Antagonistic Anti-CD40 humanized antibody

Abstract

The present invention relates to a novel antagonistic anti-CD40 humanized antibody and, more specifically, to a novel humanized antibody specifically binding to CD40, which, by comprising a complementarity-determining region of a specific sequence, exhibits an excellent antagonistic effect by blocking CD40-CD40L signaling. The novel anti-CD40 humanized 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 suppressing immune rejection upon transplantation.

Description

Antagonistic anti-CD40 humanized antibody

[0001] The present invention relates to a novel antagonistic anti-CD40 humanized antibody, and more particularly, to a novel humanized antibody that specifically binds to CD40 and exhibits an excellent antagonistic effect by blocking CD40-CD40L signaling by including a complementarity determining region of a specific sequence.

[0002] 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.

[0003] 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 of CD40 to its cognate ligand, CD40L (also known as CD154), stimulates B-cell proliferation and differentiation into plasma cells, antibody production, isotype switching, and B-cell memory replenishment. During B cell differentiation, CD40 is expressed on precursor B cells but disappears when they differentiate into plasma cells. CD40 is involved in the differentiation, survival, and proliferation of B lymphocytes, macrophages, antigen-presenting cells, and some endothelial 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 in T cells (Leukoc Bil 67:2-17, 2000).

[0004] CD40L is found on the cell surface of activated T cells (Fenslow et al (1992) J Immunol 149:655; Lane et al (1992) Eur J Imr Rauraol 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 results in 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. Immunlo., 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).

[0005] 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).

[0006] CD40-CD40L signaling is known to play a pivotal role in the production of pathogenic autoantibodies in systemic rheumatic diseases such as SLE, lupus nephritis, Sjogren's syndrome, myositis, systemic sclerosis, ANCA vasculitis, rheumatoid arthritis, IgG4-related diseases, and Pemphigus, where autoantibodies are known to play a significant role in disease progression.

[0007] 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).

[0008] 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 mechanisms such as suppression of B-cell antibody production and suppression of T-cell activation (Am J Transplant. 2012 Aug;12(8):2079-87).

[0009] 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 urgently needed.

[0010] 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.

[0011] 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, not the CD40 ligand, and exhibits excellent antagonistic effects by blocking CD40-CD154 signaling without stimulating platelets, and uses thereof. In addition, Korean Patent Publication No. 10-2018-0044422 discloses an anti-CD40 humanized antibody that can be used in various therapeutic, preventive, and diagnostic methods.

[0012] However, there is still a need for the development of anti-CD40 antibodies that can effectively inhibit the CD40 / CD40L interaction and be used to treat patients.

[0013] Against this backdrop, the inventors of the present invention have made extensive efforts to develop an antibody that blocks CD40-CD40L signaling, and as a result, have confirmed that a novel anti-CD40 humanized antibody exhibits an excellent antagonistic effect by targeting CD40 and blocking CD40-CD40L signaling, thereby completing the present invention.

[0014]

[0015] *One object of the present invention is an anti-CD40 humanized antibody or an antigen-binding fragment thereof, comprising (i) an HCDR1 consisting of a sequence having 80 to 100% homology with the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein the variant is characterized in that valine (V) at position 4 of the amino acid sequence of SEQ ID NO: 1 is modified to isoleucine (I); (ii) an HCDR2 consisting of a sequence having 80 to 100% homology with the amino acid sequence of SEQ ID NO: 2 or a variant thereof, wherein the variant is one in which alanine (A) at position 12 of the amino acid sequence of SEQ ID NO: 2 is modified to proline (P), phenylalanine (F) at position 14 is modified to leucine (L), and glycine (G) at position 16 is modified to serine (S), or one in which alanine at position 12 of the amino acid sequence of SEQ ID NO: 2 is modified to proline, alanine at position 13 is modified to serine, phenylalanine at position 14 is modified to leucine, and glycine at position 16 is modified to serine; Or an HCDR2 characterized in that asparagine (N) at position 11 of the amino acid sequence of SEQ ID NO: 2 is modified to aspartic acid (D), alanine at position 12 is modified to proline, alanine at position 13 is modified to serine, phenylalanine at position 14 is modified to leucine, and glycine at position 16 is modified to serine; (iii) an HCDR3 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 3; (iv) LCDR1 consisting of a sequence having 80 to 100% homology with the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant is LCDR1 characterized in that valine at position 6 of the amino acid sequence of SEQ ID NO: 4 is modified to isoleucine or arginine (R) at position 1 is modified to glycine;(v) LCDR2 consisting of a sequence having 80 to 100% homology with the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant is characterized in that threonine (T) at position 2 of the amino acid sequence of SEQ ID NO: 5 is modified to alanine (A); and (vi) LCDR3 consisting of a sequence having 80 to 100% homology with the amino acid sequence of SEQ ID NO: 6 or a variant thereof, wherein the variant is characterized in that threonine at position 6 of the amino acid sequence of SEQ ID NO: 6 is modified to serine (S), providing an anti-CD40 humanized antibody or an antigen-binding fragment thereof comprising complementarity determining regions (CDRs) of LCDR3.

[0016] Another object of the present invention is to provide a nucleic acid encoding the antibody.

[0017] Another object of the present invention is to provide a recombinant vector comprising the nucleic acid.

[0018] Another object of the present invention is to provide a host cell transformed with the recombinant vector.

[0019] Another object of the present invention is to provide a method for producing an anti-CD40 humanized antibody, which comprises a step of culturing the host cell.

[0020] 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.

[0021] To achieve the above object, one embodiment of the present invention is an anti-CD40 humanized antibody or an antigen-binding fragment thereof, comprising: (i) an HCDR1 consisting of a sequence having 80 to 100% homology with the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein the variant is characterized in that valine (V) at position 4 of the amino acid sequence of SEQ ID NO: 1 is modified to isoleucine (I); (ii) an HCDR2 consisting of a sequence having 80 to 100% homology with the amino acid sequence of SEQ ID NO: 2 or a variant thereof, wherein the variant is one in which alanine (A) at position 12 of the amino acid sequence of SEQ ID NO: 2 is modified to proline (P), phenylalanine (F) at position 14 is modified to leucine (L), and glycine (G) at position 16 is modified to serine (S), or one in which alanine at position 12 of the amino acid sequence of SEQ ID NO: 2 is modified to proline, alanine at position 13 is modified to serine, phenylalanine at position 14 is modified to leucine, and glycine at position 16 is modified to serine; Or an HCDR2 characterized in that asparagine (N) at position 11 of the amino acid sequence of SEQ ID NO: 2 is modified to aspartic acid (D), alanine at position 12 is modified to proline, alanine at position 13 is modified to serine, phenylalanine at position 14 is modified to leucine, and glycine at position 16 is modified to serine; (iii) an HCDR3 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 3;(iv) LCDR1 consisting of a sequence having 80 to 100% homology with the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant is characterized in that valine at position 6 of the amino acid sequence of SEQ ID NO: 4 is modified into isoleucine, or arginine (R) at position 1 is modified into glycine; (v) LCDR2 consisting of a sequence having 80 to 100% homology with the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant is characterized in that threonine (T) at position 2 of the amino acid sequence of SEQ ID NO: 5 is modified into alanine (A); And (vi) LCDR3 consisting of a sequence having 80 to 100% homology with the amino acid sequence of SEQ ID NO: 6 or a variant thereof, wherein the variant is characterized in that the threonine at position 6 of the amino acid sequence of SEQ ID NO: 6 is modified to serine (S), providing an anti-CD40 humanized antibody or antigen-binding fragment thereof comprising complementarity determining regions (CDRs) of LCDR3.

[0022] In the present invention, the anti-CD40 humanized antibody may preferably be an anti-CD40 humanized antibody or an antigen-binding fragment thereof comprising complementarity determining regions of (i) HCDR1 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 1; (ii) HCDR2 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 2; (iii) HCDR3 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 3; (iv) LCDR1 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 4; (v) LCDR2 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 5; and (vi) LCDR3 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 6.

[0023] In the present invention, the anti-CD40 humanized antibody may preferably be an anti-CD40 humanized antibody or an antigen-binding fragment thereof comprising complementarity determining regions of (i) HCDR1 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 1; (ii) HCDR2 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 2; (iii) HCDR3 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 3; (iv) LCDR1 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 13; (v) LCDR2 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 5; and (vi) LCDR3 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 14.

[0024] 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, and IgE 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 modified antibodies having the characteristics according to the present invention.

[0025] The term "humanized antibody" as used herein means an antibody in which the backbone of the antibody is a human antibody, but 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 backbone of an antibody derived from a monkey or mouse, excluding the portions essential for specific antigen binding, are replaced with those of a human antibody. In the present invention, a humanized antibody is an antibody from a non-human species in which the amino acid sequence in the non-antigen binding region (and / or the antigen-binding region) has been altered so that the antibody more closely resembles a human antibody, but still retains its original binding ability.

[0026] The humanized antibodies of the present invention can be produced by methods known in the art. For example, the humanized antibody may have one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be accomplished by substituting hypervariable region sequences for corresponding sequences of a human antibody, according to the method of Winter and co-workers (Jones et al., Nature 321:522-5, 1986; Riechmann et al., Nature 332:323-7, 1988; Verhoeyen et al., Science 239:1534-6, 1988). Thus, in such humanized antibodies, substantially less than an intact human variable domain is substituted with the corresponding sequence from a non-human species. In the present invention, a humanized antibody is a human antibody in which at least some of the hypervariable region residues as well as other variable region residues are substituted by residues from analogous sites in a non-human antibody.

[0027] The selection of human variable domains, both light and heavy, used to generate humanized antibodies can reduce antigenicity. According to the "best-fit" method, the sequence of the variable domain of a non-human (e.g., rodent, e.g., mouse) antibody is screened against an entire library of known human variable domain sequences. The human sequence that most closely matches the non-human sequence is then accepted as the human framework for the humanized antibody. See, e.g., Sims et al, J Immunol 151:2296-308, 1993; Chothia et al, J Mol Biol 196:901-17, 1987. Another method uses a specific framework derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies. See, for example, [Carter et al, Proc Natl Acad Sci USA 89:4285-9, 1992]; [Presta et al, J Immunol 151:2623-32, 1993].

[0028] Humanized antibodies can be produced by replacing sequences in the variable region that are not directly involved in antigen binding with equivalent sequences from human variable regions. Such methods involve isolating, manipulating, and expressing a nucleic acid sequence encoding all or a portion of the variable region from at least one of the heavy or light chains. Sources of such nucleic acids are well known to those skilled in the art and can be obtained, for example, from hybridomas producing antibodies against CD40. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

[0029] The CDRs of antibodies are composed of numerous individual heavy chain variable regions (VDRs), each with a different number of amino acids for each antibody. H ) or light chain variable region (VL ) It is necessary to assign numbers starting from the N-terminus to the conserved amino acid sequence (e.g., frame portion) and variable portion according to a certain rule. Representative examples include the Kabat, Chothia, and IMGT numbering systems, each of which differs depending on the order in which the amino acids in the CDR portion are numbered. The present invention uses the Kabat numbering system.

[0030] 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 length. Functionally, it contains at least a part of the activity or function of the intact antibody or the parent antibody, and examples thereof include, but are not limited to, Fab (Fragment for antigen binding), Fab', F(ab')2, Fv, or single chain antibody (SCA) (e.g., scFv or dsFv), bispecific scFv, and diabody.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] In the present invention, the anti-CD40 humanized antibody may be an anti-CD40 humanized antibody or an antigen-binding fragment thereof, which preferably comprises a heavy chain variable region consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 17; and a light chain variable region consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 23.

[0035] In the present invention, the anti-CD40 humanized antibody may be an anti-CD40 humanized antibody or an antigen-binding fragment thereof, which preferably comprises a heavy chain variable region consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 17; and a light chain variable region consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 25.

[0036] The present invention includes amino acids in which conservative substitutions have occurred in SEQ ID NOs: 15 to 25. 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.

[0037] 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 with 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.

[0038] 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.

[0039] Furthermore, according to the present invention, the anti-CD40 humanized antibody of the present invention can be produced in various forms of antibodies. 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.

[0040] Additionally, the anti-CD40 humanized 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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 -7 If 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.

[0048] In the present invention, the anti-CD40 humanized 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.

[0049] 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.

[0050] 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.

[0051] 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 one antigen or on different antigens.

[0052] 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.

[0053] Another embodiment of the present invention provides a nucleic acid encoding the anti-CD40 humanized antibody.

[0054] 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.

[0055] Another embodiment of the present invention provides a recombinant vector comprising the nucleic acid.

[0056] Another embodiment of the present invention provides a host cell transformed with the recombinant vector.

[0057] Another embodiment of the present invention provides a method for producing an anti-CD40 humanized antibody, comprising culturing the host cell. In the present invention, the method may include a step of culturing the host cell to express the anti-CD40 humanized antibody.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] Another embodiment of the present invention provides an antibody-drug complex comprising the anti-CD40 humanized antibody as an active ingredient.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] Another embodiment of the present invention relates to a pharmaceutical composition for preventing or treating an autoimmune disease, comprising the anti-CD40 humanized antibody or antigen-binding fragment as an active ingredient.

[0070] 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.

[0071] Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating a chronic inflammatory disease, comprising the anti-CD40 humanized antibody or antigen-binding fragment as an active ingredient.

[0072] 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.

[0073] 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.

[0074] Another embodiment of the present invention provides a composition for suppressing immune rejection during transplantation, comprising the anti-CD40 humanized antibody or antigen-binding fragment as an active ingredient.

[0075] 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.

[0076] 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.

[0077] The composition of the present invention is formulated with a pharmaceutically acceptable carrier, optionally an excipient or stabilizer.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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 anti-CD40 humanized antibody or antigen-binding fragment according to the present invention as an active ingredient.

[0085] In the present invention, the therapeutic agent may preferably be an antibody therapeutic agent or a gene therapeutic agent, but is not limited thereto.

[0086] 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 humanized antibody or an antigen-binding fragment thereof according to the present invention as an active ingredient.

[0087] 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.

[0088] The novel anti-CD40 humanized 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.

[0089] Figure 1 shows the results of measuring the antagonistic effect of the anti-CD40 humanized antibody of the present invention. Figure 1a shows the results measured at 3 μg / mL, Figure 1b shows the results measured at 20 μg / mL, and Figure 1c shows the results measured at 100 μg / mL.

[0090] Figure 2 shows the results of measuring the agonistic effect of the anti-CD40 humanized antibody of the present invention. Figure 2a shows the results measured at 4 μg / mL, and Figure 2b shows the results measured at 100 μg / mL.

[0091] Figure 3 shows the results of a CD40 antigen-specific binding test of anti-CD40 humanized antibodies #1-4 according to one embodiment of the present invention.

[0092] Figure 4 shows the results of a binding analysis of anti-CD40 humanized antibodies #1-4 and #1-11 according to one embodiment of the present invention, depending on the CD40 concentration.

[0093] Figure 5 shows the results of confirming the CD40-CD40L binding blocking effect using reporter cells of anti-CD40 humanized antibodies #1-4 and #1-11 according to one embodiment of the present invention.

[0094] Figure 6 illustrates the T-cell-dependent B cell activation inhibitory effect (CD86 FACS) of anti-CD40 humanized antibodies #1-4 and #1-11 according to one embodiment of the present invention. Figure 6 (a) shows a FACS histogram, and Figure 6 (b) quantifies a by CD86 expression and antagonistic inhibition.

[0095] Figure 7 shows the results of a RAMOS B cell-CHOCD40L binding blocking test of anti-CD40 humanized antibodies #1-4 and #1-11 according to one embodiment of the present invention.

[0096] Figure 8 shows the results of a PBMC non-agonist characteristic test of anti-CD40 humanized antibodies #1-4 and #1-11 according to one embodiment of the present invention. Figure 8 (a) shows a histogram of FACS, and Figure 8 (b) shows a numerically expressed as CD86 expression and CD69 expression.

[0097] Figure 9 shows the linear CD40 binding of anti-CD40 humanized antibodies #1-4 according to one embodiment of the present invention.

[0098] Figure 10 shows the results of a thermal stability (biological function test - HEK blue) test of anti-CD40 humanized antibodies #1-4 and #1-11 according to one embodiment of the present invention.

[0099] Figure 11 shows the results of a thermal stability (aggregation index - Thioflavin) test of anti-CD40 humanized antibodies #1-4 and #1-11 according to one embodiment of the present invention.

[0100] Figure 12 shows the results of interspecies cross-linking (FACS data) tests of anti-CD40 humanized antibodies #1-4 and #1-11 according to one embodiment of the present invention.

[0101] 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.

[0102]

[0103] Example 1: Production of anti-CD40 humanized antibodies

[0104] The fragment crystallizable region (Fc region) of the existing mouse antibody sequence (heavy chain (HC): QVHLKQSGPGLVQPSQSLSITCTVSGFSLSYYGVHWVRQSPGKGLEWLGVIWSGGRKDFNAAFISRLSITKDNSKSQVFIKMDSLQVDDTAIYYCASMIREYYAMDYWGQGTSVTVSS (SEQ ID NO: 26); light chain (LC): QIVLTQSPTIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSPPKVWIYSTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPWTFGGGTKLEIK (SEQ ID NO: 27)) was replaced with a human immunoglobulin G1 mutation to secure a chimeric antibody sequence (used as control 1), and humanized it through in silico, an experimental method that utilizes computer simulation. A candidate set was created by combining the acquired heavy and light chain variants. The in silico process was as follows.

[0105] A murine antibody was humanized by grafting three complementarity-determining regions (CDRs) (using the Kabat and IMGT nomenclature) from the heavy chain variable region (VL) into a selected human germline light chain variable region close to a murine antibody light chain variable region. Similarly, three complementarity-determining regions from the heavy chain variable region (VH) were grafted into a selected human germline heavy chain variable region close to a murine antibody heavy chain variable region. In addition, several amino acid residues in the framework region of the selected human germline variable region were backmutated to the corresponding murine amino acid residues. Based on the collected information on the structure of immunoglobulin variable regions and guidance from molecular models of monoclonal antibodies, these few residues in the framework region were identified as potentially key to maintaining the complementarity-determining region structure and are known to serve at the interface between the variable regions of the heavy and light chains. The light and heavy chain variable regions are two domains that interact without forming covalent bonds. The interaction between the two domains is maintained through hydrogen and electrostatic bonds. Residues involved in this interaction must also be maintained, as otherwise the antibody-binding domain may be altered and antibody affinity may be altered. Therefore, these were either retained in humanized version V1 ("low-risk") or mutated to their human germline amino acid counterparts in humanized versions V2 and V3. Using molecular models, some complementarity-determining region residues were also replaced with their human germline counterparts in humanized versions V2 and V3. Here, the combination of structural models and pure sequence analysis potentially allows for the distinction between residues in the antibody-binding domain and those in the non-antibody-binding domain of the complementarity domain. Furthermore, the use of structural models allows for the selection of backmutations to be guided by the selected germline backbone used, thereby accelerating the humanization process.

[0106] By combining the heavy and light chain variants obtained in silico, 24 candidates were produced using Chinese hamster ovary cells (using the purified CCF (cell culture fluid) produced by BioTem), and the supernatant was purified. After performing affinity tests, antagonism tests, and action tests, 3 candidates (#1, #2, #3) were selected. The variants of the 3 candidates were further combined to create 38 candidates, and among them, a total of 39 candidates, including one candidate in which the fragment crystallizable region (Fc region) of one candidate (#2) was substituted with a new human immunoglobulin G1 mutation (STR), were produced using Chinese hamster ovary cells, and the supernatant was purified. After performing affinity tests, antagonism tests, action tests, differential scanning calorimetry (DSC), isoelectric focusing (imaging capillary isoelectric focusing (icIEF), and stability tests, 2 candidates were selected. (#1-4, #1-11) were selected.

[0107] To evaluate the function of the obtained candidate sequences, small quantities of proteins of each sequence were produced and the antagonistic and agonistic properties of these candidates were measured.

[0108] To measure the antagonistic properties, the CD40L-CD40 antagonistic effect was quantified at the cell level using HEK-Blue human CD40L-SEAP Reporter cells, hereinafter HEK-BlueCD40L (InvivoGen, Cat#. hkb-cd40). That is, 2.5x10 cells were seeded in a 96-well plate. 5 / mL, and recombinant CD40L solution (InvivoGen, Cat#. rcyec-hcd40) was added at a concentration of 600 ng / mL. To measure the effect of the candidate sequence protein, antibodies were added in triplicate at concentrations of 3 μg / mL, 20 μg / mL, and 100 μg / mL, and incubated for 20 hours in an incubator at 37°C in a humidified atmosphere with 5% CO2. Afterwards, 20 μL of the cultured supernatant was transferred to a 96-well plate, and 180 μL of QUANTI-Blue™Solution (InvivoGen, Cat#. rep-qbs) was added. After 30 minutes of reaction in an incubator at 37°C in a humidified atmosphere with 5% CO2, the antagonistic effect was confirmed by the value measured at 620 nm with a microplate reader. Relative measurements were obtained and compared based on the measurement values ​​of the recombinant CD40L solution (Fig. 1; antibody concentrations in Fig. 1a are 3 μg / mL, Fig. 1b are 20 μg / mL, and Fig. 1c are 100 μg / mL).

[0109] To measure the antagonistic properties, the CD40L-CD40 agonistic effect was quantified at the cell level using HEK-Blue human CD40L-SEAP Reporter cells, hereinafter HEK-Blue CD40L (InvivoGen, Cat#. hkb-cd40). That is, 2.5x10 cells were seeded in a 96-well plate. 5 / mL HEK-Blue CD40L cell suspension was dispensed, quantified antibodies were added in triplicate, and cultured for 20 hours in a humidified 5% CO2 incubator at 37°C. 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 for 1 hour in a humidified 5% CO2 incubator at 37°C. The agonistic effect was confirmed by measuring the result at 620 nm with a microplate reader. The relative measurement value was obtained based on the measurement value of the recombinant CD40L solution and compared (Fig. 2; antibody concentration Fig. 2a is 4 μg / mL, Fig. 2b is 100 μg / mL).

[0110] To confirm the antagonistic and agonistic effects, control group 1 (ChiPB101), control group 2 (Biotem #9 - heavy chain (HC): EVQLQQSGPGLVKPSQTLSLTCAVSGFSLSYYGVHWIRQPPGKGLEWIGVIWSGGRKDYNPALISR VTISKENSKSQVSLKLSSVTAADTAVYYCASMIREYYAMDYWGQGTLVTVSS (SEQ ID NO: 28); light chain (LC): EIQLTQSPSSLSASVGERVTMTCRASSSVSSSYLHWYQQKPGKAPKVWIYSTSKLASGVPSRFSGS GSGTSYTLTISSMQPEDFATYYCHQYHRSPWTFGGGTKVEI (SEQ ID NO: 29)), control group 3 (Iscalimab, Novartis, Switzerland), and control group 4 (Ravagalimab, Abbvie, USA) were used as controls.

[0111]

[0112] Example 2: Sequence verification of anti-CD40 humanized antibodies

[0113] The CDR sequences (Kabat numbering system) of the anti-CD40 humanized antibody of the present invention are as shown in Table 1 below.

[0114] Antibody heavy chain light chain HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 #1-1 YYGVH (SEQ ID NO: 1) VIWSGGRKDYNAAFIG (SEQ ID NO: 2) MIREYYAMDY (SEQ ID NO: 3) RASSSVSSSYLH (SEQ ID NO: 4) STSKLAS (SEQ ID NO: 5) HQYHRTPWT (SEQ ID NO: 6) #1-2 YYGVH (SEQ ID NO: 1) VIWSGGRKDYNAAFIG (SEQ ID NO: 2) MIREYYAMDY (SEQ ID NO: 3) RASSSISSSYLH (SEQ ID NO: 7) SASKLAS (SEQ ID NO: 8) HQYHRTPWT (SEQ ID NO: 6) #1-3 YYGIH (SEQ ID NO: 9) VIWSGGRKDYNAAFIG (SEQ ID NO: 2) MIREYYAMDY (SEQ ID NO: 3) RASSSISSSYLH (SEQ ID NO: 7)SASKLAS(SEQ ID NO: 8)HQYHRTPWT(SEQ ID NO: 6)#1-4YYGVH(SEQ ID NO: 1)VIWSGGRKDYNAAFIG(SEQ ID NO: 2)MIREYYAMDY(SEQ ID NO: 3)RASSSVSSSYLH(SEQ ID NO: 4)STSKLAS(SEQ ID NO: 5)HQYHRTPWT(SEQ ID NO: 6)#1-5YYGVH(SEQ ID NO: 1)VIWSGGRKDYNAAFIG(SEQ ID NO: 2)MIREYYAMDY(SEQ ID NO: 3)RASSSVSSSYLH(SEQ ID NO: 4)STSKLAS(SEQ ID NO: 5)HQYHRTPWT(SEQ ID NO: 6)#1-6YYGIH(SEQ ID NO: 9)VIWSGGRKDYNAAFIG(SEQ ID NO: 2)MIREYYAMDY(SEQ ID NO: 3)RASSSVSSSYLH(SEQ ID NO: 4) STSKLAS (SEQ ID NO: 5) HQYHRTPWT (SEQ ID NO: 6) #1-7YYGVH (SEQ ID NO: 1) VIWSGGRKDYNPALIS (SEQ ID NO: 10) MIREYYAMDY (SEQ ID NO: 3) RASSSVSSSYLH (SEQ ID NO: 4) STSKLAS (SEQ ID NO: 5) HQYHRTPWT (SEQ ID NO: 6) #1-8YYGVH (SEQ ID NO: 1) VIWSGGRKDYNPSLIS (SEQ ID NO: 11) MIREYYAMDY (SEQ ID NO: 3) RASSSVSSSYLH (SEQ ID NO: 4) STSKLAS (SEQ ID NO: 5) HQYHRTPWT (SEQ ID NO: 6) #1-9YYGVH (SEQ ID NO: 1) VIWSGGRKDYDPSLIS (SEQ ID NO: 12) MIREYYAMDY (SEQ ID NO: 3) RASSSVSSSYLH (SEQ ID NO:4) STSKLAS (SEQ ID NO: 5) HQYHRTPWT (SEQ ID NO: 6) #1-10YYGVH (SEQ ID NO: 1) VIWSGGRKDYNAAFIG (SEQ ID NO: 2) MIREYYAMDY (SEQ ID NO: 3) GASSSVSSSYLH (SEQ ID NO: 13) STSKLAS (SEQ ID NO: 5) HQYHRSPWT (SEQ ID NO: 14) #1-11YYGVH (SEQ ID NO: 1) VIWSGGRKDYNAAFIG (SEQ ID NO: 2) MIREYYAMDY (SEQ ID NO: 3) GASSSVSSSYLH (SEQ ID NO: 13) STSKLAS (SEQ ID NO: 5) HQYHRSPWT (SEQ ID NO: 14) #1-12YYGIH (SEQ ID NO: 9) VIWSGGRKDYNAAFIG (SEQ ID NO: 2) MIREYYAMDY (SEQ ID NO: 3) GASSSVSSSYLH (SEQ ID NO: 13) STSKLAS (SEQ ID NO: 5) HQYHRSPWT (SEQ ID NO: 14)

[0115] The heavy chain variable region and light chain variable region of the anti-CD40 humanized antibody of the present invention are as shown in Table 2 below (the underlined portion in the table below represents the CDR sequence of each antibody according to the Kabat numbering system, and the bold portion represents the CDR sequence of each antibody according to the IMGT numbering system).

[0116] 항체중쇄 가변 영역경쇄 가변 영역#1-1EVQLVQSGAEVVKPGSSVKVSCKASGFSLSYYGVHWVRQAPGQGLEWMGVIWSGGRKDYNAAFIGRVTITKENSKSTAYIELSSLRSEDTAVYYCASMIREYYAMDYWGQGTLVTVSS(서열번호 15)EIQLTQSPSSLSASVGERVTITCRASSSVSSSYLHWYQQKPGKAPKLWIYSTSKLASGVPSRFSGSGSGTSFTLTISSLQPEDFATYYCHQYHRTPWTFGGGTKVEIK(서열번호 21)#1-2EVQLVQSGAEVVKPGSSVKVSCKASGFSLSYYGVHWVRQAPGQGLEWMGVIWSGGRKDYNAAFIGRVTITKENSKSTAYIELSSLRSEDTAVYYCASMIREYYAMDYWGQGTLVTVSS(서열번호 15)EIQMTQSPSSLSASVGERVTITCRASSSISSSYLHWYQQKPGKAPKLWIYSASKLASGVPSRFSGSGSGTSFTLTISSLQPEDFATYYCHQYHRTPWTFGGGTKVEIK(서열번호 22)#1-3EVQLVQSGAEVKKPGSSVKVSCKASGFSLSYYGIHWVRQAPGQGLEWMGVIWSGGRKDYNAAFIGRVTITKENSKSTAYIELSSLRSEDTAVYYCASMIREYYAMDYWGQGTLVTVSS(서열번호 16)EIQMTQSPSSLSASVGERVTITCRASSSISSSYLHWYQQKPGKAPKLWIYSASKLASGVPSRFSGSGSGTSFTLTISSLQPEDFATYYCHQYHRTPWTFGGGTKVEIK(서열번호 22)#1-4EVQLVQSGAEVVKPGSSVKVSCKVSGFSLSYYGVHWVRQAPGQGLEWMGVIWSGGRKDYNAAFIGRVTITKENSKSTVYIELSSLRSEDTAVYYCASMIREYYAMDYWGQGTLVTVSS(서열번호17)EIQMTQSPSSLSASVGERVTITCRASSSVSSSYLHWYQQKPGKAPKLWIYSTSKLASGVPSRFSGSGSGTSFTLTISSLQPEDFATYYCHQYHRTPWTFGGGTKVEIK(서열번호 23)#1-5EVQLVQSGAEVVKPGSSVKVSCKASGFSLSYYGVHWVRQAPGQGLEWMGVIWSGGRKDYNAAFIGRVTITKENSKSTAYIELSSLRSEDTAVYYCASMIREYYAMDYWGQGTLVTVSS(서열번호 15)EIQMTQSPSSLSASVGERVTITCRASSSVSSSYLHWYQQKPGKAPKLWIYSTSKLASGVPSRFSGSGSGTSFTLTISSLQPEDFATYYCHQYHRTPWTFGGGTKVEIK(서열번호 23)#1-6EVQLVQSGAEVKKPGSSVKVSCKASGFSLSYYGIHWVRQAPGQGLEWMGVIWSGGRKDYNAAFIGRVTITKENSKSTAYIELSSLRSEDTAVYYCASMIREYYAMDYWGQGTLVTVSS(서열번호 16)EIQMTQSPSSLSASVGERVTITCRASSSVSSSYLHWYQQKPGKAPKLWIYSTSKLASGVPSRFSGSGSGTSFTLTISSLQPEDFATYYCHQYHRTPWTFGGGTKVEIK(서열번호 23)#1-7EVQLQQSGPGLVKPSQTLSLTCAVSGFSLSYYGVHWIRQPPGKGLEWIGVIWSGGRKDYNPALISRVTISKENSKSQVSLKLSSVTAADTAVYYCASMIREYYAMDYWGQGTLVTVSS(서열번호 18)EIQMTQSPSSLSASVGERVTITCRASSSVSSSYLHWYQQKPGKAPKLWIYSTSKLASGVPSRFSGSGSGTSFTLTISSLQPEDFATYYCHQYHRTPWTFGGGTKVEIK(서열번호 23)#1-8EVQLQQSGPGLVKPSQTLSLTCAVSGFSLSYYGVHWIRQPPGKGLEWIGVIWSGGRKDYNPSLISRVTISKENSKSQVSLKLSSVTAADTAVYYCASMIREYYAMDYWGQGTLVTVSS(서열번호19)EIQMTQSPSSLSASVGERVTITCRASSSVSSSYLHWYQQKPGKAPKLWIYSTSKLASGVPSRFSGSGSGTSFTLTISSLQPEDFATYYCHQYHRTPWTFGGGTKVEIK(서열번호 23)#1-9EVQLQESGPGLVKPSQTLSLTCAVYGFSLSYYGVHWIRQPPGKGLEWIGVIWSGGRKDYDPSLISRVTISKENSKSQVSLKLSSVTAADTAVYYCASMIREYYAMDYWGQGTLVTVSS(서열번호 20)EIQMTQSPSSLSASVGERVTITCRASSSVSSSYLHWYQQKPGKAPKLWIYSTSKLASGVPSRFSGSGSGTSFTLTISSLQPEDFATYYCHQYHRTPWTFGGGTKVEIK(서열번호 23)#1-10EVQLVQSGAEVVKPGSSVKVSCKVSGFSLSYYGVHWVRQAPGQGLEWMGVIWSGGRKDYNAAFIGRVTITKENSKSTVYIELSSLRSEDTAVYYCASMIREYYAMDYWGQGTLVTVSS(서열번호 17)EIVLTQSPATLSLSPGERATMSCGASSSVSSSYLHWYQQKPGLAPRVWIYSTSKLASGIPSRFSGSGSGTSYTLTISRMEPEDFAVYYCHQYHRSPWTFGGGTKVEIK(서열번호 24)#1-11EVQLVQSGAEVVKPGSSVKVSCKVSGFSLSYYGVHWVRQAPGQGLEWMGVIWSGGRKDYNAAFIGRVTITKENSKSTVYIELSSLRSEDTAVYYCASMIREYYAMDYWGQGTLVTVSS(서열번호 17)EIVLTQSPATLSLSPGERATMSCGASSSVSSSYLHWYQQKPGLAPRVLIYSTSKLASGIPSRFSGSGSGTSYTLTISRLEPEDFAVYYCHQYHRSPWTFGGGTKVEIK(서열번호 25)#1-12EVQLVQSGAEVKKPGSSVKVSCKASGFSLSYYGIHWVRQAPGQGLEWMGVIWSGGRKDYNAAFIGRVTITKENSKSTAYIELSSLRSEDTAVYYCASMIREYYAMDYWGQGTLVTVSS(서열번호16)EIVLTQSPATLSLSPGERATMSCGASSSVSSSYLHWYQQKPGLAPRVLIYSTSKLASGIPSRFSGSGSGTSYTLTISRLEPEDFAVYYCHQYHRSPWTFGGGTKVEIK (SEQ ID NO: 25)

[0117] Example 3: Confirmation of the antagonistic effect of anti-CD40 humanized antibodies

[0118] 3-1: CD40 antigen-specific binding test

[0119] To investigate whether humanized antibodies #1-4 specifically bind to human CD40 antigen (hCD40), flow cytometry was performed using CHO cells expressing CD40 antigen and CD40-CHO cells, each at 2x10 5 For dog CHO cells and CD40-CHO cells, isotype control (mouse IgG-FITC, Biolegend) and #1-4-FITC (10 μg / mL) were treated and reacted 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 #1-4 to CD40-CHO. This confirmed that #1-4 specifically binds to the human CD40 antigen (Fig. 3).

[0120] 3-2: Analysis of binding affinity according to CD40 concentration

[0121] To confirm the concentration-dependent binding affinity of antibodies, an Enzyme-linked Immunosorbent Assay (ELISA) was performed. CD40-his (1 μg / mL, sinobiological, Cat#. 10774-H08H) diluted with 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. 100 μL of each diluted test solution was added and incubated at 37°C for 1 hour. After washing three times with PBST (10 mM sodium phosphate, 0.15 M NaCl, 0.05% Tween™20 buffer at pH 7.5), 50 μL of Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody-HRP (Invitrogen, Cat#. 62-6520, 1:5,000) solution was added and incubated at 37°C for 30 minutes. Finally, after washing three times with PBST, 50 μL of TMB Substrate Solution (Thermo Scientific, Cat#. N301) was dispensed, covered with foil to block light, and incubated at room temperature for 5 minutes. After stopping the reaction by adding 50 μL of Stop Solution (Thermo Scientific, Cat# N600), the absorbance was measured at a wavelength of 450 nm using a microplate ELISA reader (Varioskan Lux microplate reader, Thermo fisher scientific, Cat#. VLBL0TD2). Through this, it was confirmed that the antibody showed a concentration-dependent binding pattern in which the binding affinity increased depending on the amount of CD40 antigen (Fig. 4).

[0122] 3-3: Analysis of binding patterns on PMBC cells in blood

[0123] To analyze the binding properties of PBMC (peripheral blood mononuclear cell) cell subtypes in blood, flow cytometry was performed. That is, human PBMC 2X10 5 Cells were treated with Zombie aqua (Biolegend) and incubated at room temperature for 30 minutes, then washed once with FACS buffer (5% BSA, 0.01% Sodium Azide, PBS, pH 7.4). Then, anti-CD3-V450 (BD Biosciences, UCHT1), anti-CD14-APC (Biolegend, HCD14), anti-CD19-BV786 (BD Biosciences, SJ25C1), anti-CD56-PE (Invitrogen, CMSSB), and #1-4-FITC were added at a concentration of 10 μg / mL and incubated on ice for 30 minutes. After washing with FACS buffer, the cells were analyzed using a BD LSRFortessa X-20. As a result, #1-4 was confirmed to have high binding affinity to CD19+ B cells and CD14+ monocytes, which are reported to express CD40.

[0124] 3-4: Confirmation of CD40-CD40L binding blocking effect using reporter cells

[0125] The CD40L-CD40 binding blocking effect was quantified at the cell level using HEK-Blue human CD40L-SEAP Reporter cells (hereinafter referred to as HEK-Blue CD40L, InvivoGen, Cat#. hkb-cd40). That is, 2.5x10 5HEK-Blue CD40L cell suspension was dispensed to reach / mL, and recombinant CD40L solution (InvivoGen, Cat#. rcyec-hcd40) was added at a concentration of 600 ng / mL. Quantified #1-4 and #1-11 antibodies were added in triplicate and incubated for 20 hours in a humidified 5% CO2 incubator at 37°C. Afterwards, 20 μL of the cultured supernatant was transferred to a 96-well plate, and 180 μL of QUANTI-Blue™Solution (InvivoGen, Cat#. rep-qbs) was added and reacted at room temperature for 30 minutes. The binding blocking power was determined using the value measured at 620 nm with a microplate reader (Fig. 5). The equation for calculating the binding blocking power is as follows: Mathematical Formula 1.

[0126] [Mathematical Formula 1]

[0127] 100-{[(OD value of antibody-treated experimental group - OD value of non-antibody-treated experimental group) / (OD value of sCD40-treated experimental group - OD value of non-antibody-treated experimental group)] X 100}

[0128] 3-5: Evaluation of T-cell-dependent B cell activation / proliferation inhibition effect

[0129] Using an in vitro model simulating T-cell-dependent B-cell activation in human PBMCs, the inhibitory effect of specific drugs on the expression of B-cell activating factor CD86 was quantified using PBMCs from three individuals. First, 96-well U-bottom plates were coated with activating anti-CD3 antibody (OKT3) and activating anti-CD28 antibody (CD28.6) dissolved in phosphate-buffered saline (PBS, pH 7.4) at a concentration of 5 μg / mL and incubated at 4°C for 18 h. The following day, human PBMCs stained with CFSE (2.5 μM) were seeded at 2.5 × 10 to the anti-CD3 / anti-CD28 antibody-coated 96-well plates. 6After adding the cells at a concentration of / mL, #1-4 and #1-11 antibodies (10 μg / mL, 100 μg / mL) were added and cultured in a humidified 37°C, 5% CO2 incubator for 4 or 5 days. The positive control group was a plate coated with only anti-CD3 / anti-CD28 antibodies, and no drug antibodies were added, and the negative control group was a plate coated with neither anti-CD3 / anti-CD28 antibodies nor drug antibodies. After 4-5 days, the cultured cells were transferred to a tube for FACS (USA, SPL-40205) and flow cytometry staining was performed. For this, cultured cells were washed with FACS buffer (PBS pH 7.4, 0.5% BSA, 0.01% Sodium Azide), added with FC-blocker (BioLegend, US, cat. no. 422302), reacted for 15 minutes, and then 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. Finally, after washing with FACS buffer, detection was performed with BD FACS Canto II. As a result of measuring CFSE staining, treatment with #1-4 and #1-11 antibodies showed an inhibitory effect on cell proliferation at both concentrations, and this effect was more pronounced when treated with #1-4. The results of measuring CD86 expression also showed that CD86 expression was inhibited at both concentrations when treated with #1-4 and #1-11 antibodies (Fig. 6).

[0130] The rate of drug-induced activity inhibition was calculated using the difference in the expression level of CD86 between the positive control group and the negative control group, as shown in the following mathematical formula 2.

[0131] [Equation 2]

[0132] 100-{[(CD86 expression % of antibody-treated experimental group - CD86 expression % of non-antibody-treated experimental group) / (CD86 expression % of sCD40-treated experimental group - CD86 expression % of non-antibody-treated experimental group)] X 100}

[0133] 3-6: RAMOS B cell-CHOCD40L binding blocking assay

[0134] RAMOS is a human B cell with high expression of CD40. CHOCD40L is a CHO cell with overexpression of human CD40L. When the two cells are mixed and cultured, Ramos B cells are activated through CD40-CD40L binding and signal transduction, which increases the expression of ICAM-1. Therefore, measuring the increase in ICAM-1 expression by flow cytometry can quantify CD40-CD40L signal transduction and the binding blocking effect by drugs. In this study, the binding blocking effect according to the timing of drug administration was verified in the RAMOS B cell-CHOCD40L binding model. That is, 5 x 10 Ramos cells were seeded in each well of a 96-well plate at 50 μL. 4 2.5 x 10 of dog and CD40L-CHO cells 4The dogs were cultured at 37℃, 5% CO2. After that, the drug (10 μg / mL) was treated at various time points (0 h, 1 h, 3 h, 6 h, 12 h), and the ICAM-1 expression level was quantified by flow cytometry after 24 h of culture. To this end, the cells were stained with Pacific Blue™ anti-human CD54 Antibody (ICAM-1) (Biolegend cat no. 322716), CD-19 PE-Cy7 (Biolegend cat no. 303718), and Fc blocker (Biolegend cat no. 422302), washed with FACS buffer, and the ICAM-1 expression level was examined using FACSCantoII. As a result, it was confirmed that the binding blocking effect was directly proportional to the timing of administration of #1-4 and #1-11 antibodies. That is, the earlier the administration timing, the higher the binding blocking effect, and the later the administration timing, the lower the binding blocking effect (Fig. 7).

[0135] 3-7: Non-agonist trait test

[0136] Peripheral blood mononuclear cells (PBMCs) from two individuals were treated with antibodies #1-4 and #1-11 at a concentration of 100 μg / mL and cultured for 3 days in a humidified 5% CO2 incubator at 37°C. CP870,893 (Pfizer, agonist CD40 antibody) was treated at the same concentration as the positive control group, and a group without antibody treatment was used as the negative control group. 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 (PBS pH 7.4, 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 antibodies #1-4 and #1-11 have non-functional properties that do not activate human PBMCs by themselves (Fig. 8).

[0137] 3-8: Characterization of CD40 sequence recognition characteristics

[0138] To investigate whether the drug antibody recognizes the linear antigen, Western blot analysis was performed. First, CD40-CHO cells were washed 12 times with DPBS and lysed in 1X RIPA buffer (Biosesang, cat. No. R2002) on ice for 20 minutes. The supernatant was then quantified for protein concentration using a BCA assay kit (Thermo Fisher Scientific cat. No. 2161296). 2-Mercaptoethanol was added to 1X Bolt™ LDS sample buffer to prepare samples under reducing and non-reducing conditions. The samples were denatured at 100°C for 5 minutes, then separated on a 12% Bis-Tris Gel in Bolt™ 4, and transferred to a membrane. To confirm the transfer effect, the gel was stained with Coomassie Blue, and the membrane was checked with Ponceau S for 12 minutes. The membrane was washed with 1X TBST and incubated with #1-4 antibodies (concentration 10 mg / mL) at 4°C for 18 h with agitation. After incubation with secondary antibody, anti-human IgG-HRP, for 1 h, the membrane was reacted with ECL substrate (Thermo Scientific, cat. No. 32106) and the bands were confirmed with ChemiDoc MP (BIO-RAD, US). As a result, it was confirmed that #1-4 antibodies could bind to linear CD40 (Fig. 9).

[0139] 3-9: Thermal stability test (biological function test)

[0140] To confirm the thermal stability at high temperatures (45 ℃, 55 ℃, 65 ℃), which are harsh conditions, the sample (3 mg / mL) was diluted in a PBS-based stabilization buffer (0.02% Tween 80, 5% Sucrose, 20 mM Histidine, pH 7.0) and exposed to each temperature for 7 days. After 7 days, the CD40-CD40L binding blocking effect experiment using reporter cells was performed in the same manner as in Examples 3-4. As a result, it was confirmed that #1-4 and #1-11 showed very high functional thermal stability even at high temperatures (45 ℃, 55 ℃) (Fig. 10).

[0141] 3-10: CD40 thermal stability test (aggregation index test)

[0142] To confirm the thermal stability at high temperatures (45 ℃, 55 ℃, 65 ℃), which are harsh conditions, the sample (3 mg / mL) was diluted in a PBS-based stabilization buffer (0.02% Tween 80, 5% Sucrose, 20 mM Histidine, pH 7.0) and exposed to each temperature for 7 days. After 7 days, Thioflavin T (30 μM) was prepared using 50 mM Tris-HCl buffer (pH 7.5). The sample and Thioflavin T solution were added in a 1:1 ratio to a 96-well plate and reacted at room temperature for 20 minutes under light-blocking conditions. Afterwards, the fluorescence was measured using a microplate reader at a wavelength of Excitation 440 nm and Emission 490 nm. As a result, it was confirmed that #1-4 and #1-11 showed very high thermal stability even at high temperatures (45 ℃, 55 ℃) (Fig. 11).

[0143] 3-11: Interspecific cross-linking investigation

[0144] To determine whether the sample binds to other species besides human blood cells, flow cytometry was performed using marmoset PBMCs. 3 x 10 human and marmoset PBMCs were placed in a FACS tube. 5 The dogs were treated with biotinylated #1-4 antibodies (10 μg / mL), followed by treatment with FC Blocker and CD19-PE-cy7 antibodies, and incubated at 4°C for 1 hour in a darkened atmosphere. After washing with FACS buffer, the cells were treated with Streptavidin and Alexa Fluor™ 488 Conjugate S11223 (1:4000), and incubated at 4°C for another 1 hour in a darkened atmosphere. After washing twice with FACS buffer, the cells were analyzed using a BD FACSCantoII. As a result, it was confirmed that the #1-4 antibodies had cross-species cross-binding to not only humans but also marmosets (Fig. 12).

[0145]

[0146] 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.

[0147] The novel anti-CD40 humanized 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.

Claims

1. An anti-CD40 humanized antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs): (i) HCDR1 consisting of a sequence having 80 to 100% homology to the amino acid sequence of sequence number 1 or a variant thereof, The above mutant is characterized by HCDR1 in which valine (V) at position 4 of the amino acid sequence of sequence number 1 is changed to isoleucine (I); (ii) an HCDR2 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 2 or a variant thereof, The above mutant is one in which alanine (A) at position 12 of the amino acid sequence of sequence number 2 is changed to proline (P), phenylalanine (F) at position 14 is changed to leucine (L), and glycine (G) at position 16 is changed to serine (S). Or, in the amino acid sequence of sequence number 2, alanine at position 12 is modified to proline, alanine at position 13 is modified to serine, phenylalanine at position 14 is modified to leucine, and glycine at position 16 is modified to serine; Or HCDR2 characterized in that asparagine (N) at position 11 of the amino acid sequence of sequence number 2 is modified to aspartic acid (D), alanine at position 12 is modified to proline, alanine at position 13 is modified to serine, phenylalanine at position 14 is modified to leucine, and glycine at position 16 is modified to serine; (iii) HCDR3 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 3; (iv) LCDR1 consisting of a sequence having 80 to 100% homology with the amino acid sequence of sequence number 4 or a variant thereof, The above mutant is LCDR1, characterized in that valine at position 6 of the amino acid sequence of sequence number 4 is modified to isoleucine, or arginine (R) at position 1 is modified to glycine; (v) LCDR2 consisting of a sequence having 80 to 100% homology to the amino acid sequence of sequence number 5 or a variant thereof, The above mutant is LCDR2, characterized in that the threonine (T) at position 2 of the amino acid sequence of sequence number 5 is changed to alanine (A); and (vi) LCDR3 consisting of a sequence having 80 to 100% homology to the amino acid sequence of sequence number 6 or a variant thereof, The above mutant is LCDR3, characterized in that threonine at position 6 of the amino acid sequence of sequence number 6 is changed to serine (serine, S).

2. In the first paragraph, an anti-CD40 humanized antibody or antigen-binding fragment thereof comprising the following complementarity determining region: (i) HCDR1 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 1; (ii) HCDR2 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 2; (iii) HCDR3 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 3; (iv) LCDR1 consisting of a sequence having 80 to 100% homology to the amino acid sequence of sequence number 4; (v) LCDR2 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 5; and (vi) LCDR3 consisting of a sequence having 80 to 100% homology to the amino acid sequence of sequence number 6.

3. In paragraph 1, an anti-CD40 humanized antibody or antigen-binding fragment thereof comprising the following complementarity determining region: (i) HCDR1 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 1; (ii) HCDR2 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 2; (iii) HCDR3 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 3; (iv) LCDR1 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 13; (v) LCDR2 consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 5; and (vi) LCDR3 consisting of a sequence having 80 to 100% homology to the amino acid sequence of sequence number 14.

4. An anti-CD40 humanized antibody or antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 17 in the third paragraph; and a light chain variable region consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO:

23.

5. An anti-CD40 humanized antibody or antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO: 17 in paragraph 4; and a light chain variable region consisting of a sequence having 80 to 100% homology to the amino acid sequence of SEQ ID NO:

25.

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

7. A recombinant vector containing the nucleic acid of Article 7.

8. A host cell transformed with the recombinant vector of Article 8.

9. A method for producing an anti-CD40 humanized antibody, comprising a step of culturing the host cell of clause 9.

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