Anti-CD40L antibodies and their use in the treatment of human autoimmune diseases

Abstract

The present invention relates to novel antibodies and antibody fragments that specifically bind to CD40L, and to compositions comprising such antibodies or antibody fragments. Furthermore, the present invention relates to nucleic acids encoding such antibodies or antibody fragments, host cells containing them, and related uses. Moreover, the present invention relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments.

Description

[Technical Field]

[0001] The present invention relates to novel antibodies and antibody fragments that specifically bind to CD40L, and compositions comprising such antibodies or antibody fragments. The present invention further relates to nucleic acids encoding such antibodies or antibody fragments, host cells containing them, and related uses. Furthermore, the present invention relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments. [Background technology]

[0002] CD40 ligand (CD40L), also known as CD154, gp39, TNF receptor-associated protein (TRAP), 5c8 antigen, or T-BAM, is a full-length 39 kDa type II transmembrane protein. CD40L belongs to the tumor necrosis factor (TNF) superfamily and exists on the cell membrane in the form of a homotrimer. It is primarily expressed on activated T cells, but is also present on other types of immune and non-immune cells, such as epithelial cells, monocytes, dendritic cells, fibroblasts, smooth muscle cells, endothelial cells, and platelets. The CD40L / CD40 signaling pathway mediates various immune and inflammatory response processes and plays a crucial role in normal immune responses. Previous studies have revealed that the CD40L / CD40 signaling pathway not only mediates signaling between various lymphocytes but is also involved in interactions between non-immune cells. Therefore, the CD40L / CD40 signaling pathway plays a crucial pathogenic role in various chronic inflammatory diseases, such as autoimmune diseases, neurodegenerative diseases, graft-versus-host diseases, tumors, and atherosclerosis.

[0003] Currently, several antibodies or protein drugs targeting CD40 / CD40L have shown clinically active therapeutic effects. The early anti-CD40L antibody Ruplizumab (CN101072587B) showed excellent therapeutic effects in diseases such as systemic lupus erythematosus and lupus nephritis, but its development was halted due to serious safety events such as thrombosis (Nat. Med. 6(2)(2000)114). Subsequent studies have shown that thrombotic events are mainly caused by the formation of immune complexes between free CD40L and the antibody, followed by the interaction of C1q and FcgR, particularly FcgRIIa expressed on platelets, which induces platelet activation and aggregation. Therefore, completely eliminating the effector function of the Fc region is crucial for the safety of anti-CD40L antibodies.

[0004] Subsequently, anti-CD40L antibodies that entered clinical trials all had modified Fc regions, and the main technical routes for this modification can be divided into two types. One is to weaken the effector function of the Fc region through mutation, and the other is to completely remove the Fc region and extend the molecular half-life using other technical means. However, both have their problems. In the first technical route, using frexalimab (AU2016294417B2) as an example, the effector function of the Fc region was weakened using C220S / C226S / C229S / P238S and E269R / K322A mutations, respectively. However, subsequent studies have shown that none of these mutations can completely eliminate the effector function of Fc, and therefore both carry a certain level of safety risk. The second technical route, exemplified by Dapirolizumab pegol (US8293237B2) and Dazodalibep (CN101679521B), involves Dapirolizumab pegol, which retains only monovalent anti-CD40L Fab and extends its half-life through crosslinking with PEG. However, this monovalent molecular form weakens the molecule's biological activity, and the PEG crosslinking increases the difficulty of reaching the molecule's critical micelle concentration (CMC). Dazodalibep uses a different antibody-type Tn3 backbone protein as a target molecule to inhibit CD40L and extends its half-life by fusing it with albumin. However, there are certain safety risks, as molecules of this form have been clinically shown to be highly immunogenic.

[0005] Therefore, there is a demand for drugs that target CD40 / CD40L, particularly antibody drugs, that offer better safety, maintain the pharmacokinetic profile of antibody drugs, and simultaneously possess stronger biological activity. [Overview of the project]

[0006] This invention provides antibodies or fragments (e.g., antigen-binding fragments) that target CD40L. Specifically, this invention obtained novel and highly affinity CD40L-targeting antibodies by screening using mouse hybridoma technology. Furthermore, this invention further provides mutation technology and mutants produced using this technology that retain the conventional IgG molecular form while completely removing effector functions (ADCC, ADCP, CDC) mediated by the Fc region, thereby reducing safety risks. Specifically, this invention provides antibodies that target CD40L using different Fc mutation technologies (mutation sites L234A / L235A / ΔP329).

[0007] The antibody in the embodiment of the present invention exhibits improved safety. Specifically, this antibody uses the molecular form of IgG, and through Fc mutation technology, the molecule completely eliminates the ability of the Fc region to bind to all FcγRs while retaining affinity for FcRn. Therefore, this antibody and its fragments reduce the immunogenicity and safety risks of the molecule while retaining the excellent pharmacokinetic profile of the antibody molecule.

[0008] In addition, the antibodies in the embodiments of the present invention exhibit even stronger biological activity. Specifically, the high-affinity antibodies and fragments of CD40L obtained by screening can more effectively block the CD40L / CD40 signaling pathway and / or the biological functions mediated thereby. Therefore, these antibodies and fragments exhibit superior biological activity.

[0009] In some embodiments, the present invention relates to an anti-CD40L antibody or its antigen-binding fragment, wherein the antibody is (i) The three complementarity determination regions HCDR1, HCDR2 and HCDR3 included in VH shown in Sequence ID No. 4, and the three complementarity determination regions LCDR1, LCDR2 and LCDR3 included in VL shown in Sequence ID No. 10, or (ii) Includes the three complementarity determination regions HCDR1, HCDR2 and HCDR3 included in VH shown in Sequence ID No. 16, and the three complementarity determination regions LCDR1, LCDR2 and LCDR3 included in VL shown in Sequence ID No. 21.

[0010] For example, HCDR1 is determined according to a combination of Kabat and Chothia, and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are determined according to Kabat.

[0011] In some embodiments, the present invention relates to an anti-CD40L antibody or its antigen-binding fragment comprising a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), a third heavy chain complementarity-determining region (HCDR3), and a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3), wherein, (i) The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 each contain the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or each consists of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, (ii) HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 each contain the amino acid sequences shown in SEQ ID NOs. 13, 14, 15, 18, 19, and 20, respectively, or consist of the amino acid sequences shown in SEQ ID NOs. 13, 14, 15, 18, 19, and 20, respectively.

[0012] In some embodiments, the present invention relates to an anti-CD40L antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH), wherein the heavy chain variable region comprises or consists of an amino acid sequence having at least 90% identity with an amino acid sequence selected from SEQ ID NO: 4 or 16, or comprises or consists of an amino acid sequence selected from SEQ ID NO: 4 or 16.

[0013] In some embodiments, the present invention relates to an anti-CD40L antibody or an antigen-binding fragment thereof comprising a light chain variable region (VL), wherein the light chain variable region comprises or consists of an amino acid sequence having at least 90% identity with an amino acid sequence selected from SEQ ID NO: 10 or 21, or comprises or consists of an amino acid sequence selected from SEQ ID NO: 10 or 21.

[0014] In some embodiments, the present invention relates to an anti-CD40L antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein (i) The heavy chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, (ii) The heavy chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0015] In some embodiments, the present invention relates to an anti-CD40L antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein (i) The heavy chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 4, and the light chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 10, or (ii) The heavy chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 21.

[0016] In some embodiments, the present invention relates to an anti-CD40L antibody or its antigen-binding fragment comprising an Fc region. In some embodiments, the present invention relates to an anti-CD40L antibody or an antigen-binding fragment thereof, wherein the Fc region is derived from human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc.

[0017] In some embodiments, the present invention relates to an anti-CD40L antibody or its antigen-binding fragment, wherein the Fc region includes (one or more) mutations that reduce or eliminate effector function, and / or (one or more) mutations that eliminate binding to FcγR and maintain binding affinity to FcRn. In some embodiments, the mutations are L234A / L235A and P329 deletion mutations. In some embodiments, the Fc region is (i) containing or consisting of amino acid sequence number 23 or 24, (ii) an amino acid sequence having at least 90% identity with amino acid sequence SEQ ID NO: 23, for example 95%, 96%, 97%, 99%, or higher identity, or (iii) an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity, with amino acid sequence SEQ ID NO: 24, and comprising the L234A / L235A and P329 deletion mutations.

[0018] In some embodiments, the invention relates to an anti-CD40L antibody or an antigen-binding fragment thereof comprising a heavy chain constant region, wherein the heavy chain constant region is derived from the constant region of IgG1, IgG2, IgG3 or IgG4. Preferably, the heavy chain constant region (i) comprises, or consists of, an amino acid sequence selected from SEQ ID NO: 5 or 26, (ii) comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 26, or (iii) comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 5, and having the L234A / L235A and P329 deletion mutations.

[0019] In some embodiments, the invention relates to an anti-CD40L antibody or an antigen-binding fragment thereof comprising a light chain constant region, wherein the light chain constant region is a lambda or Kappa light chain constant region. Preferably, the light chain constant region (i) comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 11, or consists of said amino acid sequence, or (ii) comprises, or consists of, the amino acid sequence of SEQ ID NO: 11.

[0020] In some embodiments, the invention relates to an anti-CD40L antibody or an antigen-binding fragment thereof comprising a heavy chain, wherein the heavy chain (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence selected from SEQ ID NO: 6 or 17, or consisting of said amino acid sequence, or (ii) comprising the amino acid sequence selected from SEQ ID NO: 6 or 17, or consisting of said amino acid sequence.

[0021] In some embodiments, the present invention relates to an anti-CD40L antibody or an antigen-binding fragment thereof comprising a light chain, wherein said light chain is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence selected from SEQ ID NO: 12 or 22, or consisting of said amino acid sequence, or (ii) comprising the amino acid sequence selected from SEQ ID NO: 12 or 22, or consisting of said amino acid sequence.

[0022] In some embodiments, the present invention relates to an anti-CD40L antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein (i) said heavy chain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 6, or consisting of said amino acid sequence, and said light chain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 12, or consisting of said amino acid sequence, (ii) said heavy chain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 17, or consisting of said amino acid sequence, and said light chain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 22, or consisting of said amino acid sequence.

[0023] In some embodiments, the present invention relates to an anti-CD40L antibody or its antigen-binding fragment comprising a heavy chain and a light chain, wherein (i) The heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 6, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 12. (ii) The heavy chain comprises the amino acid sequence of SEQ ID NO: 17 or consists of the earlier sequence, and the light chain comprises the amino acid sequence of SEQ ID NO: 22 or consists of the earlier sequence.

[0024] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody or a chimeric antibody. In some embodiments, the antigen-binding fragment is an antibody fragment selected from Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibody, single-chain antibody (e.g., scFv), single-structure domain antibody (sdAb) (e.g., VHH), bivalent antibody or fragment thereof, or camel antibody or diabody.

[0025] In some embodiments, the anti-CD40L antibody or its antigen-binding fragment is a) It binds with high affinity to CD40L (e.g., human CD40L), b) Inhibiting the binding of CD40 and CD40L, c) Inhibiting the CD40 / CD40L signaling pathway, d) Inhibiting the activation or proliferation of B cells, e) Inhibiting the proliferation of B cells, f) Inhibiting the maturation and differentiation of B cells, for example, inhibiting the differentiation of B cells into plasma cells, g) Inhibiting IL-12 secretion by dendritic cells, h) It does not cause platelet activation, i) To treat or prevent autoimmune diseases, such as multiple sclerosis (MS), and / or j) For example, inhibiting antibody production to inhibit an immune response, preferably a humoral immune response, k) Having one or more properties of improving pharmacokinetics, preferably having an in vivo half-life of about 200 hours or more, more preferably about 210, 220, 230, 240, 250, 260, 270, 280 or 290 hours or more.

[0026] In some embodiments, the present invention relates to isolated nucleic acids encoding an anti-CD40 antibody or an antigen-binding fragment thereof. In some embodiments, the present invention relates to a vector comprising the nucleic acid, preferably an expression vector.

[0027] In some embodiments, the present invention relates to a host cell containing the nucleic acid or vector of the present invention. Preferably, the host cell is a prokaryotic or eukaryotic cell, and more preferably selected from yeast cells, mammalian cells (e.g., 293 cells or CHO cells, e.g., CHO-K cells or HEK293 cells) or other cells suitable for the preparation of antibodies or their antigen-binding fragments.

[0028] In some embodiments, the present invention relates to a method for preparing an anti-CD40L antibody or its antigen-binding fragment, wherein the method is l) Culturing host cells of the present invention under conditions suitable for the expression of nucleic acids encoding the anti-CD40L antibody or its antigen-binding fragment, m) Optionally, the antibody or its antigen-binding fragment is separated, n) optionally the method further comprises recovering the anti-CD40L antibody or its antigen-binding fragment from the host cells, optionally comprising purifying the antibody, for example, by Protein A.

[0029] In some embodiments, the present invention relates to an immunoconjugate, which includes an anti-CD40L antibody or its antigen-binding fragment and other substances, such as therapeutic agents, such as cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., immunoinhibitors), that can produce a biological effect. In some embodiments, the present invention relates to a drug composition comprising the anti-CD40L antibody or its antigen-binding fragment or the immunoconjugate of the present invention and an adjuvant that is optionally acceptable as a pharmaceutically acceptable agent.

[0030] In some embodiments, the present invention relates to a combination drug product comprising the anti-CD40L antibody or its antigen-binding fragment or immunoconjugate of the present invention and one or more other therapeutic agents, including but not limited to cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., immunoinhibitors), that produce a biological effect. In some embodiments, the present invention relates to a method for preventing or treating a disease or disorder associated with inappropriate activation of the CD40L / CD40-mediated pathway in an individual, the method comprising administering to the subject an effective amount of the anti-CD40L antibody or its antigen-binding fragment or immunoconjugate or drug composition or combination product of the present invention.

[0031] In some embodiments, the subject has abnormal activation of CD40L / CD40-mediated signaling pathways and / or excessive production and deposition of anti-autoantibodies compared to a healthy individual, and / or increased expression of CD40L or CD40 in the subject's cells (e.g., activated T cells or other types of immune cells or non-immune cells, e.g., epithelial cells, monocytes, dendritic cells, fibroblasts, smooth muscle cells, endothelial cells, and platelets) compared to, for example, the corresponding cells of a healthy individual. In some embodiments, the disease or disorder is selected from autoimmune diseases, lupus nephritis, immune thrombocytopenic purpura (ITP), transplant rejection, Crohn's disease, inflammatory bowel disease (IBD), colitis, asthma / allergy, atherosclerosis, myasthenia gravis, multiple sclerosis, psoriasis, rheumatoid arthritis, ankylosing spondylitis, coronary artery disease, type 1 diabetes mellitus, amyotrophic lateral sclerosis (ALS), and immune responses to recombinant drug products, such as factor VII for hemophilia. [Brief explanation of the drawing]

[0032] [Figure 1] This invention demonstrates that the antibody of the present invention inhibits the binding of CD40 and CD40L at the molecular level. [Figure 2] This invention demonstrates that the antibody of the present invention inhibits the activation of downstream signaling in CD40-overexpressing Jurkat cells at the cellular level. [Figure 3] This invention demonstrates inhibition of CD86 expression, a B cell activation marker, by the antibody of the present invention. [Figure 4] This paper demonstrates the effect of the antibody of the present invention on the proliferation of B cells isolated in vitro. [Figure 5] This demonstrates the effect of the antibody of the present invention on the differentiation of B cells into plasma cells. [Figure 6] This paper demonstrates the effect of the antibody of the present invention on IL-12 secreted by moDC cells. [Figure 7] This invention demonstrates that the antibody does not cause platelet activation in in vitro experiments. [Figure 8] This invention demonstrates that the antibody does not cause platelet activation in in vitro experiments. [Figure 9] This invention demonstrates that the antibody does not cause platelet activation in in vitro experiments. [Figure 10] The data from the present invention in an experimental autoimmune encephalomyelitis (EAE) mouse model are shown. [Figure 11] The data from the present invention in an experimental autoimmune encephalomyelitis (EAE) mouse model are shown. [Figure 12]The data from the present invention in an experimental autoimmune encephalomyelitis (EAE) mouse model are shown. [Figure 13] This invention demonstrates that the antibody of the present invention inhibits the production of anti-keyholrin pethemocyanin antibody in mice. [Figure 14] The pharmacokinetics of the antibody of the present invention in mice are shown. [Modes for carrying out the invention]

[0033] I. Definition Before detailing the present invention below, it should be understood that the present invention is not limited to the specific methodologies, schemes, and reagents described herein, as these are modifiable. Furthermore, it should be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the invention, but only to the scope of the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0034] For the purposes of interpreting this specification, the following definitions shall be used, and wherever used in the singular form, terms may also include plural forms, and vice versa. It should be understood that the terms used herein are intended solely to describe, and not to limit, specific embodiments. When used with a number, the term "approximately" is intended to refer to a range of numbers that has a lower limit of 5% less than the specified number and an upper limit of 5% more than the specified number.

[0035] As used herein, the terms “and / or” mean one of the options, or two or more or all of the options. As used herein, the terms “contains” or “includes” are intended to include the elements, integers, or steps described, but not to exclude other elements, integers, or steps. Where the terms “contains” or “includes” are used herein, unless otherwise indicated, they also encompass the combinations of elements, integers, or steps mentioned. For example, when referring to an antibody variable region that “contains” a specific sequence, it is also intended to encompass the antibody variable region consisting of that specific sequence.

[0036] The term "CD40L" refers to a ligand expressed on activated T cells. Other names known in the art include CD154, CD40 ligand (CD40L), CD40 counterreceptor (CD40CR), gp39, T-BAM, T-cell activating molecule, TRAF, TNF-related activating protein (TRAP), and member 5 of the tumor necrosis factor ligand superfamily (TNFSF5). These terms may be used interchangeably throughout the application. In some embodiments, the amino acid sequence of CD40L is as shown in Uniport P29965. In some embodiments, CD40L includes the sequence shown in Sequence ID No. 27.

[0037] The terms "whole antibody" or "full-length antibody" are interchangeable herein and refer to antibody molecules having a natural immunoglobulin molecular structure. In the case of a conventional quadruple-chain IgG antibody, a full-length antibody consists of two heavy chains (H) and two light chains (L) linked to each other by disulfide bonds. In the case of a heavy-chain antibody that has only a heavy chain but no light chain, a full-length antibody consists of two heavy chains (H) linked to each other by disulfide bonds. Compared to a conventional quadruple-chain IgG antibody, the heavy chain of a full-length antibody usually consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, where the heavy chain constant region contains at least three structural domains CH1, CH2, and CH3. The light chain of a full-length antibody consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region, where the light chain constant region consists of the structural domain CL. Each heavy chain variable region (VH) and each light chain variable region consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The term "antibody fragment" includes a portion of a complete antibody. In preferred embodiments, the antibody fragment is an antigen-binding fragment.

[0038] The term "antigen-binding fragment" of an antibody refers to a molecule distinct from the full-length antibody, which contains a portion of the full-length antibody but can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody derived from the antigen-binding fragment) for the antigen. Antigen-binding fragments can be prepared by recombinant DNA technology or by enzymatically or chemically cleaving the complete antibody. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibodies), linear antibodies, single-chain antibodies (e.g., scFv), and single-structure domain antibodies (sdAb) e.g., VHH, bivalent antibodies or fragments thereof, or camel antibodies or diabodies. For example, Fab fragments can be obtained from the digestion of a full-length antibody with papain enzyme. Furthermore, digesting a complete antibody under the disulfide bond of the hinge region with pepsin produces F(ab')2, a dimer of Fab', which is a bivalent antibody fragment. F(ab')2 can be reduced by disrupting the disulfide bond in the hinge region under neutral conditions, thereby converting the F(ab')2 dimer into the Fab' monomer. The Fv fragment consists of the VL and VH structural domains of the antibody single arm. The two structural domains of the Fv fragment, VL and VH, can be encoded by separate genes, but these two structural domains can be linked using a synthetic linker peptide and produced as a single protein chain, and the VL and VH regions in this single protein chain can be paired to form a single-stranded Fv(scFv).

[0039] "Fab fragment" or "Fab" is interchangeable herein and refers to an immunoglobulin fragment comprising two polypeptide chains, an immunoglobulin heavy chain variable structure domain VH, a heavy chain constant structure domain CH1, and a light chain variable structure domain VL and a light chain constant structure domain CL, wherein one polypeptide chain comprises VH and a constant region selected from CH1 and CL from the N-terminus to the C-terminus, and the other polypeptide chain comprises VL and another constant region selected from CL and CH1 from the N-terminus to the C-terminus, wherein the VH structural domain and the VL structural domain are paired to form an antigen-binding site. herein, a Fab polypeptide chain comprising the heavy chain constant region CH1 is also called a "Fab heavy chain," and accordingly, a Fab polypeptide chain comprising the light chain constant region CL is also called a "Fab light chain."

[0040] A "diabody" is a bivalent miniature antibody constructed by gene fusion, for example, a dimer consisting of two polypeptide chains. The VL and VH domains of each polypeptide chain in a diabody are linked by a linker, so that the VL and VH encoded by the same polypeptide chain form a dimer with different single-stranded variable region fragments. Diabodies generally have two antigen-binding sites.

[0041] The "complementarity-determining region," "CDR region," or "CDR" is a region within the antibody's variable structure domain that exhibits high sequence variability and contains structurally determined loops ("hypervariable loops") and / or antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. CDRs located within the antibody's heavy chain variable structure domain are called HCDR1, HCDR2, and HCDR3, while CDRs located within the antibody's light chain variable structure domain are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundary of each CDR can be determined by one or a combination of several well-known antibody CDR assignment schemes, such as Chothia (Chothia et al. (1989) Nature 342:877-883), Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USD Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), and International ImMunoGeneTics. This includes the database (IMGT) (available on the World Wide Web at http: / / imgt.cines.fr / ) and a North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0042] Unless otherwise specified, the term "CDR" or "CDR sequence" in this invention encompasses CDR sequences determined by any one of the above methods or combinations thereof. A CDR may be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any one of the exemplary CDRs of this invention). Unless otherwise specified, when referring to residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues) in this invention, the numbering position is based on the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0043] In some embodiments, the CDR of the heavy chain variable region of the antibody in the present invention is determined according to Kabat, Chothia, or a combination of Kabat and Chothia. In some embodiments, the CDR of the light chain variable region of the antibody in the present invention is determined according to Kabat.

[0044] In some embodiments, the heavy chain variable region CDR1 of the antibody in the present invention is determined based on a Kabat-Chothia combination scheme (hereinafter referred to as "Kabat-Chothia combination," corresponding to H26-H32 in the Kabat numbering system), and the heavy chain variable regions CDR2 and CDR3, and the light chain variable regions LCDR1, LCDR2 and LCDR3 are determined according to the Kabat scheme.

[0045] [Table 1]

[0046] An antibody that "binds to the same or overlapping epitopes as the reference antibody" refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competitive assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of this antibody to its antigen in a competitive assay. A reference antibody and an antibody that competes for the binding of its antigen refer to antibodies that block 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competitive assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of this antibody to its antigen in a competitive assay. Many types of competitive binding assays are used to determine whether an antibody competes with other antibodies, and these assays include, for example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), and sandwich competitive assays.

[0047] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. Conversely, the reference antibody inhibits the binding of this antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. The binding of an antibody to its antigen can be measured by its affinity (e.g., equilibrium dissociation constant). Methods for measuring affinity are known in this field. An antibody exhibiting the same or similar binding affinity and / or specificity as the reference antibody refers to an antibody that can have at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding affinity and / or specificity of the reference antibody. This can be measured by any method known in the art for measuring binding affinity and / or specificity.

[0048] The term "chimeric antibody" refers to an antibody molecule in which (a) the antigen-binding site is linked to a different or altered category, effector function, and / or species of constant region or an entirely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) by modifying, substituting, or exchanging the constant region or part thereof, thereby giving the chimeric antibody new properties; or (b) the variable region or part thereof, or a variable region having different or altered antigen specificity. For example, a mouse antibody can be modified by exchanging its constant region for a constant region derived from human immunoglobulin. Because it is exchanged for a human constant region, this chimeric antibody retains its specificity for recognizing the antigen and may have lower immunogenicity in humans compared to the original mouse antibody.

[0049] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a mouse monoclonal antibody) while exhibiting relatively low immunogenicity when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining parts of the antibody with the corresponding parts of the human antibody (for example, replacing parts of the constant and variable regions that do not participate in binding with the corresponding parts of the human antibody).

[0050] The terms “Fc structural domain” or “Fc region” are used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least some of the constant regions. This term includes both the native sequence Fc region and the mutant Fc region. A “Fc structural domain” of a native immunoglobulin includes two or three constant structural domains, namely the CH2 structural domain, the CH3 structural domain, and an optional CH4 structural domain. For example, in a native antibody, the immunoglobulin Fc structural domain includes the second and third constant structural domains (CH2 and CH3 structural domains) of two heavy chains derived from IgG, IgA, and IgD antibodies, or the second, third, and fourth constant structural domains (CH2, CH3, and CH4 structural domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is based on the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interests, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991, NIH Publication 91-3242. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions can undergo dimerization to form a dimer Fc, and two different Fc heterodimerizations can form a heterodimer Fc. In this specification, the terms “Fc region,” “Fc portion,” and “dimer Fc” do not include the heavy chain variable region VH and light chain variable region VL and the heavy chain constant region CH1 and light chain constant region CL of immunoglobulins, but may optionally include the hinge region or a portion of the hinge region at the N-terminus of the heavy chain constant region, for example, the IgG1 hinge region or a portion of the IgG1 hinge region, for example, sequences numbered D221 to P230 based on EU. In one embodiment, the human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from Asp231 to the carboxyl terminus of the heavy chain.

[0051] In one embodiment, the human IgG1 Fc region polypeptide (including the hinge region) is DKTHTCPPCPA It contains the amino acid sequence PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 23, the underlined part is the hinge region). The term "effector function" refers to the functional ability of an antibody's Fc or constant region to bind to proteins and / or cells of the immune system.

[0052] The terms “amino acid substitution” or “amino acid substitution” are interchangeable herein and refer to the substitution of at least one amino acid residue in a predetermined parent amino acid sequence with a different “substitution” amino acid residue. This substitution residue or set of residues may be “naturally occurring amino acid residues” (i.e., encoded by the genetic code) and may be selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile):leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). The amino acid substitution definitions herein further encompass one or more unnaturally occurring amino acid residue substitutions. “Unnaturally occurring amino acid residues” refers to residues that can covalently bond to adjacent amino acid residues in a polypeptide chain, in addition to the naturally occurring amino acid residues described above. Examples of unnaturally occurring amino acid residues include n-leucine, ornithine, n-valine, homoserine, Aib, and other amino acid residue analogues.

[0053] In some embodiments, amino acid substitutions are indicated as (original amino acid, amino acid position, mutant amino acid). For example, if the substitution site is located in the C region, "L234A" means that leucine (L) at EU position 234 is replaced with alanine (A). When a combination of mutations is described, the mutations in the combination are linked by a slash. "L234A / L235A" indicates that the mutant contains both mutations L234A and L235A simultaneously. In some embodiments, amino acid deletions are indicated by △+amino acid+mutation site, for example, △P329 indicates a deletion of proline at position 329. Amino acid deletions may also be indicated by "amino acid+mutation site+deletion", for example, a deletion of proline at position 329 may be written as "P329 deletion".

[0054] As described herein, “conservative modifications” include substitutions, deletions, or additions to polypeptide sequences that do not substantially alter the desired functional activity of the polypeptide sequence. In some embodiments, a conservative modification is a conservative substitution. A conservative substitution is when an amino acid is substituted for another amino acid within the same category, for example, an acidic amino acid being substituted for another acidic amino acid, a basic amino acid being substituted for another basic amino acid, or a neutral amino acid being substituted for another neutral amino acid. For example, in conservative substitutions, one amino acid is often substituted for a chemically similar amino acid. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. The following are eight pairs of amino acids that contain conserved substitutions with each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term “conservative modification” refers to modifying amino acids in a way that does not significantly affect or alter the target antigen-binding characteristics of the antibody molecule of the present invention, including the amino acid sequence, when applied to the amino acid sequence of an antibody molecule. For example, the conservative modifications of the variant maintain a binding affinity of at least 80%, 85%, 90%, 95%, 98%, 99%, or even 100-110% or more to the target antigen compared to the parent antibody.

[0055] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid bound to it. This term includes vectors as self-replicating nucleic acid structures and vectors bound to the genome of a host cell into which they are introduced. Some vectors can induce the expression of a nucleic acid operably bound to them. Such vectors are referred to herein as “expression vectors.” An "immunoconjugate" refers to an antibody bound to one or more other substances (including, but not limited to, labels).

[0056] As used herein, the term “therapeutic agent” encompasses all agents effective in preventing or treating diseases associated with inappropriate activation of the CD40L / CD40-mediated pathway, such as agents that produce a biological effect. These agents include, but are not limited to, cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., immunoinhibitors). The term "small molecule drugs" refers to low molecular weight organic compounds that can modulate biological processes. "Low molecules" are defined as molecules with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 kD. Low molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, low molecules have higher cell permeability, are less susceptible to degradation, and are less likely to trigger an immune response than larger molecules.

[0057] As used herein, the term “immunomodulator” refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. An immune response may be a humoral or cellular response. Immunomodulators include immunoinhibitors. As used herein, "immunoinhibitor," "immunosuppressant drug," or "immunosuppressant" refers to a therapeutic agent used to inhibit or block the activity of the immune system in immunosuppressive therapy. The term "effective dose" refers to such an amount or dosage of the antibody, fragment, conjugate, composition, or combination of the present invention, after which a desired effect is produced in a patient requiring treatment or prevention after a single or multiple doses have been administered to the patient.

[0058] The “therapeutic dose” refers to the amount that effectively achieves the desired therapeutic outcome while maintaining the required duration of action at the required dosage. The therapeutic dose is the amount in which any toxic or adverse effects of the antibody or antibody fragment or its conjugate or composition or combination outweigh the therapeutic benefits. For an untreated subject, the “therapeutic dose” preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 20%, more preferably at least about 40%, and even more preferably at least about 50%, 60%, or 70%. In some embodiments, the term “therapeutic dose” as used herein is intended to specify the amount required for a therapeutic plan to treat a condition (e.g., autoimmune disease, allergy, inflammation, GVHD, or transplantation) or to reduce or eliminate an immune response to an antigen (e.g., autoantigen, allergen, pro-inflammatory agent, transplanted cell, tissue, or organ) or an antidrug (e.g., biological agents, e.g., therapeutic antibodies, Ig fusion proteins, hormones, growth factors, or other therapeutic proteins or polypeptides), particularly a T-cell or B-cell antibody response.

[0059] The “preventive effective dose” refers to the amount that effectively achieves the desired preventive outcome while maintaining the required dose and time frame. Typically, the preventive effective dose is less than the therapeutic effective dose because the preventive dose is used earlier in the patient's disease or at an earlier stage of the disease. In some embodiments, the term “preventive effective dose” as used herein is intended to identify the amount required for a treatment plan to prevent the progression and symptoms of a condition or disease (e.g., autoimmune disease, allergy, inflammation, GVHD or anti-transplant, drug or rejection reaction). The terms “host cell,” “host cell line,” and “host cell culture” are interchangeable and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include “transformed cells” and “transformed cells,” which, regardless of passage number, include the initially transformed cells and their offspring. Offspring may not be exactly the same as the parent cells in terms of nucleic acid content, but may contain mutations. This specification includes mutant offspring having the same function or biological activity as those screened or selected from the initially transformed cells.

[0060] As used herein, the term “labeling” refers to a compound or composition that is directly or indirectly bound or fused to a reagent (e.g., a polynucleotide probe or antibody) and facilitates the detection of the bound or fused reagent. The labeling itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or may catalyze the chemical modification of a substrate compound or composition that becomes detectable when labeled by an enzyme catalyst. The term is intended to encompass both directly labeling a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirectly labeling a probe or antibody by reacting it with another reagent that is directly labeled.

[0061] "Individual" or "Subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human. A “subject / patient / individual sample” is a set of cells or fluids obtained from a patient or subject. The origin of tissue or cell samples may be solid tissues such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, interstitial fluid; and cells from any stage of pregnancy or development of the subject. Tissue samples may contain compounds that are not naturally present in tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, and antibiotics.

[0062] "Isolated" antibodies refer to antibodies isolated from components of the natural environment. In some embodiments, the antibodies are purified to a purity of 95% or 99% or higher, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). "Nucleic acid encoding an isolated anti-CD40L antibody or a fragment thereof" refers to one or more nucleic acid molecules that encode an antibody heavy chain or light chain (or a fragment thereof, e.g., a heavy chain variable region or a light chain variable region) and are contained in a single vector or separate vectors, as well as nucleic acid molecules that are present at one or more locations in a host cell.

[0063] The "percentage of amino acid sequence identity (%)" refers to the percentage of amino acid residues in the amino acid sequence of the candidate sequence that is the same as the percentage of amino acid residues in the specific amino acid sequence shown herein, after comparing the candidate sequence with the specific amino acid sequence shown herein and, if necessary, introducing gap vacancies to achieve the maximum percentage of sequence identity, and without considering conservative substitutions as part of the sequence identity. In some embodiments, the present invention considers mutations in the antibody molecule of the present invention, such mutations having substantial identity with respect to the antibody molecule and its sequence specifically disclosed herein, for example, identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more. Such mutations may include conservative modifications.

[0064] The term "pharmaceutical adjuvants" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, vectors, or stabilizers administered with a drug. The term "drug composition" refers to a composition in which the active ingredient contained herein exists in a form that enables the biological activity of the active ingredient contained herein to be effective, and which does not contain any other ingredient that is unacceptably toxic to the subject to which the composition is administered.

[0065] The term “drug combination” refers to either a non-fixed or fixed combination product, and includes, but is not limited to, cartridges and drug compositions. The term “non-fixed combination” refers to the administration of active ingredients (e.g., (i) an anti-CD40L antibody or a fragment thereof and (ii) another therapeutic agent) to a patient simultaneously in separate entities, without specific time constraints, or at the same or different time intervals, thereby providing an agent that prevents or treats two or more conditions at an effective level in vivo in the patient. In some embodiments, the anti-CD40L antibody or a fragment thereof and the other therapeutic agent used in a drug combination are administered at levels no higher than when used individually. The term “fixed combination” refers to the administration of two or more agents to a patient simultaneously as a single entity. Preferably, by selecting the doses and / or time intervals of the two or more agents, the combined effect of each part may be greater than the effect achievable by using any single component alone in treating a disease or disorder. Each component is presented in a separate formulation, which may be the same or different formulation forms.

[0066] The term "combination therapy" refers to the administration of two or more therapeutic agents or modes of treatment (e.g., radiotherapy or surgery) for the treatment of the diseases described herein. Such administrations include the administration of these therapeutic agents substantially simultaneously, for example, in a single capsule having a fixed proportion of the active ingredient. Alternatively, such administrations include the simultaneous administration of individual active ingredients contained in multiple or separate containers (e.g., tablets, capsules, powders, or liquids). Powders and / or liquids may be reconstituted or diluted to the required dose before administration. Such administrations further include the use of each type of therapeutic agent substantially simultaneously or sequentially at different times. In any case, the treatment plan provides a beneficial role for the combination of drugs in the treatment of the disorders or conditions described herein.

[0067] As used herein, “treatment” means delaying, interrupting, blocking, alleviating, stopping, reducing, or reversing the onset of any symptom, complication, or biochemical sign of a disease; relieving symptoms; or preventing or inhibiting further progression of a disease, condition, or disorder. As used herein, “prevention” includes inhibiting the onset or progression of a disease or disorder or the symptoms of a particular disease or disorder.

[0068] All publications, patent applications, patents, and other references mentioned herein are incorporated in their entirety by reference. In various embodiments of the present invention, any or all of the features described above and throughout this application may be combined. The materials, methods, and examples described herein are illustrative and not intended to be limiting. Other features, purposes, and advantages of the present invention will become apparent from this specification, the accompanying drawings, and the accompanying claims.

[0069] II. Antibodies In some embodiments, the anti-CD40L antibody of the present invention or its antigen-binding fragment binds to CD40L (e.g., human CD40L) with high affinity. In some embodiments, the binding affinity of the antibody of the present invention or its antigen-binding fragment to human CD40L is higher than that of known CD40L antibodies, such as INX-021 antibody (WO2017011544A1). In some embodiments, the antibody or antigen-binding fragment of the present invention binds to CD40L on the cell surface. In some embodiments, CD40L is expressed or overexpressed on the cell surface. In some embodiments, the cells are CHO cells that express or overexpress CD40L.

[0070] In some embodiments, the anti-CD40L antibody or its antigen-binding fragment of the present invention is a) It binds with high affinity to CD40L (e.g., human CD40L), b) Inhibiting the binding of CD40 and CD40L, c) Inhibiting the CD40 / CD40L signaling pathway, d) Inhibiting B cell activation, e) Inhibiting the proliferation of B cells, f) Inhibiting the maturation and differentiation of B cells, for example, inhibiting the differentiation of B cells into plasma cells, g) Inhibiting IL-12 secretion by dendritic cells, h) Not causing platelet activation (e.g., in vitro or in vivo), i) Treating or preventing immune diseases, such as multiple sclerosis (MS), j) Inhibiting an immune response, preferably a humoral immune response, by, for example, inhibiting antibody production, and / or k) Having one or more properties of improving pharmacokinetics, preferably having an in vivo half-life of about 200 hours or more, more preferably about 210, 220, 230, 240, 250, 260, 270, 280 or 290 hours or more.

[0071] In some embodiments, the anti-CD40L antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, derived from the heavy chain variable region. In some embodiments, the anti-CD40L antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, derived from the light chain variable region. In some embodiments, the anti-CD40L antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) derived from the heavy chain variable region and three complementarity-determining regions (LCDRs) derived from the light chain variable region.

[0072] In some embodiments, the anti-CD40L antibody or its antigen-binding fragment of the present invention includes a heavy chain variable region (VH). In some embodiments, the anti-CD40L antibody or its antigen-binding fragment of the present invention includes a light chain variable region (VH). In some embodiments, the anti-CD40L antibody or its antigen-binding fragment of the present invention includes a heavy chain variable region and a light chain variable region (VH). In some embodiments, the heavy chain variable region includes three complementarity-determining regions (CDRs), HCDR1, HCDR2, and HCDR3, derived from the heavy chain variable region. In some embodiments, the light chain variable region includes three complementarity-determining regions (CDRs), LCDR1, LCDR2, and LCDR3, derived from the light chain variable region. In some embodiments, the anti-CD40L antibody of the present invention or its antigen-binding fragment further comprises the heavy chain constant region HC of the antibody. In some embodiments, the anti-CD40L antibody of the present invention or its antigen-binding fragment further comprises the antibody light chain constant region LC. In some embodiments, the anti-CD40L antibody of the present invention or its antigen-binding fragment further comprises the heavy chain constant region HC and the light chain constant region LC.

[0073] In some embodiments, the heavy chain variable region of the present invention is (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from either SEQ ID NO: 4 or SEQ ID NO: 16, or consisting of such an amino acid sequence, (ii) comprising an amino acid sequence selected from either SEQ ID NO: 4 or SEQ ID NO: 16, or consisting of the said amino acid sequence, (iii) Having an amino acid sequence with one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from either SEQ ID NO: 4 or SEQ ID NO: 16, or consisting of such an amino acid sequence, preferably the amino acid modifications do not occur in the CDR region.

[0074] In some embodiments, the light chain variable region of the present invention is (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from either SEQ ID NO: 10 or SEQ ID NO: 21, or consisting of such an amino acid sequence, (ii) comprising an amino acid sequence selected from either SEQ ID NO: 10 or SEQ ID NO: 21, or consisting of the said amino acid sequence, (iii) Having an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from either SEQ ID NO: 10 or SEQ ID NO: 21, or consisting of such an amino acid sequence, preferably the amino acid modifications do not occur in the CDR region.

[0075] In some embodiments, the heavy chain variable region of the present invention is derived from three complementarity determination regions (HCDRs), HCDR1, HCDR2 and HCDR3, (i) The three complementarity determination regions HCDR1, HCDR2 and HCDR3 included in VH as shown in Sequence ID No. 4 or 16, (ii)(i)(ii)(ii)(ii)(ii)(ii)(ii)(ii)(ii)(ii)(ii)(ii)(ii))))))

[0076] For example, here, the HCDR is determined according to any scheme for determining the CDR, which can be determined according to, for example, the Kabat, AbM, Chothia, Contact, or IMGT scheme or a combination thereof. For example, HCDR1 is determined according to a combination scheme of Kabat and Chothia, and HCDR2 and HCDR3 are determined according to a Kabat scheme.

[0077] In some embodiments, the light chain variable region of the present invention is derived from three complementarity determination regions (LCDRs), where LCDR1, LCDR2 and LCDR3 are (i) Three complementarity determination regions LCDR1, LCDR2 and LCDR3 included in the VL shown in Sequence ID No. 10 or 21, or (ii)(i)(ii)(ii)(ii)(ii)(ii)(ii)(ii)(ii)(ii)(ii)(ii)(ii))))))

[0078] For example, here, the LCDR is determined according to any scheme for determining the CDR, which can be determined according to, for example, the Kabat, AbM, Chothia, Contact, or IMGT scheme or a combination thereof, respectively. For example, the LCDR1-3 are determined according to the Kabat scheme.

[0079] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1 or 13, or HCDR1 comprises an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 1 or 13. In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 14, or comprises an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 2 or 14.

[0080] In some embodiments, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 15, or comprises an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 3 or 15. In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 18, or LCDR1 comprises an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 7 or 18.

[0081] In some embodiments, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 8 or 19, or LCDR2 comprises an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 8 or 19. In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 9 or 20, or LCDR3 comprises an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 9 or 20.

[0082] In some embodiments, the heavy chain constant region of the antibody of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, preferably the heavy chain constant region of IgG1. In some embodiments, the light chain constant region of the antibody of the present invention is the lambda or kappa light chain constant region, preferably the kappa light chain constant region. In some embodiments, the heavy chain constant region of the antibody of the present invention includes a mutant Fc region. Therefore, the present invention further relates to the mutant Fc region.

[0083] In some embodiments, the Fc region is human-derived IgG Fc, for example, human IgG1-derived Fc, human IgG2-derived Fc, human IgG3-derived Fc, or human IgG4-derived Fc. In one embodiment, the Fc region includes amino acid sequence SEQ ID NO: 23 or 24, or is composed of an amino acid sequence having at least 90% identity thereto, for example, 95%, 96%, 97%, 99%, or higher identity.

[0084] In one embodiment, an Fc region is modified to alter its effector function characteristics (e.g., complement activation function of the Fc region). In one embodiment, the effector function is reduced or eliminated compared to the wild-type Fc region. In one embodiment, the effector function is reduced or eliminated by using an Fc isotype that naturally reduces or eliminates the effector function, and by a method selected from Fc region modifications. In a preferred embodiment, the Fc region has an effector function mediated by the reduced Fc region, such as a reduced or eliminated ADCC, ADCP, or CDC effector function, and includes, for example, one or more mutations that achieve the above function.

[0085] The Fc region may include (one or more) modifications that alter the binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region includes (one or more) mutations that reduce binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in the present invention has an L234A / L235A mutation that reduces binding to the Fcγ receptor. In a more preferred embodiment, the Fc fragment may have (one or more) mutations that result in an increased serum half-life, such as (one or more) mutations that improve the binding of the Fc fragment to FcRn.

[0086] In preferred embodiments, the Fc region of the present invention has reduced binding ability to FcγR (preferably binding ability to all FcγR completely excluded) while retaining affinity for FcRn. Therefore, the Fc region used in the present invention has L234A / L235A and P329 deletion mutations. Therefore, in a preferred embodiment, the Fc region of the present invention includes the amino acid sequence shown in SEQ ID NO: 24, or includes an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO: 24, and has L234A / L235A and P329 deletion mutations.

[0087] In some preferred embodiments, the heavy chain constant region of the antibody of the present invention is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 5 or 26, or consisting of such an amino acid sequence. (ii) comprising an amino acid sequence selected from SEQ ID NO: 5 or 26, or consisting of the said amino acid sequence, (iii) an amino acid sequence having one or more (preferably 20 or 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably amino acid conserved substitutions) compared to an amino acid sequence selected from SEQ ID NO: 5 or 26, or comprising the above amino acid sequence.

[0088] In some preferred embodiments, the heavy chain constant region of the antibody of the present invention comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 5, and having L234A / L235A and P329 deletion mutations. In some preferred embodiments, the heavy chain constant region of the antibody of the present invention comprises or consists of an amino acid sequence selected from SEQ ID NO: 5.

[0089] In some embodiments, the constant region of the antibody light chain of the present invention is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 11, or comprising the above amino acid sequence. (ii) comprising an amino acid sequence selected from Sequence ID No. 11, or consisting of the said amino acid sequence, (iii) an amino acid sequence having one or more (preferably 20 or 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably amino acid conserved substitutions) compared to an amino acid sequence selected from Sequence ID No. 11, or comprising the above amino acid sequence.

[0090] In some specific embodiments, the anti-CD40L antibody of the present invention comprises a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), a third heavy chain complementarity-determining region (HCDR3), and a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3), where, (i) The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 each contain the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or each consists of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, (ii) HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 each contain the amino acid sequence shown in SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 18, SEQ ID NO 19, and SEQ ID NO 20, respectively, or consist of the amino acid sequence shown in SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 18, SEQ ID NO 19, and SEQ ID NO 20, respectively.

[0091] In some embodiments, VH of the present invention comprises HCDR1, HCDR2, and HCDR3, and VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or each comprises the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, or each comprises the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

[0092] In some embodiments of the present invention, VH comprises HCDR1, HCDR2, and HCDR3, and VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences shown in SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15, respectively, or consist of the amino acid sequences shown in SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15, respectively, and LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences shown in SEQ ID NO. 18, SEQ ID NO. 19, and SEQ ID NO. 20, respectively, or consist of the amino acid sequences shown in SEQ ID NO. 18, SEQ ID NO. 19, and SEQ ID NO. 20, respectively.

[0093] In some embodiments, the antibody or antigen-binding fragment of the present invention is (i) comprising an amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or comprising VH consisting of the amino acid sequence, and / or comprising an amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or comprising VL consisting of the amino acid sequence, (ii) A VH comprising the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and / or a VL comprising the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith.

[0094] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, where (i) The heavy chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 4, and the light chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 10, (ii) The heavy chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 21.

[0095] In some embodiments, the anti-CD40L antibody of the present invention or its antigen-binding fragment further comprises an antibody heavy chain. In some embodiments, the anti-CD40L antibody of the present invention or its antigen-binding fragment further comprises an antibody light chain. In some embodiments, the anti-CD40L antibody of the present invention or its antigen-binding fragment further comprises a heavy chain and a light chain. In some embodiments, the heavy chain of the antibody of the present invention comprises a heavy chain variable region and a heavy chain constant region, or consists of a heavy chain variable region and a heavy chain constant region. In some embodiments, the antibody light chain of the present invention comprises a light chain variable region and a light chain constant region, or consists of a light chain variable region and a light chain constant region. In some embodiments, the antibody of the present invention comprises two heavy chains and two light chains, or consists of two heavy chains and two light chains.

[0096] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain, wherein the heavy chain is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 6 or 17, or consisting of such an amino acid sequence. (ii) comprising an amino acid sequence selected from SEQ ID NO: 6 or 17, or consisting of the said amino acid sequence, (iii) an amino acid sequence having one or more (preferably 20 or 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably amino acid conserved substitutions) compared to an amino acid sequence selected from SEQ ID NO: 6 or 17, or comprising the above amino acid sequence.

[0097] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a light chain, wherein the light chain is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 12 or 22, or consisting of such an amino acid sequence. (ii) comprising an amino acid sequence selected from Sequence ID No. 12 or 22, or consisting of the said amino acid sequence, (iii) an amino acid sequence having one or more (preferably 20 or 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably amino acid conserved substitutions) compared to an amino acid sequence selected from SEQ ID NO: 12 or 22, or comprising the above amino acid sequence.

[0098] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain and a light chain, where (i) The heavy chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 6, and / or the light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 12, (ii) The heavy chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 17, and / or the light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 22.

[0099] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain and a light chain, where (i) The heavy chain comprises the amino acid sequence of SEQ ID NO: 6 or consists of the earlier sequence, and the light chain comprises the amino acid sequence of SEQ ID NO: 12 or consists of the earlier sequence, (ii) The heavy chain comprises the amino acid sequence of SEQ ID NO: 17 or consists of the earlier sequence, and the light chain comprises the amino acid sequence of SEQ ID NO: 22 or consists of the earlier sequence.

[0100] In one embodiment of the present invention, the amino acid modifications described herein include amino acid substitutions, insertions, or deletions. Preferably, the amino acid modifications described herein are amino acid substitutions, preferably conservative substitutions. In preferred embodiments, the amino acid modification described in the present invention occurs in a region outside the CDR (e.g., within the FR). More preferably, the amino acid modification described in the present invention occurs in a region outside the heavy chain variable region and / or outside the light chain variable region.

[0101] In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of (one or more) glycosylation sites of an antibody can be readily achieved by altering the amino acid sequence to produce or remove one or more glycosylation sites. If the antibody contains an Fc region, the sugars attached to it can be altered. In some applications, modifications to enhance antibody-dependent cytotoxicity (ADCC) by removing a fucose motif, for example, or to remove an undesirable glycosylation site may be useful. In other applications, galactosylation modifications are performed to modify complement-dependent cytotoxicity (CDC). In certain embodiments, it may be desirable to produce an antibody modified with cysteine, such as "thioMab," in which one or more residues of the antibody are substituted with a cysteine ​​residue.

[0102] In certain embodiments, the antibodies provided herein may be further modified to include other non-protein moieties known in the art and readily available. Moieties suitable for antibody induction include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymer or random copolymer), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol cohopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0103] In some embodiments, the anti-CD40L antibody or its antigen-binding fragment of the present invention is (i) The antibody of the present invention exhibits the same or similar binding affinity and / or specificity to CD40L, (ii) Inhibiting the binding of the antibody of the present invention to CD40L (for example, competitively inhibiting it), (iii) Binding the same or overlapping epitopes to the antibody of the present invention, (iv) The antibody of the present invention competes with CD40L, (v) The antibody of the present invention has one or more biological properties.

[0104] In some embodiments, the anti-CD40L antibody of the present invention is an antibody of the IgG1 form, or an antibody of the IgG2 form, or an antibody of the IgG3 form, or an antibody of the IgG4 form, preferably an antibody of the IgG1 form. In some embodiments, the anti-CD40L antibody is a monoclonal antibody. In some embodiments, the anti-CD40L antibody is humanized. In some embodiments, the anti-CD40L antibody is a chimeric antibody.

[0105] In one embodiment, the anti-CD40L antibody of the present invention further comprises antibody fragments (e.g., antigen-binding fragments), preferably Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibody, single-chain antibody (e.g., scFv), and single-structure domain antibody (sdAb) e.g., VHH, bivalent antibody or its fragment, or antibody fragments selected from camel antibodies or diabodies.

[0106] In some embodiments, the anti-CD40L antibodies of the present invention further encompass multispecific antibodies, such as bispecific antibodies, that specifically bind to CD40L. In some embodiments, the anti-CD40L antibody of the present invention is a full-length antibody.

[0107] III. Nucleic acids of the present invention and host cells containing them According to one embodiment, the present invention provides a nucleic acid encoding any chain or any monomer or structural domain of the antibody or its antigen-binding fragment. Polynucleotide sequences encoding each chain can be produced by methods well known in the art. For example, when expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human CD40L antigen-binding ability. For example, in some embodiments, the nucleic acid encoding the variable regions of the heavy and / or light chain is operably linked in a reading frame to a nucleic acid encoding the constant regions of the heavy and / or light chain, thereby, when expressed from a suitable expression vector, the nucleic acid encoding the heavy and / or light chain of the antibody.

[0108] According to one embodiment, the present invention provides a nucleic acid encoding any anti-CD40L antibody or a fragment thereof as described herein. The nucleic acid may include a nucleic acid encoding the amino acid sequence of the light chain variable region and / or heavy chain variable region of the antibody, or it may include a nucleic acid encoding the amino acid sequence of the light chain and / or heavy chain of the antibody.

[0109] For example, the nucleic acids of the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 4-6, 10, 12, 16, 17, 21 and 22, or nucleic acids encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NOs: 4-6, 10, 12, 16, 17, 21 and 22. As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences. In some embodiments, the nucleic acids of the present invention include nucleic acids selected from any one of SEQ ID NOs: 28-31, or nucleic acids having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a nucleic acid selected from any one of SEQ ID NOs: 28-31.

[0110] The nucleic acid sequence encoding the molecule of the present invention can be produced by methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification. To facilitate production and purification, secretion signal peptides and / or tag peptides favorable for purification can be fused to the N-terminus of the antibody's heavy and / or light chains.

[0111] The present invention also provides vectors comprising nucleic acids of the present invention. In one embodiment, the vector is an expression vector, for example, a eukaryotic expression vector. The vector includes, but is not limited to, viruses, plasmids, mucoids, λ phages, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector of the present invention is a pcDNA vector, for example, a pcDNA3.1 expression vector.

[0112] In one embodiment, a host cell containing the vector is provided. The present invention also provides a host cell containing the nucleic acid or the vector. Host cells suitable for replicating and supporting the expression of the antibody of the present invention are known in the art. Such cells can be transfected or transduced with a specific expression vector, and sufficient quantities of the antibody for clinical application can be obtained by growing a large number of cells containing the vector and inoculating them into a large fermenter. Suitable host cells for cloning or expressing an antibody-encoding vector include prokaryotic or eukaryotic cells as described herein. For example, the antibody can be produced in bacteria, especially when glycosylation and Fc effector function are not required. After expression, the antibody can be separated from the bacterial cell paste in the soluble fraction and further purified.

[0113] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-K1, CHO-S, CHO-K, or ExpiCHO cells) or 293 cells (e.g., 293F, or HEK293 cells)) or other cells suitable for the preparation of antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, such as bacteria like Escherichia coli.

[0114] For example, eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for antibody-encoding vectors. For instance, fungal and yeast strains with "humanized" glycosylation pathways can lead to the production of antibodies with partial or complete human glycosylation patterns. Host cells suitable for the expression of glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can be used as hosts. For example, modified mammalian cell lines suitable for suspension growth can be used. Other examples of useful mammalian host cell lines include the kidney CV1 strain (COS-7) converted with SV40, and human embryonic kidney strains (HEK293, 293F, or 293T cells). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, such as CHO-K1, CHO-K, DHFR-CHO cells, CHO-S cells, ExpiCHO, and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. Mammalian host cell lines suitable for antibody production are known in this field.

[0115] IV. Production and Purification of the Antibody Molecules of the Present Invention In one embodiment, the present invention provides a method for preparing an antibody molecule or fragment thereof (preferably an antigen-binding fragment) of the present invention, wherein the method comprises culturing host cells under conditions suitable for the expression of nucleic acids encoding the antibody molecule or fragment thereof (preferably an antigen-binding fragment), and optionally separating the antibody or fragment thereof (e.g., an antigen-binding fragment). In one embodiment, the method further comprises recovering the antibody molecule or fragment thereof (e.g., an antigen-binding fragment) from the host cells.

[0116] Furthermore, for cloning and / or expression in host cells, polynucleotides encoding the polypeptide chain of the antibody of the present invention can be inserted into one or more vectors. Expression vectors can be constructed by methods well known to those skilled in the art. Once an expression vector containing one or more nucleic acid molecules of the present invention for expression is prepared, the expression vector can be transfected or introduced into suitable host cells. Various techniques, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, liposome-based transfection, or other conventional techniques can achieve this objective.

[0117] Antibody molecules prepared as described herein can be purified by known prior art, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a particular protein depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are apparent to those skilled in the art. The purity of the antibody molecules of the present invention can be determined by any one of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and high-performance liquid chromatography.

[0118] V.Measurement method Various measurement methods known in the art can be used to identify, screen, or characterize the physical / chemical properties and / or biological activity of the anti-CD40L antibodies provided herein.

[0119] The present invention further provides a method for identifying biologically active anti-CD40L antibodies. Biological activity may include, for example, binding to CD40L (e.g., human CD40L), inhibition of the CD40 / CD40L signaling pathway, blockage of CD40-CD40L binding, inhibition of B cell activation, inhibition of B cell proliferation, inhibition of B cell differentiation, inhibition of cytokine secretion by dendritic cells, inhibition of antibody production in a target, and therapeutic effects on immune system diseases. The present invention further provides antibodies possessing such biological activity in vivo and / or in vitro.

[0120] For the measurement of the above-mentioned biological activity, you can refer to the exemplary measurement methods shown in the examples. It will be understood that any of the above measurement methods can be performed using the immunoconjugate of the present invention in place of or in addition to the anti-CD40L antibody. It will be understood that any of the above measurement methods can be performed using a combination of anti-CD40L antibody and another agent.

[0121] VI. Immunoconjugates In some embodiments, the present invention provides immunoconjugates comprising any anti-CD40L antibody provided herein and other substances, such as therapeutic agents or labels. In some embodiments, the therapeutic agent may be a therapeutic agent suitable for forming an immunoconjugate with the CD40L antibody, such as a biologically effective agent or active ingredient. Here, the biologically effective agent includes, but is not limited to, cytokines, other antibodies, small molecule drugs or immunomodulators (e.g., immunoinhibitors). In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0122] VII. Drug Compositions and Drug Formulations In some embodiments, the present invention provides compositions comprising any anti-CD40L antibody or a fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof as described herein, preferably the composition being a drug composition. In one embodiment, the composition further comprises a pharmaceutically acceptable adjuvant. In one embodiment, the composition comprises, for example, a drug composition, the anti-CD40L antibody or a fragment thereof or an immunoconjugate thereof, and one or more other therapeutic agents in combination.

[0123] The present invention further encompasses compositions (including drug compositions or drug formulations) comprising an anti-CD40L antibody or its immunoconjugate, or compositions (including drug compositions or drug formulations) comprising a polynucleotide encoding an anti-CD40L antibody. In certain embodiments, the composition comprises one or more antibodies or fragments thereof that bind to CD40L, or a polynucleotide of one or more antibodies or fragments thereof that encode one or more anti-CD40L. These compositions may further comprise pharmaceutical excipients, including suitable pharmaceutically acceptable adjuvants, such as pharmaceutically acceptable carriers and buffers known in the art.

[0124] As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, isotonic agent, and absorption retarder. For information on the use and applications of pharmaceutical excipients, see "Handbook of Pharmaceutical Excipients," 8th edition, RCRowe, PJSeskey and SCOwen, Pharmaceutical Press, London, Chicago.

[0125] The compositions of the present invention may be in various forms. These forms include, for example, liquids, semi-solid and solid preparations, such as liquid solutions (e.g., injectable and intravenous solutions), powders or suspensions, liposomal preparations and suppositories. The preferred form depends on the intended administration model and therapeutic application. The antibody of the present invention having the required purity can be mixed with one or more optionally selected pharmaceutically acceptable adjuvants to prepare a drug formulation containing the antibody described herein, preferably in the form of a lyophilized formulation or an aqueous solution.

[0126] The drug composition or formulation of the present invention may further contain one or more active ingredients, the active ingredients being necessary for the specific indication being treated, and preferably having complementary activity that does not adversely affect each other. For example, it is desirable to provide formulations that include, but are not limited to, other therapeutic agents, such as cytokines, small molecule drugs, immunomodulators (e.g., immunoinhibitors), or other antibodies that exert a biological effect in preventing or treating diseases associated with inappropriate activation of the CD40L / CD40-mediated pathway. The active ingredients are present in an effective amount and in an appropriate combination for the intended use. Sustained-release formulations can be prepared. A suitable example of a sustained-release formulation is one comprising a semipermeable matrix of a solid hydrophobic polymer containing an antibody, the matrix being in the form of a molded article such as a film or microcapsule.

[0127] VIII. Drug combinations and kits In some embodiments, the present invention further provides a combination of drugs or a drug combination product comprising the anti-CD40L antibody or a fragment thereof (preferably an antigen-binding fragment), or an immunoconjugate thereof, and one or more other therapeutic agents (such as cytokines, small molecule drugs, immunomodulators (e.g., immunoinhibitors or anti-inflammatory agents), or other antibodies).

[0128] Another object of the present invention is to provide a kit comprising parts containing a combination of drugs of the present invention, preferably the cartridge being in the form of drug dose units. This makes it possible to provide dose units according to an administration scheme or drug administration interval.

[0129] In one embodiment, a kit consisting of the parts of the present invention is packaged in the same package. - A first container containing a drug composition comprising an anti-CD40L antibody or a fragment thereof, -Includes a second container containing a drug composition including other therapeutic agents.

[0130] In some embodiments, the other therapeutic agents include, but are not limited to, cytokines, small molecule drugs, immunomodulators (e.g., immunoinhibitors), or other antibodies that exert a biological effect in preventing or treating diseases associated with inappropriate activation of the CD40L / CD40-mediated pathway.

[0131] IX. Uses and Methods In some embodiments, the antibodies of the present invention are methods for preventing or treating diseases or disorders associated with abnormal activation of CD40L / CD40-mediated signaling pathways in an individual, either as a monotherapy or in combination with other agents.

[0132] In some embodiments, the disease or disorder is an abnormal disease or disorder of the immune system. In some embodiments, the CD40L / CD40-mediated immune system is hyperactivated in patients with the disease or disorder compared to healthy individuals. In some embodiments, patients with the disease or disorder have abnormal activation of the CD40L / CD40-mediated signaling pathway compared to healthy individuals. In some embodiments, patients with the disease or disorder produce or deposit excessive anti-autoantibodies compared to healthy individuals. In some embodiments, patients with the disease or disorder have abnormal activation of the CD40L / CD40-mediated signaling pathway and excessive production and deposition of anti-autoantibodies compared to healthy individuals.

[0133] In some embodiments, cells in patients with the disease or disorder (e.g., activated T cells or other types of immune or non-immune cells, e.g., epithelial cells, monocytes, dendritic cells, fibroblasts, smooth muscle cells, endothelial cells, and platelets) have increased expression of CD40L or CD40 compared to, for example, the corresponding cells in a healthy individual. In some embodiments, the disease or disorder includes, but is not limited to, autoimmune diseases, lupus nephritis, immune thrombocytopenic purpura (ITP), transplant rejection, Crohn's disease, inflammatory bowel disease (IBD), colitis, asthma / allergy, atherosclerosis, myasthenia gravis, multiple sclerosis, psoriasis, rheumatoid arthritis, ankylosing spondylitis, coronary artery disease, type 1 diabetes mellitus, amyotrophic lateral sclerosis (ALS), and immune responses to recombinant drug products, such as factor VII for hemophilia.

[0134] In some embodiments, the antibodies, antibody fragments, immunoconjugates, compositions, or products of the present invention delay the onset of disorders and / or symptoms associated with disorders. In some embodiments, the present invention provides uses for the production or preparation of the anti-CD40L antibody or a fragment thereof, or an immunoconjugate or composition containing the same, for the prevention or treatment of the drug described herein and, for example, related diseases or disorders referred to herein.

[0135] In some embodiments, the prevention or therapy described herein involves administering an antibody molecule, drug composition, or immunoconjugate disclosed herein to the subject or combination of individuals, and further comprising one or more other therapies, e.g., therapeutic schemes and / or other therapeutic agents. In some embodiments, an anti-CD40L antibody or a fragment thereof (and immunoconjugates, compositions, drug compositions, formulations, etc., containing the same) may be administered in combination with one or more other therapies, e.g., therapeutic schemes and / or other therapeutic agents, and used in the applications described herein to prevent and / or treat, for example, the related diseases or disorders mentioned herein.

[0136] In some embodiments, the therapeutic agent is selected from cytokines, small molecule drugs, immunomodulators (e.g., immunoinhibitors or anti-inflammatory agents), or other antibodies. In some embodiments, the antibodies described herein can be used in combination with other antibodies for administration, for example, by administering each antibody separately or by linking them together (e.g., as bispecific or multispecific antibody molecules).

[0137] The combination therapy of the present invention encompasses both combined administration (e.g., two or more therapeutic agents contained in the same or separate formulations) and isolated administration, in which case administration of the antibody or fragment thereof of the present invention can occur before, simultaneously with, and / or after administration of another therapeutic agent and / or drug. In some embodiments, the other therapeutic agent includes, but is not limited to, cytokines, small molecule drugs, immunomodulators (e.g., immunoinhibitors), or other antibodies that exert a biological effect in preventing or treating diseases associated with inappropriate activation of the CD40L / CD40-mediated pathway.

[0138] The antibodies or fragments thereof (and immunoconjugates, compositions, drug compositions, formulations, combination products, etc., containing them) of the present invention can be administered by parenteral administration, and, if necessary for local treatment, by any suitable method including intra-focal administration. Parenteral injection or infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous injection or infusion.

[0139] X. Methods and compositions for diagnosis and detection In one embodiment, the present invention further relates to a method for diagnosis and detection using the antibody or antigen-binding fragment of the present invention, and a composition for diagnosis and detection comprising the same. In certain embodiments, any anti-CD40L antibody or a fragment thereof (preferably an antigen-binding fragment) provided herein is used to detect the presence of CD40L in a biological sample.

[0140] When the term “detection” is used herein, it includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads compounded with antibody molecules, ELISA assay methods, and PCR techniques (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample includes cells or tissue. One embodiment provides an anti-CD40L antibody or a fragment thereof for use in a diagnostic or detection method.

[0141] In another embodiment, a method for detecting the presence of CD40L in a biological sample is provided. In a particular embodiment, the method includes detecting the presence of the CD40L protein in a biological sample. In a particular embodiment, CD40L is human CD40L. In a particular embodiment, the method includes contacting the biological sample with an anti-CD40L antibody or fragment thereof described herein under conditions that allow binding of the anti-CD40L antibody or fragment thereof to CD40L, and detecting whether a complex is formed between the anti-CD40L antibody or fragment thereof and CD40L. The formation of a complex indicates the presence of CD40L. The method may be an in vitro or in vivo method. In one embodiment, the anti-CD40L antibody or fragment thereof is used to select a target suitable for treatment using the anti-CD40L antibody or fragment thereof, for example, herein, CD40L is a biomarker for selecting the target.

[0142] In some embodiments, labeled anti-CD40L antibodies or fragments thereof are provided. The labeling includes, but is not limited to, directly detectable labels or portions (e.g., fluorescent labels, chromophore labels, electron density labels, chemiluminescent labels, and radioactive labels) and portions that are indirectly detectable by, for example, enzymes or ligands, e.g., enzymatic reactions or molecular interactions.

[0143] In some embodiments provided herein, the sample is obtained before treatment with an anti-CD40L antibody or a fragment thereof. In some embodiments, the sample is obtained before treatment with another therapy. In some embodiments, the sample is obtained during or after treatment with another therapy. In some embodiments, CD40L is detected before treatment, for example, before the start of treatment or before a treatment after a treatment interval.

[0144] In some embodiments, the present invention provides a method for treating a disease, the method comprising: testing for the presence of CD40L in a subject (e.g., a sample) (e.g., a control sample), thereby determining a CD40L value, comparing the CD40L value with a control value, and, if the CD40L value is greater than the control value, administering to the subject a therapeutically effective amount of an anti-CD40L antibody or a fragment thereof (e.g., an anti-CD40L antibody or a fragment thereof as described herein) in combination with one or more other therapies of any choice.

[0145] These and other aspects and embodiments of the present invention are described and illustrated in the accompanying drawings (a brief description of the accompanying drawings is provided below) and in the following examples in the detailed description of the invention below. Various embodiments of the present invention can be combined in any or all of the features described above and throughout this application. The following examples further illustrate the present invention, but the examples are described illustratively and not limitingly, and it should be understood that various modifications are possible for those skilled in the art. [Examples]

[0146] Example 1: Screening and humanization of anti-CD40L antibodies Using hybridoma technology, mice were immunized with a human CD40L (human CD40 ligand / TNFSF5 protein, His, Flag Tag, active trimer (MALS validated), Acro) fusion protein. The mice were sacrificed, their spleens removed, digested into a single-cell suspension, and the spleen cells were hybridized with proliferative myeloma cells. Under the action of selective media, hybridoma cells capable of expressing positive antibodies were obtained. Screening by in vitro ELISA and flow cytometry yielded mouse-derived antibodies capable of blocking the CD40-CD40L interaction.

[0147] Preparing the electroporation dish The electroporation dish was thoroughly immersed in 70% ethanol and dried in a clean bench in preparation for subsequent use.

[0148] Isolation of spleen cells: Mice were killed by cervical dislocation, their body surface was disinfected with 75% alcohol for 5 minutes, and then they were placed on a mouse dissection board in a clean bench, in the left lateral decubitus position, with their limbs fixed with a 7-gauge needle. The abdominal cavity was aseptically opened and the spleen was removed, washed with 1640 medium (containing 10% fetal bovine serum and 1% glutamac), and the surrounding connective tissue was carefully removed. The spleen was then transferred to a separate dish containing basal medium. The spleen was pushed with a curved needle, punctured with a small needle, and compressed with forceps to release the spleen cells sufficiently, thereby preparing a spleen cell suspension. The cell suspension was filtered through a 70 μM cell screen, washed once with 30 ml of 1640 medium (containing 10% fetal bovine serum and 1% glutamac), and centrifuged at 1200 rpm for 6 minutes.

[0149] Red blood cell lysis The supernatant was removed, and the cells were resuspended in 10 ml of RBC lysis buffer (GIBCO). Then, 20 ml of RBC lysis buffer was added. The suspension was allowed to stand for 5 minutes, and then centrifuged at 1100 rpm for 6 minutes. After removing the supernatant, the cells were resuspended in 10 ml of basal medium, then 30 ml of basal medium was added, and the cells were centrifuged at 1100 rpm for 6 minutes. After removing the supernatant, the cells were resuspended in 20 ml of 1640 medium (containing 10% fetal bovine serum and 1% Glutamax) and counted.

[0150] Electrofusion Mouse myeloma cells SP2 / 0 (ATCC) were resuspended in 20 ml of 1640 medium (containing 10% fetal bovine serum and 1% Glutamax) and counted. SP2 / 0 and erythrocyte-lysed spleen cells were mixed in a 1:2 to 1:1 ratio and centrifuged at 1000 rpm for 6 minutes. After removing the supernatant, the mixed cells were resuspended in 10 ml of fusion buffer (BTXpress). 15 ml of fusion buffer was added, and the cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. After repeating the above steps once, the cells were resuspended with an appropriate volume of fusion buffer, and the mixed cell density was increased to 1 × 10⁶. 7 The cell suspension was adjusted to 1 cell / ml. 2 ml of the cell suspension was added to each electroporation dish and electroporated.

[0151] Plate after electrofusion The cells were left in an electroporation dish at room temperature for 5 minutes. The cells were transferred to a centrifuge tube and centrifuged in screening medium 1640 (containing 10% fetal bovine serum, 1% Glutamax, and 1% HAT medium) for 1-2 × 10⁶ cells. 4 The cells were diluted to 100 μl / ml. 100 μl of the cell suspension was added to each well of a 96-well plate. The screening medium was changed on day 7 post-fusion. Hybridoma cells expressing specific anti-CD40L antibodies were screened using ELISA and flow cytometry after 10 days of culture (or more, depending on the cell proliferation state).

[0152] Positive hybridoma cell subcloning A 96-well plate was prepared, and 200 μl of the aforementioned 1640 medium (containing 10% fetal bovine serum and 1% Glutamax) was added to each well from the 2nd to the 8th row. Cells from the positive wells screened by the above fusion were collected in approximately 1 × 10⁶ wells. 5 At a density of cells / ml, 300 μl was taken from each well and added to the first row. Using a multichannel pipette, 100 μl of the cell suspension from the first row was taken and added to the second row, mixed thoroughly, and then 100 μl was taken and added to the next row. The above steps were repeated until the volume of the last row reached 300 μl, and the 96-well plate was left to stand for 15 minutes and observed and counted under a microscope. A volume corresponding to 100 cells was taken and added to 20 ml of the aforementioned 1640 medium (containing 10% fetal bovine serum and 1% Glutamax), mixed uniformly and seeded, with 200 μl per well. After one week, the plates were observed under a microscope, monoclonal wells were identified and marked, and positive wells for detection were selected.

[0153] In vitro screening Hybridoma cells expressing specific anti-CD40L antibodies were screened using ELISA and flow cytometry.

[0154] Hybridoma sequencing RNA extraction: Fresh cultured cells approximately 5 × 10 6Collect individual samples, centrifuge at 300 g for 5 minutes, remove the supernatant, add 500 μl LY buffer (Biomiga) to the precipitate (add 20 μl β-mercaptoethanol per ml before use), and mix until clarified. Add to a DNA removal tube and centrifuge at 13000 rpm for 2 minutes, collecting the filtration solution. Add 100% ethanol to half of the filtration solution and mix by inverting the tube 5 times until clarified. Add this clarified solution to an RNA collection tube, centrifuge at 13000 rpm for 1 minute to remove the solution, add 500 μl RB (Recovery Buffer) (Takara), centrifuge at 13000 rpm for 30 seconds, add 500 μl RNA Wash Buffer (Biomiga) (add an appropriate amount of ethanol before use), and centrifuge for 30 seconds. The above procedure was repeated once more, and after centrifugation to completely remove the ethanol, 30 μl of DEPC water was added to a collection column (PrimeScript II 1st Strand cDNA Synthesis Kit), and the eluate was collected after centrifugation at 12000 g for 2 minutes. The RNA concentration was then measured.

[0155] cDNA was obtained by reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara), incubated at 65°C for 5 minutes, and then rapidly cooled on ice. Reaction system I was added to the reverse transcription system, totaling 20 μl, and after slow mixing, reverse transcription and translation were performed under the following conditions: 42°C for 60 minutes, then 95°C for 5 minutes, followed by cooling on ice to obtain cDNA. The cDNA was ligated to a T vector, and PCR was performed using the Mighty TA-cloning Kit (Takara) to amplify the heavy chain and light chain variable regions, respectively.

[0156] transformed cells TOP10 competent cells (Tiangen Biotech (Beijing) Co., Ltd.) were removed at -80°C, thawed on ice, and 5 μl of the linked product obtained above was taken and added to the thawed TOP10 competent cells. After mixing, the mixture was incubated on ice for 30 minutes. After a 90-second heat shock at 42°C, the mixture was rapidly cooled on ice for 2 minutes. 900 μl of LB medium (Sangon Biotech (Shanghai) Co., Ltd.) was added to the EP tube, and the mixture was cultured at 37°C at 220 rpm for 1 hour with vibrations. The mixture was centrifuged at 3000 g for 2 minutes, 800 μl of the supernatant was aspirated, and the cells were resuspended in the remaining medium and seeded onto an ampicillin-resistant plate. The mixture was incubated overnight at 37°C, clones were selected, and sequencing was performed.

[0157] Construction of Chimeric Antibodies Using molecular biology techniques, the sequences of anti-CD40L antibodies produced by the above-mentioned hybridoma cells were obtained. The VH and VL region sequences of the sequenced candidate anti-CD40L antibodies were combined with the hIgG1 (mutation site L234A / L235A / ΔP329, SEQ ID NO: 5) sequence to construct human-mouse chimeric antibodies.

[0158] Chimeric antibody screening Screening using ELISA and Luciferase in vitro experiments yielded an anti-CD40L antibody that simultaneously binds to CD40L and blocks the CD40-CD40L interaction.

[0159] Humanization of chimeric antibodies The resulting chimeric antibody was humanized using the following steps. (1) Determine the CDR loop structure, (2) Find the closest homologous sequence in each V / J region of the heavy chain and light chain from a human germ cell sequence database. (3) Screen human germ cells for the most suitable heavy chain and light chain, and for the lowest amount of reverse mutations, (4) The CDR region of the chimeric antibody is constructed on the human scaffold region, (5) Using sequence and structural characteristics, the amino acid positions in the skeletal region that play a role in maintaining CDR function were determined. (6) Perform a reverse mutation at the sequence location determined to be important (return to the input amino acid type), (7) The amino acids in the risk region were optimized.

[0160] Humanized antibodies Hz33B9C5 and Hz47E2 that bind to human CD40L were ultimately obtained, and the CDR, light chain variable region and heavy chain variable region, amino acid sequences of the light chain and heavy chain, and corresponding nucleotide sequences of the two humanized antibodies (Hz33B9C5 and Hz47E2) of the example of the present invention are all shown in the sequence listing.

[0161] Example 2: Measurement of antibody-antigen binding kinetics of the present invention using ForteBio The equilibrium dissociation constant (KD) of the antibody of the present invention bound to human CD40L was measured using biofilm thin-layer interferometry (ForteBio). Based on the number of samples, the corresponding AMQ (Pall, 1506091) or AHQ (Pall, 1502051) (for positive control detection) sensor was prepared and immersed in SD buffer (PBS 1×, BSA 0.1%, Tween-20 0.05%).

[0162] 100 μl of SD buffer, antibodies (Hz33B9C5 and Hz47E2, 100 nM), and antigen (human CD40L, purchased from Acrobiosystems, 100 nM) were collected and added to 96-well black polystyrene half-volume microwell plates (Greiner, 675076) in 200 μl samples each. The plates were positioned based on the sample locations, and the sensor positions were selected. The instrument settings were as follows: execution steps: Baseline, Loading ~1 nm, Baseline, Association and Dissociation. The execution time for each step depended on the sample binding and dissociation rates, with a rotation speed of 400 rpm and a temperature of 30°C. KD values ​​were analyzed using ForteBio analysis software.

[0163] In the experiment described above using the measurement method, the affinity between Hz33B9C5 and Hz47E2 is shown in Table X:

[0164] Table 1. Detection of antigen-antibody binding affinity constants (equilibrium dissociation constants) using ForteBio. [Table 2]

[0165] Example 3: The antibody of the present invention inhibits the binding of CD40 and CD40L at the molecular level. In this application, the inventors of the present invention screened for antibodies with higher affinity to CD40L using mouse hybridoma technology, performed humanization, and ultimately obtained two humanized antibodies, Hz33B9C5 and Hz47E2. The specific sequences are shown in the sequence listing. Expression and purification in ExpiCHO cells are as follows:

[0166] The sequences of Hz33B9C5 and Hz47E2 were cloned into pcDNA3.1 vectors for subsequent use. ExpiCHO cells were collected, and the detected cell density was 8.0–10 x 10. 6 When the viable cells / mL reach 95-99%, the cell density should be 6.0 x 10⁻¹⁴. 6The solution was adjusted to live cells / mL. A container of appropriate size was selected, and transfection buffer Fectamine (supplier: Gibco) was added with an 8% transfection cell volume. The corresponding amount of plasmid was added with 0.8 μg / mL of transfection cells, and the transfection buffer containing the DNA plasmid was filtered through a 0.22 μm filter membrane for sterilization into another new, clean container. Transfection reagent FectPro (supplier: Polyplus) was added to the filtered mixture at a rate of 3.2 μL / mL and allowed to stand for 10 minutes. The complex formed by the transfection reagent and plasmid DNA was quickly added to the cells, and gently shaken while adding. The cells were cultured with vibrations at 36.5°C at 8% CO2. After 18-22 hours of culture, Enhancer (supplier: Gibco) was added at 6 μL / mL cell, and Feed (supplier: Gibco) was added at 300 μL / mL cell. Cells were harvested on day 7, and the supernatant was collected. Protein purification was performed by affinity chromatography to obtain the final antibody molecule. The purified antibody solution was centrifuged in a 15 ml ultrafiltration centrifuge tube at 4000 rpm / min for 10 minutes. After diluting the protein with PBS, centrifugation was continued and repeated several times at 4000 rpm / min for 10 minutes. The buffer was then replaced. The antibody was bound after the buffer replacement and the antibody concentration was measured. The components of the monoclonal antibody were further qualitatively analyzed using liquid chromatography-mass spectrometry (LC-MS) and volume exclusion chromatography (SEC), and quantified using a UV-Vis spectrophotometer.

[0167] In this example, the blocking effect against CD40L was tested at the molecular level. INX-021 was used as a control (see sequences in Figure 1A and Figure 1B of WO2017011544A1). Source of antigen (human CD40 ligand): [g1] Commercialized antigen: Acro human CD40 ligand / TNFSF5 protein, His, Flag Tag (active trimer) (MALS validated).

[0168] Facilities: Thermo Fisher MULTISKAN-FC

[0169] Reagents and raw materials: [Table 3]

[0170] The details of the method are as follows: a) Antigen coating: Human CD40 ligand was diluted to 1 ug / ml with 1X PBS, added to 100 ul / well of a 96-well immunoplate, and sealed overnight at 4°C. b) Blocking: Well plates were collected, the supernatant was discarded, and the plates were washed three times with PBST washing solution. Then, 300 ul / well of 1% BSA was added and the plates were blocked for 2 hours. c) Preparation of primary antibody: The antibody to be detected was diluted using a 2-fold serial dilution method with 1% BSA to prepare a standard solution, starting at a concentration of 20 ug / ml. For subsequent use, biotinylated human CD40 was diluted to 2 ug / ml with 1% BSA. d) Primary antibody incubation: Well plates were taken, the supernatant was discarded, and the plates were washed six times with PBST washing solution. First, 50 ul / well of antibody was added, followed by 50 ul / well of biotinylated human CD40, and the plates were incubated for 2 hours. e) Preparation of enzyme-labeled antibody: HRP streptavidin was diluted 1:3000 with 1% BSA for subsequent use. f) Enzyme-labeled antibody incubation: Well plates were taken, the supernatant was discarded, and the plates were washed three times with PBST washing solution. Then, 100 ul / well of diluted HRP streptavidin was added and incubated for 1 hour. g) Color development: After collecting samples from the well plate and discarding the supernatant, wash three times with PBST washing solution. Then, add 100 ul / well of TMB colorant and allow to develop for 5-10 minutes until fully colored. After complete color development, add 50 ul / well of stop solution and read at 450 nm using a microplate reader.

[0171] As shown in Figure 1, the antibody molecules Hz33B9C5 and Hz47E2 of the present invention showed a lower binding rate to the CD40 protein compared to the positive control INX-021, indicating that Hz33B9C5 and Hz47E2 better inhibit the binding of the CD40 protein to its ligand, CD40L protein. In summary, the antibody molecules Hz33B9C5 and Hz47E2 of the present invention have superior targeted blocking activity compared to the positive control INX-021.

[0172] Example 4: The antibody of the present invention inhibits the activation of downstream signaling in Jurkat cells that overexpress CD40 at the cellular level. The Jurkat-NFκB-Luc reporter system was used to detect the inhibitory activity of Hz33B9C5 and Hz47E2 on the activation of the CD40 downstream signaling pathway by anti-CD40L antibodies. In the absence of anti-CD40L antibodies, Jurkat-NFκB-Luc cells overexpressing CD40 produce luciferase and catalyze substrate luminescence under stimulation of CHO-CD40L cells. When anti-CD40L antibodies bind to CD40L overexpressed on the surface of CHO cells, they inhibit the interaction between CD40L and membrane proteins in Jurkat-NFκB-Luc cells overexpressing CD40, leading to a decrease in luciferase secretion. Therefore, the luciferase reporter system can be used to evaluate the inhibition of cell activation by Hz33B9C5 and Hz47E2.

[0173] Construction of the CHO-CD40L cell line, which overexpresses CD40L: The CD40L protein sequence (SEQ ID NO: 27) was cloned into the PXC17.4 vector, and the prepared vector plasmid was co-incubated in diluted CutSmart Buffer with Pvul-HF for enzymatic cleavage. GS-CHO cells (supplier: Lonza) were harvested, centrifuged, and the supernatant was removed. The cells were then softened and resuspended in CD CHO Medium containing 1% SP4. The enzymatically cleaved plasmid was added to the cells and mixed, then transferred to an electrolytic beaker. The electroporator program was set to 300V, 900uF, and infinite resistance, and electroporation was performed. Immediately after electroporation, the cells were transferred to preheated CD CHO Medium containing 1% SP4 and cultured at 120 rpm, 37°C, and 6% CO2 with vibration. After 1-2 days of culture, the medium containing MSX was changed and the cells were screened. Cell viability decreased and then increased, and after viability recovered to over 95%, the cells were cryopreserved for subsequent use.

[0174] Construction of the Jurkat-Luc-CD40 cell line, which overexpresses CD40: The CD40 protein sequence (SEQ ID NO: 32) was cloned into a pLVX-IRES-EGFP vector. The prepared vector plasmid was mixed with pSPAX, pMD2.G, PEIpro, and Opti-MEM, transfected into 293T cells, and viral packaging was performed. After 48 hours, the virus was collected and enriched, and used to infect NFkB Reporter Jurkat cell lines (supplier: Genomeditech (Shanghai) Co., Ltd.). Cells exhibiting fluorescence signals were screened by flow cytometry, grown in culture, and cryopreserved for subsequent use.

[0175] In a co-culture system of cells overexpressing CHO-CD40L and Jurkat-Luc-CD40, the addition of the antibody of the present invention blocked the binding of CD40L on the surface of CHO cells to CD40 on the surface of Jurkat-NFκB-Luc cells, thereby inhibiting the activation of downstream NFκB and luciferase production in Jurkat cells and reducing the fluorescence signal of the system. The experimental results are shown in Figure 2.

[0176] The details of the method are as follows: a) Cells were cultured in the logarithmic growth phase, and the cell numbers were adjusted in 1640 complete medium. At this stage, Jurkat-Luc-CD40 was adjusted to 1 * 10^5 cells / 45 ul, and CHO-CD40L was adjusted to 5000 cells / 45 ul. b) Antibody preparation: The antibody concentration was adjusted to 200 ug / ml in 1640 complete medium, and then diluted by a 2-fold serial dilution method for subsequent use. c) Sample addition: First, CHO-CD40L was gently pipetted 5-7 times and added to a 96-well plate at a rate of 45 μl / well. Subsequently, 10 μl / well of the prepared antibody dilution was added and mixed by pipetting 5 times with a multichannel pipette. Finally, Jurkat-Luc-CD40 cells were gently pipetted 5-7 times and added to the 96-well plate at a rate of 45 μl each, and mixed by pipetting 5 times with a multichannel pipette. The 96-well plate was placed in a 37°C, 5% CO2 cell culture incubator and incubated for 6 hours. d) After reacting for 6 hours, remove the 96-well plate and allow it to equilibrate to room temperature. Mix Bio-Glo in a 1:1 volume ratio. TM The Luciferase Assay System was added, and the reaction was allowed to proceed at room temperature for 10 minutes before reading the result. Equipment: SPARK Fluorescence Quantitative Microplate Reader

[0177] Reagents and raw materials: [Table 4]

[0178] The results, shown in Figure 2, demonstrate that the antibody molecules Hz33B9C5 and Hz47E2 of the present invention more strongly inhibited downstream NFκB signaling activation in CD40 cells overexpressing CD40 cells compared to the positive control INX-021, indicating that the antibody molecules of the present invention can significantly inhibit CD40L-mediated CD40 downstream signaling activation.

[0179] Example 5: Inhibition of B cell activation marker CD86 expression by the antibody of the present invention Flow cytometry (FACs) detected inhibition of B cell activation by the anti-CD40L antibodies Hz33B9C5 and Hz47E2 in an in vitro culture system of peripheral blood mononuclear lymphocytes (PBMCs). When B cells receive a stimulating signal, the expression of the activation marker CD86 on their surface rapidly increases; however, in the presence of anti-CD40L antibodies, B cell activation is inhibited. Therefore, detecting the expression rate of CD86 on the B cell surface can reflect the inhibitory effect of the antibody. The experimental results are shown in Figure 3.

[0180] The details of the method are as follows: a) PBMCs were collected, rapidly dissolved in a 37°C water bath, added to 9 ml of complete medium, centrifuged at 300 g for 5 minutes, the supernatant was discarded, and the cells were resuspended in complete medium (RPMI with 10% fetal bovine serum added) and added to a 96-well plate (U-shaped) at 5 * 10^ cells / well. b) CHO-CD40L cells were collected and added to the well plate at a rate of 5*10^3 cells / well, then uniformly pipetted using a multichannel pipette. c) After diluting the target antibodies at different concentrations, add them to the well plate described above, pipette uniformly using a multichannel pipette, and incubate for 18 hours. d) Staining, (PC5.5 CD19, PE CD86, LIVE / DEAD TM Detected using a flow cytometer (Fixable Near-IR Dead Cell Stain Kit). Equipment: Beckman Cytoflex

[0181] Reagents and raw materials: [Table 5]

[0182] As shown in Figure 3, the antibody molecules Hz33B9C5 and Hz47E2 of the present invention can dose-dependently inhibit CD86 expression in B cells, demonstrating that they easily inhibit the initial activation of B cells and that their inhibitory effect is similar to that of the positive control INX-021.

[0183] Example 6: Effect of the antibody of the present invention on the proliferation of B cells isolated in vitro B cells proliferate rapidly during the class conversion and differentiation processes into plasma cells. By isolating B cells from PBMCs and culturing them in vitro under specific conditions, B cells proliferate rapidly in a system where the first signal, second signal, and cytokines coexist. Adding Hz33B9C5 or Hz47E2 inhibits B cell proliferation by blocking the interaction between the second signal, CD40L, and CD40 on the B cell surface. The experimental results are shown in Figure 4.

[0184] The details of the method are as follows: a) PBMCs were collected, recovered, rapidly dissolved in a 37°C water bath, added to 9 ml of complete medium, centrifuged at 300 g for 5 minutes, the supernatant was discarded, and the PBMCs were resuspended in MACs buffer. b) B cells were isolated from PBMCs according to the B cell isolation kit II protocol, counted, and incubated in 10 mL of complete medium containing 0.5 μg / mL anti-IgM (AffiniPure F(ab')2 fragment goat anti-human IgM) at 4°C for 2 hours. c) The treated cells were collected, washed, counted, and B cells were diluted at 5e+5 / ml in medium containing 1 μg / mL anti-IgM, 160 ng / mL recombinant human IL-21 protein, and 2 μg / mL CD40L. d) Each antibody was started at 20 μg / ml in the first well and gradually diluted 2-fold in PBS over 12 dilutions, with 100 μL of antibody + 100 μL of B cells added to a 96-well white-bottomed immunosorbent plate. e) The culture was incubated until day 4, and a fresh culture medium was prepared with the same antibody concentration as in step d) above, and half of the culture medium was replaced. f) On day 7, after centrifugation of the well plate, 60 μl of supernatant was carefully aspirated from each well, 100 μl of CellTiter-Glo® Luminescent Cell Viability Assay was added, pipetted 3-5 times, allowed to stand at room temperature for 10 minutes, and then the plate was placed on a microplate reader to read the values. Equipment: SPARK Fluorescence Quantitative Microplate Reader

[0185] Reagents and raw materials: [Table 6]

[0186] The results are shown in Figure 4. By detecting the number of cells using the Cell Titer-Glo® kit, we found that Hz33B9C5 and Hz47E2 could dose-dependently inhibit the proliferation of B cells induced by the CD40L signal in an in vitro B cell culture system, and that the inhibitory effect was similar to that of the positive control INX-021.

[0187] Example 7: Effects of the antibody of the present invention on the differentiation of B cells into plasma cells Under appropriate conditions, B cells differentiate into plasma cells that ultimately produce antibodies, and the antibodies secreted by plasma cells are the main mode of action of humoral immunity in adaptive immunity. B cells isolated from PBMCs differentiate into plasma cells under stimulation with 3T3-CD40L cells, anti-IgM (AffiniPure F(ab')2 fragment goat anti-human IgM, Fc5μ fragment specificity) and IL-21. Furthermore, the addition of anti-CD40L antibodies Hz33B9C5 or Hz47E2 inhibits the interaction between 3T3-CD40L and B cells, thereby reducing the migration of B cells to plasma cells. The experimental results are shown in Figure 5.

[0188] Construction of a 3T3-CD40L cell line that overexpresses CD40L: a) Lentivirus (Lenti) packaging 1) 293T cells were seeded in a T175 flask. 2) When the cell density exceeded 80%, transfection was performed. Before transfection, the culture medium in the T175 flask was discarded, 20 ml of basal DMEM medium (serum-free, antibiotic-free) was added, and the flask was placed again in a 37°C, 5% CO2 culture incubator. 3) A transfection mixture was prepared, in which the ratio of plasmid:pSPAX2:pMD2.G:PEIpro was 2:2:1:15, and the total volume of the transfection mixture was equal to the total volume of 10% cultured cells. 4) The prepared transfection mixture was gently added dropwise to the culture flask, the flask was lightly shaken to distribute the mixture evenly, and the culture was performed in a 37°C, 5% CO2 culture incubator. 5) After 6 hours of transfection, aspirate the culture medium from the culture flask, add 20 ml of fresh DMEM medium containing 4% FBS, and leave it in a 5% CO2 culture incubator at 37°C.

[0189] b) The virus was collected and used to infect 3T3 cells (suppliers: Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). 1) 3T3 cells were prepared one day before infection and seeded into a 6-well plate. Here, 4 wells were used for the experimental group and 2 wells for the negative control group. 2) On the day of infection, the virus-containing supernatant obtained in a) was collected and centrifuged (500g, 10min) to remove dead cells. 3) The virus-containing supernatant after centrifugation was added to 3T3 cells, 2 ml of virus-containing supernatant was added to the experimental group wells, 2 ml of culture medium was added to the negative control group wells, and the cells were transferred to a culture incubator. 4) One day after viral infection, the culture medium was replaced with complete medium (containing 10% FBS and antibiotics).

[0190] c) Cell sorting 1) After stably amplifying the cells, they were collected by centrifugation and stained for CD40L on the cell surface using a flow cytometry antibody. 2) Cells were screened for fluorescence signals by sorting using a flow cytometry system. 3) The selected cells were cultured and grown to obtain 3T3-CD40L cells, which were then cryopreserved for future use.

[0191] The details of the method are as follows: a) 3T3-CD40L cells were treated with 10 ug / ml mitomycin C at 37°C for 30 minutes and seeded at 6250 cells / well in 96-well plates. b) The following day, a B-cell basal medium was prepared: 50 ml RPMI1640 + 0.192 μl 2-mercaptoethanol + 100 μl HEPES. The following reagents were all prepared in this medium. c) Using B cell isolation kit II, B cells were isolated from PBMCs, treated with 10 mL of B cell basal medium containing 0.5 μg / mL anti-IgM at 4°C for 2 hours, washed, counted, and the B cells were diluted to 1e+6 / ml in B cell basal medium containing 160 ng / mL IL-21. d) After preparing each antibody at an initial concentration of 40 μg / ml for the first well, it was diluted by a two-fold serial dilution method over 12 dilutions. The medium was aspirated from the 96-well plate seeded with 3T3-CD40L cells, and 100 μL of the antibody + 100 μL of B cells were added to each well, taking care to add them slowly without pipetting. e) Cultured until day 4, and half of the B cell basal medium was exchanged. f) Cells on day 7 of differentiation were stained with IgD (Brilliant Violet 785 TM anti-human CD38 antibody, 1.5:100), CD19 (CD19-PE-CF594, 1:100) and DCM (DCM APC-Cy7, 1:1000), and flow cytometry analysis was performed. Equipment: BD Symphony

[0192] Reagents and raw materials:

Table 7

[0193] As shown in Figure 5, similar to the positive controls INX-021 and MEDI4920 (shown in SEQ ID NO: 145 in CN103874501B), the antibody molecules Hz33B9C5 and Hz47E2 of the present invention inhibited the differentiation rate of plasmablasts in a concentration-dependent manner. By inhibiting the interaction between 3T3-CD40L and B cells, it can be seen that the antibody of the present invention can reduce the differentiation of B cells into plasmablasts.

[0194] Example 8: Effect of the antibody of the present invention on IL-12 secreted by moDC cells CD40 is mainly expressed in B cells. Similarly, CD40 and CD40L expressed in DC cells, which are antigen-presenting cells, often participate in immune system reactions and play roles. When IL-12 secreted by DC cells acts on receptors on T cells and NK cells, it causes the differentiation of T cells and the secretion of IFN-γ and TNF-α by NK cells, promoting the inflammatory reaction. In this experiment, monocytes were isolated from PBMCs and differentiated into moDC cells under the induction of IL-4 and GM-CSF. These cells were co-cultured in the presence of CD40L protein and the antibody of the present invention. The concentration of IL-12 secreted by DC cells was detected by the ELISA method. The experimental results are shown in Figure 6.

[0195] The details of the method are as follows. a) PBMC cells (50 million / individual) were recovered, and monocytes were negatively selected using a pan-monocyte isolation kit. b) The cells were resuspended in X-VIVO medium and inoculated into a 6-well plate. 100 ng / ml GM-CSF and 10 ng / ml IL-4 were added to the cells for induction for 7 days. Here, 1 ml was replenished on the 2nd and 4th days, and a medium containing 100 ng / ml GM-CSF and 10 ng / ml IL-4 was added. c) The induced imDC cells obtained in b) were collected, the cells were resuspended in X-VIVO medium, and the cell density was adjusted to 2.5*10^5 / ml. d) The DC cell suspension obtained in c) was added to a 96-well flat-bottom plate, 100 μl / well. e) It was prepared to 4 μg / ml CD40L using RPMI1640 medium containing 10% FBS and added at 50 μl / well to obtain a final system concentration of 1 μg / ml. f) The antibody to be detected was diluted to 20 μg / ml using X-VIVO medium, 50 μl of the diluted antibody was taken and added to the cells. After mixing well, it was incubated at 37°C. g) After culturing for 4 days, the cell culture supernatant was collected, and the content of IL-12 p40 was detected using a commercialized ELISA detection kit (human IL-12 / IL-23 p40 ELISA kit). Equipment: Thermo Fisher MULTISKAN-FC

[0196] Reagents and raw materials: [Table 8]

[0197] As shown in Figure 6, for the positive control INX-021, the antibodies Hz33B9C5 and Hz47E2 of the present invention more strongly inhibit the IL-12 secreted by DC cells. This indicates that the antibodies of the present invention can inhibit a series of inflammatory reactions mediated by IL-12 by more significantly inhibiting IL-12.

[0198] Example 9: The antibodies of the present invention do not cause platelet activation in in vitro experiments Regarding whether the antibodies of the present invention cause platelet activation, detection was performed based on two detection methods: the expression of marker molecules for platelet activation and a whole blood platelet aggregation analysis system for detecting aggregation. After treating platelets with a positive control drug and a negative control drug, the expression of activation marker molecules CD62P and PAC1 on platelets was detected by flow cytometry technology for quantitative analysis, and real-time monitoring and quantitative analysis were performed using a whole blood platelet aggregation analysis system. The experimental results are shown in Figures 7 to 9.

[0199] The details of the method are as follows. a) Human platelets were resuspended in Tyrode's buffer (sodium chloride (140 mM), potassium chloride (5 mM), HEPES (25 mM), calcium chloride dihydrate (2 mM), magnesium chloride hexahydrate (2 mM), dextrose / glucose (10 mM), water (the remaining part)), temporarily stored at room temperature, the platelet suspension was centrifuged at 3000 rpm for 2 minutes, the precipitated platelets were gently resuspended in PBS, and the platelets were counted using a three-part blood analyzer, generally 3*10 11 / L. b) The CD40L antigen and the target antibody were homogeneously mixed in a molar ratio of 1:1, and both the final concentration of CD40L and the final concentration of the antibody drug were adjusted to 3 μM. The mixture was left at room temperature for 15 minutes to activate the antibody drug and form an immune complex. c) Platelet aggregation analysis system analysis: The Chrono-Log 560Ca agglutinator was preheated to 37°C, 200 μL of platelets and 50 μL of antigen-antibody complex were gently mixed in the cuvette, the stirring speed was set to 1000 rpm, a final concentration of 1 mM CaCl2 was added, and the cuvette containing PBS was used as a control to reset the baseline, and the curve of change in cuvette permeability over time was recorded. d) Flow cytometry analysis: Antigen-antibody conjugates were prepared in 10 μL of the system in the above proportions, 1 mM CaCl2 was added, 50 μL of platelets and the 10 μL system were mixed, the mixture was bathed in water at 37°C for 10 minutes, 2.5 μL of P-selectin and PAC-1 flow antibody were added, the mixture was protected from light at room temperature for 15 minutes, diluted with 300 μL of PBS and mixed, and placed in a flow tube for analysis.

[0200] Equipment: Sysmex Corporation three-part blood analyzer (model: KX-21N), Chrono-Log Corporation whole blood platelet aggregation analyzer (model: Chrono-Log 560Ca), BD Biosciences flow cytometry analyzer (model: FACS Calibur)

[0201] Reagents and raw materials: [Table 9]

[0202] As can be seen from the FACs results shown in Figures 7-8, the antibody molecules Hz33B9C5 and Hz47E2 of the present invention had little effect on the expression of the platelet activation markers PAC-1 and CD62P compared to the positive controls Convulxin and Rupilzumab. Furthermore, the antibodies of the present invention showed performance similar to that of the Fc-mutated antibody INX-021. In addition, Figure 9 shows that the antibodies of the present invention had almost the same platelet aggregation ratio as the negative control PBS, a ratio comparable to INX-021 and significantly lower than that of Convulxin and Rupilzumab. This indicates that the antibodies of the present invention do not cause platelet aggregation and activation by the immune complex formed by CD40L.

[0203] Example 10: Data on the antibody of the present invention in an experimental autoimmune encephalomyelitis (EAE) mouse model. Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that can lead to encephalitis and demyelination. MS is thought to be an autoimmune disease caused by autoreactive T cells, and its symptoms include muscle stiffness and paralysis, visual impairment and blindness, loss of sensation and ataxia, and are characterized by recurrent episodes. Currently, various characteristic animal models of MS exist, but among them, the experimental autoimmune encephalomyelitis (EAE) model is widely used in MS research because it has pathological features of inflammation and demyelination similar to those of MS. Myelin protein (myelin oligodendrocyte glycoprotein [MOG] used in this experiment) or peptides of these proteins were used for immunization to induce the EAE model. This is thought to be because myelin-specific T cells are activated in the periphery, cross the blood-brain barrier and enter the central nervous system, where they are reactivated, triggering a series of inflammatory responses that lead to demyelination and apoptosis of axonal cells, ultimately resulting in nerve damage and loss of function. The incidence of disease can be measured by evaluating clinical symptoms using a standardized scoring system.

[0204] The scoring criteria for the experimental autoimmune encephalomyelitis (EAE) model mice used in this experiment are as follows: 0 points: The mouse is healthy. 1 point: The mouse is dragging its tail. 2 points: The mouse has paralysis in one hind limb. 3. The mouse has paralysis in both hind limbs. 4 points: The mice have lost more than 20% of their body weight. 5 points: The mouse is dead or on the verge of death.

[0205] Furthermore, measuring the body weight of the mice can indirectly indicate their health status, and detecting the titer of anti-MOG antibodies in mouse peripheral blood serum reflects the inhibitory effect of the antibody of the present invention on the antibody production function of mice. The experimental results are shown in Figures 10 to 12.

[0206] The details of the method are as follows: a) Preparation of reagents: 1. Inactivated Mycobacterium tuberculosis concentrate (H37Ra concentrate) (100 mg / ml): 10 ml complete Freund's adjuvant + 100 mg H37Ra, ultrasonic dissolution. 2. MOG35-55 undiluted solution (10 mg / ml): 5 ml complete Freund's adjuvant + 50 mg MOG35-55. 3. Pertussis toxin stock solution (PTX stock solution) (100 ug / ml): 50 ug PTX+500 ul DPBS (dispense, store frozen in -40℃ refrigerator), 4. Inactivated Mycobacterium tuberculosis standard solution (H37Ra): 500 µl complete Freund's adjuvant + 500 µl undiluted inactivated Mycobacterium tuberculosis solution. 5. MOG35-55 emulsifier: 2 ml MOG35-55 emulsifier = 200 ul MOG35-55 stock solution + 800 ul DPBS + 1 ml H37Ra standard solution. Use after thorough emulsification. 6. Pertussis toxin standard solution (PTX standard solution): 30 ul PTX stock solution + 1970 ul DPBS (ready to use after preparation)

[0207] b) Modeling method: 1. Five groups of female mice, each group consisting of 5 mice at 8 - 10 weeks of age, were administered with control (PBS), hIgG, INX - 021, Hz33B9C5, and Hz47E2 respectively, with the dosage of each being 10 mg / kg. 2. On day 0: 100 μl of MOG35 - 55 emulsifier was injected into two dorsal points respectively, 200 μl of PTX standard solution was injected into the tail vein, and antibodies or controls were administered by IP injection. 3. On day 3: PTX standard solution was injected into the tail vein, and antibodies or controls were administered by IP injection. 4. On days 7 and 10, antibodies or controls were administered by IP. 5. On days 0, 3, 7, 10, 12 - 21, the degree of the disease was evaluated based on a 0 - 5 scoring system, and the body weights of the mice were recorded. 6. On days 7, 14, and 21, blood was collected from the orbital venous plexus of the mice to collect serum, and the titer of anti - MOG antibodies in the serum was detected by ELISA. Equipment: Thermo Fisher MULTISKAN - FC

[0208] Reagents and raw materials:

Table 10

[0209] As shown in Figures 10 - 11, by administering the antibodies Hz33B9C5 and Hz47E2 of the present invention at 10 mg / kg, the clinical score of experimental EAE can be significantly reduced, and it has no effect on the body weight of the mice. These effects are close to those of the non - MOG - induced control group and are superior to the positive control INX - 021. These results indicate that Hz33B9C5 and Hz47E2 have the ability to improve experimental EAE and autoimmune diseases such as MS.

[0210] As shown in Figure 12, administration of the antibodies Hz33B9C5 and Hz47E2 of the present invention at a dose of 10 mg / kg resulted in the antibodies of the present invention exhibiting lower anti-MOG antibody titers compared to INX-021 14 days after immunization, particularly 21 days after immunization. This indicates that the antibodies of the present invention have a stronger humoral immune inhibitory effect against INX-021, and preferably a stronger inhibitory effect on antibody production.

[0211] Example 11: The antibody of the present invention inhibits the production of anti-keyforlin pethemocyanin antibody in mice. Because peptides, small proteins, and some drug molecules are haptens, which are usually non-immunogenic and require the help of vector proteins like hemocyanin to stimulate an immune system response in the form of antibody production, hemocyanin (KLH) is widely used in research, biotechnology, and therapeutic applications. At the same time, because hemocyanin originates from gastropods (snails) and their growth and development differ significantly from that of mammals, it is less likely to produce false positive results in some types of immunological studies based on mammals.

[0212] Humanized CD40 / CD40L mice were injected with the antibody of the present invention and then immunized with hemocyanin. The antibody of the present invention inhibits the interaction between CD40L and CD40, thereby inhibiting B cell maturation and differentiation and suppressing the production of anti-KLH antibodies. The experimental results are shown in Figure 13.

[0213] The details of the method are as follows: a) Reagent preparation: 250 ug of KLH was prepared per mouse, and for subsequent use, it was dissolved in 100 ug of PBS in the corresponding amount. b) Modeling method: 1. Each group consisted of 5 mice, approximately 8-10 weeks old, and was administered with control hIgG, INX-021 (3 mpk), and Hz33B9C5 (0.3 mpk, 1 mpk, and 3 mpk), respectively. 2. Day 0: KLH stock solution and alum adjuvant were mixed in a 1:1 volume ratio and injected into the peritoneal cavity, while the antibody drug was administered to the mice by IP injection. Days 3 and 7: The mice were administered antibody drugs by IP injection. 4. On days 7, 14, and 21, serum was collected from the orbital venous plexus of mice, and the titer of anti-MOG antibodies in the serum was detected using the ELISA method. Facilities: Thermo Fisher MULTISKAN-FC

[0214] Reagents and raw materials: [Table 11]

[0215] As shown in Figure 13, in humanized CD40 / CD40L mice, the concentration of anti-KLH IgG in mouse serum was lower in the Hz33B9C5 antibody group compared to INX-021. This suggests that the antibody of the present invention can inhibit the maturation and differentiation of B cells in the T cell-dependent pathway by more strongly inhibiting the interaction between CD40L and CD40, ultimately reducing the production of anti-KLH antibodies. Furthermore, since the titer of anti-KLH IgG in mouse serum showed a dose-response trend in different Hz33B9C5 dose groups, it is suggested that the efficacy of Hz33B9C5 in this animal model is dose-dependent.

[0216] Example 12: Pharmacokinetics of the antibody of the present invention in mice Experimental materials

[0217] Reagent and consumable information [Table 12]

[0218] Equipment information [Table 13]

[0219] Preparation for the experiment 2.1 1x PBS buffer Dissolve 100 mL of 10×PBS buffer in 1 L of water and mix thoroughly.

[0220] 2.2 Coating Liquid One packet of carbonate powder was dissolved in 400 mL of ultrapure water, the volume was adjusted to 500 mL, and it was mixed thoroughly. 2.3 Cleaning Solution 1 mL of Tween-20 was added to 1999 mL of 1× PBS buffer and mixed thoroughly.

[0221] 2.4 Blocking Solution 5 g of skim milk powder was weighed out, dissolved in 100 mL of washing solution, and mixed thoroughly. 2.5 Sample Diluent (Antibody Diluent) 1 g of BSA was weighed out, dissolved in 100 mL of washing solution, and thoroughly mixed.

[0222] Experimental process 3.1.1 Pre-coating: 96-well immunoplates were coated one day prior. CD40L of protein was diluted to 1 μg / mL using the coating solution. 100 μL was added per well, the plates were sealed with blocking membranes, and incubated overnight at 4°C. 3.1.2 Plate washing: The pre-coat solution was poured into the 96-well immunoplate, and after wiping off the excess water with absorbent paper, 300 μL of washing solution was added to each well, the mixture was shaken for 10 seconds, and after wiping off the excess washing solution, this washing step was repeated three times.

[0223] 3.1.3 Blocking: Using a multichannel pipette, 200 μL of blocking solution was added per well, the plate was sealed with a blocking membrane, and incubated overnight at 4°C or held at room temperature for 2 hours. 3.1.4 Plate cleaning: The steps described in 3.1.2 were repeated. 3.1.5 Addition of samples: Diluted standards (Hz33B9C5 and Hz47E2, concentrations ranging from 1 to 512 ng / ml, 10 different concentrations obtained by 2-fold dilution)), quality control samples (Hz33B9C5 and Hz47E2, 5.12 to 200 ng / ml, 5 different concentrations obtained by 2-fold dilution) and the sample to be detected (serum obtained after blood collection from the orbital venous plexus of mice for a certain period of time) were added to each well in 100 μL portions, and the mixture was incubated in the dark at room temperature for 2 hours.

[0224] 3.1.6 Plate washing: The solution was poured into the 96-well immunoplate, and after wiping off the excess water with absorbent paper, 300 μL of washing solution was added to each well, the mixture was shaken for 10 seconds, and after wiping off the excess washing solution, this washing step was repeated six times. 3.1.7 Enzyme-labeled antibody incubation: Diluted Goat anti-human IgG Fc-HRP (25 ng / mL) was added to each well at a rate of 100 μL, and incubated in a light-shielded environment at room temperature for 1 hour.

[0225] 3.1.8 Plate cleaning: The steps described in 3.1.6 were repeated. 3.1.9 Color development: 100 μL of TMB substrate was added per well to a 96-well immunoplate, and the plate was allowed to develop color at room temperature in the dark for 5-10 minutes. 3.1.10 End: Add 50 μL of ELISA stop solution to each well, shake at medium speed for 10 seconds, and read the OD450 nm / 620 nm values ​​within 30 minutes.

[0226] Analysis of results 4.1 Concentration measurements were recorded to four decimal places, and the concentration average was recorded to two decimal places, and expressed as "Mean". 4.2 The absorbance values ​​at light densities of 450 nm and 620 nm were provided by the microplate reader and could not be changed. The OD of each sample was calculated as (A450-A620) for that sample. Absorbance values ​​were retained to four decimal places, and each sample was added in a double well. Precision (used to evaluate the relative error to the theoretical value, hereinafter referred to as %RE) and precision (used to evaluate the relative standard deviation, hereinafter referred to as %CV) were retained to one decimal place. The calculation formula is as follows: Precision (%CV) = Standard Deviation (SD) / Mean Measurement Concentration × 100% Accuracy (%RE) = (Average measured concentration - Theoretical concentration) / Theoretical concentration × 100%

[0227] 4.3 Experimental Data: Concentration measurements were obtained by fitting a 4-parameter logistic curve using Skant 3.1 analytical software (Thermo). The calculation of mean concentration, accuracy, and precision was performed using the built-in functions of Excel. The correction curve equation is y = d + (ad) / (1 + (x / c)^b). See Figure 14 for the results. As shown in Table 2 and Figure 14 below, Hz33B9C5 and Hz47E2 have relatively long in vivo half lives, suggesting that the antibodies of the present invention improve the pharmacokinetics of anti-CD40L antibodies and have superior pharmacokinetic properties.

[0228] Table 2. Pharmacokinetics of the anti-CD40L antibody of the present invention [Table 14]

[0229] Sequence information: [Table 15-1] [Table 15-2] [Table 15-3] Table 15-4 Table 15-5 Table 15-6 Table 15-7 Table 15-8 Table 15-9 Table 15-10

Claims

1. An anti-CD40L antibody or its antigen-binding fragment, wherein the antibody is (i) The three complementarity determination regions HCDR1, HCDR2 and HCDR3 included in VH shown in Sequence ID No. 4, and the three complementarity determination regions LCDR1, LCDR2 and LCDR3 included in VL shown in Sequence ID No. 10, or (ii) An anti-CD40L antibody or its antigen-binding fragment, comprising three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO: 16, and three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:

21.

2. An anti-CD40L antibody or its antigen-binding fragment comprising a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), a third heavy chain complementarity-determining region (HCDR3), and a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3), wherein, (i) HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 each contain the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or each consists of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, (ii) Anti-CD40L antibodies or antigen-binding fragments thereof, wherein HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 each contain the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, or each consist of the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:

20.

3. The anti-CD40L antibody or antigen-binding fragment according to claim 1 or 2, comprising a heavy chain variable region (VH), wherein the heavy chain variable region comprises or consists of an amino acid sequence having at least 90% identity with an amino acid sequence selected from SEQ ID NO: 4 or 16, or comprises an amino acid sequence selected from SEQ ID NO: 4 or 16, or comprises a light chain variable region (VL), wherein the light chain variable region comprises or consists of an amino acid sequence having at least 90% identity with an amino acid sequence selected from SEQ ID NO: 10 or 21, or comprises an amino acid sequence selected from SEQ ID NO: 10 or 21, or comprises a light chain variable region (VL), wherein the light chain variable region comprises an amino acid sequence having at least 90% identity with

4. An anti-CD40L antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, (i) The heavy chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or (ii) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and an anti-CD40L antibody or its antigen-binding fragment.

5. It includes a heavy chain variable region and a light chain variable region, where, (i) The heavy chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 4, and the light chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO: 10, or (ii) The anti-CD40L antibody or antigen-binding fragment according to claim 1 or 2, wherein the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:

21.

6. An anti-CD40L antibody or its antigen-binding fragment according to any one of claims 1 to 5, comprising an Fc region.

7. The anti-CD40L antibody or its antigen-binding fragment according to claim 6, wherein the Fc region is derived from human IgG Fc, for example, from human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc.

8. The anti-CD40L antibody or its antigen-binding fragment according to claim 7, wherein the Fc region includes (one or more) mutations that reduce or eliminate effector function, and / or (one or more) mutations that eliminate binding to the Fcγ receptor and maintain binding affinity to FcRn.

9. The anti-CD40L antibody or its antigen-binding fragment according to claim 8, wherein the mutation is an L234A / L235A and P329 deletion mutation.

10. The aforementioned Fc region is (i) containing or consisting of amino acid sequence sequence number 23 or 24, (ii) an amino acid sequence having at least 90%, for example 95%, 96%, 97%, 99%, or higher identity with amino acid sequence SEQ ID NO: 23, or (iii) An anti-CD40L antibody or antigen-binding fragment according to any one of claims 6 to 9, comprising an amino acid sequence having at least 90%, for example 95%, 96%, 97%, 99%, or higher identity with amino acid sequence SEQ ID NO: 24, and comprising the L234A / L235A and P329 deletion mutation.

11. It includes a heavy chain steady region derived from the steady region of IgG1, IgG2, IgG3, or IgG4, and preferably the heavy chain steady region is (i) comprising or consisting of an amino acid sequence selected from Sequence ID No. 5 or 26, (ii) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of Sequence ID No. 26, or (iii) An anti-CD40L antibody or antigen-binding fragment according to any one of claims 1 to 10, comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 5, and having an L234A / L235A and P329 deletion mutation.

12. It includes a light chain steady region which is a lambda or Kappa light chain steady region, preferably the light chain steady region is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of Sequence ID No. 11, or consisting of the said amino acid sequence, (ii) An anti-CD40L antibody or antigen-binding fragment according to any one of claims 1 to 11, comprising or consisting of the amino acid sequence of SEQ ID NO:

11.

13. It includes a heavy chain, where the heavy chain is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 6 or 17, or consisting of such an amino acid sequence, (ii) comprising an amino acid sequence selected from Sequence ID No. 6 or 17, or consisting of the said amino acid sequence, It includes a light chain, where the light chain is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 12 or 22, or consisting of such an amino acid sequence, (ii) An anti-CD40L antibody or antigen-binding fragment according to any one of claims 1 to 12, comprising or consisting of an amino acid sequence selected from SEQ ID NO: 12 or 22.

14. An anti-CD40L antibody comprising a heavy chain and a light chain, or an antigen-binding fragment thereof, (i) The heavy chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 6, and the light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 12, or (ii) An anti-CD40L antibody or its antigen-binding fragment, wherein the heavy chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 17, and the light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:

22.

15. It includes heavy chains and light chains, and here (i) The heavy chain comprises the amino acid sequence of SEQ ID NO: 6 or consists of the earlier sequence, and the light chain comprises the amino acid sequence of SEQ ID NO: 12 or consists of the earlier sequence. (ii) The anti-CD40L antibody or antigen-binding fragment according to any one of claims 1 to 14, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 17 or consists of the earlier sequence, and the light chain comprises the amino acid sequence of SEQ ID NO: 22 or consists of the earlier sequence.

16. The antibody is a monoclonal antibody, according to any one of claims 1 to 15, wherein the antibody is an anti-CD40L antibody or its antigen-binding fragment.

17. The antibody for anti-CD40L according to any one of claims 1 to 16, wherein the antibody is a humanized antibody or a chimeric antibody, or an antigen-binding fragment thereof.

18. The antigen-binding fragments are Fv, Fab, Fab', Fab'-SH, F(ab') ２ The anti-CD40L antibody or antigen-binding fragment according to any one of claims 1 to 17, wherein the antibody fragment is selected from dAb (domain antibody), linear antibody, single-chain antibody (e.g., scFv), and single-structure domain antibody (sdAb) e.g., VHH, bivalent antibody or fragment thereof, or camel antibody or diabody.

19. The antibody or its antigen-binding fragment is a) It binds with high affinity to CD40L (e.g., human CD40L), b) Inhibiting the binding of CD40 and CD40L, c) Inhibiting the CD40 / CD40L signaling pathway, d) Inhibiting the activation or proliferation of B cells, e) Inhibiting the proliferation of B cells, f) Inhibiting the maturation and differentiation of B cells, for example, inhibiting the differentiation of B cells into plasma cells, g) Inhibiting IL-12 secretion by dendritic cells, h) It does not cause platelet activation, i) Treating or preventing immune diseases, such as multiple sclerosis (MS), and / or j) Inhibiting an immune response, preferably a humoral immune response, for example, inhibiting antibody production, k) An anti-CD40L antibody or antigen-binding fragment according to any one of claims 1 to 18, having one or more properties of improving pharmacokinetics and preferably having an in vivo half-life of about 200 hours or more, more preferably about 210, 220, 230, 240, 250, 260, 270, 280 or 290 hours or more.

20. An isolated nucleic acid encoding an anti-CD40L antibody or its antigen-binding fragment according to any one of claims 1 to 19.

21. Preferably, the vector is an expression vector, comprising the nucleic acid according to claim 20.

22. A host cell comprising the nucleic acid described in claim 20 or the vector described in claim 21, preferably the host cell being a prokaryotic or eukaryotic cell, more preferably selected from yeast cells, mammalian cells (e.g., 293 cells or CHO cells, e.g., CHO-K cells or HEK293 cells) or other cells suitable for the preparation of antibodies or their antigen-binding fragments.

23. A method for preparing an antibody or antigen-binding fragment that binds to CD40L, a) Culturing the host cells according to claim 22 under conditions suitable for the expression of the anti-CD40L antibody according to any one of claims 1 to 19 or the nucleic acid encoding the antigen-binding fragment thereof, b) Optionally, separate the antibody or its antigen-binding fragment, c) The method optionally further comprises recovering the anti-CD40L antibody or its antigen-binding fragment from the host cells, and optionally the antibody being purified, for example, by Protein A.

24. An immunoconjugate comprising an anti-CD40L antibody or its antigen-binding fragment according to any one of claims 1 to 19 and an agent that can produce a biological effect comprising, but not limited to, a therapeutic agent, such as a cytokine, another antibody, a small molecule drug, or an immunomodulator (such as an immunoinhibitor).

25. A drug composition comprising an anti-CD40L antibody or its antigen-binding fragment according to any one of claims 1 to 19 or an immunoconjugate according to claim 24, and an adjuvant that is acceptable as an optional pharmaceutically acceptable agent.

26. A combination drug product comprising an anti-CD40L antibody or its antigen-binding fragment according to any one of claims 1 to 19 or an immunoconjugate according to claim 24, and one or more other therapeutic agents, such as cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., immunoinhibitors), but not limited to those, that produce a biological effect.

27. A method for preventing or treating a disease or disorder associated with inappropriate activation of the CD40L / CD40-mediated pathway in an individual, comprising administering to the subject an effective amount of an anti-CD40L antibody or its antigen-binding fragment according to any one of claims 1 to 19, or an immunoconjugate according to claim 24, or a drug composition according to claim 25, or a combination product of drugs according to claim 26.

28. The method according to claim 27, wherein the subject has abnormal activation of CD40L / CD40-mediated signaling pathways and / or excessive production and deposition of anti-autoantibodies compared to a healthy individual, and / or increased expression of CD40L or CD40 in the cells of the subject (e.g., activated T cells or other types of immune cells or non-immune cells, e.g., epithelial cells, monocytes, dendritic cells, fibroblasts, smooth muscle cells, endothelial cells and platelets) compared to, for example, the corresponding cells of a healthy individual.

29. The method according to claim 27 or 28, wherein the disease or disorder is selected from autoimmune diseases, lupus nephritis, immune thrombocytopenic purpura (ITP), transplant rejection, Crohn's disease, inflammatory bowel disease (IBD), colitis, asthma / allergy, atherosclerosis, myasthenia gravis, multiple sclerosis, psoriasis, rheumatoid arthritis, ankylosing spondylitis, coronary artery disease, type 1 diabetes mellitus, amyotrophic lateral sclerosis (ALS), and immune responses to recombinant drug products, such as factor VII for hemophilia.

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