Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Hormones – e.g. – prolactin – thymosin – growth factors – etc.
Reexamination Certificate
1995-05-31
2002-07-16
Saoud, Christine J. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Hormones, e.g., prolactin, thymosin, growth factors, etc.
C530S395000, C930S010000, C435S069400, C435S243000, C435S325000
Reexamination Certificate
active
06420531
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to growth factors, particularly to isolation of a polypeptide growth factor similar to a family of factors including known fibroblast growth factors (FGFs). This invention also relates to construction of complementary DNA (cDNA) segments from messenger RNA (mRNA) encoding the novel growth factor. Further, this invention pertains to synthesis of products of such DNA segments by recombinant cells, and to the manufacture and use of certain other novel products enabled by the identification and cloning of DNAs encoding this growth factor.
ABBREVIATIONS USED IN THIS APPLICATION
aFGF
acidic fibroblast growth factor
bFGF
basic fibroblast growth factor
EGF
epidermal growth factor
HSAC
heparin-Sepharose affinity chromatography
kb
kilobases
kDa
kilodaltons
KGF
keratinocyte growth factor
NaDodSO
4
/PAGE
Sodium dodecylsulphate (SDS)/polyacryl-
amide gel electrophoresis
RP-HPLC
reversed-phase high performance liquid
chromatography
TGF&agr;
transforming growth factor &agr;
BACKGROUND OF THE INVENTION
Growth factors are important mediators of intercellular communication. These potent molecules are generally released by one cell type and act to influence proliferation of other cell types (James, R. and Bradshaw, R. A. (1984),
Ann. Rev. Biochem.
53, 259-292). Interest in growth factors has been heightened by evidence of their potential involvement in neoplasia (Sporn, M. B. and Todaro, G. J. (1980),
N. Eng. J. Med.
303, 878-880). The v-sis transforming gene of simian sarcoma virus encodes a protein that is homologous to the B chain of platelet-derived growth factor (James, R. and Bradshaw, R. A. (1984)
Ann. Rev. Biochem.
53, 259-292; Doolittle, R. F., et al. (1983)
Science
221, 275-277). Moreover, a number of oncogenes are homologous of genes encoding growth factor receptors (James, R. and Bradshaw, R. A. (1984)
Ann. Rev. Biochem.
53, 259-292). Thus, increased understanding of growth factors and their receptor-mediated signal transduction pathways is likely to provide insights into mechanisms of both normal and malignant cell growth.
One known family of growth factors affecting connective tissue cells includes acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and the related products of the hst, and int-2 oncogenes.
Further, it is known that some growth factors, including the following, have heparin-binding properties: aFGF (Maciag, T., Mehlman, T., Friesel, R. and Schreiber, A. B. (1984)
Science
225, 932-935; Conn, G and Hatcher, V. B. (1984)
Biochem. Biophys. Res. Comm.
124, 262-268); bFGF (Gospodarowicz, D., Cheng, J., Lui, G. -M., Baird, A. and Bohlen, P. (1984)
Proc. Natl. Acad. Sci. USA
81, 6963-6967; Maciag, T., Mehlman, T., Friesel, R. and Schreiber, A. B. (1984)
Science
225, 932-935); granulocyte/machrophage colony stimulating factor (James, R. and Bradshaw, R. A. (1984)
Ann. Rev. Biochem.
53, 259-292); and interleukin 3 (James, R. and Bradshaw, R. A. (1984)
Ann. Rev. Biochem.
53, 259-292). Each of these polypeptide factors is produced by stromal cells (James, R. and Bradshaw, R. A. (1984)
Ann. Rev. Biochem.
53, 259-292, Doolittle, R. F., Hunkapiller, M. W., Hood, L. E., Devare, S. G., Robbins, K. C., Aaronson, S. A. and Antoniades, M. N. (1983)
Science
221, 275-277, Roberts, R., Gallagher J., Spooncer, E., Allen, T. D., Bloomfield, F. and Dexter, T. M. (1988)
Nature
332, 376-378). Such factors appear to be deposited in the extracellular matrix, or on proteoglycans coating the stromal cell surface (James, R. and Bradshaw, R. A. (1984)
Ann. Rev. Biochem.
53, 259-292, Roberts, R., Gallagher, J., Spooncer, E., Allen, T. D., Bloomfield, F. and Dexter, T. M. (1988)
Nature
332, 376-378). It has been postulated that their storage, release and contact with specific target cells are regulated by this interaction (Roberts, R., Gallagher, J., Spooncer, E., Allen, T. D., Bloomfield, F. and Dexter, T. M. (1988)
Nature
332, 376-378, Vlodavsky, I., Folkman, J., Sullivan, R., Fridman, R., Ishai-Michaeli, R., Sasse, J. and Klagsburn, M. (1987)
Proc. Natl. Acad. Sci. USA
84, 2292-2296).
It is widely recognized, however, that the vast majority of human malignancies are derived from epithelial tissues (Wright, N. and Allison, M. (1984)
The Biology of Epithelial Cell Populations
(Oxford University Press, New York) Vol. 1, pp. 3-5). Effectors of epithelial cell proliferation derived from mesenchymal tissues have been described (James, R. and Bradshaw, R. A. (1984)
Ann. Rev. Biochem.
53, 259-292, Doolittle, R. F., Hunkapiller, M. W., Hood, L. E., Devare, S. G., Robbins, K. C., Aaronson, S. A. and Antoniades, M. N. (1983)
Science
221, 275-2772, Waterfield, M. D., Scrace, G. J., Whittle, N., Strooband, P., Johnson, A., Wasteton, A., Westermark, B., Heldin, C. -H., Huang, J. S. and Deuel, T. F. (1983) Nature 304, 35-39), however, their molecular identities and structures have not been elucidated.
In light of this dearth of knowledge about such mesenchymal growth factors affecting epithelial cells, it is apparent that there has been a need for methods and compositions and bioassays which would provide an improved knowledge and analysis of mechanisms of regulation of epithelial cell proliferation, and, ultimately, a need for novel diagnostics and therapies based on the factors involved therein.
This invention contemplates the application of methods of protein isolation and recombinant DNA technologies to fulfill such needs and to develop means for producing protein factors of mesenchymal origin, which appear to be related to epithelial cell proliferation processes and which could not be produced otherwise. This invention also contemplates the application of the molecular mechanisms of these factors related to epithelial cell growth processes.
SUMMARY OF THE INVENTION
The present invention relates to developments of protein isolation and recombinant DNA technologies, which include production of novel growth factor proteins affecting epithelial cells, free of other peptide factors. Novel DNA segments and bioassay methods are also included.
The present invention in particular relates to a novel protein having structural and/or functional characteristics of a known family of growth factors which includes acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and the related products of the hst, and int-2 oncogenes. This new member of the FGF polypeptide family retains the heparin-binding properties of the FGFs but has evolved a unique target cell specificity. This growth factor appears to be specific for epithelial cells and is particularly active on keratinocytes. Therefore, this novel factor has been designated “keratinocyte growth factor” (KGF). Notwithstanding its lack of activity on fibroblasts, since it is the sixth known member of the FGF polypeptide family, KGF may also be referred to as FGF-6.
Accordingly, this invention relates, in part, to purified KGF or KGF-like proteins and methods for preparing these proteins. Such purified factors may be made by cultivation of human cells which naturally secrete these proteins and application of isolation methods according to the practice of this invention. These proteins can be used for biochemical and biological studies leading, for example, to isolation of DNA segments encoding KGF or KGF-like polypeptides.
The present invention also relates to such DNA segments which encode KGF or KGF-like proteins. In a principal embodiment, the present invention relates to DNA segments, which encode KGF-related products, consisting of: human cDNA clones 32 or 49, derived from polyadenylated RNA extracted from the human embryonic lung fibroblast cell line M426; recombinants and mutants of these clones; and related DNA segments which can be detected by hybridization to any of the above human DNA segments, which relates segments encode KGF-like proteins or portions thereof.
In the practice of one embodiment of this invention, the DNA segments of the invention are capable of being expressed in suitable host cells, thereby producing KGF
Aaronson Stuart A.
Finch Paul W.
Rubin Jeffrey S.
Heller Ehram White and McAuliffe
Saoud Christine J.
The United States as represented by the Department of Health and
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