Anti-CD3 antibodies and uses thereof

Abstract

The present invention provides anti-CD3 antibodies and uses thereof. Specifically, the anti-CD3 antibody of the invention can specifically bind to human and monkey CD3 proteins, and has similar binding properties. In addition, the CD3 antibodies of the invention are directed to a variety of different forms of human CD3E proteins (including hCD3Eamp; ), has excellent biological activity, and has significantly improved safety. The invention also provides a humanized anti-CD3 antibody, and the humanized antibody also has the properties of low immunogenicity, high hydrophilicity, high stability and the like, and is suitable for drug development.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically, to anti-CD3 antibodies and their uses. Background Art

[0002] CD3 (Cluster of Differentiation 3), a co-receptor of T cells, is a protein complex. CD3 has five peptide chains, namely, γ chain, δ chain, ε chain, ζ chain and η chain, and all these five chains are transmembrane proteins. The transmembrane region of the CD3 molecule is connected to the transmembrane regions of two TCR peptide chains through salt bridges to form a TCR-CD3 complex, which jointly participates in the recognition of antigens by T cells. The activation signal generated by TCR recognition of antigens is transduced into T cells by CD3.

[0003] CD3 and TCR jointly form a CD3-TCR complex, which plays a key role in regulating most functions of T cell innate and acquired immune responses, as well as cell immune function and humoral immune function. These functions include eliminating pathogens and controlling tumor growth through various cytotoxic effects.

[0004] Mouse monoclonal antibodies specifically targeting human CD3, such as OKT3 (Kung et al. (1979) Science 206: 347-9), are the first generation of CD3 antibodies for treatment. Although OKT3 has strong immunosuppressive potency, the severe side effects associated with its strong immunogenicity and mitogenic potential have hindered its clinical use (Chatenoud (2003) Nature Reviews 3: 123-132). Due to the immunogenicity of OKT3, it can induce the production of neutralizing antibodies in the human body, resulting in the rapid clearance and neutralization of OKT3 itself (Chatenoud et al. (1982) Eur. J. Immunol. 137: 830-8).

[0005] In addition, OKT3 induces T cell proliferation and the in vitro production of cytokines, and causes the in vivo release of a large amount of cytokines (Hirsch et al. (1989) J. Immunol 142: 737-43, 1989). The release of cytokines (also known as "cytokine storm") in turn causes "flu-like" symptoms, which are characterized by fever, chills, headache, nausea, vomiting, diarrhea, dyspnea, septic meningitis and hypotension (Chatenoud, 2003). These severe side effects limit the wider use of OKT3 in transplantation and its extension to other clinical fields (such as autoimmunity).

[0006] CD3 bispecific antibodies developed based on anti-human CD3 antibodies have currently become important therapeutic drugs in clinical practice. However, there are still some common problems with CD3 bispecific antibodies at present. For example, the CD3 antibody sequence L2K-07 used in the first CD3 bispecific antibody Blinatumomab developed by Amgen is an antibody that only binds to human CD3 but not to monkey CD3, resulting in the use of chimpanzees as relevant species animals in the preclinical safety studies of this drug.

[0007] Basically, the CD3 antibody sequences used in some other CD3 bispecific antibodies all face the problem of too high affinity, leading to more cytokine release and bringing significant safety risks (Karin Staflin et al., JCI Insight. 2020; 5(7):e133757.). For example, in the phase I clinical study of the Xmab1405 (CD123-CD3) bispecific antibody of Xencor in the United States, a subject died due to cytokine storm, and then the clinical trial was suspended by the US FDA. Coincidentally, the odronextamab (CD20-CD3) bispecific antibody of Regeneron in the United States also caused the death of a subject due to serious adverse reactions, and thus was once suspended from clinical trials by the US FDA. After the CD3 affinity is too high, the therapeutic window of the drug is reduced. The same problem also occurred in the BCMA-CD3 bispecific antibody project of Regeneron. Its first-generation product, linvoseltamab, has currently been applied for marketing in Europe. However, due to the problems encountered in clinical development, a second-generation product, REGN5459, was developed. Compared with the first-generation product, linvoseltamab, the CD3 cell affinity of REGN5459 is reduced by more than 10 times, so it is expected to bring a larger therapeutic window.

[0008] Therefore, there is still an urgent need in this field to develop new anti-CD3 antibodies with good safety, suitable for preclinical studies, and excellent biological activity. Summary of the Invention

[0009] The object of the present invention is to provide a new anti-CD3 antibody (including humanized antibody) with good safety, suitable for preclinical studies, and excellent biological activity.

[0010] In the first aspect of the present invention, there is provided an anti-CD3 antibody or its antigen-binding fragment, which comprises a heavy-chain variable region and a light-chain variable region. Among them, the heavy-chain variable region and the light-chain variable region have 6 complementarity-determining regions CDRs selected from the following group:

[0011] (1a) Three complementarity-determining regions VH-CDR of the heavy-chain variable region and three complementarity-determining regions VL-CDR of the light-chain variable region defined based on the Kabat rule:

[0012] The VH-CDR1 shown in SEQ ID NO:5,

[0013] The VH-CDR2 shown in SEQ ID NO:6 or 33,

[0014] The VH-CDR3 shown in SEQ ID NO:7,

[0015] The VL-CDR1 shown in SEQ ID NO:11,

[0016] The VL-CDR2 shown in SEQ ID NO:12,

[0017] The VL-CDR3 shown in SEQ ID NO:13; or

[0018] (1b) Three complementarity-determining regions VH-CDR of the heavy-chain variable region and three complementarity-determining regions VL-CDR of the light-chain variable region defined based on IMGT rules:

[0019] The VH-CDR1 shown in SEQ ID NO:8,

[0020] The VH-CDR2 shown in SEQ ID NO:9 or 34,

[0021] The VH-CDR3 shown in SEQ ID NO:10,

[0022] The VL-CDR1 shown in SEQ ID NO:14,

[0023] The VL-CDR2 shown in SEQ ID NO:15,

[0024] The VL-CDR3 shown in SEQ ID NO:16; or

[0025] (2a) Three complementarity-determining regions VH-CDR of the heavy-chain variable region and three complementarity-determining regions VL-CDR of the light-chain variable region defined based on Kabat rules:

[0026] The VH-CDR1 shown in SEQ ID NO:17,

[0027] The VH-CDR2 shown in SEQ ID NO:18 or 35,

[0028] The VH-CDR3 shown in SEQ ID NO:19,

[0029] The VL-CDR1 shown in SEQ ID NO:23,

[0030] The VL-CDR2 shown in SEQ ID NO:24,

[0031] the VL-CDR3 shown in SEQ ID NO:25; or

[0032] (2b) three complementarity-determining regions VH-CDR of the heavy-chain variable region and three complementarity-determining regions VL-CDR of the light-chain variable region defined according to the IMGT rules:

[0033] VH-CDR1 shown in SEQ ID NO:20 or 36,

[0034] VH-CDR2 shown in SEQ ID NO:21

[0035] VH-CDR3 shown in SEQ ID NO:22,

[0036] VL-CDR1 shown in SEQ ID NO:26,

[0037] VL-CDR2 shown in SEQ ID NO:27,

[0038] VL-CDR3 shown in SEQ ID NO:28;

[0039] wherein any one of the above amino acid sequences further includes a derivative sequence which is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain the CD3 binding affinity.

[0040] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-5 (such as 1-3, preferably 1-2, more preferably 1).

[0041] In another preferred embodiment, the derivative sequence which is added, deleted, modified and / or substituted with at least one amino acid and can retain the CD3 binding affinity is an amino acid sequence with a homology or sequence identity of at least 96%.

[0042] In another preferred embodiment, the antibody or its antigen-binding fragment is partially or fully humanized.

[0043] In another preferred embodiment, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain of the antibody comprises the three heavy-chain CDRs and a heavy-chain framework region for connecting the heavy-chain CDRs, and the light chain of the antibody comprises the three light-chain CDRs and a light-chain framework region for connecting the light-chain CDRs.

[0044] In another preferred embodiment, the sequence of the heavy-chain variable region is as shown in SEQ ID NO:29, 31, 1 or 3.

[0045] In another preferred embodiment, the heavy chain of the antibody or its antigen-binding fragment further comprises a heavy chain constant region.

[0046] In another preferred embodiment, the heavy chain constant region is human or murine, preferably human.

[0047] In another preferred embodiment, the sequence of the light chain variable region is as shown in SEQ ID NO: 30, 32, 2 or 4.

[0048] In another preferred embodiment, the light chain of the antibody or its antigen-binding fragment further comprises a light chain constant region.

[0049] In another preferred embodiment, the light chain constant region is human or murine, preferably human.

[0050] In another preferred embodiment, the antibody or its antigen-binding fragment specifically binds to CD3.

[0051] In another preferred embodiment, the CD3 is from human or cynomolgus monkey.

[0052] In another preferred embodiment, the antibody is a diabody or a single-chain antibody (scFv).

[0053] In another preferred embodiment, the antibody is a monoclonal antibody.

[0054] In another preferred embodiment, the antibody comprises a monospecific, bispecific, or trispecific antibody.

[0055] In another preferred embodiment, the bispecific antibody comprises:

[0056] (1) an anti-CD3 antibody as described in the first aspect of the present invention;

[0057] (2) an antibody that binds to other targets.

[0058] In another preferred embodiment, the other targets are selected from the group consisting of: BCMA, GPRC5D, EGFR, CD38, CD123, CD19, CD20, CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD47, CTLA4, PD1, PDL1, DLL3, SEZ6, B7-H6, MUC-16, FRa, ALPPL2, ALPP, B7-H4, STEAP1 and STEAP2.

[0059] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 30; or the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 32; or the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2; or the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 4.

[0060] In a second aspect of the present invention, there is provided a recombinant protein, which comprises:

[0061] (i) an anti-CD3 antibody or its antigen-binding fragment as described in the first aspect of the present invention; and

[0062] (ii) optionally, a tag sequence for assisting expression and / or purification.

[0063] In another preferred embodiment, the tag sequence includes a 6His tag.

[0064] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0065] In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

[0066] In another preferred embodiment, the recombinant protein is a monospecific, bispecific, or trispecific recombinant protein.

[0067] In another preferred embodiment, the recombinant protein is a monospecific, bispecific, or trispecific antibody.

[0068] In another preferred embodiment, the recombinant protein further includes an additional fusion element (or fusion polypeptide fragment) fused to the element (i).

[0069] In another preferred embodiment, the recombinant protein comprises:

[0070] (i) an antibody selected from the group consisting of, wherein,

[0071] the heavy chain variable region of the antibody contains the amino acid sequence as shown in SEQ ID NO: 29; and the light chain variable region of the antibody contains the amino acid sequence as shown in SEQ ID NO: 30; or

[0072] The antibody contains the amino acid sequence shown in SEQ ID NO: 31; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 32; or

[0073] The heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 2; or

[0074] The heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 4;

[0075] and

[0076] (ii) Optionally, a tag sequence for assisting expression and / or purification.

[0077] In the third aspect of the present invention, a CAR construct is provided. The scFv segment of the antigen-binding region of the CAR construct is a binding region specifically binding to CD3, and the scFv segment has a heavy chain variable region and a light chain variable region. Among them, the heavy chain variable region and the light chain variable region have 6 complementarity-determining regions CDR selected from the following group:

[0078] (1a) Three complementarity-determining regions VH-CDR of the heavy chain variable region and three complementarity-determining regions VL-CDR of the light chain variable region defined based on Kabat rules:

[0079] (1a) Three complementarity-determining regions VH-CDR of the heavy chain variable region and three complementarity-determining regions VL-CDR of the light chain variable region defined based on Kabat rules:

[0080] VH-CDR1 shown in SEQ ID NO: 5,

[0081] VH-CDR2 shown in SEQ ID NO: 6 or 33,

[0082] VH-CDR3 shown in SEQ ID NO: 7,

[0083] VL-CDR1 shown in SEQ ID NO: 11,

[0084] VL-CDR2 shown in SEQ ID NO: 12,

[0085] VL-CDR3 shown in SEQ ID NO: 13; or

[0086] (1b) The three complementarity-determining regions VH-CDR of the heavy-chain variable region and the three complementarity-determining regions VL-CDR of the light-chain variable region defined based on the IMGT rules:

[0087] VH-CDR1 shown in SEQ ID NO:8,

[0088] VH-CDR2 shown in SEQ ID NO:9 or 34,

[0089] VH-CDR3 shown in SEQ ID NO:10,

[0090] VL-CDR1 shown in SEQ ID NO:14,

[0091] VL-CDR2 shown in SEQ ID NO:15,

[0092] VL-CDR3 shown in SEQ ID NO:16; or

[0093] (2a) The three complementarity-determining regions VH-CDR of the heavy-chain variable region and the three complementarity-determining regions VL-CDR of the light-chain variable region defined based on the Kabat rules:

[0094] VH-CDR1 shown in SEQ ID NO:17,

[0095] VH-CDR2 shown in SEQ ID NO:18 or 35,

[0096] VH-CDR3 shown in SEQ ID NO:19,

[0097] VL-CDR1 shown in SEQ ID NO:23,

[0098] VL-CDR2 shown in SEQ ID NO:24,

[0099] VL-CDR3 shown in SEQ ID NO:25; or

[0100] (2b) The three complementarity-determining regions VH-CDR of the heavy-chain variable region and the three complementarity-determining regions VL-CDR of the light-chain variable region defined based on the IMGT rules:

[0101] VH-CDR1 shown in SEQ ID NO:20 or 36,

[0102] VH-CDR2 shown in SEQ ID NO:21

[0103] VH-CDR3 shown in SEQ ID NO:22,

[0104] VL-CDR1 as shown in SEQ ID NO:26,

[0105] VL-CDR2 as shown in SEQ ID NO:27,

[0106] VL-CDR3 as shown in SEQ ID NO:28.

[0107] In a fourth aspect of the present invention, there is provided a recombinant immune cell that expresses an exogenous CAR construct as described in the third aspect of the present invention.

[0108] In another preferred embodiment, the immune cell is selected from the group consisting of NK cells and T cells.

[0109] In another preferred embodiment, the immune cell is derived from a human or non-human mammal (such as a mouse).

[0110] In a fifth aspect of the present invention, there is provided an antibody-drug conjugate that contains:

[0111] (a) an antibody moiety selected from the group consisting of an antibody as described in the first aspect of the present invention or an antigen-binding fragment thereof, or a recombinant protein as described in the second aspect of the present invention, or a combination thereof; and

[0112] (b) a conjugate moiety conjugated to the antibody moiety, the conjugate moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0113] In another preferred embodiment, the antibody moiety is conjugated to the conjugate moiety via a chemical bond or a linker.

[0114] In a sixth aspect of the present invention, there is provided a use of an active ingredient selected from the group consisting of an antibody as described in the first aspect of the present invention or an antigen-binding fragment thereof, or a recombinant protein as described in the second aspect of the present invention, a CAR construct as described in the third aspect of the present invention, an immune cell as described in the fourth aspect of the present invention, an antibody-drug conjugate as described in the fifth aspect of the present invention, or a combination thereof, wherein the active ingredient is used for:

[0115] (a) preparing a detection reagent or kit;

[0116] (b) preparing a drug or preparation for preventing and / or treating CD3-related diseases; and / or

[0117] (c) preparing a drug or preparation for preventing and / or treating CD3-related cancers or tumors.

[0118] In another preferred embodiment, the cancer or tumor is selected from the group consisting of: lung cancer, melanoma, colon cancer, pancreatic cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, liver cancer, lymphoma, myeloma, leukemia.

[0119] In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

[0120] (i) An active ingredient selected from the group consisting of: an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, a CAR construct as described in the third aspect of the present invention, an immune cell as described in the fourth aspect of the present invention, an antibody-drug conjugate as described in the fifth aspect of the present invention, or a combination thereof; and

[0121] (ii) A pharmaceutically acceptable carrier.

[0122] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0123] In another preferred embodiment, the pharmaceutical composition is an injection.

[0124] In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating a tumor selected from the group consisting of: lung cancer, melanoma, colon cancer, pancreatic cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, liver cancer, lymphoma, myeloma, leukemia.

[0125] In an eighth aspect of the present invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

[0126] (1) An antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; or

[0127] (2) A recombinant protein as described in the second aspect of the present invention;

[0128] (3) A CAR construct as described in the third aspect of the present invention.

[0129] In a ninth aspect of the present invention, there is provided a vector containing the polynucleotide as described in the eighth aspect of the present invention.

[0130] In another preferred embodiment, the vector includes: a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.

[0131] In a tenth aspect of the present invention, there is provided a genetically engineered host cell, which contains the vector as described in the ninth aspect of the present invention or the polynucleotide as described in the eighth aspect of the present invention integrated into its genome.

[0132] In another preferred embodiment, the host cell is a mammalian cell or a prokaryotic cell.

[0133] In another preferred embodiment, the mammalian cell is a Chinese hamster ovary (CHO) cell.

[0134] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0135] In an eleventh aspect of the present invention, there is provided a method for non-diagnostically detecting CD3 protein in a sample in vitro, the method comprising the steps of:

[0136] (1) In vitro, contacting the sample with the antibody as described in the first aspect of the present invention;

[0137] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD3 protein in the sample.

[0138] In a twelfth aspect of the present invention, there is provided a test plate, which comprises a substrate (support plate) and a test strip, and the test strip contains the antibody as described in the first aspect of the present invention or the antibody-drug conjugate as described in the fifth aspect of the present invention.

[0139] In a thirteenth aspect of the present invention, there is provided a kit, which comprises:

[0140] (1) A first container, which contains the antibody as described in the first aspect of the present invention; and / or

[0141] (2) A second container, which contains a secondary antibody against the antibody as described in the first aspect of the present invention;

[0142] Alternatively, the kit contains the test plate as described in the twelfth aspect of the present invention.

[0143] In a fourteenth aspect of the present invention, there is provided a method for preparing a recombinant polypeptide, the method comprising:

[0144] (a) Culturing the host cell as described in the tenth aspect of the present invention under suitable expression conditions;

[0145] (b) Isolating the recombinant polypeptide from the culture, and the recombinant polypeptide is the antibody as described in the first aspect of the present invention or the recombinant protein as described in the second aspect of the present invention.

[0146] In a fifteenth aspect of the present invention, there is provided a method for treating a disorder associated with CD3 molecules, comprising the step of administering to a subject in need of inhibition or treatment an antibody as described in the first aspect of the present invention, or a recombinant protein as described in the second aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect of the present invention.

[0147] In another preferred embodiment, the diseases associated with CD3 molecules include tumors or antagonizing immune rejection of organ transplantation.

[0148] In another preferred embodiment, the subject is a mammal (including humans).

[0149] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] Figure 1 shows the ELISA binding curves of anti-CD3 murine antibodies to human CD3E, human CD3E&G protein, and human CD3E&D protein respectively. Figure 1A Showing the ELISA binding curve of anti-CD3 murine antibody to human CD3E protein; Figure 1B Showing the ELISA binding curve of anti-CD3 murine antibody to human CD3E&G protein; Figure 1C Showing the ELISA binding curve of anti-CD3 murine antibody to human CD3E&D protein.

[0151] Figure 2 shows the ELISA binding curves of anti-CD3 murine antibodies to cynomolgus monkey CD3E&D protein and cynomolgus monkey CD3E&G protein respectively. Figure 2A Showing the ELISA binding curve of anti-CD3 murine antibody to cynomolgus monkey CD3E&D protein; Figure 2B Showing the ELISA binding curve of anti-CD3 murine antibody to cynomolgus monkey CD3E&G protein.

[0152] Figure 3 Showing the FACS binding curve of anti-CD3 murine antibody to human Jurkat cells.

[0153] Figure 4 Showing the FACS binding curve of anti-CD3 murine antibody to human peripheral blood mononuclear cells.

[0154] Figure 5 Showing the FACS binding curve of anti-CD3 murine antibody to cynomolgus monkey peripheral blood mononuclear cells.

[0155] Figure 6 shows the ELISA binding curves of the anti-CD3 humanized antibody with human CD3E protein, human CD3E&D protein, human CD3E&G, monkey CD3E&D protein, and monkey CD3E&G protein respectively. Figure 6A Showing the ELISA binding curve of the anti-CD3 humanized antibody with human CD3E protein; Figure 6B Showing the ELISA binding curve of the anti-CD3 humanized antibody with human CD3E&D protein; Figure 6C Showing the ELISA binding curve of the anti-CD3 humanized antibody with human CD3E&G protein; Figure 6D Showing the ELISA binding curve of the anti-CD3 humanized antibody with monkey CD3E&D protein; Figure 6E Showing the ELISA binding curve of the anti-CD3 humanized antibody with monkey CD3E&G protein.

[0156] Figure 7 Showing the FACS binding curve of the anti-CD3 humanized antibody with human Jurkat cells.

[0157] Figure 8 Showing the FACS binding curve of the anti-CD3 humanized antibody with human peripheral blood mononuclear cells.

[0158] Figure 9 Showing the FACS binding curve of the anti-CD3 humanized antibody with monkey peripheral blood mononuclear cells.

[0159] Figure 10 Showing the kinetic binding and dissociation curves of the anti-CD3 humanized antibody with human CD3E&G protein and monkey CD3E&G protein.

[0160] Figure 11 Showing the activity curve of the anti-CD3 humanized antibody in the Jurkat reporter system.

[0161] Figure 12 Showing the activity curve of the anti-CD3 humanized antibody in activating human T cells. Detailed implementation manners

[0162] Through extensive and in-depth research and a large number of screenings, the inventors of the present invention unexpectedly obtained an anti-CD3 antibody with similar binding properties and high biological activities to CD3 of humans and other primates (such as monkeys). Experiments show that the CD3 antibody of the present invention can not only specifically bind to human and monkey CD3, but also has similar binding properties (such as KD). In addition, the CD3 antibody of the present invention also has excellent biological activities against different forms of human CD3E protein (including hCD3E&D heterodimer, hCD3E&G heterodimer, and CD3E protein on the cell surface), has moderate affinity, and has significantly improved safety. Based on this, the present invention was completed.

[0163] In addition, the present invention also provides a humanized anti-CD3 antibody. The humanized antibody of the present invention not only retains various properties of the murine antibody, for example, specifically binds to human and monkey CD3 with similar binding properties, and has excellent biological activity against different forms of human CD3E protein, but also has high safety, low human immunogenicity, excellent hydrophilicity and stability, etc., and is suitable for drug development.

[0164] Term

[0165] As used herein, the terms "administer" and "treat" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer" and "treat" can refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with the cells, as well as contact of a reagent with a fluid, and contact of a fluid with the cells. "Administer" and "treat" also mean in vitro and ex vivo treatment by a reagent, diagnostic, binding composition, or by another cell. "Treat" when applied to a human, animal, or research subject refers to therapeutic treatment, prophylactic or preventive measures, research, and diagnosis; including contact of a CD3 antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

[0166] As used herein, the term "treatment" refers to the administration to a patient of a therapeutic agent, either internally or externally, including any one of the CD3 antibodies of the present invention and its composition, to a patient having one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the patient is administered an amount of the therapeutic agent (therapeutically effective amount) effective to relieve one or more symptoms of the disease.

[0167] As used herein, the term "optional" or "optionally" means that the subsequent described event or circumstance may occur but is not required to occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may or may not be present, and may be 1, 2, or 3.

[0168] Antibody

[0169] As used herein, the term "antibody" refers to an immunoglobulin, which is a four - peptide chain structure composed of two identical heavy chains and two identical light chains linked by inter - chain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or different isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE. The constant regions of the heavy chains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins. Due to differences in chemical structure and biological function, it can be further divided into 4 subclasses: IgG1, IgG2, IgG3, and IgG4. The light chains are divided into κ or λ chains according to differences in the constant region. The subunit structures and three - dimensional configurations of different classes of immunoglobulins are well - known to those skilled in the art.

[0170] The sequences of approximately 110 amino acids near the N - terminus of the antibody heavy and light chains vary greatly and are the variable regions (V regions); the remaining amino acid sequences near the C - terminus are relatively stable and are the constant regions (C regions). The variable region includes 3 hypervariable regions (HVRs) and 4 relatively conserved framework regions (FRs). The amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. The 3 hypervariable regions determine the specificity of the antibody and are also called complementarity - determining regions (CDRs). Each light - chain variable region (LCVR) and heavy - chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the order from the amino - terminus to the carboxy - terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain, i.e., the light - chain hypervariable regions (LCDRs), refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain, i.e., the heavy - chain hypervariable regions (HCDRs), refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen - binding fragment described in the invention conform to the known Kabat numbering rules (LCDR1 - 3, HCDR2 - 3) or the numbering rules of Kabat and Chothia (HCDR1). The four FR regions in the natural heavy - and light - chain variable regions are generally in a β - sheet configuration, connected by three CDRs forming linker loops, and in some cases, a partial β - sheet structure can be formed. The CDRs in each chain are closely juxtaposed by the FR regions and together with the CDRs of the other chain form the antigen - binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type. The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody - dependent cell cytotoxicity.

[0171] As used herein, the term "antigen-binding fragment" refers to a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a single Fv fragment having antigen-binding activity. An Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of an antibody, but no constant region, and is the smallest antibody fragment having all antigen-binding sites. Generally, an Fv antibody also includes a polypeptide linker between the VH and VL domains and is capable of forming the structure required for antigen binding. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) scFv molecules; (vi) dAb fragments; and (vii) the smallest recognition unit composed of amino acid residues that mimic the hypervariable regions of an antibody (e.g., an independent complementarity-determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. As used herein, the expression "antigen-binding fragment" also encompasses other engineered molecules within, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

[0172] As used herein, the term "epitope" refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the present invention.

[0173] The present invention includes not only intact antibodies, but also fragments of antibodies having immunological activity or fusion proteins formed by antibodies and other sequences. Thus, the present invention also includes fragments, derivatives, and analogs of the antibodies.

[0174] In the present invention, antibodies include murine, chimeric, humanized, or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using DNA recombination techniques well known in the art.

[0175] As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone derived from a single cell source. Monoclonal antibodies are highly specific and directed against a single antigenic epitope. The cells may be eukaryotic, prokaryotic, or phage-derived cloned cell lines.

[0176] As used herein, the term "chimeric antibody" is an antibody molecule in which the V-region gene of a murine antibody is spliced with the C-region gene of a human antibody to form a chimeric gene, which is then inserted into a vector and transfected into a host cell for expression. It retains both the high specificity and affinity of the parental murine antibody and enables its human Fc segment to effectively mediate biological effector functions.

[0177] As used herein, the term "humanized antibody" is a modified form of the murine antibody of the present invention, having CDR regions derived from (or substantially derived from) a non-human antibody (preferably a murine monoclonal antibody), and FR regions and constant regions substantially derived from a human antibody sequence; that is, the CDR region sequences of the murine antibody are grafted onto different types of human germline antibody framework sequences. Since the CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of specific naturally occurring antibodies can be expressed by constructing expression vectors.

[0178] In the present invention, the antibody can be monospecific, bispecific, trispecific, or more multispecific.

[0179] In the present invention, the antibodies of the present invention also include their conservative variants, which refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids in the amino acid sequence of the antibody of the present invention with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0180] Table A

[0181] Initial residue Representative substitution Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0182] Anti-CD3 antibody

[0183] As used herein, the term "CD3" generally refers to native or recombinant human CD3, as well as non-human homologs of human CD3. CD3 is a homodimeric or heterodimeric antigen expressed on T cells that associates with the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is a dimer formed by two of four different chains (ε, ζ, δ, and γ). CD3 dimer arrangements include γ / ε, δ / ε, and ζ / ζ. Thus, unless explicitly stated to be from a non-human species, such as "mouse CD3", "monkey CD3", etc., the term "CD3" refers to human CD3.

[0184] As used herein, "CD3E" refers to the extracellular domain of CD3ε.

[0185] The present invention provides a single-chain antibody (scFv) with high specificity for CD3, which includes a heavy-chain variable region (VH), a light-chain variable region (VL), and a connecting linker.

[0186] The present invention also provides an antibody with high specificity for CD3, which includes a heavy chain and a light chain, the heavy chain containing the amino acid sequence of the heavy-chain variable region (VH), and the light chain containing the amino acid sequence of the light-chain variable region (VL).

[0187] Preferably, the heavy chain variable region and the light chain variable region have 6 complementarity determining regions (CDRs) defined based on the Kabat rules or the IMGT rules, as shown in Table 4 for details.

[0188] Preferably, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 29; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 30; or

[0189] the antibody contains the amino acid sequence shown in SEQ ID NO: 31; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 32; or

[0190] the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 2; or

[0191] the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 4.

[0192] Among them, any one of the above amino acid sequences further includes a derivative sequence with CD3 binding affinity that has been added, deleted, modified, and / or substituted with at least one (such as 1-5, 1-3, preferably 1-2, more preferably 1) amino acid.

[0193] In another preferred example, the sequence formed by adding, deleting, modifying, and / or substituting at least one amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0194] The antibody of the present invention can be a double-stranded or single-chain antibody, and can be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.

[0195] As used herein, the term "scFv" refers to a single-chain antibody (single chain antibody fragment, scFv), which is usually formed by connecting the heavy chain variable region and the light chain variable region of an antibody through a linker of 15-25 amino acids.

[0196] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for the light and heavy chain domains to fold into an exchanged dual variable domain immunoglobulin. In the present invention, the preferred linker refers to a linker that connects the VH and VL of a single-chain antibody (scFv), or is used to link the scFv to the heavy chain of another antibody.

[0197] Examples of suitable linkers include single glycine (Gly), or serine (Ser) residues, and the identity and sequence of the amino acid residues in the linker can vary with the type of secondary structure elements to be achieved in the linker. For example, (G4S)n, where n is an integer from 1 to 5.

[0198] The antibody derivatives of the present invention can be single-chain antibodies, and / or antibody fragments, such as: Fab, Fab', (Fab')2 or other known antibody derivatives in the art, etc., and any one or several of IgA, IgD, IgE, IgG and IgM antibodies or antibodies of other subtypes.

[0199] Among them, the animal is preferably a mammal, such as a mouse.

[0200] The antibody of the present invention can be a murine antibody, chimeric antibody, humanized antibody, CDR grafted and / or modified antibody targeting human CD3.

[0201] The antibody of the present invention can be monospecific, bispecific, trispecific or multispecific. For example, the antibody of the present invention can form a bispecific antibody together with an antibody or active fragment that binds to other targets. The antibody that binds to other targets can be an antibody or its active fragment targeting CD19, CD47, CD73, CD47, CTLA4, PD-1, PD-L1, CD28.

[0202] Preparation of Antibodies

[0203] Any method suitable for generating monoclonal antibodies can be used to generate the anti-CD3 antibody of the present invention. For example, an animal can be immunized with a linked or naturally occurring CD3 homodimer or a fragment thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.

[0204] Any suitable form of CD3 can be used as an immunogen (antigen) for generating non-human antibodies specific to CD3 and screening the biological activities of said antibodies. The immunogen for immunization can be full-length mature human CD3, including the native homodimer, or a peptide containing single / multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenic adjuvants known in the art. The immunogen can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in origin. The DNA encoding the immunogen can be expressed using suitable genetic vectors, including but not limited to adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, plasmids, and non-viral vectors.

[0205] An exemplary method for producing the anti-human CD3 antibody of the present invention is described in Example 1.

[0206] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. In the present invention, the antibody is an IgG antibody and the IgG1 subtype is used. Optimization of the required constant domain sequences to produce the desired biological activities is readily achieved by screening the antibody using the biological assays described in the examples below.

[0207] Similarly, any class of light chains can be used in the compounds and methods herein. Specifically, κ, λ chains or their variants are usable in the compounds and methods of the present invention.

[0208] An exemplary method for humanizing the anti-human CD3 antibody of the present invention is described in Example 2.

[0209] The sequence of the DNA molecule of the antibody or its fragment of the present invention can be obtained by conventional techniques, such as methods using PCR amplification or screening of genomic libraries. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0210] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods.

[0211] In addition, the relevant sequence can also be synthesized by artificial synthesis methods, especially when the fragment length is short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. Then the DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.

[0212] The present invention also relates to vectors comprising the appropriate DNA sequences described above and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.

[0213] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.

[0214] The step of transforming the host cell with recombinant DNA described in the present invention can be carried out by techniques well known in the art. The obtained transformants can be cultured by conventional methods, and the transformants express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured in a conventional medium under suitable conditions.

[0215] Generally, the host cells obtained by transformation are cultured under conditions suitable for the expression of the antibodies of the present invention. Then, the antibodies of the present invention are purified by conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other conventional separation and purification means well known to those skilled in the art.

[0216] The obtained monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0217] Antibody-drug conjugate (ADC)

[0218] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0219] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, the antibody is conjugated to the effector molecule, and is preferably chemically conjugated. Among them, the effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule can be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.

[0220] The antibody of the present invention and the effector molecule can be conjugated through a coupling agent. Examples of the coupling agent can be any one or several of non-selective coupling agents, coupling agents using carboxyl groups, peptide chains, and coupling agents using disulfide bonds. The non-selective coupling agent is a compound that forms a covalent bond connection between the effector molecule and the antibody, such as glutaraldehyde, etc. The coupling agent using carboxyl groups can be any one or several of cis-aconitic anhydride-based coupling agents (such as cis-aconitic anhydride) and acylhydrazone-based coupling agents (the coupling site is acylhydrazone).

[0221] Certain residues on the antibody (such as Cys or Lys, etc.) are used to connect with various functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers), and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. The functional agent (such as a drug, detection reagent, stabilizer) is conjugated (covalently linked) to the antibody. The functional agent can be directly or indirectly connected to the antibody through a linker.

[0222] The antibody can be conjugated with a drug to form an antibody-drug conjugate (ADC). Typically, the ADC contains a linker located between the drug and the antibody. The linker can be a degradable or non-degradable linker. The degradable linker typically degrades easily in the intracellular environment. For example, the linker degrades at the target site, so that the drug is released from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-based linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptide-based linker can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide bond linkers). The non-degradable linker typically releases the drug under the condition that the antibody is hydrolyzed by protease.

[0223] Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive groups. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodo-, bromo- or chloro-substituted); haloesters (e.g., iodo-, bromo- or chloro-substituted); halomethyl ketones (e.g., iodo-, bromo- or chloro-substituted), benzyl halides (e.g., iodo-, bromo- or chloro-substituted); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurimethyl) dioxane, and the counterion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonates. The linker can include, for example, a maleimide attached to the antibody through a succinimide.

[0224] The drug can be any cytotoxic, cell growth inhibitory or immunosuppressive drug. In an embodiment, the linker attaches the antibody and the drug, and the drug has a functional group capable of bonding to the linker. For example, the drug can have an amino, carboxyl, thiol, hydroxyl, or keto group capable of bonding to the linker. In the case where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0225] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In the present invention, the drug-linker can be used to form an ADC in a single step. In other embodiments, a bifunctional linker compound can be used to form an ADC in a two-step or multi-step process. For example, a cysteine residue reacts with the reactive moiety of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0226] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkyne groups on the drug moiety. The drug is covalently attached to the linker through a 1,3-dipolar cycloaddition between the azide and the alkyne. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazines and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other conjugation strategies, such as those described in Bioconjugate Techniques, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for the selective reaction of the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for either the linker or the drug.

[0227] Applications

[0228] The present invention provides uses of the antibodies of the present invention, such as for preparing diagnostic agents, or for preparing drugs for preventing and / or treating CD3-related diseases. The CD3-related diseases include inflammatory diseases, autoimmune diseases, etc., including but not limited to psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory bowel disease (such as Crohn's disease, ulcerative colitis, etc.), osteoarthritis, rheumatoid arthritis (RA), rheumatic arthritis or osteoporosis, inflammatory fibrosis (such as scleroderma, pulmonary fibrosis and sclerosis), asthma (including allergic asthma), allergy and cancer.

[0229] Pharmaceutical compositions

[0230] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or the corresponding CAR-T cells, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substances being formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0231] The antibody of the present invention can also be used for cell therapy by intracellular expression of the nucleotide sequence. For example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T), etc.

[0232] The pharmaceutical composition of the present invention can be directly used to bind to CD3 protein molecules, and thus can be used for the prevention and treatment of CD3-related diseases. In addition, other therapeutic agents can also be used simultaneously.

[0233] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or its conjugate) of the present invention as described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by a conventional method with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0234] When using the pharmaceutical composition, a safe and effective amount of the pharmaceutical composition is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight. Preferably, the dosage is about 10 micrograms / kg body weight to about 20 milligrams / kg body weight. Of course, the specific dosage should also consider factors such as the administration route and the health status of the patient, which are within the scope of the skills of a skilled physician.

[0235] Detection uses and kits

[0236] The antibody of the present invention can be used for detection applications, for example, for detecting samples to provide diagnostic information.

[0237] In the present invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsies used in the present invention can include, for example, tissue samples prepared by endoscopic methods or puncture or needle biopsies of organs.

[0238] The samples used in the present invention include fixed or preserved cell or tissue samples.

[0239] The present invention also provides a kit containing the antibody (or its fragment) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, an instruction manual, a buffer, etc. In the preferred embodiment, the antibody of the present invention can be fixed on a detection plate.

[0240] The main advantages of the present invention include

[0241] (1) The CD3 antibody of the present invention specifically binds to human CD3 and has excellent biological activity against different forms of human CD3E protein.

[0242] (2) The CD3 antibody of the present invention has excellent affinity, a larger therapeutic window, and in vivo safety.

[0243] (3) The ability of the CD3 antibody of the present invention to bind to monkey CD3 protein is similar to that of binding to human CD3 protein, which is suitable for preclinical safety studies using monkeys as the species of animals.

[0244] (4) The CD3 antibody of the present invention can be used to construct bispecific antibodies with other antibodies.

[0245] (5) The CD3 antibody of the present invention has excellent hydrophilicity and is suitable for drug development.

[0246] (6) The CD3 antibody of the present invention has good thermal stability and is suitable for drug development.

[0247] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions such as those described by Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0248] Abbreviations

[0249] ELISA: Enzyme-linked immunosorbent assay

[0250] FACS: Fluorescence-activated cell sorting

[0251] TCR: T cell receptor

[0252] CDR: Complementary determining region

[0253] Kabat: An immunoglobulin alignment and numbering system proposed by Elvin A. Kabat

[0254] IMGT: A numbering system based on the international immunogenetics information system initiated by Lefranc et al.

[0255] EC 50 : Half maximal effective concentration, that is, the concentration that can cause 50% of the maximum effect

[0256] ELISA: Enzyme-linked immunosorbent

[0257] PCR: Polymerase Chain Reaction

[0258] HRP: Horseradish Peroxidase

[0259] Example 1. Immunization of Mice with Anti-Human CD3 Antibody

[0260] 1.1 Preparation of CD3 Antigen

[0261] Purchase human CD3E&D heterodimeric protein (Kactus Biosystems, CD3-HM105), human CD3E&G heterodimeric protein (Kactus Biosystems, CD3-HM157), monkey CD3E&D heterodimeric protein (AcroBiosystems, CDD-C52W4), and monkey CD3E&G heterodimeric protein (AcroBiosystems, CDG-C52W6) proteins.

[0262] Human CD3E-ECD-his protein (purchased from Sichuan Sibowo Biotechnology Co., Ltd., Accession#

[0263] P07766-1) was codon-optimized and gene-synthesized onto the PTT5 vector, transiently expressed in HEK293E cells by PEI transfection, and then purified by Ni column.

[0264] Human CD3E / CD3D mRNA Lipid Nanoparticles: The nucleotide sequences encoding amino acids Asp 23-Asp 126 (CD3E) & Phe 22-Ala 105 (CD3D) (Accession# P07766-1 (CD3E) & P04234-1 (CD3D)) were synthesized by Anhui General Biology into an in vitro mRNA synthesis backbone vector (Sichuan Sibowo Biotechnology Co., Ltd.). mRNA synthesis was completed using T7 RNA polymerase (Novizan, product number DD4202-01). After purification, 1 mg of mRNA was dissolved in citrate buffer, and then lipid nanoparticles were prepared by a microfluidic chip (Fluidiclab, LNP-B0). The volume ratio of the mRNA solution to the lipid solution was 3:1. The prepared lipid nanoparticles were dialyzed and exchanged into PBS + 2% sucrose solution, concentrated, sterilized, and then aliquoted and stored at -80 °C for later use.

[0265] 1.2 Immunization of Mice

[0266] Group 1: Three FVB mice and three KM mice were immunized intramuscularly with mRNA lipid nanoparticles encoding human CD3E / CD3D, boosted once every two weeks, for a total of three immunizations. Then, protein ELISA and cell FACS were used to detect the titers of the mice. For the mice with excellent performance, subcutaneous immunization was boosted once with a monkey CD3E&D heterodimer protein. One week before fusion, the mice to be fused were finally boosted with 20 μg of monkey CD3E&G heterodimer protein.

[0267] Group 2: Three Babl / c mice and three CD01 mice were initially immunized with human CD3E&D heterodimer protein or human CD3E&G heterodimer protein emulsified with CFA, and then boosted subcutaneously or abdominally with IFA every two weeks, for a total of four immunizations. Protein ELISA and cell FACS were used to detect the titers of the mice. For the mice with excellent performance, subcutaneous immunization was boosted once with a monkey CD3E&D heterodimer protein. One week before fusion, the mice received an intraperitoneal injection of 1x10 7 Jurkat cells for final boost.

[0268] Group 3: Two Babl / c mice, two CD01 mice, two KM mice, and two FVB mice were immunized with human Jurkat cells. The mice were immunized intraperitoneally once every two weeks for 6 weeks. After 6 weeks, protein ELISA and cell FACS were used to detect the titers of the mice. For the mice with excellent performance, intramuscular immunization was boosted twice with mRNA lipid nanoparticles encoding human CD3E&D. One week before fusion, the mice to be fused were finally boosted with 20 μg of monkey CD3E&G heterodimer protein.

[0269] Example 2. Screening of anti-human CD3 antibody hybridomas

[0270] 2.1 Hybridoma fusion

[0271] The mice with the best titers in mouse immunization groups 1, 2, and 3 were selected for hybridoma fusion. Two different protocols were used. In Protocol 1, mouse spleens and lymphocyte suspensions were directly isolated and prepared, then mixed with SP2 / 0 mouse myeloma cells at a ratio of 1:1, resuspended with cell electrofusion buffer, and an electrofusion reaction was carried out using BTX-ECM2001. They were resuspended with complete fusion medium (RPMI-1640 + 15% FBS + 1×HAT), seeded at 20,000 cells per well into 96-well cell culture plates, and cultured at 37°C and 5% CO2 for 7 days before screening. Protocol 1 was carried out for two rounds of fusion. In the first round, 40 plates were fused, and in the second round, 81 plates were fused. In Protocol 2, after isolating and preparing mouse spleens and lymphocyte suspensions, mouse CD138-positive plasma cells were separated by magnetic beads and then mixed with SP2 / 0 mouse myeloma cells at a ratio of 1:1, resuspended with cell electrofusion buffer, and an electrofusion reaction was carried out using BTX-ECM2001. They were resuspended with complete fusion medium (RPMI160 + 15% FBS + 1×HAT), seeded at 8,000 cells per well into 96-well cell culture plates, and cultured at 37°C and 5% CO2 for 7 days before screening. Protocol 2 was carried out for 2 rounds of fusion, and 10 plates were seeded for each round of fusion.

[0272] 2.2 Hybridoma screening

[0273] 1) ELISA primary screening was carried out using human CD3E&D heterodimer protein. The human CD3E&D heterodimer protein was coated with CBS coating solution at a coating concentration of 0.5 μg / mL and a volume of 100 μL, and coated overnight at 4°C; the next day, the coating solution was removed, each well was washed once with 300 μL of PBS, 100 μL of 2% BSA (in PBS) was added to each well, and blocked at 37°C for 2 hours; 30 μL of the hybridoma supernatant to be screened was added to the enzyme-linked immunosorbent assay (ELISA) plate. Mouse serum was added to well A1 of each ELISA plate as a positive control, and blank medium was added to well A2 as a negative control well, and incubated at 37°C for 2 hours; the supernatant was removed, each well was washed three times with 300 μL of PBST, 100 μL of anti-mouse HRP secondary antibody (Jackson, 115-035-003) (in 2% BSA) was added to each well, and incubated at 37°C for 1 hour; the secondary antibody was removed, each well was washed five times with 300 μL of PBST, then 100 μL of TMB substrate was added to each well, and the color was developed for 1 - 2 minutes; 50 μL of ELISA stop solution (Solarbio, C1058) was added to each well to terminate the reaction, and the OD450 nm reading was measured with an ELISA reader. Wells with signal values greater than 3 times that of the negative wells were selected as positive hybridoma clones for primary screening.

[0274] 2) ELISA rescreening was performed using cynomolgus CD3E&D heterodimer protein. The rescreening process was similar to the primary screening. 30 μL of the hybridoma supernatant of the primary screening positive clones was taken for the cynomolgus CD3E&D heterodimer protein ELISA rescreening, and the positive clones were further screened by Jurkat cell binding FACS. The method for Jurkat cell FACS screening was as follows: Jurkat cells were collected by centrifugation, washed three times with pre-cooled PBS, resuspended with 1% BSA (in PBS), and 2x10 5 cells (50 μL) were plated into each well of a 96-well V-bottom plate with a volume of 50 μL. 50 μL of the 20 positive monoclonal supernatants determined by ELISA rescreening were added to new PCR V-bottom plates respectively. Negative and positive controls with the same volume were added to wells A1 and A2 respectively. The positive control was mouse serum diluted 1:2000, and the negative control was blank hybridoma medium. After pipetting and mixing evenly, it was incubated at 4 °C for 1 hour. After washing three times with pre-cooled PBS, 100 μL of 1% BSA (containing 1 μL of Anti-mouse IgG-PE fluorescent secondary antibody, BioLegend, catalog number 405307) was added to each well, and it was incubated at 4 °C for 0.5 hour. After washing three times with pre-cooled PBS, the cells were resuspended and detected by flow cytometry (CytoFLEX).

[0275] A total of 10 parental clones were selected for subcloning by the above method.

[0276] Subcloning screening: ELISA screening was performed by coating human CD3E&D and cynomolgus CD3E&D heterodimer proteins successively, and cell FACS screening was performed using Jurkat cells. The experimental methods were as described above, and the experimental results are shown in Table 1.

[0277] Among the numerous antibodies, 2 antibodies unexpectedly had excellent binding properties to human CD3E&D, cynomolgus CD3E&D, and Jurkat cells, namely the positive clones 9B5C8 and 17H2B3. The clones of these 2 antibodies were selected and amplified in serum-free medium, and were named CD3-01 and CD03-02 respectively.

[0278] Table 1 Statistical table of anti-human CD3 antibody hybridoma screening results

[0279]

[0280]

[0281] Example 3. Expression and evaluation of anti-human CD3 murine antibodies

[0282] 3.1 Expression and purification of anti-human CD3 murine antibodies

[0283] Hybridoma cells were cultured in serum-free medium, and each clone was cultured in 8 - 10 mL of medium for about 10 days for expression. The monoclonal supernatant was collected by centrifugation, purified by gravity column using ProA resin, eluted with sodium acetate buffer, neutralized with Tris-HCl, and ultrafiltered and exchanged into PBS buffer. The protein concentration was measured by Nanodrop.

[0284] 3.2 Detection of the affinity of mouse-derived anti-human CD3 antibody protein

[0285] To detect the affinity of the mouse-derived CD3 antibody of the present invention for human CD3 protein, human CD3E&D heterodimer protein (0.5 μg / mL), human CD3E&G heterodimer protein (0.5 μg / mL), and human CD3E recombinant protein (0.5 μg / mL) were respectively coated, and coated overnight at 4°C; the next day, the coating solution was removed, each well was washed once with 300 μL of PBS, and 100 μL of 2% BSA (Biotopped, A6020) (in PBS) was added to each well and blocked at 37°C for 2 hours. The antibody to be tested was diluted with 2% BSA, with an initial concentration of 2 μg / mL and 4-fold dilution, for 8 dilution gradients. The diluted antibody was added to the ELISA plate, 100 μL / well, in duplicate. After incubation at 37°C for 2 hours, each well was washed 3 times with 300 μL of PBS. Then 100 μL of Anti-mouse IgG HRP (1:10000) was added to each well, and after incubation at 37°C for 1 hour, each well was washed 5 times with 300 μL of PBS. Subsequently, 100 μL of TMB substrate (Wuxi Infecure, EF-T1) was added to each well, and the color was developed for about 2 minutes; 50 μL of ELISA stop solution (Solarbio, C1058) was added to each well to terminate the reaction, and the OD450 nm reading was measured with an ELISA reader (manufacturer: MD, model: SpectraMax). The absorbance values at 450 nm corresponding to different concentrations of the antibody were fitted with 4 parameters using GraphPad Prism software.

[0286] The results are shown in Figure 1A 、 Figure 1B and Figure 1C , and the specific affinity is shown in Table 2. The CD3 antibody of the present invention has excellent affinity for different forms of human CD3E protein.

[0287] Table 2. Affinity of mouse-derived anti-CD3 antibody for human CD3 protein

[0288]

[0289] 3.3 Cross-detection of mouse-derived anti-human CD3 antibody protein in monkeys

[0290] To detect the cross-reactivity and affinity of the murine CD3 antibody of the present invention with cynomolgus monkey CD3E&D and CD3E&G heterodimer proteins, the cynomolgus monkey CD3E&D heterodimer protein and the cynomolgus monkey CD3E&G heterodimer protein were diluted to 0.5 μg / mL with CBS coating solution, and 100 μL per well was added to the enzyme-linked immunosorbent assay (ELISA) plate and coated overnight at 4°C. Then, the affinity was determined in a similar manner to Example 1.5.2. The absorbance values at 450 nm corresponding to different concentrations of the antibody were subjected to four-parameter fitting using GraphPad Prism software.

[0291] The results are shown in Figure 2A and Figure 2B , and the specific affinity is shown in Table 3. The CD3 antibody of the present invention has excellent affinity for both monkey CD3 heterodimer proteins.

[0292] Surprisingly, based on the data in Table 2 and Table 3, it can be seen that the binding properties of the two antibodies to the monkey CD3 heterodimer protein are basically the same as those to the human CD3 heterodimer protein, indicating that they are very suitable for preclinical studies.

[0293] Table 3. Affinity of anti-CD3 murine antibody and monkey CD3 protein

[0294]

[0295] 3.4 Detection of the affinity of anti-human CD3 murine antibody for Jurkat cells

[0296] To detect the cross-reactivity and affinity of the anti-CD3 murine antibody with the target protein CD3 on the human natural cell surface, flow cytometry was used to detect the affinity of the antibody to be tested. First, Jurkat cells were centrifuged and collected, washed twice with PBS, and resuspended to a density of 8x10 6 / mL. 50 μL per well, the murine CD3 antibody was diluted with 1% BSA, starting from 20 μg / mL, with a four-fold gradient and eight dilution gradients, and added to the cell wells, 50 μL per well, mixed well, and incubated at 4°C for 50 minutes; washed twice with PBS, and the cells were finally resuspended with 1% BSA solution (containing PE anti-human IgG), 100 μL per well (containing 1 μL of fluorescent secondary antibody), mixed well, and reacted at 4°C in the dark for 0.5 hour, washed twice with PBS, and the fluorescence signal value and positive rate were detected by flow cytometry. The fluorescence signal values corresponding to different concentrations of the antibody were subjected to curve fitting using GraphPad Prism software.

[0297] The results are shown in Figure 3 , the CD3 antibody of the present invention has good binding ability to the CD3 protein on the surface of human natural cells Jurkat, and CD3-01 and CD3-02 bind to EC 50They are 9.04 nM and 1.75 nM respectively.

[0298] 3.5 Affinity Detection of Murine Anti-human CD3 Antibody for Human Peripheral Blood Mononuclear Cells

[0299] To detect the cross-reactivity and affinity of the murine anti-CD3 antibody with the target protein CD3 on the human natural cell surface, flow cytometry was used to detect the affinity of the antibody to be tested. First, human peripheral blood mononuclear cells (hPBMC) were isolated from the peripheral blood of healthy volunteers through Ficoll reagent, and then the hPBMC cells were collected by centrifugation, washed twice with PBS, and resuspended to a density of 8x10 6 / mL. 50 μL per well, the murine anti-CD3 antibody was diluted with 1% BSA, starting from 20 μg / mL, with a 4-fold gradient and 8 dilution gradients, and added to the cell wells, 50 μL per well, mixed, and incubated at 4 °C for 50 minutes; washed twice with PBS, and the cells were finally resuspended with 1% BSA solution (containing PE anti-human IgG), 100 μL per well (containing 1 μL of fluorescent secondary antibody), mixed, and reacted in the dark at 4 °C for 0.5 hour, washed twice with PBS, and the fluorescence signal value and positive rate were detected by flow cytometry. The fluorescence signal values corresponding to different concentrations of the antibody were curve-fitted using GraphPad Prism software.

[0300] The results are shown in Figure 4 , and the experimental results show that the CD3 antibody of the present invention has good binding ability to the CD3 protein on the surface of human natural cell PBMC. The binding EC 50 of CD3-01 and CD3-02 are 20.52 nM and 6.07 nM respectively. 3.6 Affinity Detection of Murine Anti-human CD3 Antibody for Rhesus Monkey Peripheral Blood Mononuclear Cells

[0301] To detect the affinity of anti-CD3 murine antibody to CD3 protein on cynomolgus monkey peripheral blood mononuclear cells (cynoPBMC) and evaluate the FACS affinity, peripheral blood collected from cynomolgus monkeys aged 3 - 5 years old was separated by Ficoll reagent to obtain cynomolgus monkey peripheral blood mononuclear cells (cynoPBMC). The PBMC was centrifuged and collected, washed twice with PBS, and resuspended in 1% BSA buffer. HumanTruStain FcX (Biolegend) was added to the cynoPBMC cell suspension and incubated at room temperature for 20 min. After incubation, the cell suspension was added to a 96-well V-bottom plate, 50 μL per well. The anti-CD3 murine antibody was diluted with 1% BSA starting from 20 μg / mL, with a 4-fold gradient and 8 dilution gradients, and added to the cell wells, 50 μL per well. After mixing, it was incubated at 4°C for 50 minutes; washed twice with PBS, and finally the cells were resuspended in 1% BSA solution (containing PE anti-human IgG), 100 μL per well (containing 1 μL of fluorescent secondary antibody), mixed well, and reacted in the dark at 4°C for 0.5 hour. Washed twice with PBS, and the fluorescence signal value and positive rate were detected by flow cytometry. The fluorescence signal values corresponding to different concentrations of the antibody were curve-fitted using GraphPad Prism software.

[0302] The results are shown in Figure 5 , and the experimental results show that the CD3 antibody of the present invention has good binding ability to CD3 protein on the surface of cynomolgus monkey natural cells PBMC, and at the same time, it also shows that the CD3 antibody of the present invention has good cynomolgus monkey cross-reactivity. The binding EC 50 of CD3-01 and CD3-02 are 12.64 nM and 2.32 nM respectively.

[0303] Example 4. Obtaining and humanizing the anti-human CD3 antibody sequence

[0304] 4.1 Retrieving murine sequences: Approximately 1×10 5 candidate hybridoma cells CD03-01 (9B5C8) and CD03-02 (17H2B3) were collected, RNA was extracted by Trizol, and then cDNA was obtained by reverse transcription using the PrimeScript RT reagent kit through PolyA; upstream primers were designed respectively at the upstream of the heavy chain and light chain, and downstream primers were designed in the CH1 region of the heavy chain and the CL region of the light chain. The PCR amplification products were recovered by an agarose gel recovery kit; sequencing was performed. The specific variable region sequences are shown in Table 13 and the sequence listing.

[0305] 4.2 Antibody humanization: Using the CDR grafting method, first, the human germline sequence with the highest homology to the original murine sequence is found by the conventional BLAST method and used as a template; the CDRs of the murine antibody are transplanted onto the human template to construct a chimera; according to the structural analysis of the FR amino acids in the murine antibody that can retain its original conformation, the corresponding amino acids in the chimera are reverted to murine amino acids to maintain the original affinity; the constructed humanized antibody is analyzed for immunogenicity, and highly immunogenic fragments are found and replaced with low immunogenic fragments; at the same time, by performing druggability analysis, obvious post-translational modification sites in the sequence are removed to eliminate unfavorable sites (such as deamidation, isomerization, or glycosylation sites, etc.). In addition, during the humanization process, while ensuring the affinity of the antibody itself, the impact of post-translational modifications on antibody binding and stability is minimized.

[0306] Performance testing and screening were carried out on a variety of different humanized antibodies, and finally, two humanized antibodies CD3-01-Hz and CD3-02-Hz with excellent comprehensive performance were selected. The specific variable region sequences are shown in Table 13 and the sequence listing.

[0307] For the humanized antibody CD3-01-Hz based on CD3-01, at position 55 (Chothia numbering) of the heavy chain, the amino acid G in the corresponding murine antibody was mutated to amino acid A.

[0308] For the humanized antibody CD3-02-Hz based on CD3-02, at position 28 (Chothia numbering) of the heavy chain, the amino acid G in the corresponding murine antibody was mutated to amino acid S.

[0309] The SEQ ID NO numbers of the VH, VL, and 6 CDRs of the murine antibodies CD3-01 and CD3-02, as well as the humanized CD3-01-Hz and CD3-02-Hz, are shown in Table 4 below:

[0310] Table 4 CDR region encoding of anti-human CD3 antibodies

[0311]

[0312] Example 5. Evaluation of anti-human CD3 humanized antibodies

[0313] 5.1 Expression of anti-human CD3 humanized antibodies

[0314] The heavy chain variable region sequences (SEQ NO: 29) and light chain variable region sequences (SEQ NO: 30) of the humanized antibody CD3-01-Hz, the heavy chain variable region sequences (SEQ NO: 31) and light chain variable region sequences (SEQ NO: 32) of CD3-02-Hz, as well as the heavy chain variable region amino acid sequences of the reference antibodies (abbreviated as Roche CD3, the sequence is from KEGG-D11463; REGN CD3, the sequence is from KEGG-D11534) plus the IgG1 mutant constant region sequence (SEQ ID NO: 37) and the light chain variable region sequence plus the Kappa sequence (SEQ ID NO: 38) were submitted to General Biosystems for gene synthesis. After codon optimization, they were constructed into the PTT5 vector. After the plasmid was synthesized, HEK293E cells were transfected with PEI Max and expressed for about 7 days. The supernatant was collected by centrifugation. The supernatant was purified using ProA packing material. All the purified antibodies were ultrafiltered and replaced with PBS buffer, the concentration was measured, and they were stored at -20°C. 5.2 Evaluation of the ELISA affinity of the humanized anti-human CD3 antibody protein

[0315] For the ELISA affinity evaluation, the antigen proteins human CD3E&D heterodimer, human CD3E&G heterodimer, cynomolgus monkey CD3E&D heterodimer, cynomolgus monkey CD3E&G heterodimer, and human CD3E protein were coated with CBS at 0.5 μg / mL. The experimental method was as described in Examples 2 and 3. The antibody to be tested was serially diluted with 2% BSA (starting from 2 μg / mL, 4-fold dilution, 8 concentration points), incubated at 37°C for 2 hours, HRP-labeled anti-human secondary antibody (Jackson, 109-035-088) was added, incubated at 37°C for 1 hour, TMB substrate was added for color development, and the reaction was terminated with ELISA stop solution, then the absorbance at 450 nm was read on the machine.

[0316] The experimental results are shown in Table 5 and Figure 6A , Figure 6B , Figure 6C , Figure 6D and Figure 6E as shown.

[0317] The 2 humanized antibodies have high affinity for human and cynomolgus monkey CD3E and CD3D heterodimer proteins. At the same time, compared with the control antibody Roche CD3, the CD3 antibody of the present invention has comparable ability to bind different forms of CD3.

[0318] Table 5. Evaluation of the ELISA affinity of the humanized anti-human CD3 antibody protein

[0319]

[0320] 5.3 Flow cytometry affinity evaluation of the humanized anti-human CD3 antibody

[0321] To detect the cross-reactivity and affinity of the anti-CD3 humanized antibody with the target protein CD3 on the human natural cell surface, flow cytometry was used to detect the affinity of the antibody to be tested. First, Jurkat cells were collected by centrifugation, washed twice with PBS, and resuspended to a density of 8x10 6 / mL, 50 μL per well. The anti-CD3 humanized antibody was diluted with 1% BSA, starting from 40 μg / mL, with a 4-fold gradient and 8 concentration points, and added to the cell wells, 50 μL per well. After mixing, it was incubated at 4°C for 50 min; washed twice with PBS, and finally the cells were resuspended with 1% BSA solution (containing APC anti-human IgG), 100 μL per well (containing 1 μL of fluorescent secondary antibody). After mixing, it was reacted in the dark at 4°C for 0.5 hour, washed twice with PBS, and the fluorescence signal value and positive rate were detected by flow cytometry. The fluorescence signal values corresponding to different concentrations of the antibody were curve-fitted using GraphPad Prism software.

[0322] The experimental results are shown in Table 6 and Figure 7 as follows. The CD3 antibody of the present invention has good binding ability to human Jurkat cells, and the binding EC 50 values are 17.47 nM and 16.12 nM respectively. The affinity of the CD3 antibody of the present invention for Jurkat cells is significantly lower than that of the Roche CD3 antibody, and is also 2-3 times weaker than REGN CD3. The CD3 antibody of the present invention may provide better safety.

[0323] Table 6. Affinity of humanized antibody for Jurkat cells

[0324] Serial number Number <![CDATA[EC 50 (nM)]]> Maximum signal value 1 CD3 - 01 - Hz 17.47 9099 2 CD3 - 02 - Hz 16.12 7696 3 Roche CD3 1.225 14970 4 REGN CD3 5.271 7890

[0325] 5.4 Flow cytometry affinity evaluation of anti-human CD3 humanized antibody on human peripheral blood mononuclear cells

[0326] To detect the cross-reactivity and affinity of the anti-CD3 humanized antibody with the target protein CD3 on the human natural cell surface, flow cytometry was used to detect the affinity of the antibody to be tested. First, human peripheral blood mononuclear cells were collected by centrifugation, washed twice with PBS, and resuspended to a density of 8x10 6Dilute the anti-CD3 humanized antibody with 1% BSA at a density of 50 μL / well, starting from 80 μg / mL, with a 4-fold gradient and 8 concentration points, and add 50 μL per well to the cell wells. Mix well and incubate at 4°C for 50 min; wash twice with PBS. Finally, resuspend the cells with 1% BSA solution (containing APC anti-human IgG), 100 μL per well (containing 1 μL of fluorescent secondary antibody), mix well, react in the dark at 4°C for 0.5 h, wash twice with PBS, and detect the fluorescence signal value and positive rate by flow cytometry. Curve fitting of the fluorescence signal values corresponding to different concentrations of the antibody was performed using GraphPad Prism software.

[0327] Experimental results Figure 8 As shown in Table 7, the CD3 antibody of the present invention has good binding ability to human peripheral blood mononuclear cells, and the binding EC 50 values are 59.7 nM and 80.23 nM, respectively. Compared with the Roche CD3 antibody, the affinity of the CD3 antibody of the present invention is significantly reduced, and it is also 2-3 times weaker than the REGN CD3. The CD3 antibody of the present invention is expected to provide better safety.

[0328] Table 7. Affinity of humanized antibody to human peripheral blood mononuclear cells

[0329] Serial number Number <![CDATA[EC 50 (nM)]]> Maximum signal value 1 CD3 - 01 - Hz 59.7 19114 2 CD3 - 02 - Hz 80.23 11098 3 Roche CD3 3.71 24538 4 REGN CD3 28.3 14269

[0330] 5.5 Flow cytometry affinity evaluation of anti-human CD3 humanized antibody for rhesus monkey peripheral blood mononuclear cells

[0331] To detect the cross-reactivity and affinity of the anti-CD3 humanized antibody with the target protein CD3 on the surface of rhesus monkey natural cells, flow cytometry was used to detect the affinity of the antibody to be tested. First, centrifuge and collect rhesus monkey peripheral blood mononuclear cells, wash twice with PBS, and resuspend to a density of 8x10 6 / mL, 50 μL / well. Dilute the anti-CD3 humanized antibody with 1% BSA, starting from 80 μg / mL, with a 4-fold gradient and 8 concentration points, add to the cell wells, 50 μL per well, mix well, incubate at 4°C for 50 min; wash twice with PBS. Finally, resuspend the cells with 1% BSA solution (containing APC anti-human IgG), 100 μL per well (containing 1 μL of fluorescent secondary antibody), mix well, react in the dark at 4°C for 0.5 h, wash twice with PBS, and detect the fluorescence signal value and positive rate by flow cytometry. Curve fitting of the fluorescence signal values corresponding to different concentrations of the antibody was performed using GraphPad Prism software.

[0332] Experimental results Figure 9 As shown in Table 8, the CD3 antibody of the present invention has good cross-reactivity with rhesus monkey peripheral blood mononuclear cells, and the binding EC 50The values are 18.89 nM and 5.14 nM respectively.

[0333] Table 8. Affinity of the humanized antibody for rhesus peripheral blood mononuclear cells

[0334] Serial number Number <![CDATA[EC 50 (nM)]]> Emax 1 CD3 - 01 - Hz 18.89 23847 2 CD3 - 02 - Hz 5.14 24820 3 Roche CD3 1.371 33143

[0335] 5.6 Evaluation of the kinetic affinity of the anti-human CD3 humanized antibody

[0336] The kinetic affinity of the humanized antibody was determined by ForteBio. The experimental procedure was as follows: 1. Sensor preparation: Take out the ProA sensor and pre-wet the sensor with PBST diluent (pH 7.4) for 10 minutes; 2. Sample dilution: Dilute the antibodies to be immobilized to 5 μg / mL respectively. The antigen human CD3E and CD3G heterodimer protein and rhesus CD3E and CD3G heterodimer protein were serially diluted 2-fold starting from 500 nM, with 5 concentration points, and a 0 concentration point was set; 3. Set the program, place the sensor plate and sample plate, start the program, and regenerate the sensor with 20 mM glycine solution (pH 1.7); 4. Analyze the data using Octet analysis software and export the result graph.

[0337] The experimental results are shown in Table 9. Figure 10 As shown, the CD3 antibody of the present invention has good kinetic binding and dissociation rates. Among them, the kinetic affinity for the human CD3E&G heterodimer protein is comparable to that of the Roche CD3 antibody, and the ability of the CD3 antibody of the present invention to bind the rhesus CD3E&G heterodimer protein is similar to that of binding the human CD3E&G heterodimer protein.

[0338] Table 9. Detection of the kinetic affinity of the anti-human CD3 humanized antibody

[0339]

[0340] 5.7 Detection of the activity of the anti-human CD3 humanized antibody on Jurkat reporter cells

[0341] To detect the activation of Jurkat cells by anti-CD3 humanized antibodies, Jurkat-NFTA-Luciferase (Novoprotein, product number XCC20) reporter cells were used for evaluation. CD3&CD28 magnetic beads (Beijing Proteintech Group Co., Ltd., product number MBS-C001) were used as a positive reference with agonist activity, REGN CD3 antibody was used as a control antibody, and anti-chicken lysozyme IgG1 wild-type antibody was used as an isotype control antibody. All test CD3 humanized antibodies started at an initial concentration of 200 nM, and RPMI1640 + 10% FBS medium was used as the diluent. They were serially diluted 4-fold at 8 concentration points, and the last point was a zero-concentration well. The IgG1 antibody was only set at the highest concentration. 100 μL of the diluted samples were added to a 96-well white plate, and all samples were set up in duplicate. The CD3&CD28 magnetic bead positive control was 2 μL of magnetic beads added to 100 μL of RPMI1640 + 10% FBS medium. The Jurkat-NFAT-Luciferase cells were also diluted to 5x10 5 / mL with 1640 + 10% FBS, and 100 μL of the cell suspension was added to the above-mentioned well plate and cultured at 37 °C for 6 hours. After 6 hours, the cell culture plate was placed at room temperature for 20 minutes, and then 50 μL of Oneglu reagent (Vazyme, product number: DD1201-02) pre-equilibrated to room temperature was added to each well. After vibrating and incubating for 3 minutes, the Luminance signal value was read using a microplate reader. The signal values corresponding to different concentrations of antibodies were curve-fitted using GraphPad Prism software.

[0342] The experimental results are as shown in Figure 11 and Table 10. The experimental results show that the positive control CD3&CD28 group was able to induce strong activation of Jurkat reporter cells, and the isotype control antibody did not have activation activity. After treatment with the CD3 antibody, two CD3 humanized antibodies of the present invention had a certain degree of activation activity on the Jurkat reporter system. The EC 50 of CD3-01-Hz and CD3-02-Hz were 25.29 nM and 18.02 nM respectively, and the EC 50 of REGN CD3 was 1.03 nM. Therefore, the CD3 humanized antibodies of the present invention induced significantly weaker activation of Jurkat cells compared to the REGN CD3 antibody, and the CD3 antibodies of the present invention are expected to provide better safety.

[0343] Table 10. Activity of humanized antibodies on the Jurkat reporter system

[0344] Serial number Number <![CDATA[EC 50 (nM)]]> 1 CD3 - 01 - Hz 25.29 2 CD3 - 02 - Hz 18.02 3 REGN CD3 1.03

[0345] 5.8 Detection of the T cell activation activity of a humanized anti-human CD3 antibody

[0346] To detect the activation of T cells by the anti-CD3 humanized antibody, flow cytometry was used to evaluate the positive rates of CD25 and CD69 expression in PBMC cells, and the activation status of T cells was reflected by observing the changes in the positive rates of CD25 and CD69 in PBMC cells. CD3&CD28 magnetic beads (Beijing Proteintech Group Co., Ltd., product number MBS-C001) were used as a positive reference with agonist activity, Roche CD3 and REGN CD3 antibodies were used as control antibodies, and anti-chicken lysozyme IgG1 wild-type antibody was used as an isotype control antibody (Isotype). All test CD3 humanized antibodies started at an initial concentration of 200 nM, and RPMI1640 + 10% FBS medium was used as the diluent, diluted in a 4-fold gradient, with 8 concentration points, and the last point was a zero-concentration well. The IgG1 antibody was only set at the highest concentration. 100 μL of the diluted sample was added to a 96-well white plate, and all samples were set up in duplicate. The CD3&CD28 magnetic bead positive control was 2 μL of magnetic beads added to 100 μL of 1640 + 10% FBS medium. PBMC cells were resuscitated into 1640 + 10% FBS medium the afternoon before the experiment, and on the day of the test, PBMC was diluted to 2x10 6 / mL with 1640 + 10% FBS, and 100 μL of the cell suspension was added to the above well plate and cultured at 37 °C for 24 hours.

[0347] After 24 hours, the cell suspension in the well plate was transferred to a 96-well V-bottom plate, centrifuged at 500 g for 3 min, and after removing the supernatant, 200 μL of PBS was added to wash the cells twice. The cell pellet was resuspended in 1% BSA containing Human TruStain FcX (Biolegend), incubated at room temperature for 20 minutes, then 2 μL of CD69-APC direct-labeled antibody was added to each well, incubated at 4 °C for 45 minutes, and then the cells were washed twice with PBS. After the cell pellet was resuspended in PBS, it was analyzed by flow cytometry to detect the positive rate of CD69. The curve fitting of the CD69 positive rates corresponding to different concentrations of antibodies was performed using GraphPad Prism software.

[0348] The experimental results are as shown in Figure 12 and Table 11. The experimental results show that the positive control CD3&CD28 group can induce a strong upregulation of CD69 expression on the surface of T cells, and the isotype control antibody does not have activation activity. After treatment with the CD3 antibody, the expression level of CD69 on the surface of T cells increased significantly. For the induction of CD69 expression, the EC 50 of CD3-01-Hz and CD3-02-Hz were 7.13 nM and 37.71 nM, respectively, while the EC 50It is 3.39 nM. Therefore, the CD3 humanized antibody of the present invention has weaker T cell activation activity compared to Roche CD3, and the CD3 antibody of the present invention is expected to provide better safety.

[0349] Table 11. T cell activation activity of anti-human CD3 humanized antibodies

[0350] Serial number Number <![CDATA[CD69 EC 50 (nM)]]> 1 CD3 - 01 - Hz 7.13 2 CD3 - 02 - Hz 37.71 3 Roche CD3 3.39

[0351] 5.9 Hydrophobicity detection of anti-human CD3 humanized antibodies

[0352] The hydrophilicity and hydrophobicity of the humanized antibody were detected by HIC. The detection method is as follows: The hydrophilicity and hydrophobicity were detected on an Agilent HPLC device using a hydrophobic chromatography column (TOSOH Tskgel Buty-NPR(2.5), 4.6*100) from Tosoh Corporation. Mobile phase A was 1.5 M (NH4)2SO4, and mobile phase B was 25 mM Na2HPO4 (pH = 7.0) + 20% isopropanol. The instrument parameters were set as follows: sample chamber temperature: 8 °C, column temperature: 30 °C, flow rate: 0.5 mL / min, detection wavelength: 280 nm. The sample to be tested was diluted with mobile phase A to a final concentration of 1 mg / mL, and 20 μL was injected for gradient elution. The elution gradient is shown in Table 12:

[0353] Table 12. Liquid phase running program for hydrophobicity analysis

[0354] T / min 0 2 39 41 42 45 A％ 95 95 0 0 95 95 B％ 5 5 100 100 5 5

[0355] Finally, the elution times of CD3-01-Hz and CD3-02-Hz were 12.33 minutes and 11.22 minutes respectively. Compared with the strong hydrophobic (22 minutes) and weak hydrophobic (12 minutes) internal standard molecules, both CD3-01-Hz and CD3-02-Hz have good hydrophilicity.

[0356] 5.10 Tm value detection of anti-human CD3 humanized antibodies

[0357] The melting temperature (Tm) of the humanized antibody was determined by DSF to reflect the thermal stability of the antibody. DSF determination was performed using real-time fluorescence quantitative PCR (QuantStudio 7Flex, Thermo Fisher Scientific). Briefly, 19 μL of the antibody solution at a concentration of 1 mg / ml was mixed with 1 μL of 62.5X SYPRO Orange solution (Invitrogen) and added to a 96-well plate (Biosystems). The plate was heated from 25 °C to 95 °C at a rate of 2 °C / min, and the resulting fluorescence data were collected. The negative derivative of the fluorescence change with respect to different temperatures was calculated, and the maximum value was defined as the melting temperature Tm. Data collection and Tm calculation were automatically performed by the operating software (QuantStudio Real-Time PCR Software v1.3).

[0358] The results showed that the Tm values of CD3-01-Hz and CD3-02-Hz in PBS buffer were approximately 63.5 °C and 68.6 °C, respectively, indicating that the two humanized anti-CD3 monoclonal antibodies, CD3-01-Hz and CD3-02-Hz, had good thermal stability.

[0359] The antibody sequences obtained in the present invention are shown in Table 14 below:

[0360] Table 14 Antibody Sequences

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. An anti-CD3 antibody or an antigen-binding fragment thereof, characterized in that, Comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have 6 complementarity determining regions CDR selected from the following groups: (1a) Three complementarity determining regions VH-CDR of the heavy chain variable region and three complementarity determining regions VL-CDR of the light chain variable region defined based on Kabat rules: VH-CDR1 shown in SEQ ID NO:5, VH-CDR2 shown in SEQ ID NO:6 or 33, VH-CDR3 shown in SEQ ID NO:7, VL-CDR1 shown in SEQ ID NO:11, VL-CDR2 shown in SEQ ID NO:12, VL-CDR3 shown in SEQ ID NO:13; or (1b) Three complementarity determining regions VH-CDR of the heavy chain variable region and three complementarity determining regions VL-CDR of the light chain variable region defined based on IMGT rules: VH-CDR1 shown in SEQ ID NO:8, VH-CDR2 shown in SEQ ID NO:9 or 34, VH-CDR3 shown in SEQ ID NO:10, VL-CDR1 shown in SEQ ID NO:14, VL-CDR2 shown in SEQ ID NO:15, VL-CDR3 shown in SEQ ID NO:16; or (2a) Three complementarity determining regions VH-CDR of the heavy chain variable region and three complementarity determining regions VL-CDR of the light chain variable region defined based on Kabat rules: VH-CDR1 shown in SEQ ID NO:17, VH-CDR2 shown in SEQ ID NO:18 or 35, VH-CDR3 shown in SEQ ID NO:19, VL-CDR1 shown in SEQ ID NO:23, VL-CDR2 shown in SEQ ID NO:24, VL-CDR3 shown in SEQ ID NO:25; or (2b) Three complementarity determining regions VH-CDR of the heavy chain variable region and three complementarity determining regions VL-CDR of the light chain variable region defined based on IMGT rules: VH-CDR1 shown in SEQ ID NO:20 or 36, VH-CDR2 shown in SEQ ID NO:21, VH-CDR3 shown in SEQ ID NO:22, VL-CDR1 shown in SEQ ID NO:26, VL-CDR2 shown in SEQ ID NO:27, VL-CDR3 shown in SEQ ID NO:28; Wherein, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and / or substituted with at least one amino acid and can retain the CD3 binding affinity.

2. A recombinant protein, characterized in that, The recombinant protein comprises: (i) The anti-CD3 antibody or its antigen-binding fragment as described in claim 1; and (ii) Optionally, a tag sequence for assisting expression and / or purification.

3. A CAR construct, characterized in that, The scFv segment of the antigen-binding region of the CAR construct described above is a binding region that specifically binds to CD3, and the scFv segment has a heavy-chain variable region and a light-chain variable region. Among them, the heavy-chain variable region and the light-chain variable region have 6 complementarity-determining regions CDR selected from the following groups: (1a) Three complementarity-determining regions VH-CDR of the heavy-chain variable region and three complementarity-determining regions VL-CDR of the light-chain variable region defined based on the Kabat rules: (1a) Three complementarity-determining regions VH-CDR of the heavy-chain variable region and three complementarity-determining regions VL-CDR of the light-chain variable region defined based on the Kabat rules: VH-CDR1 shown in SEQ ID NO:5, VH-CDR2 shown in SEQ ID NO:6 or 33, VH-CDR3 shown in SEQ ID NO:7, VL-CDR1 shown in SEQ ID NO:11, VL-CDR2 shown in SEQ ID NO:12, VL-CDR3 shown in SEQ ID NO:13; or (1b) Three complementarity-determining regions VH-CDR of the heavy-chain variable region and three complementarity-determining regions VL-CDR of the light-chain variable region defined based on the IMGT rules: VH-CDR1 shown in SEQ ID NO:8, VH-CDR2 shown in SEQ ID NO:9 or 34, VH-CDR3 shown in SEQ ID NO:10, VL-CDR1 shown in SEQ ID NO:14, VL-CDR2 shown in SEQ ID NO:15, VL-CDR3 shown in SEQ ID NO:16; or (2a) Three complementarity-determining regions VH-CDR of the heavy-chain variable region and three complementarity-determining regions VL-CDR of the light-chain variable region defined based on the Kabat rules: VH-CDR1 shown in SEQ ID NO:17, VH-CDR2 shown in SEQ ID NO:18 or 35, VH-CDR3 shown in SEQ ID NO:19, VL-CDR1 shown in SEQ ID NO:23, VL-CDR2 shown in SEQ ID NO:24, VL-CDR3 shown in SEQ ID NO:25; or (2b) Three complementarity-determining regions VH-CDR of the heavy-chain variable region and three complementarity-determining regions VL-CDR of the light-chain variable region defined based on the IMGT rules: VH-CDR1 shown in SEQ ID NO:20 or 36, VH-CDR2 shown in SEQ ID NO:21, VH-CDR3 shown in SEQ ID NO:22, VL-CDR1 shown in SEQ ID NO:26, VL-CDR2 shown in SEQ ID NO:27, VL-CDR3 shown in SEQ ID NO:

28.

4. A recombinant immune cell, characterized in that, The immune cells described above express the exogenous CAR construct as described in claim 3.

5. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate described above contains: (a) An antibody portion, and the antibody portion is selected from the following groups: The antibody or antigen-binding fragment thereof as claimed in claim 1, or the recombinant protein as claimed in claim 2, or a combination thereof; and (b) a conjugate moiety conjugated to the antibody moiety, the conjugate moiety being selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

6. Use of an active ingredient, characterized in that, The active ingredient is selected from the group consisting of: The antibody or antigen-binding fragment thereof as claimed in claim 1, or the recombinant protein as claimed in claim 2, the CAR construct as claimed in claim 3, the immune cell as claimed in claim 4, the antibody-drug conjugate as claimed in claim 5, or a combination thereof, and the active ingredient is used for: (a) preparing a detection reagent or kit; (b) preparing a drug or preparation for preventing and / or treating CD3-related diseases; and / or (c) preparing a drug or preparation for preventing and / or treating CD3-related cancers or tumors.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) an active ingredient, the active ingredient being selected from the group consisting of: The antibody or antigen-binding fragment thereof as claimed in claim 1, or the recombinant protein as claimed in claim 2, the CAR construct as claimed in claim 3, the immune cell as claimed in claim 4, the antibody-drug conjugate as claimed in claim 5, or a combination thereof; and (ii) a pharmaceutically acceptable carrier.

8. A polynucleotide, characterized in that, The polynucleotide encodes a polypeptide selected from the group consisting of: (1) the antibody or antigen-binding fragment thereof as claimed in claim 1; or (2) the recombinant protein as claimed in claim 2; (3) the CAR construct as claimed in claim 3.

9. A carrier, characterized in that, The vector contains the polynucleotide as claimed in claim 8.

10. A genetically engineered host cell, characterized in that, The host cell contains the vector as claimed in claim 9 or the polynucleotide as claimed in claim 8 is integrated into the genome.

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