A cd3 antibody and uses thereof

Abstract

The application discloses a CD3 antibody and application thereof, and relates to the technical field of biological medicine.The antibody provided by the application is a full human antibody, which fundamentally reduces immunogenicity risk, and lays a material foundation for developing safer T cell redirection therapy.The antibody has cross-reactivity with cynomolgus monkey CD3, can be directly used for a cynomolgus monkey model to perform systematic pharmacodynamics, pharmacokinetics and safety evaluation, greatly improves the prediction value of preclinical data, and accelerates the conversion process of a candidate molecule to clinical research.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a CD3 antibody and its applications. Background Technology

[0002] CD3-targeted T-cell retargeting therapy has demonstrated significant clinical potential in hematologic malignancies and solid tumors, leading to the successful launch of several bispecific antibody drugs (such as Blincyto® and Mosunetuzumab). However, with the deepening of research and the expansion of indications in this field, the existing technology system still faces a series of key challenges in terms of accessibility, optimization potential, and clinical translation efficiency, mainly including: a) Immunogenicity risk remains a potential concern for long-term safety: Although humanized and fully human antibody technologies have significantly reduced immunogenicity, some early-developed or structurally complex TCEs may still contain murine sequences or non-natural frameworks, posing a risk of inducing anti-drug antibodies. This could affect the long-term efficacy of the drug and lead to potential safety issues.

[0003] b) The industrial-scale production of bispecific antibodies still faces challenges related to efficiency and cost: Light chain mismatch is a classic challenge in the production of bispecific antibodies. Although various technical platforms (such as Knob-into-Hole, common light chain, scFv-Fc fusion, etc.) have been developed to facilitate correct pairing, these approaches are often accompanied by problems such as complex production processes, reduced expression levels, or increased purification steps, thereby increasing production costs and, to some extent, limiting the wider accessibility of such drugs.

[0004] c) The limitations of preclinical translational research models restrict the development efficiency of some candidate molecules: Many investigational TCEs target human-specific epitopes, which do not cross-react with targets in mainstream preclinical animal models (such as cynomolgus monkeys). This limitation prevents researchers from comprehensively evaluating their in vivo efficacy, pharmacokinetics, and potential toxicity in these key animal models, increasing the uncertainty of clinical development and potentially delaying the development of some promising candidate molecules. Summary of the Invention

[0005] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide a CD3 antibody and its applications. The antibody provided by this invention is a fully human antibody, fundamentally reducing the risk of immunogenicity and laying the material foundation for developing safer T-cell redirection therapies. Furthermore, it exhibits cross-reactivity with cynomolgus monkey CD3, allowing it to be directly used in cynomolgus monkey models for systematic efficacy, pharmacokinetics, and safety assessments, greatly enhancing the predictive value of preclinical data and accelerating the translation of candidate molecules into clinical research.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides a CD3 antibody or its antigen-binding fragment, comprising: A heavy chain variable region (VH), the heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region contains an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity with the selected VH CDR1 amino acid sequence, the VH CDR2 region contains an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity with the selected VH CDR2 amino acid sequence, and the VH CDR3 region contains an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity with the selected VH CDR3 amino acid sequence; and The light chain variable region (VL) comprises CDR 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity with the selected VL CDR1 amino acid sequence, the VLCDR2 region comprises an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity with the selected VL CDR2 amino acid sequence, and the VLCDR3 region comprises an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity with the selected VL CDR3 amino acid sequence; The selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1, 2, and 3, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4, 5, and 6, respectively; (2) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 11, 12, and 13, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 14, 15, and 16, respectively; (3) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 21, 22, and 23, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 24, 25, and 26, respectively; (4) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 31, 32, and 33, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 34, 35, and 36, respectively; (5) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 41, 42, and 43, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 44, 45, and 46, respectively; (6) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 51, 52, and 53, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 54, 55, and 56, respectively; (7) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 61, 62, and 63, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 64, 65, and 66, respectively; (8) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 71, 72, and 73, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 74, 75, and 76, respectively. In one embodiment, the VH comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 21, 22, and 23, respectively, and the VL comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 24, 25, and 26, respectively; In one embodiment, the VH comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 71, 72, and 73, respectively, and the VL comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 74, 75, and 76, respectively.

[0007] In one embodiment, the antibody or antigen-binding fragment binds specifically to human CD3.

[0008] In one embodiment, the antibody or antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

[0009] In one embodiment, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).

[0010] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain framework region and / or a light chain framework region.

[0011] In one embodiment, at least a portion of the heavy chain framework region and / or light chain framework region is derived from mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, and / or their mutants.

[0012] In one embodiment, at least a portion of the heavy chain framework region and / or light chain framework region is derived from a human antibody.

[0013] In one embodiment, the antibody or its antigen-binding fragment comprises: The heavy chain variable region (VH) and the light chain variable region (VL), wherein the heavy chain variable region (VH) comprises an amino acid sequence having at least 90% (e.g., 90%, 95%, 96%, 97%, 98%, or 99%) identity with a selected VH sequence, and the light chain variable region (VL) comprises an amino acid sequence having at least 90% (e.g., 90%, 95%, 96%, 97%, 98%, or 99%) identity with a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 7, and the selected VL sequence is SEQ ID NO: 8; (2) The selected VH sequence is SEQ ID NO: 17, and the selected VL sequence is SEQ ID NO: 18; (3) The selected VH sequence is SEQ ID NO: 27, and the selected VL sequence is SEQ ID NO: 28; (4) The selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is SEQ ID NO: 38; (5) The selected VH sequence is SEQ ID NO: 47, and the selected VL sequence is SEQ ID NO: 48; (6) The selected VH sequence is SEQ ID NO: 57, and the selected VL sequence is SEQ ID NO: 58; (7) The selected VH sequence is SEQ ID NO: 67, and the selected VL sequence is SEQ ID NO: 68; (8) The selected VH sequence is SEQ ID NO: 77, and the selected VL sequence is SEQ ID NO: 78.

[0014] In one embodiment, the antibody or its antigen-binding fragment has: The heavy chain variable region shown in SEQ ID NO: 27 and the light chain variable region shown in SEQ ID NO: 28; And / or the heavy chain variable region shown in SEQ ID NO: 77 and the light chain variable region shown in SEQ ID NO: 78.

[0015] In one embodiment, the antibody or antigen-binding fragment binds specifically to human CD3.

[0016] In one embodiment, the antibody or antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

[0017] In one embodiment, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).

[0018] In one embodiment, the CD3 antibody or its antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region.

[0019] In one embodiment, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, and / or their mutants.

[0020] In one embodiment, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and / or IgD.

[0021] In one embodiment, the light chain constant region includes κ-type and / or λ-type light chain constant regions; In one embodiment, both the light chain constant region and the heavy chain constant region are derived from human antibodies or their mutants.

[0022] In one embodiment, the antibody includes a full-length monoclonal antibody, a chimeric antibody, a humanized antibody, Fv, scFv, Fab, Fab', Fab'-SH and / or F(ab')2; In one embodiment, the antigen-binding fragment of the antibody includes an F(ab')2 fragment, a Fab' fragment, a Fab fragment, an F(ab)2 fragment, an Fv fragment, a scFv fragment, and / or a scFv-Fc fusion protein.

[0023] In one embodiment, the antibody or antigen-binding fragment comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any of the following combinations of heavy and light chains: The heavy chain shown in SEQ ID NO: 29 and the light chain shown in SEQ ID NO: 30; And / or the heavy chain shown in SEQ ID NO: 79 and the light chain shown in SEQ ID NO: 80.

[0024] In another aspect, the present invention provides a nucleic acid comprising a polynucleotide encoding a polypeptide, said polypeptide comprising: (1) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 1, 2 and 3 respectively, and wherein the VH binds to CD3 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 8. (2) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO: 4, 5 and 6, and wherein the VL binds CD3 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 7; (3) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 11, 12 and 13 respectively, and wherein the VH binds to CD3 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 18; (4) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO: 14, 15 and 16, and wherein the VL binds CD3 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 17; (5) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 21, 22 and 23 respectively, and wherein the VH binds to CD3 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 28; (6) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO: 24, 25 and 26, and wherein the VL binds CD3 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 27; (7) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 31, 32 and 33 respectively, and wherein the VH binds to CD3 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 38; (8) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO: 34, 35 and 36, and wherein the VL binds CD3 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 37; (9) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 41, 42 and 43 respectively, and wherein the VH binds to CD3 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 48; (10) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises CDR 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 44, 45 and 46 respectively, and wherein the VL binds CD3 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO: 47; (11) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 51, 52 and 53 respectively, and wherein the VH binds to CD3 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 58; (12) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises CDR 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 54, 55 and 56 respectively, and wherein the VL binds CD3 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO: 57; (13) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 61, 62 and 63 respectively, and wherein the VH binds to CD3 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 68; (14) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises CDR 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 64, 65 and 66 respectively, and wherein the VL binds CD3 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO: 67; (15) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 71, 72 and 73 respectively, and wherein the VH binds to CD3 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 78; (16) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO: 74, 75 and 76, and wherein the VL binds CD3 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 77.

[0025] In one embodiment, the polypeptide comprises an immunoglobulin heavy chain or a fragment thereof containing VH, wherein the VH comprises CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 21, 22 and 23.

[0026] In one embodiment, the polypeptide comprises an immunoglobulin light chain or a fragment thereof containing a VL, wherein the VL comprises CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 24, 25 and 26.

[0027] In one embodiment, the polypeptide comprises an immunoglobulin heavy chain or a fragment thereof containing VH, wherein the VH comprises CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 71, 72 and 73.

[0028] In one embodiment, the polypeptide comprises an immunoglobulin light chain or a fragment thereof containing a VL, wherein the VL comprises CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 74, 75 and 76.

[0029] In one embodiment, the VH specifically binds to human CD3 when paired with the VL, or the VL specifically binds to human CD3 when paired with the VH.

[0030] In one embodiment, the immunoglobulin heavy chain or a fragment thereof is a humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or a fragment thereof is a humanized immunoglobulin light chain or a fragment thereof.

[0031] In one embodiment, the nucleic acid encodes a single-stranded variable fragment (scFv).

[0032] In one embodiment, the nucleic acid is cDNA.

[0033] In another aspect, the present invention also provides a carrier comprising the above-mentioned nucleic acid.

[0034] In one embodiment, the carrier encodes VL and VH regions, which together are combined with CD3.

[0035] In another aspect, the present invention also provides a vector pair, wherein each vector contains one of the above-described nucleic acids, wherein the vector pair together encodes a VL region and a VH region, the VL region and the VH region together binding CD3.

[0036] In another aspect, the present invention provides a cell that expresses the aforementioned nucleic acid, vector and / or vector pair.

[0037] In another aspect, the present invention also provides cells comprising the two aforementioned nucleic acids.

[0038] In one embodiment, the two nucleic acids together encode the VL region and the VH region, which together bind to CD3.

[0039] In another aspect, the present invention also provides a method for generating an antibody or an antigen-binding fragment thereof, comprising: The cells were cultured under conditions sufficient to induce them to produce antibodies or antigen-binding fragments; and Collect antibodies or antigen-binding fragments produced by the cells.

[0040] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) A therapeutically effective amount of the above-mentioned antibodies or their antigen-binding fragments, nucleic acids, vectors, vector pairs and / or cells; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0041] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0042] In another aspect, the present invention also provides a pharmaceutical product comprising the above-described pharmaceutical preparation.

[0043] In one embodiment, the pharmaceutical product is a vial or box.

[0044] In another aspect, the present invention also provides a detection reagent comprising the antibody or its antigen-binding fragment described above.

[0045] In another aspect, the present invention also provides a detection kit comprising the detection reagents described above.

[0046] In another aspect, the present invention also provides the application of the above-mentioned detection reagents and / or detection kits in the detection of CD3.

[0047] This invention relates only to detection for non-diagnostic purposes.

[0048] In another aspect, the present invention also provides the use of the above-mentioned antibodies or their antigen-binding fragments, nucleic acids, carriers, carrier pairs, cells, pharmaceutical compositions, pharmaceutical preparations and / or pharmaceutical products in the preparation of medicaments for the prevention and / or treatment of CD3-mediated diseases; In one embodiment, the CD3-mediated diseases include hyperproliferative disorders, particularly involving cancers of any tissue or organ, and are specifically used to treat cancers of the head, neck, breast, liver, skin, stomach, bladder, kidney, esophagus, gynecology, bronchi, nasopharynx, thyroid, prostate, colorectal, ovarian, pancreas, lung, and fibrosarcoma.

[0049] (III) Beneficial Effects This invention provides a CD3 antibody and its application. Compared with the prior art, it has the following advantages: 1. Achieving an optimized balance between safety and functionality: All eight fully human antibodies obtained in this invention have undergone functional screening, confirming their effective T-cell activation capabilities. Their fully human nature fundamentally reduces the risk of immunogenicity, laying a material foundation for developing safer T-cell retargeting therapies.

[0050] 2. Provides an ideal module for efficiently constructing bispecific antibodies: Six antibodies are in a fully human co-light chain format. As a "plug-and-play" module, the co-light chain antibodies can efficiently and correctly pair with heavy chains of different targets to form single, correct bispecific antibodies, fundamentally solving the industrial problem of light chain mismatch and providing a core solution for achieving low-cost, high-purity large-scale production.

[0051] 3. Created a highly valuable preclinical translation bridge: Successfully obtained two fully human co-light chain antibodies that cross-react with CD3 in cynomolgus monkeys, which can be directly used in cynomolgus monkey models for comprehensive efficacy and safety evaluation. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0053] Figure 1 This is a graph showing the results of the antibody-specific binding assay to the Jurkat cell line; Figure 2 This is a graph showing the results of the antibody's specific binding to the CD3E-KO Jurkat cell line; Figure 3 This is a graph showing the results of the antibody-specific binding detection to human PBMC cell lines; Figure 4 This is a graph showing the results of the antibody-specific binding detection to cynomolgus monkey-derived PBMC cell lines; Figure 5 This is a graph analyzing the effect of antibodies on T cell activation activity. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] Terms and Definitions As used herein, the term "antibody" generally refers to an antibody that recognizes one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, triple-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854 4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy-chain, full-length light-chain, or intact immunoglobulin molecules; or the immunologically active portion of any of these polypeptides, i.e., a molecule or portion thereof containing an antigen-binding site that specifically binds to a target antigen of interest, such targets including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases.

[0056] Within the variable region, certain areas exhibit a higher degree of variation in amino acid composition and sequence, termed "hypervariable region (HVR)." The hypervariable region is the site where antigens and antibodies bind, and is therefore also called the complementarity-determining region (CDR). Both the heavy chain and light chain variable regions contain three CDR regions. For example, these typically include amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., near 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk). J. Mol. Biol. 196:901-917 (1987)).

[0057] As used in this article, the term "CD3 antibody" refers to an antibody that can bind to CD3.

[0058] As used herein, the term “antigen-binding fragment” is equivalent to “antibody fragment” or “antigen-binding antibody fragment” and can include a portion of a complete antibody, typically a binding region or variable region. This includes, but is not limited to: Fv, scFv, Fab, Fab’, Fab’-SH, F(ab’)2, scFv-Fc fragments, or bispecific antibodies (BsAbs), linear antibodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol (“PEGylated”) (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab’)2-PEG, or Fab’-PEG) (“PEG” stands for polyethylene glycol).

[0059] As used herein, the term "human antibody" refers to an antibody encoded by an endogenous nucleic acid present in humans (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, human antibodies are collected from humans or produced in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacterial or yeast cells. In some embodiments, human antibodies are produced in transgenic non-human animals (e.g., cattle) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0060] As used herein, the term "humanized antibody" refers to a nonhuman antibody that contains a minimal sequence derived from a nonhuman (e.g., mouse) immunoglobulin and a sequence derived from a human immunoglobulin. In some non-limiting examples, a humanized antibody is a human antibody (receptor antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced with hypervariable (e.g., CDR) region residues of a nonhuman antibody (e.g., donor antibody) (e.g., mouse, rat, or rabbit antibody) having the desired specificity, affinity, and performance. In some embodiments, Fv framework residues of a human immunoglobulin are replaced with corresponding nonhuman (e.g., mouse) immunoglobulin residues. In some embodiments, a humanized antibody may contain residues not present in the recipient antibody or donor antibody. These modifications may be made to further improve antibody performance. In some embodiments, a humanized antibody contains at least one and typically substantially all of two variable domains, wherein all or substantially all of the hypervariable loops (CDRs) correspond to those of nonhuman (e.g., mouse) immunoglobulins, and all or substantially all of the framework regions are those of human immunoglobulins. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region (Fc) of human immunoglobulins. Humanized antibodies can be generated using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0061] As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of the heavy or light chains of mammalian immunoglobulins) capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0062] As used herein, when referring to antibodies, the phrases "specifically binds to" and "specifically binds to..." mean that because the interaction depends on the presence of a specific structure (i.e., antigenic determinant or epitope) on the target molecule, the antibody preferentially interacts with its target molecule (e.g., CD3) relative to other molecules; in other words, the reagent recognizes and binds to molecules containing a specific structure, rather than all molecules in general. Antibodies that specifically bind to a target molecule can be called target-specific antibodies. For example, antibodies that specifically bind to CD3 molecules can be called CD3-specific antibodies or anti-CD3 antibodies.

[0063] As used herein, the terms “homology,” “identity,” or “similarity” are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of “sequence homology” between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings and known computer algorithms for sequence alignment, such as NCBIBlast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0967284, EP 1085089, WO00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0064] For peptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0065] As used herein, the terms "vector" and "recombinant expression vector" are used interchangeably, referring to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0066] Methods well known to those skilled in the art can be used to construct expression vectors containing CD3 antibody coding sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0067] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0068] Vectors containing the appropriate DNA sequence and suitable promoters or control sequences can be used to transform suitable host cells to enable them to express proteins or peptides. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples include: *Escherichia coli*, *Streptomyces*, *Agrobacterium*; fungal cells such as yeast; and animal cells.

[0069] The polynucleotides disclosed in this invention, when expressed in higher eukaryotic cells, will enhance transcription when an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0070] As used herein, the term "pharmaceutical composition" refers to a composition comprising a CD3 antibody or an antigen-binding fragment thereof formulated with one or more pharmaceutically acceptable carriers.

[0071] The formulation of the pharmaceutical composition can be tailored to the application. In particular, pharmaceutical compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals. For example, the formulation can be selected from any of the following: liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, tablets, suppositories, injections, alcoholic preparations, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0072] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0073] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0074] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0075] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0076] For example, compounds or agents can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). Compounds or agents can also be suitably introduced via rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide prolonged, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more prolonged periods.

[0077] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0078] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0079] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0080] Example 1: Screening of CD3 antibodies: 1. Immunization and Cell Preparation CD3 antigen immunization was performed using the HUGO-Ab fully human antibody mouse platform. The endogenous antibody gene in HUGO-Ab fully human antibody mice has been replaced by a human antibody gene library, enabling them to directly produce fully human antibodies. The mRNA encoding human CD3 and TCR sequences was used as the immunogen: ionizable lipids SM102, cholesterol, DSPC, and DMG-PEG-2kJ were dissolved in ethanol at a molar ratio of 50:38.5:10:1.5 to form a lipid phase. Simultaneously, the in vitro transcribed and purified mRNA was dissolved in 20 mM Tris-HCl buffer (pH 7.5) to form an aqueous phase. The lipid and aqueous phases were rapidly mixed at a volume ratio of 1:3 using microfluidic technology, allowing the lipids and mRNA to self-assemble into lipid nanoparticles. These nanoparticles were then subjected to buffer replacement, ultrafiltration concentration, and sterile filtration through a 0.22 μm filter membrane to obtain the CD3 mRNA-LNP immunogen.

[0081] HUGO-Ab mice aged 6-8 weeks were immunized intramuscularly with 40 μg of CD3 mRNA-LNP immunogen. After three booster immunizations with mRNA-LNP (40 μg each), the spleens of the mice were harvested, ground, and passed through a 70 μm cell sieve to prepare a single-cell suspension of spleen cells. The total B cell population was isolated from the spleen cell suspension using a commercially available mouse B cell isolation kit (Stemcell #19844).

[0082] mRNA sequence(SEQ ID NO:81):Replicon+AUGCAGAGUGGUACUCACUGGAGAGUUUUAGGGCUGUGUCUUCUCUCCGUGGGUGUGUGGGGACAGGACGGGAAU GAAGAAAUGGGAGGAAUAACCCAGACCCCUAUAAGGUAUCCAUCAGUGGCACCACAGUCAUCCUGACCUGUCCUCAGUAUCCAGGAGAGU GAGAUUUUGUGGCAGCACAACGACAAGAACAUCGGUGGGACGAGGAUGAUAAAAACAUUGGGAGCGAUGAAGACCAUCUCUCUGAA GAAUUUUUCGAACUGGAACAGUCAGGCUACUAUGUCUGUUACCCAAGGGGGUUCCAAGCCUGAAGAUGCUAACUUUAUCUCUACCUGAGA GCCAGGGGUGUGCGAGAACUGCAUGGAAAUGGACGUCAUGUCUGUGGCCACAAUCGUGAUUGUGGACAAUCUGCAUCACUGGAGGGCUGU UGCUGCUGGUCUAUUAUCUGGUCUAAAAAUAGAAAGCUAAAGGCAAAACCUGUGACAAGGGAGCUGGUGCGGGGGGCAGACAGAGAGGG CAGAAUAAGGAGCGGCCACCUCCGGUGCCCAACCCUGACUAUGAGCCCAUCAGGAAAGGCCAGCGUGACUUGUACUCCGGAUUAAUCAG CGGCGCAUUGGAAGUGGGGGCAACCAACUUUUCGCUGCUGAAGCAAGCAGGUGUUGGAAGAAAAUCCAGGCCCAAUGGAGCACAGUAC AUUUCUGUCAGGCCCUGGUACUUGCAACUCUGUUAUCUCAAGUGUCCCUUUUCAAGAUCCCCAUUGAGGAGCUGGAGGAUCGGGGUUUUG UGAACUGUAACACUAGCAUUACCUGGGUGGAAGGGACUGUGGGCACACUUCUCUCAGAUAUAACUCGGCUGGACCUGGGCAAACGUAUC UUGGAUCCUCGCGGUAUAUACCGAUGCAAUGGAACAGACAUCUAUAAGGACAAAGAAAGCACAGUUCAGGUUCAUUACAGGAUGUGUCAG AGUUGUGUCGAGCUGGACCCAGCAACCGUGGCAGGUAUUAUUGUUACAGAUGUCAUUGCCACUCUGCUACUGGCUCUCGGAGUAUUCUG CUUCGCAGGGCACGAAACCGGCAGACUUUCAGGUGCAGCCGACACCCAAGCUCUGUUGCGGAAUGAUCAGGUGUACCAGCCACUCAGGG ACCGCGAUGACGCUCAAUACAGCCAUUUGGGCGGUAACUGGGCCCGCAACAAAGGCUCUGGCGCUACGAAUUUCUCCCUCCUCAAGCAG GCGGGCGACGUGGAAGAGAACCCUGGGCCCAUGGAGCAGGGGAAGGGGCUUGCCGUACUUAUCCUAGCCAUCAUAUUACUCCAAGGUAC GCUGGCCCAAUCCAUAAAGGGAAACCAUCUUGUCAAAGUAUAUGACUAUCAAGAAGAUGGGUCCGUGUUGCUGACUUGUGACGCGGAAGC UAAAAAUAUAACAUGGUUCAAAGAUGGAAAAAUGAUUGGUUUCCUCACUGAGGACAAGAAGAAAUGGAACCUGGGCAGCAAUGCCAAAGAC CCGCGUGGCAUGUACCAGUGCAAGGGCAGCCAGAACAAGUCAAAACCACUGCAGGUCUACUACCGCAUGUGCCAGAACUGUAUUGAGCUC AAUGCAGCCACGAUCUCCGGCUUCCUGUUUGCUGAAAUAGUCAGCAUCUUUGUGCUCGCUGUGGGUGUUUACUUCAUCGCUGGGCAAGA UGGUGUGCGGCAGAGCCGAGCCUCUGACAAGCAGACCUUGCUCCCAAAUGACCAGCUUUAUCAGCCUCUGAAGGACCGGGAGGACGACCAGUACAGUCACCUGCAGGGCAAUCAGCUACGAAGAAAUGGGAGCGGAGCGACCAACUUCUCAUUGCUUAAACAGGCUGGAGAUGUUGAG GAGAACCCCGGCCCGAUGAAGUGGAAGGCCCUGUUCACAGCGGCCAUUUUGCAGGCCCAGCUGCCAAUUACAGAGGCCCAGAGCUUCGG ACUUCUUGAUCCCAAGCUGUGCUACCUUCUGGAUGGCAUCCUCUUCAUUUAUGGGGUUAUCCUGACGGCACUUUUUCUCCGAGUCAAGU UUUCGAGGUCAGCAGAUGCACCCGCCUACCAGCAAGGACAAAACCAACUAUACAACGAGCUGAACCUCGGGCGCAGGGAGGAGUACGAUG UGCUUGAUAAGAGAAGAGGCAGGGACCCUGAGAUGGGCGGCAAGCCCCAGCGCAGGAAGAAUCCACAGGAGGGGCUCUACAAUGAGUUA CAGAAGGACAAGAUGGCCGAGGCUUAUUCCGAGAUCGGCAUGAAAGGAGAGAGAAGAAGAGGGAAAGGACAUGAUGGAUUGUAUCAAGGC UUAAGUACUGCCACCAAGGAUACCUAUGAUGCCCUGCACAUGCAGGCACUGCCUCCCCGGUGAUAA + UTR + PolyA).

[0083] 2. Markers of antigen - specific B cells To specifically identify B cells capable of binding the target antigen, the prepared B cells were incubated with fluorescently labeled CD3D&E probes at 4°C in the dark for 30 minutes. After incubation, the cells were washed twice with PBS containing 2% fetal bovine serum to remove unbound probes.

[0084] 3. Flow cytometry single-cell sorting Sorting was performed using a flow cytometer (e.g., SONY MA900). Based on the negative control (B cells not incubated with the fluorescently labeled CD3D&E probe), only individual B cells with significantly higher fluorescence signal intensity than the negative control group were collected. Using a single-cell sorting mode, each antigen-binding positive B cell was directly sorted into a single well of a 96-well PCR plate, with cell lysis buffer and RNase inhibitor pre-filled in the well.

[0085] Example 2: Recombinant expression of CD3 antibody: Reverse transcription and PCR amplification: The sorted single-cell lysates were subjected to reverse transcription. Using a universal primer set targeting the variable regions of the mouse antibody heavy chain (IgH) and light chain (Igκ) genes, cDNA fragments of the antibody variable regions were amplified by nested PCR.

[0086] Gene cloning and sequencing: The purified PCR product was cloned into a sequencing vector, transformed into competent cells, and clones were selected for Sanger sequencing. The VH and VL genes were then inserted into the mammalian expression vector pcDNA3.4, containing the constant regions (heavy chain) and κ chain (light chain) of mouse IgG1, respectively. CHO cells were co-transfected, expressed, and purified to obtain complete recombinant monoclonal antibodies. ELISA and flow cytometry confirmed that the recombinant antibodies specifically bind to CD3D&E proteins without binding to irrelevant proteins. The amino acid sequences of the recombinant antibodies (A0005, A0033, A0041, 444, A1441, A0010, A0013, A0018) are shown below: 444HCDR1 (SEQ ID NO: 1): NYGML; 444HCDR2 (SEQ ID NO: 2): IISYDGSNKYYADSVKG; 444HCDR3 (SEQ ID NO: 3): GPMVRGVPYNHYYGMDV; 444LCDR1 (SEQ ID NO: 4):RASQGIRNDLG; 444LCDR2 (SEQ ID NO: 5):AASSLQS; 444LCDR3 (SEQ ID NO: 6): LQHNSYPWT; 444VH(SEQ ID NO:7):QVQLVESGGGVVQPGRSLRLSCAASGFTFNNYGMLWVLQAPGKGLEWVAIISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGPMVRGVPYNHYYGMDVWGQGTTVTVSS; 444VL(SEQ ID NO:8):DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIK; 444H(SEQ ID NO:9):QVQLVESGGGVVQPGRSLRLSCAASGFTFNNYGMLWVLQAPGKGLEWVAIISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGPMVRGVPYNHYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; 444L(SEQ ID NO:10):DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; A1441HCDR1(SEQ ID NO:11):NYGMH; A1441HCDR2(SEQ ID NO:12):VILHDGSYKYYADSVKG; A1441HCDR3(SEQ ID NO:13):GSMVRGVPYNYYYGMDV; A1441LCDR1(SEQ ID NO:14):RASQGIRNDLG; A1441LCDR2(SEQ ID NO:15):AASRLQS; A1441LCDR3(SEQ ID NO:16):LQHNSYPPT; A1441VH(SEQ ID NO:17):QVQVVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVILHDGSYKYYADSVKGRFAISRDNSKNTLYLQMNSLRAEDTAVYYCARGSMVRGVPYNYYYGMDVWGQGTTVTVSS; A1441VL(SEQ ID NO:18):DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKLRKAPKRLIYAASRLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPPTFGQGTKVEIK; A1441H(SEQ ID NO:19):QVQVVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVILHDGSYKYYADSVKGRFAISRDNSKNTLYLQMNSLRAEDTAVYYCARGSMVRGVPYNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; A1441L(SEQ ID NO:20):DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKLRKAPKRLIYAASRLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; A0005HCDR1(SEQ ID NO:21):GYYWS; A0005HCDR2(SEQ ID NO:22):EIKQSGRTNYNPSLKS; A0005HCDR3(SEQ ID NO:23):EELYNYSWYFDY; A0005LCDR1(SEQ ID NO:24):RASQGISSYLA; A0005LCDR2(SEQ ID NO:25):AASSLQS; A0005LCDR3(SEQ ID NO:26):QQHYTTPPT; A0005VH(SEQ ID NO:27):QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIKQSGRTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCAREELYNYSWYFDYWGQGTLVTVSS; A0005VL(SEQ ID NO:28):DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK; A0005H(SEQ ID NO:29):EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYVMNWVRQAPGKGLEWVSTISSSGGSTYYADSVKGRFTISRDSSKNTLYLQMNSLRAEDTALYYCAKGGITMVRGWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; A0005L(SEQ ID NO:30):DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; A0010HCDR1(SEQ ID NO:31):RYNMN; A0010HCDR2(SEQ ID NO:32):YISGTGRTIFYADSVKG; A0010HCDR3(SEQ ID NO:33):WEYYESSGWDY; A0010LCDR1(SEQ ID NO:34):RASQGISSYLA; A0010LCDR2(SEQ ID NO:35):AASSLQS; A0010LCDR3(SEQ ID NO:36):QQHYTTPPT; A0010VH(SEQ ID NO:37):EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYNMNWVRQTPGRGLEWVSYISGTGRTIFYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTALYYCARWEYYESSGWDYWGQGVLVTVSS; A0010VL(SEQ ID NO:38):DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK; A0010H(SEQ ID NO:39):EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYNMNWVRQTPGRGLEWVSYISGTGRTIFYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTALYYCARWEYYESSGWDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; A0010L(SEQ ID NO:40):DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; A0013HCDR1(SEQ ID NO:41):RHSMN; A0013HCDR2(SEQ ID NO:42):SISSSSSYIYYADSVKG; A0013HCDR3(SEQ ID NO:43):RSPYFDY; A0013LCDR1(SEQ ID NO:44):RASQGISSYLA; A0013LCDR2(SEQ ID NO:45):AASSLQS; A0013LCDR3(SEQ ID NO:46):QQHYTTPPT; A0013VH(SEQ ID NO:47):EVQLVESGGGLVKPGGSLRLSCAASGVTFSRHSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGRSPYFDYWGQGTLVTVSS; A0013VL(SEQ ID NO:48):DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK; A0013H(SEQ ID NO:49):EVQLVESGGGLVKPGGSLRLSCAASGVTFSRHSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGRSPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; A0013L(SEQ ID NO:50):DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; A0018HCDR1(SEQ ID NO:51):RYNMN; A0018HCDR2(SEQ ID NO:52):YISGSSRTIFYADSVKG; A0018HCDR3(SEQ ID NO:53):WDYYDSSGWDY; A0018LCDR1(SEQ ID NO:54):RASQGISSYLA; A0018LCDR2(SEQ ID NO:55):AASSLQS; A0018LCDR3(SEQ ID NO:56):QQHYTTPPT; A0018VH(SEQ ID NO:57):EVQLVESGGGLVQPGGSLRLSCVASGFTFSRYNMNWVRQAPGRGLEWVSYISGSSRTIFYADSVKGRFIISRDNDKNSLYLQMNSLRDEDTALYYCARWDYYDSSGWDYWGQGALVTVSS; A0018VL(SEQ ID NO:58):DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK; A0018H(SEQ ID NO:59):EVQLVESGGGLVQPGGSLRLSCVASGFTFSRYNMNWVRQAPGRGLEWVSYISGSSRTIFYADSVKGRFIISRDNDKNSLYLQMNSLRDEDTALYYCARWDYYDSSGWDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; A0018L(SEQ ID NO:60):DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; A0033HCDR1(SEQ ID NO:61):GYYWS; A0033HCDR2(SEQ ID NO:62):EIKHSGRTNYNPSLKS; A0033HCDR3(SEQ ID NO:63):EELYNSSWYFDY; A0033LCDR1(SEQ ID NO:64):RASQGISSYLA; A0033LCDR2(SEQ ID NO:65):AASSLQS; A0033LCDR3(SEQ ID NO:66):QQHYTTPPT; A0033VH(SEQ ID NO:67):QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIKHSGRTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREELYNSSWYFDYWGQGTLVTVSS; A0033VL(SEQ ID NO:68):DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK; A0033H(SEQ ID NO:69):QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIKHSGRTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREELYNSSWYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; A0033L(SEQ ID NO:70):DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; A0041HCDR1(SEQ ID NO:71):GYYWS; A0041HCDR2(SEQ ID NO:72):EIKHSGRTNYNPSLKS; A0041HCDR3(SEQ ID NO:73):TSVTTWNFDY; A0041LCDR1(SEQ ID NO:74):RASQGISSYLA; A0041LCDR2(SEQ ID NO:75):AASSLQS; A0041LCDR3(SEQ ID NO:76):QQHYTTPPT; A0041VH(SEQ ID NO:77):QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIKHSGRTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARTSVTTWNFDYWGQGTLVTVSS; A0041VL(SEQ ID NO:78):DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK; A0041H (SEQ ID NO: 79): QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIKHSGRTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARTSVTTWNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; A0041L (SEQ ID NO: 80): DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0087] Example 3 Verification of the target binding specificity of the CD3 antibody: 1. Binding of the antibody to the Jurkat cell line and the CD3E-KO Jurkat cell line: To detect the specific binding ability of the recombinant candidate antibody expressed in Example 2 to the CD3E antigen, flow cytometry was used for analysis. Wild-type human Jurkat cells (expressing CD3E) and CD3E knockout (CD3E-KO) Jurkat cells (not expressing CD3E) were used. The specific steps were as follows: Wild-type and CD3E-KO Jurkat cells in logarithmic growth phase were collected, washed and resuspended in phosphate-buffered saline (PBS) containing 1% fetal bovine serum, and the cell density was adjusted to 1 × 10⁶ cells / year. 6 Cells / tubes. Eight candidate fully human antibodies expressed recombinantly in Example 2 were added, with an isotype control antibody (human IgG1, kappa-Isotype Control) used as a negative control (IgG1). Cells were incubated at 4°C in the dark for 30 minutes. After incubation, cells were washed twice with PBS to remove unbound antibodies. Then, an appropriate amount of fluorescein-labeled anti-human IgG secondary antibody was added, and the cells were incubated at 4°C in the dark for 30 minutes. After washing the cells twice again, they were resuspended in PBS and immediately analyzed by flow cytometry. Flow cytometry data were analyzed, and antibody binding capacity was assessed using mean fluorescence intensity (MFI).

[0088] The results are as follows Figure 1 and Figure 2 As shown, flow cytometry analysis revealed that all eight screened antibodies exhibited specific binding signals on the surface of wild-type Jurkat cells, while their binding signals to CD3E knockout Jurkat cells decreased to background levels. This indicates that all eight fully human antibodies screened specifically recognize CD3E.

[0089] 2. Binding of antibodies to human PBMC cell lines: To verify whether the candidate antibody could recognize the CD3E antigen expressed on the surface of innate immune cells, flow cytometry analysis was performed using human peripheral blood mononuclear cells (PBMCs). PBMCs were washed and resuspended in phosphate-buffered saline (PBS) and the cell density was adjusted to approximately 1 × 10⁻⁶ cells / mL. 6 Cells / tubes. Eight candidate fully human antibodies expressed recombinantly as described in Example 2 were added, with an isotype control antibody used as a negative control. Cells were incubated at 4°C in the dark for 30 minutes. After incubation, cells were washed twice with PBS to remove unbound antibodies. Then, an appropriate amount of fluorescein-labeled anti-human IgG secondary antibody was added, and the cells were incubated at 4°C in the dark for 30 minutes. After washing the cells twice more, they were resuspended in PBS and immediately analyzed by flow cytometry. The binding ability of the antibodies to the native CD3 molecules on the surface of PBMCs was assessed by analyzing the mean fluorescence intensity (MFI) of the lymphocyte population or the percentage of positive cells.

[0090] The results are as follows Figure 3As shown, flow cytometry analysis revealed significant binding signals on the surface of human PBMCs for all eight screened antibodies. This indicates that all eight antibodies specifically bind to naturally expressed CD3 molecules on the surface of human peripheral blood mononuclear cells, confirming their ability to recognize natively conformed CD3 antigens.

[0091] 3. Binding of antibodies to cynomolgus monkey-derived PBMC cell lines: To assess whether candidate antibodies cross-react with cynomolgus monkey CD3 antigen and to screen suitable candidate molecules for subsequent preclinical animal experiments, flow cytometry analysis was performed using cynomolgus monkey peripheral blood mononuclear cells (PBMCs). Cynomolgus monkey PBMCs were washed and resuspended in phosphate-buffered saline (PBS) and the cell density was adjusted to approximately 1 × 10⁻⁶ cells / mL. 6 Cells / tubes. Eight candidate fully human antibodies recombinantly expressed in Example 2 were added, with an isotype control antibody used as a negative control. Cells were incubated at 4°C in the dark for 30 minutes. After incubation, cells were washed twice with PBS to remove unbound antibodies. Then, an appropriate amount of fluorescein-labeled (e.g., FITC or APC) anti-human IgG secondary antibody was added, and the cells were incubated at 4°C in the dark for 30 minutes. After washing the cells twice more, they were resuspended in PBS and immediately analyzed by flow cytometry. The cross-binding ability of each antibody to CD3 molecules on the surface of cynomolgus monkey PBMCs was assessed by analyzing the mean fluorescence intensity (MFI) of the lymphocyte population.

[0092] The results are as follows Figure 4 As shown, flow cytometry analysis revealed significant binding signals of anti-CD3 antibodies A0005 and A0041 on the surface of cynomolgus monkey PBMC cells; however, no significant binding signals were detected for antibodies 444, A1441, A0010, A0013, A0018, and A0033 on the surface of cynomolgus monkey PBMC cells.

[0093] In summary, A0005 and A0041 specifically bind to the CD3 antigen on the surface of peripheral blood mononuclear cells of cynomolgus monkeys, demonstrating their cross-reactivity with cynomolgus monkey CD3. This supports the use of cynomolgus monkeys as the relevant animal species for preclinical efficacy and safety evaluation. The remaining antibodies (444, A1441, A0010, A0013, A0018, and A0033) did not show any binding activity with cynomolgus monkey CD3 under the experimental conditions, indicating species-specific limitations of their binding epitopes.

[0094] Example 4: Analysis of the effect of CD3 antibody on T cell activation activity: Assay of CD3 antibody T cell activation activity based on the Jurkat-NFAT-Luc reporter gene system: The T-cell agonistic activity of anti-CD3 antibodies was assessed using the NFAT-Luc Jurkat reporter gene cell line. This cell line was stably transfected with an NFAT (activated T cell nuclear factor) response element-driven luciferase reporter gene, and its luciferase expression level was positively correlated with the activation intensity of the T cell receptor (TCR) / CD3 signaling pathway.

[0095] The specific experimental steps are as follows: (1) Cell seeding: One day in advance, NFAT-Luc Jurkat reporter cells in the logarithmic growth phase were seeded into 96-well white cell culture plates at a density of 5 × 10⁶ cells per well. 4 Cells were cultured overnight at 37°C in a 5% CO2 incubator to ensure that the cell confluence reached 50-70% during the experiment.

[0096] (2) Antibody treatment: The eight fully human anti-CD3 antibodies screened in this invention were diluted with serum-free culture medium. Negative controls (isotype control antibodies) and positive controls (Tarlatamab and OKT3) were also established. The old culture medium was discarded, and 50 μL of the diluted antibody solution was added to each well. The solutions were incubated at 37°C in a 5% CO2 incubator for 6 hours.

[0097] (3) Luciferase activity assay: After incubation, remove the culture plate and bring it to room temperature. Add an equal volume of luciferase substrate assay reagent to each well and immediately use a multi-functional microplate reader to detect the chemiluminescence value (RFLU, relative fluorescence unit). The experimental results are expressed as luminescence value (RFLU), reflecting the T cell activation activity of different antibodies.

[0098] The results are as follows Figure 5 ( Figure 5 B in the text is Figure 5 As shown in the enlarged view of A in the image, in the Jurkat-NFAT-Luc reporter gene detection system, the antibody treatment groups A0005, A0033, and A0041 showed significantly higher NFAT luciferase signal responses than the background, indicating that they can effectively induce the activation of the signaling pathway of the reporter system. Although the antibody treatment groups A0005, A1441, A0010, A0013, and A0018 could detect signaling pathway responses, their activation intensity was significantly lower than that of the A0005, A0033, and A0041 treatment groups.

[0099] The CD3 antibodies provided by this invention exhibit significant differentiation characteristics in T cell activation: antibodies A0005, A0033, and A0041 possess potent T cell agonistic activity and can significantly activate the NFAT signaling pathway; while antibodies 444, A1441, A0010, A0013, and A0018 only show weak agonistic activity. This suggests that different CD3 antibodies can regulate the intensity of T cell activation by recognizing different epitopes or having differentiated cross-linking efficiencies, providing a key functional classification basis for developing CD3-targeted therapies with controllable immunomodulatory functions.

[0100] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0101] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A CD3 antibody or its antigen-binding fragment, characterized in that, The amino acid sequences of VH CDR 1, 2, and 3 are shown in SEQ ID NO: 71, 72, and 73, and the amino acid sequences of VL CDR 1, 2, and 3 are shown in SEQ ID NO: 74, 75, and 76.

2. The CD3 antibody or its antigen-binding fragment according to claim 1, characterized in that, Include: VH having at least 90% identity with the selected VH amino acid sequence, and VL having at least 90% identity with the selected VL amino acid sequence; The selected VH amino acid sequence is SEQ ID NO: 77, and the selected VL amino acid sequence is SEQ ID NO:

78.

3. The CD3 antibody or its antigen-binding fragment according to claim 2, characterized in that, The VH has the amino acid sequence shown in SEQ ID NO: 77, and the VL has the amino acid sequence shown in SEQ ID NO:

78.

4. A nucleic acid, characterized in that, Encodes the CD3 antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

5. A carrier, characterized in that, It includes the nucleic acid described in claim 4.

6. A carrier pair, characterized in that, The vector contains the nucleic acid of claim 4, wherein the vector pairs encode VL and VH together, and the VL and VH together bind to CD3.

7. A cell, characterized in that, Expressing the nucleic acid of claim 4, the vector of claim 5, and / or the vector pair of claim 6.

8. A pharmaceutical composition for treating hyperproliferative disorders, characterized in that, include: (1) A therapeutically effective amount of the CD3 antibody or its antigen-binding fragment as described in any one of claims 1 to 3, the nucleic acid as described in claim 4, the vector as described in claim 5, the vector pair as described in claim 6, and / or the cell as described in claim 7; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

9. A pharmaceutical preparation for treating hyperproliferative disorders, characterized in that, Includes the pharmaceutical composition of claim 8.

10. A pharmaceutical product for treating hyperproliferative disorders, characterized in that, Includes the pharmaceutical preparation described in claim 9.

11. A detection reagent, characterized in that, Includes the CD3 antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

12. A test kit, characterized in that, Includes the detection reagent as described in claim 11.

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