Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1995-05-12
2001-07-03
Kunz, Gary L. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S395000, C435S069100
Reexamination Certificate
active
06255454
ABSTRACT:
DESCRIPTION
1. Technical Field
This invention relates to the fields of molecular biology and pharmaceutical research. More specifically, this invention relates to the recombinant expression of a human high-affinity fibroblast growth factor (FGF) receptor, and its use in combination with glycosaminoglycans to model compounds capable of mimicking FGF binding.
2. Background of the Invention
The fibroblast growth factors (FGFs) are a family of structurally related polypeptides that regulate the growth and differentiation of a diverse number of cell types. Acidic and basic FGFs are mitogenic for cell types of mesenchymal, epithelial and neural origin (K. Thomas,
FASEB J
(1987) 1:434-440; D. Gospodarowicz,
Meth Enzymol
(1987) 147:106-119). The more recently discovered members of the FGF family have been implicated in early developmental processes and in epithelial cell growth and maintenance (R. Moore et al,
EMBO J
(1986) 5:919-924; A. Jakobovits et al,
Proc Natl Acad Sci USA
(1986) 83:7806-7810; P. W. Finch et al, Science (1989) 245:752-755). Currently, the FGF family consists of seven distinct gene products including acidic and basic FGFs (M. Jaye et al,
Science
(1986) 233:541-545; J. A. Abraham et al, Science (1986) 233:545-548; J. A. Abraham et al,
EMBO J
(1986) 5:2523-2528), the product of the int-2 oncogene (R. Moore et al, supra; A. Jakobovits et al, supra), a growth factor identified from Kaposi's sarcoma DNA (hst-1 or KS-FGF) (P. D. Bovi et al,
Cell
(1987) 50:729-737; M. Taira et al,
Proc Natl Acad Sci USA
(1987) 84:2980-2984), FGF-5 (X. Zhan et al,
Mol Cell Biol
(1988) 8:3487-3495), FGF-6 (I. Marics et al,
Oncogene
(1989) 4:335-340) and keratinocyte growth factor, KGF or FGF-7 (P. W. Finch et al, supra).
The large number of FGFs and their diverse spectrum of activities suggests that several receptors may mediate their effects on cells. Indeed, for the acidic and basic FGFs themselves, two classes of receptors have been well documented which are distinguished by their affinities for FGF. For example, the binding of bFGF to a high affinity site on baby hamster kidney (BHK) cells occurs with a dissociation constant in the 20 pM range, whereas bFGF binding to the low affinity site occurs with a dissociation constant in the 2 nM range, and is released with 2M NaCl. The FGF receptor has been implicated as the entry portal for Herpes simplex virus (HSV). Several high affinity FGF receptor cDNAs have been cloned (P. L. Lee et al,
Science
(1989) 245:57-60; E. Pasquale & S. J. Singer,
Proc Natl Acad Sci USA
(1989) 86:5449-5453; M. Ruta et al,
Oncogene (
1988) 3:9-15; H. H. Reid et al,
Proc Natl Acad Sci USA
(1990) 87:1596-1600; A. Isacchi et al,
Nuc Acids Res
(1990) 18:1906; D. E. Johnson et al,
Mol Cell Biol
(1990) 10:4728-4736) and shown by structural homology to be members of the cell surface protein-tyrosine kinase family of proteins. This group of membrane-bound proteins are thought to play an important role in the regulation of cell growth. They include the receptors for epidermal growth factor, platelet-derived growth factor, colony stimulating factor-1, insulin, and insulin-like growth factor-1 (for recent review see A. Ullrich & J. Schlessinger, Cell (1990) 61:203-212).
Structural analyses of the extracellular regions of the chicken FGF receptor cDNA suggests that the FGF receptors also belong to the immunoglobulin supergene family (P. L. Lee et al, supra). Accordingly, Reid et al, (supra) have found several forms of the bFGF receptor mRNA in developing mouse brain that contain either two or three immunoglobulin-like domains. Moreover, they detected a region of sequence variability between the first and second immunoglobulin-like domains. In this case, amino acids 148 and 149 are sometimes deleted in the predicted sequences for proteins that contain 2 immunoglobulin-like domains. Recently, four forms of the cDNA encoding the human two immunoglobulin-like domain FGF receptor have been identified (D. E. Johnson et al, supra). Two of these forms are homologous to the mouse two immunoglobulin-like domain FGF receptor in that they vary at amino acids 148 and 149 (H. H. Reid et al, supra). While the other two forms of the human FGF receptor also vary at these amino acids, they are unique in that they lack a transmembrane domain and the cytoplasmic tyrosine kinase domain. More recently, a fifth form of the human FGF receptor cDNA has also been isolated (A. Isacchi et al, supra), and is homologous to the mouse three immunoglobulinlike-domain FGF receptor. In addition to the five forms of the FGF receptor, Southern blot analysis and the cloning of two related cDNAs, bek (H. H. Reid et al, supra; S. Kornbluth et al,
Mol Cell Biol
(1988) 8:5541-5544) and a bek-related molecule (H. H. Reid et al, supra), indicate that FGF receptors are members of a multigene family.
A number of researchers have recently reported expression of various FGF receptors. See R. J. Kaner et al,
Science
(1990) 248:1410-13; A. Mansukhani et al,
Proc Nat Acad Sci USA
(1990) 87:4378-82; C. A. Dionne et al,
EMBO J
(1990) 9:2685-92; and D. P. Mirda & L. T Williams,
Clin Res
(1990) 38:310A. However, the reported experiments in general do not disclose the expression of human FGF receptor in quantity sufficient for study.
In order to usefully study the binding of FGF analogs to the FGF receptor, one must have available sufficient quantities of active receptor for study. Further, the receptor must be in a useful form.
DISCLOSURE OF THE INVENTION
A new human FGF receptor has now been cloned and expressed using cDNA obtained from a human liver cell line. The expression of high levels of the extracellular region of this FGF receptor in a baculovirus/insect cell system yields a high affinity FGF-binding protein that is active in radioreceptor assays, inhibits cell growth and that can be used to study the ligand-receptor interaction. Furthermore, four forms of the cDNAs that encode the FGF receptor have now been identified in several tissues and cell lines, suggesting there exists an extensive distribution of alternate forms that are generated by differential RNA splicing.
Thus, one aspect of the invention is a recombinant FGF receptor (rFGF-R), which is capable of binding aFGF and/or bFGF. Another aspect of the invention is a recombinant fragment of FGF-R comprising the extracellular domain (soluble FGF-R, or “sFGF-R”), which is capable of binding aFGF and/or bFGF.
Another aspect of the invention is a method for detecting FGF in a sample, by employing rFGF-R in a manner analogous to an anti-FGF antibody in any form of immunoassay. For example, one may detect FGF by providing a support comprising rFGF-R bound to a solid surface, contacting the support with a sample to be assayed for FGF, removing the portion of the sample which does not bind to the support, and detecting the presence of bound FGF on the support (e.g., by using a labeled anti-FGF antibody, by competition with labeled FGF, etc.).
Another aspect of the invention is a method for inhibiting the activity of FGF, using rFGF-R. Thus, rFGF-R may be used to inhibit FGF-mediated activities. For example, one method of the invention is the inhibition of FGF-dependent tumor growth by administering an effective amount of sFGF-R. Another method of the invention is the method of inhibiting angiogenesis (e.g., of a tumor) by administering an effective amount of sFGF-R. Another method of the invention is the method of inhibiting FGF-dependent cell growth in vitro by administering rFGF-R.
Another aspect of the invention is the use of rFGF-R to screen and identify compounds which mimic FGF binding. Compounds identified in this manner may be agonists or antagonists. Agonists are useful in situations in which FGF activity is beneficial, e.g., for acceleration of wound healing, nerve outgrowth, and the like. Antagonists are useful for inhibiting the activity of FGF, for example, to inhibit the growth of FGF-dependent malignancies, and the like. Compounds may be screened by providing a support having bound rFGF-R, contacting the suppor
Barr Philip J.
Keifer Michael C.
Valenzuela Pablo D. T.
Blackburn Robert P.
Chiron Corporation
Guth Joseph H.
Hemmendinger Lisa M.
Kunz Gary L.
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