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
1995-06-07
2003-05-20
Kunz, Gary L. (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...
C435S320100, C435S325000, C435S252300, C536S023510
Reexamination Certificate
active
06566098
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to naturally occurring truncated forms of Hepatocyte Growth Factor (HGF), which are encoded by alternative HGF mRNA transcripts. In particular, the present invention relates to a small HGF variant, HGF/NK2, which is a competitive antagonist of HGF, and HGF/NK1, which is a partial agonist of HGF.
2. Background Information
Hepatocyte growth factor has hormone-like activity and is released in response to partial hepatectomy and liver injury and is presumed to be an important mediator of liver regeneration (Nakamura et al.,
FEBS Lett
. 224: 311 (1987); Gohda et al.,
J. Clin. Invest
. 81: 414-419 (1988); R. Zarnegar and G. Michaelopoulous,
Cancer Research
49: 3314-3320 (1989)). Its ubiquitous expression by stromal fibroblasts and demonstrated ability to stimulate DNA synthesis in melanocytes and endothelial cells as well as epithelial cells suggest that this factor plays a role in paracrine regulation of cell growth as well (Rubin et al.,
Proc. Natl. Acad. Sci. USA
88: 415 (1991)). Reports showing that scatter factor has high amino acid sequence identity to HGF over restricted regions, suggested that HGF may also be involved in modulating cell-cell interactions and migration (E. Gherardi and M. Stoker,
Nature
346: 288 (1990); Weidner et al.,
J. Cell Biology
111: 2097-2108 (1990)). This has been borne out by subsequent studies that verify the identity of scatter factor and HGF.
Structurally, HGF resembles plasminogen in that it possesses characteristic kringle domains (Patthy et al.,
FEBS Lett
. 171: 131-136 (1984)) and a serine protease-like domain (Miyazawa et al.,
Biochem. Biophys. Res. Commun
., 163: 967-973 (1989); Nakamura et al.,
Nature
342: 440-443 (1989)). Like plasminogen, HGF can be processed by proteolytic cleavage, generating a heterodimeric molecule comprised of a heavy- and light-chain covalently linked by disulfide bonds (Nakamura et al.,
Nature
342: 440-443 (1989) and Miyazawa et al.,
Biochem. Biophys. Res. Commun
. 163: 967-973 (1989)) The possibility that its actions might be mediated by a receptor tyrosine kinase was suggested by its rapid stimulation of tyrosine phosphorylation of cellular proteins in target cells (Rubin et al.,
Proc. Natl. Acad. Sci. U.S.A
. 88: 415 (1991)). Recent studies have directly identified the HGF receptor as the c-met protooncogene product (Bottaro et al.,
Science
251: 802 (1991)), whose structure resembles that of a membrane-spanning tyrosine kinase (Park et al.,
Proc. Natl. Acad. Sci. USA
84: 6379-6383 (1987); Chan et al.,
Oncogene
2: 593-599 (1988)).
There is accumulating evidence that the positive effects of growth factors on cell proliferation can be counteracted at a variety of levels both intracellularly (Moses et al.,
Cell
63: 245-247 (1990) and at the cell surface (Hannum et al.,
Science
343: 336-340 (1990), Eisenberg, et al.,
Nature
343: 341-346 (1990); Carter et al.,
Nature
344: 633-637 (1990)). Thus, the potential exists to find an antagonist to HGF which would negatively regulate the growth factor's proliferation effects. The invention described herein relates to small HGF variants and their corresponding transcripts. Characterization of one of these HGF variants, HGF/NK2, has revealed that it is a competitive antagonist of HGF action and thus establishes a novel regulatory mechanism whereby the same gene encodes both an agonist and antagonist of growth factor action. Characterization of another HGF variant, HGF/NK1, revealed that it is a partial agonist of HGF in vitro.
SUMMARY OF THE INVENTION
Although those of skill in the art of the invention possessed knowledge of HGF, no one knew about or could have predicted the existence of alternative mRNA transcripts encoding the claimed variants. Indeed, the inventors discovered HGF/NK1 and HGF/NK2, none of the work related to HGF even remotely suggested that a small transcript, particularly one, such as HGF/NK2, that is an HGF antagonist, or HGF/NK1, that is a partial HGF agonist, existed.
Thus, in one embodiment, the invention relates to a substantially pure HGF variant that is a truncated form of HGF comprising the N-terminal and first two kringle domains of HGF, and that specifically inhibits HGF-induced mitogenesis.
In another embodiment, the invention relates to a substantially pure HGF variant that is a truncated form of HGF comprising the N-terminal and the first kringle domain of HGF, and that is a partial HGF agonist.
In yet another embodiment, the invention relates to a method of inhibiting HGF induced mitogenesis in cells expressing the receptor for HGF, comprising contacting the cells with a mitogenesis-inhibiting amount of the HGF variant HGF/NK2, such that HGF induced mitogenesis is inhibited when the HGF variant binds the receptor for HGF on the cells.
In another embodiment, the invention relates to a method of stimulating mitogenesis in cells expressing the receptor for HGF, comprising contacting the cells with a mitogenesis-stimulating amount of HGF/NK1, such that mitogenesis is stimulated when the HGF variant binds the receptor for HGF on the cells.
In another embodiment, the invention relates to an isolated and substantially pure DNA molecule that encodes HGF/NK2.
In another embodiment, the invention relates to an isolated and substantially pure DNA molecule that encodes HGF/NK1.
In another embodiment, the invention relates to a recombinant vector comprising the above DNAs.
In another embodiment, the invention relates to a host cell stably or transiently transfected with the above described DNA in a manner allowing expression of the protein encoded by the DNA.
Another embodiment of the invention relates to a method of producing a recombinant HGF variant, comprising culturing the above host cell in a manner allowing expression of a protein and isolating the protein from the host cell.
In another embodiment, the invention relates to a method of producing the above described substantially pure HGF variant protein, the method comprising the following steps:
(i) culturing HGF variant-producing cells in a culture medium under conditions such that HGF variant is produced;
(ii) concentrating the culture medium so that a concentrate is formed;
(iii) contacting the concentrate with heparin under conditions such that HGF variant in the concentrate binds to the heparin, whereby a heparin-HGF variant complex is formed;
(iv) separating the heparin-HGF variant complex from the concentrate;
(v) treating the heparin-HGF variant complex under conditions such that the HGF variant dissociates from the heparin so that a solution of free HGF variant is formed;
(vi) fractionating the solution by sizing chromatography and/or reverse phase HPLC so that HGF variant is separated from the remaining components.
In another embodiment, the invention relates to a method of producing a substantially pure and biologically active Hepatocyte Growth Factor (HGF) variant comprising the steps of:
(i) disrupting HGF variant-producing bacteria that have been cultured in a culture medium under conditions such that HGF variant is expressed, so as to produce a first HGF variant protein-containing suspension;
(ii) recovering the protein from the first suspension and, washing and solubilizing the recovered protein, wherein the solubilizing is performed with a denaturant and reducing agent, and wherein a second protein-containing suspension is produced;
(iii) fractionating the second suspension by sizing chromatography with a solvent containing a denaturant and a reducing agent;
(iv) removing the denaturant from the fractions of step (iii) and pooling selected fractions containing denatured HGF variant;
(v) purifying said HGF variant in the pooled fractions by reverse phase chromatography;
(vi) lyophilizing the purified HGF variant of step (v) and redissolving the lyophilized HGF variant with denaturing and reducing agents;
(vii) serially diluting and then incubating the redissolved lyophilized proteins in refolding buffer, and then removing the denaturant by dialysis, so as to produce
Aaronson Stuart A.
Bottaro Donald P.
Chan Andrew M. L.
Rubin Jeffrey S.
Hayes Robert C.
Kunz Gary L.
Needle & Rosenberg P.C.
The United States of America as represented by the Department of
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