Fibroblast growth factor receptor-like molecules and uses thereof

Abstract

The present invention provides Fibroblast Growth Factor Receptor-Like (FGFR-L) polypeptides and nucleic acid molecules encoding the same. The invention also provides selective binding agents, vectors, host cells, and methods for producing FGFR-L polypeptides. The invention further provides pharmaceutical compositions and methods for the diagnosis, treatment, amelioration, and/or prevention of diseases, disorders, and conditions associated with FGFR-L polypeptides.

Description

[0001] This application is a continuation of U.S. Provisional Patent Application No. 60/191,379, filed on Mar. 22, 2000, the disclosure of which is explicitly incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates to Fibroblast Growth Factor Receptor-Like (FGFR-L) polypeptides and nucleic acid molecules encoding the same. The invention also relates to selective binding agents, vectors, host cells, and methods for producing FGFR-L polypeptides. The invention further relates to pharmaceutical compositions and methods for the diagnosis, treatment, amelioration, and/or prevention of diseases, disorders, and conditions associated with FGFR-L polypeptides.

BACKGROUND OF THE INVENTION

[0003] Technical advances in the identification, cloning, expression, and manipulation of nucleic acid molecules and the deciphering of the human genome have greatly accelerated the discovery of novel therapeutics. Rapid nucleic acid sequencing. techniques can now generate sequence information at unprecedented rates and, coupled with computational analyses, allow the assembly of overlapping sequences into partial and entire genomes and the identification of polypeptide-encoding regions. A comparison of a predicted amino acid sequence against a database compilation of known amino acid sequences allows one to determine the extent of homology to previously identified sequences and/or structural landmarks. The cloning and expression of a polypeptide-encoding region of a nucleic acid molecule provides a polypeptide product for structural and functional analyses. The manipulation of nucleic acid molecules and encoded polypeptides may confer advantageous properties on a product for use as a therapeutic.

[0004] In spite of the significant technical advances in genome research over the past decade, the potential for the development of novel therapeutics based on the human genome is still largely unrealized. Many genes encoding potentially beneficial polypeptide therapeutics or those encoding polypeptides, which may act as “targets” for therapeutic molecules, have still not been identified. Accordingly, it is an object of the invention to identify novel polypeptides, and nucleic acid molecules encoding the same, which have diagnostic or therapeutic benefit.

SUMMARY OF THE INVENTION

[0005] The present invention relates to novel FGFR-L nucleic acid molecules and encoded polypeptides.

[0006] The invention provides for an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:

[0007] (a) the nucleotide sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO: 4;

[0008] (b) the nucleotide sequence of the DNA insert in ATCC Deposit No. ______;

[0009] (c) a nucleotide sequence encoding the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0010] (d) a nucleotide sequence which hybridizes under moderately or highly stringent conditions to the complement of any of (a)-(c); and

[0011] (e) a nucleotide sequence complementary to any of (a)-(c).

[0012] The invention also provides for an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:

[0013] (a) a nucleotide sequence encoding a polypeptide which is at least about 70 percent identical to the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, wherein the encoded polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0014] (b) a nucleotide sequence encoding an allelic variant or splice variant of the nucleotide sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO: 4, the nucleotide sequence of the DNA insert in ATCC Deposit No. ______, or (a);

[0015] (c) a region of the nucleotide sequence of either SEQ ID NO: 1 or SEQ ID NO: 4, the DNA insert in ATCC Deposit No. ______, (a), or (b) encoding a polypeptide fragment of at least about 25 amino acid residues, wherein the polypeptide fragment has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or is antigenic;

[0016] (d) a region of the nucleotide sequence of either SEQ ID NO: 1 or SEQ ID NO: 4, the DNA insert in ATCC Deposit No. ______, or any of (a)-(c) comprising a fragment of at least about 16 nucleotides;

[0017] (e) a nucleotide sequence which hybridizes under moderately or highly stringent conditions to the complement of any of (a)-(d); and

[0018] (f) a nucleotide sequence complementary to any of (a)-(d).

[0019] The invention further provides for an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:

[0020] (a) a nucleotide sequence encoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one conservative amino acid substitution, wherein the encoded polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0021] (b) a nucleotide sequence encoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one amino acid insertion, wherein the encoded polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0022] (c) a nucleotide sequence encoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one amino acid deletion, wherein the encoded polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0023] (d) a nucleotide sequence encoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 which has a C- and/or N-terminal truncation, wherein the encoded polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0024] (e) a nucleotide sequence encoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, C-terminal truncation, and N-terminal truncation, wherein the encoded polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0025] (f) a nucleotide sequence of any of (a)-(e) comprising a fragment of at least about 16 nucleotides;

[0026] (g) a nucleotide sequence which hybridizes under moderately or highly stringent conditions to the complement of any of (a)-(f); and

[0027] (h) a nucleotide sequence complementary to any of (a)-(e).

[0028] The present invention provides for an isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

[0029] (a) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; and

[0030] (b) the amino acid sequence encoded by the DNA insert in ATCC Deposit No. ______.

[0031] The invention also provides for an isolated polypeptide comprising the amino acid sequence selected from the group consisting of

[0032] (a) the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 6, optionally further comprising an amino-terminal methionine;

[0033] (b) an amino acid sequence for an ortholog of either SEQ ID NO: 2 or SEQ ID NO: 5;

[0034] (c) an amino acid sequence which is at least about 70 percent identical to the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5, wherein the polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0035] (d) a fragment of the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 comprising at least about 25 amino acid residues, wherein the fragment has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or is antigenic; and

[0036] (e) an amino acid sequence for an allelic variant or splice variant of the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, the amino acid sequence encoded by the DNA insert in ATCC Deposit No. ______, or any of (a)-(c).

[0037] The invention further provides for an isolated polypeptide comprising the amino acid sequence selected from the group consisting of:

[0038] (a) the amino acid sequence as se t forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one conservative amino acid substitution, wherein the polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0039] (1) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one amino acid insertion, wherein the polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0040] (c) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one amino acid deletion, wherein the polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;

[0041] (d) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 which has a C- and/or N-terminal truncation, wherein the polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; and

[0042] (e) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, C-terminal truncation, and N-terminal truncation, wherein the polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.

[0043] Also provided are fusion polypeptides comprising FGFR-L amino acid sequences.

[0044] The present invention also provides for an expression vector comprising the isolated nucleic acid molecules as set forth herein, recombinant host cells comprising the recombinant nucleic acid molecules as set forth herein, and a method of producing an FGFR-L polypeptide comprising culturing the host cells and optionally isolating the polypeptide so produced.

[0045] A transgenic non-human animal comprising a nucleic acid molecule encoding an FGFR-L polypeptide is also encompassed by the invention. The FGFR-L nucleic acid molecules are introduced into the animal in a manner that allows expression and increased levels of an FGFR-L polypeptide, which may include increased circulating levels. Alternatively, the FGFR-L nucleic acid molecules are introduced into the animal in a manner that prevents expression of endogenous FGFR-L polypeptide (i.e., generates a transgenic animal possessing an FGFR-L polypeptide gene knockout). The transgenic non-human animal is preferably a mammal, and more preferably a rodent, such as a rat or a mouse.

[0046] Also provided are derivatives of the FGPR-L polypeptides of the present invention.

[0047] Additionally provided are selective binding agents such as antibodies and peptides capable of specifically binding the FGFR-L polypeptides of the invention. Such antibodies and peptides may be agonistic or antagonistic.

[0048] Pharmaceutical compositions comprising the nucleotides, polypeptides, or selective binding agents of the invention and one or more pharmaceutically acceptable formulation agents are also encompassed by the invention. The pharmaceutical compositions are used to provide therapeutically effective amounts of the nucleotides or polypeptides of the present invention. The invention is also directed to methods of using the polypeptides, nucleic acid molecules, and selective binding agents.

[0049] The FGFR-L polypeptides and nucleic acid molecules of the present invention may be used to treat, prevent, ameliorate, and/or detect diseases and disorders, including those recited herein.

[0050] The present invention also provides a method of assaying test molecules to identify a test molecule that binds to an FGFR-L polypeptide. The method comprises contacting an FGFR-L polypeptide with a test molecule to determine the extent of binding of the test molecule to the polypeptide. The method further comprises determining whether such test molecules are agonists or antagonists of an FGFR-L polypeptide. The present invention further provides a method of testing the impact of molecules on the expression of FGFR-L polypeptide or on the activity of FGFR-L polypeptide.

[0051] Methods of regulating expression and modulating (i.e., increasing or decreasing) levels of an FGFR-L polypeptide are also encompassed by the invention. One method comprises administering to an animal a nucleic acid molecule encoding an FGFR-L polypeptide. In another method, a nucleic acid molecule comprising elements that regulate or modulate the expression of an FGFR-L polypeptide may be administered. Examples of these methods include gene therapy, cell therapy, and anti-sense therapy as further described herein.

[0052] The FGFR-L polypeptide can be used for identifying ligands thereof. Various forms of “expression cloning” have been used for cloning ligands for receptors (e.g., Davis et al., 1996, Cell, 87:1161-69). These and other FGFR-L polypeptide ligand cloning experiments are described in greater detail herein. Isolation of an FGFR-L polypeptide ligand allows for the identification or development of novel agonists or antagonists of the FGFR-L polypeptide signaling pathway. Such agonists and antagonists include FGFR-L polypeptide ligands, anti-FGFR-L polypeptide ligand is antibodies and derivatives thereof, small molecules, or antisense oligonucleotides, any of which can be used for potentially treating one or more diseases or disorders, including those recited herein.

BRIEF DESCRIPTION OF THE FIGURES

[0053] FIGS. 1A-1C illustrate the nucleotide sequence of the murine FGFR-L gene (SEQ ID NO: 1) and the deduced amino acid sequence of murine FGFR-L polypeptide (SEQ ID NO: 2). The predicted signal peptide (underline) and transmembrane domain (double-underline) are indicated;

[0054] FIGS. 2A-2B illustrate the amino acid sequence alignment of murine FGFR-L polypeptide (Smaf2-00017-f4; SEQ ID NO: 2) and Iberian ribbed newt (Pleurodeles waltlii) Fibroblast Growth Factor Receptor-4 (PIR:B49151; SEQ ID NO: 7);

[0055] FIGS. 3A-3B illustrate the nucleotide sequence of a cDNA clone encoding the N-terminal portion of the human FGFR-L gene (SEQ ID NO: 4) and the deduced amino acid sequence of the N-terminal portion of the human FGFR-L polypeptide (SEQ ID NO: 5). The predicted signal peptide (underline) and transmembrane domain (double-underline) are indicated;

[0056] FIG. 4 illustrates the amino acid sequence alignment of murine FGFR-L polypeptide (SEQ ID NO: 2) and a virtual human FGFR-L polypeptide sequence (SEQ ID NO: 8) constructed from residues 1-472 of SEQ ID NO: 5 and residues 473-504 of GenBank Accession No. AJ277437. The predicted signal peptide (underline), transmembrane domain (double-underline), and N-linked glycosylation sites (bold) are indicated;

[0057] FIG. 5 illustrates the expression of FGFR-L MRNA as detected by Northern blot analysis in day 7, 11, 15, and 17 mouse embryos;

[0058] FIG. 6 illustrates the expression of FGFR-L mrRNA as detected by Northern blot analysis in murine heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis;

[0059] FIG. 7 illustrates the expression of FGFR-L mRNA as detected by Northern blot analysis in NIH 3T3 cells and F10, F4, and D3 mouse bone marrow-derived stromal cell lines;

[0060] FIG. 8 illustrates the expression of FGFR-L mRNA as detected by Northern blot analysis in human brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, and peripheral blood leukocytes;

[0061] FIG. 9 illustrates the expression of FGFR-L mRNA as detected by Northern blot analysis in promyelocytic leukemia HL-60 cells, HeLa S3 cells, chronic myelogenous leukemia L-562 cells, lymphoblastic leukemia MOLT-4 cells, Burkitt's lymphoma Raji cells, colorectal adenocarcinoma SW480 cells, lung carcinoma A549 cells, and melanoma G361 cells;

[0062] FIG. 10 illustrates the expression of FGFR-L mRNA as detected by Northern blot analysis in human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas;

[0063] FIG. 11 illustrates the expression of FGFR-L mRNA as detected by Northern blot analysis in 266-6 cells, AR42J cells, CaPan I cells, HIG-82 cells, OHS4 cells, SW 1353 cells, SW 872 cells, K562 (old, i.e., later passage) cells, K562 (new, i.e., earlier passage) cells, Jurkat cells, and F4 cells;

[0064] FIGS. 12A-12B illustrate the expression of FGFR-L mRNA as detected by Northern blot analysis in human adipose tissue (using a human FGFR-L-derived probe) and murine adipose tissue (using a murine FGFR-L-derived probe);

[0065] FIG. 13 illustrates the expression of FGFR-L mRNA in a number of murine tissues as detected in an RNAse protection assay. The absence of the cyclophilin band in the pancreas RNA sample suggests that thi sample was degraded;

[0066] FIG. 14 illustrates the expression of FGFR-L mRNA as detected by in situ hybridization in the peri-renal, white, and brown adipose tissue of a normal adult mouse (H&E=hematoxylin and eosin counterstaining; ISH=in situ hybridization);

[0067] FIG. 15 illustrates the. expression of FGFR-L mRNA as detected by in situ hybridization in the duodenum, ileum, colon, and pancreas of a normal adult mouse (H&E=hematoxylin and eosin counterstaining; ISH=in situ hybridization);

[0068] FIG. 16 illustrates the expression of FGFR-L mRNA as detected by in situ hybridization in the trachea, articular cartilage of the knee joint, spleen, and uterus of a normal adult mouse (H&E=hematoxylin and eosin counterstaining; ISH=in situ hybridization);

[0069] FIG. 17 illustrates the induction of FGFR-L MRNA in osteoblastic ST2 cells under conditions of osteoclastogenesis (i.e., 5-day exposure to vitamin D3 and dexamethasone);

[0070] FIG. 18 illustrates the results of Western blot analysis of E. coli-derived Des7-FGFR-L/ECD and CHO-derived FGFR-L/ECD-Fc proteins using FGFR-L polypeptide antiserum;

[0071] FIG. 19 illustrates the results of Western blot analysis of murine eye (lane 1) and adipose tissue (lane 2) using FGFR-L polypeptide antiserum;

[0072] FIGS. 20A-20B illustrate the results of FACS analysis on F4 and D3 bone marrow stromal cells using FGFR-L polypeptide antiserum;

[0073] FIGS. 21A-21D illustrate the results of proliferation assays using D3 bone marrow stromal cells (either untransduced or transduced with a construct encoding FGFR-L polypeptide) following 72 hour exposure to rhuPDGF (panel A), rhuFGF-2 (panel B), rhuFGF-4 (panel C), or rhuFGF-6 (panel D);

[0074] FIG. 22 illustrates the results of proliferation assays using A5-F bone marrow stromal cells following exposure to E. coli-derived Des7-FGFR-L/ECD protein and serum, PDGF, FGF-2, FGF-4, or FGF-6;

[0075] FIG. 23 illustrates the results of proliferation assays using A5-F bone marrow stromal cells following exposure to CHO-derived FGFR-L/ECD-Fc protein and serum, PDGF, FGF-4, or FGF-6;

[0076] FIG. 24 illustrates the expression of the neomycin resistance gene as detected by Northern blot analysis of peripheral blood mononuclear cell (PBMN) RNA from two FGFR-L/neo-transduced mice (lanes 1 and 2) and two neo-transduced control mice (lanes 3 and 4).

DETAILED DESCRIPTION OF THE INVENTION

[0077] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references cited in this application are expressly incorporated by reference herein.

[0078] Definitions

[0079] The terms “FGFR-L gene” or “FGFR-L nucleic acid molecule” or “FGFR-L polynucleotide” refer to a nucleic acid molecule comprising or consisting of a nucleotide sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO: 4, a nucleotide sequence encoding the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, a nucleotide sequence of the DNA insert in ATCC Deposit No. ______, and nucleic acid molecules as defined herein.

[0080] The term “FGFR-L polypeptide allelic variant” refers to one of several possible naturally occurring alternate forms of a gene occupying a given locus on a chromosome of an organism or a population of organisms.

[0081] The term “FGFR-L polypeptide splice variant” refers to a nucleic acid molecule, usually RNA, which is generated by alternative processing of intron sequences in an RNA transcript of FGFR-L polypeptide amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.

[0082] The term “isolated nucleic acid molecule” refers to a nucleic acid molecule of the invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the “isolated nucleic acid molecule” is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence. Preferably, the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use.

[0083] The term “nucleic acid sequence” or “nucleic acid molecule” refers to a DNA or RNA sequence. The term encompasses molecules formed from any of the known base analogs of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylaminomethyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, -2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0084] The term “vector” is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell.

[0085] The term “expression vector” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control the expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.

[0086] The term “operably linked” is used herein to refer to an arrangement of flanking sequences wherein the flanking sequences so described are configured or assembled so as to perform their usual function. Thus, a flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription and/or translation of the coding sequence. For example, a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of that coding sequence. A flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

[0087] The term “host cell” is used to refer to a cell which has been transformed, or is capable of being transformed with a nucleic acid sequence and then of expressing a selected gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present.

[0088] The term “FGFR-L polypeptide” refers to a polypeptide comprising the amino acid sequence of either SEQ ID NO.: 2 or SEQ ID NO: 5 and related polypeptides. Related polypeptides include FGFR-L polypeptide fragments, FGFR-L polypeptide orthologs, FGFR-L polypeptide variants, and FGFR-L polypeptide derivatives, which possess at least one activity of the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. FGFR-L polypeptides may be mature polypeptides, as defined herein, and may or may not have an amino-terminal methionine residue, depending on the method by which they are prepared.

[0089] The term “FGFR-L polypeptide fragment” refers to a polypeptide that comprises a truncation at the amino-terminus (with or without a leader sequence) and/or a truncation at the carboxyl-terminus of the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. The term “FGFR-L polypeptide fragment” also refers to amino-terminal and/or carboxyl-terminal truncations of FGFR-L polypeptide orthologs, FGFR-L polypeptide derivatives, or FGFR-L polypeptide variants, or to amino-terminal and/or carboxyl-terminal truncations of the polypeptides encoded by FGFR-L polypeptide allelic variants or FGFR-L polypeptide splice variants. FGFR-L polypeptide fragments may result from alternative RNA splicing or from in vivo protease activity. Membrane-bound forms of an FGFR-L polypeptide are also contemplated by the present invention. In preferred embodiments, truncations and/or deletions comprise about 10 amino acids, or about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than about 100 amino acids. The polypeptide fragments so produced will comprise about 25 contiguous amino acids, or about 50 amino acids, or about 75. amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids, or more than about 200 amino acids. Such FGFR-L polypeptide fragments may optionally comprise an amino-terminal methionine residue. It will be appreciated that such fragments can be used, for example, to generate antibodies to FGFR-L polypeptides.

[0090] The term “FGFR-L polypeptide ortholog” refers to a polypeptide from another species that corresponds to FGFR-L polypeptide amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. For example, mouse and human FGFR-L polypeptides are considered orthologs of each other.

[0091] The term “FGFR-L polypeptide variants” refers to FGFR-L polypeptides comprising amino acid sequences having one or more amino acid sequence substitutions, deletions (such as internal deletions and/or FGFR-L polypeptide fragments), and/or additions (such as internal additions and/or FGFR-L fusion polypeptides) as compared to the FGFR-L polypeptide amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 (with or without a leader sequence). Variants may be naturally occurring (e.g., FGFR-L polypeptide allelic variants, FGFR-L polypeptide orthologs, and FGFR-L polypeptide splice variants) or artificially constructed. Such FGFR-L polypeptide variants may be prepared from the corresponding nucleic acid molecules having a DNA sequence that varies accordingly from the DNA sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO: 4. In preferred embodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, or more than 100 amino acid substitutions, insertions, additions and/or deletions, wherein the substitutions may be conservative, or non-conservative, or any combination thereof.

[0092] The term “FGFR-L polypeptide derivatives” refers to the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, FGFR-L polypeptide fragments, FGFR-L polypeptide orthologs, or FGFR-L polypeptide variants, as defined herein, that have been chemically modified. The term “FGFR-L polypeptide derivatives” also refers to the polypeptides encoded by FGFR-L polypeptide allelic variants or FGFR-L polypeptide splice variants, as defined herein, that have been chemically modified.

[0093] The term “mature FGFR-L polypeptide” refers to an FGFR-L polypeptide lacking a leader sequence. A mature FGFR-L polypeptide may also include other modifications such as proteolytic processing of the amino-terminus (with or without a leader sequence) and/or the carboxyl-terminus, cleavage of a smaller polypeptide from a larger precursor, N-linked and/or O-linked glycosylation, and the like. An exemplary mature FGFR-L polypeptide is depicted by the amino acid sequence of either SEQ ID NO: 3 or SEQ ID NO: 6.

[0094] The term “FGFR-L fusion polypeptide” refers to a fusion of one or more amino acids (such as a heterologous protein or peptide) at the amino- or carboxyl-terminus of the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, FGFR-L polypeptide fragments, FGFR-L polypeptide orthologs, FGFR-L polypeptide variants, or FGFR-L derivatives, as defined herein. The term “FGFR-L fusion polypeptide” also refers to a fusion of one or more amino acids at the amino- or carboxyl-terminus of the polypeptide encoded by FGFR-L polypeptide allelic variants or FGFR-L polypeptide splice variants, as defined herein.

[0095] The term “biologically active FGFR-L polypeptides” refers to FGFR-L polypeptides having at least one activity characteristic of the polypeptide comprising the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5. In addition, an FGFR-L polypeptide may be active as an immunogen; that is, the FGFR-L polypeptide contains at least one epitope to which antibodies may be raised.

[0096] The term “isolated polypeptide” refers to a polypeptide of the present invention that (1) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is naturally found when isolated from the source cell, (2) is not linked (by covalent or noncovalent interaction) to all or a portion of a polypeptide to which the “isolated polypeptide” is linked in nature, (3) is operably linked (by covalent or noncovalent interaction) to a polypeptide with which it is not linked in nature, or (4) does not occur in nature. Preferably, the isolated polypeptide is substantially free from any other contaminating polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic or research use.

[0097] The term “identity,” as known in the art, refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”).

[0098] The term “similarity” is a related concept, but in contrast to “identity,” “similarity” refers to a measure of relatedness which includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, {fraction (10/20)} identical amino acids, and the remainder are all non-conservative substitutions, then the percent identity and similarity would both be 50%. If in the same example, there are five more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% ({fraction (15/20)}). Therefore, in cases where there are conservative substitutions, the percent similarity between two polypeptides will be higher than the percent identity between those two polypeptides.

[0099] The term “naturally occurring” or “native” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials which are found in nature and are not manipulated by man. Similarly, “non-naturally occurring” or “non-native” as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man.

[0100] The terms “effective amount” and “therapeutically effective amount” each refer to the amount of an FGFR-L polypeptide or FGFR-L nucleic acid molecule used to support an observable level of one or more biological activities of the FGFR-L polypeptides as set forth herein.

[0101] The term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of the FGFR-L polypeptide, FGFR-L nucleic acid molecule, or FGFR-L selective binding agent as a pharmaceutical composition.

[0102] The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen may have one or more epitopes.

[0103] The term “selective binding agent” refers to a molecule or molecules having specificity for an FGFR-L polypeptide. As used herein, the terms, “specific” and “specificity” refer to the ability of the selective binding agents to bind to human FGFR-L polypeptides and not to bind to human non-FGFR-L polypeptides. It will be appreciated, however, that the selective binding agents may also bind orthologs of the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, that is, interspecies versions thereof, such as mouse and rat FGFR-L polypeptides.

[0104] The term “transduction” is used to refer to the transfer of genes from one bacterium to another, usually by a phage. “Transduction” also refers to the acquisition and transfer of eukaryotic cellular sequences by retroviruses.

[0105] The term “transfection” is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell has been “transfected” when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et al., Basic Methods in Molecular Biology (Elsevier, 1986); and Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

[0106] The term “transformation” as used herein refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA. For example, a cell is transformed where it is genetically modified from its native state. Following transfection or transduction, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been stably transformed when the DNA is replicated with the division of the cell.

[0107] Relatedness of Nucleic Acid Molecules and/or Polypeptides

[0108] It is understood that related nucleic acid molecules include allelic or splice variants of the nucleic acid molecule of either SEQ ID NO: 1 or SEQ ID NO: 4, and include sequences which are complementary to any of the above nucleotide sequences. Related nucleic acid molecules also include a nucleotide sequence encoding a polypeptide comprising or consisting essentially of a substitution, modification, addition and/or deletion of one or more amino acid residues compared to the polypeptide in either SEQ ID NO: 2 or SEQ ID NO: 5. Such related FGFR-L polypeptides may comprise, for example, an addition and/or a deletion of one or more N-linked or O-linked glycosylation sites or an addition and/or a deletion of one or more cysteine residues.

[0109] Related nucleic acid molecules also include fragments of FGFR-L nucleic acid molecules which encode a polypeptide of at least about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids, or more than about 200 amino acid residues of the FGFR-L polypeptide of either SEQ ID NO: 2 or SEQ ID NO: 5.

[0110] In addition, related FGFR-L nucleic acid molecules also include those molecules which comprise nucleotide sequences which hybridize under moderately or highly stringent conditions as defined herein with the fully complementary sequence of the FGFR-L nucleic acid molecule of either SEQ ID NO: 1 or SEQ ID NO: 4, or of a molecule encoding a polypeptide, which polypeptide comprises the amino acid sequence as shown in either SEQ ID NO: 2 or SEQ ID NO: 5, or of a nucleic acid fragment as defined herein, or of a nucleic acid fragment encoding a polypeptide as defined herein. Hybridization probes may be prepared using the FGFR-L sequences provided herein to screen cDNA, genomic or synthetic DNA libraries for related sequences. Regions of the DNA and/or amino acid sequence of FGFR-L polypeptide that exhibit significant identity to known sequences are readily determined using sequence alignment algorithms as described herein and those regions may be used to design probes for screening.

[0111] The term “highly stringent conditions” refers to those conditions that are designed to permit hybridization of DNA strands whose sequences are highly complementary, and to exclude hybridization of significantly mismatched DNAs. Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of “highly stringent conditions” for hybridization and washing are 0.015 M sodium FGFR-Loride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodium FGFR-Loride, 0.0015 M sodium citrate, and 50% formamide at 42° C. See Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory, 1989); Anderson et al., Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL Press Limited).

[0112] More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agent) may also be used—however, the rate of hybridization will be affected. Other agents may be included in the hybridization and Washing buffers for the purpose of reducing non-specific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO4, (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL Press Limited).

[0113] Factors affecting the stability of DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by one skilled in the art in order to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:

Tm(° C.)=81.5+16.6(log[Na+])+0.41(% G+C)−600/N−0.72(% formamide)

[0114] where N is the length of the duplex formed, [Na+] is the molar concentration of the sodium ion in the hybridization or washing solution, % G+C is the percentage of (guanine+cytosine) bases in the hybrid. For imperfectly matched hybrids, the melting temperature is reduced by approximately 1° C. for each 1% mismatch.

[0115] The term “moderately stringent conditions” refers to conditions under which a DNA duplex with a greater degree of base pair mismatching than could occur under “highly stringent conditions” is able to form. Examples of typical “moderately stringent conditions” are 0.015 M sodium FGFR-Loride, 0.0015 M sodium citrate at 50-65° C. or 0.015 M sodium FGFR-Loride, 0.0015 M sodium citrate, and 20% formamide at 37-50° C. By way of example, “moderately stringent conditions” of 50° C. in 0.015 M sodium ion will allow about a 21% mismatch.

[0116] It will be appreciated by those skilled in the art that there is no absolute distinction between “highly stringent conditions” and “moderately stringent conditions.” For example, at 0.015 M sodium ion (no formamide), the melting temperature of perfectly matched long DNA is about 71° C. With a wash at 65° C. (at the same ionic strength), this would allow for approximately a 6% mismatch. To capture more distantly related sequences, one skilled in the art can simply lower the temperature or raise the ionic strength.

[0117] A good estimate of the melting temperature in 1M NaCl* for oligonucleotide probes up to about 20nt is given by:

Tm=2° C. per A−T base pair+4° C. per G−C base pair

[0118] *The sodium ion concentration in 6× salt sodium citrate (SSC) is 1M. See Suggs et al., Developmental Biology Using Purified Genes 683 (Brown and Fox, eds., 1981).

[0119] High stringency washing conditions for oligonucleotides are usually at a temperature of 0-5° C. below the Tm of the oligonucleotide in 6× SSC, 0.1% SDS.

[0120] In another embodiment, related nucleic acid molecules comprise or consist of a nucleotide sequence that is at least about 70 percent identical to the nucleotide sequence as shown in either SEQ ID NO: 1 or SEQ ID NO: 4, or comprise or consist essentially of a nucleotide sequence encoding a polypeptide that is at least about 70 percent identical to the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. In preferred embodiments, the nucleotide sequences are about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percent identical to the nucleotide sequence as shown in either SEQ ID NO: 1 or SEQ ID NO: 4, or the nucleotide sequences encode a polypeptide that is about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percent identical to the polypeptide sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.

[0121] Related nucleic acid molecules encode polypeptides possessing at least one activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.

[0122] Differences in the nucleic acid sequence may result in conservative and/or non-conservative modifications of the amino acid sequence relative to the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5.

[0123] Conservative modifications to the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5 (and the corresponding modifications to the encoding nucleotides) will produce a polypeptide having functional and chemical characteristics similar to those of FGFR-L polypeptides. In contrast, substantial modifications in the functional and/or chemical characteristics of FGFR-L polypeptides may be accomplished by selecting substitutions in the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5 that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0124] For example, a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for “alanine scanning mutagenesis.”

[0125] Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics, and other reversed or inverted forms of amino acid moieties.

[0126] Naturally occurring residues may be divided into classes based on common side chain properties:

[0127] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0128] 2) neutral hydrophilic: Cys, Ser, Thr;

[0129] 3) acidic: Asp, Glu;

[0130] 4) basic: Asn, Gln, His, Lys, Arg;

[0131] 5) residues that influence chain orientation: Gly, Pro; and

[0132] 6) aromatic: Trp, Tyr, Phe.

[0133] For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the human FGFR-L polypeptide that are homologous with non-human FGFR-L polypeptides, or into the non-homologous regions of the molecule.

[0134] In making such changes, the hydropathic index of amino acids may be considered.

[0135] Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. The hydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

[0136] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al., 1982, J. Mol. Biol. 157:105-31). It is known that certain amino acids maybe substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0137] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functionally equivalent protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0138] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions.”

[0139] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the FGFR-L polypeptide, or to increase or decrease the affinity of the FGFR-L polypeptides described herein. Exemplary amino acid substitutions are set forth in Table I. 1 TABLE I Amino Acid Substitutions Original Residues Exemplary Substitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe Lys Arg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, Norleucine

[0140] A skilled artisan will be able to determine suitable variants of the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 using well-known techniques. For identifying suitable areas of the molecule that may be changed without destroying biological activity, one skilled in the art may target areas not believed to be important for activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of an FGFR-L polypeptide to such similar polypeptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of the FGFR-L molecule that are not conserved relative to such similar polypeptides would be less likely to adversely affect the biological activity and/or structure of an FGFR-L polypeptide. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (conservative amino acid residue substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[0141] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in an FGFR-L polypeptide that correspond to amino acid residues that are important for activity or structure in similar polypeptides. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues of FGFR-L polypeptides.

[0142] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of FGFR-L polypeptide with respect to its three dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each amino acid residue. The variants could be screened using activity assays known to those with skill in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change would be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

[0143] A number of scientific publications have been devoted to the prediction of secondary structure. See Moult, 1996, Curr. Opin. Biotechnol. 7:422-27; Chou et al., 1974, Biochemistry 13:222-45; Chou et al., 1974, Biochemistry 113:211-22; Chou et al., 1978, Adv. Enzymol. Relat. Areas Mol. Biol. 47:45-48; Chou et al., 1978, Ann. Rev. Biochem. 47:251-276; and Chou et al., 1979, Biophys. J 26:367-84. Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40%, often have similar structural topologies. The recent growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within the structure of a polypeptide or protein. See Holm et al., 1999, Nucleic Acids Res. 27:244-47. It has been suggested that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will become dramatically more accurate (Brenner et al., 1997, Curr. Opin. Struct. Biol. 7:369-76).

[0144] Additional methods of predicting secondary structure include “threading” (Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl et al., 1996, Structure 4:15-19), “profile analysis” (Bowie et al., 1991, Science, 253:164-70; Gribskov et al., 1990, Methods Enzymol. 183:146-59; Gribskov et al., 1987, Proc. Nat. Acad. Sci. U.S.A. 84:4355-58), and “evolutionary linkage” (See Holm et aL, supra, and Brenner et al., supra).

[0145] Preferred FGFR-L polypeptide variants include glycosylation variants wherein the number and/or type of glycosylation sites have been altered compared to the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. In one embodiment, FGFR-L polypeptide variants comprise a greater or a lesser number of N-linked glycosylation sites than the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional preferred FGFR-L variants include cysteine variants, wherein one or more cysteine residues are deleted or substituted with another amino acid (e.g., serine) as compared to the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. Cysteine variants are useful when FGFR-L polypeptides must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0146] In other embodiments, related nucleic acid molecules comprise or consist of a nucleotide sequence encoding a polypeptide as set forth in either Seq Id No: 2 or SEQ ID NO: 5 with at least one amino acid insertion and wherein the polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or a

Claims

1. An isolated nucleic acid molecule comprising:

(a) the nucleotide sequence as set forth in SEQ ID NO: 4;

(b) a nucleotide sequence encoding the polypeptide as set forth in SEQ ID NO: 5;

(c) a nucleotide sequence which hybridizes under at least moderately stringent conditions to the complement of the nucleotide sequence of either (a) or (b), wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO: 5; or

(d) a nucleotide sequence complementary to the nucleotide sequence of any of (a)-(c).

2. An isolated nucleic acid molecule comprising:

(a) a nucleotide sequence encoding a polypeptide that is at least about 70 percent identical to the polypeptide as set forth in SEQ ID NO: 5, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 5;

(b) a nucleotide sequence encoding an allelic variant or splice variant of the nucleotide sequence as set forth in SEQ ID NO: 4, or the nucleotide sequence of (a);

(c) a region of the nucleotide sequence of SEQ ID NO: 4, or the nucleotide sequence of either (a) or (b), encoding a polypeptide fragment of at least about 25 amino acid residues, wherein the polypeptide fragment has an activity of the encoded polypeptide as set forth in SEQ ID NO: 5, or is antigenic;

(d) a region of the nucleotide sequence of SEQ ID NO: 4, or the nucleotide sequence of any of (a)-(c) comprising a fragment of at least about 16 nucleotides;

(e) a nucleotide sequence that hybridizes under at least moderately stringent conditions to the complement of the nucleotide sequence of any of (a)-(d), wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO: 5; or

(f) a nucleotide sequence complementary to the nucleotide sequence of any of (a)-(e).

3. An isolated nucleic acid molecule comprising:

(a) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO: 5 with at least one conservative amino acid substitution, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 5;

(b) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO: 5 with at least one amino acid insertion, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 5;

(c) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO: 5 with at least one amino acid deletion, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 5;

(d) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO: 5 which has a C- and/or N-terminal truncation, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 5;

(e) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO: 5 with at least one modification that is an amino acid substitution, amino acid insertion, amino acid deletion, C-terminal truncation, or N-terminal truncation, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 5;

(f) a nucleotide sequence of any of (a)-(e) comprising a fragment of at least about 16 nucleotides;

(g) a nucleotide sequence that hybridizes under at least moderately stringent conditions to the complement of the nucleotide sequence of any of (a)-(f), wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO: 5; or

(h) a nucleotide sequence complementary to any of (a)-(g).

4. A vector comprising the nucleic acid molecule of any of claims 1, 2, or 3.

5. A host cell comprising the vector of claim 4.

6. The host cell of claim 5 that is a eukaryotic cell.

7. The host cell of claim 5 that is a prokaryotic cell.

8. A process of producing an FGFR-L polypeptide comprising culturing the host cell of claim 5 under suitable conditions to express the polypeptide, and optionally isolating the polypeptide from the culture.

9. The process of claim 8, wherein the nucleic acid molecule comprises promoter DNA other than the promoter DNA for the native FGFR-L polypeptide operatively linked to the DNA encoding the FGFR-L polypeptide.

10. The isolated nucleic acid molecule according to claim 2, wherein the percent identity is determined using a computer program that is GAP, BLASTN, FASTA, BLASTA, BLASTX, BestFit, or the Smith-Waterman algorithm.