CDC Platform Antibodies

Abstract

A platform technology for enhancing CDC effect. Specifically, the present invention relates to an antibody or a fragment thereof or a fusion protein thereof having antigen binding activity and CDC activity, comprising a binding functional domain targeting a specific antigen and a heavy chain Fc region element, the heavy chain Fc region element comprising (1) an amino acid residue selected from the group consisting of W, Y, E, F, H, C, D, N, Q, R, S, T, or K at the 309th position of the Fc region, preferably further comprising R at the 345th position of the Fc region, and / or (2) an antibody or a fragment thereof or a fusion protein thereof comprising a tail-piece element at the C-terminus. The antibody or a fragment thereof or a fusion protein thereof can significantly improve the CDC performance of the antibody or a fragment thereof or a fusion protein of the present invention by site-specific mutation of the 309th amino acid or addition of a tail-piece element to the C-terminus, and can improve the therapeutic effect of existing antibodies or fragments thereof or fusion proteins, thereby treating many diseases.

Description

[Technical Field]

[0001] The present invention relates to the field of antibodies, and in particular to complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) platform antibodies. [Background technology]

[0002] Complement (C) is a set of proteins present in the serum and tissue fluids of normal humans and animals that exhibit enzymatic activity upon activation. Bordet demonstrated at the end of the 19th century that fresh blood contains heat-resistant components that can assist and supplement specific antibodies and mediate immunolytic and hemolytic activities, hence the name complement. Complement is a multimolecular system consisting of over 30 soluble proteins, membrane-bound proteins, and complement receptors, known as the complement system. Under normal conditions, complement is a component of plasma proteins. Each component of the complement system exists in plasma as an inactive precursor. When needed, it is activated sequentially by the action of activators, such as antigen-antibody complexes, and ultimately exerts its function. Depending on the biological function of each component of the complement system, it can be divided into complement-specific components, complement regulatory components, and complement receptors.

[0003] Complement-specific components are divided into four types: 1. C1, C2, and C4 of the classical activation pathway; 2. Factors B, D, and P of the alternative activation pathway; 3. Mannose-binding lectin (MBL) and serine proteases of the MBL activation pathway; and 4. C3, C5, C6, C7, C8, and C9 involved in the common terminal pathway.

[0004] The process of complement activation is divided into three pathways based on the order of initiation: 1) the classical pathway from C1q-C1r2-C1s2, in which the antigen-antibody complex is the major activator; 2) the alternative pathway from C3, which is antibody-independent; and 3) the mannose-binding lectin (MBL) pathway, which recognizes complement by its glucosyl group. The above three pathways share a common terminal pathway, namely, the formation of the membrane attack complex and its cytolytic effect.

[0005] The classical pathway of complement activation is a cascade of enzyme-catalyzed reactions in which C1q binds to immune complexes, subsequently activating C1r, C1s, C4, C2, and C3, forming C3 convertase (C4b 2b) and C5 convertase (C4b2b3b). It is the main mode of action of antibody-mediated humoral immune responses.

[0006] Immune complexes are primarily composed of IgG and IgM molecules bound to antigens. Each C1q molecule must bind to the Fc portion of two or more immunoglobulin molecules; free or soluble antibodies do not activate complement. The complement components involved in classical pathway activation are C1, C4, C2, C3, C5, C6, C7, C8, C9, and C9, respectively.

[0007] The activation process of the classical pathway is divided into three major stages: 1) recognition stage; 2) activation stage; and 3) membrane attack stage. During the activation process of the complement system, many biologically active substances are generated, causing a series of biological effects. The biological effects of complement include: 1) enhancement of phagocytosis and chemotaxis of phagocytes; 2) enhancement of vascular permeability; 3) virus neutralization; 4) cytolysis; and 5) regulation of immune responses.

[0008] Research (reference: Complement Is Activated by IgG Hexamers Assembled at the Cell Surface[J]. Science, 2014, 343(6176):1260-3.) has shown that hexamers are the most effective form for binding C1q and initiating the classical activation pathway. Genmab developed the HexaBody platform based on this principle (reference: Jong R, Beurskens FJ, Verploegen S, et al., A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface[J]. PLOS Biology, 2016, 14(1):e1002344.). A feature of this platform technology is that by inducing mutations at specific sites in Fc (e.g., E345R), it is possible to enhance the interaction between Fc and Fc, promoting the antibody's tendency to form hexamers after binding to antigens on the cell surface. The hexamers formed can effectively improve immune response functions, particularly antibody complement-dependent cytotoxicity (CDC). However, existing technologies are still not satisfactory.

[0009] ADCC, or antibody-dependent cell-mediated cytotoxicity, is the direct mediation of target cell killing by an antibody through the binding of its Fab region to an antigen on the surface of tumor cells or other target cells and its Fc region to FcγRs on the surface of killer cells (e.g., NK cells, macrophages). NK cells are the primary effector cells through which antibodies exert their ADCC effect. When the Fc region of an antibody binds to FcγRs on the surface of NK cells, the latter are activated. Activated NK cells release cytotoxic substances such as perforin and granzymes to induce apoptosis in target cells, thereby killing them. Therefore, the ADCC effect is one of the main mechanisms by which related antibodies exert their biological activity. Currently, in the field of biomedicine, the most widely developed antibody subtype for ADCC activity is wild-type IgG1.

[0010] Therefore, there is still a need in the art for the development of platform antibodies with good CDC and / or ADCC activity. Summary of the Invention

[0011] An object of the present invention is to provide a platform for enhancing the CDC activity of an antibody or a fragment thereof or a fusion protein thereof, preferably a platform for enhancing the CDC and / or ADCC activity of an antibody or a fragment thereof or a fusion protein thereof.

[0012] In a first aspect of the present invention, there is provided an antibody or a fragment thereof or a fusion protein thereof having antigen-binding activity and CDC activity, the antibody comprising a binding functional domain targeting a predetermined antigen and a heavy chain Fc region element, the heavy chain Fc region element comprising: (1) the Fc region contains an amino acid residue selected from the group consisting of W, Y, E, F, H, C, D, N, Q, R, S, T, or K at position 309; and / or (2) Contains a tailpiece element at the C-terminus Provide something.

[0013] In another preferred embodiment, the heavy chain Fc region element and the tail-piece element are connected via a linker.

[0014] In another preferred embodiment, the heavy chain Fc region element comprises two heavy chain Fc regions, each of which independently comprises: (1) an amino acid residue at position 309 of the Fc region selected from the group consisting of W, Y, E, F, H, C, D, N, Q, R, S, T, or K; and / or (2) It contains a tail-piece element at the C-terminus.

[0015] In another preferred example, each heavy chain Fc region further comprises R at position 345.

[0016] In another preferred embodiment, the antibody, or fragment or fusion protein thereof, further has ADCC activity.

[0017] In another preferred embodiment, the predetermined antigen-targeting binding domain binds to an antigen molecule selected from the group consisting of CD38, CD3, CD47, CD19, CD20, HER2, EGFR, CD123, Glypican-3, CD25, Trop-2, EpCAM, or a combination thereof.

[0018] In another preferred embodiment, the binding functional domain targeting the predetermined antigen comprises a domain selected from the group consisting of VH, VL, Fab, F(ab')2, Fab', scFv, or VHH.

[0019] In another preferred embodiment, the heavy chain Fc region element comprises a tail-piece element at the C-terminus of the CH3 domain.

[0020] In another preferred embodiment, the heavy chain Fc region element is derived from IgG.

[0021] In another preferred embodiment, the IgG has an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:34.

[0022] In another preferred embodiment, the IgG is of mammalian origin, preferably murine, cynomolgus monkey or human, more preferably human.

[0023] In another preferred embodiment, the heavy chain Fc region element comprises a CH2 and / or CH3 domain derived from IgG1, IgG2, IgG3, or IgG4.

[0024] In another preferred embodiment, the heavy chain Fc region element is derived from human IgG1.

[0025] In another preferred embodiment, the heavy chain Fc region element comprises CH2 and CH3 domains from IgG1.

[0026] In another preferred embodiment, the antibody or fragment or fusion protein thereof is a monospecific, bispecific, trispecific or multispecific antibody or fragment or fusion protein thereof.

[0027] In another preferred embodiment, the antibody or fragment thereof or fusion protein is a dimer, trimer, or multimer.

[0028] In another preferred embodiment, the multimer is a hexamer.

[0029] In another preferred embodiment, the heavy chain Fc region element comprises an amino acid residue selected from the group consisting of F, C, W, or Y at position 309 of the Fc region.

[0030] In another preferred embodiment, the heavy chain Fc region element further comprises amino acid substitutions at other positions.

[0031] In another preferred embodiment, the heavy chain Fc region element further comprises a substitution of the glutamic acid residue at position 345 of the Fc region with an arginine residue (E345R).

[0032] In another preferred embodiment, the heavy chain Fc region element comprises an amino acid residue substitution at an Fc region position selected from the group consisting of: (1)L309W+E345R; (2)L309Y+E345R; (3)L309E+E345R; (4)L309F+E345R; (5)L309H+E345R; (6)L309C+E345R; (7)L309D+E345R; (8)L309N+E345R; (9)L309Q+E345R; (10)L309R+E345R; (11)L309S+E345R; (12) L309T+E345R; or (13)L309K+E345R.

[0033] In another preferred embodiment, the heavy chain Fc region element has the following amino acid residue substitutions at Fc region positions: L309W+E345R.

[0034] In another preferred embodiment, the numbering of the amino acids of the heavy chain Fc region elements is according to the EU numbering system.

[0035] In another preferred embodiment, the tailpiece element is derived from human IgM.

[0036] In another preferred embodiment, the tailpiece element comprises one or more amino acid mutations.

[0037] In another preferred embodiment, the sequence of the tailpiece element is set forth in SEQ ID NO: 7 or 8.

[0038] In another preferred embodiment, the binding domain that targets a predetermined antigen is a binding domain that binds to CD38 and comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (1) A heavy chain variable region comprising the three complementarity-determining regions (CDRs) shown below: H-CDR1 shown in SEQ ID NO: 11; H-CDR2 shown in SEQ ID NO: 12; H-CDR3 as set forth in SEQ ID NO: 13; and A light chain variable region comprising the three complementarity determining regions CDRs shown below: L-CDR1 shown in SEQ ID NO: 14; L-CDR2 shown in SEQ ID NO: 15; L-CDR3 as set forth in SEQ ID NO: 16; or (2) A heavy chain variable region comprising the three complementarity-determining regions (CDRs) shown below: H-CDR1 shown in SEQ ID NO: 35; H-CDR2 shown in SEQ ID NO: 36; H-CDR3 as set forth in SEQ ID NO: 37; and A light chain variable region comprising the three complementarity determining regions CDRs shown below: L-CDR1 shown in SEQ ID NO: 38; L-CDR2 shown in SEQ ID NO: 39; L-CDR3 shown in SEQ ID NO: 40.

[0039] In another preferred embodiment, the antibody or fragment thereof or fusion protein is selected from the group consisting of: (a) an antibody or fragment or fusion protein thereof having an amino acid sequence selected from the group consisting of: a heavy chain comprising a VH region containing H-CDR1 set forth in SEQ ID NO: 35, H-CDR2 set forth in SEQ ID NO: 36, and H-CDR3 set forth in SEQ ID NO: 37, and a heavy chain constant region having a W or Y mutation at position 309 of human IgG1 according to EU numbering, and a light chain comprising a VL region containing L-CDR1 set forth in SEQ ID NO: 38, L-CDR2 set forth in SEQ ID NO: 39, and L-CDR3 set forth in SEQ ID NO: 40; or a heavy chain comprising a VH region containing H-CDR1 set forth in SEQ ID NO: 11, H-CDR2 set forth in SEQ ID NO: 12, and H-CDR3 set forth in SEQ ID NO: 13, and a heavy chain constant region having a W or Y mutation at position 309 of human IgG1 according to EU numbering, and a light chain comprising a VL region containing L-CDR1 set forth in SEQ ID NO: 14, L-CDR2 set forth in SEQ ID NO: 15, and L-CDR3 set forth in SEQ ID NO: 16; or a heavy chain comprising a VH region containing H-CDR1 set forth in SEQ ID NO: 35, H-CDR2 set forth in SEQ ID NO: 36, and H-CDR3 set forth in SEQ ID NO: 37, and a heavy chain constant region having a W or Y mutation at position 309 and an R mutation at position 345 of human IgG1 according to EU numbering, and a light chain comprising a VL region containing L-CDR1 set forth in SEQ ID NO: 38, L-CDR2 set forth in SEQ ID NO: 39, and L-CDR3 set forth in SEQ ID NO: 40; or a heavy chain comprising a VH region containing H-CDR1 set forth in SEQ ID NO: 11, H-CDR2 set forth in SEQ ID NO: 12, and H-CDR3 set forth in SEQ ID NO: 13, and a heavy chain constant region having a W or Y mutation at position 309 and an R mutation at position 345 of human IgG1 according to EU numbering, and a light chain comprising a VL region containing L-CDR1 set forth in SEQ ID NO: 14, L-CDR2 set forth in SEQ ID NO: 15, and L-CDR3 set forth in SEQ ID NO: 16; or a heavy chain comprising a VH region containing H-CDR1 set forth in SEQ ID NO: 35, H-CDR2 set forth in SEQ ID NO: 36, and H-CDR3 set forth in SEQ ID NO: 37, and a heavy chain constant region having the sequence set forth in SEQ ID NO: 31, and a light chain comprising a VL region containing L-CDR1 set forth in SEQ ID NO: 38, L-CDR2 set forth in SEQ ID NO: 39, and L-CDR3 set forth in SEQ ID NO: 40; or a heavy chain comprising a VH region containing H-CDR1 shown in SEQ ID NO: 11, H-CDR2 shown in SEQ ID NO: 12, and H-CDR3 shown in SEQ ID NO: 13, and a heavy chain constant region having the sequence shown in SEQ ID NO: 31; and a light chain VL region containing L-CDR1 shown in SEQ ID NO: 14, L-CDR2 shown in SEQ ID NO: 15, and L-CDR3 shown in SEQ ID NO: 16; (b) A polypeptide derived from (a), which has an antigen-binding function and the CDC activity, and in which the amino acid sequence in (a) is obtained by substitution, deletion or addition of one or more amino acid residues.

[0040] In another preferred embodiment, the antibody or fragment thereof or fusion protein is selected from the group consisting of: (a) an antibody or fragment or fusion protein thereof having an amino acid sequence selected from the group consisting of: a heavy chain comprising a VH region having the sequence set forth in SEQ ID NO: 1 and a heavy chain constant region with a W or Y mutation at position 309 of human IgG1 according to EU numbering, and a light chain comprising a VL region having the sequence set forth in SEQ ID NO: 2; or a heavy chain comprising a VH region having the sequence set forth in SEQ ID NO: 21 and a heavy chain constant region with a W or Y mutation at position 309 of human IgG1 according to EU numbering, and a light chain comprising a VL region having the sequence set forth in SEQ ID NO: 22; or a heavy chain comprising a VH region having the sequence set forth in SEQ ID NO: 1 and a heavy chain constant region having a W or Y mutation at position 309 and an R mutation at position 345 of human IgG1 according to EU numbering, and a light chain comprising a VL region having the sequence set forth in SEQ ID NO: 2; or a heavy chain comprising a VH region having the sequence set forth in SEQ ID NO: 21 and a heavy chain constant region having a W or Y mutation at position 309 and an R mutation at position 345 of human IgG1 according to EU numbering, and a light chain comprising a VL region having the sequence set forth in SEQ ID NO: 22; or a heavy chain comprising a VH region having the sequence set forth in SEQ ID NO: 1 and a heavy chain constant region having the sequence set forth in SEQ ID NO: 31, and a light chain comprising a VL region having the sequence set forth in SEQ ID NO: 2; or a heavy chain comprising a VH region having the sequence set forth in SEQ ID NO: 21 and a heavy chain constant region having the sequence set forth in SEQ ID NO: 31, and a light chain VL region having the sequence set forth in SEQ ID NO: 22; (b) A polypeptide derived from (a), which has an antigen-binding function and the CDC activity, and in which the amino acid sequence in (a) is obtained by substitution, deletion or addition of one or more amino acid residues.

[0041] In another preferred embodiment, the antibody or fragment thereof or fusion protein is selected from the group consisting of: (a) an antibody or fragment or fusion protein thereof having an amino acid sequence selected from the group consisting of: a heavy chain comprising a VH region having the sequence set forth in SEQ ID NO: 1 and a heavy chain constant region having the sequence set forth in SEQ ID NO: 31, and a light chain comprising a VL region having the sequence set forth in SEQ ID NO: 2; or a heavy chain comprising a VH region having the sequence set forth in SEQ ID NO:21 and a heavy chain constant region having the sequence set forth in SEQ ID NO:31, and a light chain comprising a VL region having the sequence set forth in SEQ ID NO:22; or a heavy chain comprising a VH region having the sequence set forth in SEQ ID NO: 1 and a heavy chain constant region having the sequence set forth in SEQ ID NO: 32, and a light chain comprising a VL region having the sequence set forth in SEQ ID NO: 2; or a heavy chain comprising a VH region having the sequence set forth in SEQ ID NO:21 and a heavy chain constant region having the sequence set forth in SEQ ID NO:32, and a light chain comprising a VL region having the sequence set forth in SEQ ID NO:22; or a heavy chain comprising a VH region having the sequence set forth in SEQ ID NO: 1 and a heavy chain constant region having the sequence set forth in SEQ ID NO: 34, and a light chain comprising a VL region having the sequence set forth in SEQ ID NO: 2; or a heavy chain comprising a VH region having the sequence set forth in SEQ ID NO: 21 and a heavy chain constant region having the sequence set forth in SEQ ID NO: 34, and a light chain VL region having the sequence set forth in SEQ ID NO: 22; (b) A polypeptide derived from (a), which has an antigen-binding function and the CDC activity, and in which the amino acid sequence in (a) is obtained by substitution, deletion or addition of one or more amino acid residues.

[0042] In a second aspect of the present invention, there is provided a method for improving CDC of an antibody, or a fragment thereof, or a fusion protein thereof, wherein the antibody, or the fragment thereof, or the fusion protein thereof comprises an immunoglobulin Fc region and a functional binding domain that targets a predetermined antigen, the method comprising the steps of: (S1a) introducing mutations at one or more amino acid residues into the antibody, or fragment thereof, or fusion protein thereof, wherein the mutations include mutation of position 309 of the Fc region of the IgG heavy chain to an amino acid selected from the group consisting of W, Y, E, F, H, C, D, N, Q, R, S, T, or K; and / or (S1b) A tailpiece element shown in SEQ ID NO: 7 or 8 is fused to the C-terminus of the Fc region.

[0043] In another preferred example, L at position 309 in the Fc region of the heavy chain of the IgG is mutated to an amino acid selected from the group consisting of W, Y, E, F, H, C, D, N, Q, R, S, T, or K.

[0044] In another preferred embodiment, the method further comprises the steps of: (S2) The CDC performance of the mutated antibody, or its fragment, or fusion protein is compared with that of the unmutated antibody, or its fragment, or fusion protein.

[0045] In another preferred embodiment, the method further comprises the step of improving the ADCC activity of the antibody, or fragment thereof, or fusion protein thereof.

[0046] In another preferred example, (S1a) and (S1b) are performed simultaneously, one after the other, or in reverse order.

[0047] In another preferred embodiment, the mutation comprises mutating the 309th amino acid in the Fc region of the IgG heavy chain to an amino acid selected from the group consisting of W and Y.

[0048] In another preferred example, in step (S1a), if position 309 in the heavy chain Fc region of the antibody, fragment, or fusion protein thereof is neither Y nor W, it is mutated to Y or W.

[0049] In another preferred example, in step (S1a), if position 309 in the heavy chain Fc region of the antibody, fragment, or fusion protein thereof is not Y, it is mutated to Y.

[0050] In another preferred example, in step (S1a), if position 309 in the heavy chain Fc region of the antibody, fragment, or fusion protein thereof is not W, it is mutated to W.

[0051] In another preferred embodiment, in step (S1a), the mutation further comprises mutating residue 345 in the Fc region of the IgG heavy chain to R.

[0052] In another preferred embodiment, in step (S1a), the mutation comprises mutating L at position 309 in the Fc region of the IgG heavy chain to an amino acid selected from the group consisting of W, Y, E, F, H, C, D, N, Q, R, S, T, or K; and This includes a mutation of E to R at position 345 in the Fc region of the IgG heavy chain.

[0053] In a third aspect of the invention, there is provided a heavy chain Fc region element comprising: (1) the Fc region contains an amino acid residue selected from the group consisting of W, Y, E, F, H, C, D, N, Q, R, S, T, or K at position 309; and / or (2) Contains a tailpiece element at the C-terminus Provide something.

[0054] In another preferred embodiment, the heavy chain Fc region element and the tail-piece element are connected via a linker.

[0055] In another preferred embodiment, the heavy chain Fc region element comprises a tail-piece element at the C-terminus of the CH3 domain.

[0056] In another preferred embodiment, the heavy chain Fc region element is derived from IgG.

[0057] In another preferred embodiment, the heavy chain Fc region element is derived from human IgG1.

[0058] In another preferred embodiment, the heavy chain Fc region element comprises CH2 and CH3 domains derived from IgG1, IgG2, IgG3, or IgG4.

[0059] In another preferred embodiment, the heavy chain Fc region element comprises CH2 and CH3 domains from IgG1.

[0060] In another preferred embodiment, the heavy chain Fc region element comprises an amino acid residue selected from the group consisting of F, C, W, or Y at position 309 of the Fc region.

[0061] In another preferred embodiment, the heavy chain Fc region element further comprises amino acid substitutions at other positions.

[0062] In another preferred embodiment, the heavy chain Fc region element further comprises a substitution of the glutamic acid residue at position 345 of the Fc region with an arginine residue (E345R).

[0063] In another preferred embodiment, the heavy chain Fc region element comprises an amino acid residue substitution at an Fc region position selected from the group consisting of: (1)L309W+E345R; (2)L309Y+E345R; (3)L309E+E345R; (4)L309F+E345R; (5)L309H+E345R; (6)L309C+E345R; (7)L309D+E345R; (8)L309N+E345R; (9)L309Q+E345R; (10)L309R+E345R; (11)L309S+E345R; (12) L309T+E345R; or (13)L309K+E345R.

[0064] In another preferred embodiment, the numbering of the amino acids of the heavy chain Fc region elements is according to the EU numbering system.

[0065] In another preferred embodiment, the tailpiece element is derived from human IgM.

[0066] In another preferred embodiment, the tailpiece element comprises one or more amino acid mutations.

[0067] In another preferred embodiment, the sequence of the tailpiece element is set forth in SEQ ID NO: 7 or 8.

[0068] In a fourth aspect of the invention, there is provided an isolated nucleic acid molecule encoding an antibody, or fragment thereof, or fusion protein thereof, according to the first aspect of the invention, or a heavy chain Fc region element according to the third aspect of the invention.

[0069] In a fifth aspect of the invention, there is provided an expression vector containing a nucleic acid molecule according to the fourth aspect of the invention.

[0070] In a sixth aspect of the invention, there is provided a host cell containing an expression vector according to the fifth aspect of the invention.

[0071] In a seventh aspect of the present invention, there is provided a method for producing an antibody or fragment thereof or fusion protein according to the first aspect of the invention, or a heavy chain Fc region element according to the third aspect of the invention, the method comprising the steps of: (a) expressing the antibody or fragment thereof or fusion protein, or the heavy chain Fc region element, by culturing a host cell according to the sixth aspect of the invention under conditions for expression; (b) isolating and purifying the antibody or fragment or fusion protein thereof described in (a) or the heavy chain Fc region element.

[0072] In an eighth aspect of the present invention there is provided an immunoconjugate comprising: (a) an antibody, or a fragment or fusion protein thereof, according to the first aspect of the invention; and (b) a conjugate moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0073] In a ninth aspect of the present invention, there is provided a pharmaceutical composition comprising an antibody or a fragment thereof or a fusion protein thereof according to the first aspect of the present invention, or an immunoconjugate according to the eighth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0074] In a tenth aspect of the invention, there is provided the use of an antibody or fragment thereof or fusion protein according to the first aspect of the invention, or an immunoconjugate according to the eighth aspect of the invention, or a pharmaceutical composition according to the ninth aspect of the invention, in the manufacture of a medicament for treating cancer or an immune-related disease.

[0075] In another preferred embodiment, the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, multiple myeloma, and other neoplastic malignancies.

[0076] In another preferred embodiment, the immune-related disease is an autoimmune disease, preferably an autoimmune kidney disease (immune nephritis, autoimmune kidney disease), lupus erythematosus, systemic lupus erythematosus (SLE), Sjogren's syndrome, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, multiple sclerosis, Guillain-Barré syndrome, or the like. syndrome), atopic dermatitis, autoimmune hepatitis, ankylosing spondylitis, fibrosing alveolitis, Graves' disease, idiopathic thrombocytopenic purpura (ITP), Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, other autoimmune disorders, pancreatic adenitis, wounds (surgery), graft-versus-host disease, transplant rejection, heart disease (including ischemic diseases, e.g., myocardial infarction and atherosclerosis), intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochloremia, infertility associated with defective fetal-maternal tolerance, vitiligo, myasthenia gravis (MG), systemic sclerosis, inflammatory bowel disease, gastritis or IgG4-related disease.

[0077] In another preferred embodiment, the immune-related disease is an autoimmune kidney disease, preferably immunoglobulin A nephropathy (IgAN), membranous nephropathy (MN) or nephropathic monoclonal gammopathy (MGRS), lupus nephritis or purpura nephritis.

[0078] In an eleventh aspect of the present invention, there is provided a method of treating a disease, the method comprising the step of administering to a subject in need thereof an antibody or fragment thereof or a fusion protein thereof according to the first aspect of the invention, a pharmaceutical composition according to the ninth aspect of the invention or an immunoconjugate according to the eighth aspect of the invention.

[0079] In another preferred embodiment, the disease comprises cancer or an immune-related disease.

[0080] 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.

[0081] DESCRIPTION OF THE DRAWINGS [Brief explanation of the drawings]

[0082] [Figure 1] Figure 1 shows the CDC activity of antibodies containing site-directed mutations at position 309 of the Fc region (L309 E / F / H / C / D / N / Q / R / S / T / K / W / Y) against Raji cells. [Figure 2] Figure 2 shows the CDC activity of antibodies containing site-directed mutations at position 309 of the Fc region (L309 E / F / H / C / D / N / Q / R / S / T / K / W / Y) against Daudi cells. [Figure 3] Figure 3 shows the CDC activity against Daudi cells of antibodies containing a site-directed mutation at position 309 of the Fc portion (L309 F / C / W / Y) and antibodies with modified ends of the Fc portion (Pep and Pep-CS). [Figure 4] FIG. 4 shows the CDC activity of OKT10-Hu-IgG1 and its mutants against Daudi cells. [Figure 5] Figure 5 shows the binding ability of the 50G12-Hu-IgG1, OKT10-Hu-IgG1, Daratumab-IgG1, and Isatuximab-IgG1 antibodies to cynomolgus monkey CD38. [Figure 6] Figure 6 shows the killing activity of Daudi cells mediated by complement activation mediated by 50G12-L309W-E345R, single-site mutant antibody 50G12-L309W, single-site mutant antibody 50G12-E345R, unmutated antibody 50G12-Hu-IgG1, and control antibody daratumumab. [Figure 7] Figure 7 shows the killing activity of complement activation mediated by 50G12-L309W-E345R, single-site mutated antibody 50G12-L309W, single-site mutated antibody 50G12-E345R, unmutated antibody 50G12-Hu-IgG1, and control antibody daratumumab against Roma cells. [Figure 8] Figure 8 shows the killing activity of complement activation mediated by 50G12-L309W-E345R, single-site mutated antibody 50G12-L309W, single-site mutated antibody 50G12-E345R, unmutated antibody 50G12-Hu-IgG1, and control antibody daratumumab against Raji cells. [Figure 9] Figure 9 shows the killing activity of Daudi cells of the ADCC effect mediated by 50G12-L309W-E345R, single-site mutant antibody 50G12-L309W, single-site mutant antibody 50G12-E345R, unmutated antibody 50G12-Hu-IgG1, and control antibody daratumumab. [Figure 10] Figure 10 shows the killing activity of NCI-H929 cells of the ADCC effect mediated by 50G12-L309W-E345R, single-site mutated antibody 50G12-L309W, single-site mutated antibody 50G12-E345R, unmutated antibody 50G12-Hu-IgG1, and control antibody daratumumab. [Figure 11] Figure 11 shows the killing activity of Roman cells in the ADCC effect mediated by 50G12-L309W-E345R, the unmutated antibody 50G12-Hu-IgG1, the single-site mutated antibody 50G12-L309W, the single-site unmutated antibody 50G12-E345R, and the control antibody daratumumab. [Figure 12]Figure 12 shows the killing activity of MOLP8 cells of the ADCC effect mediated by 50G12-L309W-E345R, single-site mutant antibody 50G12-L309W, single-site mutant antibody 50G12-E345R, unmutated antibody 50G12-Hu-IgG1, and control antibody daratumumab. [Figure 13] Figure 13 shows the killing activity of Raji cells of the ADCC effect mediated by 50G12-L309W-E345R, single-site mutated antibody 50G12-L309W, single-site mutated antibody 50G12-E345R, unmutated antibody 50G12-Hu-IgG1, and control antibody daratumumab. [Figure 14] Figure 14 shows the apoptosis-inducing effects of 50G12-L309W-E345R, the unmutated antibody 50G12-Hu-IgG1, and the control antibody daratumumab. Specific Embodiments

[0083] The present inventors have conducted extensive and in-depth research and have unexpectedly found for the first time that modifying L309 (preferably L309 and E345) in the Fc portion of an antibody heavy chain constant region and / or fusing a human IgM tailpiece to the Fc portion of the antibody heavy chain constant region significantly enhances the immune effects of the antibody, particularly improving complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Based on this finding, the present invention has been completed.

[0084] term In the present invention, the terms "antibody (abbreviated as Ab)" and "immunoglobulin G (abbreviated as IgG)" refer to heterotetrameric glycoproteins with similar structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between heavy chains depends on the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a constant region, the heavy chain constant region consisting of three domains, CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end, the light chain constant region containing one domain, CL. The light chain constant region corresponds to the CH1 domain of the heavy chain constant region, and the light chain variable region corresponds to the heavy chain variable region. The constant regions are not directly involved in antibody-antigen binding but exhibit different effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include subtypes such as IgG1, IgG2, IgG3, and IgG4, while light chain constant regions include kappa (Kappa) and lambda (Lambda) domains. The heavy and light chains of an antibody are covalently bonded via a disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of an antibody are covalently bonded via an interpolypeptide disulfide bond formed between the hinge regions. In the present invention, the terms "Fab" and "Fc" refer to the digestion of an antibody with papain into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain. The Fc fragment, or fragment crystallizable (Fc) fragment, consists of the CH2 and CH3 domains of an antibody. The Fc fragment lacks antigen-binding activity and is the site where the antibody interacts with effector molecules or cells.In the present invention, "variable" refers to the fact that certain portions of the variable regions of antibodies differ in sequence, thereby determining the binding and specificity of each particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable regions of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy and light chain variable regions. Relatively conserved portions of the variable regions are called framework regions (FRs). Natural heavy and light chain variable regions each contain four FR regions, each of which essentially has a β-sheet structure and is connected by three CDRs that form connecting loops, and which sometimes have 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 (see Kabat et al., NIH Publication No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0085] As used herein, the term "framework region" (FR) refers to the amino acid sequences interposed between the CDRs, i.e., those portions of the light and heavy chain variable regions of a globulin that are relatively conserved among different globulins in a single species. The light and heavy chains of a globulin each have four FRs, designated FR1-L, FR2-L, FR3-L, and FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H, respectively. Correspondingly, the light chain variable domain is designated as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain is designated as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is almost similar to the naturally occurring human antibody FR, i.e., the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0086] The amino acid sequences of the CDRs are known and one skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0087] As used herein, the term "human framework region" refers to a framework region that is substantially similar (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

[0088] As used herein, the terms "tail-piece element" and "TP element" are used interchangeably and refer to a tailpiece sequence derived from IgM, preferably from human IgM. The TP element of the present invention may comprise any suitable amino acid sequence. The TP element may be a tailpiece found in a naturally occurring antibody, or, optionally, a modified tailpiece sequence that differs from the native tailpiece in length and / or composition. The modification may be a mutation of one or more amino acids, for example, a cysteine ​​to serine mutation in the tailpiece of IgM.

[0089] In one specific embodiment of the present invention, the sequence of the TP element is as set forth in SEQ ID NO:7 or 8.

[0090] The TP element may be directly fused to the C-terminus of the heavy chain Fc region, or a short linker sequence may be provided between the TP element and the heavy chain Fc region.

[0091] As used herein, the term "linker" refers to a short linker sequence between the Fc region and the TP element, preferably a flexible linker. Examples of suitable linkers include a single glycine (Gly) or serine (Ser) residue, although the sequence and number of amino acid residues in the linker will vary depending on the type of secondary structure element to be achieved in the linker.

[0092] Antibodies or fragments or fusion proteins thereof The antibody, or fragment thereof, or fusion protein thereof of the present invention is an antibody, or fragment thereof, or fusion protein thereof, having antigen-binding activity and CDC activity, and comprising a binding domain targeting a predetermined antigen and a heavy chain Fc region element, wherein the heavy chain Fc region element comprises two heavy chain Fc regions, each of which (1) contains an amino acid residue selected from the group consisting of W, Y, E, F, H, C, D, N, Q, R, S, T, or K at position 309 of the Fc region, and / or (2) contains a tailpiece element at the C-terminus. As used herein, a "heavy chain Fc region element" refers to two heavy chain Fc regions. Unless otherwise specified, in the present invention, a mutation site in a heavy chain Fc region element refers to a mutation site contained in the Fc region of each heavy chain in the heavy chain Fc region element.

[0093] The specific CDC-enhancing mutation at position 309 in the heavy chain Fc region element of an antibody, or fragment, or fusion protein thereof of the present invention can be combined with techniques for improving other antibody performance (e.g., CDC, specificity, affinity, etc.), such as the E345R mutation. Preferably, when the antibody, or fragment, or fusion protein thereof of the present invention contains the mutation combination L309W+E345R in the Fc region of each heavy chain in the heavy chain Fc region element, it has the unexpected effect of clearly enhancing CDC, more preferably the effect of clearly enhancing CDC and ADCC activity.

[0094] As used herein, the terms "CDC platform antibody," "antibody of the invention," "antibody of the invention or fragment thereof," "fusion protein of the invention," and "antibody of the invention or fragment or fusion protein thereof" are used interchangeably and refer to an antibody, fragment thereof, or fusion protein with enhanced CDC function as described in the first aspect of the invention. These terms also encompass active fragments of the antibody, fragment thereof, or fusion protein of the invention, which retain not only the antigen-binding activity but also the specific mutation at position 309 and / or the TP (tail-piece) element.

[0095] In the present invention, unless otherwise specified, the EU numbering system is used to designate residues in antibody domains. This system was first designed by Edelman et al. in 1969 and is described in detail in the following references: Kabat et al., 1987 (Edelman et al., 1969; "The covered structure of an entire γG immunoglobulin molecule," PNAS Biochemistry, Vol. 63, pp. 78-85; Kabat et al., 1987; Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA). It is known to those skilled in the art that when a position number and / or amino acid residue is assigned to a particular antibody isoform, it can also be applied to the corresponding position and / or amino acid residue in any other antibody isoform. For example, the wild-type residue found at position 309 in naturally occurring human IgG1, IgG3, and IgG4 is a leucine residue, and the wild-type residue found in naturally occurring IgG2 is a valine residue. When referring to amino acid residues in the tails from IgM or IgA, the position numbers indicated are those of the residues in naturally occurring IgM or IgA, in accordance with common practice in the art. As used herein, the "amino acid residue at position 309" refers to the residue at position 309 in naturally occurring human IgG1, and the "amino acid residue at position 345" refers to the residue at position 345 in naturally occurring human IgG1.

[0096] The present invention also provides a method for constructing a CDC platform antibody, or a fragment thereof, or a fusion protein thereof, i.e., a method for constructing an antibody, or a fragment thereof, or a fusion protein thereof of the present invention, as described in the second aspect of the present invention. The method for constructing a CDC platform antibody, or a fragment thereof, or a fusion protein thereof of the present invention can improve the CDC activity of an antibody, or a fragment thereof, or a fusion protein targeting a specific antigen, and can further significantly improve the CDC and / or ADCC activity of the fusion protein.

[0097] In the present invention, the binding domain targeting a predetermined antigen contained in the antibody, or fragment thereof, or fusion protein thereof of the present invention can target different antigen targets, and the binding domain targeting a predetermined antigen binds to an antigen molecule selected from the group consisting of CD38, CD3, CD47, CD19, CD20, HER2, EGFR, CD123, Glypican-3, CD25, Trop-2, EpCAM, or a combination thereof.

[0098] Taking CD38 as an example, the sequence of the anti-CD38 antibody of the present invention is preferably as described in patent application CN202010805420.2. Those skilled in the art can use techniques well known in the art to modify or alter the antigen-binding domain of the present invention, for example, by adding, deleting, and / or substituting one or more amino acid residues, to further increase the affinity or structural stability of the antigen-binding domain, and can also use conventional measurement methods to obtain the results after modification or alteration.

[0099] In the present invention, the antibody, or fragment thereof, or fusion protein thereof further includes conservative variants thereof, and refers to polypeptides in which, compared to the amino acid sequence of the antibody, or fragment thereof, or fusion protein thereof, 10 or less, preferably 8 or less, more preferably 5 or less, and most preferably 3 or less 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 1]

[0100] In the present invention, the terms "antibody" and "binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its corresponding antigen. Typically, an antibody binds to a target antigen at a rate of about 10 -7 Less than M, for example, about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 It binds to an antigen with a dissociation equilibrium constant (KD) of less than M or less. The term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction and is used to describe the binding affinity between the antibody and the antigen. The smaller the dissociation equilibrium constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity of an antibody and antigen can be measured using a BIACORE instrument using surface plasmon resonance (SPR) technology, or the relative affinity of antibody-antigen binding can be measured using ELISA assays.

[0101] The antibodies or fragments or fusion proteins thereof of the present invention may be used alone, or may be conjugated or coupled to a detectable marker (for diagnostic purposes), a therapeutic agent, or a combination of any one or more of these substances.

[0102] Encoding nucleic acids and expression vectors The present invention also provides polynucleotide molecules encoding the above-described antibodies, or fragments or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. Examples of DNA include cDNA, genomic DNA, and artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding or non-coding strand. In the present invention, the term "expression vector" refers to a vector carrying an expression cassette for expressing a specific target protein or other substance, such as a plasmid, a viral vector (e.g., adenovirus, retrovirus), a phage, a yeast plasmid, or other vector. Examples include expression vectors commonly used in the art that contain appropriate regulatory sequences, such as promoters, terminators, and enhancers. Examples of such expression vectors include, but are not limited to, viral vectors (e.g., adenovirus, retrovirus), plasmids, phage, yeast plasmids, and other vectors. The expression vector preferably includes pDR1, pcDNA3.4(+), pDHFR, or pTT5.

[0103] 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.

[0104] 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.

[0105] In the present invention, the term "host cell" refers to various host cells commonly used in the art, which can stably replicate vectors and effectively express the polynucleotide molecules carried therein. Among these, the host cells include prokaryotic expression cells and eukaryotic expression cells, and preferably include COS, CHO, NS0, sf9, sf21, DH5α, BL21(DE3), TG1, BL21(DE3), 293F, or 293E cells.

[0106] Drug Composition The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the antibody, or its active fragment, or fusion protein, 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 4 to 8, preferably about 5 to 7, 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, intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavity injection.

[0107] In the present invention, the term "drug composition" refers to a pharmaceutical formulation in which the bifunctional antibody, or a fragment thereof, or a fusion protein thereof of the present invention is combined with a pharmaceutically acceptable carrier to form a pharmaceutical formulation, which can more stably exert a therapeutic effect, and which can maintain the conformational integrity of the amino acid core sequence of the bifunctional antibody, or a fragment thereof, or a fusion protein disclosed in the present invention, while preventing degradation of many functional groups of the protein (including, but not limited to, aggregation, deamination, or oxidation).

[0108] The pharmaceutical composition of the present invention contains 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 above-mentioned bifunctional antibody of the present invention, or a fragment thereof, or a fusion protein thereof (or a conjugate thereof) and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, water, 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 be an injection, which can be prepared by a conventional method 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 10 μg / kg body weight to about 50 mg / kg body weight daily. The bifunctional antibody of the present invention, or a fragment thereof, or a fusion protein thereof can also be used in combination with other therapeutic agents.

[0109] When using the pharmaceutical composition, a safe and effective amount of the bifunctional antibody, or its fragment or fusion protein, or immunoconjugate thereof is administered to a mammal, and this safe and effective amount is usually at least about 10 μg / kg body weight, and in many cases not more than about 50 mg / kg body weight, preferably about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage should be determined taking 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.

[0110] In the present invention, the term "effective amount" refers to the amount or dosage of the pharmaceutical composition of the present invention that, when administered to a subject, produces the expected effect in the treated individual, including the improvement of the individual's symptoms. The term "subject" includes, but is not limited to, mammals, such as humans, non-human primates, rats, and mice.

[0111] application The present invention also provides the use of an antibody or fragment thereof or fusion protein according to the first aspect of the invention, or an immunoconjugate according to the eighth aspect of the invention, or a pharmaceutical composition according to the ninth aspect of the invention, for example in the manufacture of a diagnostic preparation or a drug.

[0112] Preferably, the drug is a drug for the prevention and / or treatment of cancer or an immune-related disease.

[0113] In the present invention, the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, multiple myeloma and other neoplastic malignant diseases.

[0114] In one preferred embodiment of the present invention, the drug is a drug for preventing and / or treating a disease associated with abnormal expression or function of CD38.

[0115] In the present invention, the disease associated with abnormal CD38 expression or function is a disease associated with abnormal CD38 expression or function commonly known in the art, preferably a tumor / cancer or an immune-related disease.

[0116] Preferably, the disease is suitably an immune-related disease, such as an autoimmune disease. More preferably, the medicament is for the treatment and / or prevention of an autoantibody-mediated autoimmune disease. Further, the autoimmune disease includes autoimmune kidney disease (immune nephritis, autoimmune kidney disease), lupus erythematosus, systemic lupus erythematosus (SLE), Grave's disease, myasthenia gravis (MG), idiopathic thrombocytopenic purpura (ITP), autoimmune kidney disease (immune nephritis, autoimmune kidney disease), lupus erythematosus, systemic lupus erythematosus (SLE), Sjogren's syndrome, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, Disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, multiple sclerosis, Guillain-Barr syndrome, atopic dermatitis, autoimmune hepatitis, ankylosing spondylitis, fibrosing alveolitis, Graves' disease, idiopathic thrombocytopenic purpura (ITP), Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis granulomatosis), other autoimmune disorders, pancreatic adenitis, wounds (surgery), graft-versus-host disease, transplant rejection, heart disease (including ischemic diseases, e.g., myocardial infarction and atherosclerosis), intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochloremia, infertility associated with defective fetal-maternal tolerance, vitiligo, myasthenia gravis (MG), systemic sclerosis, inflammatory bowel disease, gastritis or IgG4-related disease, preferably immunoglobulin A nephropathy (IgAN), membranous nephropathy (MN) or nephropathic monoclonal gammopathy (MGRS), lupus nephritis or purpura nephritis, which are caused by excessive proliferation and secretion of antibodies and immune complexes formed from immunoglobulins by plasma cells.

[0117] Currently, there is a significant unmet clinical need for treatment of the three autoimmune kidney diseases, IgAN, MN, and MGRS, particularly due to a lack of specific targeted therapeutic options. Rituximab, which targets CD20, is the only approved treatment for MN. However, because CD20 is expressed only on activated B cells, which produce small amounts of autoantibodies, clinical studies have shown that anti-CD20 therapy is ineffective in up to 40% of MN patients. On the other hand, CD38 is highly expressed on plasma cells. The antibodies specifically targeting CD38 (e.g., 50G12-L309W-E345R) of the present invention can induce plasma cell degradation and apoptosis through ADCC, ADCP, and CDC, making them useful for the treatment of IgAN, MN, MGRS, and multiple myeloma (MM).

[0118] The main advantages of the present invention include:

[0119] (1) The present invention provides a new technology for enhancing CDC and / or ADCC. This technology has the potential to convert antibody drugs with lacking or limited CDC and / or ADCC activity into antibody drugs with enhanced CDC and / or ADCC. This technology can be used to directly engineer existing antibodies to act on different targets / target cells. This technology can be used to improve the therapeutic effects of existing antibodies and treat many diseases.

[0120] (2) Site-specific mutation of amino acid 309 can significantly improve the CDC and / or ADCC performance of the antibody, or fragment or fusion protein thereof of the present invention. Furthermore, the specific CDC and / or CDC-enhancing mutation at amino acid 309 of the present invention can be combined with techniques to improve other antibody performances (e.g., CDC, specificity, affinity, etc.), such as the E345R mutation.

[0121] 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.

[0122] Related reagents and materials: EZ-link NHS-LC-Biotin (Thermo Fisher Scientific, catalog number 21343); Biotin CAPture Kit, Series S (Cytiva, catalog number 28920234); HBS-EP+ pH 7.4 buffer (GE Healthcare, catalog number BR-1006-69); Daudi, Raji, Romas, and NCI-H929 cells were purchased from the American Type Culture Collection (ATCC), and MOLP8 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.; ADCC Bioassay effector cells (Suzhou Ruian Biotechnology Co., Ltd., catalog number RA-CK01); Cell Titer-Glo Luminescent Assay (Promega, catalog number G7570); Normal Human Serum complement (Quidel Corporation, catalog number A11); Bio-Glo Luciferase Assay System (Promega, catalog number G7940); Annexin V-FITC / PI Apoptosis Detection Kit (Yeasen, Catalog number 40302ES60).

[0123] A description of the protein expression and purification methods used in the examples follows.

[0124] The target gene was constructed in the expression vector pcDNA3.4, and the PEI (Polyethylenimine)-constructed expression vector or a combination of expression vectors was introduced into FreeStyle® 293-F Cells (hereinafter abbreviated as HEK293F, purchased from Thermo Fisher Scientific) to express the antibody or recombinant protein. After culturing the HEK293F cells in FreeStyle 293 Expression Medium (purchased from Thermo Fisher Scientific) for 5 days, the cell supernatant was collected and the antibody was further purified by Protein A affinity chromatography, and the recombinant protein was purified by Ni-NTA affinity chromatography.

[0125] A description of the ELISA detection method used in the following examples follows.

[0126] Microplates were coated with the corresponding recombinant proteins and blocked with 1% bovine serum albumin-containing PBST (PBST is a phosphate buffer solution containing 0.05% Tween-20). Test antibodies were gradient diluted and transferred to the recombinant protein-coated microplates and incubated at room temperature for 0.5 hours. After washing the plate, appropriately diluted HRP (Horseradish Peroxidase)-conjugated sheep anti-human antibody (Fc specific, purchased from Sigma) was added and incubated at room temperature for 0.5 hours. After washing the plate, 100 μL of a color development solution using TMB (3,3',5,5'-tetramethylbenzidine) as a substrate was added to each well and incubated at room temperature for 1 to 5 minutes. The reaction was terminated by adding 50 μL of stop solution (2 M H2SO4). OD450 was read using a microplate reader (SpectraMax 190). Blotting and data analysis were performed using GraphPad Prism 7, and EC 50 / I C 50 was calculated.

[0127] The methods for detecting physical and chemical properties used in the following examples are described below.

[0128] HPLC-SEC Antibodies are high-molecular-weight proteins with highly complex secondary and tertiary structures. Due to changes such as post-translational modifications, assembly, and degradation, antibodies have heterogeneous biochemical and biophysical properties. When analyzing trispecific antibodies using separation techniques, variants, aggregates, and degradation fragments are commonly observed, and their presence may compromise safety and efficacy. During antibody production and storage, aggregates, degradation fragments, and incompletely assembled molecules are likely to appear. In this study, the content of the above impurities in samples was detected using high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC). Because the molecular weight of aggregates is larger than that of the monomer, the corresponding peaks have shorter retention times. Because the molecular weight of degradation fragments or incompletely assembled molecules is smaller than that of the monomer, the corresponding peaks have longer retention times. The chromatography equipment used for HPLC-SEC was a Dionex Ultimate 3000. The mobile phase was prepared as follows: an appropriate amount of 20 mM sodium dihydrogen phosphate mother liquor was taken and the pH was adjusted to 6.8 ± 0.1 with 20 mM disodium hydrogen phosphate. The loading amount was 20 μg. The chromatography column was a TSK G3000SWXL with dimensions of 7.8 × 300 mm and 5 μm. The flow rate was 0.5 mL / min, the elution time was 30 min, the column temperature was 25°C, the sample chamber temperature was 10°C, and the detection wavelength was 214 nm.

[0129] Antibody sequences of the present invention: [Table 2] TIFF0007735545000003.tif253169TIFF0007735545000004.tif255166TIFF0007735545000005.tif252167

[0130] Example 1: Production of anti-human CD38 monoclonal antibody (50G12) 50G12-Humanized (hereinafter referred to as 50G12-Hu-IgG1) is an anti-CD38 humanized monoclonal antibody, the amino acid sequences of its heavy and light chains are as set forth in SEQ ID NOs: 11 and 12 of CN202010805420.2 (i.e., SEQ ID NOs: 5 and 6 in the present invention). The amino acid sequences of the heavy chain variable region and light chain variable region of 50G12-Hu-IgG1 are as set forth in SEQ ID NOs: 1 and 2. The amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 of the heavy chain of 50G12-Hu-IgG1 are as set forth in SEQ ID NOs: 35, 36, and 37, respectively, and the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 of the light chain of 50G12-Hu-IgG1 are as set forth in SEQ ID NOs: 38, 39, and 40, respectively. The heavy chain constant region of 50G12-Hu-IgG1 is human IgG1 (amino acid sequence as shown in SEQ ID NO: 3), and the light chain constant region is human κ (amino acid sequence as shown in SEQ ID NO: 4). Here, the genes encoding the heavy and light chains of 50G12-Hu-IgG1 are designated 50G12-Hu-IgG1-HC and 50G12-Hu-IgG1-LC, respectively. The genes for 50G12-Hu-IgG1-HC and 50G12-Hu-IgG1-LC were each constructed in a pcDNA3.4 expression vector, and the two vectors were combined and expressed to produce an antibody, which was then purified and designated 50G12-Hu-IgG1.

[0131] Example 2 Modification of the Fc portion of the heavy chain constant region of an antibody As shown in the literature (Rowley TF, Peters SJ, Aylott M, et al., Engineered hexavalent Fc proteins with enhanced Fc-gamma receptor avidity provide insights into immune-complex interactions[J]. Communications biology, 2018, 1(1): 1-12.), human IgG1 monoclonal antibodies are prone to hexameric antibody complexes after L309 (Eu numbering scheme) is mutated to cysteine ​​(abbreviated as C) and the human IgM tailpiece is fused to the Fc terminus. This indicates that the L309C mutation in human IgG1 and the human IgM tailpiece have the potential to promote the formation of human IgG1 into hexamers.

[0132] 1. Site-directed mutation at position 309 of the Fc region In this example, site-directed mutagenesis was performed on the L309 site of the heavy chain gene of 50G12-Hu-IgG1, mutating L309 to (E / F / H / C / D / N / Q / R / S / T / K / W / Y), resulting in a series of mutants, which were then expressed and purified according to the method described above. The resulting antibodies were named 50G12-Hu-IgG1-L309X (X represents E / F / H / C / D / N / Q / R / S / T / K / W / Y).

[0133] The method for measuring CDC is as follows: Raji and Daudi cells were purchased from ATCC (American Type Culture Collection), and routine culture and subculture were performed according to the method recommended by ATCC. Human serum (Allcells; catalog number: PB022-C) was added to RPMI-1640 (Gibco; catalog number: 11835-030) to a final concentration of 5% human serum. Logarithmic-phase Raji / Daudi cells were resuspended in this medium and seeded into a 96-well plate (Corning; catalog number: CLS3599). 50 μL of the cell suspension (containing 100,000 cells) was seeded into each well. The test antibody was diluted in a gradient and added to the 96-well plate at 50 μL per well. After uniform mixing, the plate was incubated for 2.5 hours in a carbon dioxide cell incubator. CCK-8 (Dojindo, catalog number: CK04) was added to the plate at 20 μL per well, followed by incubation for 2 hours. The OD450 of the 96-well plate was measured using a SpectraMax 190 (Molecular Devices). GraphPad Data analysis and blotting were performed in Prism7, and IC 50 was calculated.

[0134] The stronger the CDC activity, the lower the cell vitality and the lower the OD450. The results in Figures 1 and 2 both show that 50G12-Hu-IgG1-L309F / C / W / Y has significantly enhanced CDC compared to the parent antibody 50G12-Hu-IgG1. The isotype control used here is a human IgG1 monoclonal antibody unrelated to the target.

[0135] 2. Modification of the Fc terminal Here, the coding sequence for the IgM tailpiece (amino acid sequence: PTLYNVSLVMSDTAGTCY (SEQ ID NO: 7); this short peptide is abbreviated as Pep) was genetically engineered to link it to the end of the heavy chain gene of 50G12-Hu-IgG1, and the heavy chain gene was named 50G12-Hu-IgG1-Pep-HC (the amino acid sequence is SEQ ID NO: 9). To prevent disulfide bond formation between antibody molecules during expression, the cysteines in the two tailpieces were mutated to serine (the amino acid sequence of the mutated tailpiece: PTLYNVSLVMSDTAGTCY). S Y (SEQ ID NO: 8), this short peptide is abbreviated as Pep-CS), and the gene for this heavy chain was designated 50G12-Hu-IgG1-Pep-CS-HC (its amino acid sequence is shown in SEQ ID NO: 10). The genes for 50G12-Hu-IgG1-Pep-HC and 50G12-Hu-IgG1-Pep-CS-HC were each constructed in a pcDNA3.4 expression vector, and the two vectors were each expressed in combination with the 50G12-Hu-IgG1-LC gene to produce purified antibodies, which were designated 50G12-Hu-IgG1-Pep and 50G12-Hu-IgG1-Pep-CS, respectively.

[0136] CDC of 50G12-Hu-IgG1-L309F / C / W / Y, 50G12-Hu-IgG1-Pep, and 50G12-Hu-IgG1-Pep-CS was detected by the above method. R Cell vitality was detected using a Luminescent Cell Viability Assay (Promega, catalog number: G7572, abbreviated as CTG). Triton X-100 (0.1%) was added to some wells to fully lyse the cells, and the signal generated after adding these cells to CTG was the base value. The signal generated after adding cells not treated with antibody to CTG was the maximum value. CDC was calculated using the following formula: cytotoxicity (%) = (maximum value - experimental value) / (maximum value - base value) × 100. Data analysis and blotting were performed using GraphPad Prism7 and are shown in Figure 3. EC50 was calculated and is shown in Table 2. [Table 3]

[0137] The stronger the CDC, the greater the EC 50 The peak (top) of the curve (height of the plateau) is higher. The results in Figure 3 and Table 2 show that, compared with the parent antibody 50G12-Hu-IgG1, 50G12-Hu-IgG1-L309F / C / W / Y has significantly enhanced CDC, with the CDC of 50G12-Hu-IgG1-L309F / Y being weaker than that of 50G12-Hu-IgG1-L309C / W. 50G12-Hu-IgG1-Pep and 50G12-Hu-IgG1-Pep-CS have similar CDC, and their CDC is essentially equivalent to that of 50G12-Hu-IgG1-L309C / W. 50G12-Hu-IgG1-L309C / W, 50G12-Hu-IgG1-Pep, and 50G12-Hu-IgG1-Pep-CS all had Top values ​​greater than 99, indicating that these antibodies, at high concentrations, were sufficient to degrade target cells.

[0138] Example 3 Analysis of a collection of antibody mutants The purity of the preferred antibody mutants was determined by HPLC-SEC, with the following results: [Table 4]

[0139] The HPLC-SEC results showed that the ratios of the major peaks of 50G12-Hu-IgG1-L309F / W / Y and 50G12-Hu-IgG1-Pep-CS were both greater than 98%, indicating high purity with little size heterogeneity.The ratios of the major peaks of 50G12-Hu-IgG1-L309C and 50G12-Hu-IgG1-Pep were 79.7% and 74.0%, respectively, indicating that the purity of both antibodies was less than 80%, and the spectra indicated the presence of many aggregates in these two antibodies.

[0140] Example 4 Modification of anti-human CD38 monoclonal antibody OKT10 1. Production of OKT10 humanized monoclonal antibody OKT10 is an anti-human CD38 monoclonal antibody derived from a mouse hybridoma. The amino acid sequences of its heavy and light chain variable regions (SEQ ID NOs: 19 and 20) are from NCBI GenBank (ABA42888.1 and ABA42887.1).

[0141] The DNA encoding the variable regions was synthesized by Shanghai Zhengong Bioengineering Co., Ltd. The amino acid sequences of the heavy and light chain variable regions of OKT10 were analyzed, and the antigen complementarity determining regions and framework regions of the heavy and light chains of OKT10 were determined using Kabat numbering. The amino acid sequences of the heavy chain CDRs of OKT10 are H-CDR1: RSWMN (SEQ ID NO: 11), H-CDR2: EINPDSSTINYTTSLKD (SEQ ID NO: 12), and H-CDR3: YGNWFPY (SEQ ID NO: 13). The amino acid sequences of the light chain CDRs of OKT10 are L-CDR1: KASQNVDTNVA (SEQ ID NO: 14), L-CDR2: SASYRYS (SEQ ID NO: 15), and L-CDR3: QQYDSYPLT (SEQ ID NO: 16).

[0142] https: / / www.ncbi.nlm.nih.gov / igblast / In this study, the heavy chain variable region of murine OKT10 was compared for homology with the human IgG embryonic sequence, IGHV3-48*01 was selected as the heavy chain CDR-grafting template, the heavy chain CDRs of murine OKT10 were grafted into the framework region of IGHV3-48*01, and WGQGTLVTVSS (SEQ ID NO: 17) was added as the fourth framework region after H-CDR3 to obtain the sequence of the CDR-grafted heavy chain variable region. Similarly, the light chain variable region of murine OKT10 was compared for homology with the human IgG embryonic sequence, IGKV1-16*01 was selected as the light chain CDR-grafting template, the light chain CDRs of murine OKT10 were grafted into the framework region of IGKV1-16*01, and FGQGTKVEIK (SEQ ID NO: 18) was added as the fourth framework region after L-CDR3 to obtain the sequence of the CDR-grafted light chain variable region. Based on the CDR-grafted variable region, some amino acid positions in the framework region were backmutated (backmutation is the process of replacing some amino acids in the human-derived framework region with amino acids at the same positions in the murine-derived framework region, and the backmutation positions are generally very important for maintaining the structure and / or affinity). For backmutations, the amino acid sequence is numbered using Kabat numbers, and the positions of the positions are indicated by the Kabat numbers.

[0143] Preferably, for the CDR-grafted heavy chain variable region, T at position 28 is backmutated to D. For the CDR-grafted light chain variable region, F at position 36 is backmutated to Y and S at position 46 is backmutated to A.

[0144] The heavy chain variable region and light chain variable region containing the backmutation sites were defined as the OKT10 humanized heavy chain variable region (amino acid sequence: SEQ ID NO: 21) and light chain variable region (amino acid sequence: SEQ ID NO: 22), respectively. DNA encoding the humanized heavy and light chain variable regions was synthesized by Shanghai Zhengong Bioengineering Co., Ltd. The synthesized humanized heavy chain variable region was linked to a human IgG1 constant region (amino acid sequence: SEQ ID NO: 3) to obtain a full-length humanized heavy chain gene designated OKT10-Hu-HC (amino acid sequence: SEQ ID NO: 23). The humanized light chain variable region was linked to a human κ chain constant region (amino acid sequence: SEQ ID NO: 4) to obtain a full-length humanized light chain gene designated OKT10-Hu-LC (amino acid sequence: SEQ ID NO: 24).

[0145] The OKT10-Hu-HC and OKT10-Hu-LC genes were each constructed in the pcDNA3.4 expression vector, and the two vectors were combined and expressed to produce an antibody, which was named OKT10-Hu-IgG1.

[0146] 2. Preparation of OKT10-Hu-IgG1 Mutants Here, site-directed mutation was performed on the L309 position of the OKT10-Hu-IgG1 heavy chain gene to mutate it to W or Y, respectively, and the mutated genes were then combined with OKT10-Hu-LC, respectively, and expressed to produce purified antibodies, which were designated OKT10-Hu-IgG1-L309W and OKT10-Hu-IgG1-L309Y, respectively. Here, the coding sequence for Pep-CS was linked to the end of the OKT10-Hu-IgG1 heavy chain gene by genetic engineering, and the heavy chain gene was designated OKT10-Hu-IgG1-Pep-CS-HC (the amino acid sequence is set forth in SEQ ID NO: 25). This was combined with OKT10-Hu-LC, expressed using the above method, and purified to produce an antibody, which was designated OKT10-Hu-IgG1-Pep-CS.

[0147] 3. Measuring CDC of OKT10-Hu-IgG1 Mutants The CDC of OKT10-Hu-IgG1 and its mutants was measured by the method described in the above Examples, and the results are shown in Figure 4.

[0148] The results in Figure 4 show that compared with the parent antibody OKT10-Hu-IgG1, OKT10-Hu-IgG1-L309W, OKT10-Hu-IgG1-L309Y, and OKT10-Hu-IgG1-Pep-CS have significantly enhanced CDC activity, and their EC 50 The Top values ​​were 0.5942 nM, 5.902 nM, and 1.715 nM, respectively, with plateau values ​​of 99.85, 69.84, and 99.43, respectively. OKT10-Hu-IgG1-L309W and OKT10-Hu-IgG1-Pep-CS both had Top values ​​above 99, indicating that these antibodies can sufficiently degrade target cells at high concentrations. The above results indicate that among the three, OKT10-Hu-IgG1-L309W had the strongest CDC.

[0149] 4. Binding ability of OKT10-Hu-IgG1 to cynomolgus monkey CD38 Daratumumab is a commercially available anti-human CD38 monoclonal antibody, and the amino acid sequences of its heavy and light chain variable regions (SEQ ID NOs: 26 and 27) were obtained from WHO Drug Information, Vol. 24, No. 1, 2010.

[0150] Isatuximab is another commercially available anti-human CD38 monoclonal antibody, and its heavy and light chain variable region amino acid sequences (SEQ ID NOs: 28 and 29) were obtained from WHO Drug Information, Vol. 29, No. 3, 2015.

[0151] The DNA for the heavy and light chain variable regions was synthesized by Shanghai Synchro Bioengineering Co., Ltd. The synthesized daratumumab heavy chain variable region gene was linked to a human IgG1 heavy chain constant region gene to obtain a full-length heavy chain gene. The daratumumab light chain variable region gene was linked to a human κ chain constant region gene to obtain a full-length light chain gene. The antibody was expressed and purified using the method described in the above examples, and the resulting antibody was named daratumumab-IgG1 (also referred to as daratumumab). The antibody isatuximab-IgG1 was also obtained using a similar experimental method.

[0152] The amino acid sequence of cynomolgus monkey CD38 (SEQ ID NO: 30) was obtained from https: / / www.uniprot.org / uniprot / Q5VAN0. DNA encoding the extracellular portion of cynomolgus monkey CD38 was synthesized by Shanghai Syngong Bioengineering Co., Ltd. A polyhistidine coding sequence was added to the end of the gene, and the recombinant gene was then constructed into an expression vector. The recombinant protein was expressed using the method described in the above Examples, and the recombinant protein in the culture supernatant was purified using a Ni-NTA affinity chromatography column. The resulting recombinant protein was designated CD38-ECD-Cyno.

[0153] Microplates were coated with CD38-ECD-Cyno (10 ng / well), and the binding ability of 50G12-Hu-IgG1, OKT10-Hu-IgG1, Daratumab-IgG1, and Isatuximab-IgG1 to CD38-ECD-Cyno was measured by ELISA.

[0154] As shown in Figure 5, OKT10-Hu-IgG1 can effectively bind to CD38 in cynomolgus monkeys and inhibits EC 50 The antibody titer was 0.1324 nM. None of 50G12-Hu-IgG1, Daratumab-IgG1, or Isatuximab-IgG1 can recognize cynomolgus monkey CD38.

[0155] Example 5 Production of monoclonal antibody 50G12-L309W-E345R Using genetic engineering and molecular cloning techniques, the L309 position of the 50G12-Hu-IgG1 heavy chain constant region was site-specifically mutated to W, and simultaneously the E345 position was site-specifically mutated to R, and then constructed into a pcDNA3.4 expression vector. The resulting heavy chain was designated 50G12-Hu-IgG1-L309W-E345R. This heavy chain was transfected into HEK-293F cells together with 50G12-Hu-IgG1-LC and expressed for 5 days. The resulting antibody was purified using a Protein A affinity chromatography column and designated 50G12-L309W-E345R. Single-site mutant antibodies 50G12-L309W (also designated 50G12-Hu-IgG1-L309W) and 50G12-E345R were obtained using a similar method.

[0156] Example 6 Measurement of affinity of 50G12-L309W-E345R for human CD38 Here, the binding and dissociation constants of 50G12-L309W-E345R, 50G12-Hu-IgG1, and daratumumab to human CD38 protein were measured using a Biacore 8K (purchased from GE Healthcare). The specific method is as follows.

[0157] 1) Human CD38 protein was biotinylated according to the EZ-Link NHS-Biotin Reagent instructions (purchased from Thermo Fisher Scientific, catalog number 21343) and named CD38-biotin.

[0158] 2) CAP chip coupling: 50 μg / mL Biotin CAPture Reagent was mixed with HBS-EP+ pH 7.4 Buffer at a ratio of 1:1, and the operating parameters were a contact time of 60 s and a flow rate of 2 μL / min.

[0159] 3) Biotin-CD38 was captured, and the operating parameters were as follows: biotin-CD38 concentration 2 μg / mL, contact time 15 s, flow rate 10 μL / min, and regeneration contact time 30 s.

[0160] 4) Each test antibody was diluted with HBS-EP+ pH 7.4 buffer to a maximum concentration of 200 nM, then diluted twice to 1.5625 nM and 0 concentration. A regeneration solution was prepared according to the Biotin CAPture Kit instructions and loaded into a Biacore 8K with the following parameters: binding time 180 s, dissociation time 600 s, flow rate 30 μL / min, regeneration contact time 30 s, flow rate 30 μL / min.

[0161] 5) The data was analyzed using Biacore 8K Evaluation Software, and the "1:1 binding kinetics model" formula was selected and fitted in "Kinetics" mode to obtain data on the affinity of each antibody to CD38.

[0162] As shown in Table 4, 50G12-L309W-E345R and 50G12-Hu-IgG1 have stronger affinity for CD38 protein than daratumumab. Specifically, the dissociation constants kd of 50G12-L309W-E345R and 50G12-Hu-IgG1 are better than those of daratumumab, and the equilibrium dissociation constants Kd of the three antibodies are 2.99 × 10 -9 M, 3.58 x 10 -9 M and 2.60 x 10 -8 It was shown to be M. [Table 5]

[0163] Example 7 Measurement of CDC activity of 50G12-L309W-E345R Logarithmic-phase human Burkitt's lymphoma cells, Daudi, Raji, and Roma, were washed once with DPBS and adjusted to a cell density of 2E5 / mL in serum-free, phenol red-free 1640 medium. 50 μL / well of the cells were plated into a white 96-well cell culture plate. Antibodies were diluted to the required concentrations in serum-free, phenol red-free 1640 medium, followed by 12 serial dilutions in a 3-fold gradient. 50 μL of the diluted antibody was added to the cell culture plate at a final antibody concentration of 50 nM. Two parallel replicate wells were then added to the plate at 50 μL / well and incubated for 30 min in a 37°C, 5% CO2 cell incubator. 20 μL of normal human serum complement was added to the plate at 20 μL / well and incubated for 2 h in a 37°C, 5% CO2 cell incubator. The cell culture plate was equilibrated at room temperature for 15 minutes, and 80 μL / well of Cell Titer-Glo Luminescent Assay coloring agent equilibrated at room temperature was added. After incubation at room temperature for 8 minutes, the plate was detected and collected using a microplate reader i3 (purchased from Molecular Devices, model number SPECTRA MAX i3). Data analysis and blotting were performed using GraphPad Prism9, and IC 50 was calculated.

[0164] As shown in Figure 6, the complement activation mediated by 50G12-L309W-E345R was significantly superior to the unmutated antibody 50G12-Hu-IgG1 and the control antibody daratumumab in killing activity against Roman cells, and slightly superior to the single-site mutant antibody 50G12-L309W and the single-site mutant antibody 50G12-E345R. As shown in Table 5, the complement activation mediated by 50G12-L309W-E345R had an IC of killing against Roman cells. 50 was approximately 8 times more potent than daratumumab, the absolute value of maximum killing activity (Bottom) was approximately 98 times more potent than daratumumab, and IC 50 is about 1.7 times better than the 50G12-L309W and about 1.2 times better than the 50G12-E345R.

[0165] As shown in Figure 7, the complement activation mediated by 50G12-L309W-E345R clearly showed superior killing activity against Daudi cells to the single-site mutant antibody 50G12-L309W, the unmutated antibody 50G12-Hu-IgG1, and the control antibody daratumumab, and was slightly superior to the single-site mutant antibody 50G12-E345R. As shown in Table 6, the complement activation mediated by 50G12-L309W-E345R showed an IC of killing against Daudi cells. 50 was approximately 19 times more potent than daratumumab, the absolute value of maximum killing activity (Bottom) was approximately 25 times more potent than daratumumab, and IC 50 is about 3.2 times better than 50G12-L309W, and IC 50 is about 1.4 times better than 50G12-E345R.

[0166] As shown in Figure 8, the complement activation mediated by 50G12-L309W-E345R clearly exhibited superior killing activity against Raji cells to the single-site mutant antibody 50G12-L309W, the single-site mutant antibody 50G12-E345R, the unmutated antibody 50G12-Hu-IgG1, and the control antibody daratumumab. However, 50G12-Hu-IgG1 and daratumumab had little CDC activity on Raji cells, which may be related to the relatively low expression level of CD38 on the surface of Raji cells. As shown in Table 7, the complement activation mediated by 50G12-L309W-E345R exhibited an IC of killing against Raji cells. 50 was approximately 2 times better than 50G12-E345R, and the absolute value of the maximum killing activity (Bottom) was approximately 2.4 times better than 50G12-E345R.

[0167] These data indicate that antibodies exert different CDC effects on different cells, which is related not only to the expression level of CD38 but also to the expression levels of complement regulatory proteins CD55 and CD59 in each cell (see: CD38 expression and complement inhibitors affect response and resistance to daratumumab therapy in myeloma [J]. Blood, 2016, 128(7):959-970). However, we have confirmed that the simultaneous introduction of two mutations, L309W and E345R, into the heavy chain constant region of an antibody can effectively enhance antibody-mediated CDC activity. The introduction of these two mutations confers unexpectedly superior activity to antibodies with either a single mutation (L309W or E345R) or no mutations, and this enhanced CDC effect is more pronounced in tumor cells with relatively low CD38 expression levels. [Table 6] [Table 7] [Table 8]

[0168] Example 8 Measurement of ADCC activity of 50G12-L309W-E345R Log-phase human Burkitt's lymphoma cells (Daudi, Ramos, Raji), human myeloma cells (NCI-H929), and human multiple myeloma cells (MOLP8) were washed once with DPBS and then adjusted to a cell density of 4.8E5 / mL with serum-free 1640 medium. Cells were plated in 25μL / well in a white 96-well cell culture plate. Each antibody was diluted to the required concentration in serum-free 1640 medium, then serially diluted 3-fold to 10 concentrations. The diluted antibodies were added to the cell culture plate in 25μL / well and incubated in a cell incubator for 45 minutes. Log-phase ADCC effector cells were washed once with DPBS and then adjusted to a cell density of 3E6 / mL with serum-free 1640 medium. Cells were plated in 25μL / well and incubated in a 37°C, 5% CO2 cell incubator for 6 hours.

[0169] The cell culture plate was equilibrated at room temperature for 15 minutes in advance, and 60 μL of Bio-glo coloring agent was added to each well. After incubation at room temperature for 4 minutes, the plate was detected and collected using a microplate reader i3. Data analysis and blotting were performed using GraphPad Prism9, and IC was calculated. 50 was calculated.

[0170] As shown in Figure 9, the killing activity of the ADCC effect mediated by 50G12-L309W-E345R against Daudi cells was significantly better than that of the unmutated antibody 50G12-Hu-IgG1, the single-site mutant antibody 50G12-L309W, the single-site mutant antibody 50G12-E345R, and the control antibody daratumumab. As shown in Table 8, the ADCC effect mediated by 50G12-L309W-E345R had an IC of killing against Daudi cells of 1.54. 50 was approximately 1.5 times higher than daratumumab, the absolute value of maximum killing activity (Top) was approximately 1.3 times higher than daratumumab, and IC 50 is about twice as good as 50G12-E345R, and IC 50 is about 1.7 times better than the 50G12-L309W. [Table 9]

[0171] As shown in Figure 10, the killing activity of the ADCC effect mediated by 50G12-L309W-E345R against NCI-H929 cells was significantly better than that of the unmutated antibody 50G12-Hu-IgG, the single-site mutant antibody 50G12-L309W, the single-site mutant antibody 50G12-E345R, and the control antibody daratumumab. As shown in Table 9, the ADCC effect mediated by 50G12-L309W-E345R had an IC of killing against NCI-H929 cells of 1.0181. 50 was approximately 1.5 times higher than daratumumab, the absolute value of maximum killing activity (Top) was approximately 1.9 times higher than daratumumab, and IC 50 is about 1.9 times better than 50G12-E345R, and IC 50 is about 1.3 times better than the 50G12-L309W. [Table 10]

[0172] As shown in Figure 11, the killing activity of the ADCC effect mediated by 50G12-L309W-E345R against Roman cells was significantly better than that of the unmutated antibody 50G12-Hu-IgG1, the single-site mutant antibody 50G12-L309W, the single-site mutant antibody 50G12-E345R, and the control antibody daratumumab. As shown in Table 10, the ADCC effect mediated by 50G12-L309W-E345R showed an IC of killing against Roman cells. 50 was approximately 2.7 times more potent than daratumumab, the absolute value of maximum killing activity (Top) was approximately 1.2 times more potent than daratumumab, and IC 50 is about 2.4 times better than 50G12-E345R, and IC 50 is about twice as good as the 50G12-L309W. [Table 11]

[0173] As shown in Figure 12, the killing activity of the ADCC effect mediated by 50G12-L309W-E345R against MOLP8 cells was clearly superior to that of the unmutated antibody 50G12-Hu-IgG1, the single-site mutant antibody 50G12-L309W, the single-site mutant antibody 50G12-E345R, and the control antibody daratumumab. As shown in Table 11, the ADCC effect mediated by 50G12-L309W-E345R had an IC of killing against MOLP8 cells of 1.01. 50 was approximately 3.5 times higher than daratumumab, the absolute value of maximum killing activity (Top) was approximately 1.5 times higher than daratumumab, and IC 50 is about 3.3 times better than 50G12-E345R, and IC 50 is about 2.5 times better than the 50G12-L309W. [Table 12]

[0174] As shown in Figure 13, the killing activity of the ADCC effect mediated by 50G12-L309W-E345R against Raji cells was clearly better than that of the unmutated antibody 50G12-Hu-IgG1, the single-site mutant antibody 50G12-L309W, the single-site mutant antibody 50G12-E345R, and the control antibody daratumumab. As shown in Table 12, the ADCC effect mediated by 50G12-L309W-E345R showed an IC of killing against Raji cells. 50 was approximately 9.2 times more potent than daratumumab, the absolute value of maximum killing activity (Top) was approximately 1.3 times more potent than daratumumab, and IC 50 is about 2.7 times better than 50G12-E345R, and IC 50 is about three times better than the 50G12-L309W. [Table 13]

[0175] The above data indicate that simultaneous introduction of the L309W and E345R mutations into the heavy chain constant region of an antibody can effectively enhance antibody-mediated ADCC activity, and that this activity is superior to that of antibodies with only one L309W or E345R mutation or no mutations. Furthermore, the L309W and E345R mutations also exhibit a superior ADCC enhancement effect on NCI-H929, which is not sensitive to CDC (see reference: CD38 expression and complement inhibitors affect response and resistance to daratumumab therapy in myeloma [J]. Blood, 2016, 128(7):959-970.).

[0176] Example 9 Apoptosis-inducing effect of 50G12-L309W-E345R Log-phase human Burkitt's lymphoma cells, Daudi, were washed once with 2% FBS-containing RPMI-1640 medium and plated at a cell density of 1E5 / 150 μL per well in a 96-well cell culture plate. Antibodies were diluted to 12 nM in 2% FBS-containing RPMI-1640 medium, then serially diluted to 8 concentrations in a 3-fold gradient. 50 μL of the diluted antibodies were added to the 96-well cell culture plate at a final antibody concentration of 3 nM and incubated for 24 h in a cell incubator. Cells were washed twice with chilled PBS (200 μL per well) and centrifuged at 400 g for 5 min at 4°C. Apoptotic cells were stained with the FITC-labeled Annexin V Apoptosis Detection Kit. Cells were resuspended in 100 μL of 1x binding buffer and FITC-Annexin V was added at 1.5 μL per well. The mixture was gently mixed and incubated at room temperature for 15 min, protected from light. 100 μL of 1× binding buffer was added and mixed evenly. The mean fluorescence intensity of the FITC channel was measured within 1 hour using a flow cytometer (purchased from Beckman, model number Cytoflex). The data was analyzed and the number of stained cells was calculated. Data analysis and blotting were performed using GraphPad Prism 9, and IC50 was calculated.

[0177] As shown in Figure 14, the apoptotic effect of Daudi cells induced by 50G12-L309W-E345R was comparable to that of the unmutated antibody 50G12-Hu-IgG1, and both were significantly better than the control antibody daratumumab. As shown in Table 13, the maximum activity (Top) of apoptosis of Daudi cells induced by 50G12-L309W-E345R was approximately 2.1-fold higher than that of daratumumab. [Table 14]

[0178] 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 antibody or a fragment or fusion protein thereof having antigen-binding activity and CDC activity, comprising a binding functional domain targeting a predetermined antigen and a heavy chain Fc region element, wherein the heavy chain Fc region element is derived from IgG; (1) the amino acid residue at position 309 of the Fc region is W; and (2) the amino acid residue at position 345 of the Fc region is R; wherein the numbering of the amino acids in the heavy chain Fc region elements is according to the EU numbering system. An antibody, or a fragment thereof, or a fusion protein thereof, characterized in that:

2. The antibody, or fragment thereof, or fusion protein thereof according to claim 1, further having ADCC activity.

3. The antibody or fragment thereof or fusion protein according to any one of claims 1 to 2, wherein the heavy chain Fc region element comprises a tailpiece element at the C-terminus, and the sequence of the tailpiece element is the sequence shown in SEQ ID NO: 7 or 8.

4. The antibody, or fragment thereof, or fusion protein thereof according to any one of claims 1 to 3, wherein the binding functional domain targeting the predetermined antigen binds to an antigen molecule selected from the group consisting of CD38, CD3, CD47, CD19, CD20, HER2, EGFR, CD123, Glypican-3, CD25, Trop-2, EpCAM, or a combination thereof.

5. The antibody or fragment or fusion protein thereof according to any one of claims 1 to 4, characterized in that the heavy chain Fc region element has the amino acid sequence shown in SEQ ID NO:

34.

6. 6. The antibody, or fragment thereof, or fusion protein thereof according to any one of claims 1 to 5, characterized in that the antibody, or fragment thereof, or fusion protein thereof comprises a binding functional domain targeting CD38 and has an amino acid sequence selected from the following group: a heavy chain comprising a VH region containing H-CDR1 shown in SEQ ID NO: 35, H-CDR2 shown in SEQ ID NO: 36, and H-CDR3 shown in SEQ ID NO: 37, and a heavy chain constant region having a W or Y mutation at position 309 and an R mutation at position 345 of human IgG1 according to EU numbering, and a light chain comprising a VL region containing L-CDR1 shown in SEQ ID NO: 38, L-CDR2 shown in SEQ ID NO: 39, and L-CDR3 shown in SEQ ID NO: 40; or A heavy chain comprising a VH region containing H-CDR1 shown in SEQ ID NO: 11, H-CDR2 shown in SEQ ID NO: 12, and H-CDR3 shown in SEQ ID NO: 13, and a heavy chain constant region having a W or Y mutation at position 309 and an R mutation at position 345 of human IgG1 according to EU numbering; and a light chain comprising a VL region containing L-CDR1 shown in SEQ ID NO: 14, L-CDR2 shown in SEQ ID NO: 15, and L-CDR3 shown in SEQ ID NO:

16.

7. A method for improving CDC of an antibody, or a fragment thereof, or a fusion protein thereof, wherein the antibody, or a fragment thereof, or a fusion protein thereof comprises an Fc region of an immunoglobulin and a binding functional domain that targets a predetermined antigen, the method comprising the steps of: (S1a) introducing mutations at one or more amino acid residues into the antibody, or fragment or fusion protein thereof, wherein the mutations comprise mutating amino acid 309 to W and amino acid 345 to R in the Fc region of the IgG heavy chain; Here, the numbering of the amino acids in the heavy chain Fc region elements is according to the EU numbering system.

8. (S1b) The method described in claim 7, further comprising fusing a tailpiece element shown in sequence number 7 or 8 to the C-terminus of the Fc region.

9. A heavy chain Fc region element comprising: (1) comprising a W amino acid residue at position 309 of the Fc region; and (2) the Fc region contains an R amino acid residue at position 345; wherein the numbering of the amino acids in the heavy chain Fc region elements is according to the EU numbering system. A heavy chain Fc region element characterized by:

10. An isolated nucleic acid molecule characterized by encoding an antibody or fragment thereof or fusion protein described in any one of claims 1 to 6, or a heavy chain Fc region element described in claim 9.

11. An expression vector, characterized in that it contains the nucleic acid molecule of claim 10.

12. A host cell comprising the expression vector of claim 11.

13. A method for producing the antibody or fragment thereof or fusion protein according to any one of claims 1 to 6, or the heavy chain Fc region element according to claim 9, comprising the steps of: (a) expressing the antibody or fragment thereof or fusion protein or the heavy chain Fc region element by culturing the host cell of claim 12 under expression conditions; (b) isolating and purifying the antibody or fragment or fusion protein thereof described in (a) or the heavy chain Fc region element.

14. An immunoconjugate comprising: (a) an antibody, or a fragment or fusion protein thereof, according to any one of claims 1 to 6; and (b) a conjugate moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

15. A pharmaceutical composition comprising the antibody or fragment thereof or fusion protein thereof according to any one of claims 1 to 6, or the immune complex according to claim 14, and a pharmaceutically acceptable carrier.

16. Use of an antibody or fragment thereof or fusion protein according to any one of claims 1 to 6, or an immunoconjugate according to claim 14, or a pharmaceutical composition according to claim 15, in the manufacture of a drug for treating cancer or an immune-related disease.

17. 17. The use according to claim 16, characterized in that the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer, esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, multiple myeloma and other neoplastic malignancies.

18. 17. The use according to claim 16, wherein the immune-related disease is an autoimmune disease.

19. The autoimmune disease is selected from the group consisting of autoimmune kidney disease, lupus erythematosus, systemic lupus erythematosus (SLE), Sjogren's syndrome, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, surgical adhesions, stroke, type 1 diabetes, and Lyme disease. disease), meningoencephalitis, autoimmune uveitis, multiple sclerosis, Guillain-Barr syndrome, atopic dermatitis, autoimmune hepatitis, ankylosing spondylitis, fibrosing alveolitis, Graves' disease, idiopathic thrombocytopenic purpura (ITP), Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis 17. The use according to claim 16, characterized in that the conditions treated are selected from the group consisting of: inflammatory bowel disease, gastritis, inflammatory bowel disease, inflammatory bowel disease (IGD), inflammatory bowel disease (IGN ...

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