Anti-CD3 antibody and use thereof

Abstract

Provided are an anti-CD3 antibody and a use thereof. Specifically, the provided anti-CD3 antibody can specifically bind to human and monkey CD3 proteins, and has similar binding properties. In addition, the provided anti-CD3 antibody has excellent bioactivity on various human CD3E proteins (including hCD3E&D heterodimer, and the like), and has significantly improved safety. Also provided is a humanized anti-CD3 antibody. The humanized antibody has properties of low immunogenicity, high hydrophilicity, and high stability, and is suitable for drug development.

Description

Anti-CD3 antibodies and uses thereof Technical Field

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

[0002] The CD3 (cluster of differentiation 3) T cell co-receptor is a protein complex. CD3 consists of five peptide chains: the γ chain, the δ chain, the ε chain, the ζ chain, and the η chain, all of which are transmembrane proteins. The transmembrane region of the CD3 molecule connects to the transmembrane regions of the two peptide chains of the TCR via salt bridges, forming the TCR-CD3 complex. Together, they participate in antigen recognition by T cells. The activation signal generated by TCR recognition of antigens is transduced into the T cell by CD3.

[0003] CD3, together with the TCR, forms the CD3-TCR complex, which plays a key role in regulating most functions of T cell innate and adaptive immune responses, as well as cellular and humoral immunity. These functions include eliminating pathogens and controlling tumor growth through various cytotoxic effects.

[0004] Mouse monoclonal antibodies specific for human CD3, such as OKT3 (Kung et al. (1979) Science 206:347-9), are the first generation of anti-CD3 antibodies used for treatment. Although OKT3 has strong immunosuppressive efficacy, its strong immunogenicity and serious side effects related to its mitogenic potential have hindered its clinical use (Chatenoud (2003) Nature Reviews 3:123-132). Due to its immunogenicity, OKT3 induces the production of neutralizing antibodies in humans, resulting in its rapid clearance and neutralization (Chatenoud et al. (1982) Eur. J. Immunol. 137:830-8).

[0005] In addition, OKT3 induces T cell proliferation and cytokine production in vitro, and causes large-scale cytokine release in vivo (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, characterized by fever, chills, headache, nausea, vomiting, diarrhea, dyspnea, septic meningitis and hypotension (Chatenoud, 2003). These serious side effects limit the wider use of OKT3 in transplantation and its expansion into other clinical areas (such as autoimmunity).

[0006] CD3 bispecific antibodies developed based on anti-human CD3 antibodies have become important clinical therapeutic drugs. However, some common problems still exist in CD3 bispecific antibodies. For example, the CD3 antibody sequence L2K-07 in Blinatumomab, the first CD3 bispecific antibody developed by Amgen, is an antibody that only binds to human but not monkey CD3. As a result, chimpanzees were forced to be used as the relevant species in the drug's preclinical safety studies.

[0007] The CD3 antibody sequences used in some other CD3 bispecific antibodies basically face the problem of excessive affinity, which triggers excessive cytokine release, thus bringing significant safety risks (Karin Staflin et al., JCI Insight. 2020; 5(7): e133757.). For example, in a Phase I clinical study, Xencor's Xmab1405 (CD123-CD3) bispecific antibody caused the death of a subject due to a cytokine storm, which led to the suspension of clinical trials by the US FDA. Coincidentally, Regeneron's odronextamab (CD20-CD3) bispecific antibody also caused the death of a subject due to serious adverse reactions, which led to the suspension of clinical trials by the US FDA. Excessively high CD3 affinity reduces the drug treatment window. The same problem also occurred in Regeneron's BCMA-CD3 bispecific antibody project. Its first-generation product, linvoseltamab, has been submitted for marketing approval in Europe. However, based on the problems encountered in clinical development, the 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 treatment window.

[0008] Therefore, there is still an urgent need in the art to develop new anti-CD3 antibodies that are safe, suitable for preclinical studies, and have excellent biological activity. Summary of the Invention

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

[0010] In a first aspect of the present invention, an anti-CD3 antibody or antigen-binding fragment thereof is provided, 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 six complementarity determining regions (CDRs) selected from the group consisting of:

[0011] (1a) The three complementarity determining regions (VH-CDRs) of the heavy chain variable region and the three complementarity determining regions (VL-CDRs) of the light chain variable region are defined based on the Kabat rules:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0032] (2b) Three complementarity determining regions (VH-CDRs) of the heavy chain variable region and three complementarity determining regions (VL-CDRs) of the light chain variable region defined based on 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 of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain 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 that has been subjected to addition, deletion, modification and / or substitution of at least one amino acid and is capable of retaining 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 antigen-binding fragment thereof is partially or fully humanized.

[0043] In another preferred embodiment, the antibody or antigen-binding fragment thereof 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 example, the sequence of the heavy chain variable region is shown in SEQ ID NO: 29, 31, 1 or 3.

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

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

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

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

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

[0050] In another preferred embodiment, the antibody or antigen-binding fragment thereof 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 double-chain antibody or a single-chain antibody (scFv).

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

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

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

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

[0057] (2) Antibodies that bind to other targets.

[0058] In another preferred embodiment, the other targets are selected from the following group: 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 example, the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 30; or the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32; or the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; or the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

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

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

[0062] (ii) optionally a tag sequence to facilitate 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, a dimer, or a 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 comprises an additional fusion element (or fusion polypeptide fragment) fused with the element (i).

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

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

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

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

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

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

[0075] as well as

[0076] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0077] In a third aspect of the present invention, a CAR construct is provided, wherein the scFv segment of the antigen binding region of the CAR construct 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, wherein the heavy chain variable region and the light chain variable region have 6 complementary determining regions (CDRs) selected from the following group:

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

[0079] (1a) The three complementarity determining regions (VH-CDRs) of the heavy chain variable region and the three complementarity determining regions (VL-CDRs) of the light chain variable region are defined based on the 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) Three complementarity determining regions (VH-CDRs) of the heavy chain variable region and three complementarity determining regions (VL-CDRs) 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-CDRs) of the heavy chain variable region and the three complementarity determining regions (VL-CDRs) of the light chain variable region are 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) Three complementarity determining regions (VH-CDRs) of the heavy chain variable region and three complementarity determining regions (VL-CDRs) 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 shown in SEQ ID NO: 26,

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

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

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

[0108] In another preferred embodiment, the immune cells are selected from the following group: NK cells and T cells.

[0109] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

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

[0111] (a) an antibody portion, the antibody portion being selected from the group consisting of the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the recombinant protein according to the second aspect of the invention, or a combination thereof; and

[0112] (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is 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 portion and the coupling portion are coupled via a chemical bond or a linker.

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

[0115] (a) preparing detection reagents or kits;

[0116] (b) preparing a drug or preparation for preventing and / or treating a CD3-related disease; 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 following group: 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, and leukemia.

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

[0120] (i) an active ingredient selected from the group consisting of the antibody or antigen-binding fragment thereof according to the first aspect of the invention, the recombinant protein according to the second aspect of the invention, the CAR construct according to the third aspect of the invention, the immune cell according to the fourth aspect of the invention, the antibody-drug conjugate according to the fifth aspect of the 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 to prepare a drug for treating tumors, and the tumors are selected from the following group: 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, and leukemia.

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

[0126] (1) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; or

[0127] (2) the recombinant protein according to the second aspect of the present invention;

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

[0129] In the ninth aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide according to the eighth aspect of the present invention.

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

[0131] In the tenth aspect of the present invention, a genetically engineered host cell is provided, wherein the host cell 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 is 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 cells are Chinese hamster ovary (CHO) cells.

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

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

[0136] (1) contacting the sample with the antibody according to the first aspect of the present invention in vitro;

[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 the twelfth aspect of the present invention, a detection plate is provided, which comprises: a substrate (support plate) and a test strip, wherein 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, a kit is provided, comprising:

[0140] (1) a first container containing the antibody according to the first aspect of the present invention; and / or

[0141] (2) a second container containing a secondary antibody against the antibody according to the first aspect of the present invention;

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

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

[0144] (a) culturing the host cell according to the tenth aspect of the present invention under conditions suitable for expression;

[0145] (b) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody according to the first aspect of the present invention or the recombinant protein according to the second aspect of the present invention.

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

[0147] In another preferred embodiment, the diseases associated with CD3 molecules include tumors or antagonistic organ transplant immune rejection reactions.

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

[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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0151] Figures 2A-B show the ELISA binding curves of anti-CD3 mouse antibodies to monkey CD3 E&D protein and monkey CD3 E&G protein, respectively. Figure 2A shows the ELISA binding curve of anti-CD3 mouse antibodies to monkey CD3 E&D protein; Figure 2B shows the ELISA binding curve of anti-CD3 mouse antibodies to monkey CD3 E&G protein.

[0152] FIG3 shows the FACS binding curve of anti-CD3 mouse antibody to human Jurkat cells.

[0153] FIG4 shows the FACS binding curve of anti-CD3 mouse antibody to human peripheral blood mononuclear cells.

[0154] FIG5 shows the FACS binding curve of anti-CD3 mouse antibody to monkey peripheral blood mononuclear cells.

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

[0156] FIG7 shows the FACS binding curve of anti-CD3 humanized antibody to human Jurkat cells.

[0157] FIG8 shows the FACS binding curve of anti-CD3 humanized antibodies to human peripheral blood mononuclear cells.

[0158] FIG9 shows the FACS binding curve of anti-CD3 humanized antibodies to monkey peripheral blood mononuclear cells.

[0159] FIG10 shows the dynamic binding and dissociation curves of anti-CD3 humanized antibodies with human CD3 E&G protein and monkey CD3 E&G protein.

[0160] FIG11 shows the activity curve of the anti-CD3 humanized antibody Jurkat reporter system.

[0161] FIG12 shows the human T cell activation activity curve of anti-CD3 humanized antibody. DETAILED DESCRIPTION

[0162] After extensive and intensive research and extensive screening, the inventors unexpectedly discovered anti-CD3 antibodies with similar binding properties and high biological activity to human and other primate CD3 proteins (such as monkeys). Experiments have shown that the anti-CD3 antibodies of the present invention not only specifically bind to human and monkey CD3, but also exhibit similar binding properties (e.g., KD). Furthermore, the anti-CD3 antibodies of the present invention exhibit excellent biological activity against different forms of human CD3E protein (including hCD3E & D heterodimers, hCD3E & G heterodimers, and cell surface CD3E protein), with moderate affinity and significantly improved safety. This is the basis for the completion of the present invention.

[0163] The present invention also provides humanized anti-CD3 antibodies. These humanized antibodies retain many of the properties of murine antibodies, such as specific binding to human and monkey CD3 with similar binding properties and excellent biological activity against different forms of human CD3E protein. Furthermore, they exhibit high safety, low human immunogenicity, excellent hydrophilicity, and stability, making them suitable for drug development.

[0164] the 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 therapy, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, and contact of a fluid with a cell. "Administer" and "treat" also mean in vitro and ex vivo treatment with an agent, diagnostic, binding composition, or with another cell. "Treatment," when applied to a human, animal, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; including contact of an anti-CD3 antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

[0166] As used herein, the term "treatment" refers to administering an internal or external therapeutic agent, including any of the anti-CD3 antibodies and compositions of the present invention, to a patient experiencing one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (a therapeutically effective amount).

[0167] As used herein, the term "optionally" or "optionally" means that the event or situation described subsequently may occur but need not 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 have but need not have, and may have 1, 2, or 3.

[0168] Antibody

[0169] As used herein, the term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and order of the constant region of the heavy chains of immunoglobulins differ, resulting in different antigenicity. Consequently, immunoglobulins can be divided into five classes, or different types of immunoglobulins: IgM, IgD, IgG, IgA, and IgE. The heavy chain constant regions corresponding to these different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins and, due to differences in chemical structure and biological function, is further divided into four subclasses: IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains, depending on the constant region. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0170] The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (V region). The remaining amino acid sequence near the C-terminus is relatively stable and constitutes the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four relatively conserved FR regions (FRs). The amino acid sequences of these four FRs are relatively conserved and not directly involved in binding. The three hypervariable regions determine the specificity of the antibody and are also known as the complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged from the N-terminus to the C-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDRs of the light chain, known as the light chain hypervariable regions (LCDRs), are LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain, known as the heavy chain hypervariable regions (HCDRs), are HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present invention conform to the known Kabat numbering convention (LCDR1-3, HCDR2-3), or conform to the Kabat and Chothia numbering convention (HCDR1). The four FR regions in the native heavy and light chain variable regions generally have a β-sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form a partial β-sheet structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region. The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in the antibody's antibody-dependent cellular toxicity.

[0171] As used herein, the term "antigen-binding fragment" refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment that has antigen-binding activity. The Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of the antibody, but does not have the constant region, and is the smallest antibody fragment with all antigen-binding sites. Generally, the Fv antibody also contains 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) minimal recognition units composed of amino acid residues that mimic the hypervariable region of an antibody (e.g., independent complementarity determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. As used herein, the expression "antigen-binding fragment" also encompasses other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs) and shark variable IgNAR domains.

[0172] As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0173] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, 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 using 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 recombinant DNA 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. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. The cell may be a eukaryotic, prokaryotic, or phage clone.

[0176] As used herein, the term "chimeric antibody" refers to an antibody molecule that is constructed by splicing the V region genes of a murine antibody with the C region genes of a human antibody. This chimeric gene is then inserted into a vector and transfected into a host cell for expression. This antibody retains the high specificity and affinity of the parent murine antibody while enabling the human Fc region to effectively mediate biological effector functions.

[0177] As used herein, the term "humanized antibody" refers to a modified form of the murine antibody of the present invention, having CDR regions derived (or substantially derived) from a non-human antibody (preferably a mouse monoclonal antibody), and FR regions and constant regions substantially derived from human antibody sequences; that is, the CDR region sequences of the murine antibody are grafted onto different types of human germline antibody framework sequences. Because CDR sequences are responsible for most 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 antibodies can be monospecific, bispecific, trispecific, or more multispecific.

[0179] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0180] Table A

[0181] anti-CD3 antibodies

[0182] As used herein, the term "CD3" generally refers to natural or recombinant human CD3, as well as non-human homologs of human CD3. CD3 is a homodimer or heterodimer 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. Therefore, unless explicitly stated to be from a non-human species, such as "mouse CD3", "monkey CD3", etc., the term "CD3" refers to human CD3.

[0183] As used herein, "CD3E" refers to the CD3ε extracellular domain.

[0184] The present invention provides a highly specific single-chain antibody (scFv) against CD3, which comprises a heavy chain variable region (VH), a light chain variable region (VL) and a connected linker.

[0185] The present invention also provides a highly specific antibody against CD3, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) amino acid sequence, and the light chain comprises a light chain variable region (VL) amino acid sequence.

[0186] Preferably, the heavy chain variable region and light chain variable region have 6 complementarity determining regions (CDRs) defined based on the Kabat rule or the IMGT rule, as shown in Table 4.

[0187] Preferably, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 29; and the light chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 30; or

[0188] The antibody comprises the amino acid sequence shown in SEQ ID NO: 31; and the light chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 32; or

[0189] The heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 2; or

[0190] The heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 4.

[0191] Among them, any of the above amino acid sequences also includes a derivative sequence with CD3 binding affinity after adding, deleting, modifying and / or replacing at least one (such as 1-5, 1-3, preferably 1-2, more preferably 1) amino acid.

[0192] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing 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%.

[0193] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

[0194] As used herein, the term "scFv" refers to a single-chain antibody fragment (scFv), which is composed of the antibody heavy chain variable region and the light chain variable region connected by a linker of usually 15 to 25 amino acids.

[0195] 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 domains of the light and heavy chains to fold into an exchange dual variable region immunoglobulin. In the present invention, a preferred linker refers to a linker connecting the VH and VL of a single-chain antibody (scFv), or for connecting an scFv to the heavy chain of another antibody.

[0196] Suitable examples of linkers include single glycine (Gly) or serine (Ser) residues, and the identity and sequence of amino acid residues in the linker can vary with the type of secondary structural elements desired to be achieved in the linker, e.g., (G4S)n, where n is an integer from 1 to 5.

[0197] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other antibody derivatives known in the art, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0198] The animal is preferably a mammal, such as a mouse.

[0199] The antibodies of the present invention may be murine antibodies, chimeric antibodies, humanized antibodies, CDR-grafted and / or modified antibodies targeting human CD3.

[0200] The antibodies of the present invention may be monospecific, bispecific, trispecific, or multispecific. For example, the antibodies of the present invention may be combined with antibodies or active fragments that bind to other targets to form bispecific antibodies. The antibodies that bind to other targets may be antibodies or active fragments thereof that target CD19, CD47, CD73, CD47, CTLA4, PD-1, PD-L1, or CD28.

[0201] Antibody preparation

[0202] Any method suitable for producing monoclonal antibodies can be used to produce the anti-CD3 antibodies of the present invention. For example, animals can be immunized with linked or naturally occurring CD3 homodimers or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes of administration.

[0203] Any suitable form of CD3 can be used as an immunogen (antigen) to produce non-human antibodies specific for CD3 and screen the biological activity of the antibodies. The stimulating immunogen can be full-length mature human CD3, including natural homodimers, or peptides containing single / multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen can be purified from a natural source 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 a suitable genetic vector, including but not limited to adenoviral vectors, adeno-associated viral vectors, baculoviral vectors, plasmids, and non-viral vectors.

[0204] An exemplary method for producing the anti-human CD3 antibodies of the present invention is described in Example 1.

[0205] Humanized antibodies 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 necessary constant domain sequences can be readily achieved by screening the antibodies using the biological assays described in the examples below to produce the desired biological activity.

[0206] Likewise, any type of light chain can be used in the compounds and methods herein. Specifically, kappa, lambda chains, or variants thereof can be used in the compounds and methods of the invention.

[0207] An exemplary method for humanizing the anti-human CD3 antibodies of the invention is described in Example 2.

[0208] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form a single-chain antibody.

[0209] Once the relevant sequence is obtained, it 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 propagated host cells by conventional methods.

[0210] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then connecting them, very long fragments of sequence can be obtained. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0211] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0212] Host cells can be prokaryotic cells, such as bacterial cells, lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.

[0213] The steps of transforming host cells with recombinant DNA described in the present invention can be carried out using techniques well known in the art. The transformants obtained can be cultured using conventional methods, and the transformants express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, conventional culture medium is used under appropriate conditions.

[0214] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0215] The resulting monoclonal antibodies can be characterized by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0216] Antibody-drug conjugates (ADCs)

[0217] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0218] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0219] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0220] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0221] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs contain a linker positioned between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers typically readily degrade in the intracellular environment, for example, at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. Peptide linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug when the antibody is hydrolyzed by proteases.

[0222] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0223] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0224] 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, and the like. In the present invention, drug-linkers can be used to form ADCs in a single step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue reacts with a reactive portion of a linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0225] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides 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 linking strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, when a complementary pair of reactive functional groups is selected for selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0226] application

[0227] The present invention provides uses of the antibodies of the present invention, for example, for preparing diagnostic preparations or preparing medicaments for preventing and / or treating CD3-related diseases. Such CD3-related diseases include inflammatory diseases, autoimmune diseases, and the like, including but not limited to psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), osteoarthritis, rheumatoid arthritis (RA), rheumatoid arthritis or osteoporosis, inflammatory fibrosis (such as scleroderma, pulmonary fibrosis, and sclerosis), asthma (including allergic asthma), allergies, and cancer.

[0228] Pharmaceutical composition

[0229] 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 corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary with the properties of the formulated substance and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0230] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0231] The pharmaceutical composition of the present invention can be used to directly bind to CD3 protein molecules, and thus can be used to prevent and treat CD3-related diseases. In addition, other therapeutic agents can also be used simultaneously.

[0232] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0233] When using a pharmaceutical composition, a safe and effective amount of the pharmaceutical composition is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.

[0234] Detection Uses and Kits

[0235] The antibodies of the present invention can be used in detection applications, for example, for detecting a sample to provide diagnostic information.

[0236] In the present invention, the samples used include cells, tissue samples and biopsy specimens. The term "biopsy" as used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention can include, for example, tissue samples prepared by endoscopic methods or puncture or needle biopsy of an organ.

[0237] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0238] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0239] The main advantages of the present invention include

[0240] (1) The anti-CD3 antibody of the present invention specifically binds to human CD3 and has excellent biological activity against different forms of human CD3E protein.

[0241] (2) The anti-CD3 antibody of the present invention has suitable affinity, a larger therapeutic window and in vivo safety.

[0242] (3) The anti-CD3 antibody of the present invention has an ability to bind to monkey CD3 protein that is similar to that of binding to human CD3 protein, and is suitable for preclinical safety studies using monkeys as the species.

[0243] (4) The anti-CD3 antibodies of the present invention can be combined with other antibodies to construct bispecific and multispecific antibodies.

[0244] (5) The anti-CD3 antibody of the present invention has excellent hydrophilicity and is suitable for drug development.

[0245] (6) The anti-CD3 antibody of the present invention has good thermal stability and is suitable for drug development.

[0246] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in 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.

[0247] Abbreviations ELISA: enzyme-linked immunosorbent assay FACS: fluorescence-activated cell sorting TCR: T cell receptor CDR: complementarity-determining region Kabat: immunoglobulin alignment and numbering system proposed by Elvin A. Kabat IMGT: numbering system based on the International Immunogenetics Information System initiated by Lefranc et al. 50 : Half-maximal effect concentration, the concentration that causes 50% of the maximum effect ELISA: Enzyme-linked immunosorbent assay PCR: Polymerase chain reaction HRP: Horseradish peroxidase

[0248] Example 1. Immunization of mice with anti-human CD3 antibodies

[0249] 1.1 Preparation of CD3 antigen

[0250] Human CD3E&D heterodimer protein (Kactus Biosystems, CD3-HM105), human CD3E&G heterodimer protein (Kactus Biosystems, CD3-HM157), monkey CD3E&D heterodimer protein (AcroBiosystems, CDD-C52W4), and monkey CD3E&G heterodimer protein (AcroBiosystems, CDG-C52W6) proteins were purchased.

[0251] Human CD3E-ECD-his protein (Sichuan Sibowo Biotechnology Co., Ltd., Accession#P07766-1) was codon-optimized and synthesized into a PTT5 vector, transiently expressed in HEK293E cells transfected with PEI, and then purified using a Ni column.

[0252] Human CD3E / CD3D mRNA lipid nanoparticles: The nucleotide sequence encoding amino acids Asp 23-Asp 126 (CD3E) & Phe 22-Ala 105 (CD3D) (Accession # P07766-1 (CD3E) & P04234-1 (CD3D)) was synthesized by Anhui General Biotechnology Co., Ltd. into an mRNA in vitro synthesis backbone vector (Sichuan Sibowo Biotechnology Co., Ltd.). mRNA synthesis was completed using T7 RNA polymerase (Novozymes, Catalog No. DD4202-01). After purification, 1 mg of mRNA was dissolved in citrate buffer and lipid nanoparticles were prepared using a microfluidic chip (Fluidiclab, LNP-B0) at a volume ratio of 3:1 between the mRNA solution and the lipid solution. The prepared lipid nanoparticles were dialyzed into PBS + 2% sucrose solution, concentrated and sterilized, and then aliquoted at -80°C for later use.

[0253] 1.2 Immunization of mice

[0254] Group 1: Three FVB and three KM mice were immunized intramuscularly with lipid nanoparticles encoding human CD3E / CD3D mRNA, with booster doses every two weeks for a total of three immunizations. The titers of the mice were then assessed using protein ELISA and cell FACS. High-performing mice received a single subcutaneous booster dose of monkey CD3E&D heterodimer protein. One week prior to fusion, the mice to be fused received a final booster dose of 20 μg of monkey CD3E&D heterodimer protein.

[0255] Group 2: Babl / c and CD01 mice were primed with human CD3E&D heterodimer protein or human CD3E&G heterodimer protein emulsified in CFA, and then boosted with IFA subcutaneously or abdominally every two weeks for a total of four immunizations. The titers of mice were tested by protein ELISA and cell FACS. Mice with excellent performance were boosted with monkey CD3E&D heterodimer protein subcutaneously once. One week before fusion, mice received 1x10 7 A final boost was performed by intraperitoneal injection of 10 Jurkat cells.

[0256] Group 3: Two Babl / c, CD01, KM, and FVB mice were immunized with human Jurkat cells, intraperitoneally every two weeks for six weeks. After six weeks, the titers of the mice were assessed using protein ELISA and cell FACS. High-performing mice received two intramuscular boosts with human CD3E&D mRNA lipid nanoparticles. One week before fusion, the mice received a final boost with 20 μg of monkey CD3E&G heterodimer protein.

[0257] Example 2. Screening of anti-human CD3 antibody hybridomas

[0258] 2.1 Hybridoma Fusion

[0259] Hybridoma fusions were performed on mice with the highest titers from immunization groups 1, 2, and 3. Two different protocols were used. Protocol 1 directly isolated mouse spleen and lymphocyte suspensions, mixed with SP2 / 0 mouse myeloma cells at a 1:1 ratio, resuspended in electrofusion buffer, and electrofused using BTX-ECM2001. Cells were resuspended in complete fusion medium (RPMI-1640 + 15% FBS + 1× HAT), aliquoted at 20,000 per well into 96-well cell culture plates, and cultured at 37°C, 5% CO2 for 7 days before selection. Protocol 1 involved two rounds of fusion: 40 plates in the first round and 81 plates in the second. Protocol 2 isolated mouse spleen and lymphocyte suspensions, followed by magnetic separation of CD138-positive plasma cells, mixed with SP2 / 0 mouse myeloma cells at a 1:1 ratio, resuspended in electrofusion buffer, and electrofused using BTX-ECM2001. Resuspend in complete fusion medium (RPMI160 + 15% FBS + 1× HAT), aliquot 8000 cells per well into a 96-well cell culture plate, and culture at 37°C, 5% CO2 for 7 days before screening. In protocol 2, perform two rounds of fusion, with 10 plates plated per round.

[0260] 2.2 Hybridoma Screening

[0261] 1) Primary screening was performed by ELISA using human CD3E&D heterodimer protein. Human CD3E&D heterodimer protein was coated with CBS coating solution at a coating concentration of 0.5μg / mL and a volume of 100μL, and the coating was carried out overnight at 4°C. The coating solution was removed on the next day, each well was washed once with 300μL PBS, 100μL 2% BSA (in PBS) was added to each well, and the plates were blocked at 37°C for 2 hours. 30μL of the supernatant of the hybridoma to be screened was added to the ELISA plate, mouse serum was added to well A1 of each ELISA plate as a positive control, and blank culture medium was added to well A2 as a negative control well, and the plates were incubated at 37°C for 2 hours. The supernatant was removed, each well was washed three times with 300μL PBST, 100μL anti-mouse HRP secondary antibody (Jackson, 115-035-003) (in 2% BSA) was added to each well, and the plates were incubated at 37°C for 1 hour. The secondary antibody was removed, each well was washed five times with 300μL PBST, and 100μL anti-mouse HRP secondary antibody (Jackson, 115-035-003) (in 2% BSA) was added to each well. TMB substrate was added and color was developed for 1-2 minutes. 50 μL of ELISA stop solution (Solabo, C1058) was added to each well to terminate the reaction. The OD450 nm reading was read on a microplate reader. The wells with signal values ​​3 times greater than the negative wells were selected as positive hybridoma clones in the initial screening.

[0262] 2) ELISA rescreening using cynomolgus macaque CD3E&D heterodimer protein. The rescreening process was similar to the primary screening. 30 μL of hybridoma supernatant was taken from the clones that were positive in the primary screening. Positive clones from the cynomolgus macaque CD3E&D heterodimer protein ELISA rescreening were then subjected to Jurkat cell-based FACS screening. The Jurkat cell FACS screening method was as follows: Jurkat cells were collected by centrifugation, washed three times with pre-chilled PBS, and resuspended in 1% BSA (in PBS). 2x10 cells were added to each well. 5 Each well of the PCR agar plate was plated with 50 μL of cells (50 μL) in a 96-well conical bottom plate. 50 μL of the supernatant of 20 positive monoclonal clones identified by ELISA rescreening was added to a new PCR agar plate. Equal volumes of negative and positive controls were added to wells A1 and A2, respectively. The positive control was mouse serum diluted 1:2000, and the negative control was blank hybridoma culture medium. The cells were mixed by pipetting and incubated at 4°C for 1 hour. After washing three times with pre-chilled PBS, 100 μL of 1% BSA (containing 1 μL of Anti-mouse IgG-PE fluorescent secondary antibody, BioLegend, Cat. No. 405307) was added to each well. The cells were incubated at 4°C for 0.5 hour. After washing three times with pre-chilled PBS, the cells were resuspended and detected by flow cytometry (CytoFLEX).

[0263] A total of 10 mother clones were selected for subcloning using the above method.

[0264] Subclone screening: ELISA screening was performed by coating human CD3E&D and cynomolgus monkey CD3E&D heterodimer proteins, and FACS screening was performed on Jurkat cells. The experimental methods are as described above, and the experimental results are shown in Table 1.

[0265] Among the numerous antibodies, two unexpectedly showed excellent binding to human CD3E&D, monkey CD3E&D, and Jurkat cells. These positive clones, 9B5C8 and 17H2B3, were selected and amplified in serum-free culture medium. They were named CD3-01 and CD03-02, respectively.

[0266] Table 1 Statistics of anti-human CD3 antibody hybridoma screening results

[0267] Example 3. Expression and evaluation of anti-human CD3 mouse antibodies

[0268] 3.1 Expression and purification of anti-human CD3 mouse antibody

[0269] Hybridoma cells were cultured in serum-free medium. Each clone was expressed in 8-10 mL of culture medium for about 10 days. The supernatant of the single clone was collected by centrifugation and gravity purified using ProA filler. The supernatant was eluted using sodium acetate buffer, neutralized with Tris-HCl, and then ultrafiltered into PBS buffer. The protein concentration was determined by Nanodrop.

[0270] 3.2 Anti-human CD3 mouse antibody protein affinity detection

[0271] In order to detect the affinity of the mouse anti-CD3 antibody of the present invention to the human CD3 protein, human CD3E&D heterodimer protein (0.5μg / mL), human CD3E&G heterodimer protein (0.5μg / mL), and human CD3E recombinant protein (0.5μg / mL) were coated respectively and coated overnight at 4°C; the coating solution was removed the next day, each well was washed once with 300μL PBS, and 100μL 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 a starting concentration of 2μg / mL, 4-fold dilution, and 8 dilution gradients. The diluted antibody was added to the ELISA plate, 100μL / well, and duplicate wells. After incubation at 37°C for 2 hours, each well was washed 3 times with 300μL PBS. 100 μL of Anti-mouse IgG HRP (1:10,000) was then added to each well. After incubation at 37°C for 1 hour, each well was washed five times with 300 μL of PBS. 100 μL of TMB substrate (Wuxi Invitrogen, EF-T1) was then added to each well, and color was developed for approximately 2 minutes. The reaction was terminated by adding 50 μL of ELISA stop solution (Solarbio, C1058) to each well. OD450 nm readings were read using a microplate reader (MD, SpectraMax). Four-parameter fitting of the absorbance values ​​at 450 nm corresponding to different antibody concentrations was performed using GraphPad Prism software.

[0272] The results are shown in Figures 1A, 1B and 1C, and the specific affinities are shown in Table 2. The anti-CD3 antibodies of the present invention have excellent affinity for different forms of human CD3E protein.

[0273] Table 2. Affinity of anti-CD3 mouse antibodies to human CD3 protein

[0274] 3.3 Cross-detection of anti-human CD3 mouse antibody protein in monkeys

[0275] To test the cross-reactivity and affinity of the murine anti-CD3 antibodies of the present invention with cynomolgus monkey CD3E&D and CD3E&G heterodimers, cynomolgus monkey CD3E&D and CD3E&G heterodimers were diluted to 0.5 μg / mL in CBS coating buffer, 100 μL per well was added to an ELISA plate, and the plate was coated overnight at 4°C. Affinity determination was then performed using a method similar to that described in Example 1.5.2. The absorbance values ​​at 450 nm corresponding to different antibody concentrations were subjected to a four-parameter fit using GraphPad Prism software.

[0276] The results are shown in Figures 2A and 2B, and the specific affinities are shown in Table 3. The anti-CD3 antibodies of the present invention have excellent affinity for both monkey CD3 heterodimeric proteins.

[0277] Surprisingly, based on the data in Tables 2 and 3, it can be seen that the binding performance of the two antibodies to the monkey CD3 heterodimer protein is basically consistent with that to the human CD3 heterodimer protein, suggesting that they are very suitable for use in preclinical studies.

[0278] Table 3. Affinity of anti-CD3 mouse antibodies to monkey CD3 protein

[0279] 3.4 Anti-human CD3 mouse antibody affinity detection on Jurkat cells

[0280] In order to detect the cross-reactivity and affinity of anti-CD3 mouse antibodies with the target protein CD3 on the surface of human natural cells, flow cytometry was used to perform affinity tests on the antibodies to be tested. First, Jurkat cells were collected by centrifugation, washed twice with PBS, and resuspended to 8x10 6 CD3 mouse antibody was diluted in 1% BSA at a density of 50 μL / well, starting at 20 μg / mL. A four-fold, eight-fold dilution gradient was added to the cells, 50 μL per well, mixed, and incubated at 4°C for 50 minutes. The cells were washed twice with PBS. 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, and reacted at 4°C in the dark for 0.5 hours. The cells were washed twice with PBS, and the fluorescence signal and positive rate were measured by flow cytometry. Fluorescence signal values ​​corresponding to different antibody concentrations were curve fitted using GraphPad Prism software.

[0281] The results are shown in Figure 3. The anti-CD3 antibodies of the present invention have good binding ability with the CD3 protein on the surface of human natural cell Jurkat. CD3-01 and CD3-02 bind to EC 50 9.04nM and 1.75nM respectively.

[0282] 3.5 Anti-human CD3 mouse antibody affinity test on human peripheral blood mononuclear cells

[0283] In order to detect the cross-reactivity and affinity of anti-CD3 mouse antibodies with the target protein CD3 on the surface of human natural cells, flow cytometry was used to perform affinity tests on the test antibodies. First, peripheral blood collected from healthy volunteers was separated by Ficoll reagent to obtain human peripheral blood mononuclear cells (hPBMCs), and then hPBMC cells were collected by centrifugation, washed twice with PBS, and resuspended in 8x10 6At a density of 100 μL / well, CD3 mouse antibody was diluted in 1% BSA starting at 20 μg / mL and added to the cells in a 4-fold, eight-fold dilution series. 50 μL was added to each well, mixed, and incubated at 4°C for 50 minutes. The cells were washed twice with PBS. 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, and reacted at 4°C in the dark for 0.5 hours. The cells were washed twice with PBS, and the fluorescence signal and positive rate were measured by flow cytometry. Fluorescence signal values ​​corresponding to different antibody concentrations were curve fitted using GraphPad Prism software.

[0284] The results are shown in Figure 4. The experimental results show that the anti-CD3 antibody of the present invention has good binding ability with the CD3 protein on the surface of human natural cell PBMC, and CD3-01 and CD3-02 bind to EC 50 20.52nM and 6.07nM respectively.

[0285] 3.6 Anti-human CD3 mouse antibody affinity test on monkey peripheral blood mononuclear cells

[0286] To test the affinity of anti-CD3 mouse antibodies for CD3 protein on monkey peripheral blood mononuclear cells (cynoPBMCs), FACS affinity assessment was performed. Peripheral blood was collected from 3-5 year old cynomolgus macaques and separated using Ficoll reagent to obtain monkey peripheral blood mononuclear cells (cynoPBMCs). PBMCs were collected by centrifugation, washed twice with PBS, and resuspended in 1% BSA buffer. Human TruStain FcX (Biolegend) was added to the cynoPBMC suspension and incubated at room temperature for 20 minutes. After incubation, the cell suspension was added to a 96-well conical-bottom plate at 50 μL per well. CD3 mouse antibody was diluted in 1% BSA, starting at 20 μg / mL, and diluted fourfold over eight dilutions. 50 μL was added to each well, mixed, and incubated at 4°C for 50 minutes. The cells were washed twice with PBS. 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, and reacted at 4°C in the dark for 0.5 hours. The cells were washed twice with PBS, and the fluorescence signal and positive rate were measured by flow cytometry. Fluorescence signal values ​​corresponding to different antibody concentrations were curve fitted using GraphPad Prism software.

[0287] The results are shown in Figure 5. The experimental results show that the anti-CD3 antibody of the present invention has good binding ability with the CD3 protein on the surface of monkey natural cell PBMC. It also shows that the anti-CD3 antibody of the present invention has good monkey cross-reactivity. CD3-01 and CD3-02 bind to EC 50 They are 12.64nM and 2.32nM respectively.

[0288] Example 4. Acquisition and humanization of anti-human CD3 antibody sequences

[0289] 4.1 Mouse sequence fishing: Collect approximately 1×10 5 RNA was extracted using Trizol, and then reverse transcribed using the PrimeScript RT reagent kit using PolyA to obtain cDNA. Upstream primers were designed upstream of the heavy and light chains, and downstream primers were designed for the heavy chain CH1 region and the light chain CL region, respectively. PCR amplification products were recovered using an agarose gel recovery kit and sequenced. Specific variable region sequences are shown in Table 13 and the sequence listing.

[0290] 4.2 Antibody humanization: Using the CDR grafting method, first use the conventional BLAST method to find the human germline sequence with the highest homology to the original mouse sequence as a template; transplant the CDR of the mouse antibody to the human template to construct a chimera; based on the structural analysis of the FR amino acids in the mouse antibody that can retain its original conformation, the corresponding amino acids in the chimera are reversed to mouse amino acids to maintain the original affinity; the constructed humanized antibody is subjected to immunogenicity analysis to find highly immunogenic fragments and replace them with low immunogenic fragments; at the same time, through drugability analysis, the obvious post-translational modification sites in the sequence are removed, thereby removing 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 effect of post-translational modifications on antibody binding and stability is minimized.

[0291] After performance testing and screening of a variety of humanized antibodies, two humanized antibodies, CD3-01-Hz and CD3-02-Hz, were selected with excellent overall performance. The specific variable region sequences are shown in Table 13 and the sequence listing.

[0292] The humanized antibody CD3-01-Hz based on CD3-01 was mutated from amino acid G to amino acid A at position 55 (Chothia numbering) of the heavy chain of the corresponding murine antibody.

[0293] The humanized antibody CD3-02-Hz based on CD3-02 had amino acid G mutated to amino acid S at position 28 (Chothia numbering) of the heavy chain from the corresponding murine antibody.

[0294] The SEQ ID NOs of the VH, VL, and six CDRs of the murine antibodies CD3-01 and CD3-02, and the humanized CD3-01-Hz and CD3-02-Hz are shown in Table 4 below:

[0295] Table 4 CDR region coding of anti-human CD3 antibodies

[0296] Example 5. Evaluation of anti-human CD3 humanized antibodies

[0297] 5.1 Expression of humanized anti-human CD3 antibody

[0298] The heavy chain variable region sequences (SEQ NO:29) and light chain variable region sequences (SEQ NO:30) of the humanized antibodies CD3-01-Hz, CD3-02-Hz (SEQ NO:31) and light chain variable region sequences (SEQ NO:32), and the heavy chain variable region amino acid sequences of reference antibodies (Roche CD3, sequence from KEGG-D11463; REGN CD3, sequence 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 Universal Bio for gene synthesis. After codon optimization, the constructs were constructed into the pTT5 vector. After plasmid synthesis, HEK293E cells were transfected with PEImax. Expression was allowed to proceed for approximately 7 days, and the supernatant was collected by centrifugation. The supernatant was purified using ProA medium. The purified antibodies were ultrafiltered into PBS buffer, the concentration was determined, and the cells were stored at -20°C.

[0299] 5.2 ELISA affinity evaluation of anti-human CD3 humanized antibody protein

[0300] ELISA affinity evaluation was performed on the antigen proteins human CD3E&D heterodimer, human CD3E&G heterodimer, cynomolgus macaque CD3E&D heterodimer, cynomolgus macaque CD3E&G heterodimer, and human CD3E protein at 0.5 μg / mL using CBS coating. The experimental method was as described in Examples 2 and 3. The test antibody was serially diluted with 2% BSA (starting at 2 μg / mL, 4-fold dilution, 8 concentration points) and incubated at 37°C for 2 hours. HRP-labeled anti-human secondary antibody (Jackson, 109-035-088) was added and incubated at 37°C for 1 hour. TMB substrate was added for color development. The reaction was terminated with ELISA stop solution and the absorbance at 450 nm was read on an analyzer.

[0301] The experimental results are shown in Table 5 and Figures 6A, 6B, 6C, 6D, and 6E.

[0302] The two humanized antibodies have high affinity for human and cynomolgus monkey CD3E and CD3D heterodimeric proteins. At the same time, the anti-CD3 antibodies of the present invention have comparable abilities to bind to different forms of CD3 compared to the control antibody Roche CD3.

[0303] Table 5. ELISA affinity evaluation of anti-human CD3 humanized antibody protein

[0304] 5.3 Flow cytometric affinity evaluation of humanized anti-human CD3 antibodies

[0305] In order to detect the cross-reactivity and affinity of anti-CD3 humanized antibodies with the target protein CD3 on the surface of human natural cells, flow cytometry was used to perform affinity tests on the antibodies to be tested. First, Jurkat cells were collected by centrifugation, washed twice with PBS, and resuspended to 8x10 6 / mL, 50 μL / well of anti-CD3 humanized antibody diluted in 1% BSA, starting at 40 μg / mL, was added to the cells in a 4-fold gradient over eight concentrations, 50 μL per well, mixed, and incubated at 4°C for 50 min. Cells were washed twice with PBS. Finally, cells were resuspended in 1% BSA solution (containing APC anti-human IgG), 100 μL per well (containing 1 μL of fluorescent secondary antibody), mixed, and reacted at 4°C in the dark for 0.5 h. After washing twice with PBS, fluorescence signal and positive rate were measured by flow cytometry. Fluorescence signal values ​​corresponding to different antibody concentrations were curve fitted using GraphPad Prism software.

[0306] The experimental results are shown in Table 6 and Figure 7. The anti-CD3 antibody of the present invention has good binding ability with human Jurkat cells. 50 The anti-CD3 antibody of the present invention has a significantly lower affinity for Jurkat cells than the Roche anti-CD3 antibody and is also 2-3 times weaker than REGN CD3. The anti-CD3 antibody of the present invention may provide better safety.

[0307] Table 6. Humanized antibody affinity to Jurkat cells

[0308] 5.4 Flow cytometric affinity evaluation of humanized anti-human CD3 antibodies on human peripheral blood mononuclear cells

[0309] In order to detect the cross-reactivity and affinity of anti-CD3 humanized antibodies with the target protein CD3 on the surface of human natural cells, flow cytometry was used to perform affinity tests on the antibodies to be tested. First, human peripheral blood mononuclear cells were collected by centrifugation, washed twice with PBS, and resuspended to 8x10 6 / mL, 50 μL / well of anti-CD3 humanized antibody diluted in 1% BSA, starting at 80 μg / mL, was added to the cells in a 4-fold gradient over eight concentrations, 50 μL per well, mixed, and incubated at 4°C for 50 min. Cells were washed twice with PBS. Finally, cells were resuspended in 1% BSA solution (containing APC anti-human IgG), 100 μL per well (containing 1 μL of fluorescent secondary antibody), mixed, and reacted at 4°C in the dark for 0.5 h. After washing twice with PBS, fluorescence signal and positive rate were measured by flow cytometry. Fluorescence signal values ​​corresponding to different antibody concentrations were curve fitted using GraphPad Prism software.

[0310] The experimental results are shown in Figure 8 and Table 7. The anti-CD3 antibody of the present invention has good binding ability with human peripheral blood mononuclear cells. 50 The values ​​were 59.7 nM and 80.23 nM, respectively. Compared with the Roche anti-CD3 antibody, the affinity of the anti-CD3 antibody of the present invention is significantly reduced, and it is also 2 to 3 times weaker than REGN CD3. The anti-CD3 antibody of the present invention is expected to provide better safety.

[0311] Table 7. Human peripheral blood mononuclear cell affinity of humanized antibodies

[0312] 5.5 Flow cytometric affinity evaluation of humanized anti-human CD3 antibodies on monkey peripheral blood mononuclear cells

[0313] In order to detect the cross-reactivity and affinity of anti-CD3 humanized antibodies with the target protein CD3 on the surface of monkey natural cells, flow cytometry was used to perform affinity tests on the antibodies to be tested. First, monkey peripheral blood mononuclear cells were collected by centrifugation, washed twice with PBS, and resuspended to 8x10 6 / mL, 50 μL / well of anti-CD3 humanized antibody diluted in 1% BSA, starting at 80 μg / mL, was added to the cell wells in a 4-fold gradient over eight concentrations, 50 μL per well, mixed, and incubated at 4°C for 50 min. Cells were washed twice with PBS. Finally, cells were resuspended in 1% BSA solution (containing APC anti-human IgG), 100 μL per well (containing 1 μL of fluorescent secondary antibody), mixed, and reacted at 4°C in the dark for 0.5 h. After washing twice with PBS, fluorescence signal and positive rate were measured by flow cytometry. Fluorescence signal values ​​corresponding to different antibody concentrations were curve fitted using GraphPad Prism software.

[0314] The experimental results are shown in Figure 9 and Table 8. The anti-CD3 antibody of the present invention has good cross-reactivity with monkey peripheral blood mononuclear cells and binds to EC 50 The values ​​were 18.89 nM and 5.14 nM, respectively.

[0315] Table 8. Affinity of humanized antibodies for monkey peripheral blood mononuclear cells

[0316] 5.6 Dynamic Affinity Evaluation of Humanized Anti-human CD3 Antibodies

[0317] The dynamic affinity of humanized antibodies was determined using ForteBio. The experimental steps are as follows: 1. Sensor preparation: Remove the ProA sensor and pre-wet it with PBST (pH 7.4) for 10 minutes. 2. Sample dilution: Dilute the antibody to be immobilized to 5 μg / mL. The antigens (human CD3E / CD3G heterodimer and monkey CD3E / CD3G heterodimer) were diluted to 5 μg / mL starting at 500 nM, using a two-fold dilution, five concentration points, and a zero concentration point. 3. Set up the program, insert 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 results graph.

[0318] As shown in Table 9 and Figure 10, the anti-CD3 antibodies of the present invention have good dynamic binding and dissociation rates, wherein the dynamic affinity for human CD3E&G heterodimeric protein is comparable to that of Roche anti-CD3 antibody, and the ability of the anti-CD3 antibodies of the present invention to bind to monkey CD3E&G heterodimeric protein is similar to that of binding to human CD3E&G heterodimeric protein.

[0319] Table 9. Dynamic affinity detection of anti-human CD3 humanized antibodies

[0320] 5.7 Anti-human CD3 humanized antibody Jurkat reporter cell activity assay

[0321] To test the Jurkat cell activation ability of humanized anti-CD3 antibodies, Jurkat-NFTA-Luciferase (Nearshore Protein, Catalog No. XCC20) reporter cells were used for evaluation. CD3 & CD28 magnetic beads (Beijing Biopsies Biotechnology Co., Ltd., Catalog No. MBS-C001) were used as a positive control with agonistic 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 humanized CD3 antibodies tested were diluted from a 200 nM concentration in RPMI1640 + 10% FBS medium, with a 4-fold serial dilution across 8 concentration points, with the last point being a zero concentration well. For IgG1 antibodies, only the highest concentration was set. 100 μL of the diluted sample was added to a 96-well white plate. All samples were set in duplicate. The CD3 & CD28 magnetic bead positive control consisted of 2 μL of magnetic beads added to 100 μL of RPMI1640 + 10% FBS medium. Jurkat-NFAT-Luciferase cells were also diluted to 5x10 cells using 1640+10% FBS. 5 / mL, 100 μL of the cell suspension was added to the well plate and incubated at 37°C for 6 hours. After 6 hours, the cell culture plate was left at room temperature for 20 minutes. 50 μL of Oneglu reagent (Novozymes, Catalog No. DD1201-02), previously equilibrated to room temperature, was added to each well. After incubation with shaking for 3 minutes, the luminance signal was read on a microplate reader. Curve fitting of the signal values ​​corresponding to different antibody concentrations was performed using GraphPad Prism software.

[0322] The experimental results are shown in Figure 11 and Table 10. The experimental results show that the positive control CD3 & CD28 group can induce strong activation of Jurkat reporter cells, and the isotype control antibody has no activation activity. After treatment with anti-CD3 antibodies, the two CD3 humanized antibodies of the present invention have a certain degree of activation activity of the Jurkat reporter system. EC of CD3-01-Hz and CD3-02-Hz 50 The EC values ​​of REGN CD3 were 25.29 nM and 18.02 nM, respectively. 50 Therefore, the activity of the humanized CD3 antibody of the present invention in inducing Jurkat cell activation is significantly weaker than that of the REGN anti-CD3 antibody, and the anti-CD3 antibody of the present invention is expected to provide better safety.

[0323] Table 10. Humanized Antibody Jurkat Reporter System Activity

[0324] 5.8 Detection of T cell activation activity of humanized anti-human CD3 antibody

[0325] In order to detect the activation of T cells by anti-CD3 humanized antibodies, the positive expression rates of CD25 and CD69 on PBMC cells were evaluated by flow cytometry. The changes in the positive expression rates of CD25 and CD69 on PBMC cells reflected the activation status of T cells. CD3 & CD28 magnetic beads (Beijing Biopsies Biotechnology Co., Ltd., catalog number MBS-C001) were used as a positive control with agonistic 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 tested CD3 humanized antibodies started at a concentration of 200 nM, and RPMI1640 + 10% FBS medium was used as the dilution medium. A 4-fold serial dilution was performed with 8 concentration points, and the last point was a zero concentration well. Only the highest concentration was set for IgG1 antibody. 100 μL of the diluted sample was added to a 96-well white plate. All samples were set in duplicate. The CD3 & CD28 magnetic bead positive control consisted of 2 μL of magnetic beads added to 100 μL of 1640 + 10% FBS medium. PBMC cells were revived in 1640 + 10% FBS medium the afternoon before the experiment. On the day of the test, PBMCs were diluted to 2x10 using 1640 + 10% FBS. 6 / mL, take 100 μL of cell suspension and add it to the above well plate, and culture at 37 degrees for 24 hours.

[0326] After 24 hours, the cell suspension was transferred to a 96-well conical-bottom plate and centrifuged at 500g for 3 minutes. The supernatant was removed and the cells were washed twice with 200μL of PBS. The cell pellet was resuspended in 1% BSA containing Human TruStain FcX (Biolegend) and incubated at room temperature for 20 minutes. Then, 2μL of CD69-APC-conjugated antibody was added to each well and incubated at 4°C for 45 minutes. The cells were then washed twice with PBS. The cell pellet was resuspended in PBS and analyzed for CD69 positivity. The CD69 positivity corresponding to different antibody concentrations was curve-fitted using GraphPad Prism software.

[0327] The experimental results are shown in Figure 12 and Table 11. The experimental results show that the positive control CD3&CD28 group can induce strong upregulation of CD69 expression on the surface of T cells, and the isotype control antibody has no activation activity. After treatment with anti-CD3 antibodies, the expression level of CD69 on the surface of T cells increased significantly. For the induction of CD69 expression, the EC of CD3-01-Hz and CD3-02-Hz 50 were 7.13 nM and 37.71 nM, respectively, while the EC values ​​of the control antibody Roche CD3 were 50Therefore, the humanized CD3 antibody of the present invention has weaker T cell activation activity than Roche CD3, and the anti-CD3 antibody of the present invention is expected to provide better safety.

[0328] Table 11. T cell activation activity of anti-human CD3 humanized antibodies

[0329] 5.9 Hydrophobicity Detection of Humanized Anti-human CD3 Antibody

[0330] The hydrophilicity of the humanized antibody was detected by HIC. The detection method is as follows: The hydrophilicity test was performed on an Agilent HPLC instrument using a Tosoh hydrophobic chromatography column (TOSOH Tskgel Buty-NPR (2.5), 4.6*100). The mobile phase A was 1.5M (NH4)2SO4, and the mobile phase B was 25mM Na2HPO4 (pH=7.0) + 20% isopropanol. The instrument parameters were set as sample chamber temperature: 8°C, column temperature: 30°C, flow rate: 0.5mL / min, and detection wavelength: 280nm. The sample to be tested was diluted with mobile phase A to a final concentration of 1mg / mL, and 20μL was injected for gradient elution. The elution gradient is shown in Table 12:

[0331] Table 12. HPLC run program for hydrophobicity analysis

[0332] 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 hydrophobicity (22 minutes) and weak hydrophobicity (12 minutes) of the internal standard molecules, both CD3-01-Hz and CD3-02-Hz have good hydrophilicity.

[0333] 5.10 Tm value detection of anti-human CD3 humanized antibody

[0334] Adopt DSF to measure the humanized antibody Tm value, to react the thermal stability of antibody.Use real-time fluorescence quantitative PCR (QuantStudio 7Flex, Thermo Fisher Scientific) to carry out DSF determination.In brief, 19 μ L concentration is the antibody solution of 1 mg / ml and 1 μ L 62.5X SYPRO Orange solution (Invitrogen) is mixed and added in 96-well plates (Biosystems).The plate is heated from 25 ° C to 95 ° C at a rate of 2 ° C / min, and the fluorescence data generated are collected.Calculate the negative derivative of fluorescence change relative to different temperatures, and the maximum value is defined as the melting temperature Tm.Data acquisition and T h calculation are automatically carried out by operating software (QuantStudio Real-Time PCR Software v1.3).

[0335] 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, suggesting that the two humanized CD3 monoclonal antibodies CD3-01-Hz and CD3-02-Hz have good thermal stability.

[0336] The antibody sequences obtained by the present invention are shown in Table 13 below:

[0337] Table 13 Antibody sequences

[0338] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

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 group: (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 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. The recombinant protein according to claim 2, wherein, The recombinant protein is a monospecific, bispecific, or trispecific antibody.

4. A multispecific antibody, characterized in that, The multi-specific antibody described above comprises: (1) the anti-CD3 antibody described in claim 1; (2) an antibody that binds to other targets.

5. 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 group: (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.

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

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

8. 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 described in claim 1, or the recombinant protein as described in claim 2, the CAR construct as described in claim 5, the immune cells as described in claim 6, the antibody-drug conjugate as described in claim 7, 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.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition described above contains: (i) an active ingredient selected from the group consisting of: the antibody or antigen-binding fragment thereof as described in claim 1, or the recombinant protein as described in claim 2, the CAR construct as described in claim 5, the immune cells as described in claim 6, the antibody-drug conjugate as described in claim 7, or a combination thereof; and (ii) a pharmaceutically acceptable carrier.

10. 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 described in claim 1; or (2) the recombinant protein as described in claim 2; (3) the CAR construct as described in claim 5.

11. A carrier, characterized in that, The vector contains the polynucleotide as described in claim 10.

12. A genetically engineered host cell, characterized in that, The host cell contains the vector as described in claim 11 or the polynucleotide as described in claim 10 is integrated into the genome.

13. A method for non-diagnostic in vitro detection of CD3 protein in a sample, the method comprising the steps of: (1) in vitro, contacting the sample with the antibody as described in claim 1; (2) detecting whether an antigen-antibody complex is formed, and the formation of the complex indicates the presence of CD3 protein in the sample.

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