Antibody specifically binding to CD40 and use thereof

Abstract

The present invention relates to a novel anti-CD40 antibody and use thereof and, more specifically, to a novel anti-CD40 antibody and use thereof, wherein the antibody comprises a complementarity determining region of a specific sequence and exhibits an excellent antagonistic effect by blocking CD40-CD40L signaling. The novel anti-CD40 antibody of the present invention exhibits an excellent antagonistic effect by blocking CD40-CD40L signaling, and thus can be widely used as a preparation for preventing or treating various diseases, such as autoimmune diseases and chronic inflammatory diseases, and as a preparation for inhibiting immune rejection at the time of transplantation.

Description

Antibodies specifically binding to CD40 and uses thereof

[0001] The present invention relates to a novel anti-CD40 antibody and its use, and more particularly, to a novel anti-CD40 antibody comprising a complementarity determining region of a specific sequence, which exhibits an excellent antagonistic effect by blocking CD40-CD40L signaling, and its use.

[0002] The CD40 signal transduction pathway relies on the coordinated regulation of many intracellular factors. Like other members of the TNF receptor family, CD40 interacts with TRAF proteins (TNF receptor factor-binding proteins), such as TRAF2 and TRAF3, which mediate intracellular signaling following binding of CD40 to CD40L (either solid CD40L or soluble CD40L). TRAFs transduce the signal to the nucleus via MAP kinases, such as NIK (NF-κB-inducing kinase) and I-kappa B kinase (IKK α / β), ultimately activating the transcription factor NF-κB (Young et al (1998) Immunol Today 19:502-06). Signaling via the Ras and MEK / ERK pathways has also been demonstrated in subsets of B cells. Additional pathways involved in CD40 cell signaling include the PI3K / Akt pathway and the P38 MAPK pathway (Craxton et al (1998) J Immunol 5:439-447).

[0003] Signaling via CD40 has been shown to prevent cell death from apoptosis (Makus et al (2002) J Immunol 14:973-982). Apoptotic signals are required to induce programmed cell death in a coordinated manner. Cell death signals can include intrinsic stimuli from within the cell, such as endoplasmic reticulum stress, or extrinsic stimuli, such as receptor binding of FasL or TNFα. The signaling pathway is complex and includes the activation of caspases, such as caspases 3 and 9, and the activation of poly(ADP ribose) polymerase (PARP). During the cascade, anti-apoptotic signaling proteins such as Mcl-1 and BCLx, and members of the IAP-family proteins such as X-linked inhibitor of apoptosis (XIAP) are downregulated (Budihardjo et al (1999) Annu Rev Cell Dev Biol 15:269-90). For example, CD40 cell signaling in dendritic cells can block apoptotic signals transduced by FasL (Bjorck et al (1997) Int'l Immunol 9:365-372).

[0004] Therefore, CD40 binding by CD40L and subsequent activation of CD40 signaling are essential steps for normal immune responses, and dysfunction of this CD40 signaling can lead to autoimmune diseases (Ichikawa et al (2002), J Immunol 169:2781-7 and Moore et al (2002) J Autoimmun 19:139-45). Furthermore, CD40 / CD40L interactions also play an important role in inflammatory processes. For example, both CD40 and CD40L are overexpressed in human and experimental atherosclerotic lesions. CD40 stimulation induces the expression of matrix-degrading enzymes and tissue factor in atherosclerotic cell types such as endothelial cells, smooth muscle cells, and macrophages. Furthermore, CD40 stimulation induces the production of proinflammatory cytokines such as IL-1, IL-6, and IL-8, and adhesion molecules such as ICAM-1, E-selectin, and VCAM. Inhibition of CD40 / CD40L interactions prevents atherogenesis in animal models.

[0005] CD40 is a 55 kDa cell-surface antigen expressed on the surface of normal and neonatal human B cells, dendritic cells, antigen-presenting cells (APCs), endothelial cells, monocytes, CD8+ T cells, and epithelial cells. The CD40 antigen is also expressed on activated T cells, activated platelets, inflamed vascular smooth muscle cells, eosinophils, synovial cells in rheumatoid arthritis, dermal fibroblasts, and other non-lymphoid cell types. Depending on the type of cell expressing CD40, ligation can induce intracellular adhesion, differentiation, activation, and proliferation. For example, binding to CD40L (also known as CD154), the ligand for CD40, stimulates B cell proliferation and differentiation into plasma cells, antibody production, isotype switching, and B cell memory recall. CD40 is expressed on precursor B cells during B cell differentiation but disappears upon differentiation into plasma cells. CD40 is involved in the differentiation, survival, and proliferation of B lymphocytes, macrophages, antigen-presenting cells, some endothelial cells, and epithelial cells. CD40-CD154 signaling is known to be involved in isotype switching and affinity maturation in antibody production in B cells, and in T cell activation (Leukoc Bil 67:2-17, 2000).

[0006] The CD40 ligand is found on the cell surface of activated T cells (Fenslow et al (1992) J Immunol 149:655; Lane et al (1992) Eur_J_Immunol 22:2573; Noelle et al (1992) Proc Natl Acad Sci USA 89:6550), but is not normally expressed on quiescent human T cells. CD40L is a type II transmembrane glycoprotein with homology to TNF-α (Armitage et al (1992) Nature 357:80 and Spriggs et al (1992) J Exp Med 176:1543). The extracellular domain of CD40L contains two arginine residues near the transmembrane region, which provide potential proteolytic cleavage sites that generate a soluble form of the ligand (sCD40L). Overexpression of CD40L induces an autoimmune disease resembling systemic lupus erythematosus in rodent models (Higuchi et al (2002) J Immunol 168:9-12). In contrast, the absence of functional CD40L on activated T cells induces the X-linked hyper-IgM syndrome (Allen et al (1993) Science 259:990; and Korthauer et al (1993) Nature 361: 539). Collectively, CD40-CD40L signaling is involved in dendritic cell maturation, inflammatory cytokine secretion, and survival; in B cell activation, isotype switch, and antibody production; and in macrophage activation and increased inflammatory cytokines. Accordingly, it is involved in the activation, proliferation, and differentiation of antigen-specific T cells (Jodi L. Karnetll et al, 2019, Advanced Drug delivery Reviews, vol 141, 92-103). CD40-CD40L signaling enhances the cytotoxic function of NK cells (G.terrazzano et al, Scand. J. Immunol., 59(4), 2-004), 356-362). CD40 signaling also promotes granulocyte survival and secretion of GMCSF (Y. Ohkawara, et. Al, J. Cli. Invest., 97(7) (1996), 1761-1766).

[0007] CD40-CD40L signaling is crucial for the pathogenesis of autoimmune and inflammatory diseases caused by autoantibodies (Jodi L. Karnetll et al, 2019, Advanced Drug delivery Reviews, vol 141, 92-103).

[0008] CD40L is expressed on follicular helper T (Tfh) cells that arise in lymphoid tissues, as well as on germinal centers in tissue ectopic follicles, and is thought to play a key role in the generation of plasma cells with autoimmune specificity. CD40-CD40L signaling is known to play a central role in the production of pathogenic autoantibodies in systemic rheumatic diseases such as SLE, lupus nephritis, Sjögren's syndrome, myositis, systemic sclerosis, ANCA vasculitis, rheumatoid arthritis, IgG4-related diseases, and Pemphigus's disease, where autoantibodies are known to play a significant role in disease progression.

[0009] CD40 signaling is also known to be essential for autoantibody production in non-rheumatic diseases with a large autoantibody component, such as myasthenia gravis, neurosurgery, NMDA receptor encephalitis, thyroid hashishmot or Graves' disease, idiopathic thrombocytopenic purpura, hemolytic anemia or IgA nephropathy.

[0010] Therefore, CD40-CD40L signaling is involved in the pathogenesis of various forms of autoimmune diseases. Representative examples include: rheumatoid arthritis (M. Harigai, et al.J. Rheumatol., 26 (5) (1999), pp. 1035-1043), sjogren's syndrome (S. Beaudreuil, Ann Transl Med, 3 (9) (2015), p. 115), autoimmune nephritis (S. Doublier, et al.,PLoS One, 12 (11) (2017)), autoimmune skin diseases such as systemic sclerosis (M. Caproni, et al.,J. Rheumatol., 34 (12) (2007), pp. 2412-2416), multiple sclerosis (D. Chen, et al.J. Immunol., 197 (11) (2016), pp. 4257-4265), type 1 diabetis (B. Balasa, et al. J. Immunol., 159 (9) (1997), pp. 4620-4627).

[0011] Blockade of CD40-CD40L signaling suppresses the production of anti-drug antibodies and is also important in the treatment of inflammatory diseases such as inflammatory bowel disease (S. Danese, et al., J. Immunol., 176 (4) (2006), pp. 2617-2624).

[0012] Blockade of CD40-CD40L signaling is crucial for suppressing rejection and long-term graft survival in allogeneic and xenogeneic islet transplantation. In a primate allogeneic islet transplantation model, both anti-CD154 and anti-CD40 antibodies are known to induce long-term islet survival through the suppression of B-cell antibody production and T-cell activation (Am J Transplant. 2012 Aug;12(8):2079-87).

[0013] However, paradoxically, immunosuppressants currently used in autoimmune diseases and allogeneic islet transplantation cause various adverse effects in recipients when used long-term. Therefore, the development of novel immunosuppressants (or immunosuppressive antibodies) with fewer side effects and specific immune cell types is needed.

[0014] In other words, both anti-CD154 and anti-CD40 antibodies can treat autoimmune diseases, inflammatory diseases, and cell / organ transplantation through mechanisms that suppress antibody production in B cells and suppress T cell activation, thereby inducing long-term survival of transplanted cells / organs. Blocking CD40-CD154 signaling in immune cells is a key immunosuppressive therapy, and blocking the CD40 / CD40L interaction can prevent transplant rejection and treat autoimmune diseases.

[0015] Korean Patent Publication No. 10-2021-0093968 discloses an antagonistic CD40 monoclonal antibody that binds to CD40 and does not exhibit CD40 agonist activity, and uses thereof; Korean Patent Publication No. 10-2019-0028508 discloses a multispecific antibody that binds to human CD40 and human CD137; and Korean Patent Registration No. 10-2198998 discloses an anti-CD40 antibody that directly targets CD40 rather than CD40 ligand, and exhibits an excellent antagonistic effect by blocking CD40-CD154 signaling without stimulating platelets, and uses thereof.

[0016] However, there is still a need for the development of therapeutically and clinically relevant anti-CD40 antibodies that can effectively inhibit the CD40 / CD40L interaction.

[0017] Against this backdrop, the inventors of the present invention have made extensive efforts to develop an antibody that blocks CD40-CD40L signaling, and have confirmed that a novel anti-CD40 antibody exhibits an excellent antagonistic effect by targeting CD40 and blocking CD40-CD40L signaling, thereby completing the present invention.

[0018] One object of the present invention is to provide an anti-CD40 antibody or antigen-binding fragment thereof, comprising complementarity determining regions (CDRs) of (i) a heavy chain complementarity determining region 1 (HCDR1) of the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 80% sequence identity therewith; (ii) a HCDR2 of the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 80% sequence identity therewith; (iii) a HCDR3 of the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 80% sequence identity therewith; (iv) a light chain complementarity determining region 1 (LCDR1) of the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 80% sequence identity therewith; (v) a LCDR2 of the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 80% sequence identity therewith; and (vi) a LCDR3 of the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 80% sequence identity therewith.

[0019] Another object of the present invention is to provide a nucleic acid encoding the antibody.

[0020] Another object of the present invention is to provide a vector comprising the nucleic acid.

[0021] Another object of the present invention is to provide a host cell comprising the nucleic acid.

[0022] Another object of the present invention is to provide a method for producing an antibody, which comprises a step of culturing the host cell to express the antibody.

[0023] Another object of the present invention is to provide an antibody-drug complex comprising the antibody as an active ingredient.

[0024] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune diseases, which comprises the antibody or antigen-binding fragment as an active ingredient.

[0025] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating chronic inflammatory diseases, which comprises the antibody or antigen-binding fragment as an active ingredient.

[0026] Another object of the present invention is to provide a composition for suppressing immune rejection during transplantation, which comprises the antibody or antigen-binding fragment as an active ingredient.

[0027] Another object of the present invention is to provide a composition for reducing or preventing the formation of anti-drug antibodies (ADAs) against a therapeutic agent comprising the antibody or antigen-binding fragment as an active ingredient.

[0028] Unless otherwise defined, scientific and technical terms used in connection with the present invention have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms include plurals, and plural terms include the singular. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization disclosed herein are well known and commonly used in the art.

[0029] In order to achieve the above object, one embodiment of the present invention comprises (i) a heavy chain complementarity determining region 1 (HCDR1) of the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 80% sequence identity therewith; (ii) a HCDR2 of the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 80% (preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, or most preferably at least 99%; hereinafter, the same applies to portions related to sequence identity) sequence identity therewith; (iii) a HCDR3 of the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 80% sequence identity therewith; (iv) a light chain complementarity determining region 1 (LCDR1) of the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 80% sequence identity therewith; (v) a LCDR2 of the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 80% sequence identity therewith; and (vi) an anti-CD40 antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR) of LCDR3 having the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 80% sequence identity thereto.

[0030] The term "antibody" as used herein refers to a protein that binds to another molecule (antigen) through the variable regions of the light and heavy chains, and includes IgG, IgD, IgA, IgE, and IgM types. Antibodies include polyclonal antibodies, monoclonal antibodies, and multispecific antibodies. In addition, the antibodies of the present invention include monoclonal antibodies having various structural forms, for example, an intact antibody comprising two full-length heavy chains and two full-length light chains, as well as a fragment thereof with or without a constant region, a chimeric antibody, a human antibody, a humanized antibody, or other genetically engineered antibodies having the characteristics according to the present invention.

[0031] The term "antigen-binding fragment" as used herein refers to a portion of the above-mentioned intact antibody, which is a sequence that is at least one amino acid sequence shorter than the amino acid sequence of the intact antibody in terms of length. Functionally, it contains at least a part of the activity or function of the intact antibody or the parent antibody, and its type is, for example, Fab (Fragment for antigen)

[0032] binding), Fab', F(ab')2, Fv or single chain antibodies (SCA) (e.g. scFv or dsFv), bispecific scFv and diabodies, but are not limited thereto.

[0033] The term "variable region" as used herein refers to an antigen-binding site formed by portions of the heavy chain and light chain, and each variable region is composed of four frameworks (FRs) with conserved sequences and three complementarity determining regions (CDRs) with highly variable sequences. The CDRs of the immunoglobulin heavy chain variable region (VH) are referred to as HCDR1 to HCDR3, and the CDRs of the immunoglobulin light chain variable region (VL) are referred to as LCDR1 to LCDR3.

[0034] The term "complementarity-determining region" as used herein refers to the region that determines the specificity and binding affinity of an antibody for its antigen, and where the most sequence variation is found among antibodies. Among these, the CDR3 region exhibits the most significant variation, consisting of amino acid residues ranging from as short as 2 amino acids to as long as 26 or more. The portions of VH and VL other than the CDRs are framework residues. The framework of the antigen-binding polypeptide of the present disclosure may be a sequence found in naturally occurring human antibodies, or a common sequence found in various antibodies.

[0035] An antibody according to the present invention comprises an antigen-binding fragment, variant or derivative thereof. An antibody according to the present invention may be of any type, for example, IgG, IgE, IgM, IgD, IgA or IgY, and may also be of any class, for example, IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2, or a subclass thereof.

[0036] In the present invention, the anti-CD40 antibody may include a heavy chain variable region of the amino acid sequence of SEQ ID NO: 13 or a sequence having at least 80% sequence identity therewith, and a light chain variable region of the amino acid sequence of SEQ ID NO: 14 or a sequence having at least 80% sequence identity therewith.

[0037] The present invention includes amino acids in which conservative substitutions have occurred in SEQ ID NOs: 1 to 8. In the present invention, substitutions have occurred in less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or 1 amino acid.

[0038] The term "conservative substitution" as used herein is a widely used term in the art to refer to the replacement of one amino acid with another amino acid of similar characteristics. Similar characteristics include, for example, size, hydrophobicity, or charge. Amino acids are typically classified based on the electrical properties of their side chains as having positively charged side chains, negatively charged side chains, uncharged side chains, or hydrophobic side chains. For example, conservative substitutions include substitutions of leucine (Leu) for isoleucine (Ile), arginine (Arg) for lysine (Lys), phenylalanine (Phe) for tryptophan (Trp), aspartic acid (Asp) for glutamic acid (Glu), or serine (Ser) for threonine (Thr), or vice versa. Conservative substitutions in CDR sequences generally do not fundamentally affect the function of the CDR.

[0039] Methods for substituting such sequences are known in the art, see, for example, Sambrook, Molecular Cloning A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory (2012) NY.

[0040] In the present invention, the anti-CD40 antibody or antigen-binding fragment thereof can be produced by a hybridoma cell line deposited under the deposit number KCLRF-BP-00523.

[0041] In the present invention, the anti-CD40 antibody may be selected from the group consisting of monoclonal antibodies, chimeric antibodies, primatized antibodies, humanized antibodies, and human antibodies, but is not limited thereto.

[0042] The anti-CD40 antibody or antigen-binding fragment thereof of the present invention may be a chimeric antibody. The term "chimeric antibody" as used herein refers to an antibody in which at least a portion of the variable region, i.e., the antigen-binding site, and the constant region (including the CL1 region for a light chain and the CH1, CH2, and CH3 regions for a heavy chain) are derived from different species. For example, the variable region may be mouse-derived and the constant region may be human-derived. Alternatively, the term also refers to a class-switched antibody, e.g., an antibody that has switched from an IgG type to an IgE type.

[0043] Additionally, the anti-CD40 antibody or antigen-binding fragment thereof of the present invention may be a humanized antibody. The term "humanized antibody" as used herein means that the antibody framework is a human antibody, and a portion of the CDR region is modified to include only the portions essential for specific antigen binding among the CDRs of the species from which the original antibody molecule was derived. For example, the CDR region and the light and heavy chain frameworks of a monkey or mouse antibody, excluding the portions essential for specific antigen binding, are replaced with those of a human antibody.

[0044] Furthermore, according to the present invention, the antibody of the present invention can be produced in various forms. The antibody of the present invention can be produced as a multifunctional fusion antibody, such as a CD40+CD40L fusion antibody, by fusing Fabs with different functions. In addition, the light and heavy chain variable regions obtained from the Fab antibody can be recombined with a human-derived constant region to provide a whole antibody.

[0045] Additionally, the anti-CD40 antibody or antigen-binding fragment thereof of the present invention may be a monoclonal antibody. The term "monoclonal antibody" or "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, wherein the monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.

[0046] Monoclonal antibodies are produced by fusing myeloma cells with spleen cells derived from immunized mammals, and can be produced by various methods known in the art.

[0047] The antibody according to the present invention may be provided conjugated with a functional substance selected from the group consisting of therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological modifiers, drugs, and PEG (polyethylene glycol), depending on the specific purpose. Furthermore, the antibody may be manufactured using various methods, depending on the type of conjugated substance.

[0048] Any of the above therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological modifiers and drugs commonly used in the art can be used as long as they can achieve the intended effect.

[0049] Antibody fragments can be obtained by treatment with pepsin or papain. An F(ab')2 fragment can be obtained by treating an intact antibody with pepsin, and subsequent treatment with a thiol reducing agent can yield a Fab fragment comprising portions of the light and heavy chains. A Fab fragment can also be obtained by treating an intact antibody with papain. For example, an antibody produced from the hybridoma of the present invention can be treated with pepsin or papain to produce an antibody fragment that specifically recognizes CD40, such as an F(ab')2 or Fab.

[0050] An Fv fragment is an antibody fragment composed only of the variable regions of the heavy and light chains, and the two variable regions can be linked by non-covalent or covalent bonds such as a chemical cross-linker or an intermolecular disulfide bond (Inbar et al (1972) PNAS 69:2659-2662). For example, an antibody that specifically recognizes CD40 can be produced by isolating only the variable regions of the heavy and light chains by enzymatic treatment of an antibody produced from the hybridoma of the present invention or by using recombinant DNA technology.

[0051] The SCA fragment can be produced by enzymatic treatment or genetic engineering, and is an antibody fragment in which a light chain variable region and a heavy chain variable region are linked by a linker such as a polypeptide. For a method for producing ScFv, reference can be made to those described in, for example, US Patent No. 4,936,778 or US Patent No. 5,892,019, and an antibody that specifically recognizes CD40 can be produced by enzymatic treatment of an antibody produced from the hybridoma of the present invention or by recombinant DNA technology, for example, by producing a vector containing a nucleic acid sequence encoding the heavy chain and / or light chain variable region of the antibody and expressing it in an appropriate cell.

[0052] As used herein, the term "binding" or "specific binding" refers to the affinity of the antibody or antibody composition of the present invention for the antigen. In antigen-antibody binding, "specific binding" typically means a dissociation constant (Kd) of 1x10 -5 Less than M or 1x10 -6 Less than M or 1x10 -7If it is less than M, it can be distinguished from nonspecific background binding. Specific binding can be detected by methods known in the art, such as ELISA, SPR (Surface plasmon resonance), immunoprecipitation, coprecipitation, etc., and includes an appropriate control that can distinguish between nonspecific binding and specific binding.

[0053] In the present invention, the anti-CD40 antibody may be selected from the group consisting of a multimeric antibody, a heterodimeric antibody, a semidimeric antibody, a tetravalent antibody, a bispecific antibody, and a single-chain antibody, but is not limited thereto.

[0054] The antibodies of the present invention, including intact antibodies or fragments thereof as described above, may exist as multimers, such as dimers, trimers, tetramers, and pentamers, which possess at least a portion of the antigen-binding capacity of monomers. Such multimers also include homomultimers or heteromultimers. Antibody multimers possess superior antigen-binding capacity compared to monomers because they contain multiple antigen-binding sites. Antibody multimers are also convenient for producing multifunctional (bifunctional, trifunctional, and tetrafunctional) antibodies.

[0055] The term "multifunctional" as used herein refers to an antibody or antibody composition having two or more activities or functions (e.g., antigen binding ability, enzymatic activity, ligand or receptor binding ability), for example, the antibodies of the present invention can be linked to polypeptides having enzymatic activity, such as luciferase, acetyltransferase, galactosidase, etc. Multifunctional antibodies also include antibodies in multivalent or multispecific (bispecific, trispecific, etc.) forms.

[0056] The term "multispecific" as used herein refers to a variable region capable of binding to two or more different epitopes. The two or more epitopes may be present on a single antigen or on different antigens.

[0057] In the present invention, the antigen binding fragment may be selected from the group consisting of Fab, F(ab)2, Fab', F(ab)2, F(ab')3, Fd, Fv, and domain antibodies, but is not limited thereto.

[0058] Another embodiment of the present invention provides a nucleic acid encoding the antibody.

[0059] Nucleic acids include, for example, DNA, cDNA, RNA, or recombinant or synthetic DNA or RNA. In one embodiment, the nucleic acid molecule is cDNA. The nucleic acid may also be a corresponding genomic DNA or a fragment thereof. Nucleic acid sequences encoding an antibody or a portion thereof or a fragment thereof according to the present invention may differ due to redundancy in the nucleic acid sequence encoding amino acids, and such sequences are also encompassed herein.

[0060] Another embodiment of the present invention provides a vector comprising the nucleic acid.

[0061] Another embodiment of the present invention provides a host cell comprising the nucleic acid.

[0062] Another embodiment of the present invention provides a method for producing an antibody, comprising a step of culturing the host cell to express the antibody.

[0063] Vectors that can be used herein include, for example, phage, plasmid, replication-competent or replication-deficient viral or retroviral vectors. Nucleic acid molecules according to the present invention can be introduced into various known vectors. For example, vectors for prokaryotic cells include, but are not limited to, pUC series vectors, pBluescript (Stratagene), pET series vectors (Novagen), or pCRTOPO (Invitrogen) vectors, and vectors for eukaryotic cells include, but are not limited to, pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMCI neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, plZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems), pTriEx-Hygro (Novagen), and pCINeo (Promega) vectors.

[0064] The vector according to the present invention can be introduced into various known prokaryotic or eukaryotic cells by known transformation or transfection methods. Upon introduction into the cell, it can be integrated into the genome of the host cell or can exist in the form of extrachromosomes.

[0065] Prokaryotic cells that can be used include cells belonging to the Escherichia, Bacillus, Streptomyces and Salmonella genera; eukaryotic cells include, but are not limited to, mammalian cells such as Hela, HEK293, H9, Jurkat, mouse NIH3T3, C127, Cos1, Cos7 and CV1, mouse C2C12, BHK, CHO cells; fungal cells such as Saccharomyces cerevisiae or Pichia pastoris; and insect cells such as Drosophila S2 and Spodoptera Sf9.

[0066] The antibody of the present invention can be produced using a recombinant method according to a known method. In the case of a recombinant method, a nucleic acid sequence encoding a heavy chain of an antibody according to the present invention and an antibody encoding a light chain of the antibody are cloned into one or two expression vectors, and then the vectors are transferred into a eukaryotic host cell to express the antibody, and the antibody can be obtained from the host cell or medium. Recombinant methods including the production of such vectors, expression of proteins in cells from the produced vectors, and isolation of the proteins are known in the art, and reference can be made to those described in, for example, Kaufman, RJ, Mol (2000) Biotechnol 16:151-160. A vector encoding an antibody of the present invention can be expressed in an appropriate host cell, such as CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, yeast, or Escherichia coli, and the antibody can be obtained from a cell lysate or medium.

[0067] The nucleic acid sequence encoding the antibody of the present invention or a fragment thereof can be isolated from the hybridoma cells disclosed herein by a conventional method, and then the sequence can be analyzed. The isolated nucleic acid sequence can then be cloned into an appropriate expression vector as described above, and then transfected into HEK293 cells, CHO cells, or NS0 cells that do not produce antibodies, to produce a recombinant antibody in the host cells. The nucleic acid encoding the antibody of the present invention or a fragment thereof is introduced into an expression vector comprising a promoter, a translation initiation site, a 3' untranslated site, a polyadenylation signal, and a transcription termination signal. The light chain and heavy chain can be introduced into a single vector or separate vectors.

[0068] Another embodiment of the present invention provides an antibody-drug complex comprising the antibody as an active ingredient.

[0069] The term "antibody-drug conjugate (ADC)" used in the present invention refers to a form in which a drug and an antibody are chemically linked without reducing the biological activity of the antibody and the drug. In the present invention, the antibody-drug conjugate refers to a form in which a drug is bound to an amino acid residue at the N-terminus of the heavy chain or / and light chain of an antibody, specifically, a form in which a drug is bound to an N-terminal α-amine group of the heavy chain or / and light chain of an antibody.

[0070] The term "drug" used in the present invention can mean any substance that has a specific biological activity in cells, and this is a concept that includes compounds, DNA, RNA, peptides, etc. It can be in a form that includes a reactive group that can react with an α-amine group to crosslink, and also includes a form in which a linker that includes a reactive group that can react with an α-amine group to crosslink is connected. In this case, the drug can be position-specifically bound to the N-terminal amino acid residue of the antibody by the linker, but is not limited thereto. The linker refers to a chemical part that includes an atomic chain that covalently binds the drug to the antibody. The linker is manufactured in a form that is linked to the drug, and has a reactive group that can link to the antibody at the end of the linker.

[0071] In the present invention, the drug includes all substances that can cause activation of specific signal transduction, including cell death, cell proliferation, immune activation, and immune suppression, or inhibition of specific signal transduction, and the drug may be a cytotoxic drug or an immunosuppressant in particular.

[0072] In the present invention, the cytotoxic drug includes a chemotherapeutic agent capable of functioning as a microtubulin structure formation inhibitor, a meiosis inhibitor, an RNA polymerase inhibitor, a topoisomerase inhibitor, a DNA intercalator, a DNA alkylator, a ribosome inhibitor, a protein toxin capable of functioning enzymatically, and a radioisotope.Examples include maytansinoids, auristatin, dolastatin, tubulosin, calicheamicin, pyrrolobenzodiazepines, doxorubicin, duocamycin, carboplatin (paraplatin), cisplatin, cyclophosphamide, ifosfamide, nidran, nitrogen mustard (mechlorethamine HCL), bleomycin, mitomycin C, cytarabine, flurouracil, gemcitabine, Trimetrexate, methotrexate, etoposide, vinblastine, vinorelbine, alimta, altretamine, procarbazine, taxol, taxotere, topotecan, irinotecan, trichothecene, CC1065, alpha-amanitin, other enediyne antibiotics, extramycotic toxins and plant toxins. In addition, in the case of a compound, its stereoisomers and derivatives are also included. In addition, the auristatin may be, but is not limited to, monomethyl auristatin E or monomethyl auristatin F.

[0073] In the present invention, the drug may be selected from the group consisting of an anticancer agent, an aptamer, an antisense oligonucleotide, a small interfering ribonucleic acid, and a microRNA, but is not limited thereto.

[0074] Another embodiment of the present invention relates to a pharmaceutical composition for preventing or treating an autoimmune disease, comprising the antibody or antigen-binding fragment as an active ingredient.

[0075] In the present invention, the autoimmune disease is type 1 diabetes, new-onset type 1 diabetes, lymphoma, chronic lymphocytic leukemia, alopecia areata, ankylosing spondylitis, Sjogren's syndrome, Evans syndrome, lupus, autoimmune nephritis 2, antiphospholipid syndrome, autoimmune Addison's disease, adrenal autoimmune disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune disorder syndrome, chronic inflammatory demyelinating polyneuropathy, Chorionic-Strauss syndrome, cicatricial pemphigoid, Crest syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, combined cryoglobulinemia, fibromyalgia-fibromyositis, Glomerulonephritis, focal segmental glomerulosclerosis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, inflammatory bowel disease, IgA neuritis, neuromyelitis optica, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, amyotrophic lateral sclerosis, multiple sclerosis, hidradenitis suppurativa, myasthenia gravis, pemphigus vulgaris, pernicious anemia, microscopic polyangiitis, polyarteritis nodosa, polychondritis, autoimmune polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, hepatic fibrosis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, It may be a disease selected from the group consisting of, but not limited to, scleroderma, stiff-person syndrome, systemic lupus erythematosus, lupus erythematosus, Tagayasu arteritis, transient arteritis, giant cell arteritis, ulcerative colitis, uveitis, vitiligo, graft versus host disease, opsoclonus-myoclonus syndrome, Addison's disease, and Wegener's granulomatosis.

[0076] Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating a chronic inflammatory disease, comprising the antibody or antigen-binding fragment as an active ingredient.

[0077] As used herein, the term "prevention" may refer to any action that inhibits or delays the onset of a disease by administering the composition. Furthermore, "treatment" may refer to any action that improves or beneficially alters the symptoms of a disease by administering the composition.

[0078] In the present invention, the chronic inflammatory disease may be a disease selected from the group consisting of inflammatory bowel disease, Crohn's disease, ulcerative colitis, pancreatitis, chronic hepatitis, esophagitis, gastritis, colitis, pneumonia, bronchitis, pharyngitis, myocardial infarction, heart failure, Alzheimer's, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, asthma, renal failure, psoriasis, anemia, diabetes, and fibrosis, but is not limited thereto.

[0079] Another embodiment of the present invention provides a composition for suppressing immune rejection during transplantation, comprising the antibody or antigen-binding fragment as an active ingredient.

[0080] The term "immune rejection during transplantation" used herein refers to the destruction of foreign cells or tissues inoculated or transplanted from the donor to the recipient due to a specific immune response. Grafts transplanted from donors with different graft antigens cannot engraft using the recipient's immunological mechanisms, and this rejection is caused by cellular and humoral immunity. In skin or solid tumor transplants, the main cause of rejection is cellular immunity. That is, the infusion of serum from an animal that rejected the transplanted skin piece does not promote rejection of the same graft as the recipient animal, but the infusion of lymphoid cells shortens the period of graft engraftment. In addition, animals that have had their thymocytes removed during their neonatal period do not show skin rejection. On the other hand, humoral antibodies also act as the main cause of renal rejection, and especially when the recipient already has antibodies against donor lymphocytes, microthrombi are formed in the glomeruli, causing hyperacute rejection. Acute rejection occurs between 7 and 21 days after transplantation, involving both cellular immunity and humoral antibodies. Chronic rejection occurs several months to years after transplantation, and is known to be caused by the deposition of immunoglobulins and C3 in the glomerular basement membrane.

[0081] In the present invention, the transplantation may be a transplantation of at least one selected from the group consisting of allogeneic cells, xenogeneic cells, allogeneic tissues, xenogeneic tissues, allogeneic organs, and xenogeneic organs.

[0082] In the present invention, the transplant may suppress immune rejection during transplantation of skin, blood, cornea, liver, lung, intestine, pancreas, heart, kidney, bone marrow, stem cells or progenitor cells.

[0083] The composition of the present invention is formulated with a pharmaceutically acceptable carrier, optionally an excipient or stabilizer.

[0084] The term "pharmaceutically acceptable carrier" used in the present invention refers to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the administered compound. In a composition formulated as a liquid solution, acceptable pharmaceutical carriers include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0085] The above composition may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0086] The composition according to the present invention can be administered via various routes known in the art, and it will be apparent to those skilled in the art that the method and route of administration may vary depending on the desired effect. The composition of the present invention can be administered orally or parenterally, and is preferably administered parenterally. When administered parenterally, the composition of the present invention can be administered via intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. It is preferable that the route of administration of the composition of the present invention be determined depending on the type of disease to which it is applied.

[0087] The effective dosage and administration period of the composition according to the present invention may vary depending on the desired therapeutic effect, taking into account the specific patient, the type of antibody contained in the composition, the administration method, etc., and must not cause toxicity to the patient. The actual dosage for each patient should be selected by taking into account various factors such as the activity of the composition used, the administration route, the administration time, the secretion rate, other drugs used together, gender, age, body weight, general health condition, underlying disease, etc. In one embodiment, the antibody of the present invention may be administered in an amount of about 1 to 100 mg / kg body weight, for example, about 10, 20, 30, 40, or 50 mg / kg body weight, for the treatment or prevention of a disease, but in some cases, it may be administered in an amount as high as about 100 mg / kg.

[0088] The administration interval of the composition according to the present invention can be administered at an appropriate interval, such as daily, weekly, or monthly, taking into account the half-life of the antibody to be administered.

[0089] In addition, the composition according to the present invention can be formulated into a pharmaceutically acceptable appropriate dosage form, for example, a hydrated form, for example, an aqueous solution, or a lyophilized form, regardless of the route of administration.

[0090] Another embodiment of the present invention provides a composition for reducing or preventing the formation of anti-drug antibodies (ADAs) to a therapeutic agent comprising an antibody or antigen-binding fragment according to the present invention as an active ingredient.

[0091] The term "anti-drug antibody" as used herein refers to an antibody that can be directed against any region of a drug, such as, for example, the variable domain, constant domain, or glycostructure of the drug. Such anti-drug antibodies can arise as an immunogenic response in a patient during antibody therapy (see Pan, Y., et al., FASEB J. 9 (1995) 43-49). Most anti-drug antibodies bind to one or more complementarity-determining regions of the drug. The affinity of the anti-drug antibody for the drug's antigen is generally lower than the affinity of the drug for its target antigen.

[0092] The term "drug" as used herein refers to a therapeutic protein or a therapeutically effective portion thereof that can be administered to a subject for the treatment of a disease.

[0093] In the present invention, the therapeutic agent may preferably be an antibody therapeutic agent, a cell therapeutic agent, a gene therapeutic agent, a biologic agent, or a peptide therapeutic agent, but is not limited thereto.

[0094] Another embodiment of the present invention provides a chimeric antigen receptor (CAR) comprising an anti-CD40 antibody or an antigen-binding fragment thereof according to the present invention as an active ingredient, and a T cell (CAR-T) or NK cell (CAR-NK) engineered to express the same.

[0095] The term "chimeric antigen receptor" as used herein refers to a fusion protein comprising an extracellular domain that binds an antigen, a transmembrane domain derived from a polypeptide different from the extracellular domain, and at least one intracellular domain. The chimeric antigen receptor is also called a chimeric receptor, a T-body, or a chimeric immune receptor (CIR). The extracellular domain that binds an antigen refers to any oligo or polypeptide that can bind to any antigen, and the intracellular domain refers to any oligo or polypeptide that is known to function as a domain that transmits a signal that results in the activation or inhibition of a biological process within a cell.

[0096] Another embodiment of the present invention provides a method for preventing or treating an autoimmune disease, a chronic inflammatory disease, etc., comprising administering to a subject in need of treatment a composition comprising an anti-CD40 antibody or an antigen-binding fragment thereof according to the present invention as an active ingredient.

[0097] In the present invention, the subject is an animal that is the subject of treatment, observation or experiment, preferably a mammal including a cow, pig, sheep, chicken, dog, human, etc., a bird, etc., and an subject whose disease is treated by administration of the composition of the present invention is included without limitation.

[0098] The novel anti-CD40 antibody of the present invention exhibits an excellent antagonistic effect by blocking CD40-CD40L signaling, and can be widely utilized as a preparation for preventing or treating various diseases such as autoimmune diseases and chronic inflammatory diseases, and as a preparation for suppressing immune rejection during transplantation.

[0099] Figure 1 shows the CDR sequence of the 8G1-35-31 antibody according to one embodiment of the present invention.

[0100] Figure 2 shows the results of a CD40 antigen-specific binding test according to one embodiment of the present invention.

[0101] Figure 3 shows the results of confirming binding to a CD40 expressing cell line and a Raji B cell line according to one embodiment of the present invention.

[0102] Figure 4 shows the results of confirming the concentration-dependent binding affinity of the 8G1-35-31 antibody to the human CD40 antigen according to one embodiment of the present invention.

[0103] Figure 5 shows the CD40-CD40L binding blocking effect of the 8G1-35-31 antibody according to one embodiment of the present invention.

[0104] Figure 6 shows the effect of inhibiting T-dependent B cell activation by treatment with 8G1-35-31 antibody according to one embodiment of the present invention.

[0105] Figure 7 shows the effect of inhibiting T-dependent B cell proliferation by treatment with 8G1-35-31 antibody according to one embodiment of the present invention.

[0106] Figure 8 shows the results of a non-agonistic property test of the 8G1-35-31 antibody according to one embodiment of the present invention.

[0107] Figure 9 shows the results of a binding affinity test for the linear CD40 antigen of the 8G1-35-31 antibody according to one embodiment of the present invention.

[0108] Figure 10 shows the results of an interspecies cross-linking strength test according to one embodiment of the present invention.

[0109] Figure 11 shows the KLH-specific antibody secretion inhibitory effect of the 8G1-35-31 antibody according to one embodiment of the present invention.

[0110] Figure 12 shows the inhibitory effect of 8G1-35-31 antibody on KLH-specific Th1 cell activation according to one embodiment of the present invention.

[0111] Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended solely to illustrate the present invention and should not be construed as limiting the scope of the present invention. The following examples are provided to more fully explain the present invention to those of average skill in the art.

[0112]

[0113] Example 1: Production of hybridomas producing anti-CD40 antibodies

[0114] The present inventors prepared, screened, and analyzed novel anti-CD40 antibodies that target CD40 and block CD40-CD154 (CD40L) signaling as follows.

[0115] To develop novel anti-CD40 antibodies, 1x10 human CD40 antigen (100 μg / mouse) or CD40-expressing CHO cell line was injected into each 6-week-old Balb / c female mouse. 7 The mice were injected intraperitoneally (IP) three times at three-week intervals, and blood samples were collected from a vein to separate the serum. The serum diluted with CD40-expressing CHO cells was added to the separated serum, reacted at 4°C for 30 minutes, and then 3 ml of PBS was added and centrifuged at 1500 rpm for 3 minutes to wash away unbound antibodies. To confirm the bound antibodies, a 200-fold dilution of the secondary antibody goat anti-Mouse Ig-FITC (Invitrogen) was added, reacted at 4°C for 15 minutes, washed with 3 ml of PBS in the same manner as above, and then measured using a flow cytometer to confirm the titer for the CD40-expressing CHO cell line. As a result of confirming the titer for the CD40-expressing CHO cell line using a flow cytometer, it was confirmed that the positivity for the CD40-expressing CHO cell line was high in the serum immunized with the CD40-expressing CHO cell line.

[0116] As described above, the spleen of the immunized mouse was excised to obtain a single cell suspension, which was washed twice with RPMI (GIBCO), and then mixed with 0.4% trypan blue (Sigma) in a 1:1 (v / v) ratio. The number of cells was counted using the trypan blue staining method, which measures unstained cells under a microscope. X63 moue myeloma cell line (ATCC CRL-1580) was used as the cell fusion partner cell, and the number of cells was counted after washing in the same manner as the spleen cells.

[0117] The above bone marrow (myeloma) cells and spleen cells were mixed in a 1:5 ratio, centrifuged, and the supernatant was removed. 1 ml of 50% PEG (polyethylene glycol) 1500, preheated to 37°C, was slowly added over 1 minute. After standing for about 1 minute, RPMI medium was slowly added and diluted stepwise. After centrifugation, the cells were suspended in RPMI (20% FBS) containing 1x HAT (hypoxanthine-aminopterin-thymidine), dispensed into a 96-well plate at 150 μl / well, and cultured in a 5% CO2 incubator at 37°C. After fusion, HAT feeding was performed for a certain period of time, and when wells forming colonies were observed, 150 μl of HT medium was added and cultured for 2-7 days in a 5% CO2 incubator at 37°C to obtain the culture solution. The culture medium was subjected to fluorescent staining using a CD40-expressing CHO cell line and analyzed using a flow cytometer.

[0118] 100 ㎕ of hybridoma culture supernatant was added to CD40 expressing CHO cell line and reacted at 4℃ for 30 minutes. Then, 3 ml of PBS was added and centrifuged at 1500 rpm for 3 minutes to wash away unbound antibodies. To confirm bound antibodies, goat anti-Mouse Ig-FITC (Invitrogen), a secondary antibody diluted 200 times, was added and reacted at 4℃ for 15 minutes. After washing with 3 ml of PBS in the same manner as above, measurements were taken using a flow cytometer.

[0119] In the above method, a monoclonal antibody that is negative for CHO cells but positive for CD40-expressing CHO cell lines or B cell lines (Raji) was selected, and finally, a single colony of 8G1-35-31 hybridoma cells was secured by repeating the limiting dilution experiment three times.

[0120] The above hybridoma was selected as the optimal hybridoma and designated as 8G1-35-31.

[0121] The above hybridoma 8G1-35-31 was deposited with the Korean Cell Line Research Foundation on June 1, 2023, and was assigned the following accession number: KCLRF-BP-00523.

[0122]

[0123] Example 2: Sequence verification of anti-CD40 antibodies

[0124] To analyze the structure of the antibody produced according to the present invention, the inventors cloned nucleic acids encoding heavy and light chain fragments from hybridomas producing anti-CD40 antibodies.

[0125] Cloning and sequencing were performed as follows.

[0126] For gene cloning, RNA was extracted from the 8G1-35-31 hybridoma cells obtained in Example 1 using an RNA miniprep kit (Qiagen) according to the manufacturer's instructions, and then cDNA was synthesized by performing PCR.

[0127] PCR conditions and cloning methods for heavy chain cDNA and light chain cDNA synthesis are as follows:

[0128] The CD40 antigen-specific antibody, RM8G-1 antibody gene, was cloned using the Mouse Ig-Primer Set (Millipore, Cat #: 69831). PCR was performed using the Mouse Ig-Primer Set from RNA isolated from RM8G-1 hybridomas, which was then inserted into the pGem-T vector (Promega, Cat #: A3600). The DNA base sequence was confirmed through sequencing, and the mouse antibody gene was confirmed through the IMGT site (wwwimgtorg).

[0129] The sequences of the heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the sequences of the light chain variable regions LCDR1, LCDR2, and LCDR3 are as shown in Table 1 below. In addition, the entire sequences of the heavy chain variable region and the light chain variable region are as shown in SEQ ID NOs: 13 and 14, respectively (Fig. 1).

[0130] IMGT methodKabat methodHCDR1GFSLSYYG(SEQ ID NO: 1)YYGVH(SEQ ID NO: 7)HCDR2IWSGGRK(SEQ ID NO: 2)VIWSGGRKDFNAAFIS(SEQ ID NO: 8)HCDR3ASMIREYYAMDY(SEQ ID NO: 3)MIREYYAMDY(SEQ ID NO: 9)LCDR1SSVSSSY(SEQ ID NO: 4)TASSSVSSSYLH(SEQ ID NO: 10)LCDR2STS(SEQ ID NO: 5)STSKLAS(SEQ ID NO: 11)LCDR3HQYHRSPWT(SEQ ID NO: 6)HQYHRSPWT(SEQ ID NO: 12)

[0131] Example 3: Confirmation of the antagonistic effect of anti-CD40 antibodies

[0132] 3-1: CD40 antigen-specific binding test

[0133] To investigate whether the 8G1-35-31 antibody specifically binds to human CD40 antigen (hCD40), flow cytometry analysis was performed using Chinese hamster ovary cells (CHO) and hCD40 antigen-expressing CD40-CHO cells. CHO cells and 2X10 5 CD40-CHO cells were treated with isotype control (mouse IgG-FITC, Biolegend), commercial anti-CD40 antibody (5C3, BD), 8G1-35-31-FITC (1 ug / ml), and 8G1-35-31-FITC (10 ug / ml), respectively, and incubated on ice for 30 minutes. After washing with FACS buffer (5% BSA, 0.01% Sodium Azide, PBS, pH 7.4), the cells were analyzed using a BD FACSCANTOII. As a result, CD40 staining was not observed in CHO cells that do not express CD40, but CD40 antigen was detected in samples treated with the commercial antibody, 8G1-35-31, to CD40-CHO cells. This confirmed that 8G1-35-31 specifically binds to the human CD40 antigen (Fig. 2).

[0134] 3-2: Confirmation of binding to CD40-expressing human B cell line (Raji B cells)

[0135] To confirm whether the 8G1-35-31 antibody binds to human B cells expressing CD40 antigen, flow cytometry analysis was performed using Raji B (ATCC) cells. 2X10 5Raji B cells were treated with 8G1-35-31 at concentrations of 1 μg / ml and 10 μg / ml, respectively, and incubated on ice for 30 minutes. After washing twice with FACS buffer, anti-mouse IgG-FITC (Sigma) was treated at a concentration of 1 μg / ml and incubated on ice for 40 minutes. After washing twice with FACS buffer, analysis was performed using a BD FACSCANTOII. As a result, it was confirmed that the 8G1-35-31 antibody binds to CD40-expressing Raji B cells (Fig. 3).

[0136] 3-3: Confirmation of concentration-dependent binding affinity

[0137] To confirm the concentration-dependent binding affinity of the 8G1-35-31 antibody, an Enzyme-linked Immunosorbent Assay (ELISA) was performed. CD40-his (1 μg / mL, sinobiological, Cat#. 10774-H08H) diluted in ELISA Phosphate Coating Buffer (Thermo Scientific, Cat#. CB07100) was added to a 96-well ELISA plate (Thermo Scientific, Cat#. 439454) and incubated at 37°C for 1 hour. The diluted 8G1-35-31 sample solution to be used in the experiment (0–20 μg / mL) was added at a rate of 100 μL and incubated at 37°C for 1 hour. After washing three times with 200 μl of PBST (10 mM sodium phosphate, 0.15 M NaCl, 0.05% Tween™20 buffer at pH 7.5), 50 μl of diluted goat anti-human IgG Fc cross-adsorbed secondary antibody (IgG Fc Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody)-HRP (1:5000) solution was added and incubated at 37°C for 30 minutes. After washing three times with 200 μl of PBST, 50 μL of TMB substrate solution was dispensed each time, covered with foil to block light, and incubated at room temperature for 5 minutes. Afterwards, 50 μl of stop solution (Thermo Scientific, Cat# N600) was added to stop the reaction, and the absorbance was measured at a wavelength of 450 nm using a microplate ELISA reader (Varioskan Lux microplate reader, Thermo fisher scientific, Cat#. VLBL0TD2). As a result, it was confirmed that the 8G1-35-31 antibody showed a concentration-dependent binding pattern in which the binding affinity increased depending on the amount of CD40 antigen (Fig. 4).

[0138] 3-4: CD40-CD40L binding blocking effect using reporter cells

[0139] The CD40L-CD40 binding blocking effect was quantified at the cell level using HEK-Blue human CD40L-SEAP reporter cells (HEK-Blue CD40L, InvivoGen, Cat#. hkb-cd40). That is, 2.5x10 5 / ml HEK-Blue CD40L cell suspension was dispensed, and recombinant CD40L solution (InvivoGen, Cat#. rcyec-hcd40) was added at a concentration of / ml. Human-mouse chimeric 8G1-35-31 antibody was manufactured and produced by combining the base sequence of the variable region of the mouse 8G1-35-31 antibody and the base sequence of the human Fc region. The quantified 8G1-35-31 antibody and chimeric 8G1-35-31 antibody were added at various concentrations to the binding blocking experiment model using the reporter cells (three replicates for each experimental group) and cultured in an incubator at 37°C in a humidified atmosphere with 5% CO2 for 20 hours. Afterwards, 20 μl of the cultured supernatant was transferred to a 96-well plate, 180 μl of QUANTI-Blue™Solution (InvivoGen, Cat#. rep-qbs) was added, and the reaction was performed at room temperature for 30 minutes. The binding blocking power was measured at 620 nm using a microplate reader. The equation for calculating the binding blocking power is as follows: Mathematical Formula 1.

[0140] [Mathematical Formula 1]

[0141] 1-[(OD value of the experimental group treated with both antibody and sCD40L - OD value of the experimental group treated with neither antibody nor sCD40L) / (OD value of the experimental group treated with only sCD40L - OD value of the experimental group treated with neither antibody nor sCD40L)] X 100

[0142] As a result, it was confirmed that the 8G1-35-31 antibody could effectively block CD40-CD40L signaling in a concentration-dependent manner in a binding blocking experiment using CD40-CD40L binding reporter cells (Fig. 5).

[0143] 3-5: Inhibitory effect on T-cell-dependent B cell activation and proliferation using PBMC

[0144] The degree of inhibition of CD86 expression, a B cell activating factor, by 8G1-35-31 drug in a T cell-dependent B cell activation model in human PBMC was quantified and confirmed. That is, activating anti-CD3 antibody (OKT3) and activating anti-CD28 antibody (CD28.6) were dissolved in phosphate-buffered saline (PBS, pH 7.4), added to a 96-well U-bottom plate at a concentration of 5 ug / ml, and coated at 4°C for 18 hours. The next day, human PBMCs stained with CFSE (2.5 uM) were seeded at 2.5 x 10 in a 96-well plate coated with anti-CD3 / anti-CD28 antibodies. 6 / ml concentration, and 8G1-35-31 antibody (10ug / ml, 100ug / ml) at each concentration was added and cultured for 4 or 5 days in a 37℃ humidified, 5% CO2 incubator. The positive control plate was coated with only anti-CD3 / anti-CD28 antibody and 8G1-35-31 was not added, and the negative control plate was coated with neither anti-CD3 / anti-CD28 antibody nor 8G1-35-31 antibody. After 4-5 days, the cultured cells were transferred to a FACS tube (USA, SPL-40205) and flow cytometry staining was performed. That is, FC-blocker (BioLegend, US, cat. no. 422302) was added to cultured cells washed with FACS buffer (PBS7.4, 0.5% BSA, 0.01% Sodium Azide), and after 15 minutes of reaction, they were stained with Live / Dead staining reagent (Thermofisher, US, cat. no. 65-0864-18), anti-mouse CD19 antibody (BioLegend, US, cat. no. 302216), and anti-mouse CD86 antibody (BD, US, cat. no. 560357) for 30 minutes. Afterwards, they were washed with FACS buffer and detected with BD FACS Canto II.

[0145] As a result, it was confirmed that the 8G1-35-31 antibody could very effectively block the activity and proliferation of human B cells (Fig. 6, Fig. 7).

[0146] 3-6: Test for non-agonistic properties of 8G1-35-31 antibody

[0147] To determine the effect of the drug itself on CD40-mediated signaling on the surface of immune cells, the following experiment was performed. Peripheral blood mononuclear cells (PBMCs) from two individuals were treated with 8G1-35-31 antibody at a concentration of 100 μg / ml and cultured in a humidified 5% CO2 incubator at 37°C for 3 days. CP870.893 (Pfizer) was treated at the same concentration as a positive control, and a group not treated with the antibody served as a negative control. After 3 days, the cultured cells were transferred to FACS tubes (USA, SPL-40205) and stained for flow cytometry analysis. That is, FC-blocker (BioLegend, USA, cat. no. 422302) was added to cultured cells washed with FACS buffer (PBS7.4, 0.5% BSA, 0.01% Sodium Azide), and after reaction for 15 minutes, they were stained with Live / Dead staining reagent (Thermofisher, USA, cat. no. 65-0864-18), anti-mouse CD19 antibody (BioLegend, USA, cat. no. 302216), and anti-mouse CD86 antibody (BD, USA, cat. no. 560357) for 30 minutes. The samples were detected with BD FACS Canto II. The experimental results confirmed that the 8G1-35-31 antibody has a non-functional property that does not activate human PBMCs by itself (Fig. 8).

[0148] 3-7: Characteristic analysis of CD40 sequence recognition

[0149] To investigate whether the 8G1-35-31 antibody recognizes the linear antigen, Western blot analysis was performed. CD40-CHO cells were washed once or twice with DPBS and lysed in 1X RIPA buffer (Biosesang, cat. No. R2002) on ice for 20 minutes. The protein concentration of the supernatant was quantified using a BCA assay kit (Thermo fisher scientific cat. No. 2161296), and samples under non-reducing and reducing conditions were prepared by adding 2-mercaptoethanol to 1X Bolt™LDS sample buffer. The samples were denatured at 100°C for 5 minutes, separated on a 12% Bis-Tris gel in Bolt™4, and transferred to a membrane. To confirm the transcription effect, the gel was stained with Coomassie Blue, the membrane was checked with Ponceau S for 1–2 minutes, the membrane was washed with 1X TBST, and then incubated with 8G1-35-31 antibody (5 ug / ml) at 4°C for 18 hours with agitation. After incubation with secondary antibody, anti-mouse IgG-HRP, for 1 hour, the membrane was reacted with ECL substrate (Thermo Scientific, cat. No. 32106) and the band was confirmed with ChemiDoc (BIO-RAD, US). As a result, it was confirmed that 8G1-35-31 antibody could bind to linear CD40 (Fig. 9).

[0150] 3-8: Investigation of interspecies cross-linking ability

[0151] To determine whether the 8G1-35-31 antibody binds to blood cells of other species besides human, flow cytometry was performed using PBMCs from primates such as cynomolgus, rhesus monkey, marmoset, and pig. Each cell was treated with Zombie Aqua (Biolegend), incubated at room temperature for 30 minutes, and then washed once with FACS buffer. The cells were then treated with the 8G1-35-31-FITC antibody (10 μg / ml) and incubated on ice for 30 minutes. After washing twice with FACS buffer, the cells were analyzed using a BD LSRII. The results confirmed that the 8G1-35-31 antibody exhibited cross-species binding to not only humans but also cynomolgus and rhesus monkeys (Fig. 10).

[0152] 3-9: Inhibitory effect of antigen-specific antibody production by B cells in hCD40 / hCD40L-expressing mice

[0153] The hCD40 / hCD40L double knock-in mice, which were transferred from Model organism (China), are mice that express human CD40 and CD40L, and are mice in which mouse CD40 and mouse CD40L are genetically replaced with human CD40 and CD40L. These mice (9 weeks old) were intraperitoneally treated with 10 mg / kg KLH (Sigma), and the following day, the control group was intraperitoneally treated with PBS, and the experimental group was intraperitoneally treated with 8G1-35-31 antibody (50 mg / kg). After one week, the control group was treated with KLH (10 mg / kg) and PBS (100 ul), and the experimental group was treated with KLH (10 mg / kg) and 8G1-35-31 antibody (50 mg / kg). After one week, plasma was isolated from peripheral blood and the production of anti-KLH antibodies was analyzed by ELISA. That is, KLH (100 ug) / PBS 100 ul was added to the ELISA plate and coated at 4℃ for 18 hours. The next day, it was washed with 1XPBST (PBS, Tween, Thermofisher scientific) and reacted with blocking buffer (Super Block, Thermofisher scientific) at 37℃ for 20 minutes. After washing three times with PBST, 100 ul of anti-KLH antibody standard solution (0-2.00 ng / ml) and plasma dilution solution (1: 1,000) were added and reacted at 37℃ for 1 hour. After washing three times with PBST again, 100 ul of goat-anti-mouse IgG (FC-specific)-HRP antibody dilution solution (1: 2,000) was added and reacted at 37℃ for 1 hour and the reaction was stopped with stop solution. 50 μl of TMB substrate (Thermofisher scientific) was added, reacted at room temperature for 10 minutes, and the optical density (OD) value was measured using a fluorometer (450 nm, Thermofisher scientific). As a result, it was confirmed that the 8G1-35-31 antibody could fundamentally block the production of anti-KLH antibodies in a mouse model immunized with KLH.Thus, it was confirmed through an animal model that the 8G1-35-31 antibody can antigen-specifically inhibit the activity and proliferation of B cells in vivo (Fig. 11).

[0154] 3-10: Inhibitory effect of IFN-r-secreting Th1 cell activity in hCD40 / hCD40L expressing mice

[0155] hCD40 / hCD40L expressing mice (9 weeks old) were intraperitoneally treated with 10 mg / kg KLH (Sigma). The following day, the control group was intraperitoneally treated with PBS, and the experimental group was intraperitoneally treated with 8G1-35-31 antibody (50 mg / kg). One week later, the control group was treated with KLH (10 mg / kg) and PBS (100 μl), and the experimental group was treated with KLH (10 mg / kg) and 8G1-35-31 antibody (50 mg / kg). After one week, splenocytes were obtained from the spleens of the mice, and the generation of KLH-specific Th1 cells was confirmed using the Enzyme-Linked immunoSpot (ELISpot) assay. That is, anti-IFN-r capture antibody (15 μg / ml) was added to the ELISPOT plate and coated for 18 hours at 4°C. The next day, after washing three times with phosphate buffered saline (PBS, Ph. 7.4), spleen cells from the control and experimental groups were seeded at 1 x 10 6The plate was placed in a concentration of 100 ng / ml and treated with KLH antigen (100 ng / ml) and reacted for 24 hours at 37°C and 5% CO2. The cultured plate was removed and washed three times with PBST, and then a detection antibody (Biotin-conjugated) dilution (1:10,000) was added and reacted at room temperature for 2 hours. After washing three times with PBST, 100 μl of streptavidin-HRP (1:000) dilution was treated and reacted at room temperature for 1 hour. The plate was washed three times with PBST again, and 100 μl of TMB substrate was treated and reacted for 20 minutes, and then washed with running water. The ELISPOT panel was dried at room temperature and read with an ELISPOT reader. As a result, it was confirmed that 8G1-35-31 inhibits the activity and proliferation of KLH-antigen-specific Th1 cells in vivo (Fig. 12).

[0156]

[0157] The foregoing description of the present invention is provided for illustrative purposes only, and it will be apparent to those skilled in the art that various modifications and variations are possible without departing from the technical spirit of the present invention. The scope of the present invention is indicated by the claims that follow rather than the detailed description set forth above, and all changes or modifications derived from the claims and their equivalents should be construed as being included within the scope of the present invention.

[0158]

[0159] The novel anti-CD40 antibody of the present invention exhibits an excellent antagonistic effect by blocking CD40-CD40L signaling, and is expected to be widely utilized as a preparation for preventing or treating various diseases such as autoimmune diseases and chronic inflammatory diseases, and as a preparation for suppressing immune rejection during transplantation, and thus has industrial applicability.

[0160]

[0161] Name of depositor: Korean Cell Line Research Foundation

[0162] Accession number: KCLRF-BP-00523

[0163] Date of acceptance: June 1, 2023

[0164] [Correction pursuant to Rule 91, February 6, 2025]

Claims

1. An anti-CD40 antibody or antigen-binding fragment thereof, comprising the following complementarity determining region (CDR): (i) HCDR1 of the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 80% sequence identity thereto; (ii) HCDR2 of the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 80% sequence identity thereto; (iii) HCDR3 of the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 80% sequence identity thereto; (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 80% sequence identity thereto; (v) LCDR2 of the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 80% sequence identity thereto; and (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 80% sequence identity thereto.

2. In the first paragraph, the anti-CD40 antibody comprises a heavy chain variable region of the amino acid sequence of SEQ ID NO: 13 or a sequence having at least 80% sequence identity thereto, and a light chain variable region of the amino acid sequence of SEQ ID NO: 14 or a sequence having at least 80% sequence identity thereto, or an antigen-binding fragment thereof.

3. In the first paragraph, the anti-CD40 antibody or antigen-binding fragment thereof is an anti-CD40 antibody or antigen-binding fragment thereof produced by a hybridoma cell line deposited under deposit number KCLRF-BP-00523.

4. In the first paragraph, the anti-CD40 antibody is an anti-CD40 antibody or an antigen-binding fragment thereof selected from the group consisting of a monoclonal antibody, a chimeric antibody, a primatized antibody, a humanized antibody, and a human antibody.

5. In the first paragraph, the anti-CD40 antibody is an anti-CD40 antibody or an antigen-binding fragment thereof selected from the group consisting of a multimeric antibody, a heterodimeric antibody, a semi-dimeric antibody, a tetravalent antibody, a bispecific antibody, and a single-chain antibody.

6. In the first paragraph, the antigen-binding fragment is an anti-CD40 antibody or an antigen-binding fragment thereof selected from the group consisting of Fab, F(ab)2, Fab', F(ab)2, F(ab')3, Fd, Fv, and domain antibodies.

7. A nucleic acid encoding an antibody according to any one of claims 1 to 6.

8. A vector containing the nucleic acid of paragraph 7.

9. A host cell containing the nucleic acid of paragraph 7.

10. A method for producing an antibody, comprising a step of culturing the host cell of clause 9 to express an antibody.

11. An antibody-drug complex comprising an antibody according to any one of claims 1 to 6 as an active ingredient.

12. An antibody-drug complex according to claim 11, wherein the drug is selected from the group consisting of an anticancer agent, an aptamer, an antisense oligonucleotide, a small interfering ribonucleic acid, and a microRNA.

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