Androgen induced growth factor (AIGF) is a factor isolated in 1992 from a culture supernatant of a mouse mammary tumor cell line SC-3 [J. Steroid Biochem., 27 459 (1987)] which shows sex hormone-dependent growth. AIGF is a growth factor which is induced and produced by androgen stimulation and activates growth of SC-3 cells in an autocrine manner [Proc. Natl. Acad. Sci., 89, 8928-8932, (1992)]. As a result of gene cloning, it was found that it has a homology of 30 to 40% with fibroblast growth factor (FGF) family at the amino acid sequence, and it was named FGF-8. Thereafter, human FGF-8 gene was cloned from a human placenta genomic library using mouse FGF-8 gene as a probe, and it was shown that it coincided with the mouse FGF-8 at 85% in the nucleotide sequence and at 100% in the amino acid sequence [FEBS Letters, 363, 226 (1995)]. It has been assumed that sex hormone induced growth factors would play an autocrine role in cancers such as prostate cancer and breast cancer which show sex hormone-dependent growth, and the finding of FGF-8, though in a mouse system, was the first evidence of such a mechanism. It has been assumed that FGF-8 may also relate to generation and growth of cancers in human by a similar mechanism, but the it has not been demonstrated. However, it was reported that when FGF-8 was added, growth of certain human prostate cancer cell line was accelerated [FEBS Letters, 363, 226 (1995)], expression of mRNA in various human cancer cell lines such as prostate cancer, breast cancer and ovarian cancer was confirmed [Cell Growth & Differ., 7, 1425 (1996), Oncogene, 18, 1053 (1999), Int. J Cancer, 88, 718 (2000)] and FGF-8s presented in human cancer tissues of prostate cancer, breast cancer and ovarian cancer was over-expressed compared to FGF-8s presented in normal tissues [Cancer Res., 58, 2053, Oncogene, 18, 2755 (1999), Oncogene, 18, 1053 (1999), Int. J. Cancer, 88, 718 (2000)]. Thus, such a possibility has been pointed out that FGF-8 also plays an autocrine and paracrine role on the sex hormone-dependent growth of cancer cells in human. On the other hand, since high frequency expression of FGF-8 has also been observed in various hormone-independent human prostate cancer cells, prostate cancer tissues and breast cancer cells [Cell Growth & Differ., 7, 1425 (1996), Oncogene, 18, 2755 (1999), Oncogene, 18, 1053 (1999)], there is a high possibility that expression of FGF-8 is controlled by sex hormone independent fashion. Furthermore, since there is a report that antisense DNA for FGF-8 inhibited in vitro and in vivo growth of a hormone-independent prostate cancer cell line [Oncogene, 16, 1487 (1998)], the presence of an FGF-8-dependent growth mechanism is also suggested in cancers which lost hormone-dependency.
Based on these facts, antibodies against FGF-8 are effective in analyzing biological function of FGF-8 for cancer cells and also in diagnosing cancer cells such as prostate cancer and breast cancer using an immunological detection method. Furthermore, in neutralizing antibodies which inhibits functions of FGF-8, it is expected that they are useful for analyzing the biological function of FGF-8, diagnosing cancers such as prostate cancer, breast cancer and ovarian cancer and treating sex hormone-dependent cancers and sex hormone-independent cancers. Thus, the inventors of the present invention conducted preparation of an antibody against FGF-8, and as a result, succeeded in preparing a mouse monoclonal antibody KM1334 which specifically reacts with FGF-8 and inhibits the function of FGF-8 (Japanese Published Unexamined Patent Application No. 271391/97).
It is known in general that when an antibody derived from a non-human animal, e.g., a mouse antibody, is administered to human, it is recognized as an foreign substance and induces a human antibody against the mouse antibody (human anti-mouse antibody hereinafter referred to as “HAMA”) in the human body. It is known that the HAMA reacts with the administered mouse antibody to cause side effects [J. Clin. Oncol., 2, 881 (1984), Blood, 65, 1349 (1985), J. Natl. Cancer Inst., 80 932 (1988), Proc. Natl. Acad. Sci. U.S.A., 82, 1242 (1985)], to quicken disappearance of the administered mouse antibody from the body [J. Nucl. Med., 26, 1011 (1985), Blood, 65, 1349 (1985), J. Natl. Cancer Inst., 80, 937 (1988)], and to reduce therapeutic effects of the mouse antibody [J. Immunol., 135, 1530 (1985), Cancer Res., 46, 6489 (1986)].
In order to solve these problems, attempts have been made to convert an antibody derived from a non-human animal into a humanized antibody such as a human chimeric antibody or a human CDR-grafted antibody using genetic engineering technique.
The human chimeric antibody consists of an antibody the V region derived from a non-human animal antibody and the C region derived from a human antibody [Proc. Natl. Acad. Sci. U.S.A., 81, 6851 (1984)]. The human CDR-grafted antibody consists of the amino acid sequence of CDR in the V region derived from a non-human animal antibody is grafted into an appropriate position of a human antibody [Nature, 321, 522 (1986)]. In comparison with antibodies derived from non-human animals such as mouse antibodies and the like, these humanized antibodies have various advantages for clinical applications. For example, regarding immunogenicity and stability in blood, it has been reported that blood half-life of a human chimeric antibody became about 6 times as long as a mouse antibody when administered to human [Proc. Natl. Acad. Sci. U.S.A., 86, 4220 (1989)]. In the case of a human CDR-grafted antibody, it has been reported that its immunogenicity was reduced and its serum half-life was prolonged in comparison with a mouse antibody in experiment using a monkey [J. Immunol., 147, 1352 (1991)]. Thus, it is expected that the humanized antibodies have less side effects and their therapeutic effects continue for a longer time than antibodies derived from non-human animals.
Furthermore, since the human CDR-grafted antibody is prepared using genetic engineering technique, molecules in various forms can be prepared. For example, when the 1 subclass is used as an H chain C region (CH) of a human antibody, a humanized antibody having a high effector function such as antibody-dependent cell-mediated cytotoxic activity can be prepared [Cancer Res., 56, 1118 (1996)]. A humanized antibody having a high effector function is quite effective when an antigen exists on the surface of cells such as cancer cells and destruction of the target cell is desired. On the other hand, in the case where only function which neutralizes the target molecule is required or in the case where side effects by the destruction of the target cell might be caused, the side effects can be avoided and a prolonged blood serum half-time in comparison with a mouse antibody is expected by using the 4 subclass as CH of the human antibody [Immunol., 85, 668 (1995)] because the 4 subclass usually has a low effector function [J. Exp. Med., 166, 1351 (1987), J. Exp. Med., 168, 1351 (1998)]. Furthermore, according to the recent advances in protein engineering and genetic engineering, antibody fragments having a smaller molecular weight such as Fab, Fab′, F(ab′)2, scFv [Science, 242, 423 (1988)], diabody [Nature Biotechnol., 15, 629 (1997)], dsFv [Molecular Immunol., 32, 249 (1995)] and a peptide containing CDR [J. Biol. Chem., 271, 2966 (1996)] can be prepared as humanized antibodies. The antibody fragments are excellent in transitional activity into target tissue in comparison with complete antibody molecules [Cancer Res., 52, 3402 (1992)].
It is considered that the humanized antibodies are more desirable than antibodies derived from non-human animals such as mouse antibodies, when used in clinical applications to human.
As discussed above, if humanized antibodies which specifically react with FGF-8 and inhibit function of FGF-8 or antibody fragments thereof are prepared, they are expected to be used as effective diagnostic agents or therapeutic agents for diseases relating to FGF-8 such as cancer. However, such antibodies or antibody fragments have not been prepared yet until now.
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