Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-07-15
2001-08-07
Spector, Lorraine (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C435S252300, C435S254110, C435S320100, C536S023100, C536S023400, C536S023500
Reexamination Certificate
active
06270993
ABSTRACT:
TECHNICAL FIELD
The present invention relates to polypeptides which are useful as neovascularization inhibitors, and a method of producing them.
BACKGROUND ART
It is known that pathological neovascularization is closely related to the symptoms or causes of certain diseases. Solid tumors are representative of such diseases. For the growth of tumor tissue beyond the diameter of 1 to 2 mm, newly formed blood vessels need to extend from the existing blood vessels to reach the tumor tissue (J. Folkman, J. Natl. Cancer Inst., 82:4 (1990)). When the blood vessel reaches the tumor tissue, its growth is explosively accelerated. Diabetic retinopathy is accompanied by pathological neovascularization of the retina, which often leads to the loss of eyesight. Moreover, pathological neovascularization is also seen in such diseases as chronic rheumatoid arthritis, psoriasis, hemangioma, scleroderma, and neovascular glaucomas, and it is considered to be one of the main symptoms (J. Folkman and N. Engle, J. Med., 320:1211 (1989)). Therefore, it may be possible to use substances that inhibit neovascularization for the treatment of tumors and other diseases mentioned above.
Vascular endothelial cells are the cells that constitute the innermost layer of the blood vessel. Neovascularization occurs when vascular endothelial cells proliferate upon stimulation by growth factors, physiologically active substances, or mechanical damages. Known growth factors that can directly or indirectly stimulate the proliferation of vascular endothelial cells include bFGF (basic Fibroblast Growth Factor), aFGF (acidic Fibroblast Growth Factor), VEGF (Vascular Endothelial cell Growth Factor), PD-ECGF (Platelet-Derived Endothelial Cell Growth Factor), TNF-&agr; (Tumor Necrosis Factor-&agr;), PDGF (Platelet-Derived Growth Factor), EGF (Epidermal Growth Factor), TGF-&agr; (Transforming Growth Factor-&agr;), and HGF (Hepatocyte Growth Factor) (L. Diaz-Flores et al., Histol. Histopath., 9:807 (1994)). Particularly, VEGF can be distinguished from the other growth factors by the fact that its action is very specific to vascular endothelial cells. In other words, the VEGF receptor is found in very few cells other than vascular endothelial cells.
VEGF is a glycoprotein whose molecular weight is 40,000-45,000, and exists as a dimer (P. W. Leung et al., Science, 246:1306 (1989), P. J. Keck et al., Science, 246: 1319 (1989)). VEGF acts, by binding to the VEGF receptor, to promote cell proliferation and enhance membrane permeability.
The following reports suggest the involvement of VEGF in tumor. Many tumor cells secrete VEGF (S. Kondo et al., Biochem. Biophys. Res. Commun., 194:1234 (1993)). When tumor tissue sections are stained with an anti-VEGF antibody, the tumor tissue is strongly stained as well as the newly formed blood vessels surrounding it (H. F. Dvorak et al., J. Exp. Med. 174:1275 (1991), L. F. Brown et al., Cancer Res., 53:4727 (1993)). Growth of a transplanted tumor is suppressed in mice in which one of the VEGF receptors is genetically inactivated (B. Millauer et al., Nature, 367:576 (1994)). Anti-VEGF neutralizing antibodies exhibit anti-tumor activities in tumor-bearing mice (K. J. Kim et al., Nature, 362:841 (1993), S. Kondo et al., Biochem. Biophys. Res. Commun., 194:1234 (1993)). From these facts, it is considered that VEGF secreted by tumor cells plays a major role in neoplastic neovascularization.
In humans there are two known VEGF receptors, FLT (M. Shibuya et al., Oncogene, 5:519 (1990)) and KDR (B. I. Terman et al., Biochem. Biophys. Res. Commun., 187:1579 (1992)). The extracellular domain of FLT (also known in the art as FLT-1) has seven immunoglobulin-like domains as shown in
FIG. 1
(C. DeVries et al., Science, 255:989 (1992)). Regarding FLT, a cDNA of a soluble-type receptor has been cloned (R. L. Kendal and K. A. Thomas, Proc. Natl. Acad. Sci. U.S.A., 90:10705 (1993)). The polypeptide encoded by this cDNA corresponds to the first through sixth immunoglobulin-like domains of the seven immunoglobulin-like domains of the FLT extracellular domain, and it inhibited the VEGF activities by binding to VEGF with an affinity comparable to that of the full-length FLT. Regarding KDR, it is also known that the genetically engineered first through sixth immunoglobulin-like domains of the extracellular domain bind to VEGF (R. L. Kendal et al., Biochem. Biophys. Res. Commun., 201:326(1994)).
DISCLOSURE OF THE INVENTION
Since the mouse anti-VEGF neutralizing antibodies exhibit antitumor activity, they are expected to be useful as anti-cancer agents. However, when a mouse antibody is administered to humans, human antibodies against the mouse antibody may be produced, which could lead to neutralization of the mouse antibody or might cause anaphylactic shock. In order to avoid these undesirable effects, it is necessary to modify the amino acid sequence of the mouse antibody to be closer to that of the human antibody through chimeralization (S. L. Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81:6851 (1989)) or humanization without reducing the neutralizing activity of the mouse antibody. Since this method requires advanced techniques and knowledge, experience, and labor, the results depend on individual cases and are not always successful. Furthermore, 100%-humanized antibodies cannot be obtained by these methods. Another method utilizes transgenic mice that produce human antibodies for immunization (S. Wagner et al., Nucleic Acid Res., 22:1389 (1994)), but here again highly specialized techniques are required.
As described above, since the extracellular domain of the VEGF receptor specifically binds to VEGF with high affinity, thereby inhibiting the VEGF activity, it can be considered useful as an inhibitor against neovascularization. Moreover, the possibility of antibody production in a human recipient is expected to be low because it is a polypeptide of human origin. However, it has been reported that when a polypeptide that does not naturally exist much in the human body is administered, it is metabolized very rapidly. For example, the plasma half-life of soluble CD4, which is a receptor for HIV, is 15 minutes (D. J. Capon et al., Nature, 337:525 (1989)), and that of interferon &ggr; is 30 minutes (I. Rutenfranz and H. Kirchner, J. Interferon Res., 8:573 (1988)).
As a method for prolonging the plasma half-life, it is known to utilize a fusion polypeptide genetically engineered by combining the polypeptide of interest with a molecule having a long plasma half-life, such as an antibody molecule. In the case of CD4, the plasma half-life was increased from 15 min to 48 hr when it was chimerilized with the Fc domain of IgG1 (D. J. Capon et al., Nature, 337:525 (1989)). Such a fusion polypeptide with the Fc domain of an antibody is also expected to provide an effect to induce the effector functions that the antibody possesses, i.e., complement-dependent cytotoxicity (D. B. Amos et al., Transplantation, 7:220 (1969)) and antibody-dependent cytotoxicity (A. Y. Liu et al., Proc. Natl. Acad. Sci. U.S.A., 84:3439 (1987)). Furthermore, it is expected to drastically improve the apparent affinity when the fusion polypeptide binds to a ligand on a solid phase, such as the surface of a membrane or the extracellular matrix, since the dimerization via the Fc domain enables each molecule to bind to the ligand at two sites.
When a fusion polypeptide constructed with an antibody is utilized, it is desirable to select a polypeptide with a low molecular weight as a starting material because the molecular weight increases through the fusion. This is because, if a high molecular weight polypeptide is used, the molecular weight of the corresponding DNA is also high, which is to be handled by gene manipulation upon production of the recombinant host that produces the fusion polypeptide. In general, the larger the molecular weight of the DNA to be introduced, the less efficient the transfection of the host becomes, thereby reducing the productivity of the recombinant host. Also in general, the larger the molecular weight of the recombinan
Asano Makoto
Matsumoto Tomoe
Niwa Mikio
Okamoto Masaji
Segawa Tosiaki
Fish & Richardson P.C.
Spector Lorraine
Toa Gosei Co., Ltd.
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