1. CD40
CD40 is an antigen which has a molecular weight of 50 kDa and is present on the surface of cell membrane, and expressed in B cells, dendritic cells (DCs), some types of cancer cells, and thymic epithelial cells. CD40 is known to play an important role in proliferation and differentiation of B cells and DCs. CD40 was identified as an antigen expressed on the surface of human B cells (Non-Patent Documents 1 and 2) and has been considered as a member of the TNF receptor family to which low-affinity NGF receptors, TNF receptors, CD27, OX40, CD30 and the like belongs. A ligand (CD40L) to human and murine CD40s has been found to be a type II membrane proteins expressed in activated CD4+T cells. CD40L has been also found to introduce strong signals for activation into human or murine B cells.
It is considered that the expression of CD40 in DC is higher than that in B cell and it has become clear that CD40 plays an important role. Binding of CD40 to CD40L activates an antigen presenting cell (APC). Namely, it activates the expression of costimulator molecules such as CD80 (B7-1) and CD86 (B7-2) or enhances the production of IL-2 (Non-Patent Documents 3 and 4). DC has a strong antigen-presenting activity and a strong capacity to activate helper T (Th) cells. DC is also considered to control differentiation of naive Th cells into Th1 or Th2 cells. When peripheral blood monocytes which are myeloid dendritic cells are cultured in the presence of GM-CSF and IL-4, and matured by CD40L, the resulting matured dendritic cells (DC1) can produce IL-12 in vitro, and stimulate and activate allogeneic naive Th cells to induce IFNγ-producing T cells (i.e., to promote their differentiation into Th1). This function is inhibited by anti-IL-12 antibody and hence may be a reaction mediated by IL-12. On the other hand, when plasmacytoid T cells which are present in lymphoid T regions and peripheral blood are cultured in the presence of IL-3 and CD40L, the resulting lymphoid dendritic cells (DC2) are shown to be unable to produce IL-12, and stimulate and activate allogeneic naive Th cells to induce IL-4-producing T cells, which indicates promotion of their differentiation into Th2. It is considered that Th1 cells are involved in activation of cellular immunity, while Th2 cells are associated with enhancement of humoral immunity as well as restriction of cellular immunity. When cytotoxic T cells (CTL) are activated with the help of Th1 cells, they may eliminate pathogens (various virus, listeria, tuberculosis bacteria, toxoplasma protozoa, etc.) growing in the cytoplasm and tumor cells.
The monoclonal anti-CD40 antibody which recognizes CD40 expressed on the membrane surface has been demonstrated to have different biological activities to B cells. The monoclonal anti-CD40 antibody is generally classified into agonistic substance (antagonistic antibody) or antagonistic substance (antagonistic antibody) against CD40.
2. Agonistic Antibodies
As function of an agonistic antibody, the activation of B cells is known. For example, the anti-CD40 antibody has been reported to induce cell adhesion (Non-Patent Documents 5 and 6), increase cell size (Non-Patent Documents 6 and 7), induce cell division of B cells activated only by an anti-IgM antibody, anti-CD20 antibody or phorbol ester (Non-Patent Documents 8 to 10), induce cell division of B cells in the presence of IL-4 (Non-Patent Documents 7 and 11), induce expression of IgE by cultured cells stimulated with IL-4 and deprived of T cells (Non-Patent Documents 12 and 13), induce expression of IgG and IgM by those cultured cells (Non-Patent Documents 13), secrete soluble CD23/FceRII from cells due to IL-4 (Non-Patent Documents 14 and 15), enhance expression of soluble CD23/FceRII on the cells due to IL-4 (Non-Patent Documents 16), and promote IL-6 production (Non-Patent Document 17).
Furthermore, it has been reported that addition of IL-4 and an anti-CD40 antibody to human primary culture B cells in the presence of CDw32+ adhesive cells led to establishment of cloned B cells derived therefrom (Non-Patent Document 18), and apoptosis of germinal center cells was inhibited by CD40 regardless of whether its antigen receptor was active or inactive (Non-Patent Document 19). As described above, since CD40 has been identified as antigen expressed on the surface of human B cells, most of the isolated antibodies have been mainly evaluated by their induction potency for proliferation and/or differentiation of human B cells or their induction activity for cell death of cancer cells, as an index (Non-Patent Documents 20, 21 and 22).
In addition, the anti-CD40 antibody has been demonstrated to mature DC (Non-Patent Document 23). Furthermore, the role of CD4 T cells in priming antigen-specific CD8 T cells has been reported to be the activation of DC via CD40-CD40L signaling, and the anti-CD40 monoclonal antibody (mAb) has been found to be able to substitute CD40 helper T cells in activation of DC (Non-Patent Document 24). Also, administration of an anti-CD40 antibody in mice has been found to be able to protect the animal body from CD40-expressing tumor cells as well as CD40-non-expressing tumor cells (Non-Patent Document 25).
An anti-CD40 antibody having an agonist activity is expected to be effective for treatment of infectious diseases due to such as bacteria and virus; malignancy; and the like, based on their functions described above.
As an anti-CD40 antibody having an agonist activity, the antibody KM341-1-19 is disclosed in Patent Document 1. The hybridoma KM341-1-19 producing the antibody KM341-1-19 (Accession Number: FERM BP-7759) was deposited on 27, Sep. 2001 for international deposit under the Budapest Treaty, to International Patent Organisms Depositary, National Institute of Advanced Industrial Science and Technology (central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan). The heavy chain constant region of the antibody KM341-1-19 and the antibody 341G2Ser having a heavy chain constant region which is IgG2 in which proline at position 331 is substituted with serine (this substitution is represented as P331S; hereinafter, represented as the same; numbering is based on the EU index of Non-Patent Document 26) are disclosed in Patent Document 2.
The anti-CD40 antibody 21.4.1 having an agonist activity is disclosed in Patent Document 3.
3. Mutation of Amino Acid
It has been reported that a region at positions 233-299 of a lower hinge region of IgG (numbering is based on the EU index of Kabat et al.) is one of the binding regions to an Fcγ receptor, which is a member of immunoglobulin Fc receptor (Non-Patent Document 27). The immunoglobulin Fc receptor plays an important role in antibody-mediated immune response. Specifically, it includes phagocytosis, ADCC activity (Non-Patent Documents 28 and 29) and the like. The Fcγ receptor is expressed on surfaces of leukocytes, and is divided into three classes of FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). Further, FcγRII is subdivided into FcγRIIA and FcγRIIB, and FcγRIII is subdivided into FcγRIIIA and FcγRIIIB.
It has been reported that binding of an Fcγ receptor is lowered by substituting a lower hinge region of IgG1 with IgG2 which is a subclass having a weak effector function activity of an antibody through an Fcγ receptor. Specifically, examples of E233P, L234V, L235A, deletion of G236 and the like (numbering is based on the EU index of Kabat et al. (Non-Patent Documents 30 to 33). As discussed above, it is known that an effector function activity of an antibody through an Fcγ receptor is weak for an antibody having IgG2 as a subclass, and it has been reported that lysis of a target cell by an effector cell can be further inhibited by the substitution of V234A and G237A (numbering is based on the EU index of Kabat et al.) (Non-Patent Document 34). However, the effects of V234A and G237A on the agonist activity of an anti-CD40 antibody are not disclosed. Alternatively, the effects of V234A and G237A in IgG2 subclass on the blood kinetics of an anti-CD40 antibody are not disclosed. Still further, the effects of V234A and G237A in IgG2 subclass of an anti-CD40 antibody on the liver are not disclosed.
For example, L235, D265, D270, K322, P331 and P329 (numbering is based on the EU index of Kabat et al.) have been considered to play an important role in the complement-activating capacity of human lgG and the CDC activity can be reduced by substituting these sites with other amino acids (Non-Patent Documents 35 to 40). Specifically, the reduction in CDC activity can be achieved by substituting D270, K322, P329 and/or P331 with A. Alternatively, the reduction in CDC activity can be achieved by substituting P331 with S or G.