MXPA06006760A - Mutants of anti-cd40 antibody - Google Patents

Abstract

It is intended to provide mutants of anti-CD40 antibody, which is expected as therapeutically efficacious, having been optimized as a drug by lowering the ADCC and CDC activities thereof. Namely, an agonistic anti-CD40 monoclonal antibody mutant in which at least the hinge region is a hinge region originating in human IgG2 and which has a mutation and/or a substitution of one or more amino acids causing lowering in the ADCC and/or CDC activities in the constant region;and an antagonistic anti-CD40 antibody mutant which has at least one mutation and/or substitution causing lowering in the ADCC and/or CDC activities in the constant region.

Description

ANTI-CD40 ANTIBODY MUTANTS Technical Field The present invention relates to an anti-CD40 antibody which recognizes CD40 which is a type of cell membrane molecules associated with immunity. In addition, the present invention relates to an antibody with a mutation in the constant region of the human antibody or with a subclass having its portion substituted in order to decrease an activity of the ADCC and / or the CDC, while maintaining a agonist or antagonist activity.

BACKGROUND OF THE ART 1. CD40 CD40 is an antigen that has a molecular weight of 50 kDa which is present on the surface of the cell membrane, and is expressed in B cells, dendritic cells (DCs), some types of cancer cells and thymic epithelial cells. CD40 is known to play an important role in the proliferation and differentiation of B cells and DCs. CD40 was identified as an antigen expressed on the surface of human B cells (EA Clark et al., Proc. Nati, Acad. Sci USA 83: 4494, 1986, and I. Stamenkovic et al., EMBO J. 8: 1403, 1989) and has been considered as a member of the TNF receptor family which includes low affinity NGF receptors, TNF receptor, CD27, OX40 and CD30. Ligands (CD40L) for human and murine CD40s have recently been cloned and found to be type II membrane proteins and are expressed on activated CD4 + T cells. It has also been found that CD40L introduces strong signals for activation within human or murine B cells. It has been observed that CD40 is expressed more highly in dendritic cells than in B cells, and it has become obvious that CD40 plays an important role in dendritic cells. The binding of CD40 to CD40L activates cells that present antigens (the APC), that is, they express the co-stimulatory molecules such as CD80 (B7-1) and CD86 (B7-2) or improve the production of IL-2 (Caux , C., et al .: Activation of human dendritic cells through CD40 cross-linking, J. Exp. Med., 180: 1263, 1994, and - Shu, U., et al .: Activated T cells induces interleukin-12 production by monocyte via CD40-CD40 ligand interaction, Eur. J. Immunol., 25: 1125, 1995). The dendritic cells have a strong capacity that presents antigens and a strong capacity to activate helper T cells (Th). It is also believed that dendritic cells control the differentiation of blank Th cells into Thl or Th2 cells. When peripheral blood monocytes, which are myeloid dendritic cells are grown in the presence of GM-CSF and IL-4 and mature by CD40L, the resulting matured dendritic cells (DC1) can produce IL-12 in vitro, and stimulate and activate alogeneic virgin Th cells that induce T cells that produce IFNα. (that is, they promote their differentiation in Thl). This action is inhibited by the anti-IL-12 antibody and can therefore be effected through IL-12. On the other hand, when plasmacytoid T cells, which occur in the T-lymphoid and peripheral blood regions, are cultured in the presence of IL-3 and the CD40 ligand, the resulting lymphoid dendritic cells (DC2) demonstrate that they are unable to produce IL-12, and stimulate and activate alogeneic virulent Th cells that induce T cells that produce IL-4, which indicate the promotion of Th2 differentiation. It is believed that Thl cells are involved in the activation of cellular immunity, whereas Th2 cells are associated with the enhancement of humoral immunity as well as the restriction of cellular immunity. When the cytotoxic T cells (CTL) are activated with the help of Thl cells, they can eliminate pathogens (a number of virus types, listeria, tuberculosis bacteria, toxoplasma protozoa, etc.) that grow in the cytoplasm and the cells tumor It has been shown that monoclonal anti-CD40 antibodies, which recognize CD40 expressed on the surface of the membrane, have different biological activities to B cells. Monoclonal anti-CD40 antibodies are generally classified as agonist or antagonist antibodies against the interaction between CD40 and CD40L. 2. Antibody Agonists It is known that agonist antibodies activate B cells. For example, anti-CD40 antibodies are reported to induce cell adhesion (Barrett et al., J. Immunol., 146: 1722, 1991; and Gordon et al., J. I munol. 140: 1425, 1998), increase cell size (Gordon et al., J. Immunol., 140: 1425, 1998; and Valle et al., Eur. J. Immunol., 19: 1463, 1989), induce cell division. of B cells activated only by an anti-IgM antibody, anti-CD20 antibody or phorbol ester (Clark and Ledbetter, Proc Nati, Acad Sci USA 83: 4494, 1986, Gordon et al., LEUCOCYTE TYPING III, AJ McMichael Oxford University Press, Oxford, P.426, and Paulie et al., J. Immunol., 142: 590, 1989), induce cell division of B cells in the presence of IL4 (Valle et al., Eur. J Immunol., 19: 1463, 1989 and Gordon et al., Eur. J. Immunol., 17: 1535, 1987), induce the expression of IgE by cultured cells stimulated with IL-4 and deprived of T cells (Jabara et al. ., J. Exp. Med. 172: 1861, 1990, and Gasean et al., J. I munol 147: 8, 1991) induce the expression of IgG and IgM by those cultured cells (Gasean et al., J. Immunol., 147: 8, 1991), secrete soluble CD23 / FceRII from cells at raves of IL-4 (Gordon and Guy, Immunol. Today 8:39, 1987; and Cairns et al., Eur. J. Immunol. 18: 349, 1988), improve the expression of soluble CD23 / FceRII in cells through IL4 (Challa, A., Allergy, 54: 576, 1999) and promote the production of IL-6 (Clark and Shu, J Immunol., 145: 1400, 1990). Furthermore, it is reported that the addition of IL-4 and an anti-CD40 antibody to the B cells of human primary culture in the presence of CDw32 + adhesive cells leads to the establishment of cloned B cells derived therefrom (Bancherauet et al., Science 241: 70, 1991), and germ cell apoptosis was inhibited through CD40 without taking into account whether its antigen receptor was active or inactive (Liu et al., Nature 342: 929, 1989). As described above, CD40 has been identified as an antigen expressed on the surface of human B cells, and consequently, most of the isolated antibodies have been evaluated, as an index, using mainly their induction potency for proliferation and / or the differentiation of human B cells, or their induction activity for cell death of cancer cells (Katira, A. et al., LEUKOCYTE TYPING VSF Schlossossman, et al., eds P. 547, Oxford University Press, Oxford; WC Flansow et al., LEUKOCYTE TYPING VSF Schlossossman, et al., Eds., P 555. Oxford University Press, Oxford, and JD Pound et al., International Immunology, 11:11, 1999). It has been shown that the anti-CD40 antibody matures DC (Z. H. Zhou et al., Hybridoma, 18: 471, 1999). In addition, the role of CD4 T cells in the priming of antigen-specific CD8 T cells was reported to be in DC activation through the signaling of CD40-CD40L and the anti-CD40 monoclonal antibody (mAb). it is capable of replacing CD40 helper cells in the activation of dendritic cells (DC) (Shoenberg, SP, et al .: T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions, Nature, 480, 1998). Also, it has been found that the administration of an anti-CD40 antibody in mice is able to protect the body of the animal from tumor cells expressing CD40 as well as tumor cells that do not express CD40 (French, RR, et. Al .: CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help, Nature Medicine, 5, 1999). The anti-CD40 agonist antibodies are expected to be effective for the treatment of infectious diseases, due to bacteria, viruses, etc., cancer and others, based on their functions described above. Anti-CD40 antibodies with higher agonist activities are described in WO 02/088186. Representative examples of those agonist antibodies are antibodies KM341-1-19 and 2105. Hybridoma KM341-1-19 which produces antibody KM341-1-19 and hybridoma 2105 which produces antibody 2105 were presented on September 27, 2001 and on April 17, 2002, respectively, for international deposit under the Budapest Treaty, to the Depositary of International Patent Organizations, National Institute of Advanced Industrial Science and Technology (central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan). Their access numbers are FERM BP-7759 (KM341-1-19) and FERM BP-8024 (2105). 3. Antagonist Antibodies Taking into consideration on the other hand, that CD40 plays an important role in immunological responses, as mentioned above, it is expected that the inhibition of CD40 binding to its ligands would lead to the development of therapeutic agents for immune suppression in organ transplantation and autoimmune diseases. Sawada, Hase and others have reported that the peripheral blood of patients suffering from Crohn's disease has a higher rate of monocytes that express highly CD40. However, such antibodies have not yet been well known as inhibiting the binding of CD40 to their ligands. Those inhibitory antibodies would be useful in functional analyzes of CD40 and in the treatment of diseases that require the activation of CD40. Inhibitory antibodies to CD40 ligands are also suggested to be effective against diseases involving the binding of CD40 to CD40 ligands. However, it was reported that CD40L is expressed in activated platelets (V. Henn et al., Nature 391: 591, 1998) and if an anti-CD40L antibody is used as a therapeutic agent, thrombus formation can presumably occur (T Kawai et al., Nat. Med. 6: 114, 2000). From this point of view, antibodies to CD40 are expected to be safe in place of anti-CD40L antibodies as the therapeutic antibody agent to inhibit the binding of CD40 to its ligands. Anti-CD40 antibodies would be required to inhibit binding of CD40L to CD40 and still would not activate CD40 by themselves. Such anti-CD40 antagonist antibodies can be used for the treatment of autoimmune diseases and the suppression of immunological rejections in transplantation of organs, bone marrow, etc., in view of their functions described above. Anti-CD40 antibodies with higher antagonist activities are described in WO 02/088186. The representative example of those antagonist antibodies is the 4D11 antibody. The 4D11 hybridoma producing 4D11 antibody was submitted on September 27, 2001 for international deposit under the Budapest Treaty, to the Depositary of International Patent Organizations, National Institute of Advanced Industrial Science and Technology (central 6, 1-1 , Higashi 1, Tsukuba, Ibaraki, Japan). The access number is FERM BP-7758.

Patent Document 1 WO 02/088186 Description of the Invention The object of the present invention is to create mutants from the potentially therapeutic anti-CD40 antibodies described in WO 02/088186, which mutants are optimally designed as a pharmaceutical agent. As a result of extensive and intense research, novel mutants of agonist or antagonist antibodies, whose mutants may have a higher therapeutic effect against diseases than the known anti-CD40 antibodies, have been successfully created, and complete the present invention based on the same The basic idea in the modification of anti-CD40 antibodies according to the present invention will be described in detail later. The present specification will encompass the description in the specification and / or the drawings of JP Patent Publication (Kokai) No. 2003-431408 which is the basis for the priority of the present application.

Brief Description of the Drawings Figure 1A-1 shows binding site peptides prepared based on the CD40 sequence, to which the anti-CD40 agonist antibodies bind; Figure 1A-2 shows binding site peptides prepared based on the CD40 sequence, to which the anti-CD40 agonist antibodies bind (a continuation to Figure 1A-1); Figure 1B-1 shows binding site peptides prepared based on the CD40 sequence, to which the anti-CD40 antagonist antibodies bind; Figure IB-2 shows binding site peptides prepared based on the CD40 sequence, to which anti-CD40 antagonist antibodies are linked (a continuation to Figure 1B-1); Figure 2A illustrates the binding of anti-CD40 antibodies to the CD40 mutant; Figure 2B illustrates the binding of anti-CD40 antibodies to the CD40 mutant; Figure 2C illustrates the binding of anti-CD40 antibodies to the CD40 mutant; Figure 3A shows diagrams indicating that antibody KM341-1-19 having a P331S mutation is as active as the original KM341-1-19 antibody with respect to binding to Ramos cells; Figure 3B shows diagrams indicating that antibody KM341-1-19 having a P331S mutation is as active as the original KM341-1-19 antibody with respect to the enhancement of CD95 expression of Ramos cells; Figure 4A shows a diagram indicating that antibody KM341-1-19 which has a P331S mutation has a lower CDC activity through rabbit complement; Figure 4B shows a diagram indicating that the G2 / 4 antibody has a lower complement activity when the human complement is used. Figure 5A-1 shows diagrams indicating that the conversion of sub-class of antibody 2105 from IgG2 into different subclasses has no effect on its binding to Ramos cells; Figure 5A-2 shows diagrams indicating that conversion of subclass of antibody KM341-1-19 from IgG2 into different subclasses has no effect on its binding to Ramos cells; Figure 5B-1 shows diagrams indicating that conversion of subclass of antibody 2105 from IgG2 into different subclasses reduces activity to improve CD95 expression of Ramos cells; Figure 5B-2 shows diagrams indicating that conversion of subclass of antibody KM341-1-19 from IgG2 into different subclasses decreases an activity to improve CD95 expression of Ramos cells; Figure 6A-1 shows diagrams indicating that the binding capacity of KM341-1-19 antibodies to Ramos cells is independent of the variable structure of the joint region; Figure 6A-2 shows diagrams indicating that the binding capacity of Ramos 2105 antibodies is independent of the variable structure of the joint region; Figure 6B-1 shows diagrams indicating that the upper and middle joints of the joint region are important for the activity of KM341-1-19 antibodies to improve CD95 expression of Ramos cells; Figure 6B-2 shows diagrams indicating that the upper and middle joints of the joint region are important for the activity of the 2105 antibodies to improve CD95 expression of Ramos cells; Figure 7A shows diagrams indicating that conversion of subclass of antibody F72 to IgG2 has no effect on its binding to Ramos cells; Figure 7B shows diagrams indicating that conversion of subclass of antibody F72 to IgG2 raises an activity to improve CD95 expression of Ramos cells; Figure 8A shows diagrams indicating that conversion of the subclass of the 4D11 antibody from IgG1 to IgG4 has no effect on its binding to Ramos cells; Figure 8B shows diagrams indicating that conversion of the subclass of antibody 4D11 from IgG1 to IgG4 inhibits the enhancement by CD40 ligand of CD95 expression of Ramos cells, to the same extent as under other circumstances. Figure 9 shows a diagram indicating that conversion of the subclass of antibody 4D11 from IgG1 to IgG4 or IgG4PE reduces the activity of ADCC; Figure 10 shows a diagram indicating that conversion of the subclass of antibody 4D11 from IgG1 to IgG4P reduces the activity of CDC; Figure 11 illustrates a variation in the number of B cells in the blood (B220 positive cells between peripheral blood lymphocytes) over time during 4D11G1, 4D11G4P or 4D11G4PE was administered in human CD40 transgenic mice; Figure 12A illustrates a higher expression of CD23 of splenic B cells (CD23 positive cells between splenic B cells) after each anti-CD40 antibody was administered in human CD40 transgenic mice; Figure 12B illustrates a higher expression of CD86 of splenic B cells (CD86 positive cells among splenic B cells) after each anti-CD40 antibody was administered in human CD40 transgenic mice; Figure 12C illustrates a higher expression of CD95 of splenic B cells (CD95 positive cells among splenic B cells) after each anti-CD40 antibody was administered in human CD40 transgenic mice; Figure 13A illustrates the suppressive activity of the production of antigen-specific antibody (IgGl) by 4D11 and 281-1-10 in human CD40 transgenic mice; Figure 13B illustrates the suppressive activity of the production of antigen-specific antibody (IgM) by 4D11 and 281-1-10 in human CD40 transgenic mice; Figure 14A illustrates numbers of B cells in blood (B220 positive cells between peripheral blood lymphocytes) during the suppression test of the production activity of antigen-specific antibodies; Figure 14B illustrates numbers of splenic B cells (B220 positive cells between splenic lymphocytes) during the suppression test of antigen-specific antibody production activity; Figure 15 illustrates a variation in the number of B cells in blood (B220 positive cells between peripheral blood lymphocytes) with the time after 4D11G4P or 4D11G4PE was administered in a dose of 30 mg / kg in cynomologous monkeys; Figure 16 illustrates blood IL-12 levels during the assay shown in Figure 15; Figure 17 shows the suppressive effect of 4D11G4PE on DTH (delayed type hypersensitivity in male cynomologo monkeys); Figure 18 shows the titers of the tetanus IgG toxin during the assay with the results shown in Figure 17; Figure 19 shows the titers of tetanus IgM toxin during the test with the results shown in Figure 17; Figure 20A illustrates the respective influences of 4D11G4PE and 5C8 (anti-CD40L Ligand antibody) on platelet aggregation; Figure 20B shows the respective influences of 4D11G4PE and 5C8 (anti-CD40 Ligand antibody) on platelet aggregation; Figure 21 illustrates a variation in the oligomeric content of 4D11G4P, 4D11G4PE, 4DllG2Ser or 4D11G4 / 2/4 with time after it was incubated at pH 2.7 and 37 ° C; Figure 22 illustrates the suppression of rejection of skin grafts by the anti-CD40 antagonist antibody; Figure 23 illustrates the change in tumor volume over time from the cellular implant, in a case where 341G2Ser was administered to mice suffering from tumor with Ramos cells implanted therein; Figure 24 illustrates the change in tumor volume over time from the cellular implant, in a case where 341G2Ser was administered to mice suffering from tumor with T24 cells implanted therein; Figure 25 illustrates the change in tumor volume over time from the cellular implant, in a case where 341G2Ser was administered to mice suffering from tumor with Hs 766T cells implanted therein; and Figure 26 illustrates the change in tumor volume over time from the cellular implant, in a case where 341G2Ser was administered to mice suffering from tumor with Capan-2 cells implanted therein.

Best Mode for Carrying Out the Invention 1. Modification of Agonist Antibodies Antibodies are essentially molecules that function to protect living bodies against foreign bodies, such as microorganisms and viruses, and cancer, and can therefore kill and eliminate such cells that are linked to them. Lethal activity is composed of two different activities, called Antibody-dependent Cellular Cytotoxicity (abbreviated as ADCC below) and Complementary Dependent Cytotoxicity (abbreviated as CDC below). ADCC refers to a type of cytotoxicity induced by the activation of macrophages, NK cells, neutrophilic cells, etc., which recognize dianocytes by binding to the constant region of the antibody through Fc receptors expressed on its surface. In contrast, the CDC refers to a type of cytotoxicity induced by the activation of a complement system which occurs through the binding of an antibody to an antigen. These activities are known to vary depending on a subclass of the antibody, which has been found to be due to a structural difference in the constant region of the antibodies (Charles A. Janeway et al., Immunology, 1997, Current Biology Ltd./ Garland Publishing Inc.). Anti-CD40 agonist antibodies will be more preferable as therapeutic agents if they do not have ADCC and / or CDC activities which can induce cell death of cells that express CD40, in terms of the mechanism of immunoactive action. If cells expressing CD40 are damaged by ADCC and / or CDC activities, immunosuppression may occur instead of the desired immunoactivation, resulting in exacerbation of the disease. In addition, patients suffering from infectious diseases may have higher ADCC and / or CDC activities. Therefore, when such antibodies are applied to infectious diseases, it is necessary to evaluate them for safety more carefully, for example, using more active rabbit supplements than those present in healthy human serum or peripheral blood which could not be effective to detect the previous activities in this situation. Accordingly, mutants and recombinants were created which had no ADCC or CDC activity and were examined for their activity. Since the activities of ADCC and / or CDC are known to vary depending on a subclass of the antibody of interest, the conversion of the subclass can reduce activities of ADCC and / or CDC. For human IgG subclasses, for example, IgG4 is generally known to be a subclass with low activities of both ADCC and CDC, and it is reported that IgG2 is active CDC, but poorly active in ADCC, while IgG1 is highly active. in both ADCC and CDC (Charles A. Janeway et al., Immunology, 1997, Current Biology Ltd./Garland Publishing Inc.). The selection of a particular subclass by taking advantage of the above characteristics can create a less cytotoxic antibody from the original antibody. A combination of specific subclasses of the antibody with such point mutation as described below can create an antibody with a desired activity. In addition, the reduction in ADCC and / or CDC activities of an antibody is reported to be obtained by the incorporation of a mutation within its constant region. For example, L235, D265, D270, K322, P331 and P329 (each alphabetic letter denotes an amino acid by the annotation of a single letter, and each number denotes an EU index proposed by Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition); such symbols will be used later.) may play an important role in complement activation by human IgG, and replacement of one of those sites by another amino acid may reduce CDC activity. Esohe Idusogie et al. J. Immunol. 2000, 164: 4178-4184, Yuanyuan Xu et. to the. J. Biol. Chem. 1994, 269: 3469-3474, Brekke, O. H. et. Al. Eur. J. Immunol. 1994, 24: 2542, Morgan, A., et. al., Immunology 1995, 86: 319, Lund, J., et. to the.; J. Immunol. , 1996, 157: 4963, Tao, M.H. , et al., J. Exp. Med. 1993, 178: 661). Specifically, substituting D270, K322, P329 or P331 for A can reduce the activity of CDC. Substituting P331 for S or G can also induce the same thing. It is believed that Glu233-Ser239, Gly316-Lys338, Lys274-Arg301, Tyr407-Arg416, Asn297, Glu318, Leu234-Ser239, Asp265-Glu269, Asn297-Thr299 and Ala327-Ile332 take a part in the binding of IgG to FcR (Duncan , AR, Wolf, JM, Partridge, L., J., Burton, DR, and Winter, G. (1988) Nature 332, 563-564, Gessner, JE, Heiken, H., Tamm, A., and Schmidt , RE (1998) Ann Hematol 76, 231-248, Gavin, A., Hulett, M, and Hogarth, PM (1998) in The Immunoglobulin Receptors and Their Physiological and Pathological Roles in Immunity (van de Winkel, JGJ, and Hogarth, PM, eds), pp. 11-35, Kluwer Academic Publishers Group, Dordrecht, The Netherlands, Sautes, C. (1997) in Cell-Mediated Effects of Immunoglobulins (Fridman, WH, and Sautes, C, eds) , pp. 29-66, RG Landes Co., Austin, TX, Daron, M. (1997) Annu., Rev. Immunol., 15, 203-234, Canfield, SM, and Morrison, SL (1991) J. Exp. Med. 173, 1483-1491, Chappel, MS, Isenman, DE, Everett, M., Xu, Y. -Y., Dorrington, KJ, and Klein, MH (19). 91) Proc. Nati Acad. Sci. U.S.A. 88, 9036-9040, Wolf, J.M., Partridge, L.J., Jefferis, R., and Burton, D.R. (1986) Mol. Immunol. 23, 319-330, Wines, B. D., Powell, M.S., Parren, P.W.H.I., Barnes, N., and Hogarth, P.M. (2000) J. Immunol. 164, 5313-5318) and thus the incorporation of a mutation in one of these regions can reduce the activity of ADCC. Specifically, a substitution of L235 for E or G237 for A can reduce the binding of IgG to FcR. The antibody according to the present invention has at least one amino acid mutation to reduce the activities of ADCC and / or CDC, preferably 1-20, 1-17, 1-16, 1-15, 1-14, 1- 13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1 or 2 mutations. The present invention has revealed that in some anti-CD40 antibodies, the articulation region of IgG2 is important in the expression of its strong agonist activities. The replacement of the variable region or the constant region, except the region of articulation by a counterpart of any different subclass, or the incorporation of a point mutation in it is expected to module not only the activities of ADCC and / or CDC, but also increase the productivity of the antibody, its stability during purification and storage, and its blood kinetics. To produce an antibody drug, the stability of the antibody during purification and storage is very important. Since the antibodies developed up to now belong mainly to the subclass IgG1, the conversion of the variable region or the constant region except the region of articulation to a sequence derived from subclass IgG1 will also be effective in improving the physical properties of the anti-agonist antibodies. -CD40 described above. The present invention provides mutants of anti-CD40 agonist antibodies and others as follows: [1] A heavy chain of a monoclonal antibody having an agonist activity, which binds to CD40, wherein the heavy chain comprises an upper link and a medium joint derived from a human IgG2, and a constant region with at least one amino acid deleted or substituted, or with at least one amino acid added thereto, such elimination, substitution or addition are capable of increasing or decreasing ADCC and / or CDC. [2] The heavy chain according to [1], wherein the constant region is derived from a human IgG. [3] The heavy chain according to [2], wherein the human IgG is a human IgGl. [4] The heavy chain according to [2], wherein the human IgG is a human IgG2: [5] The heavy chain according to [2], wherein the Human IgG is a human IgG3. [6] The heavy chain according to [2], wherein the human I G is a human IgG4. [7] The heavy chain according to any of [3] to [5], wherein the substitution of amino acids in the constant region is the substitution of proline with serine at position 331 which is indicated by the EU index as in Kabat et al. [8] A monoclonal antibody comprising the heavy chain according to any of [1] to [7]. [9] The heavy chain according to any of [1] to [7], wherein the heavy chain comprises a variable region from a heavy chain of a monoclonal antibody produced by the hybridoma KM341-1-19 (Accession No. FERM BP-7759). [10] A monoclonal antibody consisting of a heavy chain according to [9] and a light chain comprising a variable region from a light chain of a monoclonal antibody produced by the hybridoma KM341-1-19 (Access Number FERM BP-7759).

[11] The heavy chain according to any of [1] to [7], wherein the heavy chain comprises a variable region of the polypeptide represented by SEC. FROM IDENT. DO NOT. : 38. [12] A monoclonal antibody consisting of the heavy chain according to [11] and a light chain of a monoclonal antibody, wherein the light chain comprises a variable region of the polypeptide represented by the SEC. FROM IDENT. DO NOT. : 40. [13] The heavy chain according to [1], wherein the heavy chain consists of a remaining portion provided by removing the signal sequence from the polypeptide represented by the SEC. FROM IDENT. NO .: 132. [14] A monoclonal antibody consisting of the heavy chain according to [13] and a light chain of a monoclonal antibody, wherein the light chain consists of a remaining portion provided by removing the signal sequence to from the polypeptide represented by SEC. FROM IDENT. DO NOT. : 134. [15] The heavy chain according to [1], wherein the heavy chain is produced by a host comprising an expression vector having the polynucleotide represented by SEQ. FROM IDENT. NO .: 131. [16] The monoclonal antibody according to [8], wherein the monoclonal antibody is produced by a host comprising an expression vector having the polynucleotide represented by SEQ. FROM IDENT. NO .: 131 and the polynucleotide represented by SEC. FROM IDENT. NO .: 133. [17] The heavy chain according to any of [1] to [7], wherein the heavy chain comprises a variable region from a heavy chain of a monoclonal antibody produced by hybridoma 2105 (Accession Number FERM BP-8024). [18] A monoclonal antibody consisting of the heavy chain according to [17] and a light chain comprising a variable region from a light chain of a monoclonal antibody produced by hybridoma 2105 (Accession Number FERM BP-8024). [19] The heavy chain according to any of [1] to [7], wherein the heavy chain comprises a variable region of the polypeptide represented by SEC. FROM IDENT. DO NOT. : 42. [20] A monoclonal antibody consisting of the heavy chain according to [19] and a light chain of a monoclonal antibody, wherein the light chain comprises a variable region of the polypeptide represented by SEC.

FROM IDENT. DO NOT. : 44. [21] The heavy chain according to [1], wherein the heavy chain consists of a remaining portion provided by removing the signal sequence from the polypeptide represented by the SEC. FROM IDENT. NO .: 136. [22] A monoclonal antibody consisting of a heavy chain according to [21] and a light chain of a monoclonal antibody, wherein the light chain consists of a remaining portion provided by removing the signal sequence at from the polypeptide represented by SEC. FROM IDENT. DO NOT. : 138. [23] The heavy chain according to [1], wherein the heavy chain is produced by a host comprising an expression vector having the polynucleotide represented by SEQ. FROM IDENT. NO .: 135. [24] The monoclonal antibody according to [8], wherein the monoclonal antibody is produced by a host comprising an expression vector having the polynucleotide represented by SEQ. FROM IDENT. DO NOT. : 135 and the polynucleotide represented by SEC. FROM IDENT. NO .: 137. [25] A polynucleotide represented by SEC. FROM IDENT. DO NOT. : 131. [26] A polynucleotide represented by SEC. FROM IDENT. DO NOT. : 133. [27] An expression vector that has the polynucleotide according to [25]. [28] An expression vector that has the polynucleotide according to [26]. [29] An expression vector that has polynucleotides according to [25] and [26]. [30] A host comprising the expression vector according to [27]. [31] A host that comprises the expression vector according to [28]. [32] A host comprising the expression vector according to [29]. [33] A process for producing a heavy chain of a monoclonal antibody, comprising the steps of culturing the host according to [30] in a culture medium; and obtaining a heavy chain of a monoclonal antibody from the culture and / or the host. [34] A process for producing a monoclonal antibody, comprising the steps of: culturing the host according to [32] in a culture medium; and obtaining a monoclonal antibody from the culture and / or the host. [35] A polynucleotide represented by SEC. FROM IDENT. DO NOT. : 135. [36] A polynucleotide represented by SEC. FROM IDENT. DO NOT. : 137. [37] An expression vector that has the polynucleotide according to [35]. [38] An expression vector that has the polynucleotide according to [36]. [39] An expression vector that has polynucleotides according to [35] and [36]. [40] A host comprising the expression vector according to [37]. [41] A host that comprises the expression vector according to [38]. [42] A host comprising the expression vector according to [39]. [43] A process for producing a heavy chain of a monoclonal antibody, comprising the steps of culturing the host according to [40] in a culture medium; and obtaining a heavy chain of a monoclonal antibody from the culture and / or the host. [44] A process for producing a monoclonal antibody, comprising the steps of: culturing the host according to [42] in a culture medium; and obtaining a monoclonal antibody from the culture and / or the host. [45] A process for producing a heavy chain of a monoclonal antibody having an agonist activity capable of binding to CD40, comprising the step of replacing the upper joint and the middle joint of an antibody, which is neither a superior joint nor a median joint derived from a human IgG2, with an upper joint and a middle joint derived from a human IgG2, respectively. [46] A process for producing a heavy chain of a monoclonal antibody comprising a variable region, and an upper joint and a middle joint derived from a human IgG2, comprising the step of identifying a polypeptide that forms the variable region, which is from a heavy chain of a monoclonal antibody capable of binding to CD40. [47] A process for producing a monoclonal antibody having an agonist activity capable of binding to CD40, which comprises the step of replacing the upper joint and the middle joint of an antibody, which is neither an upper joint nor a middle joint derived from a human IgG2, with an upper joint or a middle joint derived from a human IgG2, respectively. [48] A process for producing a monoclonal antibody comprising a variable region, and an upper joint and a middle joint derived from a human IgG2, comprising the step of identifying a polypeptide that forms the variable region, which is from a heavy chain of a monoclonal antibody capable of binding to CD40. [49] A pharmaceutical composition comprising the monoclonal antibody according to [8], [10], [12], [14], [16], [18], [20], [22] or [24] as an active ingredient [50] The pharmaceutical composition according to [49] used for the prevention or treatment of a malignant tumor, a pathogen or an autoimmune disease. [51] A method for the prevention or treatment of a malignant tumor, a pathogen or an autoimmune disease, comprising the administration of the pharmaceutical composition according to [49] in a mammal. [52] The use of the monoclonal antibody according to [8], [10], [12], [14], [16], [18], [20], [22] or [24] for the production of a pharmaceutical composition used for the prevention or treatment of a malignant tumor, a pathogen or an autoimmune disease. [89] A polynucleotide provided by removing the portion encoding the signal sequence from the polynucleotide represented by SEC. FROM IDENT. NO .: 131. [90] A polynucleotide provided by removing the portion encoding the signal sequence from the polynucleotide represented by SEC. FROM IDENT. NO .: 133. [91] A polynucleotide provided by removing the portion encoding the signal sequence from the polynucleotide represented by SEC. FROM IDENT. NO .: 135. [92] A polynucleotide provided by removing the portion encoding the signal sequence from the polynucleotide represented by SEC. FROM IDENT. NO .: 137. The present invention provides an antibody produced by the modification of an anti-CD40 agonist antibody belonging to human IgG2, wherein the modified antibody is a mutant having the constant region, exclusive of the upper and middle joints , substituted with a sequence derived from a different subclass. The subclass is preferably IgGl. . The present invention provides an antibody produced by modification of an anti-CD40 agonist antibody belonging to human IgG2, wherein the modified antibody is a mutant having the constant region, exclusive of the region of articulation, substituted with a derived sequence of a different subclass. The subclass is preferably IgGl. Here, the reduction in ADCC and CDC activities means reduction in those activities when compared to the corresponding activities of an anti-CD40 monoclonal antibody different from the mutants described above, for example, when compared to the corresponding activities of a monoclonal antibody produced by hybridoma KM341-1-19 (Accession Number FERM BP-7759) or 2105 (Accession No. FERM BP-8024). The activities of ADCC and CDC can be evaluated by any known method, for example, the method described in the Examples herein. The sequences of the variable regions in the heavy and light chains of a monoclonal antibody will be presented later which is produced by the hybridoma KM341-1-19 (Accession No. FERM BP-7759) or 2105 (Accession No. FERM BP -8024). The variable regions encoding the DNA in the heavy and light chains of the KM341-1-19 antibody and the amino acid sequences of the heavy and light chains will be presented later. In the heavy chain nucleotide sequence (SEQ ID NO: 37) of antibody KM341-1-19, the signal sequence is initiated with adenine (A) at position 50. The boundary between the signal sequence and the variable region is between " adenine "([A]) at position 109 and cytosine (C) at position 110, and the boundary between the variable region and the constant region lies between adenine (A) at position 493 and guanine (G) ) at position 494 (genetic prediction software was used) (Signal P ver.2)). In the heavy chain amino acid sequence (SEQ ID NO: 38) of antibody KM341-1-19, the boundary between the signal sequence and the variable region is located between serine (S) at position 20 and glutamine (Q) at position 21 and the boundary between the variable region and the constant region is located between serine (S) at position 148 and alanine (A) at position 149. Consequently, the variable region in the heavy chain of antibody KM341-1-19 has a nucleotide sequence that varies from cytosine (C) at position 110 to adenine (A) at position 493, as seen in SEC. FROM IDENT. NO .: 37. In addition, the variable region in the heavy chain of antibody KM341-1-19 has an amino acid sequence that varies from glutamine (Q) at position 21 to serine (S) at position 148, as shown in the SEC. FROM IDENT. DO NOT. : 38. In the light chain nucleotide sequence (SEQ ID NO: 39) of antibody KM341-1-19, the signal sequence is initiated with adenine (A) in position 29. The boundary between the signal sequence and the variable region is located between "adenine" ([A]) at position 88 and guanine (G) at position 89, and the boundary between the variable region and the constant region is between adenine (A) at position 400 and "cytosine" ([C]) at position 401 (genetic prediction software (Signal P ver.2) was used). In the light chain amino acid sequence (SEQ ID NO: 40) of antibody KM341-1-19, the boundary between the signal sequence and the variable region is between glycine (G) at position 20 and glutamic acid (E) at the position 21, and the boundary between the variable region and the constant region is located between lysine (K) at position 124 and "arginine" ([R]) at position 125. Therefore, the variable region in the light chain of the antibody KM341-1-19 has a nucleotide sequence that varies from guanine (G) at position 89 to adenine (A) at position 400, as observed in SEC. FROM IDENT. DO NOT . : 39 In addition, the variable region in the light chain of antibody KM341-1-19 has a 5 amino acid sequence ranging from glutamic acid (E) at position 21 to lysine (K) at position 124, as observed in the SEC . FROM IDENT. DO NOT . : 40 The nucleotide sequence of heavy chain (SEQ ID NO: 37...) Antibody KM341 - 1 to 19: 0 GTCGACGCTGAATTCTGGCTGACCAGGGCAGCCACCAGAGCTCCAGACAATGTCTGTCTCCTTCCTCATCTT CCTGCCCGTGCTGGGCCTCCCATGGGGTGTCCTGTCACAGGTCCAACTGCAGCAGTCAGGTCCAGGACTGGT GAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTACTTG GAACTGGATCAGGCAGTCCCCATCGAGAGACCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTA TCGTGATTATGTAGGATCTGTGAAAAGTCGAATAATCATCAACCCAGACACATCCAACAACCAGTTCTCCCT GCAGCTGAACTCTGTGACTCCCGAGGACACGGCTATATATTACTGTACAAGAGCACAGTGGCTGGGAGGGGA 15 TTACCCCTACTACTACAGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTTCAGCCTCCACCAA GGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCT GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACAC CTTCCCAGCTGTCCTACAGTGCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTT CGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCG CAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCC 20 AA AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGA AGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGA GGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAA GGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGG GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCT _ _ GACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA ? O CAACTACAAGACCACACCTCCCATGCTGGACTCAGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGA CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGGATCC The heavy chain amino acid sequence (SEQ ID NO: 38) of antibody KM341-1-19: MSVSFLIFLPVLGLPWGVLSQVQLQQSGPGLVKPSQTXSLTCAISGDSVSSNSATWNWIRQSPSKDLEWLGR TYYRSKWYRDYVGS ^ RIIINPDTSNNQFSLQ T SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVmpPAVLQSSGLYSLSSV VTWSSjNTFGTQTYTCNVDHia, S? rrK ^ VVVDVSHEDPEVQFNWYVDGVEVHNAK? O'REEQFNSTFRVVSVLTVVHQDWLNGKEY ^ K? SKTKGQPREPQVYTLPPSIffiEMTKNQVSLTCL ^ LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The light chain nucleotide sequence (SEQ.

FROM IDENT. DO NOT . : 39) of antibody KM341-1-19: ACTGCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCT CCCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGC CACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGC TCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTC TGGGACAGACTrCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCG TAGCAACACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACG The amino acid sequence of light chain (SEQ ID NO:... 40) of the KM341-1-19 antibody: IvffiAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS TG ^ p] ^ PARFSGSGSGTDFTLTISSLEPEDFAWYCQQRSNTFGPGTK KRT The variable regions of DNA coding in heavy and light chains of antibody 2105 and the amino acid sequences of heavy and light chains will be presented later. In the heavy chain nucleotide sequence (SEQ ID NO: 41) of antibody 2105, the signal sequence is initiated with adenine (A) at position 70. The boundary between the signal sequence and the variable region it is located between "thymine" ([T]) at position 126 and guanine (G) at position 127, and the boundary between the variable region and the constant region lies between adenine (A) at position 495 and guanine ( G) at position 496 (genetic prediction software (Signal P ver.2) was used). In the heavy chain amino acid sequence (SEQ ID NO: 42) of antibody 2105, the boundary between the signal sequence and the variable region is between cysteine (C) at position 19 and glutamic acid (E) ) at position 20, and the boundary between the variable region and the constant region is located between serine (S) at position 142 and alanine (A) at position 143. Therefore, the variable region in the heavy chain of the antibody 2105 has a nucleotide sequence that varies from guanine (G) at position 127 to adenine (A) at position 495, as observed in SEC. FROM IDENT. DO NOT. : 41. In addition, the variable region in the heavy chain of antibody 2105 has an amino acid sequence that ranges from glutamic acid (E) at position 20 to serine (S) at position 142, as observed in SEC. FROM IDENT. NO .: 42. In the light chain nucleotide sequence (SEQ ID NO: 43) of antibody 2105, the signal sequence is initiated with adenine (A) at position 28. The boundary between the sequence of signal and the variable region is located between "adenine" ([A]) at position 87 and guanine (G) at position 88, and the boundary between the variable region and the constant region lies between adenine (A) in the position 405 and "cytosine" ([C]) at position 406 (genetic prediction software (Signal P ver.2) was used). In the light chain amino acid sequence (SEQ ID NO: 44) of antibody 2105, the boundary between the signal sequence and the variable region is between glycine (G) at position 20 and glutamic acid ( E) at position 21, and the boundary between the variable region and the constant region lies between lysine (K) at position 126 and "arginine" ([R]) at position 127. Therefore, the variable region in the light chain of antibody 2105 has a nucleotide sequence that varies from guanine (G) at position 88 to adenine (A) at position 405, as observed in SEC. FROM IDENT. DO NOT. : 43. In addition, the variable region in the light chain of antibody 2105 has an amino acid sequence that ranges from glutamic acid (E) at position 21 to lysine (K) at position 126, as observed in SEC. FROM IDENT. NO .: 44 The nucleotide sequence of heavy chain (SEQ ID NO:... 41) of the 2105 antibody: CTGAACACAGACCCGTCGACTCCCAGGTGTTTCCATTCAGTGATCAGCACTGAACACAGAGGACTCACCATG GAGTGGGGACTGAGCTGGATTTTCCTTTTGGCTAITITAAAAGGTGTCCAGTGTGA ^ TCTGGGGGAGGC1TGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGAT GATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAAT AGTGGTAGCTTGGTGCATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCC CTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAGAGATAGGCTATTT CGGGGAGTTAGGTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACC The heavy chain amino acid sequence (SEQ.

FROM IDENT. DO NOT . : 42) Antibody 2105: ELGLSW1FLLAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISW NSGSLVHADSVKGKF? SRDNAKNSLYLQMNSLRAEDTALYYCARDRLFR The light chain nucleotide sequence (SEQ ID NO: 43) of the 2105 antibody: CTGCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTC CCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCT CCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCT GGGACAGACT CACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGT AGCCACTGGCTCACTTTCGGCGGGGGGACCAAGGTGGAGATCAAACGTACGGTG The amino acid sequence of light chain (SEQ ID NO:... 44) of the 2105 antibody: MEAPAQLLFLLLLWLPDTTGEIVITQSPA'ILSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS IATGIPARFSGSGSGTDF'IL SSLEPEDFAVYYCQQRSHWLTFGGG'I VEIKRTV In the nucleotide sequence of heavy chain (SEQ ID. NO .: 131) of the 341G2Ser, the boundary between the signal sequence and the variable region is located between "adenine" ([A]) at position 60 and cytosine (C) at position 61, and the boundary between the variable region and the constant region is located between adenine (A) at position 444 and guanine (G) at position 445 (genetic prediction software (Signal P ver.2) was used). In the heavy chain nucleotide sequence (SEQ ID NO: 132) of 341G2Ser, the boundary between the signal sequence and the variable region is between serine (S) at position 20 and glutamine (Q) at position 21, and the boundary between the variable region and the constant region is located between serine (S) at position 148 and alanine (A) at position 149. Consequently, the variable region in the heavy chain of 341G2Ser has a nucleotide sequence varying from cytosine (C) at position 61 to adenine (A) at position 444, as observed in SEC. FROM IDENT. NO .: 131. In addition, the variable region in the heavy chain of 341G2Ser has an amino acid sequence that varies from glutamine (Q) at position 21 to serine (S) at position 148, as observed in SEC. FROM IDENT. NO .: 132. The complete heavy chain nucleotide sequence of 341G2Ser (SEQ ID NO: 131): ATGTCTGTCTCCTTCCTCATCTTCCTGCCCGTGCTGGGCCTCCCATGGGGTGTCCTGTCACAGGTCCAACTG CAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGT GTCTCTAGCAACAGTGCTACTTGGAACTGGATCAGGCAGTCCCCATCGAGAGACCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTATCGTGATTATGTAGGATCTGTGAAAAGTCGAÁTAATCATCAACCCAGAC ACATCCAACAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTATATACTACTGTACA AGAGCACAGTGGCTGGGAGGGGATTACCCCTACTACTACAGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAG AGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC GCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTG GTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACC AAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGC GTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT AATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTG CACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCTCCATCGAG AAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTC CTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA The complete amino acid sequence of heavy chain 341G2 Ser (SEQ ID NO:... 131): MSVSFLFLPVLGLPWGVLSQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRDLEWLGR l ^^ YESKWYRDYVGS'V -SRlIIN TVSSASTKGPSWPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV? WWSSNFGTQTYTCNVD! HKPSNtK VVVDVSHEDPEVQFNWY'VOGVEVHNAKTKPREEQI ^ ^ ^ K S'IPRVVSVLTVVHQDWLNGKEYKCKVSj? SKTKGQPREPQWTLPPSPJEEMTKNQVSLTCLV ^^ LYSKLTVDKSRWQQGNVFSCSVMEffiALHNHYTQKSLSLSPGK In the nucleotide sequence of light chain (SEQ. Ident. NO .: 133) of the 341G2Ser, the boundary between the signal sequence and the region variable is located between "adenine" ([A]) at position 60 and guanine (G) at position 61 and the boundary between the variable region and the constant region is between adenine (A) at position 372 and the " cytosine "([C]) at position 373 (genetic prediction software (Signal P ver.2) was used). In the light chain amino acid sequence (SEQ ID NO: 134) of the 341G2Ser, the boundary between the signal sequence and the variable region is between glycine (G) at position 20 and glutamic acid (E) at position 21, and the boundary between the variable region and the constant region is located between lysine ( K) at position 124 and "arginine" ([R]) at position 125. Accordingly, the variable region in the light chain of 341G2Ser has a nucleotide sequence that varies from guanine (G) at position 61 to adenine (A) at position 372, as noted in the SEC. FROM IDENT. NO .: 133. In addition, the variable region in the light chain of 341G2Ser has an amino acid sequence that varies from glutamic acid (E) at position 21 to lysine (K) at position 124, as observed in the SEC . FROM IDENT. NO .: 134. The complete light chain nucleotide sequence of 341G2Ser (SEQ ID NO: 133): ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTG ACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGT GTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCC AACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGC AGCCTAGAGCCTGAAGATL TGCAGTTTATTACTGTCAGCAGCGTAGCAACAC'ITRCGGCCCTGGGACCAAA GTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTGGAAATCT GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT AACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA The complete light chain amino acid sequence of 341G2Ser (SEQ ID NO: 134). MEAPAQLLFLLLLWLPD1 GEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS NRATGPARFSGSGSGTDFTLTISSLEPEDFA YCQQRSNTCGPG'TK ^ GTASWCLL? ^ YPREAKVQWK'VONALQSGNSQESVTEQ GLSSPVTKSFNRGEC In the heavy chain nucleotide sequence (SEQ ID NO: 135) of the 2105G2Ser, the boundary between the signal sequence and the variable region is located between "thymine" ([T]) at position 57 and guanine (G) at position 58 , and the limit between the variable region and the constant region is between adenine (A) at position 426 and guanine (G) at position 427 (genetic prediction software (Signal P ver. 2) was used). In the heavy chain amino acid sequence (SEQ ID NO: 136) of the 2105G2Ser, the boundary between the signal sequence and the variable region is between cysteine (C) at position 19 and glutamic acid (E) at position 20, and the The limit between the variable region and the constant region is between serine (S) at position 142 and alanine (A) at position 143. Consequently, the variable region in the heavy chain of 2105G2Ser has a nucleotide sequence that varies from guanine (G) at position 58 to adenine (A) at position 426, as observed in SEC. FROM IDENT. NO .: 135. In addition, the variable region in the heavy chain of 2105G2Ser has an amino acid sequence that varies from glutamic acid (E) at position 20 to serine (S) at position 142, as observed in the SEC. FROM IDENT. NO .: 136. The nucleotide sequence of the complete heavy chain of the 2105G2Ser (SEQ ID NO .: 135..): ATGGAGITGGGACTGAGCTGGATTITCCTTTTGGCTATTTTAAAAGGTGTCCAGTGTGA ^ GG GAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT GATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAG AATAGTGGTAGCTTGGTGCATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAC TCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAGAGATAGGCTA TTTCGGGGAGTTAGGTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGC ACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGC TGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTG CACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGC AACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTT GAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTC CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGC CACGAAGACCCCGAGGTCCAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCA CGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAAC GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAACC AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTC AGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG GAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA The complete heavy chain amino acid sequence of 2105G2Ser (SEQ ID NO: 136). MELGLSWIFLLAlLKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAlvlHWVRQAPGKGLEWVSGISW NSGSLVHADSVKGI? Sia > NAj ^ SLYLQMNSLRAEDTA ^ TKGPSVITLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS NFGTQI? TCNVDHKPSNIXVDKTVERKCCVECPPCPAPPVAGPSWLFPPKPKDT ^ HEDPEVQFNWYVDGVE'V? NAK'GKPREEQ ™ SI? RVVSVLTVVHQDWLNGKEY ^ KGQPREPQVYTLPPSREEMTO "QVSLTCLVKGFYPS VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In the nucleotide sequence light chain (SEQ ID NO: 137) of the 2105G2 Ser, the boundary between the signal sequence and the variable region is between "adenine" ([A]) at position 60 and guanine (G) at position 61, and the boundary between the variable region and the constant region is located between adenine (A) at position 378 and "cytosine" ([C]) at position 379 (genetic prediction software (Signal P ver.2) was used). In the light chain amino acid sequence (SEQ ID NO: 138) of the 2105G2Ser, the boundary between the signal sequence and the variable region is between glycine (G) in position 20 and glutamic acid (E) in position 21, and the limit between the variable region and the constant region lies between lysine (K) at position 126 and "arginine" ([R]) at position 127. Accordingly, the variable region in the light chain of 2105G2Ser has a nucleotide sequence that varies from guanine (G) at position 61 to adenine (A) at position 378, as observed in SEC. FROM IDENT. DO NOT . : 137 In addition, the variable region in the light chain of 2105G2Ser has an amino acid sequence that varies from glutamic acid (E) at position 21 to lysine (K) at position 126, as observed in SEC. FROM IDENT. DO NOT . : 138 The complete light chain nucleotide sequence of 2105G2Ser (SEQ ID NO: 137). ATGGAAGCCCCAGCTCAGCTTCTCI CCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTG ACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGT GTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCC AACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGC AGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCCACTGGCTCACITTCGG ^ ACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAG GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA The complete light chain amino acid sequence of 2105G2Ser (SEQ ID NO: 138).

NJ? APAQLIJLLLLWLPDTTGEIVLTQSPATLSLSPGERAILSC ^ NRATGIPARFSGSGSGTDFTL? SSLEPEDFAVYYCQQRSHWLIFGGG 'VEIKRTVAAPSWIFPPSDEQL KSGTASWCLL TNFYPREAKVQWKVDNALQSGNSQESVGEQ HQGLSSPVTKSFNRGEC 2. Modification of Antagonist Antibodies Anti-CD40 antagonist antibodies will be more preferable as a therapeutic agent as well as agonist antibodies, if they do not have the activities of ADCC and / or CDC, in terms of the mechanism of action. Furthermore, it is important that anti-CD40 antagonist antibodies have no activity to induce signals by cross-linking them in vivo through Fc receptors, even if the ADCC activity can not be detected. In other words, it is necessary to confirm that they do not activate the immunity, and such active antibodies can be desired as a pharmaceutical agent. Anti-CD40 antagonist antibodies are promising as a therapeutic agent to treat autoimmune diseases or to suppress rejection in organ transplantation. If they induce an agonist activity due to some effect after they are administered to patients, however, it may be weak, the symptoms may worsen in contrast to the desired therapeutic effect. Thus, an antibody without any agonist activity is more preferable as a pharmaceutical agent. In the present invention, the incorporation of a point mutation L235E (means the substitution of L at position 235 with E; similar symbols will be used later) in IgG4 has been shown to be effective for in vivo reduction in agonist activity, in animal testing using monkeys. Although IgG4 is a subclass with low activities of ADCC and CDC, it is reported that when an attempt was made to express IgG4 as a recombinant protein in CHO-like cells, their middle molecules were secreted due to a deficient SS bond between the heavy chains (Rob C. Aalberse et al., Immunology, 105, 9-19, 2002). To overcome this problem, the incorporation of a mutation in the constant region of antibodies is reported to successfully promote the formation of the S-S bond. Therefore, this type of mutation was also evaluated for its usefulness. Specifically, the mutation was incorporated to replace S at position 228 with P (S. Angal et al., Molecular Immunology, vol 30, No. 1, 105-108, 1993). For antagonist antibodies as well as agonist antibodies, the stability of the antibody during purification and storage is very important. There may be some methods to create such antibodies that are physically better although they maintain antagonistic activity. The antibody pharmaceutics hitherto commercially presented belong mainly to the subclass IgGl, and are not reported to be problematic in the pharmaceutical formulation. Based on these facts, it may be advantageous from the point of view of physical properties to derive the constant region of antibodies from IgG1. In the case of anti-CD40 antibodies, however, they are desirably lower in ADCC and CDC activities. As a consequence, antibodies which have a constant region of the IgGl type modified with some point mutations may be desired. The mutations described above are useful for creating such antibodies. The constant region of the IgG1 type may become lower in ADCC and CDC activities by incorporating the P331G point mutation in it. It is also observed that the incorporation of the L235E point mutation in IgG4 removes a light agonist activity in vivo to make it pharmaceutically more active, but it makes it physically less stable at a low pH. Thus, the substitution of L235 with an amino acid other than E can make it physically more functional. Like the 4D11 antibody, it is very similar to the 2B11 antibody with respect to the structure of its variable region. The 2B11 antibody has a lower antagonist activity, but has a higher stability at a low pH, compared to the 4D11 antibody. If some amino acids derived from the constant region of 2B11 are incorporated into the 4D11 antibody, based on the above properties, 4D11 may become more stable. Specifically, the point mutation L38V, P58R, G62W, 179M, K81Y, H87Y, S98A, K109R, V120M or T124A in the heavy chain, or N75S in the light chain, or a combination thereof may be effective for that purpose. Specifically, a mutant created by substituting L at position 38 in the variable region of the heavy chain of antibody 4D11 with V (abbreviated as L38V; similar symbols will be used below), a P58R mutant, a G62W mutant, an I79M mutant, a K81Y mutant, a H87Y mutant, an S98A mutant, a K109R mutant, a V120M mutant or a T124A mutant, or a N75S mutant in the Light chain, or a combination thereof may be provided for that purpose. The antibody according to the present invention has at least one amino acid mutation to reduce the activities of ADCC and / or CDC, preferably 1-15, 1-13, 1-12, 1-11, 1-10, 1- 9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1 or 2 mutations. The present invention provides antagonist anti-CD40 antibody mutants and others as follows: [53] A heavy chain of a monoclonal antibody having an antagonist activity capable of binding to CD40, wherein the heavy chain comprises a constant region with at least one amino acid deleted or substituted, or with at least one amino acid added thereto, such removal, substitution or addition is capable of increasing or decreasing ADCC and / or CDC.

[54] The heavy chain according to [53], wherein the constant region is derived from a human IgG. [55] The heavy chain according to [54], wherein the human IgG is a human IgGl. [56] The heavy chain according to [54], wherein the human IgG is a human IgG2. [57] The heavy chain according to [54], wherein the human IgG is a human IgG3. [58] The heavy chain according to [54], wherein the human I G is a human IgG4. [59] The heavy chain according to any of

[55], [57] or [58], wherein the substitution of amino acids in the constant region is the substitution of leucine with glutamic acid at position 235 which is indicated by the EU index as in Kabat et al. [60] A heavy chain according to any one of [53] to [58], wherein the heavy chain comprises a constant region with at least one amino acid deleted or substituted, or with at least one amino acid added thereto, such elimination , substitution or addition that is capable of promoting the formation of the SS link between the heavy chains. [61] The heavy chain of the antibody according to

[60], wherein the substitution of amino acids in the constant region is the substitution of serine with proline at position 228 which is indicated by the EU index as in Kabat et al. [62] A monoclonal antibody comprising the heavy chain to any of [53] to [61]. [63] The heavy chain according to any one of [53] to [61], wherein the heavy chain comprises a variable region from a heavy chain of a monoclonal antibody produced by hybridoma 4D11 (Accession Number FERM BP-7758) . [64] A monoclonal antibody comprising the heavy chain according to [63] and a light chain comprising a variable region from a light chain of a monoclonal antibody produced by hybridoma 4D11 (Accession No. FERM BP-7758). [65] The heavy chain according to any of [53] to [61], wherein the heavy chain comprises a variable region of the polypeptide represented by SEC. FROM IDENT. DO NOT. : 46. [66] A monoclonal antibody consisting of the heavy chain according to [65] and a light chain of a monoclonal antibody, wherein the light chain comprises a variable region of the polypeptide represented by SEC.

FROM IDENT. DO NOT. : 48. [67] The heavy chain according to [53], wherein the heavy chain consists of a remaining portion provided by removing the signal sequence from the polypeptide represented by the SEC. FROM IDENT. NO .: 140. [68] A monoclonal antibody consisting of the heavy chain according to [67] and a light chain of a monoclonal antibody, wherein the light chain consists of a remaining portion provided by removing the signal sequence to from the polypeptide represented by SEC. FROM IDENT. DO NOT. : 142. [69] "The heavy chain according to [53], wherein the heavy chain is produced by a host comprising an expression vector having the polynucleotide represented by SEQ ID NO: 139. [70] The monoclonal antibody according to [62], wherein the monoclonal antibody is produced by a host comprising an expression vector having the polynucleotide represented by SEQ ID NO: 139 and the polynucleotide represented by SEQ ID NO: 141. [71] A polynucleotide represented by SEQ ID NO: 139. [72] A polynucleotide represented by SEQ ID NO: 141. [73] An expression vector having the polynucleotide according to [71]. [74] An expression vector having the polynucleotide according to [72]. [75] An expression vector having the polynucleotide according to [71] and [72]. [76] A host comprising the expression vector according to [73]. [77] A host comprising the expression vector according to [74]. [78] A host comprising the expression vector according to [75]. [79] A process for producing a heavy chain of a monoclonal antibody, comprising the steps of: culturing the host according to [76] in a culture medium; and obtaining the heavy chain of a monoclonal antibody from the culture and / or the host. [80] A process for producing a monoclonal antibody, comprising the steps of: culturing the host according to [78] in a culture medium; and obtaining a monoclonal antibody from the culture and / or the host. [81] A pharmaceutical composition comprising the monoclonal antibody according to [62], [64], [66], [68] or [70] as an active ingredient. [82] The pharmaceutical composition according to [81] used for the prevention or treatment of transplant rejection, an autoimmune disease, allergy or inhibition VIII of the blood coagulation factor. [83] A method for the prevention or treatment of transplant rejection, an autoimmune disease, allergy or inhibition VIII of the blood coagulation factor, which comprises administering the pharmaceutical composition according to [81] in a mammal. [84] The use of the monoclonal antibody according to [62], [64], [66], [68] or [70] for the production of a pharmaceutical composition used for the prevention or treatment of transplant rejection, a autoimmune disease, allergy or inhibition VIII of the blood coagulation factor. [85] A method for producing a heavy chain of a monoclonal antibody having an antagonist activity capable of binding to CD40, wherein the agonist activity is reduced, comprising a step to make the elimination or substitution of at least one amino acid, or the addition of at least one amino acid in a constant region of a heavy chain of a human antibody. [86] The method according to [85], where the constant region is from a human IgG. [87] The method according to [86], wherein the human IgG is a human IgG4. [88] The method according to either of [85] a

[87], wherein such substitution of amino acids in the constant region is the substitution of leucine with glutamic acid at position 235 which is indicated by the EU index as in Kabat et al.

[93] A polynucleotide provided by removing the portion encoding the signal sequence from the polynucleotide represented by SEQ. FROM IDENT. NO .: 139. [94] A polynucleotide provided by removing the portion encoding the signal sequence from the polynucleotide represented by SEC. FROM IDENT. NO .: 141. Additionally, the present invention provides the materials later. A mutant of an anti-CD40 antagonist antibody, which comprises at least one substitution selected from the group consisting of the substitution of L with V at position 38, the substitution of P with R at position 58, substitution of G with W in position 62, the substitution of I with M in position 79, the substitution of K with Y in position 81, the substitution of H with Y in position 87, the substitution of S with A in position 98 , the substitution of K with R in position 109, the substitution of V with M in position 120 and the substitution of T with A in position 124, whose substitutions are carried out in a variable region of a heavy chain of a monoclonal antibody produced by hybridoma 4D11 (Accession No. FERM BP-7758) and a mutant of an anti-CD40 antagonist antibody comprising the substitution of N with S at position 75 in a variable region of a light chain of the 4D11 antibody. Here, the reduction in ADCC and CDC activities means the reduction in those activities when compared to the corresponding activities of a monoclonal anti-CD40 antibody different from the mutants described above, for example, when compared to the corresponding activities of a monoclonal antibody produced by hybridoma 4D11 (Accession No. FERM BP-7758). The activities of ADCC and CDC can be evaluated by any known method, for example, the method described in the Examples herein. The sequences of the variable regions in the heavy and light chains of a monoclonal antibody will be presented later which is produced by hybridoma 4D11 (Accession Number FERM BP-7758). The variable regions of DNA coding in the heavy and light chains of the 4D11 antibody and the amino acid sequences of the heavy and light chains will then be presented, respectively. In the heavy chain nucleotide sequence (SEQ ID NO: 45) of the 4D11 antibody, the boundary between the signal sequence and the variable region is located between "cytosine" ([C]) at position 93 and cytosine (C) at position 94, and the boundary between the variable region and the constant region is between adenine (A) at position 456 and guanine (G) at position 457 (genetic prediction software was used (Signal P ver.2) In the heavy chain amino acid sequence (SEQ ID NO: 46) of the 4D11 antibody, the boundary between the signal sequence and the variable region is located between serine (S) at the position 26 and glutamine (Q) at position 27, and the boundary between the variable region and the constant region is between serine (S) at position 147 and alanine (A) at position 148. Therefore, the variable region in the heavy chain of antibody 4D11 has a nucleotide sequence that varies from cytosine (C) in the position n 94 to adenine (A) at position 456, as observed in SEC. FROM IDENT. DO NOT. : 45. In addition, the variable region in the heavy chain of antibody 4D11 has an amino acid sequence that ranges from glutamine (Q) at position 27 to serine (S) at position 147, as observed in SEC. FROM IDENT. NO .: 46. In the light chain nucleotide sequence (SEQ ID NO: 47) of the 4D11 antibody, the boundary between the signal sequence and the variable region is between "thymine" ([T]) at position 124 and guanine (G) at position 125, and the limit between the variable region and the constant region is between adenine (A) at position 442 and "cytosine" ([C]) at position 443 (genetic prediction software was used (Signal P ver.2 )). In the light chain amino acid sequence (SEQ ID NO: 48) of the 4D11 antibody, the boundary between the signal sequence and the variable region is located between cytosine (C) at position 22 and alanine (A) at position 23, and the boundary between the variable region and the constant region lies between lysine (K) at position 128 and "arginine" ([R]) at position 129. Therefore, the variable region in the chain antibody 4D11 has a nucleotide sequence that varies from guanine (G) at position 125 to adenine (A) at position 442, as observed in SEC. FROM IDENT. NO .: 47. In addition, the variable region in the light chain of antibody 4D11 has an amino acid sequence ranging from alanine (A) at position 23 to lysine (K) at position 128, as observed in SEC. FROM IDENT. NO .: 48. The nucleotide sequence of heavy chain (SEQ ID NO .: 45..) Of the 4D11 antibody: ATATGTCGACGAGTCATGGATCTCATGTGCAAGAAAATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCG GCTCCCAGATGGGTCCTGTCCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTACTGAAGCCTTCGGAGACC CTGTCCCTCACCTGCACTGTCTCTGGCGGCTCCATCAGCAGTCCTGGTTACTACGGGGGCTGGATCCGCCAG CCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATAAAAGTGGGAGCACCTACCACAACCCGTCCCTC AAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCC GCAGACACGGCTGTGTATTACTGTACGAGACCTGTAGTACGATATTTTGGGTGGTTCGACCCCTGGGGCCAG GGAACCCTGGTCACCGTCTCCTCAGCTAGC The heavy chain amino acid sequence (SEC, FROM IDENT. DO NOT . : 46) of the 4D11 antibody: MDLMCKlOdKHLWFFLLLVAAPRWVLSQLQLQ LEWIGSIYKSGSTYHNPSLKSRV? SVDTSi QFSLKLS VSSAS The light chain nucleotide sequence (SEQ.

FROM IDENT. DO NOT . : 47) of the 4D11 antibody: AGATCTTAAGCAAGTGTAACAACTCAGAGTACGCGGGGAGACCCACTCAGGACACAGCATGGACATGAGGGT CCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTC TCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAG TGCTTGAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAATTTGGA AAGTGGGGTCCCATCAAGGTGCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAAC'ITATTACTGTCAACAGT ^ GGAAATCAAACGTACG The light chain amino acid sequence (SEQ ID NO: 48) of the 411 antibody: MDMRWAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVT1TCRASQGISSALAWYO? KPGKAPKLLITO ASNLESGWSRFSGSGSGTOFpj? SSLQPEDFA'TYYCQQFNSYPlFGQGTKVEIKRT In the heavy chain nucleotide sequence (SEQ ID NO: 139) of the 4D11G4PE antibody, the boundary between the signal sequence and the variable region is located between "cytosine" ([C]) at position 78 and the cytosine (C) at position 79, and the boundary between the variable region and the constant region lies between adenine (A) at position 441 and guanine (G) at position 442 (genetic prediction software was used ( Signal P ver.2) In the heavy chain amino acid sequence (SEQ ID NO: 140) of the 4D11 antibody, the boundary between the signal sequence and the variable region is located between serine (S) in the position 26 and glutamine (Q) at position 27, and the boundary between the variable region and the constant region is between serine (S) at position 147 and alanine (A) at position 148. Therefore, the variable region in the heavy chain of the 4D11 antibody has a nucleotide sequence that varies from cytosine (C) in the 79 to adenine (A) at position 441, as observed in SEC. FROM IDENT. DO NOT. : 139. In addition, the variable region in the heavy chain of antibody 4D11 has an amino acid sequence that varies from glutamine (Q) at position 27 to serine (S) at position 147, as observed in SEC. FROM IDENT. NO .: 140. The complete heavy chain nucleotide sequence (SEQ ID NO: 139) of the 4D11G4PE: ATGGATCTCATGTGCAAGAAAATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTC CTGTCCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTACTGAAGCCTTCGGAGACCCTGTCCCTCACCTGC ACTGTCTCTGGCGGCTCCATCAGCAGTCCTGGTTACTACGGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGG CTGGAGTGGATTGGGAGTATCTATAAAAGTGGGAGCACCTACCACAACCCGTCCCTCAAGAGTCGAGTCACC ATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTG TATTACTGTACGAGACCTGTAGTÁCGATATTTTGGGTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCAGCTAGCACCAAGGGGCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGC ACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG ACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAG GTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCGAGGGGGGA CCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGC GTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCAT AATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG CACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAG AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCAT GAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA The complete amino acid sequence of heavy chain (SEQ ID NO:... 140) of the 4D11G4PE: MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGPGLLIO ^ ^ SElLSLTCTVSGGSISSPGYYGGWpiQPPGKG LEWIGSIYKSGSTYHNPSLKSRV SVDTSKNQFSLK VSSAS GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVi SWNSGALTSGVHTFPAVL ^ TWSSSLG TYTCNVDHKPSN VDK-RVESKYGPPCPPCPAPEFEGGPS-v ^ LFPPKPKD VVVDVSQEDPEVQFNWYVDGVEVHNAKTj REEQFNSTYRWSV ^^ K??? ? SKAKGQPRJi # QVYTLPPSQEEM? KNQVSLTC ^^ LYSRLTVDKSRWQEGN SCSVMHEALHNHYTQKSLSLSLGK In the light chain nucleotide sequence (SEQ ID NO: 141) of 4D11G4PE, the boundary between the signal sequence and the variable region is between "thymine" ([T]) in position 66 and guanine (G) in position 67, and the boundary between the variable region and the constant region lies between adenine (A) at position 384 and "cytosine" ([C]) at position 385 (genetic prediction software was used (Signal P ver. 2)) . In the light chain amino acid sequence (SEQ ID NO: 142) of 4D11G4PE, the boundary between the signal sequence and the variable region is between cytosine (C) at position 22 and alanine (A) at position 23, and the limit between the variable region and the constant region is located between lysine (K) at position 128 and "arginine" ([R]) at position 129. Accordingly, the variable region in the light chain of 4D11G4PE has a nucleotide sequence that varies from guanine (G) at position 67 to adenine (A) at position 384, as observed in SEC. FROM IDENT. DO NOT . : 141 In addition, the variable region in the light chain of antibody 4D11 has an amino acid sequence ranging from alanine (A) at position 23 to lysine (K) at position 128, as observed in SEC. FROM IDENT. DO NOT . : 142 The complete light chain nucleotide sequence (SEQ ID NO: 141) of 4D11G4PE: ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATC CAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGAT GCCTCCAATTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACC ATCAGCAGCCTGCAGCCTGAAGATTI GCAACTTATTACTGTCAACAGTTTAATAGTTACCCGACGTTCGGC CAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG TGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC ACCTACAGCCTCAGCAGCACCCTGACGGTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA The complete amino acid sequence of light chain (SEQ ID NO:... 142) of the 4D11G4PE: MD IRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPi LIYD ASMJiSGVPSFJSGSGSGTDFIL SSLQPEDFA'rmíQQ QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE ^? HIVQGLSSPVTKSFNRGEC 3. Definition The terms used herein will be defined later. "CD40" described herein refers to a polypeptide having the amino acid sequence described in E. A. Clark et al., Proc. Nati Acad. Sci. USA 83: 4494, 1986, or I. Stamenkovic et al., EMBO J. 8: 1403, 1989, and particularly an antigenic polypeptide expressed on the surface of B cells, DC, macrophages, endothelial cells, epithelial cells or tumor cells derived therefrom. "An anti-CD40 antibody" refers to any monoclonal antibody to a CD40 expressed in cells, a full-length CD40 or a part-length CD40. In addition, "an antibody" of the invention is derived from genes (collectively called antibody genes) which encode a heavy chain variable region and a heavy chain constant region, as well as a light chain variable region and a chain constant region light that together constitute an immunoglobulin. Human immunoglobulins are grouped into 5 different classes consisting of IgG, IgA, IgM, IgD and IgE. In addition, IgG is composed of 4 different subclasses, IgGl, IgG2, IgG3 and IgG4, while IgA is composed of 2 different subclasses, IgAl and IgA2. IgGl, IgG2, IgG3 and IgG4 are located in 14g32, 33 of the human chromosomes. The fundamental structure of the immunoglobulin consists of two homologous L chains (light chains) and two homologous H chains (heavy chains). The class and subclass of an immunoglobulin is determined by its H chains. The antibody according to the present invention can comprise any class, any subclass or any isotype of immunoglobulin. "A functional fragment" of the inventive antibody refers to a portion (partial fragment) of the antibody defined above that is active individually or multiply in an antigen to the antibody, including for example, F (ab ') 2, Fab', Fab, Fv, disulfide stabilized FV, single chain FV (scFV) and a multimer thereof (DJ King, Applications and Engineering of Monoclonal Antibodies, 1998, TJ International Ltd.). So far, it has been known that IgGl includes J00228, Z17370 and Y14737, IgG2 includes J00230, AJ250170, AF449616, AF449617, AF449618, Z49802 and Z49801, IgG3 includes M12958, K01313, X16110, X99549, AJ390236, AJ390237, AJ390238, AJ390241, AJ390242 , AJ390246, AJ390247, AJ390252, AJ390244, AJ390254, AJ390260, AJ390262, AJ390272, AJ390276 and AJ390279 and IgG4 includes K01316, AJ001563 and AJ001564 (the symbols listed above indicate accession numbers of the genes). In the present invention, CH1, joint, CH2 and CH3 each denotes a portion of the heavy chain constant region of any antibody, and are based on the EU index as in Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 1991 Fifth edition). By definition CH1 varies from 118 to 215 for the EUR index, the articulation varies from 216 to 230 for the EU index, CH2 varies from 231 to 340 for the EU index, and CH3 varies from 341 to 446 for the index of the EU. The "human antibody" of the present invention means an antibody which is an expression product of an antibody gene derived from humans. "Agonist" refers to an action to improve the binding of a ligand to CD40 expressed on the surface of such cells as B cells, tumor cells or dendritic cells, or an action to provide cells expressing CD40 with at least one effect which the CD40 ligand does in cells expressing CD40. An "agonist antibody" refers to an antibody that has such an agonist action. An example of the effects provided by cells expressing CD40 is to promote the expression of CD95. "Antagonist" refers to an action to inhibit the binding of the ligand to CD40 expressed on the surface of such cells as B cells, tumor cells or dendritic cells, or a neutralizing action of at least one effect to which the CD40 ligand does in cells expressing CD40. An "antagonist antibody" refers to an antibody which has such an antagonistic action. An example of the effects provided for cells expressing CD40 is to suppress B-cell proliferation or antibody production. The present application clearly presents antibodies, or heavy chain or light chain variable regions thereof, by amino acid sequences. The present invention also encompasses amino acid sequences with at least one amino acid removed or substituted, or added thereto, preferably 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1 or 2 amino acids. The present application clearly presents genes encoding antibodies, or heavy chain or light chain variable regions thereof by nucleotide sequences. The present invention also encompasses nucleotide sequences with at least one nucleotide removed or substituted, or added thereto, preferably 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1 -4, 1-3, or 1 or 2 amino acids. The anti-CD40 antibody according to the present invention can be provided by incorporating an antibody gene into an expression vector, transfecting the vector into a suitable host cell, harvesting the antibody from the cultured cells or the supernatant, and purifying it. . The vector can be a phage or a plasmid which can replicate in the host cell by itself or can be integrated into the chromosome of the host cell. The plasmid DNA can be derived from Escherichia coli, Bacillus subtilis or a yeast, while the phage DNA can be from? phage The host cell for transformation is not particularly limited if it can express the target gene.

Examples of the host cell may include bacteria (Escheria coli, Bacillus subtilis, etc.), yeasts, animal cells (COS cell, CHO cell, etc.) and insect cells. There are known modes for transferring genes into host cells, including any mode, such as mediation by calcium ion, electroporation, spheroplast fusion, lithium acetate mediation, transfection or lipofection with calcium phosphate. In order to transfer the gene into an animal, as described below, the modes include microinjection; electroporation or lipofection for ES cells; and nuclear transplant. In the present invention, "culture" refers to (a) culturing the supernatant, (b) cultured cells, cultured biomass or interrupted matter thereof, or (c) secretion of the transformant. In order to cultivate the transformant, a suitable medium for the host is used and the static culture, the cultivation in rotating bottles or something else can be used. After cultivation, if the desired antibody protein is produced within the biomass or cells, the antibody is harvested by disrupting the biomass or cells. If the desired antibody is produced outside the biomass or cells, the culture solution is used as it is or after it is separated from the biomass or cells by centrifugation or other means. Below, a biochemical process using any chromatography, which is suitable for protein separation / purification, is used alone or optionally in combination with another to separate / isolate the desired antibody from the culture. In addition, the technology stops. creating a transgenic animal can be used to produce a transgenic animal that is a host animal having the gene integrated into an endogenous gene, such as a transgenic bovine, a transgenic goat, a transgenic sheep or a transgenic pig (Wright, G., et al., (1991) Bio / Technology 9, 830-834) and a large amount of a monoclonal antibody derived from the antibody gene can be obtained from milk secreted from the transgenic animal.

The culture of a hybridoma in vi tro can be done using a known nutrient medium or any nutrient medium prepared in a derivative form from known basic means such as is used to develop, maintain and store the hybridoma and to produce a monoclonal antibody in the supernatant, depending on the properties of the cultivated hybridoma, the purpose of the study and the culture method. 4. Properties of the Antibody (1) Antibody Agonist The mutant of the agonist antibody according to the present invention can activate the immune system without damaging the immunocompetent cells, since it has an ADCC and / or CDC activity equal to or lower than the original antibody , while an agonist activity is maintained. It is thus expected that the mutant exhibits the immunoactivation action which is equal to or higher than the original antibody and the cytotoxicity to cells expressing CD40 which is equal to or lower than the original antibody. (2) Agonist Antibodies The mutant of the anti-CD40 antagonist antibody according to the present invention has the activity of ADCC and / or reduced CDC compared to the unmodified antibody, while maintaining a suppressive activity against immunoactivation signals induced by CD40L. It is also expected to decrease the activity of induction of signal in vivo which is considered to occur through Fc receptors. 5. Pharmaceutical Compositions A pharmaceutical composition, containing a formulation of the purified antibody according to the present invention is also within the scope of the present invention. The pharmaceutical composition may preferably contain a pharmaceutically acceptable diluent or carrier in addition to the antibody, and may be a mixture thereof with a different antibody or a different drug such as an antibiotic agent. The right carrier can include, but not limited to, physiological saline solution, physiological saline solution of phosphate buffer, glucose solution of physiological saline of phosphate buffer, and physiological saline solution of buffer. Alternatively, the antibody can be freeze dried for storage and when used, reconstituted in an aqueous buffer as described above. The pharmaceutical composition can be administered through the oral route, or the parenteral route, such as injection or intravenous, intramuscular, subcutaneous or intraperitoneal dosage. A single effective dose, which is a combination of the antibody of the present antibody with a suitable diluent and a physiologically acceptable carrier, is from 0.0001 mg to 100 mg per weight of body weight, and can be taken in a time interval from 2 days. to 8 weeks. When the pharmaceutical composition of the present antibody is an agonist antibody, it is used as: immunostimulant (antiviral or anti-infective agent) for pathogens which include, for example, hepatitis viruses, A, B, C, D or E, HIV , influenza, herpes simplex virus, cytomegalovirus, EB virus, papilloma virus, chlamydia, mycoplasma, toxoplasma, malaria, trypanosome and tubercle bacillus; antitumor agent for malignant tumors having cancer cells with expressed CD40, including for example, pancreatic cancer, bladder cancer, lymphoma (e.g., Hodgkin's lymphoma), leukemia, malignant melanoma, pancreatic cancer, lung cancer, ovarian cancer, cancer of bladder, breast cancer, colon cancer, prostate cancer and cancer of the head and neck; and therapeutic agent for autoimmune diseases such as rheumatism. The pharmaceutical composition can be used for a combination of the above diseases. This can also be used in combination as an adjuvant for a cancer-specific peptide. When the pharmaceutical composition is an antagonistic antibody, on the other hand, it is used as: immunosuppressant in organ transplantation (preventive or therapeutic agent for transplant rejection of pancreatic islets, kidney or something else, GVHD), therapeutic agent for autoimmune diseases (for example, rheumatism, psoriasis, chronic ulcerative colitis, Crohn's disease, systemic lupus erythematosus, multiple sclerosis, myasthenia, scleroderma, antiphospholipid antibody syndrome, autoimmune hepatitis, idiomatic thrombocytopenic purpura, Behcet's syndrome, arteriosclerosis, nephritis and respiratory distress), therapeutic agent for allergy (for example, asthma) and therapeutic agent for inhibition VIII of the blood coagulation factor. The pharmaceutical composition can be used for a combination of the above diseases. 6. Epitopes The CD40 binding epitopes were determined for antibodies KM341-1-19 and 2105 which have a higher agonist activity, and for the antibody 4D11 which has a higher antagonist activity, respectively (Example 2). The present invention provides antibodies having an agonist or antagonist activity having a different variable region sequence from those of the above antibodies, but recognizing the same epitope as one of the above antibodies. These antibodies can be obtained in such a procedure as described below. When an agonist anti-CD40 antibody that recognizes the same epitope as the antibody KM341-1-19 is sought, for example, mice or the like are immunized with CD40 to provide monoclonal antibodies, from which some monoclonal antibodies compete with the antibody. the antibody KM341-1-19 for binding to CD40 are selected according to the standard procedure. From the selected antibodies, the antibody having the same peptide binding pattern as the antibody KM341-1-19 is selected according to the method described in Example 2. The present invention will be described in greater detail below with reference to the examples. However, the present invention is not limited to the embodiments described in the examples.

Example 1: Expression and purification of antibody and antigen proteins. A plasmid vector containing a variable region of an antibody in CHO cells (ATCC) was transfected, and cells expressing antibodies were selected by G418 to prepare a stable expression cell line. A mutant antigen was expressed by temporarily introducing a vector into the HEK cells (ATCC). An anti-CD40 antibody was purified from the above culture supernatant by the following method. The culture supernatant containing an anti-CD40 antibody was affinity purified on a Hyper D Protein A column. (manufactured by NGK Insulators, Ltd.) or in the case of the purification of mouse IgGl, a Protein G column (Amersham Pharmacia Biotech) according to the bound instruction using PBS (-) and an absorption buffer and a 0.1 M sodium citrate buffer (pH 3) as an elution buffer.

The eluted fraction was adjusted to pH 7.2 by the addition of a solution of 1 M Tris-HCl (pH 8.0) or Na2HP0. The prepared antibody solution was replaced with PBS (-) using a dialysis membrane (10,000 cuts, manufactured by Spec ': rum Laboratories, Inc.) or an SP column (Amersham Pharmecia Biotech), and filtered and sterilized using a filter of membrane MILLEX-GV with a pore diameter of 0.22 μm (manufactured by Millipore Corp.). The concentration - of the purified antibody was calculated by measuring the absorbance at 280 nm, taking 1 mg / ml as 1.450D.

Example 2: Determination of epitopes A 13-mer peptide that covers amino acid 175 (SEQ ID NO: 1) in an extracellular region of CD40 was alternated by two amino acids each to synthesize 82 peptides in total (SEQ. DE IDENT NOS .: 49 to 130) as spots from terminal C on a cellulose membrane and acetylating the N-terminal thereof (Jerini AG, Germany). The reaction was carried out further on the basis of a conventional Western analysis (see Reineke, U. et al. (2001), "Epitope mapping with synthetic peptides prepared by SPOT synthesis." Antibody Engineering (Springer Lab Manual) Eds .: Kontermann / Dubel, 433-459, for example). In the analysis, the color intensity of each spot was quantified using Lumilmager ™ (Boehringer-Mannheim Corp.) (Figures 1A-1, A-2, B-1 and B-2). The results confirmed that a 4D11 antibody strongly recognizes the twentieth, twenty-fourth and forty-first peptides, an 2105 antibody strongly recognizes the 12th, 23rd and 64th peptides, an antibody KM341-1-19 strongly recognizes the 41st and 42nd peptides, the KM643- 4-11 strongly recognizes the 43rd peptide, F72 strongly recognizes the 75th peptide, 110 strongly recognizes the 64th peptide, F4-465 strongly recognizes the 34th, 35th, 54th, 55th, 65th, 66th and 75th peptics, KM281-1-10 recognizes strongly the 21st, 24th, 64th and 75th peptides, 2B11 (novel antibody) strongly recognizes the 21st, 24th and 64th peptides and F76 (novel antibody) strongly recognizes the 21st, 35th, 51st and 52nd peptides. In order to form the binding site of the anti-CD40 antibody, a CD40-FC fusion protein having a mutation introduced therein was prepared, and the binding capacity thereof was examined by ELISA. Since the anti-CD40 antibody does not cross-react with mouse B cells, five CD40Fc fusion proteins were prepared by partially converting the amino acid sequence to that of the CD40 rat. The binding of the antibody to the antigens was examined. The method for preparing the CD40-FC fusion proteins is shown below. The mutation site was prepared by introducing a mouse CD40 sequence into a part at which the antibody strongly binds the peptide sequence. CD40mutl converted EFTE into a site corresponding to the 15th peptide in ALEK, CD40mut2 converted LDT to a site corresponding to the 21st peptide in SAQ, CD40mut3 converted TH to a site corresponding to the 24th peptide in IR, CD40mut4 converted EEGW into a site corresponding to the 42nd peptide in KEGQ, and CD40mut5 converted VSSA into a site corresponding to the 64th peptide in QSSL. Mutants were prepared according to a genetic design technique (Figures 2A, B and C). The results of the analysis confirmed that antibody 2105 has an extremely reduced binding capacity to CD40mutl. The results also confirmed that the antibody 4D11 and 2B11 have a reduced binding capacity to CD40mut2.

Example 3: Binding activity of the anti-CD40 agonist antibody to Ramos cells A Ramos cell line was suspended in a PBS (SB) staining buffer containing 0.1% NaN3 and 2% FCS at a concentration of 2 x 106 / ml . The cell suspension (100 μl / well) was distributed in a 96 well round bottom plate (manufactured by Becton, Dickinson and Company). Each hybridoma culture supernatant (50 μl) was added, and incubated at an ice temperature for 30 minutes. A human IgGl antibody to human serum albumin as a negative control was adjusted to a concentration of 2 μg / ml in a medium of hybridoma culture, added in an amount of 50 μl, and then incubated at an ice temperature for 15 minutes. After washing the plate with SB, 50 μl of an anti-human antibody fluorescently labeled with R-PE diluted 250 times (manufactured by Southern Biotechnology Associates, Inc.) was added and incubated at an ice temperature for 15 minutes. After washing the plate with SB twice, it was suspended in 300 to 500 μl of FACS buffer, and the fluorescence intensity of each well was measured using FACS (FACSort, FACScan, manufactured by Becton, Dickinson and Company).

Example 4: Evaluation of agonist activity of the anti-CD40 agonist antibody to Ramos cells 5.0 x 10 5 cells / ml of Ramos cell suspension were seeded in a 96-well plate at 100 μl / well. A supernatant of hybridoma culture or purified antibody was diluted to 20 μg / ml in medium, and the dilution was added to the 96-well plate at a concentration of 100 μl / well. After overnight culture, the cells were harvested, and the anti-CD95 antibody labeled with R-PE (Parhmingen NJ) was used for the cells. The analysis was carried out using FACScan or FACSsort (Becton, Dickinson and Company).

Example 5: Inhibition of CD95 expression by anti-CD40 antagonist antibody in Ramos cells. 1.0 x 106 cells / ml of a suspension of Ramos cells in a 96-well plate at 50 μl / well was seeded. A culture supernatant of hybridoma or purified antibody at 2 μg / ml was adjusted in a medium, and the medium was added to the 96-well plate at 100 μl / well. 4 μg / ml of a soluble CD40 ligand (Alexis Corporation) and 4 μg / ml of an anti-FLAG antibody (M2, Sigma) were added to a medium, and the medium was added to a 96-well plate at 50 μl / water well. After overnight culture, the cells were harvested, and an anti-CD95 antibody labeled with R-PE (Pharmingen NJ) was used for the cells. The analysis was carried out using FACS.

Example 6: Measurement of CDC activity in an anti-CD40 antibody In the CDC assay, 2,000 Cr51-labeled dianocytes and a complement derived from human serum (manufactured by Sigma Co.) or complement derived from rabbit serum were cultured ( Cedarlane Laboratories Limited, Ontario, Canada) at a final concentration of 5% in a 96 well round bottom plate in a total volume of 200 μl together with the antibody in various concentrations at 37 ° C in the presence of 5% C02 during two hours. After the culture, the plate was centrifuged to cause the cells to precipitate, and then 50 μl of the supernatant was transferred to a 96-well plate including a powder scintillator (Lumaplate ™ -96, manufactured by Packard Instrument Co., Inc. ) and dried at 55 ° C for 1.5 hours. After confirming that the plate dried, it was covered with a special cover (TopSeal ™ -A: 96 well microplates: manufactured by Packard Instrument Co., Inc.) and the lightning dose? was measured with a scintillation counter (TopCount: manufactured by Packard Instrument Co., Inc.).

Example 7: Measurement of ADCC activity of anti-CD40 antibody As the antibody-mediated cytotoxicity, cytotoxicity to dianocytes was measured in the presence of cells having an exterminating activity such as NK-cells or neutrophils and an antibody (Cellular Cytotoxicity Antibody dependent, later ADCC), and cytotoxicity to dianocytes in the presence of a complement and an antibody (Complementary Dependent Cytotoxicity, later CDC). HlgG was used as a control. The measurement method is simply described as follows. Radioactive chromium (Cr51) was incorporated into the cytoplasm of the dianocytes, and the amount of Cr51 released in the culture solution by cell death was measured as a dose of? Rays. Specifically, the dianocytes 105 of a Burkitt Raji lymphoma cell line (ATCC CCL-86) were suspended in 15 μl of fetal calf serum (FCS). To the suspension was added 50 μl (37 MBq / ml) of Cr51-labeled sodium chromate (manufactured by Per in Elmer, Inc.: hereinafter referred to as Cr51), and the cells were cultured at 37 ° C for one hour. Then, 10 ml of a medium was added, and the medium was discarded by centrifugation. This operation was repeated three times to remove Cr51 not incorporated in the cells. In the ADCC assay, 2,000 dianocytes labeled with Cr51 and 200,000 healthy human peripheral blood mononuclear leukocytes obtained by the method described in Example 6 were cultured in a 96-well, round bottom plate (manufactured by Falcon) in a total volume of 200 μl together with the antibody at various concentrations at 37 ° C in the presence of 5% C02 for four hours. After the culture, the plate was centrifuged to cause the cells to precipitate, and then 50 μl of the supernatant was transferred to a 96-well plate including a powder scintillator (Lumaplate ™ -96: manufactured by Packard Instrument Co., Inc. ) and dried at 55 ° C for 1.5 hours. After confirming that the plate dried, the plate was covered with a special cover (TopSeal ™ -A: 96-well microplate; manufactured by Packard Instrument Co., Inc.) and the dose of lightning? was measured with a scintillation counter (TopCount: manufactured by Packard Instrument Co., Inc.).

Example 8: Preparation and evaluation of P331S mutant activity of anti-CD40 agonist antibody Genetic cloning of KM341-1-1.9 and 2105 anti-CD40 agonists antibodies is described in WO 02/099186. It is reported that CDC activity is reduced by converting Pro to position 331 in the IgG2 constant region in Ser. To reduce the CDC activity of antibody KM341-1-19 and antibody 2105, a P331S mutation was introduced in the region constant IgG2 thereof. The human IgGl constant region of a KNK61-Val Lark vector expressing an antibody (IDEC Pharmaceuticals: later abbreviated to N5KG1) was replaced with human IgG2 to prepare N5KG2 and the Pro at position 331 of IgG2 was converted to Ser to prepare a mutation. The cDNA cloning of the IgG2 constant region was carried out by harvesting the KM341-1-19 hybridoma by centrifugation, adding TRIZOL (Gibco BRL), and extracting the total RNA according to the instruction. The variable cDNA region of the antibody was cloned using a SMART RACE cDNA amplification kit from Clontech Laboratories, Inc., according to the attached instruction. The first h-bra cDNA was prepared using 5 μg of the total RNA as a template. PCR was carried out with tnIgG3Nhe: atatGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGC (SEQ ID NO: 2) G and tnIgG2Bam: atatggatccTCATTTACCCGGAGACAGGGAGAGGCTC (SEQ ID NO: 3) as primer sequences using the ZtaqPXR (Takara) kit in 30 cycles each consisting of the reaction at 98 ° C for 1 second, at 55 ° C for 30 seconds and at 72 ° C for 1 minute to amplify the gene. After the reaction, the amplified product was purified by a QIAGEN PCR purification kit, digested with Nhel and BamHI, and incorporated into N5KG1 to confirm the sequence. This vector was defined as N5KG2. N5KG2Ser (with Pro at position 331 converted to Ser) was prepared as follows. The reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out 15 times using N5KG2 as a template and the primers IgG3Nhe: atatGCTAGCACCAAGGGCCCATCGGTCTTCCCCTGGCG (SEQ ID NO. 4) and G2Ser2: GTTTTCTCGATGGAGGCTGGGAGGCC (SEQ ID NO: 5). At the same time, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out 15 times using N5KG2 as a template and the primers IgG2Bam atatggatccTCATTTACCCGGAGACAGGGAGAGGCTC (SEQ ID. NO .: 6) and G2Serl: GGCCTCCCAGCCTCCATCGAGAAAAC (SEQ ID NO: 7). The fragments of "amplified DNA were purified using a PCR purification kit, and the same amounts of the two fragments of Purified DNA. Later, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and 72 ° C for 30 seconds was carried out five times. The IgG3Nhe and IgG2Bam primers were added to the mixture, and the same reaction was carried out 15 times. The DNA fragment amplified with Nhel and BamHI was unfolded, and replaced with the IgGl constant region of the vector N5KG1 (N5KG2Ser). The fragment containing the sequence of the variable region of the antibody digested with BglII and Nhel was incorporated into the vector N5KG2Ser. The antibody expressed and purified by the above method was evaluated in terms of binding capacity to Ramos cells (Figure 3A) and agonist activity (Figure 3B). The fluctuation in activity due to the introduction of variation P331S was not observed.

Example 9: Measurement of CDC activity of the 331st mutant of the anti-CD40 agonist antibody The activity of CDC was measured by the above method. A complement derived from rabbit serum was used, and Ramos cells were used as dianocytes. The results confirmed that, in an antibody KM341-1-19 at an antibody concentration of 1 μg / ml, IgG2ser exhibited significantly reduced CDC activity when compared to IgG2 (Figure 4A). On the other hand, when 'a human supplement was used, no change was observed (Figure 4B).

Example 10: Preparation and measurement of agonist anti-CD40 antibody activity having a transformed constant region Among the anti-CD antibodies described in WO 02/088186, two antibodies exhibiting stronger agonist activity (antibody KM341-1-19 and antibody 2105) belongs to subclass IgG2. In order to examine whether or not the subclass IgG2 is important for the activation of CD40, recombinant proteins having an antibody constant region converted into IgG1, IgG3 and IgG4, respectively, were prepared and measured in terms of binding capacity. an antigen and activity that improves the expression of CD95 in Ramos cells according to Examples 4 and 6. The IgG1 was expressed using N5KG1 and respectively IgG2 and IgG3 were expressed using expression vectors N5KG2 and N5KG3 obtained by substituting the constant region N5KG1 with IgG2 and IgG3, respectively. The cDNA cloning of the IgG3 constant region was carried out according to the partially modified IgG2 cloning method, using a specific IgG3 primer. IgG4 was expressed using N5KG4PE (IDEC Pharmaceuticals). The antibody protein was expressed according to Example 1. The binding activity to Ramos cells expressing human CD40 of antibody KM341-1-19 and antibody 2105 was not affected when converting IgG2 to IgG1, IgG3 or IgG4 (Figures 5A -1 and 5A-2). However, these antibodies were found to have an activity that improves the expression of CD95 in reduced Ramos cells by 10% or more (Figures 5B-1 and 5B-2). This demonstrates that not only the structure of the variable region that defines the binding region of the antibody, but also the structure of the constant region of the antibody are important for the strong agonist activity of antibody 2105 and antibody KM341-1-19. Thus, in order to examine which region in the IgG2 constant region is important for the agonist activity, a domain exchange mutant wherein the IgG2 structure is mixed with the IgG4 structure was prepared to measure its activity. As described below, a domain exchange mutant is prepared by substitution of a joint region. In this case, the "joint region" includes the upper joint (from Kabat EU code 216), middle joint (from Kabat EU code 226) and the lower joint (Kabat EU code 231) as described by Ole H Brekke et al. ., Immunology Today 1995, 16, 85-90. Four IgG2 / 4 domain exchange mutants (CH1 and joint region: IgG2, other IgG4 regions), IgG4 / 2/4 (joint region: IgG2, other IgG4 regions), IgG2 / 4/4 (CH1: IgG2, other regions: IgG4) and IgG4 / 2/2 (CH1: IgG4, other regions: IgG2) were prepared respectively for antibody KM341-1-19 and antibody 2105. A N5KG2 / 4 vector for expressing the IgG2 / 4 antibody was prepared using a Ztaq PCR (Takara) kit. The reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out 15 times using N5KG2 as a template and the primers IgG3Bam: atatggatccTCATTTACCCGGAGACAGGGAGAGGC (SEQ ID NO. 8) and 24CH4: AGGGGTCCGGGAGATCATGAGAGTGTCCTT (SEQ ID NO: 9). At the same time, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out 15 times using N5KG4 (IDEC Pharmaceuticals as a template and the primers 24Chi3: AAGGACACTCTCATGATCTCCCGGACCCCT ( SEQ ID NO: 10) and linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 11) The amplified DNA fragments were purified using a PCR purification kit, and the same amounts of the two fragments of Thereafter, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out five times The primers IgG3Bam and linkH2: tgatcatacgtagatatcacggc (SEQ. IDENT NO: 12) were added to the mixture, and the same reaction was carried out 15 times The amplified DNA fragment was split with Nhel and BamHI, and was replaced with the IgGl constant region of the vector N5KG1. A N5KG4 / 2/4 vector to express IgG4 / 2/4 was prepared as follows. The reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out 15 times using N5KG4 as a template and the linkH primers: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 13 ), G2Hin3: TTTGCGCTCAACTGTCTTGTCCACCTTGGTGTTGCTGGG (SEQ ID NO: 14), linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 15) and G2Hin4: ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCG (SEQ ID NO. 16). The amplified DNA fragments were purified with a PCR purification kit, and the same amounts of the two purified DNA fragments were mixed. Subsequently, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out five times using the mixture as a template. The linkH and linkH2 primers were added to the mixture, and the same reaction was carried out 15 times. The DNA fragment amplified with Nhel and Ba HI was unfolded and replaced with the IgGl constant region of the vector N5KG1. A vector NK5G2 / 4/4 to express IgG2 / 4/4 was prepared as follows. The reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out 15 times using N5KG2 as a template and the linkH primers: gggtacgtcctcacattcagtgatcag (SEQ ID NO. 17) and G4CH1-2: GGTGTTGCTGGGCTTGTGATCTACGTTGCAG (SEQ ID NO: 18). At the same time, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out 15 times using N5KG4 as a template and the primers G4CH1-1: CTGCAACGTAGATCACAAGCCCAGCAACACC (SEQ. DE IDENT NO .: 19) and linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 20). The amplified DNA fragments were purified using a PCR purification kit, and the same amounts of two purified DNA fragments were mixed. Later, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out five times. The linkH and linkH2 primers were added to the mixture, and the same reaction was carried out 15 times. The DNA fragment amplified with Nhel and BamHI was unfolded and replaced with the IgGl constant region of the vector N5KG1. A N5KG4 / 2/2 vector to express IgG4 / 2/2 was prepared as follows. The reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out using N5KG4 as a template and linkH primers: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 21) and G4CH1-2: GGTGTTGCTGGGCTTGTGATCTACGTTGCAG (SEQ ID NO: 22). At the same time, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out 15 times using N5KG2 as a template and the primers G4CH1-1: CTGCAACGTAGATCACAAGCCCAGCAACACC (SEQ. IDENTIFIER NO .: 23) and linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 24). The amplified DNA fragments were purified using a PCR purification style, and the same amounts of the two purified DNA fragments were mixed. Later, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out five times. The linkH and linkH2 primers were added to the mixture, and the same reaction was carried out 15 times. The DNA fragment amplified with Nhel and BamHI was unfolded and replaced with the IgGl constant region of the vector N5KG1.

The binding activity of the four respective domain exchange mutants of antibody KM341-1-19 and antibody 2105 was examined. As a result, no difference was observed between these and the original IgG2 in terms of binding capacity (Figures 6A-1 and 6A-2). However, only IgG2 / 4/4 of both antibody KM341-1-? 9 and antibody 2105 exhibited significantly reduced agonist activity (Figures 6B-1 and 6B-2). The results confirmed that the articulation region of IgG2 is important for agonist activity. In addition, it was examined that the sequence is important in the region of articulation. The articulation region is divided into three sites, specifically, upper joint, middle joint and lower joint (Ole H Brekke et al., Immunology Today 1995, 16, 85-90). The specific sequences of IgG2 in these regions were respectively substituted with specific sequences of IgG4. The antibodies obtained by introducing a mutation into the upper joint (from Kabat EU code 216), middle joint (from Kabat EU code 226) and lower joint (from Kabat EU code 231) were respectively defined as IgG2UH4, IgG2MH4 and IgG2LH4. Their respective expression vectors were defined as N5KG2UH4, N5KG2MH4 and N5KG2LH. "Articulation" is defined as indices of Eü 216 to 230 according to Kabat et al., Sequences of proteins of immunological interest, 1991 fifth edition. N5KG2UH4 was prepared as follows. The reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at "/ 2 ° C for 30 seconds was carried out 15 times using N5KG2 as a template and linkH primers: gggtacgtcctcacattcagtgatcag (SEQ ID NO. : 25) and UH4-2: CACAACATTTggaCTCAACTcTCTTGTCCACC (SEQ ID NO: 26) At the same time, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds carried out 15 times using N5KG2 as a template and primers UH4-1: GGTGGACAAGAgAGTTGAGtccAAATGTTGTG (SEQ ID NO: 27) and linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 28) .The DNA fragments were purified. amplified using a PCR purification method, and the same quantities of the two purified DNA fragments were mixed in. Subsequently, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 3G seconds was carried out five times.The linkH and linkH2 primers were added to the mixture, and The same reaction was carried out 15 times. The amplified DNA fragment was split with Nhel and BamHI, and replaced with the IgGl constant region of the vector N5KG1. N5KG2MH4 was prepared as follows. The reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out 15 times using N5KG2 as a template and linkH primers: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 29) ) and UM4-2: GGCACGGTGGGCAtgggggaccataTTTGCGCTC (SEQ ID NO: 30). At the same time, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out 15 times using N5KG2 as a template and primers UM4-1: GAGCGCAAAtatggtcccccaTGCCCACCGTGCC (SEQ. IDENT. NO .: 31) and linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 32). The amplified DNA fragments were purified using a PCR purification method, and the same amounts of the two purified DNA fragments were mixed. Later, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out five times. The linkH and linkH2 primers were added to the mixture, and the same reaction was carried out 15 times. The DNA fragment amplified with Nhel and BamHI was unfolded and replaced with the IgGl constant region of the vector N5KG1. N5KG2LH4 was prepared as follows. Reaction at 98 ° C for 4 seconds, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out 15 times using N5KG2 as a template and primers lirkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 33) ) and UL4-2: GAAGACTGACGGTCCccccaggaactcTGGTGCTGGGCA (SEQ ID NO: 34). At the same time, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out 15 times using N5KG2 as a template and primers UL4-1: TGCCCAGCACCAgagttcctggggGGACCGTCAGTCTTC (SEQ. IDENT. NO .: 35) and linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 36). The amplified DNA fragments were purified using the PCR purification kit, and the same amounts of the two purified DNA fragments were mixed. Subsequently, the reaction at 98 ° C for 1 second, at 60 ° C for 30 seconds and at 72 ° C for 30 seconds was carried out five times. The linkH and linkH2 primers were added to the mixture, and the same reaction was carried out 15 times. The DNA fragment amplified with Nhel and Ba HI was unfolded and replaced with the IgGl constant region of the vector N5KG1. The three respective domain exchange mutants of antibody KM341-1-19 and antibody 2105 were examined to have the same antigen binding activity (Figures 6A-1 and 6A-2). Nevertheless, IgG2UH4 and IgG2MH4 exhibited significantly reduced agonist activity to Ramos cells (Figures 6B-1 and 6B-2). It was found from the foregoing, that the structures of the upper joint and the mid joint in the joint region are important for the agonist activity dependent on the IgG2 subclass of antibodies KM341-1-19 and 2105 anti-C O. Since the IgG2 subclass was found to be important for agonist activity, antibodies of the subclass different from IgG2 were converted to those of subclass IgG2 to examine whether or not the agonist activity was improved. On examination in several clones, the agonist activity of F76 could be improved by converting subclass IgGl to subclass IgG2 (Figures 7A and B).

Example 11. Preparation of anti-CD40 antagonist antibody mutants A DNA fragment containing a heavy chain and a light chain of a 4D11 antibody gene described in WO 02/088186, whose original subclass is IgG1, was digested with BglII and Nhel , purified and then integrated into vectors N5KG4PE, N5KG4P and N5KG4 (IDEC Pharmaceuticals). N5KG4PE contains point mutations S228P and L235E in the constant region IgG4, and N5KG4P contains a point mutation S228P in the constant region IgG4. The antibody protein was expressed and purified according to the above method. The antibody was purified according to the method using the link to Ramos cells as an index. The change in the binding activity of IgG1, IgG4, IgG4P and IgG4PE to Ramos cells was not observed (Figure 8A). The IgG1 antagonist activity was compared to those of several IgG4 mutants according to the above method to find that the IgG1 antagonist activity does not differ from those of the IgG4 mutants (Figure 8B).

Example 12: Evaluation of ADCC activity and CDC activity of the anti-CD40 antagonist antibody mutants The activity of ADCC and the CDC activity of the anti-CD40 mutant antibodies were evaluated according to the above method. When human MNCs are used as executing cells and Daudi cells that express CD40 as dianocytes, two IgG4 and IgG4PE mutants were respectively observed to have significantly reduced ADCC activity when compared to IgG1 as the original subclass of the 4D11 antibody (Figure 9). The CDG activity of IgGl was compared to that of IgG4P using Daudi cells as dianocytes. It was found that IgG4P has significantly reduced CDC activity when compared to IgGl (Figure 10).

Example 13: Effect of anti-CD40 antagonist antibody on B cells 100 μg each of IgGl, IgG4P and IgG4PE of antibody 4D11 was administered to the tail vein of mice having a genetic background so they were homozygous for interrupted CD40 Endogenous mouse and a transgene of a human CD40 gene was housed (Yasui, et al. Int.

Immuno 2002 Vol 14: 319). 24 hours after the administration, the blood was collected from the orbital venous plexus. After hemolysis with 0.16 mol / 1 of ammonium chloride, an anti-B220 antibody labeled with FITC was added to the hemolysate, and analyzed using FACS. The results are shown in Figure 11. In the figure, the longitudinal axis indicates the ratio of B cells in the total lymphocytes. IgGl reduced the ratio of the majority of B cells, IgG4P reduced the ratio to a lesser extent, and IgG4PE reduced the ratio to a much smaller extent. 24 hours after administration, the spleen was removed and crushed with clear glass to prepare a cell suspension. After hemolysis of the cell suspension, an anti-B220 antibody labeled with PE and an anti-CD23, CD86 or CD95 antibody labeled with FITC were used for hemolysate and this was analyzed using FACS. The results are shown in Figures 12A, B and C. In the figures, the longitudinal axis indicates the ratio of B cells expressing each surface marker in the total lymphocytes. It was found that 4D11G1 achieved the same level of increase in expression of each marker as a commercially available mouse anti-human CD40 agonist 5C3 antibody (Pharmingen). IgG4PE achieved a smaller increase in the expression of each activation surface marker when compared to IgGl and IgG4_ ?.

Example 14: Effect to inhibit the production of the antigen-specific antibody and change in the number of B cells elicited by the anti-CD40 antagonist antibody. 100 μg (based on NP-CGG) of a conjugate complex of 4-hydroxy-3-nitrophenylacetyl-γ -globulin conjugates was administered intraperitoneally (NP-CGG: distributed by Professor Hitoshi KIKUTANI, Research Institute for Microbial Diseases, Osaka University) and alum (aluminum hydroxide gel) to mice that have a genetic background so they were homozygous for endogenous interrupted CD40 of mouse and a transgene of a human CD40 gene is housed (Yasui et al., Int. Immunol. 2002 Vol 14: 319) to sensitize the mice. Immediately prior to antigen sensitization, 50 or 100 μg of each antibody was administered to the tail vein. 100 μg of a human IgG PE antibody of anti-human albumin was administered as the negative control. 7 and 14 days after the sensitization, blood was collected from the orbital venous plexus. The amounts of IgGl and IgM antibodies specific for NP in the serum were measured by the ELISA method. The ELISA method was carried out as follows. 50 μl / well of bovine serum albumin bound to NP (NP-BSA: 2.5 μg / ml) was added to each well of a 96-well microplate for ELISA (Maxisorp, manufactured by Nunc A / S) and incubated at 4 ° C. ° C to cause NP-BSA to be absorbed in it. Then, the supernatant was discarded, and a blocking reagent (SuperBlock, manufactured by Pierce Biotechnology, Inc.) was added to each well and incubated at room temperature to carry out the blogging. Then, each well was washed with phosphate buffer (PBS-T) containing 0.1% Tween 20 three times. Then, each serum diluted with PBS-T containing 10% Block Ace (50 μl / well) was added to each well, and incubated and reacted at 37 ° C for two hours. The microplate was washed with PBS-T three times. Then, a 1,000-fold dilution of an IgGl antibody or goat anti-mouse IgM antibody labeled with alkaline phosphatase (Cosmo Bio, 1070-04 or 1020-04) with PBST-T containing 10% Block Ace (50 μg / well ) was added to each well, and incubated at 37 ° C for two hours. Then, the microplate was washed with PBS-T, and then a colored substrate solution (50 μl / well, Sigma 104, substrate-phosphatase) was added to each well. Absorbance at a wavelength of 405 nm was measured using a microplate reader. The results are shown in Figures 13a and B. In the figures, the longitudinal axis indicates values obtained by converting a 10,000-fold dilution (in the case of IgGl) or a 100-fold dilution (in the case of the IgM antibody) of serum collected from C57BL / 6 mice, to which NP-CGG was injected twice, and grouped into one unit. The 4D11 antibody and the IgG4P or IgG4PE antibody of 281 inhibited the production of NP-specific IgGl and IgM antibodies equally strongly. The change in the number of B cells in the peripheral blood and spleen in mice used to examine the effect to inhibit antibody production was measured "according to the same method as in Example 1. The results are shown in the Figures 14A and B. The 4D11 antibody and the IgG4P antibody of 281 reduce the B-cell ratio in peripheral blood significantly when compared to the IgG4PE antibody.Administration of 100 μg of IgG4PE antibody does not change the ratio of B cells in the spleen removed 14 days after antigen sensitization, however, the administration of IgG4P changes or tends to change the relationship.

Example 15: Effect of anti-CD40 antagonist antibody on cynomologo monkeys 30 mg / kg of IgG4P or IgG4PE of a 4D11 antibody was administered to the cephalic vein of the forearm of cynomologo monkeys, and blood was collected from the femoral vein after a certain period of time. In the subject analysis of the peripheral blood lymphocytes, an anti-CD3 antibody labeled with FITC, an anti-CD20 antibody labeled with PE, and an anti-CD45 antibody labeled with APC were used for each cell suspension, and the cell ratio positive was measured using FACS to calculate the ratio of CD45 positive cells. The results are shown in Figure 15. In the figure, the longitudinal axis indicates the ratio of positive cells 7D20 each time to CD20 positive cells before administration of the antibody. 1 to 7 days after administration of the antibody, CD20 positive cells were reduced by approximately 40% in individuals to whom the IgG4P antibody was administered. However, 4 days after the administration, the CD20 positive cells were reduced by only about 20% in individuals to whom the IgG4PE antibody was administered. The concentration of IL12 in serum was measured by the ELISA method. Blood was allowed to collect from the femoral vein which remained at room temperature for 20 to 60 minutes, and then centrifuged at 3,000 rpm at room temperature for 15 minutes. The concentration of IL12 in the resulting serum was measured using an IL12 ELISA kit in mono (BioSource International Inc.). The results are shown in Figure 16. No increase in IL12 production was observed by the IgG4PE antibody at any blood collection point. However, maximal IL12 production by the IgG4P antibody was observed on the fourth day.

Example 16: Effect of anti-CD40 antagonist antibody in delayed hypersensitivity model of cynomologous monkey Nine male cynomologo monkeys were sensitized intradermally and intramuscularly with Tetanus toxoid (TTx) (10 Lf / ml; Denka Seiken Co., Ltd.) to induce Delayed hypersensitivity to TTx. At the same time, 10 minutes before the start of sensitization, 0.1 and 10 mg / kg of a 4D11G4PE antibody was administered intravenously to every three animals three times (once a week) to examine the effect of 4D11G4PE on delayed hypersensitivity. Under anesthesia by intramuscular administration of ketamine, sensitization was carried out by transdermal administration of TTx again (50 μL / site x 12 sites) and intramuscular administration to TTx to the femur (0.6 ml / body), and was carried out challenge for intradermal administration of TTx to the thorax (10 μl / site, 0 to 10 Lf / ml for every three sites) 21 days after sensitization. 24 and 48 hours after the response, the skin reaction at the administration sites was observed and evaluated according to the Draize skin irritation classification. The results to determine the concentration of TTx in each three sites were respectively an average value. The results are shown in Figure 17. Administration of the 4D11G4PE antibody apparently inhibits the delayed hypersensitivity reaction observed 24 and 48 hours after administration. The effect of TTx on titers of TTx-specific IgG and IgM antibodies was examined. Blood was allowed to collect from the femoral vein with the time it remained at room temperature for 20 to 60 minutes, and then centrifuged at 3,000 rpm at room temperature for 15 minutes. The titration of antibody in the resulting serum was measured using the ELISA method. The ELISA method was carried out as follows. 100 μl / well of TTx (0.5 Lf / l) was added to each well of a 96-well ELISA microplate (Maxisorp, manufactured by Nunc A / S) and incubated at 4 ° C to cause the TTx to be absorbed by the same. Then, the supernatant was discarded, and a blocking reagent (phosphate buffer containing 0.5% BSA) was added to each well and incubated at room temperature to effect blockage. Then, each well was washed with a phosphate buffer (PBS-T) containing 0.05% Tween 20 three times. Next, each serum diluted with PBS-T containing 0.5% BSA (100 to 819,200 times dilution, dilution amplification: 2; 100 μl / well) was added to each well, and incubated and reacted at room temperature for two hours. The microplate was washed with PBS-T three times. Then, a 3,000-fold dilution of a goat anti-monkey IgG antibody or IgM antibody labeled with peroxidase (Nordic Immunology) with PBS-T containing 0.5% BSA (100 μg / well) was added to each well, and incubated at room temperature for one hour. Then, the microplate was washed with PBS-T, and then a colored substrate solution (100 μl / well, o-phenylenediamine hydrochloride + aqueous acid peroxide) was added to each well. Absorbance at a wavelength of 492 nm was measured using a microplate reader. The titration of anti-TTx antibody was defined as a maximum dilution amplification to make the absorbance 0.1 and more. The titration of the antibody was 0 when the absorbance did not reach 0.1 even at 100-fold dilution. The results are shown in Figures 18 and 19. Administration of 1 mg / kg of 4D11G4PE suppressed titers of TTx-specific IgG and IgM antibody to approximately 1/10. When 10 mg / kg of 4D11G4PE was administered, the antibody titers were below the detection sensitivity at any blood collection point.

Example 17: Effect of anti-CD40 antagonist antibody on platelet thrombus formation The blood collected from a healthy human was divided into four aliquots (every 6 ml). Control human IgG4PE, control mouse IgG2a, anti-human human CD40 IgG4PE (4D11) and mouse anti-mouse IgG2a CD154 (5C8) were added respectively to the fractions so that each fraction had a concentration blood of 100 μg / ml. A flat perfusion chamber (GlycoTech Corp.) and a Petri dish coated with collagen were assembled according to the attached instruction. The blood treated with several antibodies was caused to flow into the chamber at an index that can apply a shear stress of 1,500 / s to the blood for seven minutes. Further on, 4% of the paraformaldehyde phosphate buffer was caused to flow into the chamber at an index that can apply a shear stress of 1,500 / s to the buffer for 10 minutes. The platelet aggregate formed in the Petri dish was fixed, stained with a CD41a antibody labeled cor. Platelet-specific PE, and was observed with a fluorescence microscope. The results are shown in Figures 20A and B. The blood treated with human anti-human IgG4PE CD40 (4D11) formed a platelet aggregate in the Petri dish coated with collagen, as the blood treated with the control antibodies did. However, blood treated with mouse anti-human IgG2a CD154 did not form a plague aggregate.

Example 18: Stability evaluation of anti-CD40 antagonist antibody The antibodies modified with constant region of antibody 4D11 were compared and examined in terms of stability. In the evaluation method, the culture supernatants obtained by G4P, G4PE, G2Ser and G4 / 2A which are temporarily expressed respectively in HEK293 cells were loaded with a Protein A column (Amersham Pharmacia Biotech), eluted with a buffer. of 0.1 M citrate (pH 2.7) and then incubated at 37 ° C for 1 minute and 10 minutes, then neutralized with a 50 nM phosphate buffer (pH 7.0) .The oligomer content in the solutions of The resulting antibody was measured using a gel filtration column (Tosh Corp.) As a result, it was found that the oligomer content increases in proportion to the incubation time, and G4 / 2/4 produces a simpler oligomer, G4PE second simpler, G2Ser third easier, and G4P fourth simpler (Figure 2 ±.

Example 19: Effect to inhibit rejection of skin graft by anti-CD40 antagonist antibody A graft harvested from the tail of DBA / 2 mice was grafted into the lateral dorsal thorax of mice of C57BL / 6 background that have a genetic background consequently these were homozygous for mouse endogenous destabilized CD40 and harboring a transgene of a human CD40 gene and the graft was fixed with a patch for seven days. 100 μg of a 4D11G4PE test substance or a vehicle was administered to the tail vein 0, 2, 4, 7, 9, 11 and 14 days after skin grafting, respectively. To inhibit graft rejection by NK cells, 100 μg of an anti-asialo GML antibody was administered intraperitoneally to all mice 3 days before the operation and 1, 4 and 9 days after the operation. The results are shown in Figure 22. The delay in graft rejection was found to be significant in the group administered by 4D11G4PE when compared to the group administered per vehicle.

Example 20: Analysis of CD40 expression in human tumor cell lines Expression of CD40 in a Burkitt Ramos lymphoma cell line, a T24 bladder cancer cell line (ATCC, HTB-4), pancreatic cancer cell line Hs 766T (ATCC, HTB-134) and Capan-2 (ATCC, HTB-80) was confirmed by FACS analysis using 341G2Ser. T24, Hs 766T and Capan-2 were digested with trypsin and harvested, and Ramos was harvested as is. The cell lines were washed with PBS, and then resuspended in a staining buffer containing 1 μg / ml of 341G2Ser. The staining buffer was prepared by adding 0.05 mM EDTA, 0.05% sodium azide and 5% immobilized bovine serum to PBS.

After incubation at 4 ° C for 15 minutes, the cells were washed with the staining buffer twice, and resuspended in a 1: 250 dilution of goat antihuman IgG linked to PE (?) (Southern Biotechnology Associates , Inc) with the staining buffer. After incubation at 4 ° C for 15 minutes, the cells were washed with the staining buffer twice, and analyzed with FACSCalibur (manufactured by BD Biosciences). The same amount of a human anti-2,4-dinitrophenol antibody (DNP) was used as a negative control. The analysis was carried out using Cellquest (manufactured by BD Biosciences) as data analysis software to calculate the average fluorescence intensity. As a result, Ramos, T24, Hs766T and Capan-2 had an average fluorescence intensity obviously higher than that of the negative control when stained with 341G2Ser, and thus the expression of CD40 was confirmed.

Example 21: Effect of anti-CD40 agonist antibody on human tumor cell lines 2.5 x 103 Ramos cells, 2.5 x 102 T24 cells, 5 x 103 Hs766T cells and 5 x 103 Capan-2 cells were respectively suspended in a medium to make the total volume of 100 μl in a 96-well flat bottom plate (manufactured by Falcon). Ramos and Hs766T, Capan-2 and T24 were cultured for 66 hours, 90 hours and 114 hours, respectively, together with 341G2Ser at a concentration of 1 ng / ml at 1,000 ng / ml at 37 [deg.] C. in the presence of 5% C02 10 μl (3.7 MBq / ml) of thymidine labeled with 3 H (manufactured by Amersham Biosciences) was added and cultured at 37 ° C in the presence of 5% C02 for six hours. Ramos cells were harvested in Printed Filtermat A (manufactured by PerkinElmer, Inc.) using a 96 microcell harvester (manufactured by Skatron Instruments, Inc.) and covered with a sample bag (manufactured by PerkinElmer, Inc.). 12 ml of Betaplate Saint (manufactured by PerkinElmer, Inc.) was added and the dose of ß-rays was measured with a liquid scintillation counter (Pharmacia 1205 Betaplate: manufactured by Pharmacia Ccrp.) Hs 766T cells, T24 cells and Capan- 2 were harvested respectively in Unifilter (manufactured by PerkinElmer, Inc.) using a harvester (manufactured by PerkinElmer, Inc.) A special seal was attached to the back of each filter, and 20 μl / well of MicroScint 20 (manufactured by PerkinElmer , Inc.) was added to it. The dose of ß-rays was measured with a scintillation counter (TopCount: manufactured by Packard Instrument Co., Inc.). Data were expressed as cell survival rates (%) obtained by dividing an average of triplicate measurements obtained in three independent tests by a control value without treatment. As a result, cell survival rates were reduced in all cell lines depending on the 341G2Ser concentration (Table 1). When 100 ng / ml of 341G2Ser were added, the Ramos cell survival rate was 58%, the T24 cell survival rate was 22%, the HS 766T cell survival rate was 15%, and the Capan cell survival rate was 15%. -2 was 77%. 341G2Ser was found to have activity to inhibit the growth of Ramos cells, T24 cells, HS 766T cells and Capan-2 cells.

Table 1 Cell Survival index Example 22: Effect of anti-CD40 agonist antibody in mouse model suffering from cancer (1) Ramos cells Bare / female six-week-old Balb / c mice (purchased from CLEA Japan, Inc.) were irradiated with 3Gy radiation, and 2 x 107 cells / mouse of Ramos cells were grafted subcutaneously on the back thereof. 16 days after grafting, the size of tumors that he took was measured. Mice suffering from cancer that have a tumor size of 50 to 170 mm3 were classified into groups consisting of each of five mice. 100 μg / mouse of 341G2Ser (a solution in 200 μl of PBS containing 1% of naked mouse serum) was intravenously administered to mice suffering from cancer once on day 16, and the tumor size was measured until the day 47. An anti-human human serum albumin (HAS) antibody was used as a negative control. (2) T24 cells A T24 cell mass that had undergone cutaneous passage in the back of nude mice three times was removed, and was grafted subcutaneously on the back of six-week-old female Balb / c nude mice. (acquired from CLEA Japan, Inc.). The tumor cell mass that is grafted is appropriately about 3 square mm. 10 days after grafting, the size of the grafted tumors was measured. Mice suffering from cancer that have a tumor size of 80 to 200 mm3 were classified into groups each consisting of five mice. 100 μg / mouse of 341G2Ser (a solution in 200 μl of PBS containing 1% of naked mouse serum) was administered intravenously to mice suffering from cancer once on day 10, and the tumor size was measured until day 29 The same amount of a human anti-DNP antibody was used as a negative control. (3) HS 766T cells 7 × 10 5 cells / mouse of Hs 766T were subcutaneously grafted onto the back of eight-week-old female Balb / c nude mice (purchased from CLEA Japan, Inc.). 16 days after grafting, the size of grafted tumors was measured. Mice suffering from cancer that have a tumor size of 50 to 140 mm3 were classified into groups each consisting of five mice. 100 μg / mouse of 341G2Ser (a solution in 200 μl of PBS containing 1% of naked mouse serum) was administered intravenously to mice suffering from cancer on day 16, and the tumor size was measured until day 32. The same amount of a human anti-DNP antibody was used as a negative control. (4) Capan-2 cells 2 x 10d cells / mouse of Capan-2 cells were grafted subcutaneously on the back of six-week-old female Balb / c nude mice (purchased from CLEA Japan, Inc.). 13 days after grafting, the size of grafted tumors was measured. Mice suffering from cancer that have a tumor size of 30 to 130 mm3 were classified into groups consisting of each of five mice. 10 or 100 μg / mouse of 341G2Ser (a solution in 200 μl of PBS containing 1% of naked mouse serum) was administered intravenously to mice suffering from cancer twice a week from day 13, and tumor size was measured until day 34. A human polyclonal antibody (hlgG) (manufactured by Sigma Co.) was used as a negative control. The tumor growth inhibition ratio (TGIR) was calculated from the following formula. 100- [. { (average tumor volume of the 341G¿.3er administered group on the last day of measurement - average tumor volume of the 341G2Ser administered group on the day of initiation of antibody administration) / (average tumor volume of a group administered with control on the last day of measurement - average tumor volume of the group administered with negative control on the day of initiation of antibody administration)} x 100]. As a result, TGIR exceeded 100% in mice suffering from cancer T24, Hs766T and Capan-2 and a decrease in tumor volume was observed in the mice. On the other hand, TGIR was 73.4% in mice suffering from Ramos cancer, and an increase in tumor volume was suppressed considerably in the mice (Table 2). Figures z.3 and 26 respectively show the change in tumor volume of mice suffering from cancer to which Ramos cells, T24 cells, Hs 766T cells and Capan-2 cells were grafted respectively.

Table 2 Relationship of inhibition of tumor growth The inhibition ratio in each cell line is a value on the last day of measurement. Industrial Applicability As shown in the Examples, the anti-CD40 antibody of the present invention having a constant region wherein a mutation is introduced and an anti-CD40 antibody wherein a part of the subclass structure is replaced with that of another subclass has reduced ADCC activity and CDC activity, while maintaining its activity. Accordingly, the antibody of the present invention has reduced cytotoxicity to cells expressing CD40 when administered to a subject as a therapeutic antibody, and thus can be used safely. All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety. Sequence List Free Text SEC. FROM IDENT. NOS .: 2 to 36: the synthetic DNAs SEC. FROM IDENT. NOS .: 49 to 130: Synthetic peptides

Claims (94)

1. CLAIMS 1. A heavy chain of a monoclonal antibody which has an agonist activity, which binds to CD40, wherein the heavy chain comprises an upper joint and a middle joint derived from a human IgG2, and a constant region with at least one amino acid deleted or substituted, or with at least one amino acid added thereto, such removal, substitution or addition is capable of increasing or decreasing ADCC and / or CDC.
2. 2. The heavy chain according to claim 1, wherein the constant region is derived from a human IgG.
3. 3. The heavy chain according to claim 2, wherein the human IgG is a human IgGl.
4. 4. The heavy chain according to claim 2, wherein the human IgG is a human IgG2.
5. 5. The heavy chain according to claim 2, wherein the human IgG is a human IgG3.
6. 6. The heavy chain according to claim 2, wherein the human IgG is a human IgG4. The heavy chain according to any of claims 3 to 5, wherein the substitution of amino acids in the constant region is the substitution of proline with serine at position 331 which is indicated by the EU index as in Kabat et al. 8. A monoclonal antibody comprising the heavy chain according to any of claims 1 to 7. The heavy chain according to any of claims 1 to 7, wherein the heavy chain comprises a variable region from a heavy chain of a monoclonal antibody produced by hybridoma KM341-1-19 (Accession No. FERM BP-7759). 10. A monoclonal antibody consisting of a heavy chain according to claim 9 and a light chain comprising a variable region from a light chain of a monoclonal antibody produced by the hybridoma KM341-1-19 (Accession No. FERM BP -7759). 11. The heavy chain according to any of claims 1 to 7, wherein the heavy chain comprises a variable region of the polypeptide represented by SEQ. FROM IDENT. NO .: 38. 12. A monoclonal antibody consisting of the heavy chain according to claim 11, and a light chain of a monoclonal antibody, wherein the light chain comprises a variable region of the polypeptide represented by SEQ. FROM IDENT. NO .: 40. The heavy chain according to claim 1, wherein the heavy chain consists of a remaining portion provided by removing the signal sequence from the polypeptide represented by SEC. FROM IDENT. DO NOT. : 132. 14. A monoclonal antibody consisting of the heavy chain according to claim 13, and a light chain of a monoclonal antibody, wherein the light chain consists of a remaining portion provided by removing the signal sequence from the polypeptide represented by the SEC. FROM IDENT. NO: 134. 15. The heavy chain according to claim 1, wherein the heavy chain is produced by a host comprising an expression vector having the polynucleotide represented by SEQ. FROM IDENT. NO .: 131. 16. The monoclonal antibody according to claim 8, wherein the monoclonal antibody is produced by a host comprising an expression vector having the polynucleotide represented by SEQ. FROM IDENT. NO .: 131 and the polynucleotide represented by SEC. FROM IDENT. DO NOT. : 133. The heavy chain according to any of claims 1 to 7, wherein the heavy chain comprises a variable region of a heavy chain of a monoclonal antibody produced by hybridoma 2105 (Accession No. FERM BP-8024 ). 18. A monoclonal antibody consisting of heavy chain according to claim 17, and a light chain comprising a variable region from a light chain of a monoclonal antibody produced by hybridoma 2105 (Accession No. FERM BP-8024 ). 19. The heavy chain according to any one of claims 1 to 7, wherein the heavy chain comprises a variable region of the polypeptide represented by SEC. FROM IDENT. NO .: 42. 20. A monoclonal antibody consisting of the heavy chain according to claim 19 and a light chain of a monoclonal antibody, wherein the light chain comprises a variable region of the polypeptide represented by SEQ. FROM IDENT. NO .: 44. 21. The heavy chain according to claim 1, wherein the heavy chain consists of a remaining portion provided by removing the signal sequence from the polypeptide represented by SEC. FROM IDENT. NO .: 136. 22. A monoclonal antibody consisting of the heavy chain according to claim 21 and a light chain of a monoclonal antibody, wherein the light chain consists of a remaining portion provided by removing the signal sequence from of the polypeptide represented by the SEC. FROM IDENT. NO .: 138. The heavy chain according to claim 1, wherein the heavy chain is produced by a host comprising an expression vector having the polynucleotide represented by SEQ. FROM IDENT. NO .: 135. 24. The monoclonal antibody according to claim 8, wherein the monoclonal antibody is produced by a host comprising an expression vector having the polynucleotide represented by SEQ. FROM IDENT. NO .: 135. and the polynucleotide represented by SEC. FROM IDENT. DO NOT. : 137. 25. A polynucleotide represented by SEC. FROM IDENT. DO NOT. : 131. 26. A polynucleotide represented by SEC. FROM IDENT. NO .: 133. 27. An expression vector having the polynucleotide according to claim 25. 28. An expression vector having the polynucleotide according to claim 26. 29. An expression vector having the polynucleotides of according to claims 25 and 26. 30. A host comprising the expression vector according to claim 27. 31. A host comprising the expression vector according to claim 28. 32. A host comprising the vector of expression according to claim 29. 33. A process for producing a heavy chain of a monoclonal antibody, comprising the steps of: culturing the host according to claim 30 in a culture medium.; and obtaining a heavy chain of a monoclonal antibody from the culture and / or the host. 34. A process for producing a monoclonal antibody, comprising the steps of: culturing the host according to claim 32, in a culture medium; and obtaining a monoclonal antibody from the culture and / or the host. 35. A polynucleotide represented by SEQ. FROM IDENT. NO .: 135. 36. A polynucleotide represented by SEC. FROM IDENT. NO .: 137. 37. An expression vector having the polynucleotide according to claim 35. 38. An expression vector having the polynucleotide according to claim 36. 39. An expression vector having the polynucleotides of according to claims 35 and 36. 40. A host comprising the expression vector according to claim 37. 41. A host comprising the expression vector according to claim 38. 42. A host comprising the vector of expression according to claim 39. 43. A process for producing a heavy chain of a monoclonal antibody, comprising the steps of: culturing the host according to claim 40 in a culture medium; and obtaining a heavy chain of a monoclonal antibody from the culture and / or the host. 44. A process for producing a monoclonal antibody, comprising the steps of: culturing the host according to claim 42, in a culture medium; and obtaining a monoclonal antibody from the culture and / or the host. 45. A process for producing a heavy chain of a monoclonal antibody having an agonist activity capable of binding to CD40, comprising the step of replacing the upper joint and the middle joint of an antibody, which is neither a superior joint nor a middle joint derived from a human IgG2, with an upper joint and a middle joint derived from a human IgG2, respectively. 46. A process for producing a heavy chain of a monoclonal antibody comprising a variable region, and an upper joint and a middle joint derived from a human IgG2, comprising the step to identify a polypeptide that forms the variable region, the which is from a heavy chain of a monoclonal antibody capable of binding to CD40. 47. A process for producing a monoclonal antibody having an agonist activity capable of binding to CD40, which comprises the step to replace the upper joint and the middle joint of an antibody, which is neither an upper joint nor a middle joint derived from of human IgG2, with an upper joint and a middle joint derived from a human IgG2, respectively. 48. A process for producing a monoclonal antibody comprising a variable region, and an upper joint and a middle joint derived from a human IgG2, comprising the step to identify a polypeptide that forms the variable region, which is from a chain heavy of a monoclonal antibody capable of binding to CD40. 49. A pharmaceutical composition comprising the monoclonal antibody according to any of claims 8, 10, 12, 14, 16, 18, 20, 22 and 24 as an active ingredient. 50. The pharmaceutical composition according to claim 49, used for the prevention or treatment of a malignant tumor, a pathogen or an autoimmune disease. 51. A method for the prevention or treatment of a malignant tumor, a pathogen or an autoimmune disease, which comprises administering the pharmaceutical composition according to claim 49 in a mammal. 52. The use of the monoclonal antibody according to any of claims 8, 10, 12, 14, 16, 18, 20, 22 and 24 for the production of a pharmaceutical composition used for the prevention or treatment of a malignant tumor, a pathogen or an autoimmune disease. 53. A heavy chain of a monoclonal antibody that has an antagonistic activity capable of binding to CD40, wherein the heavy chain comprises a constant region with at least one amino acid deleted or substituted, or with at least one amino acid added thereto, such elimination, substitution or addition which is capable of increasing or decreasing ADCC and / or CDC. 54. The heavy chain according to claim 53, wherein human IgG is a human IgG. 55. The heavy chain according to claim 54, wherein the human IgG is a human IgGl. 56. The heavy chain according to claim 54, wherein the human IgG is a human IgG2. 57. The heavy chain according to claim 54, wherein the human IgG is a human IgG3. 58. The heavy chain according to claim 54, wherein the human IgG is a human IgG4. 59. The heavy chain according to any of claims 55, 57 and 58, wherein the substitution of amino acids in the constant region is the substitution of leucine with glutamic acid at position 235 which is indicated by the EU index as in Kabat et al. 60. A heavy chain according to any one of claims 53 to 59, wherein the heavy chain comprises a constant region with at least one amino acid deleted or substituted, or with at least one amino acid added thereto, such removal, substitution or addition that is able to promote the formation of the SS link between the heavy chains. 61. The heavy chain of the antibody according to claim 60, wherein the substitution of amino acids in the constant region is the substitution of serine with the proline at position 228 which is indicated by the EU index as in Kabat et al. 62. A monoclonal antibody comprising the heavy chain according to any of claims 53 to 61. 63. The heavy chain according to any of claims 53 to 61, wherein the heavy chain comprises a variable region from a Heavy chain of a monoclonal antibody produced by hybridoma 4D11 (Accession No. FERM BP-7758). 64. A monoclonal antibody consisting of the heavy chain according to claim 63, and a light chain comprising a variable region from a light chain of a monoclonal antibody produced by the hybridoma 4D11 (Accession No. FERM BP-7758) . 65. The heavy chain according to any one of claims 53 to 61, wherein the heavy chain comprises a variable region of the polypeptide represented by SEQ. FROM IDENT. NO .: 46. 66. A monoclonal antibody consisting of dt- the heavy chain according to claim 65 and a light chain of a monoclonal antibody, wherein the light chain comprises a variable region of the polypeptide represented by SEQ. FROM IDENT. NO .: 48. 67. The heavy chain according to claim 53, wherein the heavy chain consists of a remaining portion provided by removing the signal sequence from the polypeptide represented by SEQ. FROM IDENT. NO .: 140. 68. A monoclonal antibody consisting of the heavy chain according to claim 67, and a light chain of a monoclonal antibody, wherein the light chain consists of a remaining portion provided by removing the signal sequence at from the polypeptide represented by SEC. FROM IDENT. NO .: 142 1 69. The heavy chain according to claim 53, wherein the heavy chain is produced by a host comprising an expression vector having the polynucleotide represented by SEQ. FROM IDENT. NO .: 139. 70. The monoclonal antibody according to claim 62, wherein the monoclonal antibody is produced by a host comprising an expression vector having the polynucleotide represented by SEQ. FROM IDENT. NO .: 139 and the polynucleotide represented by SEC. FROM IDENT. DO NOT. : 141. 71. A polynucleotide represented by SEC. FROM IDENT. NO .: 139. 72. A polynucleotide represented by SEC. FROM IDENT. NO .: 141. 73. An expression vector having the polynucleotide according to claim 71. 74. An expression vector having the polynucleotide according to claim 72. 75. An expression vector having the polynucleotides of according to claim 71 and 72. 76. A host comprising the expression vector according to claim 73. 77. A host comprising the expression vector according to claim 74. 78. A host comprising the vector of expression according to claim 75. 79. A process for producing a heavy chain of a monoclonal antibody, comprising the steps of: culturing a host according to claim 76, in a culture medium; and obtaining a heavy chain of a monoclonal antibody from the culture and / or the host. 80. A process to produce a monoclonal antibody, comprising the steps of: cultivating the host according to claim 78, in a culture medium, obtaining a monoclonal antibody from the culture and / or the host. 81. A pharmaceutical composition comprising the monoclonal antibody according to any of claims 62, 64, 66, 68 and 70 as an active ingredient. 82. The pharmaceutical composition according to claim 81 used for the prevention or treatment of transplant rejection, autoimmune diseases, allergy or inhibition VIII of the blood coagulation factor. 83. A method for the prevention or treatment of rejection of transplantation, autoimmune diseases, allergy or inhibition VIII of the blood coagulation factor, which comprises administering the pharmaceutical composition according to claim 81 in a mammal. 84. The use of the monoclonal antibody according to any of claims 62, 64, 66, 68 and 70 for the production of a pharmaceutical composition used for the prevention or treatment of transplant rejection, autoimmune diseases, allergy or inhibition VIII of the factor of blood coagulation. 85. A method for producing a heavy chain of a monoclonal antibody having an agonist activity capable of binding to CD40, wherein the agonist activity is reduced, comprising the step to carry out the elimination or substitution of at least one amino acid, or the addition of at least one amino acid in the heavy chain constant region of a human antibody. 86. The method according to claim 85, wherein the constant region is derived from a human IgG. 87. The method according to claim 86, wherein the human IgG is a human IgG4. 88. The method according to any of claims 85 to 87, wherein the substitution of amino acids in the constant region is the substitution of leucine with glutamic acid at position 235 which is indicated by the EU index as in Kabat et al. 89. A polynucleotide provided by removing the portion encoding the signal sequence from the polynucleotide represented by SEQ. FROM IDENT. NO .: 131 90. A polynucleotide provided by removing the portion encoding the signal sequence from the polynucleotide represented by SEQ. FROM IDENT. NO .: 133. 91. A polynucleotide provided by removing the portion encoding the signal sequence from the polynucleotide represented by SEQ. FROM IDENT. NO .: 135. 92. A polynucleotide provided by removing the portion encoding the signal sequence from the polynucleotide represented by SEQ. FROM IDENT. NO .: 137. 93. A polynucleotide provided by removing the portion encoding the signal sequence from the polynucleotide represented by SEQ. FROM IDENT. NO .: 139. 94. A polynucleotide provided by removing the portion encoding the signal sequence from the polynucleotide represented by SEQ. FROM IDENT. NO .: 141