigen-binding fragment, a receptor, and a ligand.

[0153] According to an embodiment of the present invention, the bispecific antibody is an asymmetric bispecific antibody.

[0154] 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.

[0155] According to an embodiment of the present invention, the first antigen-binding region further comprises a first Fc peptide segment.

[0156] According to an embodiment of the present invention, the N-terminus of the first Fc peptide is connected to the C-terminus of the scFv fragment or the Fv fragment; or, the N-terminus of the first Fc peptide is connected to the C-terminus of the CH1 of the Fab fragment or the Fab-Linker fragment.

[0157] According to an embodiment of the present invention, the first antigen-binding region further includes a second linker peptide, wherein the N-terminus of the first Fc peptide is linked to the C-terminus of the second linker peptide, and the N-terminus of the second linker peptide is linked to the C-terminus of the scFv fragment or Fv fragment; or, the N-terminus of the first Fc peptide is linked to the C-terminus of the second linker peptide, and the N-terminus of the second linker peptide is linked to the C-terminus of CH1 of the Fab fragment or Fab-Linker fragment.

[0158] In some specific embodiments, the second linker peptide has an amino acid sequence as shown in (GGGGS)n, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0159] According to an embodiment of the present invention, the first Fc peptide is selected from human Fc peptides. Preferably, it is human IgG1 Fc peptide.

[0160] According to an embodiment of the present invention, the first Fc peptide has an amino acid sequence as shown in SEQ ID NO:118; or the first Fc peptide has an amino acid sequence as shown in SEQ ID NO:166.

[0161] According to an embodiment of the present invention, the first scFv fragment has an amino acid sequence shown in any one of SEQ ID NO: 95 to 117.

[0162] According to an embodiment of the present invention, the first Fab fragment has an amino acid sequence as shown in any of the following combinations of Fab VL-CL and Fab VH-CH1:

[0163]

[0164]

[0165] 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 NO:142 to 160.

[0166] 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 NO:49-52, 54-68.

[0167] According to an embodiment of the present invention, the biomolecule is Trop2 or PDL1.

[0168] 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.

[0169] According to an embodiment of the present invention, the first anti-Trop2 antibody or its antigen-binding fragment is a second scFab fragment or a second Fab fragment of anti-Trop2; or, the first anti-PDL1 antibody or its antigen-binding fragment is a third scFab fragment or a third Fab fragment of anti-PDL1.

[0170] According to embodiments of the present invention, the second scFab fragment has an amino acid sequence as shown in SEQ ID NO:161; or, the second Fab fragment has an amino acid sequence as 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 an amino acid sequence as shown in SEQ ID NO:164 and 72.

[0171] 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 the CH1 of the second scFab fragment, the second Fab fragment, the third scFab fragment, or the third Fab fragment.

[0172] According to an embodiment of the present invention, the second Fc peptide is selected from human Fc peptides, preferably human IgG1 Fc peptides.

[0173] According to an embodiment of the present invention, the second Fc peptide has an amino acid sequence as shown in SEQ ID NO:165.

[0174] According to embodiments of the present invention, the first Fc peptide and the second Fc peptide are connected via a knob-into-hole structure. According to embodiments 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.

[0175] In some optional embodiments of the present invention, the multispecific antibody of the present invention has Figure 5 The structure shown.

[0176] In some alternative embodiments of the present invention, such as Figure 5 As shown in A, 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 NO:49-52 and 54-68, 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.

[0177] In some alternative embodiments of the present invention, such as Figure 5 As shown in B, 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 NO:49-52, 54-68, and a second peptide chain having an amino acid sequence as shown in SEQ ID NO:161 or 163.

[0178] In some alternative embodiments of the present invention, such as Figure 5 As shown in C, 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 NO:142 to 160, 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.

[0179] In some alternative embodiments of the present invention, such as Figure 5 As shown in D, 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 NO:142 to 160, and a second peptide chain having an amino acid sequence as shown in SEQ ID NO:161 or 163.

[0180] In some alternative embodiments of the present invention, such as Figure 5As shown in E, the multispecific antibody of the present invention comprises: 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 comprising Fab VH-CH1 having an amino acid sequence as shown in SEQ ID NO:162 or 164 and scFv having amino acid sequences as shown in SEQ ID NO:99 to 117, wherein the C-terminus of the Fab VH-CH1 is connected to the N-terminus of the scFv.

[0181] Conjugate

[0182] This invention provides a conjugate. The conjugate comprises: the antibody described above or its antigen-binding fragment, or the multispecific antibody described above;

[0183] The conjugated portion is linked to the antibody or its antigen-binding fragment, or a multispecific antibody;

[0184] As described above, the anti-CD3 antibody or its antigen-binding fragment of the present invention can be used to prepare multispecific antibodies, such as bispecific antibody drugs. On the one hand, the prepared bispecific antibody drug can be distributed more extensively to the tumor site, increasing the local drug concentration and reducing the peripheral drug concentration, thereby reducing the risk of off-target toxicity. On the other hand, the bispecific antibody can reduce the production of pro-inflammatory cytokines, reducing the risk of on-target toxicity. Furthermore, drugs prepared using the anti-CD3 antibody or its antigen-binding fragment can be used to prevent and / or treat CD3-related diseases. CD3 bispecific antibody drugs developed based on the anti-CD3 antibody or its antigen-binding fragment have higher safety and can be used to prevent and / or treat cancer, tumors, infections, or autoimmune diseases. Therefore, conjugates including the anti-CD3 antibody or its antigen-binding fragment of the first aspect of the present invention and the multispecific antibody of the second aspect of the present invention have good clinical application value and drug development value.

[0185] According to embodiments of the present invention, the coupling portion includes at least one selected from carriers, drugs, toxins, cytokines, protein tags, modifiers, therapeutic agents, and chemotherapeutic agents.

[0186] Nucleic acid

[0187] This invention provides a nucleic acid. The nucleic acid encodes the aforementioned antibody or its antigen-binding fragment, or the aforementioned multispecific antibody.

[0188] According to embodiments of the present invention, the nucleic acid encoding the antibody of the first aspect of the present invention or its antigen-binding fragment, or the multispecific antibody of the second aspect of the present invention, is suitable for developing monoclonal or bispecific antibody drugs with higher safety. Furthermore, the protein encoded by the nucleic acid can be used for the prevention and / or treatment of CD3-related diseases, or for the prevention and / or treatment of cancer, tumors, infections, or autoimmune diseases.

[0189] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases, the complementary strand is also disclosed. Furthermore, the molecular sequences in this invention include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0190] Vector or transformant

[0191] This invention provides a vector or transformant. The vector or transformant contains the aforementioned nucleic acid. The vector or transformant may include an optional control sequence operatively linked to the nucleic acid molecule. The control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The vector or transformant constructed thereby can effectively express the antibody or antigen-binding fragment of the first aspect of this invention or the multispecific antibody of the second aspect of this invention, and obtain the conjugate of the third aspect of this invention.

[0192] When linking the aforementioned nucleic acid molecules to the vector or transformant, such as an expression vector, the nucleic acid molecules can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. Of course, these control elements can be directly derived from the vector itself or be exogenous, i.e., not derived from the vector itself. The nucleic acid molecules and control elements simply need to be operatively linked.

[0193] According to embodiments of the present invention, the vector may refer to a cloning vector or an expression vector, and can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in this invention is not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0194] In this document, the term "operably ligated" refers to ligating a foreign gene to a vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include viral vectors, plasmids, bacteriophages, etc. After the expression vector of some specific embodiments of the present invention is introduced into suitable recipient cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of the proteins encoded by the nucleic acid molecules.

[0195] According to an embodiment of the present invention, the carrier is a eukaryotic carrier or a prokaryotic carrier.

[0196] According to an embodiment of the present invention, the vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

[0197] cell

[0198] This invention provides a cell. The cell carries the aforementioned nucleic acid, the aforementioned vector or transformant, or expresses the aforementioned antibody or its antigen-binding fragment, or the aforementioned multispecific antibody. Thus, according to embodiments of this invention, the cell can efficiently express the antibody or its antigen-binding fragment of the first aspect of this invention, or the multispecific antibody of the second aspect of this invention, under suitable conditions. Furthermore, it yields an antibody or its antigen-binding fragment that can bind to human CD3 protein and has improved safety, or a multispecific antibody thereof.

[0199] According to an embodiment of the present invention, the cell is a prokaryotic cell, a eukaryotic cell, or a bacteriophage.

[0200] According to an embodiment of the present invention, the prokaryotic cells are Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0201] According to embodiments of the present invention, the eukaryotic cell is a fungal cell, an insect cell, a plant cell, or a mammalian cell.

[0202] According to an embodiment of the present invention, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomyces cerevisiae, or Trichoderma.

[0203] According to an embodiment of the present invention, the insect cell is a grass 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.

[0204] According to an embodiment of the present invention, the cell is a mammalian cell.

[0205] According to an embodiment of the present invention, the cells are BHK cells, CHO cells, COS cells, or NSO cells.

[0206] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the antibody or its antigen-binding fragment or multispecific antibody described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the antibody or its antigen-binding fragment or multispecific antibody include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the recombinant antibody based on the specific environment of their laboratory.

[0207] Pharmaceutical Composition

[0208] This invention provides a pharmaceutical composition. The pharmaceutical composition comprises: the antibody or antigen-binding fragment thereof described above, the multispecific antibody described above, the conjugate described above, the nucleic acid described above, or the carrier or transformant described above. The resulting drug can be further used for the prevention and / or treatment of CD3-related diseases, or further used for the prevention and / or treatment of CD3-related diseases and / or cancer or tumors or infections or autoimmune diseases.

[0209] According to embodiments of the present invention, pharmaceutically acceptable excipients are further included.

[0210] According to embodiments of the present invention, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0211] According to an embodiment of the present invention, the pharmaceutical composition is an injection.

[0212] It should be noted that the pharmaceutical composition includes combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the present invention. For example, the components contained in the composition may 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 may be administered to the subject simultaneously or sequentially.

[0213] The medicament of this invention contains a safe and effective amount of the active ingredient of this invention and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage forms of the medicament of this invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, it is prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The medicament is preferably manufactured under aseptic conditions.

[0214] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0215] The pharmaceutically acceptable excipients described in this 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 matched to the route of administration, as is well known to those skilled in the art.

[0216] Reagent test kit

[0217] This invention provides a kit. The kit comprises the antibody or its antigen-binding fragment described above, or the multispecific antibody described above. As previously stated, the antibody or its antigen-binding fragment of this invention can effectively bind specifically to CD3. The CD3-related kit developed using this characteristic can be used for CD3-related research.

[0218] The kit can effectively detect, enrich, or 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 involving the detection of CD3 by utilizing the specific binding properties of antibodies, such as immunoblotting and immunoprecipitation. 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 types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for CD3-related diseases by detecting the serum or blood of a subject.

[0219] Uses in the preparation kit

[0220] The present invention provides the use of the above-described antibody or its antigen-binding fragment or the above-described multispecific antibody in the preparation of a kit for detecting CD3.

[0221] As previously described, the antibodies or antigen-binding fragments of the present invention can specifically bind to CD3; therefore, the antibodies or antigen-binding fragments 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 involving the specific binding properties of CD3 and antibodies or their binding fragments, such as immunoblotting and immunoprecipitation. 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 different types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, CD3-related diseases can be tested by examining the serum or blood of a subject.

[0222] Uses in drug preparation

[0223] The present invention provides the use of the above-described antibody or antigen-binding fragment thereof, the above-described multispecific antibody, the above-described conjugate, the above-described nucleic acid, the above-described carrier or transformant, or the above-described pharmaceutical composition in the preparation of a medicament for regulating immune responses and / or inhibiting tumor growth, or for preventing and / or treating CD3-related diseases and / or cancer or tumors or infections or autoimmune diseases.

[0224] According to an embodiment of the present invention, the cancer or tumor or infection or autoimmune disease is a Trop2 or PDL1-related disease.

[0225] According to embodiments 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, stomach cancer, esophageal cancer, oral squamous cell carcinoma, or head and neck cancer.

[0226] According to embodiments of the present invention, optionally, 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 classic Hodgkin lymphoma.

[0227] Disease treatment methods

[0228] This invention provides a method for modulating immune responses and / or inhibiting tumor growth or for preventing and / or treating CD3-related diseases and / or cancer, tumors, infections, or autoimmune diseases.

[0229] According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the antibody or antigen-binding fragment thereof, the multispecific antibody, the conjugate, the nucleic acid, the carrier or transformant, or the pharmaceutical composition thereof.

[0230] It should be noted that the terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one implementation, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects can be humans, but also include other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0231] The effective amount of the antibody or its antigen-binding fragment, multispecific antibody conjugate, nucleic acid, carrier or transformant, or pharmaceutical composition described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0232] According to an embodiment of the present invention, the cancer or tumor or infection or autoimmune disease is a Trop2 or PDL1-related disease.

[0233] According to embodiments 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, stomach cancer, esophageal cancer, oral squamous cell carcinoma, or head and neck cancer.

[0234] According to embodiments of the present invention, optionally, 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 classic Hodgkin lymphoma.

[0235] The sequence descriptions involved in this invention are detailed in Table 1.

[0236] Table 1. Amino Acid Sequence Description

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255] The present invention will be described in detail below through examples. In the examples or test cases, experimental methods without specific conditions are performed under conventional conditions.

[0256] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0257] Example 1: Obtaining and producing antibodies with reduced affinity

[0258] During the affinity maturation process of natural antibodies, high-frequency somatic mutations are mainly concentrated in the CDR region. Generally, single-point saturation mutations at each site in the CDR region are performed in vitro to obtain sufficient mutational diversity without disrupting the protein structure. This approach achieves in vitro reproducibility most similar to the high-frequency somatic mutations of natural antibodies in vivo. While this method is generally used for antibody affinity maturation (enhancement), this invention, referencing this technical route, conversely reduces the affinity of CD3 antibodies.

[0259] Single-point saturation mutations were performed on each amino acid site in the CDR region to construct an unbiased single-point saturation mutant plasmid library of the maternal antibody. Mutation hotspots with weakened antigen-specific binding were screened, and these hotspots were then combined and screened to obtain candidate antibody mutant sequences.

[0260] Through this technical route and extensive screening experiments, 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 aforementioned low-affinity CD3 monoclonal antibodies are shown in Table 1. Compared with the parent antibody Cross3, the amino acid mutations in its CDR region are as follows: Figure 1 As shown.

[0261] The specific experimental procedures for the production of CD3 monoclonal antibodies are as follows: (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured in ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×10⁻⁶. 6 / mL, to obtain ExpiCHO cell solution. (2) The pcDNA3.4 vector containing the CD3 antibody heavy chain and light chain (Cross3 heavy chain amino acid sequence as shown in SEQ ID NO:35 and light chain amino acid sequence as shown in SEQ ID NO:33; Cross303 heavy chain amino acid sequence as shown in SEQ ID NO:37 and light chain amino acid sequence as shown in SEQ ID NO:33; Cross307 heavy chain amino acid sequence as shown in SEQ ID NO:38 and light chain amino acid sequence as shown in SEQ ID NO:33; Cross308 heavy chain amino acid sequence as shown in SEQ ID NO:39 and light chain amino acid sequence as shown in SEQ ID NO:33; Cross309 heavy chain amino acid sequence as shown in SEQ ID NO:40 and light chain amino acid sequence as shown in SEQ ID NO:33; Cross310 heavy chain amino acid sequence as shown in SEQ ID NO:41 and light chain amino acid sequence as shown in SEQ ID NO:33; Cross311 heavy chain amino acid sequence as shown in SEQ ID NO:42 and light chain amino acid sequence as shown in SEQ ID NO:33) was prepared. The heavy chain amino acid sequence of Cross312 is shown in SEQ ID NO:33 and the light chain amino acid sequence is shown in SEQ ID NO:33; the heavy chain amino acid sequence of Cross313 is shown in SEQ ID NO:32 and the light chain amino acid sequence is shown in SEQ ID NO:33; the heavy chain amino acid sequence of Cross314 is shown in SEQ ID NO:44 and the light chain amino acid sequence is shown in SEQ ID NO:33; the heavy chain amino acid sequence of Cross316 is shown in SEQ ID NO:34 and the light chain amino acid sequence is shown in SEQ ID NO:33; the heavy chain amino acid sequence of Cross317 is shown in SEQ ID NO:45 and the light chain amino acid sequence is shown in SEQ ID NO:33; the heavy chain amino acid sequence of Cross318 is shown in SEQ ID NO:46 and the light chain amino acid sequence is shown in SEQ ID NO:33; the heavy chain amino acid sequence of Cross323 is shown in SEQ ID NO:45 and the light chain amino acid sequence is shown in SEQ ID NO:47; the heavy chain amino acid sequence of Cross325 is shown in SEQ ID NO:33. The amino acid sequences shown in NO:45 and light chain amino acid sequences are shown in SEQ ID NO:36; the heavy chain amino acid sequence of Cross326 is shown in SEQ ID NO:46 and the light chain amino acid sequence is shown in SEQ ID NO:48. (Synthesized by Nanjing GenScript) were added to 2 mL of OptiSFM 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 transfection mixture, and add the entire transfection mixture to 50 mL of ExpiCHO cell solution within 5 minutes. (5) After culturing at 37℃ and 5% CO2 for 1 day, add 8 mL of Feed and 300 μL of Enhancer (purchased from Thermo Fisher), and transfer to 32℃ and 5% CO2 for 9 days. Harvest the culture supernatant, adding 8 mL of Feed on day 5. (6) Use a Protein A purification column (purchased from Nanomicro) to affinity purify the target antibody from the culture supernatant.

[0262] The specific experimental procedures for the production of bispecific antibodies are as follows: (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured in ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×10⁻⁶. 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 as shown in SEQ ID NO:52 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70; Cross303×Trop2 contains CD3 antibody as shown in SEQ ID NO:53 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70; Cross307×Trop2 contains CD3 antibody as shown in SEQ ID NO:54 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70; Cross308×Trop2 contains CD3 antibody as shown in SEQ ID NO:55 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70) As shown in NO:70; Cross309×Trop2 contains the CD3 antibody as shown in SEQ ID NO:56 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; Cross310×Trop2 contains the CD3 antibody as shown in SEQ ID NO:57 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; Cross311×Trop2 contains the CD3 antibody as shown in SEQ ID NO:58 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; Cross312×Trop2 contains the CD3 antibody as shown in SEQ ID NO:59 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; Cross313×Trop2 contains the CD3 antibody as shown in SEQ ID NO:49 and the Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:70. The heavy chain amino acid sequence is shown in SEQ ID NO:69 and the light chain amino acid sequence is shown in SEQ ID NO:70; Cross314×Trop2 contains the CD3 antibody shown in SEQ ID NO:60 and the Trop2 antibody heavy chain amino acid sequence is shown in SEQ ID NO:69 and the light chain amino acid sequence is shown in SEQ ID NO:70.Cross316×Trop2 contains the CD3 antibody shown in SEQ ID NO:50 and the Trop2 antibody heavy chain amino acid sequence shown in SEQ ID NO:69 and the light chain amino acid sequence shown in SEQ ID NO:70; Cross317×Trop2 contains the CD3 antibody shown in SEQ ID NO:61 and the Trop2 antibody heavy chain amino acid sequence shown in SEQ ID NO:69 and the light chain amino acid sequence shown in SEQ ID NO:70; Cross318×Trop2 contains the CD3 antibody shown in SEQ ID NO:62 and the Trop2 antibody heavy chain amino acid sequence shown in SEQ ID NO:69 and the light chain amino acid sequence shown in SEQ ID NO:70; Cross323×Trop2 contains the CD3 antibody shown in SEQ ID NO:63 and the Trop2 antibody heavy chain amino acid sequence shown in SEQ ID NO:69 and the light chain amino acid sequence shown in SEQ ID NO:70; Cross325×Trop2 contains the CD3 antibody shown in SEQ ID NO:51 and the Trop2 antibody heavy chain amino acid sequence shown in SEQ ID NO:70. The amino acid sequence of the CD3 antibody (SEQ ID NO:69) and the light chain amino acid sequence of the PDL1 antibody (SEQ ID NO:70) are shown in SEQ ID NO:69; Cross326×Trop2 contains the CD3 antibody (SEQ ID NO:64) and the Trop2 antibody heavy chain amino acid sequence (SEQ ID NO:70) (synthesized by Nanjing Genscript), or, pcDNA3.4 vectors containing CD3 antibody, PDL1 antibody heavy chain, and PDL1 antibody light chain (Cross3×PDL1 contains the CD3 antibody (SEQ ID NO:52) and the PDL1 antibody heavy chain amino acid sequence (SEQ ID NO:71) and the light chain amino acid sequence (SEQ ID NO:72); Cross303×PDL1 contains the CD3 antibody (SEQ ID NO:53) and the PDL1 antibody heavy chain amino acid sequence (SEQ ID NO:71) and the light chain amino acid sequence (SEQ ID NO:72); Cross307×PDL1 contains the CD3 antibody (SEQ ID NO:54) and the PDL1 antibody heavy chain amino acid sequence (SEQ ID NO:70) (SEQ ID NO:69) and the light chain amino acid sequence (SEQ ID NO:70) are shown in SEQ ID NO:69; The 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; Cross308×PDL1 contains the CD3 antibody shown in SEQ ID NO:55 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;Cross309×PDL1 contains the CD3 antibody shown in SEQ ID NO:56 and 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; Cross310×PDL1 contains the CD3 antibody shown in SEQ ID NO:57 and 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; Cross311×PDL1 contains the CD3 antibody shown in SEQ ID NO:58 and 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; Cross312×PDL1 contains the CD3 antibody shown in SEQ ID NO:59 and 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; Cross313×PDL1 contains the CD3 antibody shown in SEQ ID NO:49 and the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO:59. The heavy chain amino acid sequence of the CD3 antibody SEQ ID NO:71 and the light chain amino acid sequence of the PDL1 antibody are shown in SEQ ID NO:72; Cross314×PDL1 contains the CD3 antibody SEQ ID NO:60 and the heavy chain amino acid sequence of the PDL1 antibody as shown in SEQ ID NO:71 and the light chain amino acid sequence of the PDL1 antibody as shown in SEQ ID NO:72; Cross316×PDL1 contains the CD3 antibody SEQ ID NO:50 and the heavy chain amino acid sequence of the PDL1 antibody as shown in SEQ ID NO:71 and the light chain amino acid sequence of the PDL1 antibody as shown in SEQ ID NO:72; Cross317×PDL1 contains the CD3 antibody SEQ ID NO:61 and the heavy chain amino acid sequence of the PDL1 antibody as shown in SEQ ID NO:71 and the light chain amino acid sequence of the PDL1 antibody as shown in SEQ ID NO:72; Cross318×PDL1 contains the CD3 antibody SEQ ID NO:62 and the heavy chain amino acid sequence of the PDL1 antibody as shown in SEQ ID NO:71 and the light chain amino acid sequence of the PDL1 antibody as shown in SEQ ID NO:72; Cross323×PDL1 contains the CD3 antibody SEQ ID NO:71 and the heavy chain amino acid sequence of the PDL1 antibody as shown in SEQ ID NO:72; The heavy chain amino acid sequence of the PDL1 antibody shown in NO:63 is shown in SEQ ID NO:71, and the light chain amino acid sequence is shown in SEQ ID NO:72; Cross325×PDL1 contains the CD3 antibody shown in SEQ ID NO:51, and the heavy chain amino acid sequence of the PDL1 antibody shown in SEQ ID NO:71, and the light chain amino acid sequence is shown in SEQ ID NO:72.Cross326×PDL1 contains the CD3 antibody shown in SEQ ID NO:64 and 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 contains the CD3 antibody shown in SEQ ID NO:65 and 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 contains the CD3 antibody shown in SEQ ID NO:66 and 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 contains the CD3 antibody shown in SEQ ID NO:67 and 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 contains the CD3 antibody shown in SEQ ID NO:68 and the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO:64 and the light chain amino acid sequence shown in SEQ ID NO:72. (3) Add 160 μL of ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) to 2 mL of OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution A. (4) Then mix solution a and solution b to obtain transfection mixture, and add the entire transfection mixture to 50 mL of ExpiCHO cell solution within 5 minutes. (5) After culturing at 37℃ and 5% CO2 for 1 day, add 8 mL of feed and 300 μL of enhancer (purchased from Thermo Fisher), and culture at 32℃ and 5% CO2 for 9 days. Harvest the culture supernatant, and add 8 mL of feed on day 5. (6) The target CD3×Trop2 or CD3×PDL1 antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from Nanomicro). The antibody configuration is as follows: Figure 5 As shown.

[0263] Example 2: Antibody Affinity Detection

[0264] 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 data that are very important in drug development, such as the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD), thereby calculating the affinity of antibodies.

[0265] In the Biacore 8K (Cytiva) system, the antibody was diluted to 10 μg / mL with running buffer (HBS-EP) and conjugated to the CM5 chip (Cytiva) at a flow rate of 10 μL / min. The kinetics and affinity data of the binding between the CD3E&D antigen and the low-affinity CD3 monoclonal antibody of Example 1 of this invention were detected at a flow rate of 30 μL / min, with a binding time of 120 s and a dissociation time of 800 s. The antibody affinity test results are as follows: Figure 2 As shown.

[0266] Example 3: CD3 monoclonal antibody ELISA binding assay

[0267] This embodiment utilizes ELISA to detect the binding properties of maternal antibodies with reduced affinity for Cross3 and CD3. The inventors coated CD3E&D proteins into 96-well plates, and the intensity of the signal after antibody addition was used to determine the binding properties between the antibody and CD3E&D.

[0268] Dilute CD3E&D protein (purchased from Acro) to 2 μg / ml with PBS buffer and add 100 μL / well to each well of a 96-well plate. Incubate overnight at 4°C. Remove the PBS buffer from the 96-well plate, wash 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, and then add 200 μL / well of PBS / 10% BSA. Incubate at 37°C for 2 hours to block the plate.After removing the blocking buffer and washing the plate 6 times with PBST, add 100 μL / well of serially diluted PBST / 0.05% BSA to the following assays: Cross3 (heavy chain amino acid sequence as shown in SEQ ID NO:35 and light chain amino acid sequence as shown in SEQ ID NO:33), Cross303 (heavy chain amino acid sequence as shown in SEQ ID NO:37 and light chain amino acid sequence as shown in SEQ ID NO:33), Cross307 (heavy chain amino acid sequence as shown in SEQ ID NO:38 and light chain amino acid sequence as shown in SEQ ID NO:33), Cross308 (heavy chain amino acid sequence as shown in SEQ ID NO:39 and light chain amino acid sequence as shown in SEQ ID NO:33), Cross309 (heavy chain amino acid sequence as shown in SEQ ID NO:40 and light chain amino acid sequence as shown in SEQ ID NO:33), and Cross310 (heavy chain amino acid sequence as shown in SEQ ID NO:41 and light chain amino acid sequence as shown in SEQ ID NO:33). Cross311 (heavy chain amino acid sequence as shown in SEQ ID NO:33 and light chain amino acid sequence as shown in SEQ ID NO:42), Cross312 (heavy chain amino acid sequence as shown in SEQ ID NO:43 and light chain amino acid sequence as shown in SEQ ID NO:33), Cross313 (heavy chain amino acid sequence as shown in SEQ ID NO:32 and light chain amino acid sequence as shown in SEQ ID NO:33), Cross314 (heavy chain amino acid sequence as shown in SEQ ID NO:44 and light chain amino acid sequence as shown in SEQ ID NO:33), Cross316 (heavy chain amino acid sequence as shown in SEQ ID NO:34 and light chain amino acid sequence as shown in SEQ ID NO:33), Cross317 (heavy chain amino acid sequence as shown in SEQ ID NO:45 and light chain amino acid sequence as shown in SEQ ID NO:33), Cross318 (heavy chain amino acid sequence as shown in SEQ ID NO:46 and light chain amino acid sequence as shown in SEQ ID NO:33). (Shown NO:33), Cross323 (heavy chain amino acid sequence as shown in SEQ ID NO:45 and light chain amino acid sequence as shown in SEQ ID NO:47), Cross325 (heavy chain amino acid sequence as shown in SEQ ID NO:45 and 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 light chain amino acid sequence as shown in SEQ ID NO:48), and control IgG1 (purchased from Baiying Biotechnology) were incubated at 37°C for 1 h.Remove the reaction mixture, wash the plate 6 times with PBST, and then dilute HRP (horseradish peroxidase)-labeled anti-human antibody secondary antibody (Fab specific) (purchased from Sigma) with PBST / 0.05% BSA at 100 μL / well. Incubate at 37°C for 1 h. After washing the plate 6 times with PBST, add 80 μL / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 min, and then stop the reaction by adding 80 μL / well of 4M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0269] The results are as follows Figure 3 As shown, all CD3 antibodies of the present invention can bind to CD3E&D. However, due to the limited resolution of the ELISA experiment, the ELISA results cannot reflect the differences in affinity of each CD3 monoclonal antibody.

[0270] Example 4: CD3 monoclonal antibody flow cytometry combined with experiment

[0271] Dilute PBMC to 2×10⁻⁶ with PBS. 6 / mL, add 100μL / tube to a 1.5ml EP tube, add 10μL / tube of goat serum, and block at 4℃ for 30min. Add serially diluted versions of the following antibodies to be tested: Cross3 (heavy chain sequence SEQ ID NO: 11 and light chain sequence SEQ ID NO: 12), Cross303 (heavy chain sequence SEQ ID NO: 13 and light chain sequence SEQ ID NO: 14), Cross307 (heavy chain sequence SEQ ID NO: 13 and light chain sequence SEQ ID NO: 14), Cross308 (heavy chain sequence SEQ ID NO: 13 and light chain sequence SEQ ID NO: 14), Cross309 (heavy chain sequence SEQ ID NO: 13 and light chain sequence SEQ ID NO: 14), Cross310 (heavy chain sequence SEQ ID NO: 13 and light chain sequence SEQ ID NO: 14), Cross311 (heavy chain sequence SEQ ID NO: 11 and light chain sequence SEQ ID NO: 12), and Cross312 (heavy chain sequence SEQ ID NO: 11 and light chain sequence SEQ ID NO: 12). Cross313 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross314 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross316 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross317 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross318 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross323 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross325 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross326 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross313 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross314 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross316 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross317 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross318 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross323 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross325 antibody (heavy chain sequence shown in SEQ ID NO:11 and light chain sequence shown in SEQ ID NO:12), Cross NO:12 (as shown), control IgG1 (purchased from Baiying Biotechnology), incubated at 4℃ for 30 min. Add 1 mL PBS to the EP tube, centrifuge at 3500 rpm for 5 min at 4℃, discard the supernatant, and wash 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 antibody secondary antibody (purchased from Jackson Lab) and 0.5 μL / tube of PerCP-Cy5.5-labeled anti-human CD8 antibody to the resuspending solution, and incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μL / tube of PBS and analyze using flow cytometry.

[0272] The results are as follows Figure 4 As shown, 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 and have reduced CD3 affinity. The binding ability of the CD3 antibodies is weaker than that of the parent antibody Cross3.

[0273] Example 5: CD3×Trop2, CD3×PDL1 bispecific antibody ELISA binding assay

[0274] This embodiment utilizes ELISA to detect the binding characteristics of CD3×Trop2 and CD3×PDL1 bispecific antibodies. The inventors coated CD3E&D, Trop2, or PDL1 proteins into 96-well plates, and the intensity of the signal after antibody addition was used to determine the binding characteristics between the antibody and the corresponding protein.

[0275] (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 incubate overnight at 4°C. Remove the PBS buffer from 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 of PBS / 10% BSA, and incubate at 37°C for 2 h to block. Remove the blocking buffer, wash the plate 6 times with PBST, and then add 100 μL / well of the following bispecific antibodies serially diluted with PBST / 0.05% BSA: Cross3×Trop2 (containing the CD3 antibody SEQ ID NO:52 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), Cross303×Trop2 (containing the CD3 antibody SEQ ID NO:53 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), Cross307×Trop2 (containing the CD3 antibody SEQ ID NO:54 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), and Cross308×Trop2 (containing the CD3 antibody SEQ ID NO:55 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). (Symptoms shown in SEQ ID NO:70), Cross309×Trop2 (containing the CD3 antibody SEQ ID NO:57 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), Cross310×Trop2 (containing the CD3 antibody SEQ ID NO:57 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), Cross311×Trop2 (containing the CD3 antibody SEQ ID NO:58 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), Cross312×Trop2 (containing the CD3 antibody SEQ ID NO:59 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), Cross313×Trop2 (containing the CD3 antibody SEQ ID NO:70 and the heavy chain amino acid sequence as shown in SEQ ID NO:5 ...70), Cross311×Trop2 (containing the CD3 antibody SEQ ID NO:58 and the 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), Cross312×Trop2 (containing the CD3 antibody SEQ ID NO:59 and the heavy chain amino acid sequence as shown in SEQ ID NO:69 and the light chain amino acid sequence as shown in NO:49 and the Trop2 antibody heavy chain amino acid sequence are shown in SEQ ID.The following antibodies are listed: Cross314×Trop2 (containing the CD3 antibody SEQ ID NO:69 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), Cross316×Trop2 (containing the CD3 antibody SEQ ID NO:50 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), Cross317×Trop2 (containing the CD3 antibody SEQ ID NO:61 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), and Cross318×Trop2 (containing the CD3 antibody SEQ ID NO:62 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). Cross323×Trop2 (containing the CD3 antibody SEQ ID NO: 63 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), Cross325×Trop2 (containing the CD3 antibody SEQ ID NO: 51 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), Cross326×Trop2 (containing the CD3 antibody 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), Cross3×PDL1 (containing the CD3 antibody 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 (containing the CD3 antibody SEQ ID NO: 70), Cross323×Trop2 (containing the CD3 antibody SEQ ID NO: 70), Cross325×Trop2 (containing the CD3 antibody SEQ ID NO: 51 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), Cross326×Trop2 (containing the CD3 antibody 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), Cross3×PDL1 (containing the CD3 antibody ...25×Trop2 (containing the CD3 antibody SEQ ID NO: 51 and the Trop The following are listed: NO:53 (containing the CD3 antibody SEQ ID NO:54 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 (containing the CD3 antibody 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), and Cross308×PDL1 (containing the CD3 antibody SEQ ID NO:55 and the PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:72).The following are listed: Cross309×PDL1 (containing the CD3 antibody SEQ ID NO: 56 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), Cross310×PDL1 (containing the CD3 antibody SEQ ID NO: 57 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), Cross311×PDL1 (containing the CD3 antibody SEQ ID NO: 58 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), and Cross312×PDL1 (containing the CD3 antibody SEQ ID NO: 59 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). (Shown NO:72), Cross313×PDL1 (containing CD3 antibody SEQ ID NO:60 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross314×PDL1 (containing CD3 antibody SEQ ID NO:60 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross316×PDL1 (containing CD3 antibody SEQ ID NO:50 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross317×PDL1 (containing CD3 antibody SEQ ID NO:61 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross318×PDL1 (containing CD3 antibody SEQ ID NO:72 and PDL1 antibody heavy ...3×PDL1 (containing CD3 antibody SEQ ID NO:72 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:72), Cross314×PDL1 (containing CD3 antibody SEQ ID NO:60 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross316×PDL1 (containing CD3 antibody SEQ ID NO:50 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross317×PDL1 (containing CD3 antibody SEQ ID NO:61 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO The following are listed: NO:62 (containing the CD3 antibody SEQ ID 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), Cross323×PDL1 (containing the CD3 antibody 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), and Cross325×PDL1 (containing the CD3 antibody 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).The reaction mixture consisted of: (SEQ ID NO:72), Cross326×PDL1 (containing CD3 antibody as shown in SEQ ID NO:64 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), and control IgG1LALA (purchased from Baiying Biotechnology). The mixture was incubated at 37°C for 1 h. The reaction mixture was then removed, and the plate was washed 6 times with PBST. 100 μL / well of HRP (horseradish peroxidase)-labeled anti-human antibody secondary antibody (Fab specific) (purchased from Sigma) was diluted with PBST / 0.05% BSA and incubated at 37°C for 1 h. After washing 6 times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added, and the plate was incubated at room temperature for 3 min. The reaction was terminated by adding 80 μL / well of 4M sulfuric acid. The absorbance was read at 450 nm using a microplate reader.

[0276] like Figure 6 As shown, the CD3×Trop2 antibody of the present invention can bind to CD3E&D. Compared with the parent Cross3×Trop2 bispecific antibody, the affinity is reduced. The binding of the CD3 bispecific antibody to CD3E&D protein is weakened, which is consistent with expectations.

[0277] like Figure 14 As shown, the CD3×PDL1 antibody of the present invention can bind to CD3E&D. Compared with the parent Cross3×PDL1 bispecific antibody, the affinity is reduced. The binding of the CD3 bispecific antibody to CD3E&D protein is weakened, which is consistent with expectations.

[0278] (2) Dilute Trop2 protein (purchased from Acro) to 2 μg / ml with PBS buffer, add 100 μL / well to a 96-well plate, and incubate overnight at 4°C. Remove the PBS buffer from 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 of PBS / 10% BSA, and incubate at 37°C for 2 h to block. Remove the blocking buffer, wash the plate 6 times with PBST, and then add 100 μL / well of the following bispecific antibodies serially diluted with PBST / 0.05% BSA: Cross3×Trop2 (containing the CD3 antibody SEQ ID NO:52 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), Cross303×Trop2 (containing the CD3 antibody SEQ ID NO:53 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), Cross307×Trop2 (containing the CD3 antibody SEQ ID NO:54 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), and Cross308×Trop2 (containing the CD3 antibody SEQ ID NO:55 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). (Symptoms shown in SEQ ID NO:70), Cross309×Trop2 (containing the CD3 antibody SEQ ID NO:57 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), Cross310×Trop2 (containing the CD3 antibody SEQ ID NO:57 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), Cross311×Trop2 (containing the CD3 antibody SEQ ID NO:58 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), Cross312×Trop2 (containing the CD3 antibody SEQ ID NO:59 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), Cross313×Trop2 (containing the CD3 antibody SEQ ID NO:70 and the heavy chain amino acid sequence as shown in SEQ ID NO:5 ...70), Cross311×Trop2 (containing the CD3 antibody SEQ ID NO:58 and the 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), Cross312×Trop2 (containing the CD3 antibody SEQ ID NO:59 and the heavy chain amino acid sequence as shown in SEQ ID NO:69 and the light chain amino acid sequence as shown in NO:49 and the Trop2 antibody heavy chain amino acid sequence are shown in SEQ ID.The following antibodies are listed: Cross314×Trop2 (containing the CD3 antibody SEQ ID NO:69 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), Cross316×Trop2 (containing the CD3 antibody SEQ ID NO:50 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), Cross317×Trop2 (containing the CD3 antibody SEQ ID NO:61 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), and Cross318×Trop2 (containing the CD3 antibody SEQ ID NO:62 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). The following antibodies were incubated at 37°C for 1 hour: Cross323×Trop2 (containing the CD3 antibody SEQ ID NO:63 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), Cross325×Trop2 (containing the CD3 antibody SEQ ID NO:51 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), Cross326×Trop2 (containing the CD3 antibody 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), and control IgG1LALA (purchased from Baiying Biotechnology). Remove the reaction mixture, wash the plate 6 times with PBST, and then dilute HRP (horseradish peroxidase)-labeled anti-human antibody secondary antibody (Fab specific) (purchased from Sigma) with PBST / 0.05% BSA at 100 μL / well. Incubate at 37°C for 1 h. After washing the plate 6 times with PBST, add 80 μL / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 min, and then stop the reaction by adding 80 μL / well of 4M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0279] like Figure 7 As shown, the CD3×Trop2 antibody of the present invention can bind to the Trop2 protein, and all bispecific antibodies bind to the Trop2 protein in a similar manner.

[0280] (3) Dilute PDL1 protein (purchased from Acro) to 2 μg / ml with PBS buffer, add 100 μL / well to a 96-well plate, and incubate overnight at 4°C. Remove the PBS buffer from 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 h to block. Remove the blocking buffer, wash the plate 6 times with PBST, and then add 100 μL / well of the following bispecific antibodies serially diluted with PBST / 0.05% BSA: Cross3×PDL1 (containing the CD3 antibody 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 (containing the CD3 antibody 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 (containing the CD3 antibody SEQ ID NO:54 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), and Cross308×PDL1 (containing the CD3 antibody SEQ ID NO:55 and the PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:72). The following are listed: Cross309×PDL1 (containing the CD3 antibody SEQ ID NO: 56 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), Cross310×PDL1 (containing the CD3 antibody SEQ ID NO: 57 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), Cross311×PDL1 (containing the CD3 antibody SEQ ID NO: 58 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), and Cross312×PDL1 (containing the CD3 antibody SEQ ID NO: 59 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). (as shown in NO:72), Cross313×PDL1 (containing the CD3 antibody shown in SEQ ID NO:49 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).(Shown NO:72), Cross314×PDL1 (containing CD3 antibody SEQ ID NO:60 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross316×PDL1 (containing CD3 antibody SEQ ID NO:50 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross317×PDL1 (containing CD3 antibody SEQ ID NO:61 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross318×PDL1 (containing CD3 antibody SEQ ID NO:62 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross323×PDL1 (containing CD3 antibody SEQ ID NO:72 ...2), Cross316×PDL1 (containing CD3 antibody SEQ ID NO:72 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:72), Cross317×PDL1 (containing CD3 antibody SEQ ID NO:60 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross318×PDL1 (containing CD3 antibody SEQ ID NO:62 and The reaction mixture consisted of: NO:63 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72); Cross325×PDL1 (containing CD3 antibody as shown in SEQ ID NO:51 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72); Cross326×PDL1 (containing CD3 antibody as shown in SEQ ID NO:64 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72); and control IgG1LALA (purchased from Baiying Biotechnology). The mixture was incubated at 37°C for 1 h. The reaction mixture was then removed, and the plate was washed 6 times with PBST. HRP (horseradish peroxidase)-labeled anti-human antibody secondary antibody (Fab specific) (purchased from Sigma) was diluted 100 μL / well with PBST / 0.05% BSA and incubated at 37°C for 1 h. After washing the plate 6 times with PBST, add 80 μL / well of TMB (tetramethylbenzidine) and incubate at room temperature for 3 min. Then, add 80 μL / well of 4M sulfuric acid to terminate the reaction. Read the absorbance at 450 nm using a microplate reader.

[0281] like Figure 15 As shown, the CD3×PDL1 antibody of the present invention can bind to the PDL1 protein.

[0282] Example 6: CD3×Trop2 antibody ELISA bridging assay

[0283] ELISA assays were used to detect the bridging binding properties of CD3×Trop2 bispecific antibodies. CD3E&D antigen proteins were coated into 96-well plates. After antibody addition, biotin-labeled Trop2 protein was used for detection. The signal strength was used to determine the binding properties of the bispecific antibody to CD3E&D and Trop2 protein.

[0284] Dilute CD3E&D protein (purchased from Acro) to 2 μg / ml with PBS buffer, and add 100 μl / well to each well of a 96-well plate. Incubate overnight at 4°C. Remove the PBS buffer from the 96-well plate, wash 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, and then add 200 μl / well of PBS / 10% BSA. Incubate at 37°C for 2 hours to block the plate. Remove the blocking buffer, wash the plate 6 times with PBST, and add 100 μl / well of the following bispecific antibodies serially diluted with PBST / 0.05% BSA: Cross3×Trop2 (containing the CD3 antibody SEQ ID NO:52 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), Cross303×Trop2 (containing the CD3 antibody SEQ ID NO:53 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), Cross307×Trop2 (containing the CD3 antibody SEQ ID NO:54 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), and Cross308×Trop2 (containing the CD3 antibody SEQ ID NO:55 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). (Symptoms shown in SEQ ID NO:70), Cross309×Trop2 (containing the CD3 antibody SEQ ID NO:57 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), Cross310×Trop2 (containing the CD3 antibody SEQ ID NO:57 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), Cross311×Trop2 (containing the CD3 antibody SEQ ID NO:58 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), Cross312×Trop2 (containing the CD3 antibody SEQ ID NO:59 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), Cross313×Trop2 (containing the CD3 antibody SEQ ID NO:70 and the heavy chain amino acid sequence as shown in SEQ ID NO:5 ...70), Cross311×Trop2 (containing the CD3 antibody SEQ ID NO:58 and the 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), Cross312×Trop2 (containing the CD3 antibody SEQ ID NO:59 and the heavy chain amino acid sequence as shown in SEQ ID NO:69 and the light chain amino acid sequence as shown in NO:49 and the Trop2 antibody heavy chain amino acid sequence are shown in SEQ ID.The following antibodies are listed: Cross314×Trop2 (containing the CD3 antibody SEQ ID NO:69 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), Cross316×Trop2 (containing the CD3 antibody SEQ ID NO:50 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), Cross317×Trop2 (containing the CD3 antibody SEQ ID NO:61 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), and Cross318×Trop2 (containing the CD3 antibody SEQ ID NO:62 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). The following antibodies were incubated at 37°C for 1 hour: Cross323×Trop2 (containing the CD3 antibody as shown in SEQ ID NO:63 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), Cross325×Trop2 (containing the CD3 antibody as shown in SEQ ID NO:51 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), Cross326×Trop2 (containing the CD3 antibody as 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), and control hIgG1LALA (purchased from Bio-Tech). The reaction mixture was removed, the plate was washed 6 times with PBST, and Trop2-Biotin protein diluted to an appropriate concentration was added. The mixture was then incubated at 37°C for 1 hour. Remove the reaction mixture, wash the plate 6 times with PBST, and then dilute HRP (horseradish peroxidase)-labeled Streptavidin secondary antibody (purchased from Southern Biotech) with PBST / 0.05% BSA at 100 μl / well. Incubate at 37°C for 1 h. After washing the plate 6 times with PBST, add 80 μl / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 min, and then stop the reaction by adding 80 μl / well of 4M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0285] The results are as follows Figure 8This indicates 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-reducing CD3 bispecific antibody, which is consistent with expectations.

[0286] Example 7: Bispecific antibody flow cytometry combined with experiment

[0287] Flow cytometry is used to detect the binding properties of bispecific antibodies by adding bispecific antibodies to cells and then measuring the strength of the signal after antibody addition.

[0288] (1) Dilute PBMC with PBS to 2×10 6 / ml, add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of goat serum, and block at 4℃ for 30min. Add serially diluted bispecific antibodies Cross3×Trop2 (containing the CD3 antibody SEQ ID NO:52 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), Cross303×Trop2 (containing the CD3 antibody SEQ ID NO:53 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), Cross307×Trop2 (containing the CD3 antibody SEQ ID NO:54 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), Cross308×Trop2 (containing the CD3 antibody SEQ ID NO:55 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), and Cross309×Trop2 (containing the CD3 antibody SEQ ID NO:52 and the Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:70), SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:69, and the light chain amino acid sequence as shown in SEQ ID NO:70), SEQ ID NO:5 ... The following antibodies are listed: Cross310×Trop2 (containing CD3 antibody SEQ ID NO:57 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross311×Trop2 (containing CD3 antibody SEQ ID NO:58 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross312×Trop2 (containing CD3 antibody SEQ ID NO:59 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), and Cross313×Trop2 (containing CD3 antibody SEQ ID NO:49 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70). (Shown NO:70), Cross314×Trop2 (containing CD3 antibody as shown in SEQ ID NO:60 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross316×Trop2 (containing CD3 antibody SEQ ID NO:70).The following antibodies are listed: Cross317×Trop2 (containing CD3 antibody SEQ ID NO:61 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross318×Trop2 (containing CD3 antibody SEQ ID NO:62 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross323×Trop2 (containing CD3 antibody SEQ ID NO:63 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), and Cross325×Trop2 (containing CD3 antibody SEQ ID NO:51 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70). Cross326×Trop2 (containing the CD3 antibody 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), Cross3×PDL1 (containing the CD3 antibody 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), Cross313×PDL1 (containing the CD3 antibody SEQ ID NO: 49 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), Cross316×PDL1 (containing the CD3 antibody SEQ ID NO: 50 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 (containing the CD3 antibody SEQ ID NO: 70 and the Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO: 70), Cross326×Trop2 (containing the CD3 antibody 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), Cross325×PDL1 (containing the CD3 antibody SEQ ID NO: 70 and the Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO: 70), Cross326×Trop2 (containing the CD3 antibody SEQ ID NO: 70), Cross326×Trop2 (containing the CD3 antibody 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), Cross326×PDL1 (containing the CD3 antibody SEQ ID NO: 70 The heavy chain amino acid sequence of PDL1 antibody (shown in NO:51 and SEQ ID NO:71, and the light chain amino acid sequence (shown in SEQ ID NO:72)) and control hIgG1 LALA (purchased from Baiying Biotechnology) were incubated at 4°C for 30 min. 1 ml of [unclear text - possibly a typo, should be inserted here] was added to the EP tube.Centrifuge with PBS at 3500 rpm for 5 min at 4°C, discard the supernatant, and wash once more with PBS. After centrifugation, discard the supernatant again, resuspend the cells in 100 μl / tube of PBS, add 1 μl / tube of Alexa-647-labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson Lab) and 0.5 μl / tube of PerCP-Cy5.5-labeled anti-human CD8 antibody, and incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze using flow cytometry.

[0289] The results are as follows Figure 9 As shown, the CD3×Trop2 antibody of the present invention can bind to CD8 T cells, and compared with the parental CD3 antibody Cross3×Trop2, the CD3 affinity is reduced and the binding ability of the bispecific antibody to T cells is weakened, which is in line with expectations.

[0290] The results are as follows Figure 16 As shown, the Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 antibodies of the present invention can bind to CD8 T cells, and the binding ability of Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 to T cells is weaker than that of Cross3×PDL1, which is consistent with expectations.

[0291] (2) Dilute the tumor cells to 2×10⁻⁶ with PBS. 6 / ml, add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of goat serum, and block at 4℃ for 30min. Add serially diluted bispecific antibodies Cross3×Trop2 (containing the CD3 antibody SEQ ID NO:52 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), Cross303×Trop2 (containing the CD3 antibody SEQ ID NO:53 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), Cross307×Trop2 (containing the CD3 antibody SEQ ID NO:54 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), Cross308×Trop2 (containing the CD3 antibody SEQ ID NO:55 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), and Cross309×Trop2 (containing the CD3 antibody SEQ ID NO:52 and the Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:70), SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:69, and the light chain amino acid sequence as shown in SEQ ID NO:70), SEQ ID NO:5 ... The following antibodies are listed: Cross310×Trop2 (containing CD3 antibody SEQ ID NO:57 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross311×Trop2 (containing CD3 antibody SEQ ID NO:58 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross312×Trop2 (containing CD3 antibody SEQ ID NO:59 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), and Cross313×Trop2 (containing CD3 antibody SEQ ID NO:49 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70). (Shown NO:70), Cross314×Trop2 (containing CD3 antibody as shown in SEQ ID NO:60 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross316×Trop2 (containing CD3 antibody SEQ ID NO:70).The following antibodies are listed: Cross317×Trop2 (containing CD3 antibody SEQ ID NO:61 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross318×Trop2 (containing CD3 antibody SEQ ID NO:62 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross323×Trop2 (containing CD3 antibody SEQ ID NO:63 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), and Cross325×Trop2 (containing CD3 antibody SEQ ID NO:51 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70). Cross326×Trop2 (containing the CD3 antibody 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), Cross3×PDL1 (containing the CD3 antibody 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 (containing the CD3 antibody 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 (containing the CD3 antibody SEQ ID NO: 54 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), Cross308×PDL1 (containing the CD3 antibody SEQ ID NO: 70 and the Trop2 ...0), Cross326×Trop2 (containing the CD3 antibody SEQ ID NO: 52 and the Trop2 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), Cross308×PDL1 (containing the CD3 antibody SEQ ID NO: 70 and the Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO: 70), Cross326×Trop2 (containing the CD3 antibody SEQ ID NO: 70), Cross3×PDL1 (containing the CD3 antibody SEQ ID NO: 70), Cross The heavy chain amino acid sequence of the PDL1 antibody shown in SEQ ID NO:55 and the light chain amino acid sequence shown in SEQ ID NO:71 and SEQ ID NO:72 are shown in SEQ ID NO:55. Cross309×PDL1 (containing the CD3 antibody shown in SEQ ID NO:56 and the heavy chain amino acid sequence of the PDL1 antibody shown in SEQ ID NO:71 and the light chain amino acid sequence shown in SEQ ID NO:72) and Cross310×PDL1 (containing the CD3 antibody shown in SEQ ID NO:56 and SEQ ID NO:72 are shown in SEQ ID NO:71 and SEQ ID NO:72 are shown in SEQ ID NO:55) are shown in SEQ ID NO:55 and SEQ ID NO:72 are shown in SEQ ID NO:71 ...The following are listed: Cross311×PDL1 (containing CD3 antibody SEQ ID NO:57 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross312×PDL1 (containing CD3 antibody SEQ ID NO:59 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross313×PDL1 (containing CD3 antibody SEQ ID NO:49 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), and Cross314×PDL1 (containing CD3 antibody SEQ ID NO:60 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72). (Shown NO:72), Cross316×PDL1 (containing CD3 antibody SEQ ID NO:61 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross317×PDL1 (containing CD3 antibody SEQ ID NO:61 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross318×PDL1 (containing CD3 antibody SEQ ID NO:62 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross323×PDL1 (containing CD3 antibody SEQ ID NO:63 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross325×PDL1 (containing CD3 antibody SEQ ID NO:72 and PDL1 antibody heavy ...17×PDL1 (containing CD3 antibody SEQ ID NO:62 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross323×PDL1 (containing CD3 antibody SEQ ID NO:63 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross325×PDL1 (containing CD3 antibody SEQ ID NO:72 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:72), Cross317×PDL1 (containing CD3 antibody SEQ ID NO:72 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO The following were added to the EP tube: NO:51 (containing the CD3 antibody SEQ ID NO:64 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 (containing the CD3 antibody SEQ ID NO:64 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), and control hIgG1 LALA (purchased from Baiying Biotechnology). The mixture was incubated at 4°C for 30 min. 1 ml of the mixture was added to the EP tube.Centrifuge with PBS at 3500 rpm for 5 min at 4°C, discard the supernatant, and wash once more with PBS. After centrifugation, discard the supernatant again, resuspend the cells in 100 μl / tube of PBS, add 1 μl / tube of Alexa-647-labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson Lab), and incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze using flow cytometry.

[0292] The results are as follows Figure 10 As shown, the CD3×Trop2 antibody of the present invention can bind to A-375-Trop2 melanoma cells. Based on the average fluorescence intensity, all bispecific antibodies have similar binding ability to A-375-Trop2 cells.

[0293] The results are as follows Figure 17 , 18 As shown in Figure 19, the CD3×PDL1 antibody of the present invention can bind to A-375 melanoma cells, HCT-15 colorectal cancer cells, and A549 lung cancer cells.

[0294] Example 8: Experiment on bispecific antibody promoting PBMC killing of tumor cells

[0295] To detect the ability of bispecific antibodies to promote the killing of A-375 melanoma cells and HCT-15 colorectal cancer cells by PBMCs.

[0296] (a) Add 50 μL of complete RPMI-1450 medium to a 16-well RTCA plate and calibrate it.

[0297] (b) Dilute tumor cells to 2 × 10⁶ using complete RPMI-1450 medium. 5 / mL, at a volume of 50μL / well, were individually added to the RTCA plate obtained in step (1), and then the cell coefficient was detected for 24h using an xCELLigence RTCAMP device at 37°C and 5% CO2.

[0298] (c) Using complete RPMI-1450 medium, serially diluted bispecific antibodies Cross3×Trop2 (containing the CD3 antibody as shown in SEQ ID NO:52 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), Cross303×Trop2 (containing the CD3 antibody as shown in SEQ ID NO:53 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), Cross307×Trop2 (containing the CD3 antibody as shown in SEQ ID NO:54 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), and Cross308×Trop2 (containing the CD3 antibody as shown in SEQ ID NO:55 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) were prepared. (Symptoms shown in SEQ ID NO:70), Cross309×Trop2 (containing the CD3 antibody SEQ ID NO:56 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), Cross310×Trop2 (containing the CD3 antibody SEQ ID NO:57 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), Cross311×Trop2 (containing the CD3 antibody SEQ ID NO:58 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), Cross312×Trop2 (containing the CD3 antibody SEQ ID NO:59 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), Cross313×Trop2 (containing the CD3 antibody SEQ ID NO:70 and the heavy chain amino acid sequence as shown in SEQ ID NO:59 and the light chain amino acid sequence as shown in SEQ ID NO:70), Cross313×Trop2 (containing the CD3 antibody SEQ ID NO:70 and the heavy chain amino acid sequence as shown in SEQ ID NO:70), Cross311×Trop2 (containing the CD3 antibody SEQ ID NO:58 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), Cross312×Trop2 (containing the CD3 antibody SEQ ID NO:59 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), Cross313×Trop2 (containing the CD3 antibody SEQ ID NO:70 and the heavy chain amino acid sequence as shown in SEQ ID NO:70), Cross The heavy chain amino acid sequence of the Trop2 antibody is shown in SEQ ID NO:49 and the light chain amino acid sequence is shown in SEQ ID NO:70), Cross314×Trop2 (containing the CD3 antibody SEQ ID NO:60 and the heavy chain amino acid sequence of the Trop2 antibody as shown in SEQ ID NO:69 and the light chain amino acid sequence as shown in SEQ ID NO:70), and Cross316×Trop2 (containing the CD3 antibody SEQ ID NO:50 and the heavy chain amino acid sequence of the Trop2 antibody as shown in SEQ ID NO:49 and the light chain amino acid sequence as shown in SEQ ID NO:70).The following antibodies are listed: Cross317×Trop2 (containing the CD3 antibody SEQ ID NO:61 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), Cross318×Trop2 (containing the CD3 antibody SEQ ID NO:62 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), Cross323×Trop2 (containing the CD3 antibody SEQ ID NO:63 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), and Cross325×Trop2 (containing the CD3 antibody SEQ ID NO:51 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). (Shown NO:70), Cross326×Trop2 (containing CD3 antibody SEQ ID NO:64 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross3×PDL1 (containing CD3 antibody SEQ ID NO:52 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross303×PDL1 (containing CD3 antibody SEQ ID NO:53 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross307×PDL1 (containing CD3 antibody SEQ ID NO:54 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross308×PDL1 (containing CD3 antibody SEQ ID NO:70), Cross326×Trop2 (containing CD3 antibody SEQ ID NO:64 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:79 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross308×PDL1 (containing CD3 antibody SEQ ID NO:70), Cross326×Trop2 (containing CD3 antibody SEQ ID NO:70), Cross3 ...26×Trop2 (containing CD3 antibody SEQ ID NO:70), Cross326×PDL1 (containing CD3 antibody SEQ The following are listed: NO:55 (containing the CD3 antibody SEQ ID NO:56 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 (containing the CD3 antibody SEQ ID NO:56 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), and Cross310×PDL1 (containing the CD3 antibody SEQ ID NO:57 and the PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:55 and the PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:72).The following are listed: Cross311×PDL1 (containing the CD3 antibody SEQ ID NO: 58 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), Cross312×PDL1 (containing the CD3 antibody SEQ ID NO: 59 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), Cross313×PDL1 (containing the CD3 antibody SEQ ID NO: 49 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), and Cross314×PDL1 (containing the CD3 antibody SEQ ID NO: 60 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). (Shown NO:72), Cross316×PDL1 (containing CD3 antibody SEQ ID NO:61 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross317×PDL1 (containing CD3 antibody SEQ ID NO:61 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross318×PDL1 (containing CD3 antibody SEQ ID NO:62 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross323×PDL1 (containing CD3 antibody SEQ ID NO:63 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross325×PDL1 (containing CD3 antibody SEQ ID NO:72 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:72), Cross325×PDL1 (containing CD3 antibody SEQ ID NO:72 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:72), Cross317×PDL1 (containing CD3 antibody SEQ ID NO:62 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross323×PDL1 (containing CD3 antibody SEQ ID NO:63 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross325×PDL1 (containing CD3 antibody SEQ ID NO:72 and PDL1 antibody heavy ... The following are listed: NO:51 (containing the CD3 antibody SEQ ID NO:64 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 (containing the CD3 antibody SEQ ID NO:65 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), and Cross3ZH07×PDL1 (containing the CD3 antibody SEQ ID NO:65 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).The following antibodies were added to the RTCA plate obtained in step (2): Cross3ZH09×PDL1 (containing CD3 antibody SEQ ID NO:66 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross3ZH16×PDL1 (containing CD3 antibody SEQ ID NO:67 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross3ZH23×PDL1 (containing CD3 antibody SEQ ID NO:68 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), and control hIgG1LALA (purchased from Baiying Biotechnology). The volume of each antibody was 20 μl / well.

[0299] (4) Dilute PBMC (purchased from Miaoshun Biotechnology) to 1.25 × 10⁻⁶ using complete RPMI-1450 medium. 6 Add 80 μl / well to the RTCA plate obtained in step (3);

[0300] (5) The reaction system obtained in step (4) was subjected to 37°C and 5% CO2 for 24 h using an xCELLigence RTCA MP instrument to detect the cell coefficient.

[0301] like Figure 11 As shown, the CD3×Trop2 antibody of the present invention promotes the killing of A-375-Trop2 melanoma cells by PBMCs. The effective dose of the Cross3×Trop2 bispecific antibody is lower than that of the affinity-reduced bispecific antibody. Although the effective doses of Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 are higher, their maximum killing ratios are consistent with those of Cross3×Trop2, and all can achieve the maximum killing effect.

[0302] like Figure 12 As shown, Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 promote the killing of A-375-Trop2 melanoma cells by PBMCs. Although the effective dose of Cross313×Trop2 and Cross316×Trop2 is higher, their maximum killing ratio is consistent with that of Cross3×Trop2, and both can achieve the maximum killing effect.

[0303] like Figure 20As shown, the CD3×PDL1 of the present invention promotes the killing of A-375 melanoma cells by PBMCs, and Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 have the highest killing effect compared to other bispecific antibodies.

[0304] like Figure 21 As shown, Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 of the present invention promote the killing of A-375 melanoma cells by PBMCs, and Cross316×PDL1 has the highest killing effect than Cross313×PDL1 and Cross325×PDL1.

[0305] like Figure 22 As shown, the CD3×PDL1 of the present invention promotes the killing of HCT-15 colorectal cancer cells by PBMCs, and Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 can promote 100% killing of HCT-15 colorectal cancer cells by PBMCs.

[0306] Example 9: Experiment on bispecific antibody-induced PBMC cytokine secretion

[0307] Bispecific antibodies were added to a co-incubation system of PBMCs and tumor cells. After 48 hours of culture, the culture supernatant was collected, and the cytokine content in the supernatant was detected to determine the characteristics of cytokine release induced by the bispecific antibodies.

[0308] (1) The tumor was diluted to 1×10⁻⁶ using complete RPMI 1450 medium. 5 Add / ml to a 96-well plate and incubate at 37°C in a 5% CO2 incubator for 24 hours;

[0309] (2) Using complete RPMI 1450 medium, the following bispecific antibodies were prepared: Cross3×Trop2 (containing CD3 antibody SEQ ID NO:52 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross313×Trop2 (containing CD3 antibody SEQ ID NO:49 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross316×Trop2 (containing CD3 antibody SEQ ID NO:50 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), Cross325×Trop2 (containing CD3 antibody SEQ ID NO:51 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), and Cross3×PDL1 (containing CD3 antibody SEQ ID NO:52 and Trop2 antibody heavy chain amino acid sequence as shown in SEQ ID NO:69 and light chain amino acid sequence as shown in SEQ ID NO:70), respectively. The following antibodies were serially diluted: Cross313×PDL1 (containing CD3 antibody SEQ ID NO:52 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross316×PDL1 (containing CD3 antibody SEQ ID NO:50 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), Cross325×PDL1 (containing CD3 antibody SEQ ID NO:51 and PDL1 antibody heavy chain amino acid sequence as shown in SEQ ID NO:71 and light chain amino acid sequence as shown in SEQ ID NO:72), and control hIgG1LALA (purchased from Bio-Tech). 20 μl / well was added to each well of a 96-well plate.

[0310] (3) PBMC (purchased from Selene Biotechnology) was diluted to 1.25 × 10⁻⁶ using complete RPMI 1450 medium. 6 Add 80 μl / well to a 96-well plate;

[0311] (4) Incubate the 96-well plate at 37°C in a 5% CO2 incubator for 48 hours;

[0312] (5) Centrifuge at 300g for 10 min at room temperature and collect the cell culture supernatant;

[0313] (6) The cytokine content in the supernatant was detected using the CBA kit (purchased from BD).

[0314] like Figure 13 As shown, the Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 of the present invention promote the secretion of pro-inflammatory cytokines TNF-α, IL-6, IFN-γ, and IL-2 by PBMCs, and the antibodies of Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 promote the secretion of inflammatory factors less than Cross3×Trop2.

[0315] like Figure 23 , 24 As shown, the Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 of the present invention promote the secretion of pro-inflammatory cytokines TNF-α and IFN-γ by PBMCs, and the antibodies of Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 promote the secretion of inflammatory factors less than Cross3×PDL1.

[0316] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0317] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An anti-CD3 antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment comprises any one of the following groups: 1) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 1, 2 and 3 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 4, 5 and 6 respectively; 2) Having heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 1, 2 and 8 respectively, and having light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 4, 5 and 9 respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain framework region and a light chain framework region; At least a portion of the heavy chain framework region and the light chain framework region are derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, or mutants thereof.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, At least a portion of the heavy chain framework region and the light chain framework region are derived from at least one of human antibodies, dairy bovine antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises: Heavy chain variable regions having amino acid sequences as shown in SEQ ID NO: 10, 13 or their conserved modified forms; and / or, The light chain variable region having an amino acid sequence as shown in SEQ ID NO: 11, 14 or an amino acid sequence of a conserved modified form thereof.

5. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region has an amino acid sequence that has at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 10, 13; and / or, The light chain variable region has an amino acid sequence that has at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 11, 14.

6. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence represented by any of the following combinations of heavy chain variable regions and light chain variable regions: The heavy chain variable region shown in SEQ ID NO:10 and the light chain variable region shown in SEQ ID NO:11; Or the heavy chain variable region shown in SEQ ID NO:13 and the light chain variable region shown in SEQ ID NO:

14.

7. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes a constant region; The constant region includes at least one of the heavy chain constant region and the light chain constant region.

8. The antibody or its antigen-binding fragment according to claim 7, 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 mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, or mutants thereof.

9. The antibody or its antigen-binding fragment according to claim 7, 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 human antibodies, dairy bovine antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

10. The antibody or its antigen-binding fragment according to claim 7, characterized in that, 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 light chain constant regions selected from κ-type or λ-type.

11. The antibody or its antigen-binding fragment according to claim 7, characterized in that, The N-end of the heavy chain constant region is connected to the C-end of the heavy chain variable region; and / or, The N-end of the constant region of the light chain is connected to the N-end of the variable region of the light chain.

12. The antibody or its antigen-binding fragment according to claim 7, characterized in that, The heavy chain constant region has an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 93; and / or, The light chain constant region has an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:

94.

13. The antibody or its antigen-binding fragment according to claim 7, characterized in that, The heavy chain constant region and the light chain constant region have amino acid sequences as shown in SEQ ID NO: 93 and SEQ ID NO: 94, respectively.

14. The antibody or its antigen-binding fragment according to claim 7, characterized in that, The C-end of the heavy chain variable region is connected to the N-end of the light chain variable region, or the N-end of the heavy chain variable region is connected to the C-end of the light chain variable region.

15. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further comprises a first linker peptide, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the first linker peptide, and the C-terminus of the first linker 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 linker peptide, and the C-terminus of the first linker peptide is connected to the N-terminus of the heavy chain variable region.

16. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment includes at least one of full-length monoclonal antibody, chimeric antibody, humanized antibody, Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, scFv, scFv-Fc fusion protein, and scFv-Fv fusion protein.

17. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or antigen-binding fragment comprises: Heavy chains having an amino acid sequence that is at least 80% identical to the amino acid sequences shown in SEQ ID NO: 32, 35; and / or, A light chain having an amino acid sequence that is at least 80% identical to the amino acid sequences shown in SEQ ID NO: 33, 36.

18. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment has an amino acid sequence that is at least 80% sequence identical to the amino acid sequence shown in any of the following combinations of heavy and light chains: The heavy chain shown in SEQ ID NO:32 and the light chain shown in SEQ ID NO:33; Or the heavy chain shown in SEQ ID NO:35 and the light chain shown in SEQ ID NO:

36.

19. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment has an amino acid sequence that is at least 80% identical to the amino acid sequence shown in either SEQ ID NO: 106 or 112.

20. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment has an amino acid sequence represented by any one of the following combinations of Fab VL-CL and Fab VH-CH1, or an amino acid sequence having at least 80% sequence identity with it:

21. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment has an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:149 or 155.

22. A multispecific antibody, characterized in that, Include: A first antigen-binding region, wherein the first antigen-binding region comprises the anti-CD3 antibody or its antigen-binding fragment as described in any one of claims 1 to 21; The second antigen-binding region has biomolecule binding activity, wherein the biomolecule is not CD3, but Trop2 or PDL1; The second antigen-binding region contains a first anti-Trop2 antibody or its antigen-binding fragment, or a first anti-PDL1 antibody or its antigen-binding fragment; The first anti-Trop2 antibody or its antigen-binding fragment is a second scFab fragment or a second Fab fragment of anti-Trop2; or, The first anti-PDL1 antibody or its antigen-binding fragment is a third scFab fragment or a third Fab fragment of anti-PDL1; 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 an amino acid sequence as shown in SEQ ID NO: 164 and 72.

23. The multispecific antibody according to claim 22, characterized in that, The multispecific antibody is selected from one of bispecific antibodies, trispecific antibodies, and tetraspecific antibodies.

24. The multispecific antibody according to claim 23, characterized in that, The bispecific antibody includes symmetrical bispecific antibodies or asymmetrical bispecific antibodies.

25. The multispecific antibody according to claim 22, characterized in that, The first antigen-binding region includes a first scFv fragment, a first Fv fragment, a first Fab fragment, or a first Fab-Linker fragment.

26. The multispecific antibody according to claim 25, characterized in that, The first antigen-binding region further includes a first Fc peptide segment; The N-terminus of the first Fc peptide is connected to the C-terminus of the scFv fragment or the Fv fragment; or, The N-terminus of the first Fc peptide is linked to the C-terminus of the CH1 of the Fab fragment or Fab-Linker fragment.

27. The multispecific antibody according to claim 26, characterized in that, The first antigen-binding region further includes a second linker peptide, wherein the N-terminus of the first Fc peptide is linked to the C-terminus of the second linker peptide, and the N-terminus of the second linker peptide is linked to the C-terminus of the scFv fragment or the Fv fragment; or, The N-terminus of the first Fc peptide is connected to the C-terminus of the second linker peptide, and the N-terminus of the second linker peptide is connected to the C-terminus of CH1 of the Fab fragment or Fab-Linker fragment.

28. The multispecific antibody according to claim 26, characterized in that, The first Fc peptide is selected from human Fc peptides.

29. The multispecific antibody according to claim 28, characterized in that, The first Fc peptide is the human IgG1 Fc peptide.

30. The multispecific antibody according to claim 26, characterized in that, The first Fc peptide has the amino acid sequence shown in SEQ ID NO:118; or the first Fc peptide has the amino acid sequence shown in SEQ ID NO:

166. The first scFv fragment has the amino acid sequence shown in either SEQ ID NO:106 or 112; The first Fab fragment has the amino acid sequence shown in any of the following combinations of Fab VL-CL and Fab VH-CH1:

31. The multispecific antibody according to claim 25, characterized in that, The first Fab-Linker has an amino acid sequence as shown in either SEQ ID NO: 149 or 155; The first antigen-binding region has an amino acid sequence as shown in either SEQ ID NO: 49 or 51.

32. The multispecific antibody according to claim 22, characterized in that, The second antigen-binding region further includes a second Fc fragment; Wherein, the N end of the second Fc segment is connected to the C end of CH1 of the second scFab segment, the second Fab segment, the third scFab segment, or the third Fab segment; The second Fc peptide is selected from human Fc peptide.

33. The multispecific antibody according to claim 32, characterized in that, The second Fc peptide has the amino acid sequence shown in SEQ ID NO: 165; The first Fc peptide and the second Fc peptide are connected by a knock-into-hole structure; 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 the amino acid sequence 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.

34. A nucleic acid, characterized in that, The antibody or its antigen-binding fragment as described in any one of claims 1 to 21, or the multispecific antibody as described in any one of claims 22 to 33.

35. A carrier, characterized in that, It contains the nucleic acid as described in claim 34.

36. The carrier according to claim 35, characterized in that, The vector is a eukaryotic vector or a prokaryotic vector.

37. The carrier according to claim 35, characterized in that, The vector includes one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

38. A cell characterized by, It carries the nucleic acid of claim 34, the vector of claim 35; or expresses the antibody or its antigen-binding fragment of any one of claims 1 to 21 or the multispecific antibody of any one of claims 22 to 33.

39. The cell according to claim 38, characterized in that, The cells mentioned are prokaryotic cells and eukaryotic cells.

40. A pharmaceutical composition, characterized in that, It comprises: the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 21, the multispecific antibody as described in any one of claims 22 to 33, the nucleic acid as described in claim 34, or the vector as described in any one of claims 35 to 37; And pharmaceutically acceptable excipients.

41. The pharmaceutical composition according to claim 40, characterized in that, The excipients include one or more pharmaceutically acceptable excipients, diluents, and stabilizers.

42. The pharmaceutical composition according to claim 41, characterized in that, The pharmaceutical composition is an injectable preparation.

43. A reagent kit, characterized in that, It comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 21, or the multispecific antibody as described in any one of claims 22 to 33.

44. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 21 or the multispecific antibody according to any one of claims 22 to 33 in the preparation of a kit for detecting CD3.

45. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, the multispecific antibody according to any one of claims 22 to 33, or the pharmaceutical composition according to any one of claims 40 to 42 in the preparation of a medicament for the treatment of 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.

46. ​​Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, the multispecific antibody according to any one of claims 22 to 33, or the pharmaceutical composition according to any one of claims 40 to 42 in the preparation of a medicament for the treatment of 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 lymphoma.

Images