JP2006342173A - Anti-cd40 monoclonal antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an anti-human CD40 antibody or a functional fragment thereof substantially antagonistic even to a human CD40 antigen on the surface of a DC (a dendritic cell) by adopting an evaluation system using the DC cell and to provide an agonistic anti-human CD40 antibody expected to have higher therapeutic effects than those of conventional anti-human CD40 antibodies. <P>SOLUTION: The antibody or functional fragment thereof agonistically or antagonistically acting on the CD40 is provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an antibody or a functional fragment thereof that recognizes human CD40 antigen present on the surface of human B cells, dendritic cells (DCs) and the like. Specifically, the present invention provides an anti-human CD40 antibody or functional fragment thereof that is substantially antagonistic to human CD40 antigen on the surface of dendritic cells (DC), and compared to conventional anti-human CD40 antibodies. The present invention relates to an agonistic anti-human CD40 antibody or a functional fragment thereof that is expected to have a higher therapeutic effect.

1. CD40
CD40 is an antigen present on the surface of a cell membrane having a molecular weight of 50 kDa, and is expressed on B cells, dendritic cells (DC), certain 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 has been identified as an antigen expressed on the surface of human B cells (see Non-Patent Documents 1 and 2). From the homology of amino acid sequences, CD40 is a low affinity NGF receptor, TNF receptor, CD27, OX40, CD30, etc. Is considered as a member of the TNF receptor family to which it belongs. A ligand for human and mouse CD40 (CD40L) has recently been genetically cloned and found to be a type II membrane protein and expressed on activated CD4 + T cells. CD40L has also been shown to introduce strong activation signals into human and mouse B cells.

  In addition, dendritic cells have been confirmed to play an important role because more CD40 expression has been confirmed than B cells. When CD40 binds to CD40L, antigen-presenting cell (APC) activation, ie, expression of costimulatory molecules such as CD80 (B7-1) and CD86 (B7-2), or IL-12 production is enhanced ( (See Non-Patent Documents 3 and 4). Dendritic cells have a strong antigen-presenting ability and a strong helper T (Th) cell activation ability. In addition, it is considered that dendritic cells control the differentiation of naive Th cells into Th1 or Th2 cells. Dendritic cells (DC1) obtained by culturing peripheral blood monocytes, which are myeloid dendritic cells, with GM-CSF and IL-4 and matured with CD40L have IL-12 production ability in vitro and are allogeneic. It stimulates naive Th cells and induces IFNγ-producing T cells (ie, promotes differentiation into Th1). Since this action is inhibited by anti-IL-12 antibody, it is considered to be a reaction mediated by IL-12. On the other hand, lymphoid dendritic cells (DC2) obtained by culturing plasmacytoid T cells present in lymphoid tissue T region and peripheral blood with IL-3 and CD40 ligands do not have IL-12 production ability and It has been shown to stimulate and activate Th cells, induce IL-4 producing T cells, and promote differentiation into Th2. Th1 cells are involved in the activation of cellular immunity, and Th2 cells are thought to increase humoral immunity and at the same time suppress cellular immunity. Cytotoxic T cells (CTL) activated with the help of Th1 cells can remove pathogens (such as many viruses, Listeria monocytogenes, Mycobacterium tuberculosis, and Toxoplasma gondii) and tumor cells that grow in the cytoplasm .

  It has been shown that anti-CD40 monoclonal antibodies that recognize CD40 expressed on the membrane surface exhibit various biological activities against B cells. Anti-CD40 monoclonal antibodies are roughly classified into those that are agonistic and those that are antagonistic for the interaction between CD40 and CD40L.

2. Agonistic antibody The activation of B cells is known as the action of an agonistic antibody. For example, anti-CD40 antibody induces cell adhesion (see Non-Patent Documents 5 and 6), increases cell size (see Non-Patent Documents 6 and 7), anti-IgM antibody, anti-CD20 antibody or phorbol ester Induces the division of B cells activated only by (see Non-Patent Documents 8 to 10), induces the division of B cells in the presence of IL4 (see Non-Patent Documents 7 and 11), IL-4 stimulation Secretion of soluble CD23 / FceRII from B cells induced by IL-4 that induces the expression of IgE (see Non-patent Documents 12 and 13), IgG, and IgM (see Non-patent Document 13) of cultured cells from which T cells have been removed (See Non-Patent Documents 14 and 15), which enhances expression on cells (see Non-Patent Document 16) and promotes IL-6 production (see Non-Patent Document 17). Furthermore, by adding IL-4 and anti-CD40 antibody in the presence of CDw32 + adherent cells, B cell clones can be established from human primary cultured B cells (see Non-Patent Document 18), It has been reported that apoptosis of central cells is inhibited through CD40 regardless of the action of the antigen receptor (see Non-Patent Document 19). As described above, since CD40 was identified as an antigen expressed on the surface of human B cells, many of the isolated antibodies mainly use the function of inducing proliferation and differentiation of human B cells and the activity of inducing cell death in cancer cells. Have been evaluated (see Non-Patent Documents 20-22).

  It has been shown that anti-CD40 antibodies mature DCs (see Non-Patent Document 23). Furthermore, the role of CD4T cells in antigen-specific CD8T cell priming was reported to be DC activation through CD40-CD40L signaling, and the activity of dendritic cells (DCs) by anti-CD40 monoclonal antibody (mAb) was reported. It has been shown that the role of CD4 helper T cells can be replaced in the process (see Non-Patent Document 24). Moreover, it was shown that the living body can be protected not only from tumor cells expressing CD40 but also from non-expressing tumor cells by administration of an anti-CD40 antibody in mice (see Non-patent Document 25).

  There are few antibodies that have been reported so far, with the effect on DC cells as an index. However, from the point of functional modification of DC cells, selecting antibodies that act on B cells is likely to be insufficient as a therapeutic agent. In the monoclonal antibody of the antibody against mouse CD40, depending on the epitope recognized by the antibody, there are clones that react with DC but do not react with vascular endothelial cells, and conversely clones that do not react with DC but react with vascular endothelial cells. It is reported to exist (see Non-Patent Document 26). It is speculated that human CD40 antibodies also have different DC binding and action depending on the epitope.

  It has been found that anti-CD40 antibody or CD40 ligand can suppress the proliferation of lymphoma cell lines expressing CD40 and induce cell death (see Non-Patent Documents 23 and 27-29). What is interesting about agonistic antibodies is that the action of the antibody does not necessarily match the function of CD40L. The activation effect on B cells and the growth inhibitory effect on B cell tumors are not necessarily consistent. It is desirable to develop an antibody having both DC activation ability and tumor cell growth inhibitory action. Further, among agonistic antibodies, there are those that inhibit or do not inhibit the binding of CD40L to CD40 (see Non-Patent Document 30). For example, since the antibody produced by G28-5 (ATCC No. HB-9110) competes with CD40L, there is no combined effect with CD40L. There are also differences in the degree of activation of CD40-expressing cells depending on the antibody. Even in the case of an antibody that exhibits weak agonistic activity alone, when used in combination with CD40 ligand, the activity may be significantly promoted in the presence of the antibody than the activity of CD40 ligand alone. Conversely, even an antibody that exhibits agonistic activity alone may inhibit the activity in the presence of the antibody by inhibiting CD40 ligand (see Non-Patent Document 31). ). It has been shown that an antibody that does not compete with CD40 ligand can suppress growth of tumor cells by itself, but can suppress growth more strongly in the presence of CD40 ligand (see Non-Patent Document 29). Therefore, it is desirable to develop an antibody that binds to CD40 and suppresses cell proliferation by itself but does not inhibit the binding of CD40 ligand to CD40. By taking advantage of these characteristics, it is possible to develop therapeutic agents that are more effective than soluble CD40L. For example, soluble CD40L binds to CD40 and activates it, but at the same time suppresses the action of CD40L present in the living body. For antibodies that do not compete with CD40L, this does not happen and a better therapeutic effect can be expected due to synergistic effects.

3. Antagonistic antibody On the other hand, as mentioned above, CD40 plays an important role in the immune response, so it inhibits the binding of CD40 and its ligand, thereby suppressing immunosuppression and autoimmune diseases during organ transplantation. It is expected that a new therapeutic drug can be developed. Sawada-Hase et al. Reported that the proportion of cells that strongly express CD40 is increased in monocytes in the peripheral blood of Crohn's disease patients. However, the antibody that inhibits the binding of CD40 and its ligand is still not well understood. Such an inhibitory antibody may be effective in, for example, functional analysis of CD40 and treatment of diseases that require CD40 activation. Inhibitory antibodies against CD40 ligand have also been shown to be effective as disease drugs involving the binding of CD40 and CD40 ligand. However, since there is a report that CD40L is expressed in activated platelets (see Non-Patent Document 32), it has been reported that when anti-CD40L antibody is used as a therapeutic agent, there is a risk of causing thrombus. (See Non-Patent Document 33). From this point of view, it can be expected that an antibody against CD40 is superior in safety to an antibody therapeutic agent that inhibits the binding between CD40 and its ligand rather than an anti-CD40L antibody. The anti-CD40 antibody is required to suppress the binding of CD40L to CD40, and the antibody itself does not activate CD40.

  A report on an antibody that specifically binds to human CD40, suppresses the binding of CD40L to CD40, and does not activate CD40 has been reported in spite of a huge amount of research in the past. There is only one example of the named mouse anti-human CD40 antibody (see Non-Patent Document 34). Furthermore, it has not been known whether an antibody showing neutralizing activity against B cells can neutralize the action of CD40 ligand on DC cells as well. Furthermore, it has been reported that the action of a biotinylated anti-mouse CD40 antibody is enhanced by cross-linking with avidin (see Non-Patent Document 35). The present inventor enhanced the action of soluble CD40 ligand on B cell line (Ramos cell) by using antibody (M2) against tag (FLAG), which was previously genetically engineered to soluble ligand, and neutralizing activity thereof. As a result, it was confirmed that 5D12 (ATCC No. HB-11339) showed only a slight neutralizing activity.

  The present inventor has newly found that the antagonist antibody 5D12 has an agonistic activity due to cross-linking even in the absence of CD40L. Conventionally, it has been reported that the action of a mouse CD40 antibody is enhanced by cross-linking of biotin and avidin (see Non-Patent Document 35). In addition, it was known that the anti-immunoglobulin antibody immobilized on the plate and the CD40 antibody were immobilized, the tumor cell growth inhibitory activity was increased. It was thought. However, it has not been known that an anti-immunoglobulin antibody may be added to the culture medium and the anti-CD40 antibody may be cross-linked to show agonistic activity even with an antagonistic antibody. If the antibody used for the treatment has antigenicity, an antibody that binds to the CD40 antibody is produced in the human body, and the CD40 antibody is cross-linked. The Therefore, it is considered very important to keep the antigenicity of the antibody low from the viewpoint of the safety of the therapeutic agent. It is known that humanized antibodies are immunogenic when developed as therapeutic agents by humanization technology based on the murine antibody variable region sequences. There is a risk that it may become an agonistic antibody. Even if the antigenicity is low, the anti-CD40 antibody may be cross-linked by the antibody receptor (FcR). From these points, the antagonistic antibody is a human antibody that specifically binds to CD40, suppresses the binding of CD40L, and does not activate CD40 even by cross-linking, and has a weak binding to FcR. It is considered desirable.

E. A. Clark et. Al., Proc. Natl. Acad. Sci. USA 83: 4494, 1986 I. Stamenkovic et. Al., EMBO J. 8: 1403, 1989 Caux, C., et al., J. Exp. Med., 180: 1263,1994 Shu, U., et al., Eur. J. Immunol., 25: 1125, 1995 Barrett et al., J. Immunol. 146: 1722, 1991 Gordon et al., J. Immunol. 140: 1425, 1988 Valle et al., Eur. J. Immunol. 19: 1463, 1989 Clark and Ledbetter, Proc. Natl. Acad. Sci. USA 83: 4494, 1986 Gordon et al., LEUCOCYTE TYPING III. A. J. McMicheal ed. Oxford University Press. Oxford, p.426 Paulie et al., J. Immunol. 142: 590,1989 Gordon et al., Eur. J. Immunol. 17: 1535, 1987 Jabara et al., J. Exp. Med. 172: 1861, 1990 Gascan et al., J Immunol. 147: 8,1991 Gordon and Guy, Immunol. Today 8: 339, 1987 Cairns et al., Eur. J. Immunol. 18: 349, 1988 Challa A, Allergy, 54: 576, 1999 Clark and Shu, J. Immunol. 145: 1400, 1990 Bancherauet al., Science 241: 70,1991 Liu et al., Nature 342: 929,1989 Katira, A. et. Al., LEUKOCYTE TYPING V. S. F. Schlossossman, et. Al. Eds.p.547. Oxford University Press. Oxford W. C. Flansow et. Al., LEUKOCYTE TYPING V. S. F. Schlossossman, et. Al. Eds.p. 555. Oxford University Press. Oxford J. D. Pound et.al., International Immunology, 11: 11, 1999 Z. H. Zhou et.al., Hybridoma, 18: 471 1999 Shoenberger, S.P., et.al., Nature, 480,1998 French, R.R., et.al., Nature Medicine, 5,1999 Van Den Berg, TK, et.al., Immunology, 88: 294, 1996 Funakoshi S et al., Blood, 83: 2782, 1994 Funakoshi S et al., Journal of Immunotherapy, 19,93,1996 Joseph A et al., Cancer Research, 60: 3225, 2000 Challa A et al., Allergy, 54: 576, 1999 Pound et al., International Immunology, 11: 11,1999 V. Henn et. Al., Nature 391: 591, 1998 T. Kawai et. Al., Nat. Medi. 6: 114, 2000 J. Kwekkeboom et. Al., Immunology 79: 439, 1993 Johnson et al., Eur J Immunol, 24 ,: 1835, 1994

  As described above, functional analysis of DC cells has progressed in recent years, and CD40 has started to be recognized as an important gene that controls the function of DC cells. From such a background, the present invention adopts an evaluation system using DC cells, whereby an anti-human CD40 antibody that is substantially antagonistic to human CD40 antigen on the surface of dendritic cells (DC). Alternatively, an object thereof is to provide a functional fragment thereof, and an agonistic anti-human CD40 antibody or a functional fragment thereof that is expected to have a higher therapeutic effect than a conventional anti-human CD40 antibody.

  As a result of diligent research on the production of antibodies against human CD40, the present inventors have found that novel agonistic antibodies and antagonists, which are considered to have a higher therapeutic effect on diseases, compared to conventionally known anti-CD40 antibodies. The present invention was completed by successfully producing a gonistic antibody. That is, the present invention is as follows.

(1) An antibody against human CD40 or a functional fragment thereof having at least one property selected from the following (a) to (f):
(a) Acts on dendritic cells to produce IL-12 in the presence of LPS and IFNγ
(b) Acts on dendritic cells and has higher maturation activity than G28-5 antibody
(c) B95 cell line has higher activity to promote CD95 expression than G28-5 antibody
(d) The activity of inhibiting the growth of B cell established cell line is higher than that of G28-5 antibody
(e) Inducing cell death against B cell line
(f) Does not inhibit the binding of CD40 ligand to CD40 (2) The antibody of the present invention or the functional fragment thereof is one in which maturation of dendritic cells is performed at a concentration of 20 μg / ml or less. In addition, CD95 expression is promoted at an antibody concentration of 20 μg / ml or less for B cell established cell lines. Examples of B cell established cell lines include Ramos and HS-Sulton.

(3) The antibody of the present invention or a functional fragment thereof is 100 pg / ml or more when an antibody having a concentration of 0.1 μg / ml or more is added to dendritic cells having a concentration of 1 × 10 ⁶ cells / ml. IL-12 is produced, and when an antibody having a concentration of 1 μg / ml or more is added, it produces 1000 pg / ml or more, preferably 10,000 pg / ml or more of IL-12.

(4) Further, the antibody of the present invention or a functional fragment thereof is a control in which the expression promotion of CD95 against a B cell established cell line (Ramos cell) is within the antibody concentration range of 0.01 μg / ml to 10 μg / ml. It is about 2 to 3 times more than G28-5 antibody. For example, at an antibody concentration of 0.01 μg / ml, it is about 2 to 6 times or more than the control G28-5 antibody, and at an antibody concentration of 0.1 μg / ml, it is about 2 to 7 times that of the control G28-5 antibody. When the antibody concentration is 1 μg / ml, it is about 2 to 7 times or more of the control G28-5 antibody, and at the antibody concentration of 10 μg / ml, it is about 2 to 6 times that of the control G28-5 antibody. It is more than double.

(5) Hybridoma KM302-1 (Accession No. FERM BP-7578), KM341-1-19 (Accession No. FERM BP-7759), 2105 (Accession No. FERM BP-8024) or F1-102 (Accession No. ATCC PTA-3337) The antibody or the functional fragment thereof having the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody produced by (1).

(6) the heavy chain variable region and light chain variable region of the antibody produced by hybridoma F2-103, encoded by plasmid DNAs having accession numbers ATCC PTA-3302 and ATCC PTA-3303, accession numbers ATCC PTA-3304 and The heavy chain variable region and light chain variable region of the antibody produced by hybridoma F5-77, respectively encoded by the plasmid DNA that is ATCC PTA-3305, or the plasmid DNA that is the accession numbers ATCC PTA-3306 and ATCC PTA-3307 An antibody or functional fragment thereof having the amino acid sequence of the mature part of the heavy chain variable region and light chain variable region of the antibody produced by hybridoma F5-157, respectively encoded.

(7) Heavy chain variable region and light chain variable region of the antibody produced by hybridoma KM341-1-19 shown in SEQ ID NOs: 28 and 30, respectively, and the antibody produced by hybridoma 2105 shown in SEQ ID NOs: 32 and 34, respectively The heavy chain variable region and the light chain variable region of the antibody produced by the hybridoma 110 shown in SEQ ID NOs: 36 and 38, respectively, and the hybridoma 115 shown in the heavy chain variable region and the light chain variable region of the antibody produced by the hybridoma 110, respectively. The heavy chain variable region and light chain variable region of the antibody produced by the hybridoma KM643-4-11, shown in SEQ ID NOs: 52 and 54, SEQ ID NO: 60 and The heavy chain variable region and light chain variable region of the antibody produced by hybridoma F2-103, or SEQ ID NO: Respectively 64 and 66 shown, of the heavy and light chain variable regions of an antibody produced by hybridoma F5-77, antibody or functional fragment thereof having the amino acid sequence of the mature part.

(8) heavy chain variable region and light chain variable region encoded by the nucleic acid sequence isolated from hybridoma KM341-1-19 shown in SEQ ID NOs: 27 and 29, respectively, shown in SEQ ID NOs: 31 and 33, respectively Heavy chain variable region and light chain variable region encoded by nucleic acid sequence isolated from hybridoma 2105, heavy chain variable region encoded by nucleic acid sequence isolated from hybridoma 110, shown in SEQ ID NOs: 35 and 37, respectively And a light chain variable region, a heavy chain variable region and a light chain variable region encoded by a nucleic acid sequence isolated from hybridoma 115, shown in SEQ ID NOs: 39 and 41, respectively, and a hybridoma shown in SEQ ID NOs: 51 and 53, respectively. Heavy and light chain variable regions encoded by nucleic acid sequences isolated from KM643-4-11, SEQ ID NOs: 59 and 6 A heavy chain variable region and a light chain variable region encoded by a nucleic acid sequence isolated from hybridoma F2-103, respectively, or isolated from hybridoma F5-77, shown in SEQ ID NOs: 63 and 65, respectively. An antibody having a mature amino acid sequence of a heavy chain variable region and a light chain variable region encoded by a nucleic acid sequence, or a functional fragment thereof.

(9) An antibody against human CD40 or a functional fragment thereof having at least one property selected from the following (g) to (j).
(g) Neutralize ligand action on CD40
(h) neutralizing or mitigating one or more of the effects of a ligand for CD40 on a B cell established cell line on CD40 expressing cells, and against CD40 on the B cell established cell line by cross-linking with an anti-immunoglobulin antibody Agonistic action is weaker than 5D12
(i) CD40 ligand mitigates or neutralizes the effect of increasing CD95 expression on B cell established cell lines
(j) Has an antagonistic effect on CD40 expressed in dendritic cells

(10) The antibody or functional fragment of (9) above is obtained when an antibody at a concentration of 0.1 μg / ml is added to Ramos cells at a concentration of 1 × 10 ⁶ cells / ml to which a saturated amount of CD40L-expressing cells are added. Can suppress the expression of CD95 in Ramos cells to about 10% or less of the control. When 1 μg / ml antibody is added, CD95 expression in Ramos cells is the same level as the negative control. When an antibody having a concentration of 10 μg / ml is added, CD95 expression of Ramos cells is suppressed to the same level as the negative control.

(11) The antibody of (9) or a functional fragment thereof is added in vitro to 1 × 10 ⁵ tonsil B cells supplemented with soluble CD40L (1 μg / ml) at a concentration of 0.001 μg / ml to 10 μg / ml. When an antibody is added, proliferation of tonsil B cells is suppressed by about 80 to 95% or more. For example, when an antibody having a concentration of 0.01 μg / ml to 10 μg / ml is added, proliferation of tonsillar B cells is suppressed by about 95% or more, and in particular, an antibody having a concentration of 0.001 μg / ml When added, the proliferation of tonsil B cells is suppressed by about 80% or more.

(12) the heavy chain variable region of an antibody produced by hybridoma KM281-1-10 (Accession No. FERM BP-7579), 4D11 (Accession No. FERM BP-7758) or F4-465 (Accession No. ATCC PTA-3338) and An antibody having a light chain variable region amino acid sequence or a functional fragment thereof.

(13) The heavy chain variable region and light chain variable region of the antibody produced by hybridoma KM281-1-10 shown in SEQ ID NOs: 44 and 46, respectively, and the antibody produced by hybridoma 4D11 shown in SEQ ID NOs: 48 and 50, respectively Antibody having the amino acid sequence of the mature part of the heavy chain variable region and the light chain variable region of the antibody produced by the hybridoma F4-465 shown in SEQ ID NOs: 56 and 58, respectively, Or a functional fragment thereof.

(14) heavy chain variable region and light chain variable region encoded by nucleic acid sequences isolated from hybridoma KM281-1-10 shown in SEQ ID NOs: 43 and 45, respectively, shown in SEQ ID NOs: 47 and 49, respectively Heavy chain variable region and light chain variable region of antibody produced by hybridoma 4D11, or heavy chain variable region and light chain encoded by nucleic acid sequence isolated from hybridoma F4-465 shown in SEQ ID NOs: 55 and 57, respectively An antibody having a mature region amino acid sequence of a variable region or a functional fragment thereof.
(15) Examples of the antibodies (1) to (14) or functional fragments thereof include human antibodies.
(16) A pharmaceutical composition comprising the antibody or functional fragment thereof according to any one of (1) to (15) above as an active ingredient.
(17) An immunostimulatory agent, antitumor agent or autoimmune disease therapeutic agent comprising the antibody or functional fragment thereof according to any one of (1) to (8) as an active ingredient.
(18) An immunosuppressive agent, an autoimmune disease therapeutic agent, an allergy therapeutic agent or a blood coagulation factor VIII inhibition syndrome comprising the antibody or functional fragment thereof according to any one of (9) to (14) as an active ingredient Therapeutic agent.

(19) Here, the epitope of human CD40 recognized by the monoclonal antibody of the present invention is determined by a known method such as examining the binding to an overlapping synthetic oligopeptide obtained from the primary amino acid sequence of human CD40. (Eg, Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual , 1988 Cold Spring Harbor Laboratory Press; US Patent No. 4708871). A peptide library kit by phage display (New England Biolabs) can also be used for epitope mapping. The present invention also includes an antibody or a functional fragment thereof that recognizes a novel epitope of human CD40 that recognizes an antibody produced by the above-mentioned individual hybridoma or a functional fragment thereof.

(20) The present invention further includes a nucleic acid (RNA or cDNA) encoding at least the variable region of the heavy chain and / or light chain of an antibody isolated from the individual hybridomas described above, a vector containing the nucleic acid, the nucleic acid A host cell is provided.

  According to the present invention, an antibody against CD40 is provided. The antibodies of the present invention include both those acting on CD40 in an agonistic manner and those acting on an antagonistic one, and are therefore useful as immunostimulators, immunosuppressants, etc., respectively.

  Hereinafter, the present invention will be described in detail. This specification includes PCT application PCT / US01 / 13672 (filed on April 27, 2001), Japanese patent application 2001-142482 (filed on May 11, 2001), Japan It includes the contents described in the specification and / or drawings of patent application 2001-310535 (filed October 5, 2001) and US patent application USSN 10 / 040,244 (filed October 26, 2001).

  As described below, the inventors have disclosed that the known monoclonal antibody 5D12 (ATCC No. HB-11339), which is antagonistic against CD40 on B cells, is antagonistic against CD40 on DC cells. I got the knowledge that it was not. Furthermore, the inventor has found that even if an antagonistic antibody blocks the action of CD40L, many monoclonal antibodies themselves exhibit agonistic activity by cross-linking with an anti-immunoglobulin antibody.

1. Definitions The terms used in this specification are defined as follows.
As used herein, “human CD40” refers to Clark et al. (EA Clark et. Al., Proc. Natl. Acad. Sci. USA 83: 4494, 1986) or Stamenkovic et al. (I. Stamenkovic et. Al., EMBO). J. 8: 1403, 1989), and particularly an antigen polypeptide expressed on the surface of B cells, DCs, macrophages, endothelial cells, epithelial cells, or their tumor cells. is there.
“Anti-CD40 monoclonal antibody” means any of the monoclonal antibodies against cell-expressed CD40, full-length CD40 or partial-length CD40, but more preferably binds to the extracellular portion of CD40 and binds to cells expressing CD40. It means a monoclonal antibody that provides an agonistic or antagonistic action.

Further, in the present invention, “antibody” refers to genes encoding heavy chain variable region and heavy chain constant region, and light chain variable region and light chain constant region constituting immunoglobulin (collectively referred to as “antibody gene”). It is derived from. The antibodies of the present invention include antibodies having any immunoglobulin class and isotype. The “functional fragment” of an antibody in the present invention means a part (partial fragment) of an antibody as defined above, which retains one or more actions of an antibody on an antigen, specifically F (ab ′) ₂ , Fab ′, Fab, Fv, disulfide bond FV, single chain FV (scFV), and polymers thereof (DJKing., Applications and Engineering of Monoclonal Antibodies., 1998 TJ International Ltd) .

In the present invention, “human antibody” means an antibody that is an expression product of a human-derived antibody gene.
“Agonistic” refers to the effect of promoting the binding of the ligand to CD40 expressed on the surface of cells such as B cells, tumor cells or dendritic cells, or the effect of CD40 ligand on CD40-expressing cells. The term “agonistic antibody” refers to an antibody having such an agonistic effect.

  “Antagonic” refers to the effect of inhibiting the binding of the ligand to CD40 expressed on the surface of cells such as B cells, tumor cells or dendritic cells, or the effect of CD40 ligand on CD40-expressing cells. The term “antagonistic antibody” means an antibody having such an effect.

  The “dendritic cell (DC)” referred to in the present invention is also called a dendritic leukocyte and refers to a group of cells having a strong antigen-presenting function. The dendritic cells to be used are induced by culturing CD34 positive progenitor cells contained in, for example, bone marrow, umbilical cord blood or peripheral blood. Alternatively, it can be obtained by culturing CD14 positive monocytes in peripheral blood in the presence of GM-CSF and IL-4.

  “Immmature DC” means CD14 negative, CD1a strong positive, CD83, CD86 positive, MHC class II positive DC.

  “Mature DC” means CD14 negative, CD1a positive, CD83, CD86, MHC class II strong positive.

  In the present invention, `` activate DC '' means a change caused by DC in response to stimulation by CD40, for example, mature immature DC and highly express CD80, CD86, HLA-Class II, Means to enhance the production of IL-12, or, when T cells coexist, to stimulate T cells and promote proliferation.

  In the present invention, activating B cells and B cell lines means changes caused by cells in response to stimulation by CD40, for example, causing DNA synthesis, promoting thymidine incorporation, and increasing the expression level of CD95. Say.

2. Acquisition of antibody In order to acquire the antibody of the present invention, it is preferable to immunize mice with soluble human CD40 produced and purified in recombinant form as an antigen, or a transgenic mouse cell line expressing human CD40. . The mouse used for immunization is preferably a mouse producing a human antibody (Tomizuka. Et al., Proc Natl Acad Sci USA., 2000 Vol97: 722). Rather than selecting clones that react specifically with B cells by selecting monoclonal antibodies that bind to recombinant human-produced and purified soluble human CD40, antibodies that also react with CD40 expressed in cells other than B cells It is thought that it is easy to obtain. Hybridomas are prepared using the lymph node cells and spleen cells of immunized mice using the method of Köhler and Milstein et al. (Nature., 1975 Vol. 256: 495) commonly used in the production of monoclonal antibodies. be able to.

  In addition, the binding of soluble CD40L to CD40 is analyzed using a surface plasmon resonance measuring apparatus such as BIAcore 2000 (Biacore) to select antibodies that do not compete with CD40L. In addition, antibodies that suppress cell growth inhibition of B lymphoma with antibodies alone were selected. In addition, antibodies that have the advantage of suppressing the proliferation of CD40-expressing cancer cells by acting on dendritic cells or B cells without competing with CD40L by performing antibody selection using DC as an indicator Can be produced and sorted.

  The antibody of the present invention can be obtained by culturing the obtained hybridoma. In addition, a gene encoding a human monoclonal antibody or its variable region is cloned from an antibody-producing cell such as a B cell or a hybridoma, and incorporated into an appropriate vector, which is then used as a host (eg, mammalian cell line, E. coli, yeast cell, insect cell). Recombinant antibodies produced by gene recombination technology can be prepared (PJDelves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY, P. Shepherd and C. Dean) Monoclonal Antibodies., 2000 OXFORD UNIVERSITY PRESS, JWGoding., Monoclonal Antibodies: principles and practice., 1993 ACADEMIC PRESS). Furthermore, a transgenic bovine, goat, sheep or pig in which the gene of the target antibody is incorporated into the endogenous gene is produced using a transgenic animal production technique, and the antibody gene is derived from the milk of the transgenic animal. It is also possible to obtain a large amount of monoclonal antibodies. When hybridomas are cultured in vitro, the hybridomas are grown, maintained and stored according to various characteristics such as the characteristics of the cell type to be cultured, the purpose of the test study and the culture method, and monoclonal antibodies are produced in the culture supernatant. It can be carried out using any nutrient medium that is derived from a known nutrient medium or a known basal medium, such as that used for the preparation.

3. Screening Agonistic antibodies can be selected by analysis using human B lymphoma, and antibodies that promote CD95 expression can be selected. Add antibodies to the purified DC culture and select the antibodies to be matured. Alternatively, an antibody that exhibits T cell proliferation activity in a mixed lymphocyte reaction using immature DC cells is selected. Select antibodies that are added to mature DCs and promote IL-12 production. In addition, an antibody that suppresses the growth of tumor cells expressing CD40 or has an activity of inducing cell death is selected. Competition with CD40L can be determined by using a surface plasmon resonance measuring apparatus such as BIACore based on whether the antibody inhibits the binding of soluble CD40 and soluble CD40 ligand. Or it discriminate | determines by whether the effect | action of the CD40 ligand with respect to a B cell strain is enhanced.

  Antagonistic antibodies are selected by analysis using human B lymphoma, and soluble CD40L with FLAG as a tag is added in the presence of anti-FLAG antibody, so that soluble CD40L can be added to human B lymphoma cells. It is possible to screen for antibodies that more strongly inhibit the binding of CD40 to CD40. Instead of soluble CD40L, it is also possible to use a recombinant cell expressing a large amount of CD40L on the cell surface by introducing a gene encoding CD40L. The human antibody is then cross-linked with an anti-human IgG antibody to exclude clones that activate B-lymphoma by cross-linking. Furthermore, an antibody that exhibits T cell proliferation inhibitory activity in a mixed lymphocyte reaction using purified and matured DC cells, or an antibody that suppresses IL-12 production when CD40 ligand is added to mature DC is selected.

  The antibody obtained as described above has a property considered to be useful for at least one of the following therapeutic effects.

(1) For agonistic antibodies
(a) Acts on dendritic cells to produce IL-12 in the presence of LPS (lipopolysaccharide) and IFNγ. In this case, the LPS concentration is 10 pg / ml to 10 ng / ml, and the IFNγ concentration is 10 ⁻⁴ M to 10 ⁻² M. At an antibody concentration of 1 μg / ml or higher, preferably at an antibody concentration of 0.1 μg / ml or higher, IL-12 is produced in a larger amount than the test using G28-5 antibody, which is a known agonistic anti-CD40 antibody, as a control. IL-12 of 100 pg / ml or more is produced when dendritic cells with a concentration of 1 × 10 ⁶ cells / ml are added at an antibody concentration of 0.1 μg / ml or more, or 1000 pg / ml when 1 μg / ml or more is added. IL-12 of greater than or equal to ml, preferably greater than or equal to 10000 pg / ml is produced (see Example 9, Example 13).

  (b) It has an action to bind to dendritic cells and mature dendritic cells. Moreover, when the antibody is cultured with dendritic cells at a concentration of 20 μg / ml or less, preferably 0.1 to 15 μg / ml, more preferably 5 to 15 μg / ml, the maturation activity is higher than that of the G28-5 antibody. (See Example 9).

  (c) The activity of promoting the expression of CD95 is higher than that of the G28-5 antibody against the B cell established cell line. In this case, at an antibody concentration of 10 μg / ml or more, preferably 1 μg / ml or more, more preferably 0.1 μg / ml or more, more preferably 0.01 μg / ml or more, and most preferably 0.001 μg / ml, the G28-5 antibody The activity of promoting the expression of CD95 is higher than that of the G28-5 antibody than in the test using. Regarding the activity to promote the expression of CD95, the magnification of the test using the G28-5 antibody as a control at antibody concentrations of 10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml is as follows (Table 1). .

  The enhanced expression of CD95 means that the antibody activated the B cell established cell line. Here, examples of the B cell established cell line include Ramos cells and HS-Sulton cells. Ramos cells are Burkitt's lymphoma and are model cells of human germinal center B cells. HS-Sulton cells are Burkitt lymphoma (see Example 6, Example 12).

  (d) The activity to suppress DNA synthesis, thymidine incorporation, and proliferation is higher than that of the G28-5 antibody against B cell established cell lines (Ramos cells or HS-Sulton cells). The antibody concentration in this case is at least 0.05 μg / ml, preferably 0.1 to 15 μg / ml (see Example 8).

(e) Inducing cell death against B cell established cell lines (see Example 16).
(f) Does not inhibit the binding of CD40 ligand to CD40. “Do not inhibit” means that CD40L can bind to CD40 to the same extent as in the absence of the antibody even when the antibody is bound to CD40 in advance (that is, even in the presence of the antibody). means. Either or both of the CD40 ligand and CD40 may be membrane expressed or soluble proteins (see Example 11).

  Antibodies having the above properties are produced, for example, by hybridoma KM302-1 (FERM BP-7578) and hybridoma KM341-1-19 (FERM BP-7759).

  The base sequence and amino acid sequence of the heavy chain (H chain) and light chain (L chain) variable regions of the monoclonal antibody produced by the hybridoma KM341-1-19 were determined (Example 17). The present invention provides DNA encoding at least the heavy chain variable region or the full length of the heavy chain of the monoclonal antibody produced by the hybridoma KM341-1-19, and DNA encoding the light chain variable region. The DNA includes those described in Example 17 and other DNAs that encode the same amino acid sequence due to codon degeneracy. The present invention also provides a monoclonal antibody or a functional fragment thereof defined by at least the amino acid sequence of the heavy chain variable region or the amino acid sequence of the full length of the heavy chain and the amino acid sequence of the light chain variable region disclosed in Example 17. provide.

(2) Antagonistic antibody
(g) Neutralize ligand action on CD40. Here, “ligand action” means both the action of a ligand expressed on T cells or other cells, and the action of a free ligand on CD40 (see Examples 7 and 14). thing).

  (h) Neutralizing one or more effects of a ligand for CD40 on a B cell established cell line on CD40 expressing cells, and agonistic to CD40 on the B cell established cell line by cross-linking with an anti-immunoglobulin antibody Does not show any effect. Its action is weaker than 5D12. “Influence on CD40-expressing cells” means activation of expressing cells, and means thymidine uptake and proliferation in B cells, and enhanced expression of CD95 in B cell established cell lines. In DC, it means maturation, enhanced expression of CD86 and HLA-DR, thymidine incorporation by coexisting T cells, promotion of proliferation, production of IL-12 and IL-10, and the like. Crosslinking with an anti-immunoglobulin antibody is carried out by allowing 0.1 μg / ml or more of the anti-immunoglobulin antibody to be present in the culture medium. (See Example 7)

  (i) For a B cell established cell line, cross-linked CD40L or CD40L expressed in cells alleviates or neutralizes the activity of increasing CD95 expression. CD40L, which is cross-linked with an antibody against the tag and has an enhanced action, also reduces or neutralizes the activity. When ligands for CD40 (including both free ligands and ligands expressed in specific cells) bind to CD40-expressing cells, signal transduction occurs in the cells, and finally CD95 (Fas) is put on the cell surface. To express. Accordingly, the antagonistic antibody of the present invention neutralizes CD95 expression by binding to CD40, thereby inhibiting the above signal transduction. In this case, the antibody concentration is 1 μg / ml or more, preferably 0.1 μg / ml or more (see Examples 7 and 14).

  (j) Antagonistic to CD40 on DC. That is, CD40L relaxes or neutralizes the activity of activating DC. When stimulated by a ligand on a T cell in which DC coexists, it activates the T cell and promotes thymidine incorporation. In a mixed lymphocyte reaction in which DCs and T cells derived from different individuals coexist, activation of T cells occurs when DCs and T cells interact. The antagonistic antibody of the present invention inhibits the above interaction by binding to CD40, and as a result, thymidine incorporation is suppressed. The antibody concentration in this case is at least 0.001 μg / ml, preferably 0.1 to 10 μg / ml (see Example 10).

  The above antagonistic antibodies are, for example, hybridomas KM281-1-10 (FERM BP-7579), KM281-2-10-1-2 (FERM BP-7580) (incorporated administrative agency industrial technology dated May 9, 2001) Produced by 4D11 (FERM BP-7758), Research Center for Biological Biology (1st, 1st East, 1st Street, Tsukuba, Ibaraki).

(3) The antibodies of the present invention can be converted into those of different subclasses by genetic engineering modifications well known to those skilled in the art, that is, by substituting regions that define subclasses of antibody heavy chains with regions that define other subclasses. (See, for example, EP 314161). The variable region of the heavy chain can be directly connected to the constant region of another subclass. For example, the degree of binding to the Fc receptor can be reduced by converting the antibody subclass of the present invention into IgG2 or IgG4. Specifically, Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institute of Health, Bethesda, Md. (1991)), human antibody heavy chain, EU index 118 (Ala), 119 By introducing an NheI site (GCTAGC) into the (Ser) site and digesting with this restriction enzyme, it can be connected to other subclass IgG without changing the amino acid. Furthermore, by artificially modifying the amino acid sequence of the constant region, or by binding the constant region sequence having such a sequence to the variable region of the antibody of the present invention, the degree of binding to the Fc receptor may be reduced. (Lund J., et al., J. Immunol. 1991 vol147: 2657-2662), it is also possible to increase or decrease CDC activity (Tao M., et. Al., J. Exp. Med. 1991 vol 1025-1028, Idusogie E E., et. Al., J. Immunol. 2001 vol166: 2571-5). In addition, in order to avoid the action of ADCC, CDC, etc., it is possible to select only antibodies having subclass IgG2 or IgG4 in advance. In addition, the therapeutic effect of diseases such as cancer can be further enhanced by binding a radionuclide, a bacterial toxin, a chemotherapeutic agent, a prodrug or the like to the antibody of the present invention.

4). Pharmaceutical composition A pharmaceutical composition containing a purified preparation of the antibody of the present invention is also included in the scope of the present invention. Such a pharmaceutical composition preferably includes a physiologically acceptable diluent or carrier in addition to the antibody, and may be a mixture with other agents such as other antibodies or antibiotics. Good. Suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline, phosphate buffered saline glucose solution, and buffered saline. Alternatively, the antibody may be lyophilized (freeze-dried) and reconstituted by adding a buffered aqueous solution as described above when needed. The routes of administration are oral routes and parenteral routes including intravenous, intramuscular, subcutaneous and intraperitoneal injection or delivery.

  In this case, the effective amount administered as a combination of an effective amount of the antibody of the present invention and an appropriate diluent and a pharmacologically usable carrier is 0.1 mg to 100 mg per kg body weight at a time, and 2 days From 8 weeks.

  When the pharmaceutical composition containing the antibody of the present invention is used, the agonistic antibody is an immunostimulating agent (antiviral agent, anti-infective agent), antitumor agent, autoimmune disease therapeutic agent, Multiple diseases may occur. Alternatively, a vaccine such as a cancer-specific peptide can be used in combination as an adjuvant. Antagonistic antibodies can be used as immunosuppressants at the time of organ transplantation (rejection at the time of islet transplantation or kidney transplantation, preventive or therapeutic agents for GVHD), or autoimmune diseases (eg, rheumatism, arteriosclerosis treatment). Drugs, multiple sclerosis, systemic lupus erythematosus, idiopathic thrombocytopenia, Crohn's disease, etc.), useful as therapeutic agents for allergies such as asthma, and blood coagulation factor VIII inhibition syndrome. May be combined.

  When an anti-CD40 antibody is used as a treatment for a disease involving CD40, it can be expected that an antibody having a better therapeutic effect can be obtained by selecting the antibody using the function of DC cells as an index.

  As for an agonistic antibody, it is expected that an antibody having a strong immunity enhancing effect can be obtained by selecting an antibody that can activate DC more. Furthermore, by using IL-12 production promotion of mature DC as an index, an antibody having a strong CTL-inducing action can be obtained. By CTL induction, an antibody having a high effect of removing virus-infected cells and tumor cells can be obtained. In addition, since a synergistic effect can be expected, an antibody that binds to CD40 but does not inhibit binding of CD40 ligand to CD40 is desirable. When considering cancer treatment, if there is an antibody that directly induces cell death or suppresses proliferation of cancer cells expressing CD40 and efficiently activates DC cells, a synergistic effect is expected. It is considered to be a therapeutic agent that can be used for tumors that do not develop. This antibody is considered useful as a therapeutic agent for viral infections and an antitumor agent.

  On the other hand, an antibody that specifically binds to CD40, suppresses binding of CD40L, and does not activate CD40 not only suppresses the action of the ligand on B cells but also can suppress the action on DC cells. desired. However, since the antibodies obtained so far have been taken with the effect on B cells as an index, it is of great significance to acquire antibodies with strong inhibitory action on dendritic cells and develop them as pharmaceuticals. . Furthermore, as described above, anti-CD40 antibodies are feared to have completely opposite effects due to cross-linking, and therefore antibodies that do not activate CD40 even by cross-linking are required. Monoclonal antibodies derived from non-human mammals such as mice, chimeric antibodies consisting of variable regions of mouse monoclonal antibodies and constant regions of human immunoglobulin, and humanized antibodies by CDR grafting to human CD40 reported so far Since there is a concern about having antigenicity, a human antibody is desirable as an antibody that inhibits the binding to CD40 ligand.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Production of antigen
(1) Cell
EL-4 cells are a mouse-derived T cell establishment strain and can be easily obtained (ATCC No .: TIB-39). Ramos B cells (ATCC No .: CRL-1596) and mouse anti-CD40 antibody-producing hybridomas G28-5 (HB-9110) and 5D12 (HB-11339) were purchased from ATCC.

(2) Expression and purification of antigen Using human CD40 cDNA (Genbank Accession Number: NM_001250) as a template and the following primers, PCR (95 ° C 5 seconds, 55 ° C 30 seconds, 72 ° C 30 seconds) × 20 cycles The extracellular region was amplified under conditions.
Primer 1: 5'-CCCAGATCTGTCCATCCAGAACCACCCACTGCATGCAGAG-3 '(SEQ ID NO: 1)
Primer 2: 5'-ACAAGATCTGGGCTCTACGTATCTCAGCCGATCCTGGGGAC-3 '(SEQ ID NO: 2)

  The amplified cDNA was inserted into the pFastBac vector (Gibco BRL) after the melittin signal sequence and before the human IgG1-derived mouse or mouse IgG2a-derived FC region. For production of CD40, recombinant baculovirus was prepared according to the instructions for use. Tn5 cells were infected with the recombinant virus and then cultured for 4 days. The supernatant was treated with a 0.22 nm filter, Protein G sepharose (Amersham Pharmacia) was added, and the mixture was gently shaken at 4 ° C. After one night, sepharose was transferred to a column and washed with 20 volumes of PBS. Human CD40 FC protein was eluted with 20 mM glycine buffer (pH 3.0). A vector for expressing CD40 on the cell surface was obtained from Randolph J. Noelle (Inui, S et al., EJI, 20, 1747-1753, 1990). The full-length cDNA was digested with XbaI and inserted into pCDNA3 (INVITROGEN). The vector was introduced into EL-4 cells and cultured in the presence of 0.5 mg / ml G418 (Gibco BRL) to obtain a stable expression strain. Expression of CD40 was confirmed by FACS analysis using FITC-conjugated anti-human CD40 antibody (Pharmingen).

[Example 2] Preparation of mouse for immunization The mouse used for immunization has a genetic background of homozygote for both endogenous Ig heavy chain and κ light chain disruption, and the human Ig heavy chain gene The chromosome 14 fragment (SC20) containing the locus and the human Igκ chain transgene (KCo5) are simultaneously retained. This mouse was prepared by crossing a strain A mouse having a human Ig heavy chain locus with a strain B mouse having a human Ig kappa chain transgene. Strain A is a mouse strain that is homozygous for both endogenous Ig heavy chain and kappa light chain disruption and retains a chromosome 14 fragment (SC20) capable of offspring transmission. For example, Totsuka et al. (Tomizuka. et al., Proc Natl Acad Sci USA., 2000 Vol97: 722). The following hybridomas, F2-103 and F5-77, were obtained by immunizing strain A mice. Line B is a mouse line (transgenic mouse) that is homozygous for both endogenous Ig heavy chain and κ light chain disruption and carries the human Ig κ chain transgene (KCo5), as reported by Fishwild et al. (Nat Biotechnol., 1996 Vol 14: 845).

  Individuals with human Ig heavy chain and κ light chain detected simultaneously in serum obtained by mating of male mice of line A and female mice of line B, or female mice of line A and male mice of line B (Ishida & Lonberg , IBC's 11th Antibody Engineering, Abstract 2000) was used for the following immunization experiments. The human antibody-producing mouse can be obtained from Soba Co., Ltd. by signing a contract. By immunizing the mice, the following hybridomas, KM302-1, KM341-1-19, KM643-4-11, 2053, 2105, 3821, 3822, 285, 110, 115, KM281-1-10, KM281 -2-10-1-2, KM283-5, KM292-1-24, KM225-2-56, KM341-6-9, 4D11, 5H10, 11E1, 5G3, 3811, 3411, 3417 were obtained. A chimeric mouse (Kuroiwa et. Al., Nat Biotechnol., 2000 vol 18: 1086) carrying the human antibody Lambda chain reported by Kuroiwa et al. Was also used in the following immunization experiment. Hybridoma F4-465 was obtained from this mouse.

[Example 3] Preparation of human monoclonal antibody against human CD40 Preparation of monoclonal antibody in this example was prepared according to the general method described in the introduction to monoclonal antibody experimental procedures (authored by Ando Tamie et al., Kodansha 1991). . As human CD40 as an immunogen, human CD40 human FC and CD40-expressing EL-4 cells prepared in Example 1 were used. As the immunized animal, the human antibody-producing mouse producing human immunoglobulin prepared in Example 2 was used.

  Human antibody-producing mice were immunized with CD40: hFc at 2 to 100 μg / dose per individual. The antigen solution was mixed with an equal volume of incomplete Freund's adjuvant (Sigma) except for the first time, and injected subcutaneously at several locations. Immunization was performed 3-4 times every 3 weeks from about 10 days. The first time, incomplete Freund's adjuvant (Sigma) was used. Blood was collected from the tail of the mouse, and human antibodies γ and κ against CD40 in the serum were measured using ELISA. The final immunization was performed by injecting 20 μg of CD40: Fc dissolved in PBS from the tail vein 3-4 days before excision of the spleen.

Human antibody-producing mice were immunized with mouse EL-4 cells expressing human CD40. It was suspended in PBS at 10 ⁸ EL-4 cells / ml and gently mixed with an equal amount of RIBI adjuvant pre-emulsified with PBS. Cells were immunized 3-5 times every 3 weeks from about 10 days. When no adjuvant was used, X-ray irradiation was performed at 8000 rad.

  The spleen was surgically obtained from the immunized mouse, and the collected spleen cells were mixed with mouse myeloma SP2 / 0 (ATCC No .: CRL1581) 5: 1, and polyethylene glycol 1500 (manufactured by Boehringer Mannheim) as a fusion agent A number of hybridomas were prepared by cell fusion using Hybridomas were selected in HAT-containing DMEM medium (Gibco BRL) containing 10% fetal calf serum (Fetal Calf Serum, FCS), hypoxanthine (H), aminopterin (A), and thymidine (T). This was done by culturing. Further, single clones were made by limiting dilution using HT-containing DMEM medium. The culture was performed in a 96-well microtiter plate (Becton Dickinson). Selection (screening) of a hybridoma clone producing an anti-human CD40 human monoclonal antibody was performed by measuring with an enzyme-labeled immunosorbent assay (ELISA) and a fluorescence activated cell sorter (FACS) described later in Example 4.

  Screening of human monoclonal antibody-producing hybridomas by ELISA is based on the following three types of ELISA and FACS analysis, and has human immunoglobulin gamma chain (hIgγ) and human immunoglobulin light chain kappa and is specific to human CD40 A number of hybridomas producing human monoclonal antibodies with sex were obtained. In any of the following examples including this example, as well as in the tables or figures shown as test results in the examples, each hybridoma clone producing the human anti-human CD40 monoclonal antibody of the present invention has a symbol. Named. In addition, the ones with “antibodies” next to the symbols were produced by the host cells carrying the antibodies produced by the respective hybridomas or antibody genes (full length or variable regions) isolated from the hybridomas. Recombinant antibody is meant. In addition, the name of the hybridoma clone may represent the name of an antibody within a range that is clear from the context.

The following hybridoma clones represent single clones.
Agonistic antibodies:
KM302-1, KM341-1-19, KM643-4-11, 2053, 2105, 3821, 3822, 285, 110, 115, F1-102, F2-103, F5-77, F5-157
Antagonistic antibodies:
KM281-1-10, KM281-2-10-1-2, KM283-5, KM292-1-24, KM225-2-56, KM341-6-9, 4D11, 5H10, 11E1, 5G3, 3811, 3411, 3417, F4-465

  Three of these hybridoma clones, KM 302-1, KM 281-1-10 and KM 281-2-10-1-2, were cloned on May 9, 2001, with clones KM341-1-19 and 4D11 dated September 27, 2001, and clone 2105 dated April 17, 2002, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st East, Tsukuba City, Ibaraki) Deposited internationally at address 1 center 6) under the Budapest Treaty. Plasmids with F2-103, F5-77 and F5-157 heavy and light chain variable regions dated April 19, 2001, and hybridoma clones F1-102 and F4-465 dated April 24, 2001. (American Type Culture Collection, 10801 University Blvd., Manassas, Virginia, USA) (American National Culture Collection, Virginia 20110-2209 Manassas 10801 University Boulevard) was deposited internationally under the Budapest Treaty (Table 2).

[Example 4] Screening of hybridomas Detection of monoclonal antibody having human immunoglobulin γ chain 50 μl / well of human CD40 mouse FC (1 μg / ml) prepared in Example 1 was added to a 96-well microplate for ELISA (Maxisorp, Nunc) And CD4 mouse FC were adsorbed on a microplate. Next, the supernatant was discarded, and a blocking reagent (Block Ace, Dainippon Pharmaceutical) was added to each well and incubated at room temperature for blocking. The culture supernatant (50 μl) of each hybridoma was added to each well and reacted, and then each well was washed with a phosphate buffer solution (PBS-T) containing 0.1% Tween20. Next, a solution (50 μl / well) of a goat anti-human IgG (γ) antibody labeled with peroxidase (Sigma, A0170) diluted 5,000 times with PBS-T containing 1% FBS (50 μl / well) was added to each well and incubated. did. After the microplate was washed 3 times with PBS-T, a chromogenic substrate solution (TMB, 50 μl / well, manufactured by Sumitomo Bakelite) was added to each well and incubated at room temperature for 30 minutes. Stop solution (50 μl / well) was added to each well to stop the reaction. Absorbance at a wavelength of 450 nm was measured with a microplate reader. The culture supernatant of positive wells was analyzed by FACS, the wells that stained Ramos cells were selected, and the cells were cloned by the limiting dilution method to obtain one clone of cells per well. Hκ positive was confirmed by ELISA using human CD40 mouse FC. As a result, 173 clones of anti-human CD40 antibody were obtained from 20 mice. A part thereof is shown in Table 3 (agonistic antibody) and Table 4 (antagonistic antibody). Among the agonistic antibodies, at least KM341-1-19 and 2105 did not show significant competition with ligands in competition tests using cells expressing CD40L, cells expressing CD40 and antibodies.

  The detection of the monoclonal antibody having human immunoglobulin light chain κ (Igκ) was described above except that a goat anti-human Igκ antibody labeled with peroxidase (1,000-fold dilution, 50 μl / well, manufactured by Southern Biotechnology) was used. The same as ELISA for human immunoglobulin γ chain.

  The subclass identification of each monoclonal antibody was carried out by using a sheep anti-human IgG1 antibody, a sheep anti-human IgG2 antibody, a sheep anti-human IgG3 antibody or a sheep anti-human IgG4 antibody labeled with peroxidase (diluted 2,000 times each, 50 μl / well, The Binding) The same procedure as described above for the human immunoglobulin γ chain ELISA was performed except that Site Co.) was used.

Reaction test of each monoclonal antibody against human CD40 expressing cells
FACS analysis was conducted to examine the reactivity of each monoclonal antibody against the Ramos cell line that is reported to express CD40.
The Ramos cell line was suspended in a staining buffer (SB) containing PBS containing 0.1% NaN ₃ and 2% FCS at a concentration of 2 × 10 ⁶ / ml. The cell suspension (100 μl / well) was dispensed into a 96-well round bottom plate (Becton Dickinson). Culture supernatant (50 μl) of each hybridoma was added and incubated at ice temperature for 30 minutes. As a negative control, a human IgG1 antibody against human serum albumin was used, adjusted to a concentration of 2 μg / ml in a hybridoma culture medium, added with 50 μl, and incubated at ice temperature for 15 minutes. After washing with SB, 50 μl of R-PE fluorescently labeled anti-human antibody (manufactured by Southern Biotechnology) diluted 250-fold was added and incubated at ice temperature for 15 minutes. After washing twice with SB, the suspension was suspended in 300-500 μl of FACS buffer, and the fluorescence intensity of each cell was measured with FACS (FACSort, FACScan, Becton Dickinson). As a result, antibodies having binding activity to the Ramos cell line were selected.

[Example 5] Preparation of each antibody A culture supernatant containing a monoclonal antibody was prepared by the following method.
A G28-5 antibody-producing hybridoma was obtained from ATCC (ATCC No. HB-9110). Anti-CD40 antibody-producing hybridomas were bovine insulin (5 μg / ml, Gibco BRL), human transferrin (5 μg / ml, Gibco BRL), ethanolamine (0.01 mM, Sigma), sodium selenite (2.5 × 10 ^-5 nM, Sigma) -containing eRDF medium (Kyokuto Pharmaceutical Co., Ltd.). After culturing in a spinner flask, the culture supernatant was recovered when the viable cell rate of the hybridoma reached 90%. The collected supernatant was subjected to 10 μm and 0.2 μm filters (manufactured by Gelman Science) to remove miscellaneous wastes such as hybridomas.

Purification of the anti-CD40 antibody from the culture supernatant was performed by the following method. The culture supernatant containing anti-CD40 antibody is purified using Hyper D Protein A column (manufactured by NGK) or Protein G column (Amersham Pharmacia Biotech) for purification of mouse IgG1, and PBS (-) as an adsorption buffer according to the attached instructions. Then, affinity purification was performed using 0.1 M sodium citrate buffer (pH 3) as an elution buffer. The elution fraction was adjusted to around pH 7.2 by adding 1 MTris-HCl (pH 8.0) or Na ₂ HPO ₄ solution. The prepared antibody solution was replaced with PBS (-) using a dialysis membrane (10000 cut, manufactured by Spectrum Laboratories) or SP column (Amersham Pharmacia Biotech), and a membrane filter MILLEX-GV (manufactured by MILLIPORE) with a pore size of 0.22 μm. The solution was sterilized by filtration. The concentration of the purified antibody was calculated by measuring the absorbance at 280 nm and setting 1 mg / ml as 1.45 OD.

[Example 6] Promotion of CD95 expression by anti-CD40 agonistic antibody in Ramos cells
5.0 × 10 ⁵ cells / ml Ramos cell suspension was seeded in a 96-well plate at 100 μl / well (5 × 10 ⁴ cells per well). Hybridoma culture supernatant or purified antibody was diluted to 20 μg / ml with a medium and added to a 96-well plate at a concentration of 100 μl / well. After overnight culture, the cells were collected and analyzed using R-PE labeled anti-CD95 antibody (Pharmingen NJ) using FACSCan or FACSsort (Becton Dickinson). Figure 1 shows the results. In the figure, the horizontal axis represents the expression intensity of CD95. The addition of antibody is indicated by a bold line, and the absence of addition is indicated by a thin line. The KM302-1 antibody was shown to promote CD95 expression more than the known antibody G28-5 antibody. That is, it was shown that it is a more effective agonistic.

[Example 7] Suppression of CD95 expression by anti-CD40 antagonistic antibody in Ramos cells
1.0 × 10 ⁶ cells / ml Ramos cell suspension was seeded in a 96-well plate at 50 μl / well. Hybridoma culture supernatant or purified antibody was adjusted to 2 μg / ml with a medium, and added to a 96-well plate at 100 μl / well. 4 μg / ml of soluble CD40 ligand (ALEXIS CORPORATION) and 4 μg / ml of anti-FLAG antibody (M2, Sigma) were added to the medium, and 50 μl / well was added to a 96-well plate. After overnight culture, the cells were collected and analyzed using R-PE labeled anti-CD95 antibody (Pharmingen NJ) using FACS. The results are shown in FIG. 2A, FIG. 2B, and FIG. In the figure, the horizontal axis represents the expression intensity of CD95. The antibodies produced by the hybridomas KM281-1-10, KM281-2-10-1-2, KM283-5, KM292-1-24, and KM225-2-56 suppress the expression of CD95 to the same extent as the negative control. did.

  In FIG. 3, the KM281-1-10 antibody (lower panel) suppresses CD95 expression more than the known antibody 5D12 antibody (middle panel) suppresses CD95 expression only slightly. It was shown that. That is, it was shown that it is a more effective antagonistic. This indicated that the human monoclonal antibody is an antagonistic antibody.

Effects of cross-linking with anti-immunoglobulin antibodies
1.0 × 10 ⁶ cells / ml Ramos cell suspension was seeded in a 96-well plate at 50 μl / well. Hybridoma culture supernatant or purified antibody was adjusted to 2 μg / ml with a medium, and added to a 96-well plate at 100 μl / well. Anti-human IgG antibody (Sigma, I3382) or anti-mouse IgG antibody (Biosource, AMI3401) was added to the medium to 4 μg / ml, and 50 μl / well was added to a 96-well plate. After overnight culture, the cells were collected and analyzed using R-PE labeled anti-CD95 antibody (Pharmingen NJ) using FACS. The results are shown in FIGS. In the figure, the horizontal axis represents the expression intensity of CD95. The antibody produced by each of the hybridomas KM281-1-10 and KM281-2-10-1-2 suppressed the expression of CD95. However, the antibodies produced by the hybridomas 5D12, KM283-5, KM292-1-24, and KM225-2-56 enhanced CD95 expression on the contrary.

[Example 8] Growth inhibition by anti-CD40 agonistic antibody in Ramos cells
1.0 × 10 ⁵ cells / ml Ramos cells and HS-Sulton suspension were seeded in a 96-well plate at 100 μl / well. A purified antibody or an equal mixture of soluble CD40 ligand and anti-FLAG antibody (M2) was added to the medium, and after 2 days of culture, 10 μl of 100 μCi / ml ³ H-Thymidine (Amersham Pharmacia) was added. 18 hours later, using Macro96 Harvester (SKATRON), harvested in Print Filter A (Wallac), dried, soaked well in Betap; Scint (Wallac), packaged, and activity was measured with a 1205 BETAPLATE liquid scintillation counter. Figure 6 shows the results. In the figure, the vertical axis the uptake of ³ H-thymidine by the cells, the abscissa shows the antibody or CD40L concentration in the culture solution. In both Ramos and HS-Sulton cells, when KM302-1 antibody is added, thymidine incorporation is lower than conventional G28-5 antibody and CD40L, and KM302-1 antibody is effective in tumor cell growth. It was shown to be an agonistic antibody that can be suppressed.

[Example 9] Activation of dendritic cells by CD40 agonistic antibody
(1) Materials and Methods Recombinant human IL-4 was purchased from Genzyme techne. Anti-human CD14 MACS beads were purchased from Miltenyi Biotech GmbH. Lymphoprep was purchased from Nycomed Pharma AS. The medium used for the culture was 10% heat inactivated FCS (Cell Culture Technologies), 10 mM HEPES (Sigma), 55 μM 2-mercaptoethanol (Gibco BRL), streptomycin sulfate (Meiji Seika) when DC was induced. Used. For cell washing at the time of staining, 2% FCS (Cell Culture Technologies) and 0.02% Azaid added PBS (Sigma) were used. A cell banker manufactured by Nippon Zenyaku Kogyo Co., Ltd. was used during cell freezing.

(2) Induction of monocyte-derived DC Mononuclear cells were prepared from peripheral blood by density gradient centrifugation using Lymphoprep (PBMC). This was separated into a CD14 positive fraction and a negative fraction by positive selection with anti-human CD14 MACS beads. Recombinant human GM-CSF (50 ng / ml) and recombinant human IL-4 (100 ng / ml) were added to the positive fraction and cultured in a 6-well plate in RPMI1640 medium supplemented with 10% FCS. The cell concentration at the start of the culture was 1 × 10 ⁶ / ml, and 3 ml per well. During the culture, the medium was changed once every two days. To change the medium, centrifuge 10% of the culture solution in a centrifuge tube, remove the supernatant, suspend in a new culture solution (containing cytokines, etc. at the above concentration) twice the volume of the collected culture solution, and return to each well. It was. On the 6th day of culture, cells are collected, the number of cells is calculated, suspended in the above medium at a concentration of 1 × 10 ⁶ / ml, added with anti-CD40 antibody or its isotype control and further cultured in a 24-well plate for 4 days. Was cultured. During this period, the medium was not changed (number of cells per well: 1 × 10 ⁶ cells, cell concentration: 1 × 10 ⁶ cells / ml).

(3) Cell staining and analysis by flow cytometer Anti-HLA-DR antibody (isotype control: rat IgG2a), anti-CD86 antibody (isotype control: rat IgG1), anti-CD83 antibody (isotype control: rat IgG2b) are used for staining It was. First, the antibody was added and incubated for 30 minutes at 4 ° C., then washed three times, and analyzed using a FACS Calibur manufactured by Beckton Dickinson.

(4) Enhancement of IL-12 secretion ability against mature DC After obtaining immature DC as described above, LPS (400 pg / ml) and IFNγ (10 ^-3 M) were added and mature DC was further cultured for 2 days. Obtained. To this, 10 μg / ml of anti-CD40 antibody or isotype control was added, and IL-12 production of the supernatant after 24 hours was measured by ELISA (Pharmingen).

(5) Results and Discussion FIG. 7 shows the effect of the KM302-1 antibody, which is an agonistic antibody, on DC maturation, and FIG. 8 shows the effect of mature DC on IL-12 production. The degree of maturation was compared using the G28-5 antibody as a control. As a result of observing the expression of CD86 and HLA-DR, it was shown that the KM302-1 antibody further increased the expression, that is, the maturity level was increased, as compared with the G28-5 antibody. It was also shown that IL-12 secretion was enhanced by treatment of mature DC with the KM302-1 antibody. From the above, it was shown that the KM302-1 antibody acts as an agonistic antibody against DC.

Example 10 DC-MLR
Blood collected from normal humans (peripheral blood) was centrifuged at 2000 rpm for 10 minutes, and the serum was sucked off. The blood cell fraction was resuspended in PBS and gently placed on Ficoll (Amersham Pharmacia). Centrifuge at 2000 rpm for 30 minutes to recover the PBMC part of the intermediate layer. After washing twice with PBS, it was used for specific cell separation using MACS.

Separation of monocytes for DC culture was performed using MACS (Miltenyi Biotec GmbH) according to the attached instructions. Briefly, 800 μl of MACS Buffer and 200 μl of MACS CD14 (Miltenyi Biotec GmbH, 502-01) were added to 1 × 10 ⁸ PBMCs and treated at 4 ° C. for 15 minutes. Cells were adsorbed onto a MACS LS column and washed. Cells adsorbed on the column were collected as monocytes. MACS HLA-DR (Miltenyi Biotec GmbH, 461-01) was added to the cells that were not adsorbed on the column, and HLR-DR positive cells were removed with a BS column to obtain a T cell fraction. The ratio of CD3 positive cells was measured by FACS, and the substantial number of T cells was calculated from the number of cells in the entire T cell fraction. The resulting monocytes were placed in a 6-well culture dish at a concentration of 1 × 10 ⁶ cells / ml, 100 ng / ml IL-4 (R & D system), 50 ng / ml G-CSF (KIRIN), 10% FCS (SIGMA ) Containing R0 medium (PPMI medium supplemented with β-mercaptoethanol (Gibco), HEPES (SIGMA)). After 5 days of culture, 10 ng / ml LPS (DIFCO) was added and differentiated into mature DCs.

MLR was performed by mixing T cells and mature DCs isolated from different humans. The cell ratio of T cells / DC is 1:80, respectively, and the number of T cells is 2 × 10 ⁵ cells / well. First, add antibodies to DC and react for 30 minutes. Thereafter, T cells were added and cultured for 4 days, and then 10 μl of 100 μCi / ml ³ H-Thymidine (Amersham Pharmacia) was added. After 14 hours, using Macro96 Harvester (SKATRON), harvested on Print Filter A (Wallac), dried, soaked well in Betap; Scint (Wallac), and after packaging, activity was measured with a 1205 BETAPLATE liquid scintillation counter. In addition, MLR using immature DC was performed by mixing T cells and mature DC separated from different humans. The cell ratio of T cells / DC was set to 1:40, respectively. The results are shown in FIGS. The addition of KM281-1-10 antibody showed that thymidine incorporation was low and MLR was suppressed. Further, in FIG. 10, it was shown that the KM283-5 and 5D12 antibodies cannot suppress DC-MLR, that is, only the KM281-1-10 antibody is an antagonistic antibody that neutralizes the action of CD40 ligand on DC. . Furthermore, FIG. 11 shows the results of examining the effect of the antibody KM302-1 antibody, which is an agonistic antibody, on MLR using immature DC. Activation of DC promotes interaction with T cells and increases thymidine uptake. This showed that KM302-1 is an agonistic antibody that acts on immature DC.

[Example 11] Effect of CD40 antibody on binding of CD40L to CD40
BIAcore 2000 (Biacore) was used to bind the anti-CD40 antibody to immobilized CD40 human FC, and then the change in the amount of soluble CD40L bound to CD40 was measured. Soluble CD40 human FC was immobilized on a CM chip (CM5, Biacore) according to the instructions attached to the device. Then 25 (μg / ml) anti-CD40 antibody was added and allowed to bind to CD40. Further, 10 (μg / ml) soluble CD40L was added and bound. The difference in the amount of binding before and after the addition of CD40L was measured. When control IgG was added, the amount of CD40L bound was 100 RU. After the KM302-1 antibody was added, the binding amount of CD40L was 110 RU, and after the KM283-5 antibody was added, it was 18 RU. This showed that the KM302-1 antibody did not inhibit the binding of CD40L to CD40.

[Example 12] Promotion of CD95 expression by anti-CD40 agonistic antibody in Ramos cells The purified hybridoma antibody obtained in Example 4 was analyzed according to the method of Example 6, and a clone producing an agonist antibody was selected (1 well). Per cell 5 × 10 ⁴ cells concentration 2.5 × 10 ⁵ cells / ml). The results are shown in FIG. In the figure, the horizontal axis represents the antibody concentration in the culture solution, and the vertical axis represents the average fluorescence intensity, that is, the CD95 expression intensity. It was shown that the KM341-1-19 and 2105 antibodies promote CD95 expression on Ramos cells more than the known mouse antibody G28-5 antibody at a concentration of 0.01 μg / ml or more. That is, it was shown that it is a more effective agonistic. In addition, the agonist activity to enhance CD95 expression in Ramos cells of KM341-1-19, 2105 antibody at 0.01 μg / ml was higher than that of 10 μg / ml G28-5 antibody (FIG. 12). Table 5 summarizes how many times the expression of CD95 corresponds to the addition of G28-5 antibody at each antibody concentration.

[Example 13] Activation of dendritic cells by CD40 agonistic antibody According to the method of Example 9, the effect of CD40 agonistic antibody on IL-12 and IL-10 production of mature DC was examined. IL-10 was measured by ELISA (Pharmingen). The results are shown in FIG. 13 and FIG. It was shown that IL-12 secretion was enhanced by treatment with the KM341-1-19 antibody. On the other hand, even when CD40 ligand-expressing recombinant L cells irradiated with X-rays at 5000 rads were present at 2 × 10 ⁵ cells / ml, the concentrations of IL-12 and IL-10 in the culture medium were 254 and 51 pg / ml, and KM341 -1-19 antibody was less than when 1 μg / ml was added.

  From the above, it was shown that the KM341-1-19 antibody acts as an effective agonistic antibody against DC. The agonist activity that secretes IL-12 into mature DC of KM341-1-19 antibody at 0.1 μg / ml is higher than that of G28-5 antibody at 100 μg / ml, and mature DC of KM341-1-19 antibody at 1 μg / ml The agonist activity for secreting IL-12 was more than 100 times higher than that of 100 μg / ml G28-5 antibody (FIG. 13). In addition, the agonist activity for secreting IL-10 into mature DC of KM341-1-19 antibody at 1 μg / ml was more than 10 times higher than that of 100 μg / ml G28-5 antibody (FIG. 14). In addition, the KM341-1-19 antibody has an IgG2 subclass, has a lower binding ability to Fc receptors than IgG1 and IgG3, and sensitizes NK cell killer activity and activates the complement system. From this, it is considered that the risk that the function of the CD40-expressing cell or the cell itself is decreased by the antibody is low. In addition, since it is difficult to undergo cross-linking by the Fc receptor, it is expected that the agonist activity in the body does not vary greatly due to cross-linking and the drug efficacy is easily controlled.

[Example 14] Suppression of CD95 expression by anti-CD40 antagonistic antibody in Ramos cells
1.0 × 10 ⁶ cells / ml Ramos cell suspension was seeded in a flat bottom 96-well plate at 50 μl / well (5 × 10 ⁴ cells per well). The purified antibody diluted with the medium was added to a 96-well plate at 100 μl / well. Recombinant mouse L cells expressing human CD40 ligand (Spriggs, MK et. Al., J. Exp. Med., 176: 1543, 1992; Garrone, P. et. Al., J. Exp. Med., 182 : 1265,1995, etc.) was prepared at 1.0 × 10 ⁵ cells / ml and added at 50 μl / well (5 × 10 ⁴ Ramos cells per well, cell concentration 2.5 × 10 ⁵ cells / ml, 1 5 × 10 ³ mouse L cells per well, cell concentration 2.5 × 10 ⁴ cells / ml). After overnight culture, cells were collected and analyzed using R-PE labeled anti-CD95 antibody using FACS. FIG. 15 shows the results. In the figure, the vertical axis represents the average fluorescence intensity, that is, the expression intensity of CD95. Compared to the 5D12 antibody, which is a known antibody, only slightly suppressed, the 4D11 antibody suppressed the expression of CD95 to the same extent as the negative control in which no CD40L-expressing cells were added even at a concentration of 0.1 μg / ml. At a concentration of 1 μg / ml, 4D11, F4-465, and KM281-1-10 suppressed the expression of CD95 to the same extent as the negative control without addition of CD40L-expressing cells. This indicated that the 4D11, F4-465, and KM281-1-10 antibodies are more effective antagonistic antibodies. Table 6 shows the relative values of the average fluorescence intensity at each antibody concentration, with 100 as the control to which no antagonistic antibody was added.

[Example 15] Antitumor effect in Ramos cell transplantation model with anti-CD40 agonistic antibody Anti-Asialo GM1 antibody was intravenously injected into CB17 / Icr-scidJc1 mice (CLEA Japan) 5 weeks old. One day later, 5 × 10 ⁶ Ramos cells were injected intravenously per mouse as tumor cells. One day later, KM302-1 antibody or anti-human albumin human IgG antibody was injected intravenously as a negative control. The doses of KM302-1 antibody were 1, 10, 100 μg per mouse, and 100 μg of negative control antibody was administered once to each of 5 mice. The results are shown in FIG. In the negative control administration group, all died 34 days after transplantation, whereas in the group administered with 10 μg and 100 μg of KM302-1 antibody, all 5 animals survived, confirming the antitumor effect of KM302-1 antibody. . The KM302-1 antibody is an IgG4 subclass. Despite the weak activation of the antibody-dependent cytotoxicity mechanism (ADCC) and the complement system via Fc receptors, a single dose of 10 μg prolongs the survival of cancer-bearing mice. It was observed.

[Example 16] Inhibition of Ramos cell proliferation by anti-CD40 agonistic antibody
Distribute 100 μl of Ramos cell suspension prepared in RPMI1640 medium supplemented with 10% FBS to 1 × 10 ⁴ cells / ml into a 96-well plate, and prepare KM341-1-19 antibody or soluble ligand solution prepared using 20 μg / ml medium. Was added. The soluble ligand coexisted with anti-FLAG antibody (M2) at the same concentration as the ligand (concentration in the reaction solution was 10 μg / ml) to enhance the activity. After culturing for 5 days, 20 μl of MTS reagent (Promega) was added to each well and allowed to react for 2 to 3 hours. The difference in absorbance with a well containing only medium containing no cells or antibodies at a wavelength of 490 nm was measured to determine the number of surviving cells. Similarly, the G28-5 antibody and growth inhibitory action were compared using a 96-well U-bottom plate. KM341-1-19 antibody or G28-5 antibody prepared using a medium was added to 2 μg / ml. The results are shown in FIG. Dead cells were observed in the wells to which the KM341-1-19 antibody was added, and the number of cells was significantly less than in the wells to which the G28-5 antibody or ligand was added, the absorbance was low, and the growth of tumor cells was suppressed, Cell death was shown to be induced.

[Example 17] cDNA cloning of antibody gene
Cultivate hybridomas producing KM341-1-19, 2105, 110, 115, KM281-1-10, 4D11, KM643-4-11, F4-465, F2-103, F5-77 antibodies and centrifuge the cells collected. TRIZOL (Gibco BRL) was added to this, and TotalRNA was extracted according to the instruction manual. Cloning of the variable region of the antibody cDNA was performed using CLONTECH SMART RACE cDNA amplification Kit according to the attached instructions. 1 ^st Strand cDNA was prepared using 5 μg of total RNA as a template. KM341-1-19, 2105, 110, 115, KM281-1-10, 4D11, KM643-4-11, F2-103, F5-77 For heavy chain (H chain) amplification, Takara's Z-Taq The cycle of 98 ° C for 1 second and 68 ° C for 30 seconds was repeated 30 times with UMP and hh6 primer. Further, 1 μl of this reaction solution was used as a template, and a cycle of 98 ° C. for 1 second and 68 ° C. for 30 seconds was repeated 20 times using NNUM and hh3 primers. For amplification of F4-465 heavy chain, UMP and hh2 primers were used, and Advantage 2 PCR kit (Clonthech, cat # 1910) was used at 94 ° C for 5 seconds, 72 ° C for 3 minutes, 5 cycles, 94 ° C for 5 seconds, 70 The test was carried out at 0 ° C., 72 ° C. 3 minutes, 5 cycles, 94 ° C. 5 seconds, 68 ° C. 10 seconds, 72 ° C. 3 minutes, 25 cycles.
hh6 primer: 5'-GGT CCG GGA GAT CAT GAG GGT GTC CTT-3 '(SEQ ID NO: 3)
hh3 primer: 5'-GTG CAC GCC GCT GGT CAG GGC GCC TG-3 '(SEQ ID NO: 4)
hh2 primer: 5'-GCT GGA GGG CAC GGT CAC CAC GCT G-3 '(SEQ ID NO: 5)

Thereafter, the amplified PCR product was purified with a PCR purification kit (QIAGEN), and the nucleotide sequence was determined using hh4 as a primer. Alternatively, it was subcloned into PCRScript (Stratagene, Lajolla, CA) or PCR-Blunt (Invitrogene, Carlsbad, CA) and sequenced. Based on the sequence information, antibody heavy chain specific primers were synthesized. KM341-1-19 for 341H primer, 2105 for 2105Hsal primer, 110,115 for 110Hsal primer, KM281-1-10 for 281Hsal primer, 4D11 for 4D11Sal primer, KM643-4-11 In this case, 643Hsal primer was synthesized. In the case of F4-465, an H11-9 5 'primer was synthesized. In the case of F2-103, an F2-103H primer was synthesized. In the case of F5-77, an F5-77H primer was synthesized. CDNA was amplified from 1 ^st Strand cDNA using antibody heavy chain specific primer and hh4, and the sequence from the reverse direction was determined using antibody specific primer as a template.
hh4 primer: 5'-GGTGCCAGGGGGAAGACCGATGG-3 '(SEQ ID NO: 6)
341H primer: 5'-atatgtcgacGCTGAATTCTGGCTGACCAGGGCAG-3 '(SEQ ID NO: 7)
2105Hsal: atatgtcgacTCCCAGGTGTTTCCATTCAGTGATCAG (SEQ ID NO: 8)
110Hsal: atatgtcgacTTCCATTCGGTGATCAGCACTGAACAC (SEQ ID NO: 9)
281Hsal: atatgtcgacTTTGAGAGTCCTGGACCTCCTGTG (SEQ ID NO: 10)
4D11Sal: atatgtcgacGAGTCATGGATCTCATGTGCAAG (SEQ ID NO: 11)
643Hsal: atatgtcgacCCAGGGCAGTCACCAGAGCTCCAGAC (SEQ ID NO: 12)
H11-9 5 ': -ACC GTG TCG ACT ACG CGG GAG TGA CT (SEQ ID NO: 13)
F2-103H: accgtgtcgacgctgatcaggactgcaca (SEQ ID NO: 14)
F5-77H: accgtgtcgacggtgatcaggactgaacag (SEQ ID NO: 15)

  KM341-1-19, 2105, 110, 115, KM281-1-10, 4D11, KM643-4-11, F2-103, F5-77 light chain (L chain) using UMP and hk2 primer, Amplification was performed by repeating 30 cycles of 98 ° C. for 1 second and 68 ° C. for 30 seconds. The light chain of F4-465 was amplified by repeating 30 cycles of 98 ° C for 1 second and 68 ° C for 30 seconds using UMP and hL2 primers. The amplified PCR product was purified using a PCR purification kit, and the nucleotide sequence was determined using hk6 or hL2 primers. Based on this sequence, a light chain specific primer was synthesized. KM341-1-19 for 341K primer, 2105 for 2053KBgl primer, 110,115 for 110KBgl primer, KM281-1-10 for 281KBgl primer, 4D11 for 4D11KBgl, KM643-4-11 For 643-KBgl primer, Lamda 5 'primer for F4-465 and F2-103K primer for F-103 and F5-77.

In the case of 341-1-19, 110, 115, KM643-4-11, KM281-1-10, 4D11, 2105, amplify cDNA from 1 ^st Strand cDNA using light chain specific primer and hk6 primer, Using this as a template, the sequence from both directions was determined. F4-465, F2-103, and F5-77 were subcloned into PCRScript (Stratagene, Lajolla, CA) or PCR-Blunt (Invitrogene, Carlsbad, CA) and sequenced.
hk2 primer: 5'-GTT GAA GCT CTT TGT GAC GGG CGA GC-3 '(SEQ ID NO: 16)
hL2 primer: 5'-TCT TCT CCA CGG TGC TCC CTT CAT-3 '(SEQ ID NO: 17)
341K primer: 5'-atatagatctGAACTGCTCAGTTAGGACCCAGAGG-3 '(SEQ ID NO: 18)
2053KBgl: atatagatctCGCGGGGAAGGAGACTGCTCAGTT (SEQ ID NO: 19)
110KBgl: atatagatctAGTCAGACCCAGTCAGGACACAGC (SEQ ID NO: 20)
281KBgl: atatagatctGAGCTGCTCAGTTAGGACCCAGAGGG (SEQ ID NO: 21)
4D11KBgl: atatagatctTAAGCAAGTGTAACAACTCAGAGTAC (SEQ ID NO: 22)
643KBgl: atatagatctGAGGAACTGCTCAGTTAGGACCCAGAGG (SEQ ID NO: 23)
Lamda 5 ':-AACTCCAGATCTGCCTCAGGAAGCAGCATC (SEQ ID NO: 24)
F2-103K: aactccagatctagggcaagcagtggtaac (SEQ ID NO: 25)
hk6 primer: 5'-TGGCGGGAAGATGAAGACAGATGGTG-3 '(SEQ ID NO: 26)

The full length of the H chain of 341-1-19, the DNA encoding the L chain variable region, and the amino acid sequences of the H chain and L chain are shown below.
The translation start point of H chain DNA is an ATG codon starting from the 50th adenine (A) from the 5 ′ end of SEQ ID NO: 27, and the stop codon is TGA starting from the 1472th thymine (T). The boundary between the antibody variable region and the constant region is located between the 493rd adenine (A) and the 494th guanine (G) from the 5 ′ end. In the amino acid sequence, the H chain variable region is from the N-terminal of SEQ ID NO: 28 to the 148th serine (S) residue, and the 149th alanine (A) and subsequent are the constant regions. By the gene sequence prediction software (Signal P ver. 2), the signal sequence of the H chain was predicted from the N-terminus of SEQ ID NO: 28 to the 20th serine (S). The N-terminus of the mature body is considered to be the 21st glutamine (Q) of SEQ ID NO: 28.

  The translation start point of the L chain DNA is an ATG codon starting from the 29th A from the 5 ′ end of SEQ ID NO: 29, and the variable region is from the 5 ′ end to the 400th adenine (A). In the amino acid sequence, the variable region is from the N-terminus of SEQ ID NO: 30 to the 124th lysine (K). According to N-terminal analysis of the purified L chain protein, the signal sequence of the L chain is from the N terminus of SEQ ID NO: 30 to the 20th glycine (G), and the mature N terminus is the 21st glutamic acid of SEQ ID NO: 30. It became clear that (E).

341-1-19 H chain (SEQ ID NO: 27):
GTCGACGCTGAATTCTGGCTGACCAGGGCAGCCACCAGAGCTCCAGACAATGTCTGTCTCCTTCCTCATCTTCCTGCCCGTGCTGGGCCTCCCATGGGGTGTCCTGTCACAGGTCCAACTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTACTTGGAACTGGATCAGGCAGTCCCCATCGAGAGACCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATCGTGATTATGTAGGATCTGTGAAAAGTCGAATAATCATCAACCCAGACACATCCAACAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTATATATTACTGTACAAGAGCACAGTGGCTGGGAGGGGATTACCCCTACTACTACAGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG CCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCAGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGGATCC

341-1-19 H chain amino acid sequence (SEQ ID NO: 28)
MSVSFLIFLPVLGLPWGVLSQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRDLEWLGRTYYRSKWYRDYVGSVKSRIIINPDTSNNQFSLQLNSVTPEDTAIYYCTRAQWLGGDYPYYYSMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

341-1-19 L chain (SEQ ID NO: 29):
ACTGCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACG

341-1-19 L chain amino acid sequence (SEQ ID NO: 30)
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNTFGPGTKVDIKRT

  The DNAs encoding 2105 H chain variable region and L chain variable region, and the amino acid sequences of H chain and L chain are shown below.

  The translation start point of the H chain DNA is an ATG codon starting from the 70th adenine (A) from the 5 ′ end of SEQ ID NO: 31, and the boundary between the antibody variable region and the constant region is the 495th adenine (A ) And 496th guanine (G). In the amino acid sequence, the heavy chain variable region extends from the N-terminus of SEQ ID NO: 32 to the 142nd serine (S) residue, and the 149th alanine (A) and subsequent residues are constant regions. The signal sequence of the H chain was predicted from the N-terminus of SEQ ID NO: 32 to the 19th cystein (C) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 20th Glutamic acid (E) of SEQ ID NO: 32.

  The translation start point of the L chain DNA is an ATG codon starting from the 28th A from the 5 ′ end of SEQ ID NO: 33, and the variable region is from the 5 ′ end to the 405th adenine (A). In the amino acid sequence, the variable region is from the N-terminus of SEQ ID NO: 34 to the 126th lysine (K). The signal sequence of the L chain was predicted from the N-terminus of SEQ ID NO: 34 to the 20th Glycine (G) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 21st Glutamic acid (E) of SEQ ID NO: 34.

2105 H chain (SEQ ID NO: 31)
CTGAACACAGACCCGTCGACTCCCAGGTGTTTCCATTCAGTGATCAGCACTGAACACAGAGGACTCACCATGGAGTTGGGACTGAGCTGGATTTTCCTTTTGGCTATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCTTGGTGCATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAGAGATAGGCTATTTCGGGGAGTTAGGTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGG

2105 H chain amino acid sequence (SEQ ID NO: 32)
MELGLSWIFLLAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSLVHADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDRLFRGVRYYGMDVWGQGTTVTVSSASTK

2105 L chain (SEQ ID NO: 33)
CTGCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCCACTGGCTCACTTTCGGCGGGGGGACCAAGGTGGAGATCAAACGTACGGTG

2105 L chain amino acid sequence (SEQ ID NO: 34)
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSHWLTFGGGTKVEIKRTV

  The DNA encoding 110 H chain variable regions and L chain variable regions, and the amino acid sequences of H chain and L chain are shown below, respectively.

  The translation start point of the H chain DNA is an ATG codon starting from the 60th adenine (A) from the 5 ′ end of SEQ ID NO: 35, and the boundary between the antibody variable region and the constant region is the 479th adenine (A ) And 480th guanine (G). In the amino acid sequence, the heavy chain variable region is from the N-terminus of SEQ ID NO: 36 to the 140th serine (S) residue, and the 141st alanine (A) and subsequent are the constant regions. With the gene sequence prediction software (Signal P ver. 2), the signal sequence of the H chain was predicted from the N-terminus of SEQ ID NO: 36 to the 19th Cystein (C). The N-terminus of the mature body is considered to be the 20th glutamine (Q) of SEQ ID NO: 36.

  The translation start point of the L chain DNA is an ATG codon starting from the 35th A from the 5 ′ end of SEQ ID NO: 37, and the variable region is from the 5 ′ end to the 421st adenine (A). In the amino acid sequence, the variable region is from the N-terminus of SEQ ID NO: 38 to the 129th lysine (K). The signal sequence of the L chain was predicted from the N-terminus of SEQ ID NO: 38 to the 22nd Cystein (C) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 23rd valine (V) of SEQ ID NO: 38.

110 H chain (SEQ ID NO: 35)
CTGAACACAGACCCGTCGACTTCCATTCGGTGATCAGCACTGAACACAGAGGACTCACCATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTATTAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGCTACAATATTTTGACTGGTTATTTTGGCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGG

110 H chain amino acid sequence (SEQ ID NO: 36)
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGYNILTGYFGYWGQGTLVTVSSASTK

110 L chain (SEQ ID NO: 37)
TCACAGATCTAGTCAGACCCAGTCAGGACACAGCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGTCATCTGGATGACCCAGTCTCCATCCTTACTCTCTGCATCTACAGGAGACAGAGTCACCATCAGTTGTCGGATGAGTCAGGGCATTAGCAGTGATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTGAGCTCCTGATCTCTGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCTGCCTGCAGTCTGAAGATTTTGCAACTTATTACTGTCAACAGTATTATAGTTTTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACG

110 L chain amino acid sequence (SEQ ID NO: 38)
MDMRVPAQLLGLLLLWLPGARCVIWMTQSPSLLSASTGDRVTISCRMSQGISSDLAWYQQKPGKAPELLISAASTLQSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQYYSFPWTFGQGTKVEIKRT

  The DNAs encoding 115 H chain variable regions and L chain variable regions, and the amino acid sequences of H chain and L chain are shown below, respectively.

  The translation start point of the H chain DNA is an ATG codon starting from the 60th adenine (A) from the 5 ′ end of SEQ ID NO: 39, and the boundary between the antibody variable region and the constant region is the 479th adenine (A ) And 480th guanine (G). In the amino acid sequence, the H chain variable region is from the N-terminus of SEQ ID NO: 40 to the 140th serine (S) residue, and the 141st alanine (A) and subsequent are the constant regions. The signal sequence of the H chain was predicted from the N-terminus of SEQ ID NO: 40 to the 19th Cystein (C) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 20th glutamine (Q) of SEQ ID NO: 40.

  The translation start point of the L chain DNA is an ATG codon starting from the 35th A from the 5 ′ end of SEQ ID NO: 41, and the variable region is from the 5 ′ end to the 421th adenine (A). In the amino acid sequence, the variable region is from the N-terminus of SEQ ID NO: 42 to the 129th lysine (K). The signal sequence of the L chain was predicted from the N-terminus of SEQ ID NO: 42 to the 22nd Cystein (C) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 23rd valine (V) of SEQ ID NO: 42.

115 H chain (SEQ ID NO: 39)
CTGAACACAGACCCGTCGACTTCCATTCGGTGATCAGCACTGAACACAGAGGACTCACCATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGAATGATGGAAGTATTAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGCTACAATATTTTGACTGGTTATTTTGGCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGG

115 H chain amino acid sequence (SEQ ID NO: 40)
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWNDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGYNILTGYFGYWGQGTLVTVSSASTK

115 L chain (SEQ ID NO: 41)
TCACAGATCTAGTCAGACCCAGTCAGGACACAGCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGTCATCTGGATGACCCAGTCTCCATCCTTACTCTCTGCATCTACAGGAGACAGAGTCACCATCAGTTGTCGGATGAGTCAGGGCATTAGCAGTGATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTGAGCTCCTGATCTCTGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCTGCCTGCAGTCTGAAGATTTTGCAACTTATTACTGTCAACAGTATTATAGTTTTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACG

115 L chain amino acid sequence (SEQ ID NO: 42)
MDMRVPAQLLGLLLLWLPGARCVIWMTQSPSLLSASTGDRVTISCRMSQGISSDLAWYQQKPGKAPELLISAASTLQSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQYYSFPWTFGQGTKVEIKRT

  The DNA encoding the H chain variable region and the L chain variable region of 281-1-10 and the amino acid sequences of the H chain and L chain are shown below.

  The translation start point of the H chain DNA is an ATG codon starting from the 52nd adenine (A) of SEQ ID NO: 43, and the boundary between the antibody variable region and the constant region is the 468th adenine (A ) And 469th guanine (G). In the amino acid sequence, the H chain variable region extends from the N-terminus of SEQ ID NO: 44 to the 139th serine (S) residue, and the 140th alanine (A) and thereafter are constant regions. The signal sequence of the H chain was predicted from the N-terminus of SEQ ID NO: 44 to the 19th serine (S) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 20th glutamine (Q) of SEQ ID NO: 44.

  The translation start point of the L chain DNA is an ATG codon starting from the 41st A from the 5 ′ end of SEQ ID NO: 45, and the variable region is from the 5 ′ end to the 424th adenine (A). In the amino acid sequence, the variable region is from the N-terminus of SEQ ID NO: 46 to the lysine (K) at the 128th position. The signal sequence of the L chain was predicted from the N-terminus of SEQ ID NO: 46 to the 20th Glycine (G) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 21st Glutamic acid (E) of SEQ ID NO: 46.

281-1-10 H chain (SEQ ID NO: 43)
CTGAACACAGACCCGTCGACTTTGAGAGTCCTGGACCTCCTGTGCAAGAACATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTGGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAATCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAATTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGCCCCCTTGCACGGTGACTACAAATGGTTCCACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGG

281-1-10 H chain amino acid sequence (SEQ ID NO: 44)
MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSISGYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARAPLHGDYKWFHPWGQGTLVTVSSASTK

281-1-10 L chain (SEQ ID NO: 45)
TCACAGATCTGAGCTGCTCAGTTAGGACCCAGAGGGAACCATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAACGTACG

281-1-10 L chain amino acid sequence (SEQ ID NO: 46)
METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRT

  The DNAs encoding 4D11 H chain variable region and L chain variable region, and the amino acid sequences of H chain and L chain are shown below.

  The translation start point of the H chain DNA is the ATG codon starting from the 16th adenine (A) from the 5 ′ end of SEQ ID NO: 47, and the boundary between the antibody variable region and the constant region is the 456th adenine (A ) And 457th guanine (G). In the amino acid sequence, the H chain variable region is from the N-terminal of SEQ ID NO: 48 to the 147th serine (S) residue, and the 148th alanine (A) and subsequent are the constant regions. The signal sequence of the H chain was predicted from the N-terminus of SEQ ID NO: 48 to the 26th serine (S) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 27th glutamine (Q) of SEQ ID NO: 48.

  The translation start point of the L chain DNA is an ATG codon starting from the 59th A from the 5 ′ end of SEQ ID NO: 49, and the variable region is from the 5 ′ end to the 442nd adenine (A). In the amino acid sequence, the variable region is from the N-terminus of SEQ ID NO: 50 to the 128th lysine (K). With the gene sequence prediction software (Signal P ver. 2), the signal sequence of the L chain was predicted from the N-terminus of SEQ ID NO: 50 to the 22nd Cystein (C). The N-terminus of the mature body is considered to be the 23rd Alanine (A) of SEQ ID NO: 50.

4D11 H chain (SEQ ID NO: 47)
ATATGTCGACGAGTCATGGATCTCATGTGCAAGAAAATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTACTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGCGGCTCCATCAGCAGTCCTGGTTACTACGGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATAAAAGTGGGAGCACCTACCACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTACGAGACCTGTAGTACGATATTTTGGGTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGC

4D11 H chain amino acid sequence (SEQ ID NO: 48)
MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGPGLLKPSETLSLTCTVSGGSISSPGYYGGWIRQPPGKGLEWIGSIYKSGSTYHNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCTRPVVRYFGWFDPWGQGTLVTVSSAS

4D11 L chain (SEQ ID NO: 49)
AGATCTTAAGCAAGTGTAACAACTCAGAGTACGCGGGGAGACCCACTCAGGACACAGCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAATTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACG

4D11 L chain amino acid sequence (SEQ ID NO: 50)
MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPTFGQGTKVEIKRT

  The DNAs encoding the H chain variable region and L chain variable region of KM643-4-11 and the amino acid sequences of the H chain and L chain are shown below.

  The translation start point of the H chain DNA is the ATG codon starting from the first adenine (A) from the 5 ′ end of SEQ ID NO: 51, and the boundary between the antibody variable region and the constant region is the 447th adenine (A ) And 448th guanine (G). In the amino acid sequence, the H chain variable region is from the N-terminal of SEQ ID NO: 52 to the 149th serine (S) residue, and the 150th alanine (A) and subsequent are the constant regions. With the gene sequence prediction software (Signal P ver. 2), the signal sequence of the H chain was predicted from the N-terminus of SEQ ID NO: 52 to the 20th serine (S). The N-terminus of the mature body is considered to be the 21st glutamine (Q) of SEQ ID NO: 52.

  The translation start point of the L chain DNA is an ATG codon starting from the 38th A from the 5 ′ end of SEQ ID NO: 53, and the variable region is from the 5 ′ end to the 409th adenine (A). In the amino acid sequence, the variable region is from the N-terminus of SEQ ID NO: 54 to the 124th lysine (K). The signal sequence of the L chain was predicted from the N-terminus of SEQ ID NO: 54 to the 20th Glycine (G) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 21st Glutamic acid (E) of SEQ ID NO: 54.

KM643-4-11 H chain (SEQ ID NO: 51)
ATGTCTGTCTCCTTCCTCATCTTCCTGCCCGTGCTGGGCCTCCCATGGGGTGTCCTGTCACAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCATTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAAAGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACCCCCGAGGACACGGCTGTGTATTACTGTGCAAGAGGGTATTACTATGGTTCGGGGAGCTATCCCTACTACTACCAAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGC

KM643-4-11 H chain amino acid sequence (SEQ ID NO: 52)
MSVSFLIFLPVLGLPWGVLSQVQLQQSGPGLVKPSQTLSFTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYKDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGYYYGSGSYPYYYQMDVWGQGTTVTVSSAS

KM643-4-11 L chain (SEQ ID NO: 53)
AATTGAGGAACTGCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGTGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAAC

KM643-4-11 L chain amino acid sequence (SEQ ID NO: 54)
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGESATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNTFGGGTKVEIKR

  The DNAs encoding F4-465 H chain variable region and L chain variable region, and the amino acid sequences of H chain and L chain are shown below.

  The translation start point of H chain DNA is the ATG codon starting from the 47th adenine (A) of SEQ ID NO: 55, and the boundary between the antibody variable region and the constant region is the 484th adenine (A ) And 445th guanine (G). In the amino acid sequence, the heavy chain variable region extends from the N-terminus of SEQ ID NO: 56 to the 146th serine (S) residue, and the 147th alanine (A) and subsequent residues are constant regions. The signal sequence of the H chain was predicted from the N-terminus of SEQ ID NO: 56 to the 19th serine (S) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 20th glutamine (Q) of SEQ ID NO: 56.

  The translation start point of the L-strand DNA is an ATG codon starting from the 81st A from the 5 'end of SEQ ID NO: 57, and the variable region is from the 5' end to the 440th (C). In the amino acid sequence, the variable region is from the N-terminus of SEQ ID NO: 58 to the 120th Threonine (T). The signal sequence of the L chain was predicted from the N-terminus of SEQ ID NO: 58 to the 19th Alanine (G) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 20th Serine (S) of SEQ ID NO: 58.

F4-465 H chain (SEQ ID NO: 55)
CTGAACACAGACCCGTCGACTACGCGGGAGACCACAGCTCCACACCATGGACTGGACCTGGAGGATCCTATTCTTGGTGGCAGCAGCAACAGGTGCCCACTCCCAGGTGCAGCTGGTGCAATCTGGGTCTGAGTTGAAGAAGCCTGGGGCCTCAGTGAAGGTCCCCTGCAAGGCTTCTGGATACACCTTCACTAGCTATGCTATGAATTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACACCAACACTGGGAACCCAACGTATGCCCAGGGCTTCACAGGACGGTTTGTCTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTGCAGATCAGCAGCCTAAAGGCTGAGGACACTGCCGTGTATTACTGTGCGAGAGAGGTAGTACCAGTTGCTATGAGGGTAACTCACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAA

F4-465 H chain amino acid sequence (SEQ ID NO: 56)
MDWTWRILFLVAAATGAHSQVQLVQSGSELKKPGASVKVPCKASGYTFTSYAMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAREVVPVAMRVTHYYYGMDVWGQGTTVTVSSAST

F4-465 L chain (SEQ ID NO: 57)
CTGGGTACGGTAACCGTCAGATCGCCTGGAGACGCCATCACAGATCTGCCTCAGGAAGCAGCATCGGAGGTGCCTCAGCCATGGCATGGATCCCTCTCTTCCTCGGCGTCCTTGTTTACTGCACAGGATCCGTGGCCTCCTATGAGCTGACTCAGCCACCCTCAGTGTCCGTGGCCCCAGGACAGACAGCCAGCATCACCTGTTCTGGAGATAAATTGGGGGATAATTTTACTTGCTGGTATCAGCAGAAGCCAGGCCAGTCCCCTGTGCTGGTCATCTTTCAGGATTGGAAGCGGCGCCCAGGGATCCCTGCGCGATTCTCTGGCTCCAAGTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTATTACTGTCAGGCGTGGGACATCAGCACTGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGAATTCAGATCCGTTAACGGTTACCAACTACCTAGACTGGATTCGTGACCAACATA

F4-465 L chain amino acid sequence (SEQ ID NO: 58)
MAWIPLFLGVLVYCTGSVASYELTQPPSVSVAPGQTASITCSGDKLGDNFTCWYQQKPGQSPVLVIFQDWKRRPGIPARFSGSKSGNTATLTISGTQAMDEADYYCQAWDISTVVFGGGTKLTVLGQPKAAPSVTLFPPSSEQQNKKATLVQSSVYPGAVTVAWQTPVSK

  The DNAs encoding the H-chain variable region and the L-chain variable region of F2-103, and the amino acid sequences of the H-chain and L-chain are shown below.

  The translation start point of the H chain DNA is an ATG codon starting from the 32nd adenine (A) of SEQ ID NO: 59, and the boundary between the antibody variable region and the constant region is the 463rd adenine (A ) And 464th guanine (G). In the amino acid sequence, the H chain variable region extends from the N-terminus of SEQ ID NO: 60 to the 144th serine (S) residue, and the 145th alanine (A) and subsequent residues are constant regions. The signal sequence of the H chain was predicted from the N-terminus of SEQ ID NO: 60 to the 19th Cystein (C) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 20th Glutamic acid (E) of SEQ ID NO: 60.

  The translation start point of the L chain DNA is an ATG codon starting from the 29th A from the 5 ′ end of SEQ ID NO: 61, and the variable region is from the 5 ′ end to the 415th adenine (A). In the amino acid sequence, the variable region is from the N-terminus of SEQ ID NO: 62 to the 129th lysine (K). The signal sequence of the L chain was predicted from the N-terminus of SEQ ID NO: 62 to the 22nd Cystein (C) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 23rd Asp (D) of SEQ ID NO: 62.

F2-103 H chain (SEQ ID NO: 59)
GCTGATCAGGACTGCACACAGAGAACTCACCATGGAGTTTGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTCAGTACCTACTGGATGCACTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGTGTGGGTCTCACGTATTAATAGTGATGGGAGTAGCACAACCTACGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGAGATAGAGTACTATGGATCGGGGAGTTATCCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCT

F2-103 H chain amino acid sequence (SEQ ID NO: 60)
MEFGLSWVFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAVSGFTFSTYWMHWVRQAPGKGLVWVSRINSDGSSTTYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARDRVLWIGELSYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTS

F2-103 L chain (SEQ ID NO: 61)
GGGGAGTCAGACCCAGTCAGGACACAGCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAAATGTGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAACTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGCTCTATAAGGCATCTGGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAACAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTCTAATAGTTATTCGTGGACGTTCGGCCACGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA

F2-103 L chain amino acid sequence (SEQ ID NO: 62)
MDMRVPAQLLGLLLLWLPGAKCDIQMTQSPSTLSASVGDRVTITCRASQSISNWLAWYQQKPGKAPKLLLYKASGLESGVPSRFSGSGSGTEFTLTINSLQPDDFATYYCQQSNSYSWTFGHGTKVEIKRTVAAPSVFIFIPSPSQLKSGTASVVCLLNQYSG

  The DNAs encoding the F5-77 H chain variable region and L chain variable region, and the amino acid sequences of the H chain and L chain are shown below.

  The translation start point of the H chain DNA is an ATG codon starting from the 100th adenine (A) from the 5 ′ end of SEQ ID NO: 63, and the boundary between the antibody variable region and the constant region is the 528th adenine (A ) And 529th guanine (G). In the amino acid sequence, the H chain variable region is from the N-terminal of SEQ ID NO: 64 to the 143rd serine (S) residue, and the 144th alanine (A) and subsequent are the constant regions. With the gene sequence prediction software (Signal P ver. 2), the signal sequence of the H chain was predicted from the N-terminus of SEQ ID NO: 64 to the 19th Cystein (C). The N-terminus of the mature body is considered to be the 20th Glutamic acid (E) of SEQ ID NO: 64.

  The translation start point of L-strand DNA is the ATG codon starting from the 59th A from the 5 ′ end of SEQ ID NO: 65, and the variable region is from the 5 ′ end to the 445th adenine (A). In the amino acid sequence, the variable region is from the N-terminus of SEQ ID NO: 66 to the 129th lysine (K). The signal sequence of the L chain was predicted from the N-terminus of SEQ ID NO: 66 to the 22nd Cystein (C) by the gene sequence prediction software (Signal P ver. 2). The N-terminus of the mature body is considered to be the 23rd Asp (D) of SEQ ID NO: 66.

F5-77 H chain (SEQ ID NO: 63)
GGTCTATATAAGCAGAGCTGGGTACGTCCTCACATTCAGTGATCAGCACTGAACACAGACCCGTCGACGGTGATCAGGACTGAACAGAGAGAACTCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGTGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGGGGGGTACTATGGTTCGGGGAGTTATGGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC

F5-77 H chain amino acid sequence (SEQ ID NO: 64)
MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGYYGSGSYGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKFP

F5-77 L chain (SEQ ID NO: 65)
CAAGCAGTGGTAACAACGCAGAGTACGCGGGGGGAGTCAGACCCAGTCAGGACACAGCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAGGTTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGGATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGGATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATTTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAGCAGTTTCCCTCGGACATTCGGCCAAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA

F5-77 L chain amino acid sequence (SEQ ID NO: 66)
MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSGSVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAGSSLQSGVPSRFSGSGFGTDFTLTISSLQPEDFATYYCQQASSFPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCTLNKAY

[Example 18] Expression of antibody protein in animal cells An antibody expression vector is prepared by incorporating the DNA fragment containing the antibody variable region obtained above into an appropriate vector such as N5KG1 (IDEC Pharmaceuticals, US patent 6001358). For example, CHO-Ras (Katakura Y., et al., Cytotechnology, 31: 103-109, 1999) is preferably used as a host cell for expression. Introduction of the vector into the host cell can be performed, for example, by electroporation. About 2 μg of the antibody expression vector is linearized with a restriction enzyme, and the gene is introduced into 4 × 10 ⁷ CHO-Ras cells using a Bio-Rad electrophoreter under conditions of 350 V and 500 μF and seeded in a 96-well culture plate. The drug corresponding to the selection marker of the expression vector is added and the culture is continued. When colonies are observed, antibody-expressing strains are selected by the method shown in Example 4. Antibody purification from the sorted cells can be performed according to Example 5.

[Example 19] Inhibition of antigen-specific antibody production by CD40 antagonistic antibody A mouse having a homozygous genetic background for mouse endogenous CD40 disruption and a human CD40 gene transgene (Yasui et al. Int. Immunol. 2002 Vol14: 319) and 4-hydroxy-3-nitrophenylacetyl-chiken γ-globulin conjugates (NP-CGG: distributed by Professor Hitoshi Kikutani, Osaka University Research Institute for Microbial Diseases) and Aram (ARAM: Antigen recognition activation motif) was sensitized by intraperitoneal injection of 100 μg (as NP-CGG amount). Each monoclonal antibody was administered into the tail vein in an amount of 30 or 100 μg immediately before antigen sensitization. As a negative control, 100 μg of anti-human albumin human IgG4 antibody was administered. Seven days after sensitization, blood was collected from the orbital venous plexus, and the amounts of NP-specific IgG1 and IgM antibodies in the serum were measured by ELISA. In ELISA method, 50 μl / well of bovine serum albumin (NP-BSA: 2.5 μg / ml) conjugated with NP was added to each well of a 96-well microplate for ELISA (Maxisorp, manufactured by Nunc), and incubated at 4 ° C. NP-BSA was adsorbed. Next, discard the supernatant, add blocking reagent (Super Block, manufactured by Pierce) to each well, incubate at room temperature to block, then wash each well 3 times with 0.1% Tween20-containing phosphate buffer (PBS-T) did. Next, each serum (50 μl / well) diluted with PBS-T containing 10% Block Ace was added to each well, and incubated at 37 ° C. for 2 hours to react. After washing the microplate 3 times with PBS-T, alkaline phosphatase-labeled goat anti-mouse IgG1 or IgM antibody (Cosmo Bio, 1070-04 or 1020-04) was added 1,000 times with PBS-T containing 10% Block Ace. Diluted solution (50 μl / well) was added to each well and incubated at 37 ° C. for 2 hours. Subsequently, the microplate was washed 3 times with PBS-T, and then a chromogenic substrate solution (50 μl / well, Sigma104, phosphatase substrate) was added to each well, and the absorbance at a wavelength of 405 nm was measured with a microplate reader. The results are shown in FIGS. In the figure, the vertical axis shows sera collected after two injections of NP-CGG into C57BL / 6 mice, diluted 10,000-fold for IgG1 antibody, and 100-fold for IgM antibody. The value converted as one unit is shown. F4-465, 4D11 and KM281-1-10 antibodies strongly suppressed NP-specific IgG1 and IgM antibody production by administration of 100 μg.

[Example 20] Inhibition of Tonsil B Cell Growth by CD40 Antagonistic Antibody Human tonsils were obtained from Children's Hospital (San Diego, Calif., USA). Cell suspensions were prepared by cutting the tonsils into small pieces, minced and passed through a 70 micrometer nylon mesh cell strainer. After washing several times with PBS, the number of cells was counted and stored frozen in 90% human serum (ICN) and 10% DMSO. Cells were used after thawing and resuspended in standard RPMI medium supplemented with 10% human serum, 2.5 microgram / ml amphotericin (fungizone, Gibco / BRL).

1 × 10 ⁵ cells were added to 96 wells and anti-human CD40 antibody was added at concentrations of 0.01, 0.1, 1.0 and 10 microgram / ml. The test was performed in triplicate for each concentration. Flag-labeled CD40L (Alexis) 1 microgram / ml and CD40L enhancer antibody (Alexis) 1 microgram / ml were added to each well and cultured for 3 days. Then 1 microCi of [ ³ H] thymidine was added to each well. Added. Cells were collected after 12 to 15 hours, and proliferation of tonsillar B cells was measured by a liquid scintillation counter. The count when B cells proliferated by stimulation of CD40L and no antibody was added was 100, and the count when CD40L was not added and B cells were not stimulated was 0. For example, when 30 is measured as a relative count value, it is expressed in this experiment that 70% growth inhibition has occurred.

A known antagonistic antibody, 5D12, did not show growth inhibition exceeding 50% even at an antibody concentration of 100 microgram / ml.
F4-465 showed about 80% growth inhibition at a concentration of only 0.01 microgram / ml and about 95% growth inhibition at a concentration of 0.1-10 microgram / ml (FIG. 20).

  All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

It is a figure which shows the result that KM302-1 antibody promotes CD95 expression. It is a figure which shows that an antagonistic antibody neutralizes the function of CD40 ligand with respect to Ramos cell. It is a figure which shows that an antagonistic antibody neutralizes the function of CD40 ligand with respect to Ramos cell. It is a figure which shows that KM281-1-10 antibody neutralizes the function of CD40 ligand with respect to Ramos cell. It is a figure which shows the result by which the cross-linked KM281-1-10 antibody does not promote CD95 expression. It is a figure which shows the result in which the cross-linked 5D12 antibody promotes CD95 expression. It is a figure which shows the growth inhibitory effect with respect to the tumor cell by KM302-1 antibody. It is a figure which shows the result in which KM302-1 antibody accelerates | stimulates maturation of DC cell. It is a figure which shows the result that KM302-1 antibody promotes IL-12 production to DC cells. It is a figure which shows neutralizing the function of CD40 ligand with respect to DC by KM281-1-10 antibody. It is a figure which shows neutralizing the function of CD40 ligand with respect to DC by KM281-1-10 antibody. It is a figure which shows that immature DC-MLR by a KM302-1 antibody is activated. It is a figure which shows the result that KM341-1-19 antibody etc. promote CD95 expression of Ramos cells. It is a figure which shows the result that KM341-1-19 antibody promotes IL-12 production of mature DC cells. It is a figure which shows the result that KM341-1-19 antibody promotes IL-10 production of mature DC cells. It is a figure which shows that 4D11 antibody etc. neutralize the function of CD40 ligand with respect to Ramos cell. It is a figure in which KM302-1 antibody shows an antitumor effect in a human tumor cell transplant mouse model. It is a figure in which KM341-1-19 antibody shows the growth inhibitory effect with respect to the tumor cell. It is a figure which shows that F4-465, 4D11, and KM281-1-10 suppress the production of antigen-specific IgG. It is a figure which shows that F4-465, 4D11, and KM281-1-10 suppress the production of antigen-specific IgM. It is a figure which shows that F4-465 suppresses the proliferation of tonsil B cell.

  Sequence number 1-26: Synthetic DNA

Claims (30)

An antibody against human CD40 or a functional fragment thereof having at least one property selected from the following (a) to (f):
(a) Acts on dendritic cells to produce IL-12 in the presence of LPS and IFNγ
(b) Acts on dendritic cells and has higher maturation activity than G28-5 antibody
(c) B95 cell line has higher activity to promote CD95 expression than G28-5 antibody
(d) The activity of inhibiting the growth of B cell established cell line is higher than that of G28-5 antibody
(e) Inducing cell death against B cell line
(f) Does not inhibit the binding of CD40 ligand to CD40   The antibody or the functional fragment thereof according to claim 1, wherein maturation of dendritic cells is carried out at an antibody concentration of 20 µg / ml or less.   2. The antibody or functional fragment thereof according to claim 1, wherein the CD95 expression is promoted to a B cell established cell line at an antibody concentration of 20 μg / ml or less.   2. The antibody or functional fragment thereof according to claim 1, wherein the B cell established cell line is Ramos or HS-Sulton. The production of IL-12 of 100 pg / ml or more is caused when an antibody having a concentration of 0.1 μg / ml or more is added to dendritic cells having a concentration of 1 × 10 ⁶ cells / ml. An antibody or functional fragment thereof. The antibody according to claim 1, wherein when an antibody having a concentration of 1 µg / ml or more is added to a dendritic cell having a concentration of 1x10 ⁶ cells / ml, the antibody produces IL-12 of 1000 pg / ml or more. Or a functional fragment thereof. The antibody according to claim 1, wherein when an antibody having a concentration of 1 µg / ml or more is added to dendritic cells having a concentration of 1x10 ⁶ cells / ml, the antibody produces IL-12 of 10,000 pg / ml or more. Or a functional fragment thereof.   The promotion of CD95 expression on a B cell established cell line (Ramos cells) is about 2 to 3 times or more of the control G28-5 antibody in an antibody concentration range of 0.01 μg / ml to 10 μg / ml. 2. The antibody or functional fragment thereof according to 1.   The antibody according to claim 1, wherein the expression promotion of CD95 against a B cell established cell line (Ramos cell) is about 2 to 6 times or more that of the control G28-5 antibody at an antibody concentration of 0.01 µg / ml. Functional fragment.   The antibody according to claim 1, wherein the expression promotion of CD95 against a B cell established cell line (Ramos cell) is about 2 to 7 times or more that of the control G28-5 antibody at an antibody concentration of 0.1 µg / ml. Functional fragment.   The antibody or its function according to claim 1, wherein the CD95 expression promotion for B cell established cell line (Ramos cell) is about 2 to 7 times or more that of the control G28-5 antibody at an antibody concentration of 1 µg / ml. Fragment.   The antibody or its function according to claim 1, wherein the CD95 expression promotion for the B cell established cell line (Ramos cell) is about 2 to 6 times or more that of the control G28-5 antibody at an antibody concentration of 10 µg / ml. Fragment.   Produced by hybridoma KM302-1 (Accession number FERM BP-7578), KM341-1-19 (Accession number FERM BP-7759), 2105 (Accession number FERM BP-8024) or F1-102 (Accession number ATCC PTA-3337) An antibody or a functional fragment thereof having the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody.   The heavy chain variable region and light chain variable region of the antibody produced by hybridoma F2-103, encoded by plasmid DNAs having accession numbers ATCC PTA-3302 and ATCC PTA-3303, respectively, accession numbers ATCC PTA-3304 and ATCC PTA- The heavy chain variable region and light chain variable region of the antibody produced by hybridoma F5-77, respectively encoded by plasmid DNA 3305, or the plasmid DNAs having accession numbers ATCC PTA-3306 and ATCC PTA-3307, respectively. An antibody having a mature amino acid sequence of a heavy chain variable region and a light chain variable region of an antibody produced by hybridoma F5-157 or a functional fragment thereof.   Heavy chain variable region and light chain variable region of antibody produced by hybridoma KM341-1-19 shown in SEQ ID NOs: 28 and 30, respectively, Heavy chain of antibody produced by hybridoma 2105 shown in SEQ ID NOs: 32 and 34, respectively Production of the hybridoma 115 shown in the variable region and the light chain variable region, shown in SEQ ID NOs: 36 and 38, respectively. Antibody heavy chain variable region and light chain variable region, shown in SEQ ID NOs: 52 and 54, respectively, heavy chain variable region and light chain variable region of antibody produced by hybridoma KM643-4-11, SEQ ID NOs: 60 and 62, respectively The heavy and light chain variable regions of the antibody produced by hybridoma F2-103, or SEQ ID NO: 64 Each preliminary 66 shown, of the heavy and light chain variable regions of an antibody produced by hybridoma F5-77, antibody or functional fragment thereof having the amino acid sequence of the mature part.   Heavy chain variable region and light chain variable region encoded by nucleic acid sequences isolated from hybridoma KM341-1-19, shown in SEQ ID NOs: 27 and 29, respectively, from hybridoma 2105 shown in SEQ ID NOs: 31 and 33, respectively. Heavy chain variable region and light chain encoded by the nucleic acid sequence isolated from hybridoma 110, as shown in SEQ ID NOs: 35 and 37, respectively. Hybridoma KM643-4 shown in the variable region, heavy chain variable region and light chain variable region encoded by the nucleic acid sequence isolated from hybridoma 115, shown in SEQ ID NOs: 39 and 41, respectively, SEQ ID NOs: 51 and 53, respectively. Heavy chain variable region and light chain variable region encoded by the nucleic acid sequence isolated from -11, SEQ ID NOS: 59 and 61, respectively. Isolated from the hybridoma F5-77 shown in SEQ ID NO: 63 and 65, respectively, or the heavy and light chain variable regions encoded by the nucleic acid sequences isolated from the hybridoma F2-103 An antibody having a mature amino acid sequence of a heavy chain variable region and a light chain variable region encoded by a nucleic acid sequence, or a functional fragment thereof. An antibody against human CD40 or a functional fragment thereof having at least one property selected from the following (g) to (j):
(g) Neutralize ligand action on CD40
(h) neutralizing or mitigating one or more of the effects of a ligand for CD40 on a B cell established cell line on CD40 expressing cells, and against CD40 on the B cell established cell line by cross-linking with an anti-immunoglobulin antibody Agonistic action is weaker than 5D12
(i) CD40 ligand mitigates or neutralizes the effect of increasing CD95 expression on B cell established cell lines
(j) Has an antagonistic effect on CD40 expressed in dendritic cells Ramos cells with a concentration of 1x10 ⁶ cells / ml supplemented with a saturating amount of CD40L-expressing cells will have CD95 expression in Ramos cells less than about 10% of the control when 0.1 μg / ml of antibody is added. The antibody or the functional fragment thereof according to claim 17, which can be suppressed. When an antibody at a concentration of 1 μg / ml is added to Ramos cells at a concentration of 1 × 10 ⁶ cells / ml supplemented with a saturating amount of CD40L-expressing cells, CD95 expression in Ramos cells is suppressed to the same level as the negative control. The antibody or the functional fragment thereof according to claim 17, wherein the antibody or the functional fragment thereof is. When 10 μg / ml concentration of antibody is added to 1 × 10 ⁶ cells / ml of Ramos cells supplemented with saturating CD40L expressing cells, CD95 expression of Ramos cells is suppressed to the same level as the negative control. The antibody or the functional fragment thereof according to claim 17, wherein the antibody or the functional fragment thereof is. In vitro, when 1 × 10 ⁵ tonsillar B cells supplemented with soluble CD40L (1 μg / ml) were added with antibodies at a concentration of 0.001 μg / ml to 10 μg / ml, the proliferation of tonsillar B cells was approximately The antibody or the functional fragment thereof according to claim 17, which is suppressed by 80 to 95% or more. In vitro, when 1 × 10 ⁵ tonsillar B cells supplemented with soluble CD40L (1 μg / ml) were added with an antibody at a concentration of 0.01 μg / ml to 10 μg / ml, the proliferation of tonsillar B cells was about The antibody or functional fragment thereof according to claim 17, which is suppressed by 95% or more. In vitro, when an antibody at a concentration of 0.001 μg / ml is added to 1 × 10 ⁵ tonsil B cells supplemented with soluble CD40L (1 μg / ml), the proliferation of tonsil B cells is suppressed by about 80% or more. 18. The antibody or the functional fragment thereof according to claim 17, wherein   Heavy chain variable region and light chain variable of antibody produced by hybridoma KM281-1-10 (Accession No. FERM BP-7579), 4D11 (Accession No. FERM BP-7758) or F4-465 (Accession No. ATCC PTA-3338) An antibody having a region amino acid sequence or a functional fragment thereof.   Heavy chain variable region and light chain variable region of antibody produced by hybridoma KM281-1-10 shown in SEQ ID NOs: 44 and 46, respectively, Heavy chain of antibody produced by hybridoma 4D11 shown in SEQ ID NOs: 48 and 50, respectively The antibody having the amino acid sequence of the mature part of the variable region and the light chain variable region, or the heavy chain variable region and the light chain variable region of the antibody produced by the hybridoma F4-465 shown in SEQ ID NOs: 56 and 58, respectively, or a function thereof Fragment.   The heavy chain variable region and light chain variable region encoded by the nucleic acid sequence isolated from hybridoma KM281-1-10 shown in SEQ ID NOs: 43 and 45, respectively, and hybridoma 4D11 shown in SEQ ID NOs: 47 and 49, respectively. Of the heavy and light chain variable regions encoded by the nucleic acid sequence isolated from the hybridoma F4-465 shown in SEQ ID NOs: 55 and 57, respectively. An antibody having a mature portion amino acid sequence or a functional fragment thereof.   The antibody or functional fragment thereof according to any one of claims 1 to 26, which is a human antibody.   27. A pharmaceutical composition comprising the antibody or functional fragment thereof according to any one of claims 1 to 26 as an active ingredient.   An immunostimulatory agent, an antitumor agent or an autoimmune disease therapeutic agent comprising the antibody according to any one of claims 1 to 16 or a functional fragment thereof as an active ingredient.   An immunosuppressant, an autoimmune disease therapeutic agent, an allergy therapeutic agent or a blood coagulation factor VIII inhibition syndrome therapeutic agent comprising the antibody or functional fragment thereof according to any one of claims 17 to 26 as an active ingredient.

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