Receptors for fibroblast growth factors

Details Receptors for fibroblast growth factors Receptors for fibroblast growth factors

1992-02-13

2002-05-07

Spector, Lorraine (Department: 1646)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C536S023500, C536S023100, C435S069100, C435S091500, C435S252300, C435S325000, C435S173300, C514S002600, C514S012200

Reexamination Certificate

active

06384191

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to receptors for growth factors, specifically to the fibroblast growth factor receptor (FGF-R). More particularly, it provides various purified fibroblast growth factor receptor proteins, nucleic acids encoding the receptor proteins, methods for the production of purified FGF-R proteins, proteins made by these methods, antibodies against these proteins, and diagnostic and therapeutic uses of these various reagents.
BACKGROUND OF THE INVENTION
Polypeptide growth factors are mitogens that act on cells by specifically binding to receptors situated at the plasma membrane. These receptors usually have three major identifiable regions. The first is an extracellular region which contains the domain that binds the polypeptide growth factor (i.e. the ligand-binding domain). The second region is a transmembrane region and the third is an intracellular region. Many of these receptors contain a tyrosine kinase domain in the intracellular region.
The fibroblast growth factor receptor (FGF-R) proteins bind to a family of related growth factor ligands, the fibroblast growth factor (FGF) family. This family of growth factors are characterized by amino acid sequence homology, heparin-binding avidity, the ability to promote angiogenesis and mitogenic activity toward cells of epithelial, mesenchymal and neural origin.
The FGF family includes the following seven known FGFs:
(1, 2) acidic FGF (aFGF) and basic FGF (bFGF) O. Gospodarowicz et al.,
Mol. Cell. Endocrinol.,
46:107 (1986
);
(3) the int-2 gene product (R. Moore et al.,
EMBO. J.,
5:919 (1986);
(4) the hst gene product or Kaposi's sarcoma FGF (by J. Anderson et al.
Nature,
332:360 (1988); M. Taira et al.,
Proc. Natl. Acad. Sci. USA,
84:2980 (1987));
(5) FGF-5 (X. Zhan et al.,
Mol. Cell. Biol.,
8:3487 (1988)); and
(6) keratinocyte growth factor (J. S. Rubin et al.,
Proc. Natl. Acad. Sci. USA,
86:802 (1989)).
(7) FGF-6 (I. Marics, et al.,
Oncogene
3:335 (1989)).
The actions of acidic and basic FGF are mediated through binding to high affinity cell surface receptors of appoximately 145 and 125 kDa (G. Neufeld and D. Gospodarowicz,
J. Biol. Chem.,
261:5631 (1986)).
The reference of Imamura et al., “Purification of Basic FGF Receptors from Rat Brain,”
Biochem. Biophys. Res. Communications,
155:583 (Sep. 15, 1988) discloses the purification of nanogram amounts of a basic FGF receptor (bFGF-R) from rat brain.
While genes encoding a number of growth factor receptors have been molecularly cloned (e.g., mouse PDGF receptor, Yarden et al.,
Nature,
323:226 (1986), no clone has previously been identified as encoding a fibroblast growth factor receptor (FGF-R). Using antiphosphotyrosine antibodies to screen &lgr;gt11 cDNA expression libraries, a 2.5 kilobase cDNA encoding a novel tyrosine kinase gene, designated bek (bacterially expressed kinase), was isolated from a mouse liver cDNA library. (S. Kornbluth et al., “Novel Tyrosine Kinase Identified by Phosphotyrosine Antibody Screening of cDNA Libraries”,
Mol. Cell. Biol.
No. 8, 5541 (1988)). The bek sequence did not contain a transmembrane region and therefore could not be identified as a growth factor receptor. Another protein tyrosine kinase gene designated a (fms-like-gene) was isolated from a human endothelial cell cDNA library by hybridization under relaxed stringency with a v-fms oncogene probe. (M. Ruta et al., “A Novel Protein Tyrosine Kinase Gene Whose Expression is Modulated During Endothelial Cell Differentiation”, Oncopene, 3:9 (1988)). Those authors could not identify a transmembrane region in their isolated sequence and therefore hypothesized that flg encodes a cytoplasmic tyrosine kinase.
The purified and cloned chicken bFGF and human bFGF receptors of this invention have amino acid sequence similarity with the bek and flg clones in the regions which have been isolated. However, both the bek and flg sequences reported were incomplete and there was no recognition of their function as FGF binding receptors. Moreover, the prior reports failed to recognize many of the structural and functional features described in the present invention.
Members of the FGF family appear to have roles in tissue development, tissue repair, maintenance of neurons and in the pathogenesis of disease. Aberrant expression of FGF may cause cell transformation by an autocrine mechanism. Moreover, FGFs may enhance tumor growth and invasiveness by stimulating blood vessel growth in the tumor or by inducing production of proteins such as plasminogen activator. However, identification of the components involved and understanding of the mechanisms and interactions involved remain woefully incomplete.
Purified FGF receptors and fragments, and isolated DNA sequences encoding defined FGF receptors and defined fragments (e.g., the ligand-binding domain) will greatly accelerate the understanding of fibroblast growth factor functions. Antibodies against specific and defined regions of the FGF receptor also become available. These reagents will find both diagnostic and therapeutic uses in the aforementioned processes. The present invention fulfills these and other needs.
SUMMARY OF THE INVENTION
The present invention provides purified fibroblast growth factor receptor (FGF-R) proteins, nucleic acids encoding FGF-R proteins, methods for the production of purified FGF-R proteins, purified proteins made by these methods, antibodies against these proteins and fragments, and diagnostic and therapeutic uses of these reagents. Notably, the present invention provides soluble and secreted forms of the receptors exhibiting an unusual receptor structure.
The present invention provides a method for modifying in vivo a fibroblast growth factor receptor modulated activity comprising administering to a patient an amount of a fibroblast growth factor receptor blocking agent effective to inhibit fibroblast growth factor binding to said fibroblast growth factor receptor. Typically, the agent will be a fragment of a human fibroblast growth factor receptor, e.g., a fragment produced in a cell transformed with a nucleic acid containing at least about 15 bases of a sequence selected from the group consisting of:
a) a DNA sequence in
FIGS. 3
or
4
;
b) a sequence encoding a polypeptide of
FIGS. 3
,
4
or
7
; and
c) a sequence substantially homologous to a sequence of
FIGS. 3
or
4
.
The fragment will often be a fibroblast growth factor receptor extracellular domain without a tyrosine kinase region.
Alternatively, a method is provided for inhibiting binding between a fibroblast growth factor and a fibroblast growth factor receptor in a solution. This method will contain a step of combining an FGF-R peptide, e.g., a peptide homologous in sequence to a sequence described in
FIGS. 3
,
4
or
7
to a solution or medium containing fibroblast growth factor and fibroblast growth factor receptor, usually native fibroblast growth factor receptor. Such methods will be useful in vitro, after employing labeled FGF-R peptide in assay procedures.
Compositions containing a soluble FGF-R polypeptide having between about five and two hundred contiguous amino acids from a human FGF-R extracellular domain are described. In one embodiment, the polypeptide contains at least about 80 amino acids from residues 1 to 287 of a human fibroblast growth factor receptor of
FIG. 7
or an IgII or IgIII domain, or both. In alternative embodiments, the IgII domain will have about 7 contiguous amino acids from residues 85 to 141 of a human sequence of
FIG. 7
or may contain a carboxy-terminal sequence substantially homologous to the 79 amino acid sequence from residues 222 to 300 of a soluble human protein of FIG.
7
. Particularly preferred polypeptides consist essentially of the h4 or h5 sequences (FIG.
7
).
A further aspect of the invention is a fibroblast growth factor receptor composition containing a substantially pure polypeptide of less than about 85 KDa comprising a fibroblast growth factor-binding domain. The polypeptide may be soluble or may specifically possess a signal segment, an IgI

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