anti-CD3 humanized antibody

Abstract

The present invention provides a novel anti-CD3 humanized antibody, which can specifically bind to human and monkey CD3 proteins and has good biological activity.

Description

[Technical Field]

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

[0002] T cell activation is crucial for stimulating immune responses. T cells exhibit immune specificity and guide most cellular immune responses. T cells do not secrete antibodies, but they are required for antibody secretion by B cells.

[0003] CD3 is a homodimeric or heterodimeric antigen expressed on T cells that binds to the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is a dimer composed of two of four distinct chains (ε, ζ, δ, and γ). The CD3 dimer arrangements include γ / ε, δ / ε, and ζ / ζ. Antibodies to CD3 recruit CD3 to T cells, activating them in a manner that engages with the TCR like peptide-carrying MHC molecules. Therefore, anti-CD3 antibodies have therapeutic applications in T cell activation. Bispecific antibodies capable of binding both CD3 and a target antigen have also been proposed for therapeutic use, targeting T cell immune responses to tissues and cells expressing the target antigen.

[0004] Currently, existing CD3-binding bispecific antibodies in clinical trials for cancer treatment are limited by short half-lives and / or uncertain efficacy. Many attempts have been made to obtain anti-CD3 antibodies with particularly advantageous properties, but to date these attempts have met with limited success.

[0005] Therefore, there is a strong demand in this field for the development of novel humanized anti-CD3 antibodies. Summary of the Invention

[0006] An object of the present invention is to provide a novel humanized anti-CD3 antibody.

[0007] In a first aspect of the present invention, there is provided an anti-CD3 humanized antibody, 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: (A5) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH5-CDR3: HGNFGNTYISYWAY, SEQ ID NO: 29 huVL5-CDR1: TGAVTSGNY, SEQ ID NO: 30 huVL-CDR2: GTK, SEQ ID NO: 5 huVL5-CDR3: VLWYSKRW, SEQ ID NO: 31; (A1) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH4-CDR3: HGNFGNSYISYWEY, SEQ ID NO: 3 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL4-CDR3: VLWNSNRW, SEQ ID NO: 6; (A2) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH3-CDR3: HGNFGNSYISYWRY, SEQ ID NO: 10 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL3-CDR3: VLWYSGRW, SEQ ID NO: 11; (A3) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH2-CDR3: HGNFGNSYISYWQY, SEQ ID NO: 15 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL2-CDR3: VLWRSNRW, SEQ ID NO: 16; or (A4) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH1-CDR3: HGNFGNTYISYWAY, SEQ ID NO: 20 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL1-CDR3: VLWYSKRW, SEQ ID NO: 21.

[0008] In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence, optionally having at least one amino acid addition, deletion, modification and / or substitution, and retaining CD3 binding affinity.

[0009] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-5 (for example, 1-3, preferably 1-2, more preferably 1).

[0010] In another preferred embodiment, the derived sequence having at least one amino acid addition, deletion, modification, and / or substitution and retaining affinity for binding to CD3 has at least 96% homology or sequence identity with the amino acid sequence. In another preferred embodiment, the antibody 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 linking the heavy chain, and the light chain of the antibody comprises the three light chain CDRs and a light chain framework region for linking the light chain.

[0011] In another preferred embodiment, the sequence of the heavy chain variable region is set forth in SEQ ID NO:7.

[0012] In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.

[0013] In another preferred embodiment, the heavy chain constant region is of human, mouse or rabbit origin, preferably human origin.

[0014] In another preferred embodiment, the sequence of the light chain variable region is set forth in SEQ ID NO:8.

[0015] In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

[0016] In another preferred embodiment, the light chain constant region is of human, mouse or rabbit origin, preferably human origin.

[0017] In another preferred embodiment, the Kd of binding between the antibody and human CD3ε polypeptide is ≦250 nM, preferably ≦100 nM, preferably ≦15 nM, preferably ≦10 nM, more preferably ≦5 nM.

[0018] In another preferred embodiment, the antibody specifically binds to CD3.

[0019] In another preferred embodiment, the CD3 is derived from a human or a cynomolgus monkey.

[0020] In another preferred embodiment, the CD3 is CD3ε, CD3ζ, CD3δ or CD3γ, preferably CD3ε or CD3γ.

[0021] In another preferred embodiment, the antibody specifically binds to human CD3ε or cynomolgus CD3ε, or the antibody specifically binds to human CD3γ or cynomolgus CD3γ.

[0022] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody (scFv).

[0023] In another preferred embodiment, the antibody is a single-chain antibody 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: (A5) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH5-CDR3: HGNFGNTYISYWAY, SEQ ID NO: 29 huVL5-CDR1: TGAVTSGNY, SEQ ID NO: 30 huVL-CDR2: GTK, SEQ ID NO: 5 huVL5-CDR3: VLWYSKRW, SEQ ID NO: 31; (A1) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH4-CDR3: HGNFGNSYISYWEY, SEQ ID NO: 3 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL4-CDR3: VLWNSNRW, SEQ ID NO: 6; (A2) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH3-CDR3: HGNFGNSYISYWRY, SEQ ID NO: 10 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL3-CDR3: VLWYSGRW, SEQ ID NO: 11; (A3) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH2-CDR3: HGNFGNSYISYWQY, SEQ ID NO: 15 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL2-CDR3: VLWRSNRW, SEQ ID NO: 16; or (A4) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH1-CDR3: HGNFGNTYISYWAY, SEQ ID NO: 20 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL1-CDR3: VLWYSKRW, SEQ ID NO: 21.

[0024] In another preferred embodiment, the single chain antibody comprises, in order, a light chain variable region, a linker and a heavy chain variable region, or, in order, a heavy chain variable region, a linker and a light chain variable region.

[0025] In another preferred embodiment, the linker is (G4S)n, where n is an integer from 1 to 5, preferably the linker is (G4S)3.

[0026] In another preferred embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 32, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 33; or the heavy chain variable region of said antibody comprises the amino acid sequence set forth in SEQ ID NO:7, and the light chain variable region of said antibody comprises the amino acid sequence set forth in SEQ ID NO:8; or the heavy chain variable region of said antibody comprises the amino acid sequence set forth in SEQ ID NO:12, and the light chain variable region of said antibody comprises the amino acid sequence set forth in SEQ ID NO:13; or the heavy chain variable region of said antibody comprises the amino acid sequence set forth in SEQ ID NO:17, and the light chain variable region of said antibody comprises the amino acid sequence set forth in SEQ ID NO:18; or The heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:22, and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:23.

[0027] In another preferred embodiment, the sequence of the single chain antibody is selected from the group consisting of: SEQ ID NO:34, SEQ ID NO:9, SEQ ID NO:22, SEQ ID NO:19 or SEQ ID NO:24.

[0028] In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO: 32, 7, 12, 17 or 22.

[0029] In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO: 33, 8, 13, 18 or 23.

[0030] In another preferred embodiment, the antibody is a monoclonal antibody.

[0031] In another preferred embodiment, the antibody is a monospecific, bispecific, or trispecific antibody.

[0032] In another preferred embodiment, the bispecific antibody comprises: (1) an anti-CD3 antibody according to the first aspect of the present invention; (2) Antibodies that bind to other targets.

[0033] In another preferred embodiment, the other target is selected from the group consisting of BCMA, EGFR, CD38, CD123, CD19, CD20, CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD47, CTLA4, PD1, and PDL1.

[0034] In another preferred embodiment, the bispecific antibody comprises: a first antigen-binding domain (D1), and a second antigen-binding domain (D2), Here, D1 specifically binds to the target molecule CD3 protein, D2 specifically binds to the target molecule CD19 protein, wherein D1 is an antibody or an antigen-binding fragment thereof that specifically binds to CD3 protein, D2 is an antibody or an antigen-binding fragment thereof that specifically binds to CD19 protein, wherein D1 comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region have six complementarity determining regions (CDRs) selected from the group consisting of: (A5) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH5-CDR3: HGNFGNTYISYWAY, SEQ ID NO: 29 huVL5-CDR1: TGAVTSGNY, SEQ ID NO: 30 huVL-CDR2: GTK, SEQ ID NO: 5 huVL5-CDR3: VLWYSKRW, SEQ ID NO: 31; (A1) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH4-CDR3: HGNFGNSYISYWEY, SEQ ID NO: 3 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL4-CDR3: VLWNSNRW, SEQ ID NO: 6; (A2) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH3-CDR3: HGNFGNSYISYWRY, SEQ ID NO: 10 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL3-CDR3: VLWYSGRW, SEQ ID NO: 11; (A3) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH2-CDR3: HGNFGNSYISYWQY, SEQ ID NO: 15 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL2-CDR3: VLWRSNRW, SEQ ID NO: 16; or (A4) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH1-CDR3: HGNFGNTYISYWAY, SEQ ID NO: 20 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL1-CDR3: VLWYSKRW, SEQ ID NO: 21; wherein the structure of the antigen-binding fragment is selected from the group consisting of (i) a Fab fragment, (ii) a F(ab')2 fragment, (iii) a Fd fragment, (iv) a Fv fragment, (v) a scFv molecule, or (vi) a dAb fragment.

[0035] In another preferred embodiment, the D1 and / or the D2 is a single chain antibody (scFv).

[0036] In another preferred embodiment, D1 and D2 are linked via a linker, and preferably, the linker is a flexible linker.

[0037] In another preferred embodiment, the linker is (GGGGS)n, where n is an integer from 1 to 5, preferably the linker is GGGGS.

[0038] In another preferred embodiment, D1 is an anti-CD3 single chain antibody and D2 is an anti-CD19 single chain antibody.

[0039] In another preferred embodiment, the anti-CD3 single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 33; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:12 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:13; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:17 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:18; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:22, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:23.

[0040] In another preferred embodiment, the sequence of the bispecific antibody is shown in SEQ ID NO:37.

[0041] In a second aspect of the present invention, there is provided a recombinant protein comprising: (i) an antibody or bispecific antibody thereof according to the first aspect of the invention; and (ii) An optional tag sequence to aid in expression and / or purification.

[0042] In another preferred embodiment, the tag sequence comprises a 6His tag.

[0043] In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

[0044] In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

[0045] In another preferred embodiment, the recombinant protein is a monospecific, bispecific, or trispecific recombinant protein.

[0046] In another preferred embodiment, the recombinant protein further comprises another fusion element (or fusion polypeptide fragment) fused to element (i).

[0047] In another preferred embodiment, the recombinant protein is (i) an antibody selected from the group consisting of: the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:32, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:33; or the heavy chain variable region of said antibody comprises the amino acid sequence set forth in SEQ ID NO:7, and the light chain variable region of said antibody comprises the amino acid sequence set forth in SEQ ID NO:8; or the heavy chain variable region of said antibody comprises the amino acid sequence set forth in SEQ ID NO:12, and the light chain variable region of said antibody comprises the amino acid sequence set forth in SEQ ID NO:13; or the heavy chain variable region of said antibody comprises the amino acid sequence set forth in SEQ ID NO:17, and the light chain variable region of said antibody comprises the amino acid sequence set forth in SEQ ID NO:18; or the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:22, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:23; and (ii) An optional tag sequence to aid in expression and / or purification.

[0048] In a third aspect of the present invention, there is provided a CAR construct, wherein an scFv domain fragment of an antigen-binding domain of the CAR construct is a binding domain that specifically binds to CD3, and the scFv fragment comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain and the light chain variable domain have six complementarity-determining regions (CDRs) selected from the group consisting of: (A5) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH5-CDR3: HGNFGNTYISYWAY, SEQ ID NO: 29 huVL5-CDR1: TGAVTSGNY, SEQ ID NO: 30 huVL-CDR2: GTK, SEQ ID NO: 5 huVL5-CDR3: VLWYSKRW, SEQ ID NO: 31; (A1) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH4-CDR3: HGNFGNSYISYWEY, SEQ ID NO: 3 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL4-CDR3: VLWNSNRW, SEQ ID NO: 6; (A2) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH3-CDR3: HGNFGNSYISYWRY, SEQ ID NO: 10 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL3-CDR3: VLWYSGRW, SEQ ID NO: 11; (A3) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH2-CDR3: HGNFGNSYISYWQY, SEQ ID NO: 15 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL2-CDR3: VLWRSNRW, SEQ ID NO: 16; or (A4) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH1-CDR3: HGNFGNTYISYWAY, SEQ ID NO: 20 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL1-CDR3: VLWYSKRW, SEQ ID NO: 21.

[0049] In a fourth aspect of the invention, there is provided a recombinant immune cell, the immune cell expressing an exogenous CAR construct according to the third aspect of the invention.

[0050] In another preferred embodiment, the immune cells are selected from the group consisting of NK cells and T cells.

[0051] In another preferred embodiment, the immune cells are derived from a human or non-human mammal (eg, a mouse).

[0052] In a fifth aspect of the present invention, there is provided an antibody-drug conjugate comprising: (a) an antibody or bispecific antibody thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or a combination thereof; and (b) a coupling moiety coupled to said antibody moiety, said coupling moiety being selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0053] In another preferred embodiment, the antibody moiety and the coupling moiety are coupled via a chemical bond or linker.

[0054] In a sixth aspect of the present invention, there is provided the use of an active ingredient selected from the group consisting of an antibody or bispecific antibody thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, a CAR construct according to the third aspect of the invention, an immune cell according to the fourth aspect of the invention, an antibody-drug conjugate according to the fifth aspect of the invention, or a combination thereof, for: (a) Manufacturing of detection reagents or kits; (b) the manufacture of drugs or preparations for the prevention and / or treatment of CD3-related diseases; and / or (c) The manufacture of drugs or preparations for preventing and / or treating CD3-associated cancers or tumors.

[0055] In another preferred embodiment, the cancer or tumor is selected from the group consisting of lung cancer, melanoma, colon cancer, pancreatic cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, testicular adenocarcinoma, esophageal cancer, gastrointestinal cancer, liver cancer, lymphoma, myeloma, and leukemia.

[0056] In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising: (i) an active ingredient selected from the group consisting of an antibody or bispecific antibody thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, a CAR construct according to the third aspect of the invention, an immune cell according to the fourth aspect of the invention, an antibody-drug conjugate according to the fifth aspect of the invention, or a combination thereof; and (ii) A pharmaceutically acceptable carrier.

[0057] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0058] In another preferred embodiment, the pharmaceutical composition is an injection.

[0059] In another preferred embodiment, the pharmaceutical composition is used for the manufacture of a medicament for treating a tumor, wherein the tumor is selected from the group consisting of lung cancer, melanoma, colon cancer, pancreatic cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, testicular adenocarcinoma, esophageal cancer, gastrointestinal cancer, liver cancer, lymphoma, myeloma, and leukemia.

[0060] In an eighth aspect of the present invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of: (1) an antibody or a bispecific antibody thereof according to the first aspect of the present invention; or (2) a recombinant protein according to the second aspect of the present invention; (3) A CAR construct according to the third aspect of the present invention.

[0061] In a ninth aspect of the present invention, there is provided a vector comprising a polynucleotide according to the eighth aspect of the present invention.

[0062] In another preferred embodiment, the vector comprises a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, such as an adenovirus, a retrovirus, or other vector.

[0063] In a tenth aspect of the present invention, there is provided a genetically engineered host cell which comprises a vector according to the ninth aspect of the invention or has incorporated into its genome a polynucleotide according to the eighth aspect of the invention.

[0064] In another preferred embodiment, the host cell is a mammalian cell or a prokaryotic cell.

[0065] In another preferred embodiment, the mammalian cells are Chinese hamster ovary (CHO) cells.

[0066] In another preferred embodiment, the prokaryotic cell is E. coli.

[0067] In an eleventh aspect of the present invention, there is provided a method for non-diagnostic in vitro detection of CD3 protein in a sample, the method comprising the steps of: (1) contacting the sample in vitro with the antibody of the first aspect of the invention; (2) detecting the formation of antigen-antibody complexes, where the formation of complexes indicates the presence of CD3 protein in the sample;

[0068] In a twelfth aspect of the present invention, there is provided a detection plate comprising a base sheet (support plate) and a measurement bar containing the antibody according to the first aspect of the present invention or the antibody-drug conjugate according to the ninth aspect of the present invention.

[0069] In a thirteenth aspect of the present invention, there is provided a kit comprising: (1) a first container containing an antibody according to the first aspect of the invention, and / or (2) a second container containing a secondary antibody against the antibody of the first aspect of the invention; Alternatively, there is provided a kit containing the detection plate according to the twelfth aspect of the present invention.

[0070] In a fourteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising the steps of: (a) culturing a host cell according to the tenth aspect of the invention under conditions suitable for expression; (b) isolating a recombinant polypeptide from the culture, which is an antibody according to the first aspect of the invention or a recombinant protein according to the second aspect of the invention.

[0071] In a fifteenth aspect of the present invention, there is provided a method of treating a disease associated with a CD3 molecule, the method comprising administering to a subject in need of inhibition or treatment an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention.

[0072] In another preferred embodiment, the CD3 molecule-related disease includes immune rejection of tumors or organ transplants.

[0073] In another preferred embodiment, the subject is a mammal (including a human).

[0074] Of course, it is understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be described here one by one due to space limitations. [Brief explanation of the drawings]

[0075] [Figure 1] Figure 1 shows the affinity of CD3 antibodies to recombinant human CD3 as measured by ELISA. [Figure 2] Figure 1 shows the affinity of CD3 antibodies to recombinant monkey CD3 as measured by ELISA. [Figure 3]Figure 1 shows the affinity of full-length CD3 antibodies detected by fortebio with human CD3E protein, where A, B, C, D, and E are the affinity graphs of full-length CD3 antibodies CQ53, CQ52, CQ51, CQ50, and CQ54 with human CD3E protein, respectively. [Figure 4] 1 shows the affinity of full-length CD3 antibodies to monkey CD3E protein as detected by ELISA, where A, B, C, D, and E are graphs showing the affinity of full-length CD3 antibodies CQ53, CQ52, CQ51, CQ50, and CQ54 to monkey CD3E protein, respectively. [Figure 5] 1 shows the affinity of full-length CD3 antibodies to Jurkat cells, where A, B, C, D, and E show the affinity of full-length CD3 antibodies CQ53, CQ52, CQ51, CQ50, and CQ54 to Jurkat cells, respectively. [Figure 6] Figure 1 shows the competitive ELISA detection of full-length CD3 antibodies and OKT3, where A, B, C, D, and E are the competitive ELISA detection of full-length CD3 antibodies CQ53, CQ52, CQ51, CQ50, and CQ54 and OKT3, respectively. [Figure 7] Figure 1 shows a diagram of the changes in CD69 expression induced by CD3 antibody, where NC is the negative control. [Figure 8] Figure 1 shows a diagram of IFNγ secretion by PBMC activation induced by CD3 antibody, where NC is the negative control. [Figure 9] Figure 1 shows the affinity of bispecific antibodies to human CD3E (A) and CD19 (B) proteins as detected by fortebio. [Figure 10] FIG. 1 shows the affinity of bispecific antibodies (SC30A and BLMOA) to monkey CD3E protein as measured by ELISA. [Figure 11] Figure 1 shows a diagram of the changes in CD69 expression induced by CD3 antibody, where NC is the negative control. [Figure 12] FIG. 1 shows the tumor cell killing efficiency of bispecific antibodies (SC30A and BLMOA) detected by flow cytometry. [Figure 13]1 shows the residual Raji-GFP revealed by fluorescent scanning of the wells. [Figure 14] Figure 1 shows the direct killing efficiency of bispecific antibodies against PBMC tumor cells. [Figure 15] Figure 1 shows a diagram of a PBMC proliferation experiment. [Figure 16] The dependence of the SC30A signal detected by the reporter gene on the target cells is shown, where RLU is relative light units. [Figure 17] A diagram of the activity of SC30A detected by the reporter gene is shown, where RLU is relative light units. DETAILED DESCRIPTION OF THE INVENTION

[0076] Through extensive and in-depth research, the inventors unexpectedly discovered a CD3 antibody with high biological activity. Experiments have shown that the CD3 antibody of the present invention can specifically bind to human and monkey CD3. The CD3 antibody of the present invention can also be combined with an antibody or its binding fragment that binds to the target antigen CD19 to construct a bispecific antibody, resulting in enhanced tumor-killing activity and therapeutic use in targeting T cell immune responses to tissues and cells expressing the target antigen. Furthermore, the activation of the bispecific antibody of the present invention is target-cell dependent and safe. Based on this, the present invention has been completed.

[0077] term As used herein, the terms "administration" and "treatment" 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. "Administration" and "treatment" can refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with a cell, contact of a reagent with a fluid, and contact of a fluid with a cell. "Administration" and "treatment" also refer to in vitro and ex vivo treatment via a reagent, diagnostic, binding composition, or another cell. "Treatment," when applied to a human, animal, or research subject, refers to therapeutic treatment, prophylactic or preventative treatment, research, and diagnosis, and includes contact of a CD3 antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

[0078] As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including any one of the CD3 antibodies and compositions of the present invention, to a patient suffering from one or more disease symptoms, where the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the therapeutic agent is administered to the patient in an amount that effectively alleviates one or more disease symptoms (a therapeutically effective amount).

[0079] As used herein, the terms "any" or "optionally" mean that the subsequently described event or circumstance may be present, but is not required. For example, "optionally comprising 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may be present, but are not necessarily present, and may be 1, 2, or 3.

[0080] antibody As used herein, the term "antibody" refers to an immunoglobulin, a four-peptide chain structure consisting of two identical heavy chains and two identical light chains linked via disulfide bonds. The antigenicity of an immunoglobulin varies depending on the amino acid composition and sequence of the heavy chain constant region. Therefore, immunoglobulins are divided into five types, or classes of immunoglobulins, namely, IgM, IgD, IgG, IgA, and IgE, designated α, δ, ε, γ, and μ, respectively, depending on the heavy chain constant region of the different classes of immunoglobulins. IgG is the most important type of immunoglobulin, and is divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on their chemical structure and biological function. Light chains are classified as κ or hλ chains depending on their constant region. The subunit structure and three-dimensional structure of each class of immunoglobulin are well known to those skilled in the art.

[0081] The sequences of approximately 110 amino acids near the N-terminus of the heavy and light chains of antibodies are highly variable and constitute the variable region (V region), while the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region contains three hypervariable regions (HVR) and four relatively conserved FR regions (FR). The amino acid sequences of the four FRs are relatively conserved and are not involved in direct binding reactions. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain, i.e., the light chain hypervariable region (LCDR), are designated LCDR1, LCDR2, and LCDR3, while the three CDR regions of the heavy chain, i.e., the heavy chain hypervariable region (HCDR), are designated HCDR1, HCDR2, and HCDR3. The amino acid residues of the CDRs of the LCVR and HCVR regions of the antibodies or antigen-binding fragments described in the invention are numbered and positioned according to the known Kabat numbering system (LCDR1-3, HCDR2-3) or Kabat and Chothia numbering system (HCDR1). The four FR regions in the variable regions of naturally occurring heavy and light chains essentially have a β-sheet structure, connected by three CDRs that form connecting loops, and sometimes include four FR regions with a partial β-sheet structure. The CDRs in each chain are closely spaced by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody. The amino acids that make up the FR or CDR regions can be identified by comparing the amino acid sequences of similar antibodies. The constant region is not directly involved in binding of an antibody to an antigen, but exhibits different effector functions, such as participating in antibody-dependent cellular toxicity of the antibody.

[0082] As used herein, the term "antigen-binding fragment" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, or single Fv fragment that has antigen-binding activity. An Fv antibody is the smallest antibody fragment that contains the heavy and light chain variable regions of an antibody but lacks the constant region and contains all of the antigen-binding site. Generally, an Fv antibody further contains a polypeptide linker between the VH and VL domains and is capable of forming the structure necessary for antigen binding. Non-limiting examples of antigen-binding fragments include: (i) a Fab fragment, (ii) a F(ab')2 fragment, (iii) a Fd fragment, (iv) a Fv fragment, (v) an scFv molecule, (vi) a dAb fragment, and (vii) a minimal recognition unit consisting of amino acid residues resembling the hypervariable region of an antibody (e.g., an independent complementarity-determining region (CDR) such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. As used herein, the term "antigen-binding fragment" also includes 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 IgNAR variable domains.

[0083] As used herein, the term "antigenic determinant" refers to a discrete spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the present invention.

[0084] The present invention includes not only complete antibodies, but also immunologically active antibody fragments or fusion proteins consisting of antibodies and other sequences. Thus, the present invention further includes fragments, derivatives and analogs of said antibodies.

[0085] In the present invention, antibodies include murine, chimeric, humanized, or fully human antibodies produced by techniques familiar to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, contain human and non-human portions and can be produced by recombinant DNA techniques familiar to those skilled in the art.

[0086] 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 and directed against a single epitope. The cell may be a clonal cell line, eukaryotic, prokaryotic, or phage.

[0087] As used herein, the term "chimeric antibody" refers to an antibody molecule produced by ligating the V region genes of a mouse-derived antibody with the C region genes of a human antibody into a chimeric gene, which is then inserted into a vector and transfected into host cells to express the chimeric gene. This antibody molecule retains the high specificity and affinity of the parent mouse antibody while allowing the human-derived fragments to effectively mediate biological effects and functions.

[0088] As used herein, the term "humanized antibody" refers to a modified form of the variable region of a murine antibody of the present invention, having CDR regions derived from (or essentially derived from) a non-human antibody (preferably a murine monoclonal antibody) and FR and constant regions derived essentially from sequences of an antibody of human origin, i.e., the sequences of the CDR regions of a murine antibody are grafted onto the framework sequences of a heterologous human antibody. Because the CDR sequences are responsible for most antibody-antigen interactions, expression vectors can be constructed to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies.

[0089] In the present invention, antibodies may be monospecific, bispecific, trispecific, or of greater multispecificity.

[0090] In the present invention, the antibodies of the present invention further include conservative variants thereof, which refer to polypeptides in which, compared to the amino acid sequence of the antibodies of the present invention, 10 or fewer, preferably 8 or fewer, more preferably 5 or fewer, and most preferably 3 or fewer amino acids have been substituted with amino acids having similar or close properties. These conservatively mutated polypeptides are preferably generated by amino acid substitutions as shown in Table A. [Table 0]

[0091] Anti-CD3 antibody As used herein, the term "CD3" generally refers to native or recombinant human CD3 and non-human homologs of human CD3. CD3 is a homodimeric or heterodimeric antigen expressed on T cells that binds to the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is a dimer of two of four distinct chains (ε, ζ, δ, and γ). CD3 dimer sequences include γ / ε, δ / ε, and ζ / ζ. Therefore, "CD3" refers to human CD3 unless specifically stated to be derived from a non-human species, e.g., "mouse CD3," "monkey CD3," etc.

[0092] As used herein, "CD3E" refers to the CD3ε extracellular domain.

[0093] 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 connecting linker.

[0094] The present invention also provides a highly specific antibody against CD3, comprising a heavy chain and a light chain, wherein the heavy chain contains the amino acid sequence of a heavy chain variable region (VH), and the light chain contains the amino acid sequence of a light chain variable region (VL).

[0095] Preferably, the heavy and light chain variable regions have six complementarity determining regions (CDRs) selected from the group consisting of: (A5) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH5-CDR3: HGNFGNTYISYWAY, SEQ ID NO: 29 huVL5-CDR1: TGAVTSGNY, SEQ ID NO: 30 huVL-CDR2: GTK, SEQ ID NO: 5 huVL5-CDR3: VLWYSKRW, SEQ ID NO: 31; (A1) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH4-CDR3: HGNFGNSYISYWEY, SEQ ID NO: 3 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL4-CDR3: VLWNSNRW, SEQ ID NO: 6; (A2) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH3-CDR3: HGNFGNSYISYWRY, SEQ ID NO: 10 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL3-CDR3: VLWYSGRW, SEQ ID NO: 11; (A3) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH2-CDR3: HGNFGNSYISYWQY, SEQ ID NO: 15 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL2-CDR3: VLWRSNRW, SEQ ID NO: 16; or (A4) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH1-CDR3: HGNFGNTYISYWAY, SEQ ID NO: 20 huVL-CDR1: TGAVTSGNY, SEQ ID NO: 4 huVL-CDR2: GTK, SEQ ID NO: 5 huVL1-CDR3: VLWYSKRW, SEQ ID NO: 21.

[0096] Preferably, the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 32, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 33; or the sequence of the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7, and the sequence of the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:12 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:13; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:17 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:18; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:22, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:23.

[0097] Here, any one of the above amino acid sequences further includes a sequence having CD3 binding affinity, which has undergone addition, deletion, modification and / or substitution of at least one amino acid (for example, 1 to 5, 1 to 3, preferably 1 to 2, more preferably 1).

[0098] In another preferred embodiment, the sequence having the addition, deletion, modification and / or substitution of at least one amino acid is preferably an amino acid sequence that is at least 80% identical, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% identical.

[0099] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and even more preferably a fully humanized antibody.

[0100] As used herein, the term "scFv" refers to a single chain antibody fragment (scFv), which is composed of an antibody heavy chain variable region and a light chain variable region linked together via a linker typically consisting of 15 to 25 amino acids.

[0101] As used herein, the term "linker" refers to one or more amino acid residues inserted into a globulin domain to provide sufficient flexibility between the light and heavy chain domains so that they can fold into a dual variable region-exchanged globulin. In the present invention, a suitable linker is a linker that links the VH and VL of a single-chain antibody (scFv) or a linker for linking an scFv to the heavy chain of another antibody.

[0102] Examples of suitable linkers include a single glycine (Gly) or serine (Ser) residue, although the identity and sequence of amino acid residues in the linker will vary depending on the type of secondary structure element desired to be achieved in the linker, e.g., (G4S)n, where n is an integer from 1 to 5.

[0103] The antibody derivatives of the present invention may 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 antibody subtypes.

[0104] Here, the animal is preferably a mammal, such as a mouse.

[0105] The antibodies of the present invention may be murine, chimeric, humanized, CDR-grafted and / or modified antibodies that target human CD3.

[0106] In one preferred embodiment of the present invention, SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:10; SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:15; SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:20; or SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 29 Any one or more of the sequences listed above, or sequences thereof with at least one amino acid addition, deletion, modification and / or substitution, that have CD3-binding affinity, are located in the CDR regions of the heavy chain variable region (VH).

[0107] In one preferred embodiment of the present invention, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:11; SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:16; SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:21; or SEQ ID NO: 30, SEQ ID NO: 5 and SEQ ID NO: 31 Any one or more of the sequences listed above, or sequences thereof with at least one amino acid addition, deletion, modification and / or substitution, that have CD3-binding affinity, are located in the CDR regions of the light chain variable region (VL).

[0108] In the above-mentioned content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably 40% or less of the total number of amino acids in the original amino acid sequence, more preferably 35% or less, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20%.

[0109] In the present invention, the number of added, deleted, modified and / or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, and most preferably 1.

[0110] As used herein, the term "bispecific antibody" refers to an antibody molecule that can simultaneously and specifically bind to two different antigens (targets) or two different epitopes. Depending on symmetry, bispecific antibodies are divided into symmetric and asymmetric molecules. Depending on the number of binding sites, bispecific antibodies are divided into bivalent, trivalent, tetravalent, and multivalent molecules.

[0111] The antibodies of the present invention may be monospecific, bispecific, trispecific, or multispecific. For example, the antibodies of the present invention may form bispecific antibodies together with antibodies or active fragments that bind to other targets. 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.

[0112] In one specific embodiment of the present invention, an anti-CD3 antibody of the present invention forms a bispecific antibody with an antibody that binds to CD19, and the constructed bispecific antibody targeting both CD3 and CD19 is designated SC30A, and its sequence is set forth in SEQ ID NO: 37.

[0113] Antibody production Any method suitable for generating monoclonal antibodies can be used to generate the anti-CD3 antibodies of the present invention. For example, animals can be immunized with a conjugated or naturally occurring CD3 homodimer or fragment thereof. Any suitable immunization method, including adjuvants, immunostimulants, and booster immunizations, can be used, and one or more methods can be used.

[0114] Any suitable form of CD3 can be used as an immunogen (antigen) to generate CD3-specific non-human antibodies and screen the antibodies for biological activity. The stimulating immunogen can be full-length mature human CD3, a native homodimer, or a peptide containing one or more epitopes. The immunogen can be used alone or in combination with one or more immunogenic adjuvants known in the art. The immunogen can be purified from natural sources or produced in genetically modified cells. DNA encoding the immunogen can be of genomic or non-genomic origin (e.g., cDNA). The DNA encoding the immunogen can be expressed in an appropriate genetic vector, including, but not limited to, adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, plasmids, and non-viral vectors.

[0115] An exemplary method for generating an anti-human CD3 antibody of the invention is described in Example 1.

[0116] Humanized antibodies may be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. In the present invention, the antibody is an IgG antibody, with the IgG1 subtype. In the biological assay and screening of antibodies described in the Examples below, certain domain sequences can be easily optimized to achieve the desired biological activity.

[0117] Similarly, any light chain can be used in the compounds and methods herein. Specifically, kappa chains, lambda chains or variants thereof are useful in the compounds and methods of the invention.

[0118] An exemplary method for humanizing an anti-human CD3 antibody of the invention is described in Example 2.

[0119] The sequence of a DNA molecule encoding an antibody of the invention or a fragment thereof can be obtained by conventional techniques, such as PCR amplification or genomic library screening. The coding sequences for the light and heavy chains may also be fused together to form a single-chain antibody.

[0120] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant techniques, typically by cloning the sequence into a vector, introducing it into cells, and isolating the relevant sequence from host cells grown in the usual manner.

[0121] Alternatively, the relevant sequences may be synthesized by artificial synthesis, especially if the fragments are short. Typically, many small fragments are synthesized and then joined together to form longer fragments. The DNA sequences may then be introduced into a variety of known DNA molecules (or vectors, etc.) and cells known in the art.

[0122] Furthermore, the present invention relates to vectors containing the above-described appropriate DNA sequences and appropriate promoter or control sequences, which can be used to transform appropriate host cells so as to express the proteins.

[0123] The host cell may be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Suitable animal cells include, but are not limited to, CHO-S, CHO-K1, and HEK-293 cells.

[0124] The process of transforming host cells by DNA recombination according to the present invention may be carried out by techniques well known in the art. The resulting transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention. Depending on the host cells used, they are cultured in conventional media under appropriate conditions.

[0125] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibody of the present invention, and the antibody of the present invention is obtained by purification using conventional globulin purification techniques, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, and affinity chromatography, which are well known to those skilled in the art.

[0126] The monoclonal antibodies obtained can be identified by conventional means, for example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0127] Antibody-drug conjugates (ADCs) The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[0128] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, which is coupled, preferably chemically coupled, to the antibody, and the effector molecule is preferably a therapeutically active drug, and may be one or more of a toxin protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0129] The antibody of the present invention and the effector molecule may be coupled using a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent using a carboxy group, a peptide chain, or a coupling agent using a disulfide bond. The non-selective coupling agent is a compound that couples the effector molecule and the antibody by conjugation, such as glutaraldehyde. The coupling agent using a carboxy group may be any one or more of a cis-aconitic anhydride coupling agent (e.g., cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0130] Some residues in antibodies (e.g., Cys and Lys) are available for attachment to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorescent groups), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies may be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection agents, stabilizers) are coupled (covalently bound) to antibodies. Functional agents may be linked to antibodies directly or indirectly via linkers.

[0131] An antibody may be coupled with a drug to form an antibody-drug conjugate (ADC). Typically, an ADC includes a linker positioned between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers are typically readily degraded in the intracellular environment, e.g., by degradation at the desired site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases and endosomal proteases), or sugar linkers, such as glucuronic acid-containing linkers that can be degraded by glucuronidase. Peptidyl linkers may 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, e.g., hydrazone linkers) and linkers that degrade under reducing conditions (disulfide bond linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0132] Before linking to an antibody, the linker has an active reactive group capable of reacting with a certain amino acid residue, and linkage is achieved via the active reactive group. Active reactive groups specific for thiol groups are preferred, including, for example, maleimide-based compounds, halogenated amides (e.g., iodinated, brominated, or chlorinated), halogenated esters (e.g., iodinated, brominated, or chlorinated), halogenated methyl ketones (e.g., iodinated, brominated, or chlorinated), benzyl halides (e.g., iodinated, brominated, or chlorinated), vinyl sulfone, pyridyl disulfide, mercury derivatives such as 3,6-di(mercurymethyl)dioxane (counterion is acetate, chloride, or nitrate), and polymethylene dimethyl sulfide thiosulfonate. The linker may, for example, comprise a maleimide linked to the antibody via thiosuccinimide.

[0133] The drug may be any cytotoxic drug, cell growth inhibitory drug, or immunosuppressant drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group that can bind to the linker. For example, the drug may have an amino group, a carboxyl group, a thiol group, a hydroxyl group, or a ketone group that can bind to the linker. When the drug is directly linked to the linker, the drug has an active group that can react before being linked to the antibody.

[0134] Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In the present invention, drug-linkers can be used to form ADCs in one simple step. In another embodiment, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step method. For example, a cysteine ​​residue can react with a reactive moiety on the linker in a first step, and then a functional group on the linker can react with the drug in a later step to form an ADC.

[0135] Typically, functional groups on the linker are selected to react specifically with appropriate reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to react with reactive alkynyl groups on specific drug moieties. The drug is covalently attached to the linker by addition via a 1,3-dipole 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 azide groups), 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 means are familiar to those skilled in the art, as described, for example, in "Biological Coupling Techniques," Second Edition (Elsevier). Those skilled in the art will appreciate that, when selecting a complementary pair of reactive functional groups for selective reactivity of the drug moiety and the linker, any of the complementary pair can be used for either the linker or the drug.

[0136] application The present invention provides uses of the present invention, such as for the manufacture of diagnostic preparations or for the manufacture of medicaments for preventing and / or treating CD3-related diseases, including inflammatory diseases and autoimmune diseases, including, but not limited to, psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), osteoarthritis, rheumatoid arthritis (RA), rheumatoid arthritis or osteoporosis, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis and cirrhosis), asthma (allergic asthma), allergic reactions, and cancer.

[0137] Drug Composition The present invention also provides compositions. In a preferred embodiment, the composition is a pharmaceutical composition containing the above-mentioned antibody, or an active fragment thereof, or a fusion protein thereof, or an ADC thereof, or corresponding CAR-T cells, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, pharmaceutically acceptable aqueous carrier, and the pH value is usually about 5 to 8, preferably about 6 to 8, depending on the properties of the formulated substances and the disease to be treated. The formulated pharmaceutical composition can be administered by any conventional route, including, but not limited to, intratumoral, intraperitoneal, intravenous, or local administration.

[0138] The antibodies of the present invention may be expressed intracellularly via the nucleotide sequence and used in cell therapy, for example, the antibodies may be used in chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0139] The pharmaceutical composition of the present invention can be used directly to bind to the CD3 protein molecule, and is therefore useful for the prevention and treatment of CD3-related diseases. It may also be used in combination with other therapeutic agents.

[0140] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001 to 99 wt%, preferably 0.01 to 90 wt%, more preferably 0.1 to 80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer solution, glucose, water, glycerin, ethanol, and combinations thereof. The pharmaceutical formulation corresponds to the dosage form. The pharmaceutical composition of the present invention may also be an injection, which can be prepared by conventional methods using, for example, physiological saline or an aqueous solution containing glucose and other excipients. In the case of an injection or solution, the pharmaceutical composition is prepared 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 daily. The polypeptide of the present invention can also be used in combination with other therapeutic agents.

[0141] When using the pharmaceutical composition, a safe and effective amount of the pharmaceutical composition is administered to a mammal, and this safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases, less than about 50 mg / kg body weight, preferably about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dosage should further take into account factors such as the mode of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0142] Detection Applications and Kits The antibodies of the present invention are useful in the detection, eg, detection of analytes, and can provide diagnostic information.

[0143] In the present invention, specimens (samples) used include cells, tissue specimens, and biopsy specimens. The term "biopsy" as used in the present invention includes all types of biopsies known to those skilled in the art. Therefore, biopsies as used in the present invention include tissue specimens prepared, for example, by endoscopic methods or by puncture or needle stick of an organ.

[0144] Specimens for use in the present invention include fixed or preserved cell or tissue specimens.

[0145] The present invention also provides a kit containing the antibody of the present invention (or a fragment thereof), and in a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer solution, etc. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

[0146] The main advantages of the present invention include: (1) The CD3 antibody of the present invention specifically binds to CD3 and has high biological activity. (2) The CD3 antibody of the present invention can be combined with other antibodies to construct a bispecific antibody.

[0147] The present invention will be further described below with reference to specific examples. It should be understood that these examples are used only to illustrate the present invention and do not limit the scope of the present invention. Experimental methods for which detailed conditions are not specified in the following examples generally follow conventional conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989), or the manufacturer's recommended conditions. Unless otherwise specified, percentages and parts are calculated by weight. [Example]

[0148] Generation of humanized CD3 monoclonal antibodies Balb / C mice were immunized with recombinant human CD3E protein (C00E, Novoprotein). After immunization, B cells were isolated and a phage display library was constructed. Candidate antibodies were isolated by screening. A series of humanized antibodies was obtained through structural simulation and logical design. The heavy chain variable regions of the resulting humanized antibodies were designated huVH1, huVH2, huVH3, and huVH4, and the light chain variable regions were designated huVL1, huVL2, huVL3, and huVL4. The VH and VL sequences of the humanized sequences were linked via a (G4S)3 linker, and a 6HIS tag was added to the C-terminus. The resulting single-chain fragments (scFvs) were expressed in E. coli and purified using a nickel column to obtain the single-chain fragments. The sequences of the heavy chain variable region (VH) and light chain variable region (VL) and CDRs of the resulting humanized antibodies are as follows:

[0149] Here, VH-CDR1 and VH-CDR2 are both the same and have the following sequences:

[0150] VH-CDR1: GFTFNKYA (SEQ ID NO: 1) VH-CDR2: IRSKYNNYAT (SEQ ID NO: 2) The VH-CDR3 sequences are all different and are as follows: huVH5-CDR3:HGNLQNSYISYWAY (SEQ ID NO: 29) huVH4-CDR3:HGNFGNTYISYWAY (SEQ ID NO: 3) huVH3-CDR3:HGNFGNSYISYWQY (SEQ ID NO: 10) huVH2-CDR3:HGNFGNSYISYWRY (SEQ ID NO: 15) huVH1-CDR3:HGNFGNSYISYWEY (SEQ ID NO: 20) Here, the VL-CDR1 of huVL1-VL4 is the same and has the following sequence:

[0151] VL-CDR1:TGAVTSGNY (SEQ ID NO: 4) The VL5-CDR1 of huVL5 is altered and has the following sequence:

[0152] huVL5-CDR1:TGPVTGGNY (SEQ ID NO: 30) Here, the VL-CDR2 of huVL1-VL5 is the same and has the following sequence:

[0153] VL-CDR2: GTK (SEQ ID NO: 5) The VL-CDR3 sequences are all different and are as follows:

[0154] huVL5-CDR3:VLWESNRW (SEQ ID NO: 31) huVL4-CDR3:VLWYSKRW (SEQ ID NO: 6) huVL3-CDR3:VLWRSNRW (SEQ ID NO: 11) huVL2-CDR3:VLWYSGRW (SEQ ID NO: 16) huVL1-CDR3:VLWNSNRW (SEQ ID NO: 21) Humanized CD3 antibody heavy chain variable region huVH5 (SEQ ID NO: 32) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNLQNSYISYWAYWGQGTLVTVSS Humanized CD3 antibody heavy chain variable region huVH4 (SEQ ID NO: 7) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWEYWGQGTLVTVSS Humanized CD3 antibody heavy chain variable region huVH3 (SEQ ID NO: 12) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWRYWGQGTLVTVSS Humanized CD3 antibody heavy chain variable region huVH2 (SEQ ID NO: 17) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWQYWGQGTLVTVSS Humanized CD3 antibody heavy chain variable region huVH1 (SEQ ID NO: 22) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNTYISYWAYWGQGTLVTVSS Humanized CD3 antibody light chain variable region huVL5 (SEQ ID NO: 33) QTVVTQEPSLTVSPGGTVTLTCGSSTGPVTGGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWESNRWVFGGGTKLTVL Humanized CD3 antibody light chain variable region huVL4 (SEQ ID NO: 8) QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGDKAALTLSGVQPEDEAEYYCVLWNSNRWVFGGGTKLTVL Humanized CD3 antibody light chain variable region huVL3 (SEQ ID NO: 13) QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSGRWVFGGGTKLTVL Humanized CD3 antibody light chain variable region huVL2 (SEQ ID NO: 18) QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWRSNRWVFGGGTKLTVL Humanized CD3 antibody light chain variable region huVL1 (SEQ ID NO: 23) QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSKRWVFGGGTKLTVL Humanized CD3 single chain antibody huVH5VL5 (SEQ ID NO: 34) QTVVTQEPSLTVSPGGTVTLTCGSSTGPVTGGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWESNRWVFGGGTKLT VLTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Humanized CD3 single chain antibody huVH4VL4 (SEQ ID NO: 9) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWEYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGDKAALTLSGVQPEDEAEYYCVLWNSNRWVFGGGTKLTVL Humanized CD3 single chain antibody huVH3VL3 (SEQ ID NO: 14) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWRYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSGRWVFGGGTKLTVL Humanized CD3 single chain antibody huVH2VL2 (SEQ ID NO: 19) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWQYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWRSNRWVFGGGTKLTVL Humanized CD3 single chain antibody huVH1VL1 (SEQ ID NO: 24) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNTYISYWAYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSKRWVFGGGTKLTVL [Example]

[0155] CD3 antibody affinity detection This example mainly describes the situation of detecting the affinity of CD3 antibodies in the form of single chain antibodies with human and monkey recombinant CD3E proteins.

[0156] 2.1 Affinity with human CD3 protein The affinity of CD3 antibodies to recombinant hCD3 protein (CP19, Novoprotein) was measured by ELISA, where recombinant hCD3 was coated onto plates and CD3 antibodies were diluted in a 10-fold gradient (starting from 20 μg / ml), see FIG. 1 .

[0157] The calculated EC50 results are shown in Table 1. [Table 1]

[0158] 2.2 Affinity with monkey CD3 protein The affinity of the CD3 antibody to recombinant monkey CD3 protein (CW07, Novoprotein) was measured by ELISA, where the recombinant monkey CD3 was coated onto a plate and the CD3 antibody was diluted in a 10-fold gradient (starting from 20 μg / ml), see Figure 2 .

[0159] The calculated EC50 results are shown in Table 2. [Table 2] [Example]

[0160] Construction of full-length antibodies and huVH5VL5 bispecific antibodies Full-length CD3 antibodies were constructed by fusing the heavy chain variable region with the heavy chain constant region hIgG1 and the light chain variable region with the light chain constant region kappa. The resulting full-length CD3 antibodies are CQ50 (containing huVH1 and huVL1), CQ51 (containing huVH2 and huVL2), CQ52 (containing huVH3 and huVL3), CQ53 (containing huVH4 and huVL4), and CQ54 (containing huVH5 and huVL5), respectively. The variable region of the commercially available drug OKT3 was selected and fused to hIgG1 to serve as a control antibody. The relevant sequences are as follows:

[0161] Heavy chain of CD3 full-length antibody CQ54 (SEQ ID NO: 35) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNLQNSYISYWA YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light chain of CD3 full-length antibody CQ54 (SEQ ID NO: 36) QTVVTQEPSLTVSPGGTVTLTCGSSTGPVTGGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWESNRWVFGGGTKLT VLTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC hIgG1 constant region (SEQ ID NO: 25) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK κ chain constant region (SEQ ID NO: 26) LTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain of OKT3 antibody (SEQ ID NO: 27) QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQG TTLTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light chain of OKT3 antibody (SEQ ID NO: 28) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIN RADTAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0162] Because the current main application of CD3 antibodies is in bispecific and agonistic antibodies, we used the heavy and light chain variable region sequences of a humanized CD3 antibody (huVH5 and huVL5) and linked it to a CD19 antibody scFv via a flexible linker (GGGGS) to construct a bispecific antibody targeting both CD3 and CD19, named SC30A. Here, CD19 was derived from Amgen's commercially available bispecific antibody drug blinatumomab, and a control antibody (named BLMOA) was also constructed.

[0163] CD3 and CD19 bispecific antibody SC30A (SEQ ID NO: 37) DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGS QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGG GGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNLQNSYISYWAYWGQGTLVTV SSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGPVTGGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWESNRWVFGGGTKLTVL BLMOA (SEQ ID NO: 38) DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGGSGGGGSGGG GSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVS SGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVS SVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK [Example]

[0164] Detection of full-length antibody affinity This example demonstrates the binding of full-length antibodies to human and monkey proteins and their affinity to the human CD3+ cell line Jurkat.

[0165] 4.1 Detection of affinity between full-length CD3 antibodies and human CD3E protein by Fortebio Using the Fortebio method, a protein A sensor was selected, CD3 antibody was immobilized, and CD3E was gradient diluted, diluting two-fold over a seven-step gradient from 100 nM, to detect affinity. The results are shown in Figure 3.

[0166] The calculated affinities are shown in Table 3. [Table 3] The calculated affinity of CQ50 is 5.16 x 10 -8M, and the affinity of CQ51 is 6.24 × 10 -8 In M, The affinity of CQ52 is 3.11 x 10 -9 M, and the affinity of CQ53 is 2.02 × 10 -9 In M, The affinity of CQ53 is 0.97 x 10 -9 It's M.

[0167] 4.2 Detection of binding between full-length CD3 antibodies and monkey CD3E by ELISA The results are shown in Figure 4.

[0168] The EC50 detected for CQ50 was 0.2004 μg / ml. The EC50 detected for CQ51 was 0.3385 μg / ml. The EC50 detected for CQ52 was 0.195 μg / ml. The EC50 detected for CQ53 was 0.0823 μg / ml. The EC50 detected for CQ54 was 0.375 μg / ml.

[0169] The detection results showed that CQ50, CQ51, CQ52, CQ53 and CQ54 could bind to monkey CD3E but could not bind to OKT3.

[0170] 4.3 Binding to human CD3-positive cells 4×10 5 Jurkat cells were cultured in gradient-diluted CD3 antibodies and incubated for 1 hour. After 3 washes with PBS, anti-hFC-APC (purchased from Jackson Immunology) was added and the cells were loaded onto a flow cytometer for detection. The resulting S curve is shown in Figure 5.

[0171] When the EC50 was calculated, CQ50 is 0.01947μg / ml and OKT3 is 0.01μg / ml, and the two do not differ significantly in affinity to cells.

[0172] CQ51 is 0.0158 μg / ml and OKT3 is 0.01 μg / ml, and the two do not differ significantly in affinity to cells.

[0173] CQ52 is 0.02167 μg / ml and OKT3 is 0.01 μg / ml, and the two do not differ significantly in affinity to cells.

[0174] CQ53 is 0.013 μg / ml and OKT3 is 0.01 μg / ml, and the two do not differ significantly in affinity to cells.

[0175] CQ54 is 0.023 μg / ml and OKT3 is 0.01 μg / ml, and the two do not differ significantly in affinity to cells. [Example]

[0176] Preliminary determination of epitopes of full-length CD3 antibodies Because full-length CD3 antibodies and OKT3 antibodies are functionally similar, this example primarily investigated whether the full-length CD3 antibodies and OKT3 antibodies share the same epitope for CD3E. Specifically, a plate was coated with full-length CD3 antibodies, a predetermined amount of HIS-tagged CD3E (C578 Novoprotein) protein was added, and gradient-diluted OKT3 antibodies were added based on the EC90 value detected by ELISA. An anti-HIS secondary antibody (purchased from Biolegend) was then added for detection. The results are shown in Figure 6.

[0177] The results showed that there was no competition between CQ50 and OKT3, and the epitopes of the two antibodies did not match. There is no competition between CQ51 and OKT3, and the epitopes of the two antibodies do not match. There is no competition between CQ52 and OKT3, and the epitopes of the two antibodies do not match. There is no competition between CQ53 and OKT3, and the epitopes of the two antibodies do not match. There is no competitive relationship between CQ54 and OKT3, and the epitopes of the two antibodies do not match. [Example]

[0178] PBMC activation induced by full-length antibodies This example examines the changes in the expression of CD69, an early T cell activation marker, and IFNγ secretion when PBMCs are activated with a CD3 antibody and cultured. Specifically, PBMCs from volunteers were isolated using lymphocyte separation fluid and cultured at 1 × 10 6 The cells were seeded into a 96-well plate at a density of 1 / ml, added with 10 ng / ml of CD3 antibody, and cultured overnight. The percentage of CD69+ T cells was detected by flow cytometry. The results are shown in Figure 7 below.

[0179] The results showed that both OKT3 and CQ54 could activate T cells, and that the CD69 positivity rate after activation was 65.8% for CQ54 and 62.5% for OKT3, indicating that the two antibodies did not differ significantly in the early activation of T cells.

[0180] After separating PBMCs, 10 ng / ml of CD3 antibody was added, and the cells were cultured. Samples were taken at 24 and 48 hours, and the expression of IFNγ was detected using an IFNγ ELISA kit. The results are shown in FIG.

[0181] The results showed that CQ54 and OKT3 had similar effects in inducing IFNγ secretion. [Example]

[0182] Detection of bispecific antibody affinity This example relates to the binding of a bispecific antibody to two human target proteins and to the monkey CD3 protein.

[0183] Using the ForteBio method, Protein A sensors were selected and immobilized with Fc-tagged recombinant human CD3E (CP19 Novoprotein) and human CD19 (C572 Novoprotein), respectively, and the bispecific antibody SC30A was gradient diluted. The detected affinity plots are shown in Figure 9 (A and B).

[0184] The calculated affinities are shown in Table 4. [Table 4]

[0185] The results showed that the bispecific antibody SC30A had affinity for both targets.

[0186] The binding of the bispecific antibodies (SC30A and BLMOA) to monkey CD3E was detected by ELISA, and the results are shown in FIG.

[0187] The detection results showed that SC30A could bind to monkey CD3E with a detected EC50=1.243 μg / ml, while the reference molecule BLMOA was unable to bind. [Example]

[0188] Bispecific antibodies induce early activation of PBMCs This example examines the changes in the expression of CD69, an early T cell activation marker, when PBMCs are activated with a CD3 bispecific antibody and cultured. Specifically, PBMCs from volunteers were isolated using lymphocyte separation fluid and cultured at 1 x 10 6 The cells were seeded into a 96-well plate at a density of 1 / ml, added with 10 ng / ml of CD3 antibody, and cultured overnight. The proportion of CD69+ T cells was detected by flow cytometry. The results are shown in Figure 11.

[0189] The results showed that both SC30A and BLMOA could activate T cells, and that the CD69 positivity rate after activation was 20.7% for SC30A and 21.9% for BLMOA, indicating that the two antibodies did not differ significantly in the early activation of T cells. [Example]

[0190] Bispecific antibody-mediated tumor cell killing This example demonstrates the bispecific antibody-mediated killing of CD19+ tumor cells, Raji, under both activated and inactivated T cell conditions. Specifically, whole blood was collected from volunteers, and PBMCs were isolated using lymphocyte separation fluid. Cultured cells were added with 10 ng / ml OKT3 and 100 ng / ml IL-2. Cell counts were performed every three days. After 10 days of culture, cell phenotypes were determined to confirm that the CD3+ cell ratio exceeded 90. Activated PBMCs and Raji-GFP cells were added at an effector-target ratio of 2:1, along with gradient-diluted bispecific antibodies at concentrations of 50 ng / ml, 5 ng / ml, and 500 pg / ml, respectively. After overnight killing, the killing effect was detected by flow cytometry and fluorescence scanning.

[0191] 9.1 Flow cytometric detection As shown in Figure 12 and Table 5, in the 50 ng / ml experiment, the results of the experiments with three volunteers showed no significant difference between BLMOA and SC30A. However, in the 500 pg / ml experiment, the percentage of Raji-GFP remaining after BLMOA-mediated killing was 6.9%, 19.9%, and 2.8%, respectively, while in the SC30A group, the percentage of Raji-GFP remaining was 0.3%, 2.0%, and 0.0%, respectively. In the 5 ng / ml experiment, the percentage of Raji-GFP remaining after BLMOA-mediated killing was 1.6%, 6.8%, and 0.6%, respectively, while in the SC30A group, the percentage of Raji-GFP remaining was 0.3%, 1.4%, and 0.0%, respectively. [Table 5]

[0192] As described above, SC30A was shown to be superior to BLMOA in mediating tumor cell killing by activated PBMCs.

[0193] 9.2 Detection by Fluorescence Scanning All wells were fluorescently scanned to observe the presence of residual Raji-GFP.

[0194] The results are shown in Figure 13. The bright spots in the figure are green fluorescent. It is clear that after treatment with 500 pg / ml BLMOA, many bright spots of Raji-GFP remained, while after treatment with 500 pg / ml SC30A, almost no bright spots of Raji-GFP were observed. Therefore, the killing ability mediated by SC30A is better than that of BLMOA.

[0195] Isolated, unactivated PBMCs were mixed with Raji-GFP at an effector-target ratio of 5:1 and simultaneously incubated with gradient-diluted bispecific antibodies at concentrations of 50 ng / ml, 5 ng / ml, and 500 pg / ml, respectively, for 48 hours. The killing efficiency is shown in Figure 14. [Example]

[0196] Influence of B cells on bispecific antibody-mediated PBMC activation This example investigated the safety of bispecific antibodies by monitoring T cell activation in the presence and absence of target cells. Specifically, PBMCs were isolated and CD19+ B cells were isolated and removed from the PBMCs using magnetic beads coated with CD19 antibodies to create a B cell-PBMC group. At the same time, 5% Raji cells were added to create a B cell-PBMC Raji group. These groups, along with untreated normal PBMCs, were cultured with SC30A. After 5 days of culture, cells were counted to monitor cell proliferation. The results are shown in Figure 15.

[0197] The above results indicated that the activation of SC30A on PBMCs depends on the target cells. [Example]

[0198] Dependence of bispecific antibody-mediated intracellular signals on target cells This example examines the bispecific antibody-induced changes in downstream signaling of NFAT transcription factors in T cells and whether these signals are also dependent on CD19+ cells. Specifically, a Jurkat-NFAT-Luc reporter cell line (XCC20 Novoprotein) was constructed and transfected with 40 ng / ml SC30A. One group contained target cells, Raji, while the other group did not. After 4 hours of incubation, the cells were lysed and luciferase activity was detected. The results are shown in Figure 16.

[0199] The experimental results show that when Raji cells were added, the signal was clearly stronger than when Raji cells were not added, indicating that the activation signal of SC30A depends on the target cells.

[0200] When target cells were contained, the activity of SC30A was detected, and the results are shown in FIG.

[0201] The activity of SC30A was detected by the reporter gene method, and the calculated EC50 was 1.359 ng / ml.

[0202] All documents related to the present invention are incorporated herein by reference as if each document were individually incorporated by reference. After reading the above content of the present invention, it should be understood that those skilled in the art can make various changes and modifications to the present invention, and that equivalents thereof are within the scope of the claims of the present invention.

Claims

1. An anti-CD3 humanized antibody 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: (A5) Three complementarity-determining regions VH-CDR of the heavy chain variable region and three complementarity-determining regions VH-CDR of the light chain variable region Complementarity determining regions VL-CDR: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH5-CDR3: HGNLQNSYISYWAY, SEQ ID NO: 29 huVL5-CDR1: TGPVTGGNY, SEQ ID NO: 30 huVL-CDR2: GTK, SEQ ID NO: 5 huVL5-CDR3: VLWESNRW, SEQ ID NO:

31.

2. The antibody according to claim 1, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain of the antibody comprises a heavy chain framework region for connecting the three complementarity determining regions VH-CDR and VH-CDR, and the light chain of the antibody comprises a light chain framework region for connecting the three complementarity determining regions VL-CDR and VL-CDR, and the anti-CD3 single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32, and the light chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO:

33.

3. The antibody of claim 1, wherein the antibody is a single-chain antibody.

4. A bispecific antibody, a first antigen-binding domain D1, and a second antigen-binding domain D2; wherein D1 specifically binds to the CD3 protein of the target molecule, D2 specifically binds to the CD19 protein of the target molecule, wherein D1 is an antibody or an antigen-binding fragment thereof that specifically binds to CD3 protein, D2 is an antibody or an antigen-binding fragment thereof that specifically binds to CD19 protein, wherein D1 comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region have six complementarity determining regions (CDRs) selected from the group consisting of: (A5) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH5-CDR3: HGNLQNSYISYWAY, SEQ ID NO: 29 huVL5-CDR1: TGPVTGGNY, SEQ ID NO: 30 huVL-CDR2: GTK, SEQ ID NO: 5 huVL5-CDR3: VLWESNRW, SEQ ID NO: 31; wherein the structure of the antigen-binding fragment is (i) a Fab fragment, (ii) a F(ab')2 fragment, (iii) a Fd fragment, (iv) a Fv fragment, (v) a scFv molecule, or (vi) dAb fragments An antibody characterized by:

5. A recombinant protein characterized in that it comprises: (i) an antibody according to claim 1 or a bispecific antibody according to claim 4; and (ii) An optional tag sequence to aid in expression and / or purification.

6. A CAR construct, wherein an scFv fragment of an antigen-binding region of the CAR construct is a binding region that specifically binds to CD3, and the scFv fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region have six complementarity-determining regions (CDRs) selected from the group consisting of: (A5) 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: huVH-CDR1: GFTFNKYA, SEQ ID NO: 1 huVH-CDR2: IRSKYNNYAT, SEQ ID NO: 2 huVH5-CDR3: HGNLQNSYISYWAY, SEQ ID NO: 29 huVL5-CDR1: TGPVTGGNY, SEQ ID NO: 30 huVL-CDR2: GTK, SEQ ID NO: 5 huVL5-CDR3: VLWESNRW, SEQ ID NO:

31.

7. A recombinant immune cell characterized by expressing an exogenous CAR construct according to claim 6.

8. an antibody-drug conjugate comprising: (a) an antibody portion selected from the group consisting of the antibody of claim 1, the bispecific antibody of claim 4, or the recombinant protein of claim 5, or a combination thereof; and (b) a conjugation moiety conjugated to said antibody moiety, said conjugation moiety being selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

9. 10. Use of an active ingredient, wherein the active ingredient is selected from the group consisting of an antibody according to claim 1, a bispecific antibody according to claim 4, or a recombinant protein according to claim 5, a CAR construct according to claim 6, an immune cell according to claim 7, an antibody-drug conjugate according to claim 8, or a combination thereof, characterized in that the active ingredient is used for: (a) production of detection reagents or kits; (b) the manufacture of a drug or formulation for preventing and / or treating a CD3-associated disease; and / or (c) The manufacture of a drug or preparation for preventing and / or treating a CD3-associated cancer or tumor.

10. A pharmaceutical composition comprising: (i) an active ingredient selected from the group consisting of the antibody of claim 1, the antibody of claim 4, or the recombinant protein of claim 5, the CAR construct of claim 6, the immune cell of claim 7, the antibody-drug conjugate of claim 8, or a combination thereof; and (ii) A pharmaceutically acceptable carrier.

11. A polynucleotide encoding a polypeptide selected from the group consisting of: (1) The antibody according to claim 1; or (2) The bispecific antibody according to claim 4; (3) The recombinant protein according to claim 5; (4) The CAR construct according to claim 6.

12. A vector comprising the polynucleotide of claim 11.

13. A genetically engineered host cell, characterized in that the host cell contains the vector of claim 12 or has the polynucleotide of claim 11 integrated into its genome.

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

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