Anti-CD3 antibodies and their use

Abstract

We propose an anti-CD3 antibody or its antigen-binding fragment and its use. It comprises heavy-chain variable region CDRs and light-chain variable region CDRs, wherein the heavy-chain variable region CDRs have amino acid sequences represented by SEQ ID NO: 1-3, 7-8, and 74-86, or their conserved modified forms, and / or the light-chain variable region CDRs have amino acid sequences represented by SEQ ID NO: 4-6, 9, and 87-92, or their conserved modified forms. The provided anti-CD3 antibody or its antigen-binding fragment can bind to human CD3 protein and reduce the production of pro-inflammatory cytokines. A bispecific antibody constructed using this antibody possesses high tumor-killing activity via PBMCs and weak pro-inflammatory cytokine secretion-promoting activity. Therefore, drug compositions prepared using this anti-CD3 antibody or its antigen-binding fragment or its bispecific antibody have better safety and higher clinical use and pharmaceutical development value.

Description

[Technical Field]

[0001] This invention belongs to the field of biopharmaceuticals, and more specifically, it relates to an anti-CD3 antibody or its antigen-binding fragment and its use. [Background technology]

[0002] Cancer is a serious disease that affects the survival and development of humankind. According to the latest data, there are approximately 19 million new cases of cancer worldwide each year, and approximately 10 million deaths from cancer each year, with both incidence and mortality rates on the rise. Traditional cancer treatments other than surgical resection, such as chemotherapy and radiation therapy, 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 new hope for anti-cancer treatment. Immunotherapies, including monoclonal and bispecific antibodies, achieve their anti-cancer effects mainly by promoting the function of autoimmune cells, and have the advantages of fewer side effects and longer survival times for beneficiaries. Immunotherapy checkpoint antibodies already approved for market include PD1-1 / L1, CTLA-4, and LAG-3, while bispecific anticancer antibodies include Blinatumomab (CD3 x CD19), Kimtrak (CD3 x GP100), Mosunetuzumab (CD3 x CD20), and Tecvayli (CD3 x BCMA). Bispecific antibodies represent a significant advance in the treatment of hematological malignancies, targeting CD19, CD20, and BCMA, promoting T cell elimination of tumor cells, and potentially benefiting patients.

[0003] CD3-based bispecific antibodies (hereinafter abbreviated as "CD3 bispecific antibodies") recruit T cells and reach the tumor site, bridging the gap between T cells and the tumor, thereby promoting T cell activation and killing the tumor. Such bispecific antibodies do not require newly generated antigens and can induce T cells to kill "cold tumors." After binding to T cells and tumor cells, CD3 bispecific antibodies trigger a strong activation signal, allowing them to "ignore" the suppressive signals of immune checkpoint molecules to some extent. However, CD3 bispecific antibodies also promote the production of large amounts of pro-inflammatory cytokines, such as TNFα and IL-6, which can trigger a strong cytokine storm and an excessive immune response, causing biological damage and potentially leading to a serious life-threatening situation. Therefore, although CD3-based bispecific antibodies have a good clinical outlook, their safety needs to be further improved.

[0004] One way to address the safety issues of CD3 bispecific antibodies is to increase the affinity of the CD3 bispecific antibody to the tumor target while decreasing its affinity to CD3. This allows for greater distribution of the CD3 bispecific antibody drug to the tumor site, increasing the drug concentration at the tumor site, decreasing peripheral drug concentrations, reducing extratarget toxicity, lowering the production level of pro-inflammatory cytokines, and thus reducing targeted toxicity. Therefore, there is an urgent need to develop anti-CD3 antibodies that can bind to the human CD3 protein with low affinity, and furthermore, to obtain CD3 antibody drugs that are safer and have high clinical use and drug development value. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The present invention aims to solve, to at least some extent, one of the technical problems existing in the prior art. Therefore, one object of the present invention is to provide an anti-CD3 antibody or its antigen-binding fragment. Another object of the present invention is to provide a multispecific antibody constructed using the anti-CD3 antibody or its antigen-binding fragment. The anti-CD3 antibody or its antigen-binding fragment of the present invention can bind to human CD3 protein and reduce the production of pro-inflammatory cytokines. The bispecific antibody constructed using it exhibits high tumor-killing activity by PBMCs and weak promotion of pro-inflammatory cytokine secretion. As a result, drug compositions prepared using the anti-CD3 antibody or its antigen-binding fragment or the multispecific antibody have superior safety and high clinical use and pharmaceutical development value.

[0006] This invention was made based on the inventor's expression and recognition of the following facts and problems.

[0007] Currently, the most commonly used CD3 antibodies for bispecific antibodies are TR66 and UCHT1. Neither of these antibodies binds to monkey CD3, increasing the difficulty of nonclinical drug evaluation. Furthermore, these two antibodies have very strong binding affinity to T cells, increasing the safety risk of CD3 bispecific antibody drugs constructed based on them. The inventor modified the human-monkey cross anti-CD3 antibody Cross3, which has high CD3 affinity (KD=0.165nM), and through extensive screening, obtained a series of anti-CD3 antibodies with low affinity that can bind to human CD3 protein. Further, based on the aforementioned series of anti-CD3 antibodies, CD3 bispecific antibodies were constructed and screened, yielding 19 anti-CD3 antibodies. Compared to Cross3, the bispecific antibody tumor antigen-specific recognition and binding are not affected by the CD3 antigen-binding region. Further experimental results showed that: 1) The CD3 bispecific antibody of the present invention binds to CD3E&D proteins and can specifically bind to tumor antigens (e.g., PDL1, Trop2), and its tumor antigen binding activity is essentially equivalent to that of a bispecific antibody constructed based on Cross3; 2) Cell-level experimental results showed that the CD3 bispecific antibody of the present invention can bind to T cells and can specifically bind to tumor cells (e.g., A-375-Trop2 cells, A-375 melanoma cells, HCT-15 colorectal cancer cells, A549 lung cancer cells), and its tumor cell binding ability is essentially equivalent to that of a bispecific antibody constructed based on Cross3 antibody; and 3) Experimental results on cytokine secretion promotion by PBMCs demonstrated that the CD3 bispecific antibody of the present invention has the ability to promote the secretion of reduced pro-inflammatory cytokines.

[0008] The inventors further verified the tumor cell killing activity of the above-mentioned CD3 bispecific antibodies through tumor cell killing experiments using PBMCs. The results showed that the bispecific antibodies constructed based on the anti-CD3 antibodies Cross313, Cross316, and Cross325 of the present invention (e.g., Cross313×Trop2, Cross316×Trop2, Cross325×Trop2, Cross313×PDL1, Cross316×PDL1, Cross325×PDL1) could achieve the highest killing effect or promote 100% killing of tumor cells by PBMCs, and that the bispecific antibodies constructed based on the anti-CD3 antibody Cross3 (e.g., Cross3×Trop2, Cross3×PDL1) had a corresponding tumor cell killing effect.

[0009] As can be seen from the experimental results above, the CD3 bispecific antibody developed based on the anti-CD3 antibody or its antigen-binding fragment of the present invention has high tumor-killing activity by PBMCs and weak pro-inflammatory cytokine secretion-promoting ability, is safer, and has high value for clinical use and pharmaceutical development. [Means for solving the problem]

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

[0011] The anti-CD3 antibody or its antigen-binding fragment according to the 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 the CD3 antibody or its antigen-binding fragment has high tumor-killing activity by PBMCs and weak pro-inflammatory cytokine secretion-promoting ability. After being prepared as a drug, it can be distributed more abundantly to the tumor site, increasing the drug concentration at the tumor site, decreasing the peripheral drug concentration, and reducing the risk of off-target toxicity. On the other hand, the bispecific antibody drug can reduce the production of pro-inflammatory cytokines and reduce the risk of targeted toxicity. Furthermore, drugs prepared using the above anti-CD3 antibody or its antigen-binding fragment can be used to prevent and / or treat CD3-related diseases, and the CD3 bispecific antibody drug developed based on the above anti-CD3 antibody or its antigen-binding fragment has higher safety and can be used to prevent and / or treat cancer or tumors or infections or autoimmune diseases. Thus, the anti-CD3 antibody or its antigen-binding fragment of the present invention has good clinical use value and pharmaceutical development value.

[0012] In a second aspect of the present invention, the present invention includes a polyspecific antibody. According to an example of the present invention, the polyspecific antibody is A first antigen-binding region comprising an anti-CD3 antibody or its antigen-binding fragment according to a first aspect of the present invention, It has biomolecule binding activity, and the biomolecule comprises a second antigen-binding region that is not CD3.

[0013] As described above, a polyspecific antibody (e.g., a bispecific antibody) developed based on the anti-CD3 antibody or its antigen-binding fragment according to the first aspect of the present invention can be distributed more abundantly to the tumor site after being prepared as a drug, increasing the drug concentration at the tumor site and decreasing the peripheral drug concentration, thereby reducing the risk of off-target toxicity. At the same time, the bispecific antibody drug reduces the production of pro-inflammatory cytokines and reduces the risk of target toxicity of the polyspecific antibody (e.g., bispecific antibody). As a result, the polyspecific antibody based on the anti-CD3 antibody or its antigen-binding fragment according to the first aspect of the present invention has better safety and high clinical use value and pharmaceutical development value.

[0014] In a third aspect of the present invention, the present invention proposes a conjugate. According to an embodiment of the present invention, the conjugate comprises an antibody or its antigen-binding fragment described in the first aspect of the present invention or a polyspecific antibody described in the second aspect of the present invention.

[0015] As described above, the anti-CD3 antibody or its antigen-binding fragment according to the embodiments of the present invention can be used to prepare bispecific antibody drugs, which can be distributed more abundantly to the tumor site, increasing the drug concentration at the tumor site and decreasing the peripheral drug concentration, thereby reducing the risk of off-target toxicity. Meanwhile, the bispecific antibody can reduce the production of pro-inflammatory cytokines and reduce the target toxicity risk of polyspecific antibodies (e.g., 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, and CD3 bispecific antibody drugs developed based on the anti-CD3 antibody or its antigen-binding fragment are safer and can be used to prevent and / or treat cancer or tumors or infections or autoimmune diseases. Thus, conjugates comprising the anti-CD3 antibody or its antigen-binding fragment according to the first aspect of the present invention and the polyspecific antibody according to the second aspect of the present invention have good clinical use value and pharmaceutical development value.

[0016] In a fourth aspect of the present invention, the present invention proposes a nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes an anti-CD3 antibody or its antigen-binding fragment as described in the first aspect of the present invention or a polyspecific antibody as described in the second aspect of the present invention. According to an embodiment of the present invention, the antibody or its antigen-binding fragment as described in the first aspect of the present invention or the polyspecific antibody as described in the second aspect of the present invention encoded by the nucleic acid is suitable for developing a safer monoclonal antibody or bispecific antibody drug. Furthermore, the protein encoded by the nucleic acid can be used to prevent and / or treat CD3-related diseases, or to prevent and / or treat cancer or tumors or infections or autoimmune diseases.

[0017] In a fifth aspect of the present invention, the present invention proposes a carrier or transformant. According to an embodiment of the present invention, the carrier or transformant comprises the nucleic acid described in the fourth aspect of the present invention. By utilizing the constructed carrier or transformant, the antibody or antigen-binding fragment described in the first aspect of the present invention or the polyspecific antibody described in the second aspect of the present invention can be efficiently expressed, and the conjugate described in the third aspect of the present invention can be obtained.

[0018] In a sixth aspect of the present invention, the present invention proposes cells. According to an embodiment of the present invention, the cells carry the nucleic acid described in the fourth aspect of the present invention or the carrier or transformant described in the fifth aspect of the present invention, or express the antibody or its antigen-binding fragment described in the first aspect of the present invention or the polyspecific antibody described in the second aspect of the present invention. According to an embodiment of the present invention, the cells can efficiently express the antibody or its antigen-binding fragment described in the first aspect of the present invention or the polyspecific antibody described in the second aspect of the present invention under appropriate conditions, and furthermore, an antibody or its antigen-binding fragment or the polyspecific antibody with improved safety that can bind to human CD3 protein can be obtained.

[0019] In a seventh aspect of the present invention, the present invention proposes a drug composition. According to an example of the present invention, the drug composition comprises an antibody or antigen-binding fragment described in the first aspect of the present invention, a polyspecific antibody described in the second aspect of the present invention, a conjugate described in the third aspect of the present invention, a nucleic acid described in the fourth aspect of the present invention, or a carrier or transformant described in the fifth aspect of the present invention. The resulting drug can be used to further prevent and / or treat CD3-related diseases, or to further prevent and / or treat CD3-related diseases and / or cancer or tumors or infections or autoimmune diseases.

[0020] In an eighth aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present invention, the kit comprises an antibody or its antigen-binding fragment described in the first aspect of the present invention or a polyspecific antibody described in the second aspect of the present invention. The resulting kit is available for use in CD3-related research.

[0021] In a ninth aspect of the present invention, the present invention is used in the preparation of a kit for detecting CD3 of 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. As can be understood by those skilled in the art, the features and advantages of the above antibody or its antigen-binding fragment are similarly applicable to the use, and will not be repeatedly described herein.

[0022] In a tenth aspect of the present invention, the present invention proposes the use of the antibody or its antigen-binding fragment described in the first aspect of the present invention, the multispecific antibody described in the second aspect of the present invention, the conjugate described in the third aspect of the present invention, the nucleic acid described in the fourth aspect of the present invention, the carrier or transformant described in the fifth aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention in the preparation of a drug for preventing and / or treating CD3-related diseases and / or cancer or tumor or infection or autoimmune diseases. By using the antibody or its antigen-binding fragment, multispecific antibody and conjugate, nucleic acid, carrier or transformant or pharmaceutical composition of the present invention, it can be further prepared into a drug, and the drug can be clinically used for preventing and / or treating diseases.

[0023] As can be understood by those skilled in the art, the features and advantages described for the above antibody or its antigen-binding fragment, conjugate, nucleic acid molecule, carrier or transformant and pharmaceutical composition are similarly applicable to the use, and will not be repeatedly described herein.

Advantages of the Invention

[0024] Beneficial effects: (1) The anti-CD3 antibodies Cross303, Cross307, Cross308, Cross309, Cross310, Cross311, Cross312, Cross313, Cross314, Cross316, Cross317, Cross318, Cross323, Cross325, Cross326, Cross3ZH07, Cross3ZH09, Cross3ZH16, Cross3ZH23 obtained in the present invention can reduce the production of pro-inflammatory cytokines by the CD3 antigen-binding portion as compared with the parental antibody Cross3. (2) The bispecific antibodies constructed with the antibodies Cross313, Cross316, Cross325 obtained in the present invention have a weaker binding ability to T cells as compared with the bispecific antibodies constructed with the parental antibody Cross3. (3) The bispecific antibodies constructed with the antibodies Cross313, Cross316, Cross325 obtained in the present invention can similarly promote tumor cells by PBMC to the maximum extent as compared with the bispecific antibodies constructed with the parental antibody Cross3, and have a weaker promoting performance for the production of pro-inflammatory cytokines and better safety.

[0025] Additional aspects and advantages of the present invention are shown in part in the following description, become apparent in part from the following description, or are understood by the practice of the present disclosure.

Brief Description of Drawings

[0026] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the examples in combination with the following drawings. [Figure 1] It is a comparison diagram of the amino acid mutation situation of the affinity-reducing antibody according to the example of the present invention compared with the parental antibody Cross3. [Figure 2] It is a SPR result diagram of the affinity measurement of the parental antibody Cross3 and the affinity-reducing antibody according to the example of the present invention with CD3E&D protein. [Figure 3] It is an ELISA result diagram of the parental antibody Cross3 and the affinity-reducing antibody according to the example of the present invention binding to CD3E&D protein [Figure 4] This is a flow celometry diagram showing the binding of the progenitor antibody Cross3 and the low-affinity antibody to CD8 T cells according to the embodiments of the present invention. [Figure 5] This is a schematic diagram of the structure of CD3×Trop2 and CD3×PDL1 bispecific antibodies composed of the mother antibody Cross3 and the affinity-reducing antibody according to the examples of the present invention. [Figure 6] This is an ELISA result diagram showing that a CD3×Trop2 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, binds to the CD3E&D protein. [Figure 7] This is an ELISA result diagram showing that a CD3 × Trop2 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, binds to the Trop2 protein. [Figure 8] This figure shows the ELISA results demonstrating that a CD3×Trop2 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, bridges the gap between CD3E&D and Trop2 proteins. [Figure 9] This is a flow cell metric diagram showing the binding of a CD3 × Trop2 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, to CD8 T cells. [Figure 10] This is a flow celometry result diagram showing that a CD3×Trop2 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, binds to A-375-Trop2 melanoma cells. [Figure 11] This figure shows the results of a CD3×Trop2 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, which promotes the killing of A-375-Trop2 melanoma cells by PBMCs. [Figure 12] This figure shows the results of how the bispecific antibodies Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2, according to the examples of the present invention, promote the killing of A-375-Trop2 melanoma cells by PBMCs. [Figure 13] This figure shows the results of using the Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 bispecific antibodies according to the embodiment of the present invention to promote the secretion of pro-inflammatory cytokines by PBMCs. [Figure 14] This is an ELISA result diagram showing that a CD3×PDL1 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, binds to the CD3E&D protein. [Figure 15] This is an ELISA result diagram showing that a CD3 × PDL1 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, binds to the PDL1 protein. [Figure 16] This is a flow cell metric diagram showing the binding of a CD3×PDL1 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, to CD8 T cells. [Figure 17] This is a flow celometry result diagram showing that a CD3×PDL1 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, binds to A-375 melanoma cells. [Figure 18] This is a flow cell metric diagram showing the binding of a CD3×PDL1 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, to HCT-15 colorectal cancer cells. [Figure 19] This is a flow cell metric diagram showing the binding of a CD3×PDL1 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, to A-549 lung cancer cells. [Figure 20] This figure shows the results of a CD3×PDL1 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody, according to an example of the present invention, which promotes the killing of A-375 melanoma cells by PBMCs. [Figure 21] This figure shows the results of Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 according to the embodiment of the present invention, which promote the killing of A-375 melanoma cells by PBMCs. [Figure 22] This figure shows the results of a CD3×PDL1 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, which promotes the killing of HCT-15 colorectal cancer cells by PBMCs. [Figure 23] This figure shows the results of a CD3×PDL1 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, promoting the secretion of pro-inflammatory cytokines after co-incubation of PBMCs and A-375. [Figure 24] This figure shows the results of a CD3×PDL1 bispecific antibody, composed of the parent antibody Cross3 and a low-affinity antibody according to an example of the present invention, promoting the secretion of pro-inflammatory cytokines after co-incubation of PBMCs and HCT-15. [Modes for carrying out the invention]

[0027] The embodiments of the present invention will be described in detail below, and examples of these embodiments are shown in the drawings. The embodiments described below with reference to the drawings are illustrative and are for interpretation purposes only, and should not be understood as limiting the present invention.

[0028] Furthermore, terms such as “first” and “second” are merely descriptive and cannot be considered to indicate or imply relative importance, or to imply a number that explicitly reveals a technical feature. Thus, features that are limited as “first” or “second” may be explicitly or implicitly indicated to include one or more such features. In the description of this disclosure, unless otherwise explicitly and specifically limited, the concept of “multiple” refers to at least two, for example, two or three.

[0029] The endpoints and any values ​​of the ranges disclosed herein should be understood to include values ​​close to such exact ranges or values, and not to be limited to such exact ranges or values. In the case of numerical ranges, the intervals between the endpoint values ​​of each range, between the endpoint values ​​of each range and individual point values, and between individual point values ​​combine to obtain one or more new numerical ranges, and these numerical ranges shall be deemed to be specifically disclosed in the specification.

[0030] Terms and Definitions

[0031] To facilitate understanding of the present invention, several technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art. Amino acid residue abbreviations are standard three-letter and / or one-letter codes indicating one of the 20 common L-amino acids used in the art.

[0032] In this specification, the term “antibody” usually refers to an antibody that recognizes one or more antigen epitopes, and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, dimers, multimers, polyspecific antibodies (e.g., bispecific antibodies), heavy-chain only antibodies, tri-chain antibodies, single-chain Fv (scFv), nanoantibodies, and further includes antibody fragments as long as they exhibit the desired biological activity. Antibodies may be derived from mouse, human, humanized, chimeric, or other species. Antibodies may refer to full-length heavy chains, full-length light chains, complete immunoglobulin molecules, or molecules or portions thereof that include an immunoactive portion of any of these polypeptides, i.e., an antigen-binding site that binds immunospecifically to a target antigen of interest, and such targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases.

[0033] In this specification, certain regions within the variable region exhibit a higher degree of change in amino acid composition and sequence order and are called "hypervariable regions (HVRs)." Since hypervariable regions are the sites where antigens and antibodies bind, they are also called complementarity-determining regions (CDRs). Both the heavy chain variable region and the light chain variable region contain three CDR regions. For example, typically amino acid residues around 23-34(L1), 50-56(L2), and 89-97(L3) in the light chain variable region, and around 31-35B(H1), 50-65(H2), and 95-102(H3) in the heavy chain variable region, and / or "highly variable loops" (e.g., including amino acid residues around 26-32(LI), 50-52(L2), and 91-96(L3) in the light chain variable region, and around 26-32(H1), 53-55(H2), and 96-101(H3) in the heavy chain variable region).

[0034] In this specification, the term "anti-CD3 antibody" refers to an antibody that can bind to CD3. Such antibodies are also referred to in this specification as "CD3-binding antibodies."

[0035] In this specification, the term "Fab-Linker" refers to a polypeptide fragment obtained by linking Fab VH-CH1 and Fab VL-CL of an antibody Fab fragment with a linking peptide. This includes, but is not limited to, scFab and scFab△ (where the linking peptide does not contain a disulfide bond).

[0036] In this specification, the term “antigen-binding fragment” is equivalent to “antibody fragment” or “antigen-binding antibody fragment,” and may include a portion of a complete antibody, generally being 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 whose half-life can be increased by chemical modification or incorporation into liposomes, and such chemical modification is the addition of a poly(alkylene) glycol, such as polyethylene glycol ("polyethylene glycolized, PEGylated") (polyethylene glycolized fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" is polyethylene glycol).

[0037] In this specification, the term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of the constant region of a monoclonal antibody from one species (e.g., mouse) with the constant region of an antibody from another species (e.g., human).

[0038] In this specification, the term "humanized antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace all 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, an antibody is called a chimeric antibody when only the constant region of one antibody is humanized, and a antibody is called a humanized antibody when all non-CDR amino acid sequences in both the constant and variable regions are humanized. The humanization method can be carried out by referring to conventional antibody engineering techniques and will not be described again here.

[0039] With respect to nucleotides, the terms “homology,” “identity,” or “similarity” are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of “sequence homology” between a first sequence and a second sequence can be calculated by [dividing the number of identical nucleotides in the first sequence by the number of nucleotides at the corresponding positions in the first sequence]. Alternatively, the difference can be considered as a single nucleotide (position) difference, where each deletion, insertion, substitution, or addition of nucleotides in the second sequence is considered a single nucleotide (position) difference compared to the first sequence. 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. Several other techniques, computer algorithms, and settings for calculating the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768-A.

[0040] With respect to polypeptides, the terms “(substantial) homology,” “identity,” or “similarity” are used to describe or compare the degree of amino acid similarity when two or more polypeptides or a given sequence of them are in optimal alignment and comparison (appropriately inserting or deleting nucleotides). The homology % between two sequences varies depending on the number of identical positions that these sequences share when the sequences are optimally aligned (i.e., homology % = number of identical positions / total number of positions × 100). Here, the optimal alignment is determined by considering the number of spaces that need to be introduced and the length of each space that needs to be introduced to achieve the optimal alignment of the two sequences. The measurement of the sequence comparison and identity percentage between two sequences can be completed using mathematical algorithms, as described in the non-restrictive examples below.

[0041] In this specification, 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 the amino acid sequence, and such modification includes amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody of the present invention by standard techniques such as sentinel mutagenesis or PCR-mediated mutagenesis. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0042] In this specification, the term “conjugate” refers to an antibody or its antigen-binding fragment conjugated to a coupling portion such as a carrier, drug, toxin, cytokine, protein tag, modifier, therapeutic agent, or chemotherapeutic agent using any co- or non-co-co-co-co-co-conjugate strategy, as understood in its context.

[0043] In this specification, the term “carrier” typically refers to a nucleic acid molecule that can be inserted into a suitable host and self-replicate, and that moves the inserted nucleic acid molecule into and / or between host cells. The carrier may include carriers primarily for inserting DNA or RNA into cells, carriers primarily for duplication of DNA or RNA, and expression carriers primarily for transcription and / or translation of DNA or RNA. The carrier may further include carriers having a variety of the above functions. The carrier may also be a polynucleotide that can be transcribed into polypeptides and translated upon introduction into a suitable host cell. Typically, by culturing a suitable host cell containing the carrier, the carrier can produce the desired expression product.

[0044] In this specification, the term “drug composition” usually means in the form of a unit dose and can be prepared by any method well known in the pharmaceutical field. All methods include the step of binding the active ingredient to a carrier constituting one or more accessory components. Typically, the composition is prepared by uniformly and sufficiently binding the active compound to a liquid carrier, shredded solid carrier, or both.

[0045] In this specification, the term "pharmaceutically acceptable" means that an ingredient can be administered without causing excessive adverse effects (such as toxicity, irritation, and allergy) in humans and / or mammals, i.e., it has a reasonable benefit / risk ratio.

[0046] In this specification, the term “pharmaceutically acceptable excipient” can include any solvent, solid excipient, diluent or other liquid excipient, suitable for a specific target dosage form. Conventional excipients are also considered in the present invention, except to the extent that they are incompatible with the compounds of the present invention, for example, resulting in any undesirable biological effects or interacting in a detrimental manner with any other component of the pharmaceutically acceptable composition.

[0047] In this specification, the term “administration” means introducing a predetermined amount of a substance into a patient in a suitable manner. The antibody or antigen-binding fragment, recombinant protein, polyspecific antibody, coupling compound, or drug composition of the present invention can be administered by any common method, provided that it can reach the desired tissue. Various methods of administration are predictable and include, but the present invention is not limited to these exemplary methods of administration, such as peritoneal, intravenous, intramuscular, and subcutaneous injections. Preferably, the compositions of the present invention are administered by intravenous or subcutaneous injection.

[0048] In this specification, the term “treatment” means used to obtain a desired pharmacological and / or physiological effect. Such effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely cures a disease and / or the adverse effects caused by a disease. As used herein, “treatment” covers diseases of mammals, particularly humans, and includes (a) preventing disease or the onset of disease in individuals that are susceptible to disease but have not yet been diagnosed with the disease, (b) suppressing disease, such as by hindering the progression of the disease, or (c) alleviating disease, for example, reducing symptoms associated with the disease. As used herein, “treatment” includes, but is not limited to, any administration of a fusion protein, pharmaceutical composition, or drug to an individual to treat, cure, alleviate, improve, reduce or suppress a disease in that individual, and includes administering a drug containing a fusion protein described herein to an individual in need.

[0049] As used herein, “effective amount” or “effective dose” means an amount that can produce function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0050] The present invention provides anti-CD3 antibodies or their antigen-binding fragments, multispecific antibodies, conjugates, nucleic acid molecules, carriers or transformants, cells, drug compositions, kits, and their uses, each of which is described in detail below.

[0051] Anti-CD3 antibody or its antigen-binding fragment

[0052] The present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof. The anti-CD3 antibody or antigen-binding fragment has heavy chain variable regions (CDRs) and light chain variable regions (CDRs), wherein the heavy chain variable regions (CDRs) have an amino acid sequence shown in any of SEQ ID NO: 1-3, 7-8, and 74-86, or an amino acid sequence of a conservatively modified form thereof, and / or the light chain variable regions (CDRs) have an amino acid sequence shown in any of SEQ ID NO: 4-6, 9, and 87-92, or an amino acid sequence of a conservatively modified form thereof.

[0053] 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 the CD3 antibody or its antigen-binding fragment possesses high tumor-killing activity via PBMCs and weak promotion of pro-inflammatory cytokine secretion. After drug preparation, it can be distributed more effectively to the tumor site, increasing the drug concentration at the tumor site and decreasing the peripheral drug concentration, thereby reducing the risk of off-target toxicity. Furthermore, the bispecific antibody drug reduces the production of pro-inflammatory cytokines, thus lowering the risk of targeted toxicity. Additionally, drugs prepared using the anti-CD3 antibody or its antigen-binding fragment can be used to prevent and / or treat CD3-related diseases, and the CD3 bispecific antibody drug developed based on the anti-CD3 antibody or its antigen-binding fragment is safer and can be used to prevent and / or treat cancer or tumors or infections or autoimmune diseases. Thus, the anti-CD3 antibody or its antigen-binding fragment of the present invention has excellent clinical use value and pharmaceutical development value.

[0054] Furthermore, one or more amino acid residues in the antibody or its antigen-binding fragment CDR region may be substituted with other amino acid residues derived from the same side-chain family, and the retention function of the altered antibody can be tested using the functional measurement method described herein. Preferably, the number of conservative modifications is one or less, or two.

[0055] According to embodiments of the present invention, the heavy chain variable regions (CDRs) have amino acid sequences shown in SEQ ID NO: 1-2 or their conserved modified forms, and amino acid sequences shown in any of SEQ ID NO: 3, 7-8 and 74-86 or their conserved modified forms, and the light chain variable regions (CDRs) have amino acid sequences shown in SEQ ID NO: 4-5 or their conserved modified forms, and amino acid sequences shown in any of SEQ ID NO: 6, 9 and 87-92 or their conserved modified forms.

[0056] According to an embodiment of the present invention, the antibody or its antigen-binding fragment comprises the following: heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 1; heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 2; heavy chain variable region CDR3 has the amino acid sequence shown in any of SEQ ID NO: 3, SEQ ID NO: 7-8, and SEQ ID NO: 74-86; light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 4; light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 5; and light chain variable region CDR3 has the amino acid sequence shown in any of SEQ ID NO: 6, 9, and 87-92.

[0057] According to embodiments of the present invention, the heavy chain variable region CDR3 has an amino acid sequence shown in SEQ ID NO: 3 and 7-8, and / or the light chain variable region CDR3 has an amino acid sequence shown in SEQ ID NO: 6 or 9.

[0058] According to embodiments of the present invention, the antibody or its antigen-binding fragment comprises any of the following groups: 1) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 74, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 6. 2) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 75, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 6. 3) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 76, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 6. 4) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 77, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 6. 5) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 78, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 6. 6) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 79, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 6. 7) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 80, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 6. 8) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 81, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 6. 9) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 82, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 6. 10) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 83, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 6. 11) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 3, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 12) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 7, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 13) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 8, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 87, 14) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 8, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 88, 15) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 8, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 9. 16) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 84, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 89. 17) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 85, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 90. 18) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 86, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 92. 19) Each has heavy chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 1, 2, and 8, and each has light chain variable regions CDR1, CDR2, and CDR3, indicated by SEQ ID NO: 4, 5, and 91.

[0059] Furthermore, the antigen-binding fragment consists of a sequence from a part of the heavy chain variable region or light chain variable region of the derived antibody, or includes a sequence from a part of the light or heavy chain variable region, and the sequence is sufficient to maintain the same binding specificity and sufficient affinity as the derived antibody.

[0060] According to embodiments of the present invention, the antibody or its antigen-binding fragment includes a heavy chain framework region and / or a light chain framework region.

[0061] 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 the following: mouse-derived antibody, human-derived antibody, primate-derived antibody, cattle-derived antibody, horse-derived antibody, dairy cow-derived antibody, pig-derived antibody, sheep-derived antibody, goat-derived antibody, dog-derived antibody, cat-derived antibody, rabbit-derived antibody, camel-derived antibody, donkey-derived antibody, deer-derived antibody, mink-derived antibody, chicken-derived antibody, duck-derived antibody, goose-derived antibody, turkey-derived antibody, fighting cock-derived antibody, or a mutant thereof, preferably at least one of mouse-derived antibody, human-derived antibody, and primate-derived antibody.

[0062] Furthermore, in order to further improve the bioacceptability of the antibody, the antibody can be humanized; in other words, the antibody is a chimeric antibody or a humanized antibody.

[0063] As described above, one or more amino acid residues in the heavy chain or light chain variable region of the antibody or antigen-binding fragment of the present invention may be substituted with other amino acid residues of the same side chain family, and the retention function of the altered antibody can be tested using the functional measurement method described herein.

[0064] According to an embodiment of the present invention, the antibody or its antigen-binding fragment includes a heavy chain variable region having an amino acid sequence shown in any of SEQ ID NO: 10, 12-13, 15-24, 27, and 31 or an amino acid sequence of a conservatively modified form thereof, and / or a light chain variable region having an amino acid sequence shown in any of SEQ ID NO: 11, 14, 25-26, 28, and 30 or an amino acid sequence of a conservatively modified form thereof.

[0065] Furthermore, provided that the binding activity of the antibody or its antigen-binding fragment CD3 is not substantially affected (at least 95% activity is maintained), those skilled in the art may substitute, add, and / or delete one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) from the heavy chain or light chain variable region amino acid sequence of the antibody or its antigen-binding fragment of the present invention to obtain variants of the sequence of the antibody or its antigen-binding fragment. These are considered to fall within the scope protected by the present invention. For example, amino acids with similar properties are substituted in the heavy chain or light chain variable region. The sequences of the above variants of the present invention may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence.

[0066] Furthermore, the sequence identity described in this invention can be measured using sequence analysis software. For example, the computer program BLAST with default parameters, particularly BLASTP or TBLASTN, can be used.

[0067] 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 any 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 any of SEQ ID NO: 11, 14, 25-26, 28, and 30.

[0068] According to embodiments of the present invention, the antibody or its antigen-binding fragment comprises an amino acid sequence represented by a combination of a heavy chain variable region and a light chain variable region from any of the following groups, or an amino acid sequence having at least 90% sequence identity thereto. [Table 1]

[0069] According to embodiments of the present invention, the antibody or its antigen-binding fragment further comprises a constant region, the constant region comprising at least one of a heavy chain constant region and a light chain constant region.

[0070] According to an embodiment of the present invention, at least one portion of the heavy chain constant region and the light chain constant region is derived from at least one of the following: mouse-derived antibody, human-derived antibody, primate-derived antibody, cattle-derived antibody, horse-derived antibody, dairy cow-derived antibody, pig-derived antibody, sheep-derived antibody, goat-derived antibody, dog-derived antibody, cat-derived antibody, rabbit-derived antibody, camel-derived antibody, donkey-derived antibody, deer-derived antibody, mink-derived antibody, chicken-derived antibody, duck-derived antibody, goose-derived antibody, turkey-derived antibody, fighting cock-derived antibody, or a mutant thereof.

[0071] Furthermore, the immunoglobulins described herein may be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass, and may include engineered subclasses having modified Fc moieties that provide reduced or enhanced effector cell activity. The immunoglobulins may be derived from any species.

[0072] According to embodiments of the present invention, the heavy chain constant region is 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.

[0073] 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.

[0074] In some specific embodiments, 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.

[0075] According to embodiments of the present invention, both the light chain constant region and the heavy chain constant region are derived from a mouse-derived antibody or its mutant, or a human-derived antibody or its mutant.

[0076] Furthermore, all amino acid sequences described in this invention are shown in the N-terminal to C-terminal format.

[0077] According to an embodiment of the present invention, the N end of the heavy chain steady region is connected to the C end of the heavy chain variable region, and / or the N end of the light chain steady region is connected to the N end of the light chain variable region.

[0078] As described above, provided that the binding activity of the antibody or its antigen-binding fragment CD3 is not substantially affected (at least 95% activity is maintained), those skilled in the art can substitute, add and / or delete one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) from the amino acid sequence of the heavy chain or light chain constant region of the antibody or antigen-binding fragment of the present invention to obtain variants of the sequence of the antibody or antigen-binding fragment. These are considered to fall within the scope protected by the present invention. For example, amino acids with similar properties are substituted in the heavy chain or light chain constant region. The sequences of the above variants of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence.

[0079] According to an embodiment of the present invention, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 93 or an amino acid sequence having at least 80% identity thereto, and / or the light chain constant region has the amino acid sequence shown in SEQ ID NO: 94 or an amino acid sequence having at least 80% identity thereto.

[0080] According to an embodiment of the present invention, the heavy chain constant region and the light chain constant region have the amino acid sequences shown in SEQ ID NO: 93 and SEQ ID NO: 94, respectively.

[0081] 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.

[0082] According to embodiments of the present invention, the antibody or its antigen-binding fragment further comprises a first linking peptide, wherein the C-end of the heavy chain variable region is connected to the N-end of the first linking peptide, the C-end of the first linking peptide is connected to the N-end of the light chain variable region, or the C-end of the light chain variable region is connected to the N-end of the first linking peptide, and the C-end of the first linking peptide is connected to the N-end of the heavy chain variable region.

[0083] In some specific embodiments, the first linked peptide has the amino acid sequence shown in (GGGGS)n, where n is an integer of 1 or more, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0084] As described above, the antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies), or may consist only of antigen-binding moieties (e.g., Fab, Fab', Fab'-SH, F(ab')2 or Fv, scFv fragments), or may be modified to affect their function. The present invention includes anti-CD3 antibodies having modified glycosylation patterns. In some embodiments, modifications to remove undesirable glycosylation sites are useful, or the antibodies may lack a fucose moiety on the oligosaccharide chain to enhance antibody-dependent cytotoxicity (ADCC) function. In some other embodiments, galactose modification alters complement-dependent cytotoxicity (CDC).

[0085] 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, and the antigen-binding fragment of the antibody comprises at least one of F(ab')2 fragment, Fab' fragment, Fab fragment, F(ab')2 fragment, Fv fragment, scFv fragment, scFv-Fc fusion protein, scFv-Fv fusion protein, and minimal recognition unit.

[0086] As described above, provided that the binding activity of the antibody or its antigen-binding fragment CD3 is not substantially affected (maintaining at least 95% activity), those skilled in the art can substitute, add, and / or delete one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) from the amino acid sequence of the heavy chain or light chain, scFv fragment, Fab fragment, or scFab fragment of the antibody or its antigen-binding fragment of the present invention to obtain variants of the sequence of the antibody or its antigen-binding fragment. These are considered to fall within the scope protected by the present invention. For example, amino acids having similar properties in the heavy chain or light chain constant are substituted. The sequences of the above variants of the present invention have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence.

[0087] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises a heavy chain having an amino acid sequence shown in any of SEQ ID NO: 32, 34, 35 and 37-46 or an amino acid sequence having at least 80% identity thereto, and / or a light chain having an amino acid sequence shown in any of SEQ ID NO: 33, 36 and 47-48 or an amino acid sequence having at least 80% identity thereto.

[0088] According to embodiments of the present invention, the antibody or its antigen-binding fragment has an amino acid sequence represented by a combination of heavy and light chains from any of the following groups, or an amino acid sequence having at least 80% sequence identity thereto. [Table 2]

[0089] According to embodiments of the present invention, the antibody or its antigen-binding fragment has an amino acid sequence shown in any of SEQ ID NO: 99 to 117 or an amino acid sequence having at least 80% sequence identity thereto.

[0090] According to embodiments of the present invention, the antibody or its antigen-binding fragment has an amino acid sequence shown in any of the following Fab VL-CL and Fab VH-CH1 combinations, or an amino acid sequence having at least 80% sequence identity thereto. [Table 3]

[0091] According to an embodiment of the present invention, the antibody or its antigen-binding fragment has an amino acid sequence shown in any of SEQ ID NO: 142 to 160 or an amino acid sequence having at least 80% sequence identity thereto.

[0092] multispecific antibody

[0093] The present invention provides a polyspecific antibody. The polyspecific antibody comprises a first antigen-binding region containing the above-mentioned anti-CD3 antibody or its antigen-binding fragment, and a second antigen-binding region having binding activity for a biomolecule, wherein the biomolecule is not CD3.

[0094] As described above, a multispecific antibody (e.g., a bispecific antibody) developed based on the anti-CD3 antibody or its antigen-binding fragment described in the first aspect of the present invention can be distributed more abundantly to a part of the tumor after being prepared as a drug, increasing the drug concentration at the tumor site and decreasing the peripheral drug concentration. This reduces the risk of off-target toxicity, while simultaneously reducing the production of pro-inflammatory cytokines and lowering the target toxicity risk of the multispecific antibody (e.g., bispecific antibody). As a result, the multispecific antibody based on the anti-CD3 antibody or its antigen-binding fragment described in the first aspect of the present invention has better safety and high clinical use value and pharmaceutical development value.

[0095] According to the embodiments of the present invention, the polyspecific antibody is selected from any of a bispecific antibody, a triplicate antibody, and a quadruplicate antibody.

[0096] Preferably, it is a bispecific antibody.

[0097] According to embodiments of the present invention, the bispecific antibody includes a symmetric bispecific antibody or an asymmetric bispecific antibody.

[0098] According to embodiments of the present invention, the second antigen-binding region is a binding protein or fragment thereof for the biomolecule.

[0099] According to embodiments of the present invention, the biomolecule is selected from at least one of 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, and EPCAM.

[0100] According to embodiments of the present invention, the binding protein or fragment thereof is selected from at least one of an antibody or its antigen-binding fragment, a receptor, and a ligand.

[0101] According to the embodiments of the present invention, the bispecific antibody is an asymmetric bispecific antibody.

[0102] According to embodiments of the present invention, the first antigen-binding region includes a first scFv fragment, a first Fv fragment, a first Fab fragment, or a first Fab-Linker fragment.

[0103] According to an embodiment of the present invention, the first antigen-binding region further comprises a first Fc peptide segment.

[0104] According to embodiments of the present invention, the N-end of the first Fc peptide segment is connected to the C-end of the scFv fragment or Fv fragment, or the N-end of the first Fc peptide segment is connected to the C-end of CH1 of the Fab fragment or Fab-Linker fragment.

[0105] According to embodiments of the present invention, the first antigen-binding region further comprises a second linking peptide, wherein the N-end of the first Fc peptide segment is connected to the C-end of the second linking peptide, and the N-end of the second linking peptide is connected to the C-end of the scFv fragment or Fv fragment, or the N-end of the first Fc peptide segment is connected to the C-end of the second linking peptide, and the N-end of the second linking peptide is connected to the C-end of CH1 of the Fab fragment or Fab-Linker fragment.

[0106] In some specific embodiments, the second linked peptide has the amino acid sequence shown in (GGGGS)n, where n is an integer of 1 or more, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0107] According to embodiments of the present invention, the first Fc peptide segment is selected from a human Fc peptide segment. Preferably, it is a human IgG1 Fc peptide segment.

[0108] According to an embodiment of the present invention, the first Fc peptide segment has the amino acid sequence shown in SEQ ID NO: 118, or the first Fc peptide segment has the amino acid sequence shown in SEQ ID NO: 166.

[0109] According to an embodiment of the present invention, the first scFv fragment has an amino acid sequence shown in any of SEQ ID NO: 95 to 117.

[0110] According to an embodiment of the present invention, the first Fab fragment has an amino acid sequence represented by a Fab VL-CL and Fab VH-CH1 combination of any of the following groups. [Table 4]

[0111] According to an embodiment of the present invention, the first Fab-Linker has an amino acid sequence represented by SEQ ID NO: 142 to 160.

[0112] According to an embodiment of the present invention, the first antigen-binding region has an amino acid sequence represented by SEQ ID NO: 49-52 or 54-68.

[0113] According to the embodiments of the present invention, the biomolecule is Trop2 or PDL1.

[0114] According to embodiments of the present invention, the second antigen-binding region comprises a first anti-Trop2 antibody or its antigen-binding fragment, or a first anti-PDL1 antibody or its antigen-binding fragment.

[0115] According to embodiments 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.

[0116] According to embodiments of the present invention, the second scFab fragment has the amino acid sequence shown in SEQ ID NO: 161, or the second Fab fragment has the amino acid sequences shown in SEQ ID NO: 162 and 70, or the third scFab fragment has the amino acid sequence shown in SEQ ID NO: 163, or the third Fab fragment has the amino acid sequences shown in SEQ ID NO: 164 and 72.

[0117] According to embodiments of the present invention, the second antigen-binding region further comprises a second Fc fragment, the N end of the second Fc fragment being connected to the C end of CH1 of the second scFab fragment, the second Fab fragment, the third scFab fragment, or the third Fab fragment.

[0118] According to an embodiment of the present invention, the second Fc peptide segment is selected from human Fc peptide segments, and is preferably a human IgG1 Fc peptide segment.

[0119] According to an embodiment of the present invention, the second Fc peptide segment has the amino acid sequence shown in SEQ ID NO: 165.

[0120] According to embodiments of the present invention, the first Fc peptide segment and the second Fc peptide segment are linked by a knob-into-hole structure. According to embodiments of the present invention, the second antigen-binding region has an amino acid sequence shown in SEQ ID NO: 161 or 163, or the second antigen-binding region has an amino acid sequence shown in SEQ ID NO: 162 and 70, or the second antigen-binding region has an amino acid sequence shown in SEQ ID NO: 164 and 72.

[0121] In some selectable embodiments of the present invention, the polyspecific antibody of the present invention has the structure shown in Figure 5.

[0122] In some selectable embodiments of the present invention, as shown in Figure 5A, the polyspecific antibody of the present invention comprises a first peptide chain having an amino acid sequence shown in any of SEQ ID NO: 49-52 or 54-68, a second peptide chain having an amino acid sequence shown in SEQ ID NO: 69, and a third peptide chain having an amino acid sequence shown in SEQ ID NO: 70, or a second peptide chain having an amino acid sequence shown in SEQ ID NO: 71, and a third peptide chain having an amino acid sequence shown in SEQ ID NO: 72.

[0123] In some selectable embodiments of the present invention, as shown in Figure 5B, the polyspecific antibody of the present invention comprises a first peptide chain having an amino acid sequence shown in SEQ ID NO: 49-52 or 54-68, and a second peptide chain having an amino acid sequence shown in SEQ ID NO: 161 or 163.

[0124] In some selectable embodiments of the present invention, as shown in Figure 5C, the polyspecific antibody of the present invention comprises a first peptide chain having an amino acid sequence shown in any of SEQ ID NO: 142 to 160, a second peptide chain having an amino acid sequence shown in SEQ ID NO: 69, and a third peptide chain having an amino acid sequence shown in SEQ ID NO: 70, or comprises a second peptide chain having an amino acid sequence shown in SEQ ID NO: 71 and a third peptide chain having an amino acid sequence shown in SEQ ID NO: 72.

[0125] In some selectable embodiments of the present invention, as shown in Figure 5D, the polyspecific antibody of the present invention comprises a first peptide chain having an amino acid sequence shown in any of SEQ ID NO: 142-160, and a second peptide chain having an amino acid sequence shown in SEQ ID NO: 161 or 163.

[0126] In some selectable embodiments of the present invention, as shown in Figure 5E, the polyspecific antibody of the present invention comprises a first peptide chain having the amino acid sequence shown in SEQ ID NO: 70, a third peptide chain having the amino acid sequence shown in SEQ ID NO: 69, and a fourth peptide chain having the amino acid sequence shown in SEQ ID NO: 70, or comprises a first peptide chain having the amino acid sequence shown in SEQ ID NO: 72, a third peptide chain having the amino acid sequence shown in SEQ ID NO: 71, a fourth peptide chain having the amino acid sequence shown in SEQ ID NO: 72, and a second peptide chain, wherein the second peptide chain comprises Fab VH-CH1 of the amino acid sequence shown in SEQ ID NO: 162 or 164 and scFv of the amino acid sequences shown in SEQ ID NO: 99-117, and the C-end of Fab VH-CH1 is connected to the N-end of scFv.

[0127] conjugate

[0128] The present invention provides a conjugate. The conjugate comprises the above antibody or its antigen-binding fragment or the above polyspecific antibody, The antibody or its antigen-binding fragment, or a conjugation portion connected to a multispecific antibody, is included.

[0129] As described above, the anti-CD3 antibody or its antigen-binding fragment according to the embodiments of the present invention can be used to prepare a multispecific antibody, such as a bispecific antibody drug. The prepared bispecific antibody drug can be distributed more abundantly to the tumor site, increasing the drug concentration at the tumor site and decreasing the peripheral drug concentration, thereby reducing the risk of off-target toxicity. At the same time, the bispecific antibody can reduce the production of pro-inflammatory cytokines and lower the target toxicity risk of the bispecific antibody. Furthermore, drugs prepared using the anti-CD3 antibody or its antigen-binding fragment can be used to prevent and / or treat CD3-related diseases, and CD3 bispecific antibody drugs developed based on the anti-CD3 antibody or its antigen-binding fragment are safer and can be used to prevent and / or treat cancer or tumors or infections or autoimmune diseases. Thus, the conjugates containing the anti-CD3 antibody or its antigen-binding fragment according to the first aspect of the present invention and the multispecific antibody according to the second aspect of the present invention have good clinical use value and pharmaceutical development value.

[0130] According to embodiments of the present invention, the coupling portion is selected from at least one of a carrier, drug, toxin, cytokine, protein tag, modifier, therapeutic agent, and chemotherapeutic agent.

[0131] nucleic acid

[0132] The present invention provides nucleic acids, which encode the above-mentioned antibody or its antigen-binding fragment or the above-mentioned polyspecific antibody.

[0133] According to embodiments of the present invention, the antibody or antigen-binding fragment thereof encoded by the nucleic acid as described in the first aspect of the present invention, or the polyspecific antibody as described in 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 to prevent and / or treat CD3-related diseases, or to prevent and / or treat cancer or tumors or infections or autoimmune diseases.

[0134] Those skilled in the art should understand that the nucleic acid molecules referred to herein actually include either one or both of the complementary double helixes. For convenience, only one helix is ​​given in most cases herein, but in practice, other complementary helixes are also disclosed. Furthermore, the molecular sequences in the present invention include either DNA or RNA forms, and the disclosure of one implies the disclosure of the other.

[0135] Carrier or transformer

[0136] The present invention provides a carrier or transformant. The carrier or transformant comprises the nucleic acid described above. The carrier or transformant may also include a selectable control sequence, the control sequence being operably linked to the nucleic acid molecule. The control sequence is one or more control sequences capable of guiding the nucleic acid molecule to be expressed in a host. The constructed carrier or transformant can efficiently express the antibody or its antigen-binding fragment described in a first aspect of the present invention or the polyspecific antibody described in a second aspect of the present invention, and can also obtain the conjugate described in a third aspect of the present invention.

[0137] By linking the nucleic acid molecule to the carrier or transformant, such as an expression carrier, the nucleic acid molecule and the control elements on the expression carrier can be directly or indirectly connected, and these control elements should be able to control the translation and expression of the nucleic acid molecule. Of course, these control elements may originate directly from the carrier itself or be exogenous, that is, they may not originate from the vector itself. The nucleic acid molecule just needs to be operablely linked to the control elements.

[0138] According to embodiments of the present invention, the carrier may be a clonal carrier or an expression carrier, and can be obtained by manipulating the nucleic acid with a commercially available carrier (e.g., a plasmid or a viral carrier). The carrier in the present invention is not particularly limited, and common plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0139] In this specification, the term "manipulable linkage" refers to the ability of a foreign gene to be linked to a carrier so that regulatory elements within the carrier, such as transcriptional and translational regulatory sequences, can perform the expected transcriptional and translational functions of regulating the foreign gene. Commonly used carriers may include, for example, viral carriers, plasmids, and phages. Expression carriers according to some specific embodiments of the present invention, after being introduced into appropriate receptor cells, can efficiently achieve the expression of the aforementioned nucleic acid molecules through the mediation of a regulatory system, and further enable the acquisition of large quantities of the proteins encoded by the nucleic acid molecules in vitro.

[0140] According to embodiments of the present invention, the carrier is a eukaryotic carrier or a prokaryotic carrier.

[0141] According to embodiments of the present invention, the carrier comprises at least one selected from plasmid carriers, adenovirus carriers, lentivirus carriers, and adeno-associated virus carriers.

[0142] cell

[0143] The present invention provides cells. These cells carry the above-mentioned nucleic acids, the above-mentioned carriers or transformants, or express the above-mentioned antibodies or their antigen-binding fragments or the above-mentioned polyspecific antibodies. Thus, according to the embodiments of the present invention, the cells can efficiently express the antibody or its antigen-binding fragment described in the first aspect of the present invention or the polyspecific antibody described in the second aspect of the present invention under appropriate conditions, and furthermore, an antibody or its antigen-binding fragment or its polyspecific antibody that can bind to human CD3 protein and have improved safety can be obtained.

[0144] According to embodiments of the present invention, the cells are prokaryotic cells, eukaryotic cells, or phages.

[0145] According to embodiments of the present invention, the prokaryotic cell is Escherichia coli, Bacillus subtilis, Bacillus streptomyces, or Bacillus proteobilus.

[0146] According to embodiments of the present invention, the eukaryotic cell is a fungal, insect, plant, or mammalian cell.

[0147] According to embodiments of the present invention, the fungus is Pitia pasteris, Sacromize cerevisia, Schizosaccharomyces pombe, or Trichoderma.

[0148] According to embodiments of the present invention, the insect cells are grassland phagocytic insect cells, the plant cells are tobacco plant cells, and the mammalian cells are BHK cells, CHO cells, COS cells, myeloma cells, or human embryonic kidney 293 cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0149] According to an embodiment of the present invention, the cell is a mammalian cell.

[0150] According to embodiments of the present invention, the cells are BHK cells, CHO cells, COS cells, or NSO cells.

[0151] In the specification of the present invention, "suitable conditions" refers to conditions suitable for the expression of the antibody or its antigen-binding fragment or the polyspecific antibody described in the present invention. As will be readily apparent to those skilled in the art, the conditions suitable for the expression of the antibody or its antigen-binding fragment or the polyspecific antibody include, but are not limited to, a suitable transformation or transfection scheme, suitable transformation or transfection conditions, a healthy host cell state, a suitable host cell density, a suitable cell culture environment, and a suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the expression conditions of the recombinant antibody to the optimal level depending on the specific laboratory environment.

[0152] Drug composition

[0153] The present invention provides a drug composition comprising the above-mentioned antibody or antigen-binding fragment, the above-mentioned polyspecific antibody, the above-mentioned conjugate, the above-mentioned nucleic acid, or the above-mentioned carrier or transformant. The drug obtained thereby can be used to prevent and / or treat CD3-related diseases, or further to prevent and / or treat CD3-related diseases and / or cancer or tumors or infections or autoimmune diseases.

[0154] According to embodiments of the present invention, the invention further includes pharmaceutically acceptable adjuvants.

[0155] According to embodiments of the present invention, the auxiliary agent comprises one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers.

[0156] According to the embodiments of the present invention, the drug composition is an injectable preparation.

[0157] Furthermore, the drug composition includes combinations that are separated in time and / or space, as long as they can work together to achieve the objectives of the present invention. For example, the components of the composition may be administered to a subject as a whole or individually. When the components of the composition are administered to a subject individually, each component may be administered to the subject simultaneously or sequentially.

[0158] The drug of the present invention comprises a safe and effective amount of the active ingredient of the present invention and pharmaceutically acceptable adjuvants. Such adjuvants include (but are not limited to) saline, buffer solutions, glucose, water, glycerin, ethanol, and combinations thereof. Typically, the drug formulation should be tailored to the method of administration, and the dosage forms of the drug of the present invention are injections, oral formulations (tablets, capsules, oral solutions), transdermal preparations, and sustained-release preparations. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The drug is preferably manufactured under sterile conditions.

[0159] The effective amount of the active ingredient described in this invention varies depending on the mode of administration and the severity of the disease being treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., clinical trials). These factors include, but are not limited to, pharmacokinetic parameters such as the bioavailability, metabolism, and half-life of the active ingredient, the severity of the disease being treated in the patient, the patient's weight, the patient's immune status, and the route of administration. For example, depending on the urgent needs of the treatment situation, the drug may be administered in divided doses several times a day or the dosage may be reduced proportionally.

[0160] The pharmaceutically acceptable adjuvants described in the present invention include (but are not limited to) water, saline solution, liposomes, lipids, proteins, protein-antibody conjugates, peptide substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be in accordance with the administration method, all of which are well known to those skilled in the art.

[0161] kit

[0162] The present invention provides a kit. The kit comprises the above-mentioned antibody or its antigen-binding fragment or the above-mentioned polyspecific antibody. As described above, the antibody or its antigen-binding fragment according to the examples of the present invention can effectively and specifically bind to CD3, and a CD3-related kit developed utilizing this property can be used for CD3-related research.

[0163] The aforementioned kit can effectively detect, concentrate, or isolate and purify CD3 in biological samples, and can be used in scientific research, for example, to qualitatively or quantitatively detect CD3 protein molecules in biological samples. More specifically, it can be used in kits related to detection that utilizes the specific binding performance of CD3 to antibodies, such as immunoblotting and immunoprecipitation. These kits may contain one or more of the following: an antagonist, a CD3 antibody or drug reference material protein purification column, an immunoglobulin affinity purification buffer, and a cell measurement diluent. CD3 antibodies can be used in different types of diagnostic tests, for example, to detect the presence of various diseases or drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for CD3-related diseases by detecting the serum or blood of a subject.

[0164] Use in kit preparation The present invention provides for use in the preparation of a kit for detecting the CD3 of the above-mentioned antibody or its antigen-binding fragment or the above-mentioned polyspecific antibody.

[0165] As described above, the antibodies or antigen-binding fragments according to the embodiments of the present invention can specifically bind to CD3, and therefore, the antibodies or antigen-binding fragments can be used to detect CD3. Furthermore, they can be used to prepare CD3-related kits and can be used in scientific research, such as qualitatively or quantitatively detecting CD3 protein molecules in biological samples. More specifically, they can be used in kits related to detection that utilize the specific binding performance of CD3 and antibodies, such as immunoblotting and immunoprecipitation. These kits may contain one or more of the following: an antagonist, a CD3 antibody or drug reference material protein purification column, an immunoglobulin affinity purification buffer, and a cell measurement diluent. CD3 antibodies can be used in different types of diagnostic tests, for example, to detect the presence of various diseases or drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for CD3-related diseases by detecting the serum or blood of a subject.

[0166] Use in drug preparation

[0167] The present invention provides the use of the above-mentioned antibodies or antigen-binding fragments thereof, the above-mentioned polyspecific antibodies, the above-mentioned conjugates, the above-mentioned nucleic acids, the above-mentioned carriers or transformants, or the above-mentioned drug compositions in the preparation of drugs, the drugs being used to modulate immune responses and / or suppress tumor growth, or the drugs being used to prevent and / or treat CD3-related diseases and / or cancer or tumors or infections or autoimmune diseases.

[0168] According to embodiments of the present invention, the cancer or tumor or infection or autoimmune disease is a Trop2 or PDL1-related disease.

[0169] According to embodiments of the present invention, the cancer or tumor is at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, rectal cancer, breast cancer, pancreatic cancer, prostate cancer, neuroglioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, or head and neck cancer.

[0170] According to embodiments of the present invention, the cancer or tumor includes 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.

[0171] Treatment methods for diseases The present invention provides a method for regulating the immune response and / or suppressing tumor growth, or for preventing and / or treating CD3-related diseases and / or cancer, tumors, infections, or autoimmune diseases.

[0172] According to embodiments of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the above antibody or its antigen-binding fragment, the above polyspecific antibody, the above conjugate, the above nucleic acid, the above carrier or transformant, or the above drug composition.

[0173] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to the mammal being evaluated for treatment and / or the mammal being treated. In one implementation, the mammal is 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. A subject may be human, but may also include other mammals, particularly mammals that can be used as laboratory models for human diseases, such as mice and rats.

[0174] The effective amount of the antibody or its antigen-binding fragment, multispecific antibody conjugate, nucleic acid, carrier or transformant or drug composition described in the present invention varies depending on the mode of administration and the severity of the disease being treated. The selection of a preferred effective amount can be determined by those skilled in the art based on various factors (e.g., clinical trials). These factors include, but are not limited to, pharmacokinetic parameters such as the bioavailability, metabolism, and half-life of the active ingredient, the severity of the disease being treated in the patient, the patient's body weight, the patient's immune status, and the route of administration. For example, depending on the urgent demands of the treatment situation, the drug may be administered in divided doses several times a day or the dosage may be reduced proportionally.

[0175] According to embodiments of the present invention, the cancer or tumor or infection or autoimmune disease is a Trop2 or PDL1-related disease.

[0176] According to embodiments of the present invention, the cancer or tumor is at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, rectal cancer, breast cancer, pancreatic cancer, prostate cancer, neuroglioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, or head and neck cancer.

[0177] According to embodiments of the present invention, the cancer or tumor is at least one of non-small cell lung cancer, malignant melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, colorectal cancer, liver cancer, or classical Hodgkin lymphoma.

[0178] Table 1 shows the sequence description of the present invention.

[0179] Table 5 JPEG2026522470000006.jpg243170JPEG2026522470000007.jpg243169JPEG2026522470000008.jpg173169JPEG2026522470000009.jpg242170JPEG2026522470000010.jpg242169JPEG2026522470000011.jpg197170JPEG2026522470000012.jpg197169JPEG2026522470000013.jpg198170JPEG2026522470000014.jpg198170JPEG2026522470000015.jpg192169JPEG2026522470000016.jpg217169JPEG2026522470000017.jpg216170JPEG2026522470000018.jpg217170JPEG2026522470000019.jpg217170JPEG2026522470000020.jpg219170JPEG2026522470000021.jpg219169JPEG2026522470000022.jpg218170JPEG2026522470000023.jpg217169JPEG2026522470000024.jpg219169JPEG2026522470000025.jpg219170JPEG2026522470000026.jpg242169JPEG2026522470000027.jpg230169JPEG2026522470000028.jpg198170JPEG2026522470000029.jpg197170JPEG2026522470000030.jpg214170JPEG2026522470000031.jpg228169JPEG2026522470000032.jpg227169JPEG2026522470000033.jpg227170JPEG2026522470000034.jpg227169JPEG2026522470000035.jpg247170JPEG2026522470000036.jpg253170JPEG2026522470000037.jpg255170JPEG2026522470000038.jpg255168JPEG2026522470000039.jpg255168JPEG2026522470000040.jpg208170JPEG2026522470000041.jp g207170JPEG2026522470000042.jpg206170JPEG2026522470000043.jpg208170JPEG2026522470000044.jpg20 7169JPEG2026522470000045.jpg207169JPEG2026522470000046.jpg207170JPEG2026522470000047.jpg2061 70JPEG2026522470000048.jpg206170JPEG2026522470000049.jpg239170JPEG2026522470000050.jpg255170.

[0180] The present invention will be described in detail below with reference to examples. In examples or test examples, if specific conditions are not specified for the experimental method, the method shall be carried out under normal conditions.

[0181] The means of this disclosure will be interpreted in combination with the following examples. Those skilled in the art will understand that the following examples are used solely to illustrate this disclosure and should not be considered to limit the scope of this disclosure. Where no specific technique or conditions are shown in the examples, they should be carried out in accordance with the techniques or conditions described in the art literature or in accordance with the product specifications. Where the manufacturer of the reagents or equipment used is not indicated, they are conventional products available on the market.

[0182] Example 1: Acquisition and production of low-affinity antibodies

[0183] In the affinity maturation process of natural antibodies, somatic high-frequency mutations are mainly concentrated in the CDR region. Generally, by in vitro experiments inducing single-point saturation mutations at various sites in the CDR region, sufficient mutational diversity can be obtained while simultaneously not disrupting the protein structure. This pathway can achieve in vitro reproduction that most closely resembles somatic high-frequency mutations in natural antibodies in vivo. The above method is generally used for antibody affinity maturation (improvement), and the present invention refers to this technical approach and, conversely, reduces the affinity of a CD3 antibody.

[0184] Single-point saturation mutations were introduced at each amino acid site in the CDR region to construct an unbiased single-point saturation mutation plasmid library of the parent antibody. Mutation hotspots with weakened specific binding to the antigen were screened, and these hotspots were further screened in combination to obtain candidate antibody mutation sequences.

[0185] Following this technical approach, a large number of screening tests were conducted to reduce 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), resulting in 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 CDR region amino acid sequences and heavy and light chain amino acid sequences of the low-affinity CD3 monoclonal antibody described above are shown in Table 1, and the CDR region amino acid mutation status compared to the parent antibody Cross3 is shown in Figure 1.

[0186] The specific experimental procedure for producing CD3 monoclonal antibodies is as follows: (1) Culturing ExpiCHO cells (purchased from Thermo Fisher) in ExpiCHO Expression Medium medium (purchased from Thermo Fisher) and increasing the cell concentration to 6 × 10⁶6The ExpiCHO cell solution was obtained by adjusting the volume to / mL. (2) pcDNA3.4 carrier containing CD3 antibody heavy chain and light chain (Cross3 heavy chain amino acid sequence is shown in SEQ ID NO:35, light chain amino acid sequence is shown in SEQ ID NO:33, Cross303 heavy chain amino acid sequence is shown in SEQ ID NO:37, light chain amino acid sequence is shown in SEQ ID NO:33, Cross307 heavy chain amino acid sequence is shown in SEQ ID NO:38, light chain amino acid sequence is shown in SEQ ID NO:33, Cross308 heavy chain amino acid sequence is shown in SEQ ID NO:39, light chain amino acid sequence is shown in SEQ ID NO:33, Cross309 heavy chain amino acid sequence is shown in SEQ ID NO:40, light chain amino acid sequence is shown in SEQ ID NO:33, Cross310 heavy chain amino acid sequence is shown in SEQ ID NO:41, light chain amino acid sequence is shown in SEQ ID NO: The heavy chain amino acid sequence of Cross311 is shown in SEQ ID NO: 42, and the light chain amino acid sequence is shown in SEQ ID NO: 33. The heavy chain amino acid sequence of Cross312 is shown in SEQ ID NO: 43, 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: The heavy chain amino acid sequence of Cross318 is shown in SEQ ID NO: 46, 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, 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: 45, the light chain amino acid sequence is shown in SEQ ID NO: 36, and the heavy chain amino acid sequence of Cross326 is shown in SEQ ID(1) Solution A was obtained by adding 1:1 of the compound (shown as NO:46, with the light chain amino acid sequence shown as SEQ ID NO:48) (commissioned to Nanjing Kingsley Synthesis) to 2 mL of OptiSFM medium (purchased from Thermo Fisher). (3) Solution b was obtained by adding 160 μL of ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) to 2 mL of OptiSFM medium (purchased from Thermo Fisher). (4) Next, solution a and solution b were mixed to obtain the transfection mixture, and within 5 minutes the entire transfection mixture was added to 50 mL of ExpiCHO cell solution. (5) After culturing at 37°C under 5% CO2 conditions for 1 day, 8 mL of Feed and 300 μL of Enhancer (purchased from Thermo Fisher) were added, and the culture was changed to 32°C under 5% CO2 conditions for 9 days to obtain the culture supernatant, at which point 8 mL of Feed was added on day 5. (6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from Nabi).

[0187] The specific experimental procedure for producing bispecific antibodies is as follows: (1) Culturing ExpiCHO cells (purchased from Thermo Fisher) in ExpiCHO Expression Medium medium (purchased from Thermo Fisher) and increasing the cell concentration to 6 × 10⁶ 6The ExpiCHO cell solution was obtained by adjusting the volume to / mL. (2) pcDNA3.4 carrier containing CD3 antibody, Trop2 antibody heavy chain and Trop2 antibody light chain (Cross3×Trop2 contains the CD3 antibody shown in SEQ ID NO: 52, 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; Cross303×Trop2 contains the CD3 antibody shown in SEQ ID NO: 53, 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; Cross307×Trop2 contains the CD3 antibody shown in SEQ ID NO: 54, 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; Cross308×Trop2 contains the CD3 antibody shown in SEQ ID NO: 55, SEQ ID Cross309×Trop2 includes the heavy chain amino acid sequence of the Trop2 antibody shown at NO:69 and the light chain amino acid sequence shown at SEQ ID NO:70, Cross310×Trop2 includes the CD3 antibody shown at SEQ ID NO:57, the heavy chain amino acid sequence of the Trop2 antibody shown at SEQ ID NO:69 and the light chain amino acid sequence shown at SEQ ID NO:70, Cross311×Trop2 includes the CD3 antibody shown at SEQ ID NO:58, the heavy chain amino acid sequence of the Trop2 antibody shown at SEQ ID NO:69 and the light chain amino acid sequence shown at SEQ ID NO:70, Cross312×Trop2 includes the CD3 antibody shown at SEQ ID NO:59 and the light chain amino acid sequence shown at SEQ ID NO:70 The sequence includes the heavy chain amino acid sequence of the Trop2 antibody shown in NO:69 and the light chain amino acid sequence shown in SEQ ID NO:70, and Cross313×Trop2 includes the CD3 antibody shown in SEQ ID NO:49, the heavy chain amino acid sequence of the Trop2 antibody shown in SEQ ID NO:69, and SEQ ID NO:Cross314×Trop2 includes the light chain amino acid sequence shown in SEQ ID NO: 70, the CD3 antibody shown in SEQ ID NO: 60, 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, Cross316×Trop2 includes the CD3 antibody shown in SEQ ID NO: 50, 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 includes the CD3 antibody shown in SEQ ID NO: 61, 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 includes the CD3 antibody shown in SEQ ID NO: 62, the Trop2 antibody heavy chain amino acid sequence shown in SEQ ID NO: 69, and SEQ ID NO: (Containing the light chain amino acid sequence shown in 70, Cross323×Trop2 contains the CD3 antibody shown in SEQ ID NO:63, 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, 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, Cross326×Trop2 contains the CD3 antibody shown in SEQ ID NO:64, 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) (Nanjing Kingsley Synthesis outsourced), or a pcDNA3.4 carrier containing CD3 antibody, PDL1 antibody heavy chain and PDL1 antibody light chain (Cross3×PDL1 is SEQ ID The CD3 antibody shown in NO:52, 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 are included, and Cross303×PDL1 includes the CD3 antibody shown in SEQ ID NO:53, the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO:71, and SEQ IDCross307×PDL1 includes the light chain amino acid sequence shown in NO:72, the CD3 antibody shown in SEQ ID NO:54, 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; Cross308×PDL1 includes the CD3 antibody shown in SEQ ID NO:55, 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; Cross309×PDL1 includes the CD3 antibody shown in SEQ ID NO:56, 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 includes the CD3 antibody shown in SEQ ID NO:57, the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO:71, and SEQ ID Cross311×PDL1 includes the light chain amino acid sequence shown in NO:72, the CD3 antibody shown in SEQ ID NO:58, 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 includes the CD3 antibody heavy chain amino acid sequence shown in SEQ ID NO:59, 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 includes the CD3 antibody shown in SEQ ID NO:49, 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, Cross314×PDL1 includes the CD3 antibody shown in SEQ ID NO:60, the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO:71, and SEQ ID The light chain amino acid sequence shown in NO:72 is included, Cross316×PDL1 is the CD3 antibody shown in SEQ ID NO:50, 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 is included, Cross317×PDL1 is the CD3 antibody shown in SEQ ID NO:61, SEQ IDCross318×PDL1 includes the PDL1 antibody heavy chain amino acid sequence shown in NO:71 and the light chain amino acid sequence shown in SEQ ID NO:72, Cross323×PDL1 includes the CD3 antibody shown in SEQ ID NO:62, 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, Cross325×PDL1 includes the CD3 antibody shown in SEQ ID NO:51, 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, Cross326×PDL1 includes the CD3 antibody shown in SEQ ID NO:64 and SEQ ID Cross3ZH07×PDL1 includes the PDL1 antibody heavy chain amino acid sequence shown in NO:71 and the light chain amino acid sequence shown in SEQ ID NO:72, Cross3ZH09×PDL1 includes the CD3 antibody shown in SEQ ID NO:66, the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO:71 and the light chain amino acid sequence shown in SEQ ID NO:72, Cross3ZH09×PDL1 includes the CD3 antibody shown in SEQ ID NO:66, the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO:71 and the light chain amino acid sequence shown in SEQ ID NO:72, Cross3ZH16×PDL1 includes the CD3 antibody shown in SEQ ID NO:67, the PDL1 antibody heavy chain amino acid sequence shown in SEQ ID NO:71 and the light chain amino acid sequence shown in SEQ ID NO:72, Cross3ZH23×PDL1 includes the CD3 antibody shown in SEQ ID NO:68 and SEQ ID Solution A was obtained by adding the heavy chain amino acid sequence of the PDL1 antibody shown in NO:71 and the light chain amino acid sequence shown in SEQ ID NO:72 (commissioned to Nanjing Kingsley Synthesizer) in a 1:1:1 ratio to 2 mL of OptiSFM medium (purchased from Thermo Fisher). (3) 160 μL of ExpiFectamineCHO transfection reagent (Thermo(Purchased from Fisher) was added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) to obtain solution b. (4) Next, solution a and solution b were mixed to obtain the transfection mixture, and within 5 minutes the entire transfection mixture was added to 50 mL of ExpiCHO cell solution. (5) After culturing at 37°C under 5% CO2 conditions for 1 day, 8 mL of Feed and 300 μL of Enhancer (purchased from Thermo Fisher) were added, and the conditions were changed to 32°C under 5% CO2 conditions for 9 days to obtain the culture supernatant, where 8 mL of Feed was added on the 5th day. (6) Affinity purification was performed from the culture supernatant using a Protein A purification column (purchased from Nobi) to obtain the target CD3×Trop2 or CD3×PDL1 antibody, and the antibody structure is shown in Figure 5.

[0188] Example 2: Detection of antibody affinity

[0189] Biacore is a method for analyzing the interactions of biomolecules based on the optical surface plasmon resonance (SPR) principle. It not only detects specific binding between antigens and antibodies, but also provides data crucial for drug development, such as intermolecular binding rate constants (ka), dissociation rate constants (kd), and equilibrium dissociation constants (KD), enabling the calculation of antibody affinity.

[0190] In the Biacore 8K (Cytiva) system, the antibody was diluted to 10 μg / mL with running buffer (HBS-EP) and coupled to the CM5 chip (Cytiva) under a flow rate of 10 μL / min. Under a flow rate of 30 μL / min, the kinetics and affinity data of the binding of the CD3E&D antigen to the low-affinity CD3 monoclonal antibody of Example 1 of the present invention were detected. The set binding time was 120 s and the dissociation time was 800 s. The results of the antibody affinity analysis are shown in Figure 2.

[0191] Example 3: CD3 monoclonal antibody ELISA conjugation experiment

[0192] This example uses ELISA to detect the binding characteristics of the parent antibody Cross3 and the CD3 affinity-decreasing antibody (see Example 1 for production method and heavy and light chain amino acid sequences). The inventors coated a 96-well plate with CD3E&D protein and added the antibody. The signal strength was then used to determine the binding characteristics of the antibody and CD3E&D.

[0193] CD3E&D protein (purchased from Acro) was diluted to 2 μg / ml in PBS buffer and added to a 96-well plate at a volume of 100 μL / well, and left overnight at 4°C. The PBS buffer in the 96-well plate was aspirated, the plate was washed six times with PBST (pH 7.2, PBS-containing 0.1% Tween 20) buffer, and then 200 μL / well of PBS / 10% BSA was added and incubated at 37°C for 2 hours to block. After removing the block solution and washing the plate six times with PBST, the test Cross3 antibody, Cross303, Cross307, Cross308, Cross309, Cross310, Cross311, Cross312, Cross313, Cross314, Cross316, Cross317, Cross318, Cross323, Cross325, Cross326, and control IgG1 (purchased from Hyakuei Seibutsu) were added diluted in a PBST / 0.05% BSA gradient at 100 μL / well, and incubated at 37°C for 1 hour. After removing the reaction system and washing the plate six times with PBST, the HRP (home radish peroxidase) labeled anti-human secondary antibody (Fab specificity) (purchased from Sigma) was diluted in a PBST / 0.05% BSA gradient at 100 μL / well, and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added, incubated at room temperature for 3 minutes, and the reaction was terminated by adding 80 μL / well of 4M sulfuric acid. Absorbance values ​​were read using a microplate reader at 450 mm.

[0194] As shown in Figure 3, all CD3 antibodies of the present invention were able to bind to CD3E&D, and the ELISA results were limited in resolution and could not show differences in affinity between each CD3 monoclonal antibody.

[0195] Example 4: Flow cerometry conjugation experiment of CD3 monoclonal antibody PBMC in PBS 2 x 10 6 Diluted to / mL, added to a 1.5ml EP tube at a volume of 100μL / tube, then 10μL / tube of goat serum was added, and the mixture was blocked at 4°C for 30min. Gradient-diluted test antibodies Cross3, Cross303, Cross307, Cross308, Cross309, Cross310, Cross311, Cross312, Cross313, Cross314, Cross316, Cross317, Cross318, Cross323, Cross325, Cross326 (see Example 1 for the production method of antibodies Cross3 and CD3 affinity-decreasing antibodies and the amino acid sequences of the heavy and light chains), and control IgG1 (purchased from Hyakuei Seibutsu) were added, and the mixture was incubated at 4°C for 30min. Add 1 mL of PBS to an EP tube, centrifuge at 3500 rpm for 5 minutes at 4°C, discard the supernatant, and wash again with PBS. After centrifugation, discard the supernatant, resuspend the cells in 100 μL / tube of PBS, add 1 μL / tube of Alexa-647-labeled goat anti-human secondary antibody (purchased from Jackson Lab) and 0.5 μL / tube of PerCP-Cy5.5-labeled anti-human CD8 antibody, and incubate at 4°C for 30 minutes in the dark. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μL / tube of PBS and detect by flow cetometry.

[0196] As shown in Figure 4, the results indicate that the CD3 antibodies Cross309, Cross310, Cross311, Cross312, Cross313, Cross314, Cross316, Cross317, Cross318, Cross323, and Cross325 of the present invention were able to bind to CD8 T cells, and that their CD3 affinity was reduced, indicating that their CD3 antibody binding ability was weaker compared to the parent antibody Cross3.

[0197] Example 5: ELISA conjugation experiment of CD3×Trop2 and CD3×PDL1 bispecific antibodies

[0198] This example uses ELISA to detect the binding properties of CD3×Trop2 and CD3×PDL1 bispecific antibodies (see Example 1 for production methods and heavy and light chain amino acid sequences). The inventors coated CD3E&D, Trop2, or PDL1 proteins in 96-well plates, added the antibodies, and then used the signal strength to determine the binding properties of the antibodies to the corresponding proteins.

[0199] (1) Dilute CD3E&D protein (purchased from Acro) to 2 μg / ml with PBS buffer and add it to a 96-well plate at a volume of 100 μL / well, and leave overnight at 4°C. Aspirate the PBS buffer from the 96-well plate, wash the plate six times with PBST (pH 7.2, PBS containing 0.1% Tween 20) buffer, add 200 μL / well of PBS / 10% BSA, and incubate at 37°C for 2 hours to block. Remove the block solution, wash the plate six times with PBST, and add 100 μL / well of PBST / 0.05% Bispecific antibodies of the test sample diluted with a BSA gradient: Cross3×Trop2, Cross303×Trop2, Cross307×Trop2, Cross308×Trop2, Cross309×Trop2, Cross310×Trop2, Cross311×Trop2, Cross312×Trop2, Cross313×Trop2, Cross314×Trop2, Cross316×Trop2, Cross317×Trop2, Cross318×Trop2, Cross323×Trop2, Cross325×Trop2, Cross326×Trop2, Cro ss3×PDL1, Cross303×PDL1, Cross307×PDL1, Cross308×PDL1, Cross309×PDL1, Cross310×PDL1, Cross311×PDL1, Cross312×PDL1, Cross313×PDL1, Cross314×PDL1, Cross316×PDL1, Cross317×PDL1, Cross318×PDL1, Cross323×PDL1, Cross325×PDL1, Cross326×PDL1, and control IgG1LALA (purchased from Hyakuei Seibutsu) were added and incubated at 37°C for 1 hour. After removing the reaction system and washing the plate six times with PBST, the HRP (Home Radish Peroxidase) labeled anti-human secondary antibody (Fab specificity) (purchased from Sigma) was diluted in 100 μL / well PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes, and the reaction was terminated by adding 80 μL / well 4M sulfuric acid.The absorbance values ​​were read using a microplate reader at 450 mm.

[0200] As shown in Figure 6, the CD3×Trop2 antibody of the present invention can bind to CD3E&D, and the binding of the low-affinity CD3 bispecific antibody to the CD3E&D protein was weaker compared to the parental Cross3×Trop2 bispecific antibody, as expected.

[0201] As shown in Figure 14, the CD3×PDL1 antibody of the present invention can bind to CD3E&D, and the binding of the low-affinity CD3 bispecific antibody to the CD3E&D protein was weaker compared to the parental Cross3×PDL1 bispecific antibody, as expected.

[0202] (2) The Trop2 protein (purchased from Acro) was diluted to 2 μg / ml in PBS buffer and added to a 96-well plate at a volume of 100 μL / well, and left overnight at 4°C. The PBS buffer in the 96-well plate was aspirated, the plate was washed six times with PBST (pH 7.2, PBS containing 0.1% Tween 20) buffer, and then 200 μL / well of PBS / 10% BSA was added and incubated at 37°C for 2 hours to block. After removing the blocking solution and washing the plate six times with PBST, the following two-specific test antibodies, Cross3×Trop2, Cross303×Trop2, Cross307×Trop2, Cross308×Trop2, Cross309×Trop2, Cross310×Trop2, Cross311×Trop2, Cross312×Trop2, Cross313×Trop2, Cross314×Trop2, Cross316×Trop2, Cross317×Trop2, Cross318×Trop2, Cross323×Trop2, Cross325×Trop2, Cross326×Trop2, and control IgG1LALA (purchased from Hyakuei Seibutsu) were added and incubated at 37°C for 1 hour. After removing the reaction system and washing the plate six times with PBST, the HRP (home radish peroxidase)-labeled anti-human secondary antibody (Fab specific) (purchased from Sigma) was diluted in 100 μL / well of PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes, and the reaction was terminated by adding 80 μL / well of 4M sulfuric acid. The absorbance was read using a microplate reader at 450 mm.

[0203] As shown in Figure 7, the CD3×Trop2 antibody of the present invention was found to be able to bind to the Trop2 protein, and the binding of all bispecific antibodies to the Trop2 protein was similar.

[0204] (3) Dilute the PDL1 protein (purchased from Acro) to 2 μg / ml with PBS buffer, add it to a 96-well plate at a volume of 100 μL / well, and leave it overnight at 4°C. Aspirate the PBS buffer from the 96-well plate, wash the plate six times with PBST (pH 7.2, PBS containing 0.1% Tween 20) buffer, add 200 μL / well of PBS / 10% BSA, and incubate at 37°C for 2 hours to block. After removing the block solution and washing the plate six times with PBST, the following two-specific test antibodies, Cross3×PDL1, Cross303×PDL1, Cross307×PDL1, Cross308×PDL1, Cross309×PDL1, Cross310×PDL1, Cross311×PDL1, Cross312×PDL1, Cross313×PDL1, Cross314×PDL1, Cross316×PDL1, Cross317×PDL1, Cross318×PDL1, Cross323×PDL1, Cross325×PDL1, Cross326×PDL1, and control IgG1LALA (purchased from Hyakuei Seibutsu) were added and incubated at 37°C for 1 hour. After removing the reaction system and washing the plate six times with PBST, the HRP (home radish peroxidase)-labeled anti-human secondary antibody (Fab specific) (purchased from Sigma) was diluted in 100 μL / well of PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes, and the reaction was terminated by adding 80 μL / well of 4M sulfuric acid. The absorbance was read using a microplate reader at 450 mm.

[0205] As shown in Figure 15, it was found that the CD3×PDL1 antibody of the present invention can bind to the PDL1 protein.

[0206] Example 6: CD3 × Trop2 antibody ELISA bridging experiment

[0207] ELISA experiments were used to detect the bridging properties of a CD3 × Trop2 bispecific antibody (see Example 1 for production method and heavy and light chain amino acid sequences). CD3E&D antigen proteins were coated into a 96-well plate, and after adding the antibody, the signal strength was determined by detecting biotin-labeled Trop2 protein, indicating the binding properties of the bispecific antibody in bridging CD3E&D and Trop2 proteins.

[0208] CD3E&D protein (purchased from Acro) was diluted to 2 μg / ml in PBS buffer and added to a 96-well plate at a volume of 100 μL / well, and left overnight at 4°C. The PBS buffer in the 96-well plate was aspirated, the plate was washed six times with PBST (pH 7.2, PBS-containing 0.1% Tween 20) buffer, and then 200 μL / well of PBS / 10% BSA was added and incubated at 37°C for 2 hours to block. After removing the blocking solution and washing the plate six times with PBST, the following two-specific test antibodies, Cross3×Trop2, Cross303×Trop2, Cross307×Trop2, Cross308×Trop2, Cross309×Trop2, Cross310×Trop2, Cross311×Trop2, Cross312×Trop2, Cross313×Trop2, Cross314×Trop2, Cross316×Trop2, Cross317×Trop2, Cross318×Trop2, Cross323×Trop2, Cross325×Trop2, Cross326×Trop2, and control hIgG1LALA (purchased from Hyakuei Seibutsu) were added and incubated at 37°C for 1 hour. After removing the reaction system and washing the plate six times with PBST, Trop2-Biotin protein diluted to the appropriate concentration was added and incubated at 37°C for 1 hour. After removing the reaction system and washing the plate six times with PBST, Streptavidin secondary antibody labeled with HRP (home radish peroxidase) (purchased from Southern Biotech) was diluted in 100 μL / well PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes, and the reaction was terminated by adding 80 μL / well 4M sulfuric acid. Absorbance values ​​were read using a microplate reader at 450 mm.

[0209] As shown in Figure 8, the results indicate that the bispecific antibody of the present invention can bridge CD3E&D and the Trop2 protein, and that the bridging ability of the parental Cross3×Trop2 antibody is stronger than that of the low-affinity CD3 bispecific antibody, as expected.

[0210] Example 7: Flow cell methylation experiment of bispecific antibody binding

[0211] Flow celometry experiments are used to detect the binding characteristics of bispecific antibodies and the signal strength after adding the bispecific antibody (see Example 1 for production method and heavy and light chain amino acid sequences) to cells.

[0212] (1) PBMC in PBS 2 × 10 6Diluted to / ml, added 100μl / tube to a 1.5ml EP tube, then 10μl / tube of goat serum was added, and blocked at 4°C for 30min. Gradient-diluted bispecific antibodies Cross3×Trop2, Cross303×Trop2, Cross307×Trop2, Cross308×Trop2, Cross309×Trop2, Cross310×Trop2, Cross311×Trop2, Cross312×Trop2, Cross313×Trop2, Cross314×Trop2, Cross316×Trop 2. Add Cross317×Trop2, Cross318×Trop2, Cross323×Trop2, Cross325×Trop2, Cross326×Trop2, Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, Cross325×PDL1, and control hIgG1LALA (purchased from Hyakuei Seibutsu), and incubate at 4°C for 30 minutes. Add 1 ml of PBS to the EP tube, centrifuge at 3500 rpm for 5 minutes at 4°C, discard the supernatant, and wash once again with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100 μl / tube of PBS. 1 μl / tube of Alexa-647-labeled goat anti-human IgG secondary antibody (purchased from Jackson Lab) and 0.5 μl / tube of PerCP-Cy5.5-labeled anti-human CD8 antibody were added, and the cells were incubated at 4°C for 30 minutes in the dark. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μl / tube of PBS and detected by flow cetometry.

[0213] As shown in Figure 9, the results demonstrate that the CD3×Trop2 antibody of the present invention can bind to CD8 T cells, and that its ability to bind to T cells with reduced CD3 affinity is weaker compared to the parental CD3 antibody Cross3×Trop2, as expected.

[0214] As shown in Figure 16, the results demonstrate that the Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 antibodies of the present invention can bind to CD8 T cells, and that the binding ability of Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 to T cells is weaker than that of Cross3×PDL1, as expected.

[0215] (2) In PBS, tumor cells 2 × 10 6Diluted to / ml, added to a 1.5ml EP tube at a volume of 100μl / tube, then 10μl / tube of goat serum was added, and blocked at 4°C for 30min. Gradient-diluted bispecific antibodies Cross3×Trop2, Cross303×Trop2, Cross307×Trop2, Cross308×Trop2, Cross309×Trop2, Cross310×Trop2, Cross311×Trop2, Cross312×Trop2, Cross313×Trop2, Cross314×Trop2, Cross316×Trop2, Cross317×Trop2, Cross318×Trop2, Cross323×Trop2, Cross325×Trop2, Cross326×Trop2, Cross3× PDL1, Cross303×PDL1, Cross307×PDL1, Cross308×PDL1, Cross309×PDL1, Cross310×PDL1, Cross311×PDL1, Cross312×PDL1, Cross313×PDL1, Cross314×PDL1, Cross316×PDL1, Cross317×PDL1, Cross318×PDL1, Cross323×PDL1, Cross325×PDL1, Cross326×PDL1, and control hIgG1LALA (purchased from Hyakuei Seibutsu) were added and incubated at 4°C for 30 minutes. 1 ml of PBS was added to the EP tube, centrifuged at 3500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the tube was washed once again with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100 μl / tube of PBS. 1 μl / tube of Alexa-647-labeled goat anti-human IgG secondary antibody (purchased from Jackson Lab) was added, and the cells were incubated at 4°C for 30 minutes in the dark. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μl / tube of PBS and detected by flow cetometry.

[0216] As shown in Figure 10, the results indicate that the CD3×Trop2 antibody of the present invention can bind to A-375-Trop2 melanoma cells, and based on average fluorescence intensity, all bispecific antibodies exhibited similar binding ability to A-375-Trop2 cells.

[0217] The results, as shown in Figures 17, 18, and 19, showed that 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.

[0218] Example 8: Experiment to enhance tumor cell killing by bispecific antibody PBMCs

[0219] We detected the ability of bispecific antibodies to promote the killing of A-375 melanoma cells and HCT-15 colorectal cancer cells by PBMCs. (a) Add complete RPMI-1450 medium to a 16-well RTCA plate at a volume of 50 μL / well, set it in the instrument and perform calibration. (b) Tumor cells in complete RPMI-1450 medium 2 × 10 5 Dilute to / mL and add to each RTCA plate obtained in step (1) individually in a volume of 50 μL / well, then detect the cell count for 24 hours using an xCELLigence RTCA MP instrument at 37°C under 5% CO2 conditions. (c) Bispecific antibodies Cross3×Trop2, Cross303×Trop2, Cross307×Trop2, Cross308×Trop2, Cross309×Trop2, Cross310×Trop2, Cross311×Trop2, Cross312×Trop2, Cross313×Trop2, Cross314×Trop2, Cross316×Trop2, Cross317×Trop2, Cross318×Trop2, Cross323×Trop2, Cross325×Trop2, Cross326×Trop2, Cross3×PDL1, Cross303×PDL1, Cross307×PDL1, C ross308×PDL1, Cross309×PDL1, Cross310×PDL1, Cross311×PDL1, Cross312×PDL1, Cross313×PDL1, Cross314×PDL1, Cross316×PDL1, Cross317×PDL1, Cross318×PDL1, Cross323×PDL1, Cross325×PDL1, Cross326×PDL1, Cross3ZH07×PDL1, Cross3ZH09×PDL1, Cross3ZH16×PDL1, Cross3ZH23×PDL1, and control hIgG1LALA (purchased from Hyakuei Seibutsu) were added to the RTCA plate obtained in step (2), with an addition volume of 20 μl / well. (4) PBMC (purchased from Myojun Seibutsu) in complete RPMI-1450 medium in 1.25 × 10 6 Dilute to 1 / ml and add to the RTCA plate obtained in step (3), with an addition volume of 80 μl / well. (5) The reaction system obtained in step (4) was tested for cell coefficients for 24 hours using an xCELLigence RTCA MP instrument at 37°C and 5% CO2.

[0220] As shown in Figure 11, 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 low-affinity bispecific antibody, while the effective doses of Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 are higher. However, the maximum killing rate is consistent with that of Cross3×Trop2, and all of them can achieve maximum killing.

[0221] As shown in Figure 12, Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 promote the killing of A-375-Trop2 melanoma cells by PBMCs. While Cross313×Trop2 and Cross316×Trop2 have higher effective doses, the maximum killing rate is consistent with that of Cross3×Trop2, indicating that all can achieve maximum killing.

[0222] As shown in Figure 20, the CD3×PDL1 of the present invention promotes the killing of A-375 melanoma cells by PBMCs, and the highest killing performance of Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 is superior to that of other bispecific antibodies.

[0223] As shown in Figure 21, the Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 of the present invention promote the killing of A-375 melanoma cells by PBMCs, and the highest killing rate of Cross316×PDL1 is superior to that of Cross313×PDL1 and Cross325×PDL1.

[0224] As shown in Figure 22, 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.

[0225] Example 9: Experiment on promoting cytokine secretion using bispecific antibody PBMCs.

[0226] Add bispecific antibodies (for the production method and the amino acid sequences of the heavy and light chains, refer to Example 1) to the co-incubation system of PBMC and tumor cells. After culturing for 48 h, collect the culture supernatant, detect the cytokine content in the supernatant, and use it to determine the property that the bispecific antibody induces cytokine release. (1) Dilute the tumor to 1×10 5 / ml using complete RPMI 1450 medium, add it to a 96-well plate, and culture it in a 37°C, 5% CO2 incubator for 24 h. (2) Gradient dilute bispecific antibodies Cross3×Trop2, Cross313×Trop2, Cross316×Trop2, Cross325×Trop2, Cross3×PDL1, Cross313×PDL1, Cross316×PDL1, Cross325×PDL1, and control hIgG1LALA (purchased from Baiying Biology) using complete RPMI 1450 medium, and add 20 μl / well to a 96-well plate. (3) Dilute PBMC (purchased from Saili Biology) to 1.25×10 6 / ml using complete RPMI 1450 medium, and add 80 μl / well to a 96-well plate. (4) Culture the 96-well plate in a 37°C, 5% CO2 incubator for 48 h. (5) Centrifuge at 300 g for 10 min at room temperature, collect the cell culture supernatant. (6) Detect the cytokine content in the supernatant using a CBA kit (purchased from BD).

[0227] As shown in Fig. 13, 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 PBMC, and the promotion of the secretion of inflammatory factors by Cross313×Trop2, Cross316×Trop2, and Cross325×Trop2 antibodies is weaker than that of Cross3×Trop2.

[0228] As shown in Figures 23 and 24, 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, while the promotion of inflammatory factor secretion by Cross313×PDL1, Cross316×PDL1, and Cross325×PDL1 antibodies is weaker than that of Cross3×PDL1.

[0229] In this specification, any reference to terms such as “one embodiment,” “several embodiments,” “example,” “specific example,” or “several examples” means that a particular feature, structure, material, or property described with reference to such embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the general expressions of the above terms do not necessarily apply to the same embodiment or example. In addition, any particular feature, structure, material, or property described may be incorporated in an appropriate manner in any one or more embodiments or examples. Furthermore, those skilled in the art can combine and combine the various embodiments or examples and the features relating to the various embodiments or examples described herein without contradiction.

[0230] Although embodiments of the present invention have been presented and described, these embodiments are illustrative and should not be understood as limiting the present invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations are possible in the above embodiments within the scope of the present invention.

Claims

1. An anti-CD3 antibody or its antigen-binding fragment comprising heavy chain variable regions CDRs and light chain variable regions CDRs, An anti-CD3 antibody or its antigen-binding fragment, characterized in that the heavy chain variable regions CDRs have an amino acid sequence shown in any of SEQ ID NO: 1-3, 7-8, and 74-86, or an amino acid sequence of a conservatively modified form thereof, and / or the light chain variable regions CDRs have an amino acid sequence shown in any of SEQ ID NO: 4-6, 9, and 87-92, or an amino acid sequence of a conservatively modified form thereof.

2. The heavy chain variable regions CDRs have amino acid sequences shown in SEQ ID NO: 1-2 or their conserved modified forms, and amino acid sequences shown in any of SEQ ID NO: 3, 7-8 and 74-86 or their conserved modified forms. The antibody or antigen-binding fragment according to claim 1, characterized in that the light chain variable regions CDRs have amino acid sequences shown in SEQ ID NO: 4-5 or amino acid sequences of their conserved modified forms, and amino acid sequences shown in any of SEQ ID NO: 6, 9 and 87-92 or amino acid sequences of their conserved modified forms.

3. The antibody or its antigen-binding fragment is The heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO:

1. The heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO:

2. The heavy chain variable region CDR3 has an amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 7-8, and SEQ ID NO: 74-86. The light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO:

4. The light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO:

5. The antibody or antigen-binding fragment according to claim 2, characterized in that the light chain variable region CDR3 has an amino acid sequence shown in any of SEQ ID NO: 6, 9, and 87-92.

4. The antibody or its antigen-binding fragment comprises one of the following groups: 1) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 74, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 2) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 75, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 3) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 76, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 4) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 77, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 5) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 78, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 6) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 79, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 7) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 80, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 8) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 81, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 9) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 82, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 10) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 83, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 11) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 1, 2, and 3 respectively, and each has light chain variable regions CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 4, 5, and 6, 12) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 7, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 6, 13) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 8 respectively, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 87, 14) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 8 respectively, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 88 respectively, 15) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 8, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 9, 16) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 84, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 89, 17) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 85, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 90, 18) Each has heavy chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 1, 2, and 86, and each has light chain variable regions CDR1, CDR2, and CDR3 as indicated by SEQ ID NO: 4, 5, and 92, 19) The antibody or antigen-binding fragment according to claim 3, characterized in that it has heavy chain variable regions CDR1, CDR2, and CDR3, respectively, as indicated by SEQ ID NO: 1, 2, and 8, and light chain variable regions CDR1, CDR2, and CDR3, respectively, as indicated by SEQ ID NO: 4, 5, and 91.

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

6. The antibody or antigen-binding fragment according to claim 5, characterized in that at least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of the following: a mouse-derived antibody, a human-derived antibody, a primate-derived antibody, a cat-derived antibody, a horse-derived antibody, a dairy cow-derived antibody, a pig-derived antibody, a sheep-derived antibody, a goat-derived antibody, a dog-derived antibody, a cat-derived antibody, a rabbit-derived antibody, a camel-derived antibody, a donkey-derived antibody, a deer-derived antibody, a mink-derived antibody, a chicken-derived antibody, a duck-derived antibody, a goose-derived antibody, a turkey-derived antibody, a fighting cock-derived antibody, or a mutant thereof, and preferably derived from at least one of the mouse-derived antibody, a human-derived antibody, and a primate-derived antibody.

7. The antibody or its antigen-binding fragment is A heavy chain variable region having an amino acid sequence shown in any of SEQ ID NO: 10, 12-13, 15-24, 27, and 31, or an amino acid sequence of a conservative modified form thereof, and / or The antibody or antigen-binding fragment according to claim 5, characterized in that it comprises a light chain variable region having an amino acid sequence shown in any of SEQ ID NO: 11, 14, 25-26, 28, and 30, or an amino acid sequence of a conserved modified form thereof.

8. The heavy chain variable region has an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of SEQ ID NO: 10, 12-13, 15-24, 27, and 31, and / or The antibody or antigen-binding fragment according to claim 5, characterized in that the light chain variable region has an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of SEQ ID NO: 11, 14, 25-26, 28, and 30.

9. The antibody or its antigen-binding fragment has an amino acid sequence represented by a combination of heavy chain variable region and light chain variable region from any of the following groups, or an amino acid sequence having at least 90% sequence identity thereto. Table 1 The antibody or antigen-binding fragment according to feature 8.

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

11. The antibody or antigen-binding fragment according to claim 10, characterized in that at least one portion of the heavy chain constant region and the light chain constant region is derived from at least one of the following: a mouse-derived antibody, a human-derived antibody, a primate-derived antibody, a cattle-derived antibody, a horse-derived antibody, a dairy cow-derived antibody, a pig-derived antibody, a sheep-derived antibody, a goat-derived antibody, a dog-derived antibody, a cat-derived antibody, a rabbit-derived antibody, a camel-derived antibody, a donkey-derived antibody, a deer-derived antibody, a mink-derived antibody, a chicken-derived antibody, a duck-derived antibody, a goose-derived antibody, a turkey-derived antibody, a fighting cock-derived antibody, or a mutant thereof.

12. The heavy chain steady-state region includes a heavy chain steady-state region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD, and / or The antibody or antigen-binding fragment according to claim 10, characterized in that the light chain constant region includes a light chain constant region selected from κ-type or λ-type.

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

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

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

16. The antibody or antigen-binding fragment according to claim 10, 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.

17. The antibody or antigen-binding fragment according to claim 16, wherein the antibody or antigen-binding fragment further comprises a first linking peptide, the C-end of the heavy chain variable region being connected to the N-end of the first linking peptide, the C-end of the first linking peptide being connected to the N-end of the light chain variable region, or the C-end of the light chain variable region being connected to the N-end of the first linking peptide, and the C-end of the first linking peptide being connected to the N-end of the heavy chain variable region.

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

19. The antibody or antigen-binding fragment is A heavy chain having an amino acid sequence shown in any of SEQ ID NO: 32, 34, 35 and 37-46, or an amino acid sequence having at least 80% identity thereto, and / or The antibody or antigen-binding fragment according to claim 18, characterized in that it comprises a light chain having an amino acid sequence shown in any of SEQ ID NO: 33, 36, and 47-48, or an amino acid sequence having at least 80% identity thereto.

20. The antibody or its antigen-binding fragment has an amino acid sequence represented by a combination of heavy and light chains from any of the following groups, or an amino acid sequence having at least 80% sequence identity thereto. Table 2 The antibody or antigen-binding fragment according to feature 18.

21. The antibody or antigen-binding fragment according to claim 18, characterized in that the antibody or antigen-binding fragment has an amino acid sequence shown in any of SEQ ID NO: 99 to 117 or an amino acid sequence having at least 80% sequence identity thereto.

22. The antibody or its antigen-binding fragment has an amino acid sequence shown in any of the following combinations of Fab VL-CL and Fab VH-CH1, or an amino acid sequence having at least 80% sequence identity thereto. Table 3 The antibody or antigen-binding fragment according to feature 18.

23. The antibody or antigen-binding fragment according to claim 18, characterized in that the antibody or antigen-binding fragment has an amino acid sequence shown in any of SEQ ID NO: 142 to 160 or an amino acid sequence having at least 80% sequence identity thereto.

24. It is a polyspecific antibody, A first antigen-binding region comprising an anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1 to 23, A multispecific antibody characterized by comprising a second antigen-binding region having binding activity to a biomolecule other than CD3.

25. The polyspecific antibody according to claim 24, characterized in that the polyspecific antibody is selected from one of a bispecific antibody, a triplicate antibody, and a quadruplicate antibody, and is preferably a bispecific antibody.

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

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

28. The aforementioned biomolecule is The polyspecific antibody according to claim 24, characterized in that it is selected from at least one of PDL1, CD47, TIGIT, CD73, CD33, CEACAM1, CEACAM5, CEACAM6, STING, WNT, Betacatenin, 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, and EPCAM.

29. The polyspecific antibody according to claim 27, characterized in that the binding protein or fragment thereof is selected from at least one of an antibody or its antigen-binding fragment, a receptor, and a ligand.

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

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

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

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

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

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

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

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

166.

38. The polyspecific antibody according to claim 31, characterized in that the first scFv fragment has an amino acid sequence shown in any of SEQ ID NO: 95 to 117.

39. The first Fab fragment has an amino acid sequence represented by a combination of Fab VL-CL and Fab VH-CH1 from any of the following groups. Table 4 The polyspecific antibody according to claim 31.

40. The polyspecific antibody according to claim 31, characterized in that the first Fab-Linker has an amino acid sequence shown in any of SEQ ID NO: 142 to 160.

41. The polyspecific antibody according to claim 24, characterized in that the first antigen-binding region has an amino acid sequence shown in any of SEQ ID NO: 49-52 or 54-68.

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

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

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

45. 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 polyspecific antibody according to claim 44, characterized in that the third Fab fragment has the amino acid sequences shown in SEQ ID NO: 164 and 72.

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

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

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

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

165.

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

51. The second antigen-binding region has the amino acid sequence 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 polyspecific antibody according to claim 43, characterized in that the second antigen-binding region has the amino acid sequences shown in SEQ ID NO: 164 and 72.

52. It is a conjugated form, An antibody or its antigen-binding fragment according to any one of claims 1 to 23 or a polyspecific antibody according to any one of claims 24 to 51, A conjugate characterized by comprising the antibody or its antigen-binding fragment, or a conjugation portion connected to a polyspecific antibody.

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

54. A nucleic acid characterized by encoding an antibody or its antigen-binding fragment according to any one of claims 1 to 23 or a polyspecific antibody according to any one of claims 24 to 51.

55. A carrier or transformant characterized by containing the nucleic acid described in claim 54.

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

57. The carrier or transformant according to claim 55, characterized in that the carrier is selected from at least one of plasmid carriers, adenovirus carriers, lentivirus carriers, and adeno-associated virus carriers.

58. A cell characterized by carrying the nucleic acid described in claim 54, the carrier or transformant described in any one of claims 55 to 57, or expressing the antibody described in any one of claims 1 to 23 or its antigen-binding fragment, or the polyspecific antibody described in any one of claims 24 to 51.

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

60. A drug composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 23, a polyspecific antibody according to any one of claims 24 to 51, a conjugate according to any one of claims 52 to 53, a nucleic acid according to claim 54, or a carrier or transformant according to any one of claims 55 to 57.

61. The drug composition according to claim 60, further comprising a pharmaceutically acceptable adjuvant.

62. The drug composition according to claim 61, characterized in that the auxiliary agent comprises one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers.

63. The drug composition according to claim 60, characterized in that the drug composition is an injectable preparation.

64. A kit characterized by comprising an antibody or its antigen-binding fragment according to any one of claims 1 to 23, or a polyspecific antibody according to any one of claims 24 to 51.

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

66. Use in the preparation of a drug, wherein the drug is used to modulate an immune response and / or inhibit tumor growth, or the drug is used to prevent and / or treat CD3-related diseases and / or cancer or tumors or infections or autoimmune diseases.

67. The use according to claim 66, characterized in that the cancer or tumor or infection or autoimmune disease is a Top2 or PDL1-related disease.

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

69. A method for regulating an immune response and / or suppressing tumor growth or for the prevention and / or treatment of CD3-related disease and / or cancer or tumor or infection or autoimmune disease, the method comprising the step of administering to a subject a pharmaceutically acceptable amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, a polyspecific antibody according to any one of claims 24 to 51, a conjugate according to any one of claims 52 to 53, a nucleic acid according to claim 54, a carrier or transformant according to any one of claims 55 to 57, or a drug composition according to any one of claims 60 to 63.

70. The method according to 69, characterized in that the cancer or tumor or infection or autoimmune disease is a Top2 or PDL1-related disease.

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

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