CA2347655A1 - Growth factor related molecules - Google Patents

Abstract

The invention provides human growth factor related molecules (GFRP) and polynucleotides which identify and encode GFRP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with expression of GFRP.

Description

GROWTH FACTOR RELATED MOLECULES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human growth factor related molecules and to the use of these sequences in the diagnosis.
treatment. and prevention of developmental disorders; cell proliferative disorders including cancer; immune disorders including inflammation; reproductive and cardiovascular disorders; and infections.
BACKGROUND OF THE INVENTION
Intercellular communication is essential for the development and survival of multicellular organisms. Communication is achieved through the secretion of proteins by signaling cells and the internalization of these proteins by target cells. Growth factors are an example of secreted proteins that mediate communication between signaling and target cells. Inside a signaling cell, growth factors are synthesized anti transported through the secretory pathway. Entry into a secretory pathway is I S mediated by a signal peptide sequence, a protein sorting motif at the N-terminus of most secreted proteins. Within the secretory pathway, the signal sequence is proteolytically removed from its cognate growth factor. Most growth factors also undergo further post-translational modifications within the secretory pathway. These modifications can include glycosylation, phosphorylation, and intramolecular disulfide bond formation. Following secretion into the extracellular space, some growth factors oligomerize or associate with extracellular matrix components.
Secreted growth factors bind to specific receptors on the surfaces of their target cells, and the bound receptors trigger second messenger signal transduction pathways. These signal transduction pathways elicit specific cellular responses in target cells. These responses can include the modulation of gene expression and the stimulation or inhibition of cell division, cell differentiation, and cell motility.
<!5 Most growth factors are local mediators that act on cells in the immediate environment. Such local activity is maintained by physical proximity of a signaling cell to its target cell, sequestration of the growth factor by extracellular matrix components, internalization and degradation of the growth factor by the target cell, and exclusion of the growth factor from circulation.
Growth factors fall into three broad and overlapping classes. The first and broadest class 30 includes the large polypeptide growth factors, which are wide-ranging in their effects. These factors include epidermal growth factor (EGh), fibroblast growth factor (FGF), transforming growth factor-~i (TGF-(3), insulin-like growth factor (IGF), nerve growth factor (NGF), and platelet-derived growth factor (PDGF), each defining a family of numerous related factors. The large polypeptide growth factors generally act as mitogens on diverse cell types to stimulate wound healing, bone synthesis and 35 remodeling, extracel'lular matrix synthesis. and proliferation of epithelial, epidermal, and connective tissues. Some members of the TtiF-Vii. E:GF, and FGF families also function as inductive signals in the differentiation ofembryonic tissue. However. some of the large polypeptide growth factors carry out specific functions on a restricted. set of target tissues. For example, mouse growth/differentiation factor 9 (GDF-9) is a TGF-(i family member that is expressed solely in the ovary (McPherron, A.C.
and S.-J. Lee (199:i) J. Biol. Chem. ~ti8:3444-3449). NGF functions specifically as a neurotrophic factor, promoting neuronal growth and differentiation.
Follistatin (FS) is a protein that specifically binds and inhibits activin, a member of the transforming growth factor-(3 family of growth and differentiation factors.
Activin performs a variety of functions associated with growth and differentiation, including induction of mesoderm in the developing embryo and regulation of female sex hormone secretion in the adult (de Krester, D.M.
(1998) J. Reprod. lmmunol. 39:1-12). Both activin and FS are found in many types of cells. The interaction of FS and activin influences a variety of cellular processes in the gonadal tissues, the pituitary gland, membranes associated with pregnancy, the vascular tissues.
and the liver (reviewed in Phillips, D.J. and D.M. de Krester ( 1998) Front. Neuroendocrinol. 19:287-322). FS may also play a IS direct role in the neuralization of embryonic tissue (Hemmati-Brivanlou et al. (1994) Cell 77:283-295).
FS is conserved among diverse species such as frog, chicken, and human.
Variants of human FS include a 288 arnino acid and a 3 ( 5 amino acid isoform (McConnell, D.S.
et al. ( 1998) J. Clin.
Endocrinol. Metab. 83:851-858). Most follistatins contain a conserved domain with ten regularly ~0 spaced cysteine residues. These residues are likely involved in disulfide bond formation and the binding of cations. Similar domains are observed in Kazal protease inhibitors and osteonectin (also called SPARC or BM-40), an extrace~llular matrix-associated glycoprotein expressed in a variety of tissues during embryogenesis and repair (reviewed in Lane, T.F. and E.H. Sage ( 1994) FASEB J.
8:163-173). Osteonectin contains not only an FS-like polycysteine domain, but also other modular :!5 domains that can function independently to bind cells and matrix components and can change cell shape by selectively disrupting cellular contacts with matrix. High levels of osteonectin are associated with developing bones and teeth, principally osteoblasts, odontoblasts, and perichondrial fibroblasts of embryos. Osteonectin modulation of cell adhesion and proliferation may also function in tissue remodeling and angiogenesis (Kupprion et al. ( 1998) J. Biol. Chem.
45:29635-29640).
3~0 FS is associated with a variety of cell proliferative, reproductive, and developmental disorders. Transgenic mice lacking F'S have multiple musculoskeietal defects and die shortly after ' birth (Matzuk, M.M. et al. ( 1995) Nature 374:360-363). Abnormal expression and localization of FS
have been implicated in benign prostatic hyperplasia and prostate cancer (Thomas, T.Z. et al. (1998) Prostate 34:34-43). The Follistatin-Related Gene, which encodes a protein with a FS-like 35 polycysteine domain, is associated with chromosomal translocations that may play a role in leukemogenesis (Hayette. S. ( 1998) Oncogene 16:2949-2954). In the inflammatory response. FS
increases the macrophage foam cell formation characteristic of early atherosclerosis (Kozaki. K. et al.
(1997) Arterioscler. Thromb. Vase. l3iol. 17:2389-2394).
The bone motphogenetic proteins (BMPs) are bone-derived factors capable of inducing S ectopic bone formation (Wozney, J.M. et al. (1988) Science 242:1528-1534).
BMPs are hydrophobic glycoproteins involved in bone generation and regeneration, several of which are related to the TGF-beta superfamily. 13MP-1, for example. appears to have a regulatory role in bone formation and is characterized by procollagen C-proteinase activity and the presence of an extracellular ''CUB"
domain. The CUB domain is composed of some I 10 residues containing four cysteines which probably form two disulfide bridges, and is found in a variety of functionally diverse, mostly developmentally regulated proteins (ExPASy PROSITE document PR00908).
The second class of growth factors includes the hematopoietic growth factors, which have a narrow target specificity. These factors stimulate the proliferation and differentiation of blood cells such as B-lymphocytes, T-lymphocytes, erythrocytes, platelets. eosinophils, basophils, neutrophils, 'IS macrophages, and stem cell precursors. 'these factors include the colony-stimulating factors (e.g., G-CSF, M-CSF, GM-CSF, and CSF l -3 ), erythropoietin, and the cyrtokines.
Cytokines comprise a family of signaling molecules that modulate the immune system and the inflammatory response. Cytokines are specialized hematopoietic factors secreted by cells ofthe immune system, usually leukocytes ('white blood cells), in response to external insults, such as tissue damage and viral or microbial infection. However, other tissues are also capable of secreting cytokines in response to disease or trauma. Cytokines function in tissue repair, inflammation, and modulation of the immune response. C:ytokines act as growth and differentiation factors that primarily affect cells of the immune system such as B- and T-lymphocytes, monocvtes, macrophages, and granuiocytes. Like other signaling molecules, cytokines bind to specific plasma membrane receptors and trigger intracellular signal transduction pathways which alter gene expression patterns.
There is considerable potential for the: use of cytokines in the treatment of inflammation and immune system disorders.
Cytokine structure and function have been extensively characterized in vitro.
Most cytokines are small polypeptides of about 30 kilodaitons or less. Over 50 cytokines have been identified from human and rodent sources. Examples of cytokine subfamilies include the interferons (IFN-a, -~3, and -y), the interleukins (ILl-IL13), the tumor necrosis factors (TNF-a and -(3), and the chemokines. Many cytokines have been produced using recombinant DNA techniques. and the activities of individual cytokines have been determined in vitro. These activities include regulation of leukocyte proliferation, differentiation. and motility.
The activity of an individual c:ytokine in vitro may not reflect the full scope of that cytokine~s activity in vivo. l~vtokines are not expressed individually in vivo but are instead expressed in combination with a multitude of other cytokines when the organism is challenged with a stimulus.
Together, these c.~tokines collectively modulate the immune response in a manner appropriate for that particular stimulus. Therefore. the physiological activity of a cytokine is determined by the stimulus itself and by compleX interactive ncaworks among co-expressed cytokines which may demonstrate both synergistic and antagonistic relationships. . , Recently. a unique cytokine has been isolated that appears to have ami-tumor activity in vitro (Ridge, R.J. and N.H. Sloane ( 19960 Cytokine 8:1-5). This cytokine, anti-neoplastic urinary protein (ANUP), was orif~inally purified as a dimer from human urine. ANUP was later classified as a l0 cytokine when localization studies demonstrated that it was expressed in human granulocytes. ANUP
inhibits the growth of cell lines derived from tumors of the breast, skin, lung, bladder, pancreas. and cervix. However, ANUP does not ;effect the growth of human non-tumor cell lines. The N-terminal 22 amino acids of ANUP comprise a signal peptide which is cleaved from the mature protein. The first nine amino acids of the mature' protein retain about 10% of the anti-tumor activity. In addition, ANUP contains a Ly-6/u-PAR sequence motif that is typical of certain cell surface glycoproteins.
This motif is characterized by a distinct pattern of six cysteine residues within a 50-residue consensus sequence. The Ly-6/u-PAR motif is found in the Ly-6 T-lymphocyte surface antigen and in the receptor (u-PAR) for urokinase-type plasminogen activator, an extracellular serine protease.
Chemokines comprise a cytokine subfamily with over 30 members. (Reviewed in Wells, T.N.C. and M.C. Peitsch (1997) J. Lcukoc. Biol. 61:545-550.) Chemokines were initially identified as chemotactic proteins that recruit monocvtes and macrophages to sites of inflammation. Recent evidence indicate s that chemokines may also play key roles in hematopoiesis and HIV-1 infection.
Chemokines are small proteins which range from about 6-15 kilodaltons in molecular weight.
Chemokines are further classified as C, CC, CXC, or CX3C based on the number and position of critical cysteine reaidues. The CC c:hemokines, for example, each contain a conserved motif consisting of two consecutive cysteines followed by two additional cysteines which occur downstream at 24- and 16-residue intervals, respectively (ExPASy PROSITE database, documents PS00472 and PDOC00434). Th.e presence and spacing of these four cysteine residues are highly conserved, whereas the intervening residues diverge significantly. However, a conserved tyrosine located about 15 residues downstream of the cysteine doublet seems to be important for chemotactic activity. Most of the human gene's encoding CC chemokines are clustered on chromosome 17.
although there are a few examples of (:C chemokine genes that map elsewhere.
Recently, a novel CC chernokine has been identified in mouse and human thymus (Vicari, A.P. et al. (1997) Immunity 7:291-301). This protein, called thymus-expressed chemokine (TECK), is also expressed at lower levels in the small intestine. 'PECK likely plays a role in T-lymphocye ~t development for two reasons. First, TE(_'K is most abundantly expressed in the thymus, which is the major lymphoid ocean where 'h-lymphocyte maturation occurs. Second, the primary source of TECK
in the thymus is dendritic cells, which are leukocytic cells that help establish self tolerance in developing T-lymphocytes. In addition, TECK demonstrates chemotactic activity for activated macrophages. dendritic cells, and thymic T-lymphocytes. The cDNA encoding human TECK
(hTECK) contains an open reading frame of 453 base pairs which predicts a protein of 151 amino acids. hTECK retains the conserved features of CC chemokines described above.
including four conserved cysteines at C30, C31, C58, and C75. However, the spacing between C31 and C58 is increased by three residues, and the spacing between C~8 and C75 is increased by one residue. In addition, hTECK lacks the conserved tyrosine found in most CC chemokines.
The third class of growth facaors includes the small peptide factors, which primarily function as hormones in the regulation of highly specialized processes other than cellular proliferation. These factors. which are typically less than 20 amino acids in length, are generated by the proteolytic processing of larger precursor proteiins. Some of these factors include bombesin, vasopressin.
oxytocin. endothelin, transferrin, angiotensin I1, vasoactive intestinal peptide, bradykinin, and related peptides. (See. e.g., Pimentel, E. ( 1994) Handbook of Growth Factors, CRC
Press, Ann Arbor MI;
MeKay, 1. and I. Leigh, eds. (1993) (Jrowth Factors: A Practical Approach, Oxford University Press, New York NY: and Habenicht, A., ed. ( 1990) Growth Factors, Differentiation Factors. and Cvtokines, Springer-Verlag, New York NY.) ZO The discovery of new human growth factor related molecules and the polynucleotides encodine them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of developmental disorders; cell proliferative disorders including cancer; immune disorders including .inflammation; reproductive and cardiovascular disorders: and infections.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, human growth factor related molecules, referred to collectively as ''GFRP" and individually as "GFRP-1,"
"GFRP-2," "GFRP-3,"
and "GFRP-4.'~ In one aspect, the invention provides a substantially purified polypeptide comprising :30 an amino acid sequence selected fronn the group consisting of SEQ ID NO:1-4 and fragments thereof.
The invention also iincludes a polypeptide comprising an amino acid sequence that differs by one or more conservative amino acid substitutions from an amino acid sequence selected from the group consisting of SEQ ID NO: I-4 The invention further provides a substantially purified variant having at least 90% amino acid :35 identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID

NO:1-~1 and fragments thereof. Tire invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising: an amino acid sequence selected from the group consisting of SEQ ID NO: l-4 and fragments thereof. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprisincan amino acid sequence selected from the group consisting of SEQ ID NO:1-4 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: l -a and fragments thereof. The invention also provides an isolated and purified poiynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1-4 .and fragments thereof.
The invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising; the steps of: (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample. In one aspect, the method further <:omprises amplifying the polynucleotide prior to hybridization.
The invention also provides an isolated and purified polynucleotide comprising a 0 polynucleotide sequence selected from the group consisting of SEQ ID NO:S-8 and fragments thereof.
The invention further provides an isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ 1D NO:S-8 and fragments therec>f. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:S-8 and fragments thereof.
The invention further provides an expression vector containing at least a fragment ofthe polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-4. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing a polynucleotide of the invention under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from thc: host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: l-4 3.5 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.

The invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ 1D NO: I-4 and fragments thereof. The invention also provides a purified agonist and a purilfied antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of GFRP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amuno acid sequence selected from the group consisting of SEQ ID
NO:1-4 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder associated with increased expression or activity of GFRI', the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: l-4 and fragments thereof.
BRIEF DESCRIPTION OF THE FIGURES AND TABLE
Figure 1 shows the amino acid sequence alignment between GFRP-1 (Incyte Clone 2777282;
SEQ ID NO: l ) and ANUP (GI 1536902; SEQ ID N0:9), produced using the multisequence alignment program of LASER.GENE software (DNASTAR, Madison WI).
Figure 2 shows the amino acid sequence alignment between GFRP-2 (Incyte Clone 4185824;
SEQ ID N0:2) and hTECK (GI 2388627; SEQ ID NO:10), produced using the multisequence :?0 alignment program of LASERGENE software (DNASTAR).
Figures 3A and 3B show the amino acid sequence alignment between GFRP-3 (Incyte Clone 2484440; SEQ ID Td0:3) and chicken follistatin (GI 853834; SEQ ID NO: l 1 ), produced using the multisequence alignment program of L,ASERGENE software (DNASTAR).
Figure 4 shows the partial amino acid sequence alignment between GFRP-4 (Incyte Clone 2.5 4163378; SEQ ID rI0:4) and human bone morphogenetic protein 1, BMP-1 (GI
179500; SEQ ID
N0:12), produced using the multisequence alignment program of LASERGENE
software (DNASTAR).
Table 1 shows the tools, programs, and algorithms used to analyze GFRP, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood tlaat the terminology used herein is for the purpose of describing 3:i particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be; noted that as used herein and in the appended claims. the singular forms "a." ''an,"
and "the" include plural reference unless the content clearly dictates otherwise. Thus, for example, a reference to "a host cell'' includes a plurality of such host cells, and a reference to "an antibody' is a S reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be l0 used to practice or test the present invention. the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
I S DEFINITIONS
''GFRP" refers to the amino acid sequences of substantially purified GFRP
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural. synthetic. semi-synthetic, or recombinant.
The term ''agonist" refers tc> a molecule which intensifies or mimics the biological activity of 20 GFRP. Agonists rnay include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of GFRP either by directly interacting with GFRP or by acting; on components of the biological pathway in which GFRP
participates.
An "allelic; variant" is an alternative form of the gene encoding GFRP.
Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in 25 polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
30 "Altered'' nucleic acid sequences encoding GFRP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as GFRP or a polypeptide with at least one functional characteristic of GFRP. Included within this definition are polymorphisms which may or may riot be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding GFRP. and improper or unexpected hybridization to allelic variants, ;5 with a locus other than the normaE chromosomal locus for the polynucleotide sequence encoding GFRP. The encoded protein may also be "altered.~~ and may contain deletions, insertions. or substitutions of arnino acid residue s which produce a silent change and result in a functionally equivalent GFRP. Deliberate am ino acid substitutions may be made on the basis of similarity in polarity, charge. solubility, hydrophobicity, hydrophilicity. and/or the amphipathic nature of the p residues, as long as the biological or immunological activity of GFRP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged .
amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilirity values may include: asparagine and glutamine: and serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine. and vaGine; glycine and alanine: and phenylaianine and tyrosine.
The terms "amino acid" and ''amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native: amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried ont using polymerase chain reaction (PCR) technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of GFRP. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of GFRP either by directly interacting with GFRP or by acting on components of the biological pathway in which GFRP
participates.
'_'S The term ".antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')=, and Fv fragments, which are capable of binding an epitopic determinant.
Antibodies that bind GFRP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse. a rat. or a rabbit) can be derived from the translation of RNA, :p0 or synthesized chennically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin.
thyroglobulin, and keyhole limpet hemocyanin (KLH). Tile coupled peptide is then used to immunize the animal.
The term "antigenic determinant' refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to :;s immunize a host animal. numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e.. the immunogen used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid sequence which is complementary to the ''sense" strand of a specific nucleic acid sequence.
Antisense molecules may be produced by any :method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation ''negative"
or''minus'' can refer to the antisense strand. and the designation "positive" or ''plus" can refer to the sense strand.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" refers to the capability of the natural, recombinant, or synthetic GFRP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
The terms "complementary" and "complementarily" refer to the natural binding of polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds to the complementary sequence "3' T-C-A :i'." Complementarily between two single-stranded molecules may be "partial,'' such that only some of the nucleic acids bind, or it may be "complete," such that total complementarily exists between the single stranded molecules. The degree of complementarily between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acid strands, and in the design and use of peptide nucleic acid (PNA) molecules.
A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid) sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotide sequences encoding GFRP or fragments of GFRP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCi), detergents (e.g., sodium dodecyl sulfate; SDS), and other components. (e.g.. Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence"' refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases. extended using the XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5' and/or the 3' direction. and resequenced, or which has been assembled from the overlapping sequences of one or more Incvte Clones and, in some cases. one or more public domain ESTs, using a computer program for fragment assembly. such as the GELVIEW fragment assembly system (GCG, Madison WI). Some sequences have been both extended and assembled to produce the consensus sequence.
''Conservative amino acid substitutions'' are those substitutions that, when made, least interfere with the properties of the original protein, i.e.. the structure and especially the function of the protein is conserved and not signitic,antly chanced by such substitutions.
'rhe table below shows S amino acids which may be substituted far an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
C)riginal Residue Conservative Substitution A'la Gly, Ser A.rg His, Lys i0 A.sn Asp, Gln, His A.sp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His 15 Gly Ala His Asn, Arg, Gln, Glu Ile I_eu, Val Leu lle, Val L.ys Arg, Gln, Glu a!0 Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Sf;r Cys, T'hr Thr Ser, Val Tip Phe, Tyr 2.5 T:~r His, Phe, Trp V;al Ile, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure ofthe polypeptide backbone in the area ofthe substitution, for example, as a beta sheet or alpha helical conformation, 30 (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid rep>idues or nucleotides.
The term ''derivative" refers to the chemical modification of a polypeptide sequence, or a 3.5 polynucleotide sequence. Chemical madifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one; biological or immunological function of the polypeptide from 40 which it was derived.
A "fragment" is a unique portion of GFRP or the polynucleotide encoding GFRP
which is identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up to the entire length of the defined secauence. minus one nucleotide/amino acid residue. For example, a fragment may comprise from ~ to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer. antigen, therapeutic molecule, or for other purposes, may be at least 5. 10, 15, 20, 25, 30, 40, 50, fi0, 75. 100. 150, ~'.j0 or at least X00 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, . .
a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or fi7rst 25% or 50% of a polypeptide) as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, .I O including the Sequence Listing, tables. and figures, may be encompassed by the present embodiments.
A fragment of SEQ ID NO:S-8 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:S-8, for example, as distinct from any other sequence in the same aenome. A fragment of SEQ ID NO:~-8 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:S-8 from related polynucleotide sequences. The precise length of a fragment of SEQ ID NO:S-8 and the region of SEQ ID NO:S-8 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose: for the fragment.
A fragment of SEQ ID NO:1-4 is encoded by a fragment of SEQ ID NO:S-8. A
fragment of SEQ ID NO:1-4 comprises a region of unique amino acid sequence that specifically identifies SEQ ID
NO:1-4. For example, a fragment of SEQ ID NO:1-4 is useful as an immunogenic peptide for the development of antibodies that speciliically recognize SEQ ID NO: l-4. The precise length of a fragment of SEQ ID NO:1-4 and the region of SEQ ID NO:1-4 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
2:i The term "similarity" refers to a degree of complementarity. There may be partial similarity or complete similarity. The word "identity' may substitute for the word ''similarity." A partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as "substantially similar." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot. solution hybridization, and the like) under conditions of reduced stringency. A substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. 'This is not to say that conditions of reduced stringency are such that non-specific binding is permitted. as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction. The absence of non-specific binding may be tested by the use of a second tarzet sequence which lacks even a partial degree of complementarity (e.g., less than about 30% similarity or identity). In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.
The phrases "percent identity°~ and "% identity." as applied to polynucleotide sequences, refer to the percentage of residue matches between at least nvo polynucleotide sequences aligned using a . .
standardized algorithm. Such an algorithm may insert. in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the tvvo sequences.
Percent identity between polynucleotide sequences may be determined using the default parameters of the C'LUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignmern: program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins. D.G, and P.M. Sharp ( 1989) CABIOS 5:151-1 ~3 and in Higgins, D.G. et al. { 1992) CABIOS
I S 8:189-191. For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, ;gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selecaed as the default. Percent identity is reported by CLUSTAL V as the ''percent similarity" between aligned polynucleotide sequence pairs.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center foir Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI. Bethesda, MD, and on the Internet at http://wwv.ncbi.nim~.nih~ov/BL,ASTJ. The BLAST software suite includes various sequence analysis programs including ''blastn," that is used to align a known poiynucleotide sequence with other polynucleotide sequences from a varieay of databases. Aiso available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http:/hwvw.ncbi.nlm.nih.eovi~orf/bl2.html. The "BLAST 2 Sequence's" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, once may use blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999)~ set at default parameters. Such default parameters may be, for example:
Matrix: BLG~SUM62 Reward for match. I
Peualtv for misnratch: -?
35~ Open Gap: .i and E.rrensiort Gup: ? penalties Gup x drop-off' ~0 E.rpect: l 0 GI'ord Si:.e: l l Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a paoicular SEQ lU number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance. a fragment of at least 20. at least 30. at least 40, at least >0. at least 70, at least 100. or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases ''percent identity' and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches bexween at least two polypeptide sequences aligned using a standardized algorithm. Methods of poiypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions.
Such conservative substitutions, explained in more detail above, generally preserve the hydrophobicity and acidity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between pol;ypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment: program (described and referenced above). For pairwise alignments of ~5 poiypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and ''diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table;. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example. for a pairwise comparison of two poiypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø9 (May-07-1999) with blastp set at default parameters. Such default parameters may be, for example:
Matri_r: BLI~SUM62 Open Gap: l l and E.rterrsion Gcrp: I penalties Gcrp x drop-off 50 3 5 Erpect: I 0 Word .Sire: 3 Filter: ofr Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ IU number. or may be measured over a shorter length, for example, over the length of a fragment taken trom a larger, defined polypeptide sequence, for instance. a fragment of at least 15, at least 20, at least 30, at least 40, at least 50. at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables. figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
''Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to ) 0 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human. antibody, and still retains its original binding ability.
"Hybridiza.tion" refers to the' process by which a polynucieotide strand anneals with a complementary str;~nd through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of identity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency ofthe hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for annealing of nuclei.:. acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 pg/ml denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Generally, such wash temperatures are selected to be about ?',0 5°C to 20°C lower than the thermal melting point (T,") for the specific sequence at a defined ionic strength and pH. The T", is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a pertectly matched probe. An equation for calculating T," and conditions for nucleic acid hybridization are well known and can be found in Sambrook et al., 1989, Molecular Clonine: A Laboratory Manual, 2"° ed., vol. 1-3, Cold Sprint Harbor Press, Plainview NY;
specifically see volume 2, chapter 9.

High stringency conditions for hybridization between polvnucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1 % SDS. for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x: SSC, with SDS being present at about 0.1%. Typically, blocking reagents are used t:o block non-specific hybridization. Such blocking reagents include, for instance.
denatured salmon sperm DNA at about 100-200 p.g/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides .and their encoded polypeptides.
The term "hybridization cornplex" refers to a complex formed between t<vo nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.b., C"t or R°t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, fitters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids Ihave been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation.
trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors. e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
The term "microarray' refer:, to an arrangement of distinct polynucteotides on a substrate.
~5 The terms "element" and "array element" in a microarray context, refer to hybridizable polynucleotides arranged on the surt;~ce of a substrate.
The term "modulate" refers to a change in the activity of GFRP. For example, modulation may cause an increase or a decrease in protein activity. binding characteristics. or any other biological, functional, or immunological properties of GFRP.
..0 The phrases "nucleic acid" and ''nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand. to peptide nucleic acid (PNA), or to any DNA-like or RNA-Pike material.
"Operably linked" refers to tire situation in which a first nucleic acid sequence is placed in a 35 functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding ~;equence if tire pre.~rnoter affects the transcription or expression of the codin;
sequence. Generality, operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid'' (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about ~ nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
"Probe" refers to nucleic acid( sequences encoding GFRP, their complements, or fragments 1'.0 thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
''Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairin<~. The primer may then be extended along the target IS DNA strand by a DPJA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used ins the present invention typically comprise at least 1 S contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 20 or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these: examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook c;t al., 1989, Molecular Clonine: A Laboratory Manual, 2"d ed., vol. 1-3, Cold 2.5 Spring Harbor Press., Plainview NY; Ausubel et al.,1987, Current Protocols in Molecular Bioloev, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis et al., 1990, PCR Protocols. A
Guide to Methods and Applications, Academic Press, San Diego CA. PCR primer pairs can be derived from a known sequence, for e;rample, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991. Whitehead Institute for Biomedical Research, Cambridge MA).
30 Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to x,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the 3_~ PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center. Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for desis~ning primers on a genome-wide scope.
The Primer3 primer selection program (available to the public from the Whitehead InstitutelMIT
Center for Genome Research. Cambridge MA) allows the user to input a wmispriming library,'' in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful. in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may _ .
also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers. microarray elements. or specific probes to identify fully or I S partially complementary polynucleotides in a sample of nucleic acids.
Methods of oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or has a sequence that is made by an ,artificial combination of two or more otherwise separated segments of sequence.
This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sam brook, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to ~5 transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector.
e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunologicai response in the mammal.
The term "sample" is used in its broadest sense. A sample suspected of containing nucleic acids encoding GFR'.P, or fragments thereof, or GFRP itself, may comprise a bodily fluid; an extract from a cell. chromosome, organelle, or membrane isolated from a cell: a cell;
genomic DNA, RNA, or cDNA, in solution or bound to a substrate: a tissue; a tissue print; etc.
The terms ".specific binding'' and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein. e.~~.. the antigenic determinant or epitope.
recognized by the binding molecule. For example, if an antibody is specific for epitope "A:" the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A. in a reaction containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the antibody.
The term w'substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels. tubing, plates. polymers.
microparticles and capillaries. The substrate can have a variety of surface forms. such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
''Transforrnation" describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and rnay include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term "transformed" cells includes stably transformed cells in 'which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells vrhich express the inserted DNA or RNA for limited periods of time.
A "variant" of a particular n~ucteic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at (east 50%, at least 60%, at least '10%, at least 80%, at least 85%. at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length. A variant may be described as, for example, an ''allelic'' (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due: to alternate splicing of exons during mRNA processing.
The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants. are polynucleotide sequences that vary from one species to another. The resulting polypeptides ;;enerally will have significant amino acid identity relative to each other. A pol:~morphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of. for example. a certain population. a disease state, or a propensity for a disease state.
A "variant" of a particular poiypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the: particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show. for example. at least 50%, at least 60%, at least 70%, at least 80°~0, at least 90%, at least 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human growth factor related molecules (GFRP), the polynucleotides encoding GFRP. and the use of these compositions for the diagnosis, treatment, or prevention of developnnental disorders; cell proliferative disorders including cancer;
immune disorders including inflammation; reproductive and cardiovascular disorders; and infections.
Nucleic acids encoding the GFRP- I of the present invention were identified in Incyte Clone 2777282H1 from the ovarian tumor cDNA library (OVARTUT03) using a computer search for nucleotide and/or amino acid sequence alignments. A consensus sequence, SEQ ID
N0:5, was derived from the following overlapping and/or extended nucleic acid sequences:
Incyte Clones 2777282H1 (OVARTUT03) and 277728276 (OVARTUT03).
In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: l . GFRP- I is 125 amino acids in length and has one potential N-glycosylation site at N46; two potential casein kinase II phosphorylation sites at 744 and S80; and one :?5 potential protein kinase C phosphorylation site at S122. MOTIFS analysis indicates that GFRP-I
contains a Ly-6/u-PAR domain signature from R24 to C73. Likewise, BLOCKS
analysis indicates that all three protein blocks which are characteristic of Ly-6/uPAR domains are found in GFRP-1 from L9 to C28, from Q87 to N J OU, and from P 105 to L J 18. SPSCAN and HMM
analyses indicate that GFRP-1 contains a signal peptide sequence from M 1 to either L19 or A22.
As shown in Figure I, :>0 GFRP-1 has chemical and structural similarity with ANUP (also known as ARS; GI 1536902; SEQ ID
N0:9). GFRP-1 and ANUP share 39% identity and, as shown in bold type, all eleven cysteine residues in ANUP are conserved in GF'RI'-1. GFRP-1 also contains an additional C-terminal cvsteine at CI 12. GFRP-I and ANUP are both acidic proteins with predicted isoelectric points of 5.8 and 5.2, respectively. Furthf~rmore, GFRP-I and ANUP are both relatively small proteins of 125 and 103 ~5 amino acids in length, respectively. The Ly-6/u PAR domain signature is also conserved between the two proteins. GFR.P-1 also has chemical and structural similarity with mouse ARS component B
precursor (G1 4218459). Fragments of SEQ ID NO:S fiom about nucleotide I68 to about nucleotide 197 and from about nucleotide 390 to about nucleotide 419 are useful in hybridization or amplifcation technologies to identify SEQ ID NO:S and to distinguish between SEQ ID NO:S and a related sequence. l~Iorthern analysis shows the expression of this sequence in cDNA libraries derived from ovarian tumor tissue and thymus.
Nucleic acids encoding the (iFRP-2 of the present invention were identified in Incyte Clone 4185824H 1 from the breast tissue cDNA library (BRSTNOT31 ) using a computer search for nucleotide and/or amino acid sequence alignments. A consensus sequence, SEQ ID
N0:6, was derived from the following overlapping and/or extended nucleic acid sequences:
Incyte Clones 4185824H I (BRS'INOT31 ) and 4185824F6 (BRSTNOT3 I ).
In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID N0:2. GFRP-2 is 127 amino acids in length and has one potential N-glycosylation site at N78; one potential cAMP- and cGMP-dependent protein kinase phosphorylation I S site at S I 10; and three potential pratc:in kinase C phosphorylation sites at 539, T80, and S 110.
SPSCAN and HMM analyses indicate that GFRP-2 contains a signal peptide sequence from M1 to either A 19 or A22. Analysis of GFRP-2 suggests that this protein is a CC
chemokine. The predicted molecular weight of GFRP-2 is 14.3 kilodaltons, which is typical of chemokines. The amino acid sequence of GFRP-2 from C30 to VT4 shows strong similarity to the CC chemokine consensus 0 sequence (ExPASy PROSITE database, documents PS00472. PDOC00434). The four cysteines which are characteristic of this consensus sequence are conserved at C30, C31, C58, and C73. All but one of the remaining amino acids in this region match the consensus sequence.
The spacing between C31 and C58 and bc;tween C58 and C'73 is slightly altered from the consensus sequence. However, similar alterations in spacing have been observed in other CC chemokines such as hTECK. As .'S shown in Figure 2, GFRP-2 has chemical and structura) similarity with hTECK (GI 2388627; SEQ ID
NO:10). In particular, GFRP-2 and hTEC:K share 20% identity, including the four cysteines which are characteristic of the CC chemokine consensus sequence (shown in bold type). In addition, GFRP-2 and hTECK are both basic proteins with predicted isoelectric points of 10.1 and 10.2, respectively.
GFRP-2 also has ch~emica) and structural similarity with human Dvic-1 C-C
chemokitie (GeneSeq ID
30 W60649) and with rnouse CC chemokine ALP (GI 4140686). A fragment of SEQ ID
N0:6 from about nucleotide 28'7 to about nucleotide 316 is useful in hybridization or amplification technologies to identiy SEQ ID 1J0:6 and to distinguish between SEQ ID N0:6 and a related sequence. Northern analysis shows the expression of this sequence in five cDNA libraries, four of which are derived from normal or breast tumor tissue.
35 Nucleic acids encoding the GFRP-3 of the present invention were identified in Incyte Clone 2484440 from the smooth muscle cell cDNA library (SMCANOTOI ) using a computer search for nucleotide andior .amino acid sequence alignments. A consensus sequence. SEQ
ID N0:7, was derived from the following overlapping and/or extended nucleic acid sequences:
Inevte Clones 2484440H1 (SMCANOT01), 4763347H1 (PLACNOT05), 96185281 (BRSTTUT03), 2026240X20C1(KERANOT02), 86'7727Ri (BRAITUT03), 1340443F1 (COLNTUT03), and 151312771 (PAN(JTUTO 1 ) and 1513 I27F I (PANCTUTO 1 ).
In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID N0:3. GFRP-3 is 147 amino acids in length and has a potential N-glycosylation site at residue N99, two potential casein kinase II phosphorylation sites at residues 7130 and S139, and seven potential N-myristoylatioin sites at residues G4, G7, G8, G 10, G24, G27, and G3 I .
BLOCKS analysis indicates that the regions of GFRP-3 from residue E53 to P85 and from residue G91 to 5126 share homology with an osteonectin domain. PROFILESCAN analysis likewise indicates that the region of GFRP~3 from residue Q64 to C 106 shares homology with an osteonectin domain. PFAM analysis indicates that the region of GFRP-3 from residue C76 to C 113 shares IS homology with a K:azal-type serine protease inhibitor domain. The regions identified by BLOCKS, PROFILESCAN, and PFAM comprise a domain with similarity to the polycysteine follistatin domain.
GFRP-3 contains a predicted signal peptide from residue M 1 to G31. As shown in Figures 3A and 3B, GFRP-3 has chemical and structural similarity with chicken follistatin (GI
853834; SEQ ID
NO:11 ). In particular, GFRP-3 and chicken follistatin share 33% identity. In addition, the region of GFRP-3 from C55 to G 120 shares 6l'; % identity with the region of follistatin from C167 to 6231.
This region contains nine of the ten c:ysteine residues characteristic of the follistatin domain, with nearly identical spacing patterns. GFRP-3 also has chemical and structural similarity with human foilistatin-related protein FLRG (CSI 3764055). A fragment of SEQ ID N0:7 from about nucleotide 110 to about nucleotide 154 is useful, for example, in hybridization or amplification technologies to a5 identify SEQ ID N(~:7 and to distinguish between SEQ ID N0:7 and a related sequence. The encoded polypeptide is useful, for example, as an immunogenic peptide. Northern analysis shows the expression of this sequence in various libraries. at least 75% of which are associated with cell proliferation, and at least 23% are associated with inflammation. Of particular note, at least 29% of the libraries expressing GFRP-3 are associated with reproductive tissue.
:10 Nucleic acids encoding the GFRP-4 of the present invention were identified in lncyte Clone 4163378 from the diseased breast tissue cDNA library (BRSTNOT32) using a computer search for nucleotide and/or amino acid sequen<:e alignments. A consensus sequence. SEQ
ID N0:8, was assembled from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 4163378H I and 41 (i3378F6 (BRSTNOT32), 3598059F6 (FIBPNOTO l ). 2962366H 1 (ADRENOT09), '~5 3325672H2 (PTHYNOT03), 307370:iH 1 (BONEUNTO1 ), 1302516F1 (PLACNOT02), (OVARNOT09). 877279T1 (LUNGASTOI ), and 3094133 F6 (BRSTNOT19).
In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID N0:4. GFRP-4 is 345 amino acids in length and has three potential N-glycosylation sites at residues N25. N55, and N254: seven potential casein kinase II phosphorylation sites at T20, S34. T89, S 194. T195, 5258, and S323: and six potential protein kinase C
phosphorylation sites at S27, S3~=1, 560, T251, S258, and T302. GFRP-4 also contains a potential _ , signal peptide between residues M 1 and A14. as determined by HMM analysis: a PDGF family signature between residues F229 and Q310, as determined by ProfileScan; and a CUB domain between residues :R48 and Y 160 and a PDGF domain between residues 1269 and C337. both determined by PF.4M analysis. As shown in Figure 4, GFRP-4 has chemical and structural similarity with human bone morphogenetic protein l, BMP-1 (GI 179500; SEQ ID N0:12). In particular, GFRP-4 and BMP-1 share 27% identity near the C-terminus of BMP-1 between residues 599 and 718 of BMP-1. This region of GFRP-4 encompasses the CUB domain identified above between residues 48 to 160 and includes two cvsteine residues at Ci04 and C124, also shared by BMP-1, that are proposed to be involved in cysteine-cysteine disulfide bridging. GFRP-4 also has chemical and structural similarity with chicken bane morphogenetic protein 1 (GI 2852121).
A fragment of SEQ
ID N0:8 from about nucleotide 420 to about nucleotide 467 is useful, for example. in hybridization or amplification technologies to identify SEQ ID N0:8 and to distinguish between SEQ ID N0:8 and a related sequence. 'The encoded polypeptide is useful, for example, as an immunogenic peptide.
Northern analysis shows the expression of this sequence in various libraries, at least 57% of which are associated with cancer and at least 30% of which are associated with the inflammation and the immune response. Of particular note is the expression of GFRP-4 in reproductive and cardiovascular tissue.
The invention also encompasses GFRP variants. A preferred GFRP variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the GFRP amino acid sequence, and which contains at least one functional or structural characteristic of GFRP.
The invention also encompasses polynucleotides which encode GFRP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected :30 from the group consisting of SEQ ID~ N0:5-8, which encodes GFRP.
The invention also encompa<.;ses a variant of a polynucleotide sequence encoding GFRP. In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%. or even at least abo~.n 95% polynucleotide sequence identity to the polynucleotide sequence encoding GFRP. A particular aspect of the invention encompasses a variant of a ~~5 polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:S-?3 8 which has at least about 70%. or alternatively at least about 85%. or even at least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:S-8. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of GFRP.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of poiynucleotide sequences encoding GFRP, some bearing minimal similarity to the poiynucleotide sequences of any known and naturally occurring gene, may be produced. Thus. the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring GFRP. and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode GFRP and its variants are generally capable of hybridizing to the nucleotide sequence of the natural ly occurring GFRP under appropriately selected I S conditions of stringency, it may be advantageous to produce nucleotide sequences encoding GFRP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding GFRP and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode GFRP
and GFRP derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding GFRP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID
.'.0 NO:S-8 and fragments thereof under various conditions of stringency.
(See, e.g., Wahl, G.M. and S.L. Berger ( 1987) IVlethods Enzymol. 152:399-407; Kimmel, A.R. ( I 987) Methods Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencini; are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment ~~t of DNA polymerase 1. SEQUENAS1~ (US Biochemical. Cleveland OH). Taq polymerase (Perkin-Elmer}, thermostable T7 polymerase (Amersham Pharmacia Biotech. Piscataway NJ), or combinations of polymerases and proofreading exonucieases such as those found in the ELONGASE
amplification system (Life Technologies. Gaithersburg MD). Preferably, sequence preparation is automated with machines such as the; MICROLAB ?200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Researclh. Watertown MA) and ABl CATALYST 800 thermal cycler (Perkin-Elmer). Sequencing is then harried out using either the ABI 373 or 377 DNA sequencing system (Perkin-Elrner), the MEGAB.ACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvaie CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are wel I known in the art. ( See, e.g., Ausubel, F.M. ( 1997) Short Protocols in Molecular BioloQV, John Wiley & Sons, New York NY, unit 7.7;
Meyers, R.A. ( 1995) Molecular Biolosv and Biotechnolo~,y, Wiley VCH, New York NY, pp. 856-853.) The nucleic acid sequences encoding GFRP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, L5 such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. ( 1993) PCR Methods Applic. 2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising f.0 a known genomic locus and surrounding sequences. (See, e.g., Triglia, T.
et al. ( 1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.
(1991) PCR Methodls Applic. 1:1 1 I-I 19.) In this method. multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown 25 sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parke;r, J.D. et al. ( 1991 ) Nucleic Acids Res. 19:3055-3060).
Additionally, one m,ay use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using 30 commercially available software, such as OLIGO 4.06 Primer Analysis software (National 8iosciences. Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about ~0% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
When screening for full-length cDNAs. it is preferable to use libraries that have been 3:5 size-selected to include larger cDNAs. In addition. random-primed libraries. which often include ~J

sequences comaining the ~' regions of genes. are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into ~' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may ernploy tlowable polymers for electrophoretic separation. four different nucleotide- . , specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GE1VOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
Capillary electrophoresis is especially preferable for sequencing small DNA
fragments which may be present in limited amounts in a particular sample.
In another embodiment of~ the invention, polynucleotide sequences or fragments thereof which encode GFRP may be cloned in recombinant DNA molecules that direct expression of GFRP, or fragments or functional equivalents thereof; in appropriate host cells. Due to the inherent degeneracy of the frenetic code, other DNA sequences which encode substantially the same or a functionally equiva.ient amino acid sequence may be produced and used to express GFRP.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter GFRP-encoding sequences for a variety of purposes including, but :?0 not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA
shuffling by randorn fragmentation and PCR reassembiy of gene fragments and synthetic oligonucleotides m;ay be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosyl.ation patterns. change codon preference, produce splice variants, and so forth.
In another c;mbodiment, sequences encoding GFRP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. ( 1980) Nucleic Acids Symp. Ser. 7:21 S-223; and Horn. '1. ca al. ( 1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively, GFR;P itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques. (See, e.g., .~0 Roberge, J.Y. et al. ( 1995) Science 2<9:202-204.) Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (Perkin-Elmer). Additionally, the amino acid sequence of GFRP, or any part thereof may be altered during direct synthesis and/or combined with sequences from other proteins. or any part. thereof, to produce a variant polypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography. (See. e.g., Chiez. R.M. and F.Z. Regnier ( 1990) Methods Enzymol. I 82:392-421.) The composition o~~the synthetic peptides may be confirmed by amino acid analysis or by sequencing.
(See, e.~., Creighton. T. (1984) Proteins. Structures and Molecular Properties, WH Freeman, New York NY.) In order to express a biologically active GFRP, the nucleotide sequences encoding GFRP or derivatives thereof may be inserted into an appropriate expression vector, i.e.. a vector which contains the necessary elements for transcriptiional and translational control of the inserted coding sequence in . , a suitable host. These elements include regulatory sequences. such as enhancers. constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynueleotide sequences encoding GFRP. Such elements may vary in their strength and specificity.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding GFRP. Such signals include the ATG initiation codon and. adjacent sequences. e.g. the Kozak sequence. In cases where sequences encoding; GFRP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector. no additional transcriptional or translational control signals may be needed. However. in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translationa) control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-16 ~.) 2.0 Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding GFRP and appropriate transcriptional and trans(ational control elements. These mc;thods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g:., Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press. Plainview NY, ch. =1. 8. and 16-17; Ausubel, F.M. et al. ( 1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding GFRP. These include, but are not limited to. microorganisms such as bacteria transformed with recombinant ba.cteriophage, plasmid. or cosmid DNA expression vectors;
yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus);
3~0 plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic viru,~, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding GFRP. For example, routine cloning, 3:i subcloning. and propagation of polynucleotide sequences encoding GFRP can be achieved using a ?7 multifunctional E. coli vector such as PBLUESCRIPT (Strata~;ene, La Joila CA) or PSPORTI
plasmid (Life Technologies). Li~.:ation of sequences encodin~~ GFRP into the vector's multiple cloning site disrupts the lacZ gene. Hallowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing. single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke. G. and S.M.
Schuster ( 1989) J. Biol. . .
Chem. 264:5503-5509.) When largE: quantities of GFRP are needed, e.g. for the production of antibodies, vectors which direct high level expression of GFRP may be used.
For example, vectors containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of GFRP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia~astoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel.
1995, supra; Bitter, G.A. et al. ( 1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et al. ( I994) Bio/Technology 12:181-184.) Plant systems may also be used for expression of GFRP. Transcription of sequences encoding GFRP many be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
~0 6:307-311 ). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., C.'oruzzi, G. et al. ( 1984) EMBO J. 3:1671-1680; Brogue, R. et al.
(1984.) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hiil Yearbook of Science and Tecfrnolo~y .!5 (1992) McGraw Hill, New York NY, pp. 191-196.) In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding GFRP
may be ligated into an adenovirus transc:ription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E 1 or E3 region of the viral genome may be used to obtain 30 infective virus which expresses GFRf in host cells. (See, c.g., Logan, J, and T. Shenk ( 1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition. transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of 35 DNA than can be contained in and eY~,pressed from a plasmid. I-iACs of about 6 kb to 10 Mb are ?8 constructed and df:livered via conventional delivery methods (liposomes.
polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.<~.. Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.) For lone term production o1'recombinant proteins in mammalian systems. stable expression of GFRP in cell lines is preferred. For example. sequences encoding GFRP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent. and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine IS phosphoribosyltransferase genes, for use in tk and apr cells, respectively.
(See, e.g., Wigler, M. et al. (1977) Cell 11: 223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic.
or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. ( 1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. ( 1981 ) J. Mol. Biol. 150: I -14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan ( 1988) Proc.
Natl. Acad. Sci. USA 85:8047-80.51.) Visible markers. e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), f3 glucuronidase and its substrate f3-glucuronide, or luciferase and its substrate :?5 luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes. C.A. (1995) Methods Mol. Biol. SS:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the :30 sequence encoding GFRP is inserted within a marker gene sequence.
transformed cells containing sequences encoding: GFRP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding GFRP under the control of a single promoter. Expression of the marker ;~:ene in response to induction or selection usually indicates expression of the tandem gene as well.
?;5 In general. host cells that contain the nucleic acid sequence encoding GFRP and that express ?9 GFRP may be identitied by a variety of procedures known to those of skill in the art. These procedures include. but are not limited to. DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include membrane, solution. or chip based technoloeies for the detection and/or quantification of nucleic acid or protein sequences. .
Immunologicai methods for detecting and measuring the expression of GFRP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs). radioimmunoassays (RIAs), and fluorescence activated cell sorting: (FAGS). A two-site. monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on GFRP is preferred, but a t0 competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. ( 1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997),Current Protocols in Immunolosy, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) lmmunochemical Protocols, Humana Press, Totowa NJ. ) A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding GFRP
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, the sequences encoding GFRP, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerise such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding GFRP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence :30 and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode GFRP may be designed to contain signal sequences which direct secretion of GFRP through a prokaryotic or eukaryotic cell membrane.
In addition. a host cell strain rnay be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of :~5 the polypeptide include, but are not limited to. acetylation.
carboxylation, glycosylation, phosphorylation. lipidation. and acylatian. Post-translational processing which cleaves a "prepro'~ or "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CI-(O, HeLa. MDCK. HEK293, and WI38) are available from the S American Type Culture Collection (ATCC. Manassas VA) and may be chosen to ensure the correct modification and processing of the fbreign protein. _ .
In another embodiment of tlne invention, natural. modified, or recombinant nucleic acid sequences encoding GFRP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric GFRP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of GFRP activity.
Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thio~redoxin (Trx), calmodulin binding peptide (CBP). 6-His, FLAG, l5 c-rnyc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-nryc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a :?0 proteolytic cleavage: site located between the GFRP encoding sequence and the heterologous protein sequence, so that GFRP may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10).
A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
~:S In a further embodiment of the invention, synthesis of radiolabeled GFRP
may be achieved in vitro using the TNT rabbit reticulocyl:e lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 'SS-methionine.
30 Fragments of GFRP may be produced not only by recombinant means, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton. supra.
pp. 5~-60.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the ABI 431 A peptide synthesizer (Perkin-Eimer).
Various fragments of GFRP may be synthesized separately and then combined to produce the full length molecule.
3.5 THERAPEUTICS

Chemical and structural similarity. e.~~.. in the context of sequences and motifs. exists between regions of GFRP amd human growth factor related molecules. In particular, chemical and structural similarity exists bcaween regions of GFRP-1 and ANUP. between regions of GFRP-2 and CC
chemokines, beriveen regions of <.iFRP-3 and chicken follistatin, and behveen GFRP-4 and growth factor related molecule. In addition, the expression of GFRP-1 is closely associated with ovarian tumor and thymic tissue; the expression of GFRP-2 is closely associated with tumorous and nontumorous breast tissue: the expression of GFRP-3 is closely associated with cell proliferation and inflammation, and with reproductive; tissue: and the expression of GFRP-4 is closely associated with cancer, inflammation and the immune response, and with reproductive and cardiovascular tissue.
Therefore. GFRP appears to play a role in developmental disorders; cell proliferative disorders including cancer: immune disorders including inflammation; reproductive and cardiovascular disorders; and infeeaions. In the treatment of disorders associated with increased GFRP expression or activity, it is desirable to decrease the expression or activity of GFRP. In the treatment of disorders associated with decreased GFRP expression or activity. it is desirable to increase the expression or activity of GFRP.
Therefore. in one embodiment, GFRP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of GFRP. Lxamples of such disarders include, but are not limited to, a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwa~sm, Duchenne and Beck:er muscular dystrophy, epilepsy, gonadal dysgenesis. WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myeiodysplastic syndrome, hereditary mucoepitheiial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-'Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spins bifida, ?.5 anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and a cancer including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular. a cancer of the adrenal gland. bladder, bone, bone marrow, brain.
breast, cervix, gall bladder, ganglia. gastrointestinal tract, heart, kidney, liver. lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen. testis. thymus, thyroid, and uterus; an immune disorder, such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease. adult respiratory distress syndrome. allergies, ankylosing spondylitis. amyloidosis, anemia, 3:i arteriosclerosis. asthma. atherosclerosis, autoimmune hemolytic anemia.
autoimmune thyroiditis, 3~

WO 00/2477f. PCT/US99/25458 bronchitis, bursitis. cholecystitis. ciirrhosis. contact dermatitis, Crohn's disease. atopic dermatitis.
derrrratomyositis. diabetes mellitus. emphysema. erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomeru~lonephritis. Goodpasture~s syndrome, gout. Graves' disease, Hashimoto's thyroiditis. paroxysmal nocturnal hernoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome. episodic lymphopenia with lymphocytotoxins. mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome. rheumatoid arthritis, sclexoderma. Sjogren's syndrome, systemic anaphylaxis, systemic Lupus erythematosus, systemic sclerosis, primary thrombocythemia.
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation. trauma, amd hematopoietic cancer including lymphoma, leukemia, and myeloma: a reproductive disorder, such as a disorder of prolactin production, infertility, including tubal disease, ovuLatory defects, and endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial I S or ovarian tumor, a~ uterine fibroid, autoimmune disorders, an ectopic pregnancy. and teratogenesis;
cancer of the breast, fibrocystic breast disease, and galactorrhea; a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis. Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia; a cardiovascular disorder, such as conl;estive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, conl;enitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of :'S cardiac transplantation; an infection such as that caused by a viral agent classified as adenovirus, arenavirus. bunyavirus, calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus. rhabdovirus, or togavirus; an infection such as that caused by a bacterial agent classified as pneumococcus, staphylococcus, streptococcus. bacillus, corynebacterium, clostridium, meningococcus, gonococcus, listeria, moraxella, kingella. haemophilus, legionella, bordetella, gram-negative enterobacte;rium including shigella, salmonella, and campylobacter, pseudomonas, vibrio, brucella, francisella. yersinia, bartonella, norcardium, actinomyces, mycobacterium. spirochaetale, rickettsia, chlamydur, or mycoplasma;: an infection such as that caused by a fungal agent classified as aspergillus, blastomyces. dermatophytes, cryptococcus. coccidioides, malasezzia. histoplasma, or other fungal agents causing various mycoses: and an infection such as that caused by a parasite classified as plasmodium or malaria-causing. parasitic entamoeba. leishmania, trypanosoma, toxoplasma, pneumocystis carinii, intestinal protozoa such as ~iardia.
trichomonas, tissue nematodes such as trichinella. intestinal nematodes such as ascaris, lymphatic filarial nematodes, trematodes such as schistosoma, or cestrodes such as tapeworm.
In another embodiment. a vector capable of expressing GFRP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of GFRP including, but not limited to, those described above.
In a further embodiment. a pharmaceutical composition comprising a substantially purified GFRP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of GFRP
including, but not limited to, those provided above.
In still another embodiment. an agonist which modulates the activity of GFRP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of GFRP including, but not Limited to, those listed above.
IS In a further embodiment, an antagonist of GFRP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of GFRP.
Examples of such disorders include, but are not limited to, those developmental disorders; cell proliferative disorders including cancer; immune disorders including inflammation; reproductive and cardiovascular disorders; and infecaions described above. In one aspect, an antibody which specifically binds GFRP
?0 may be used directlly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express GFRP.
In an additional embodiment, a vector expressing the complement of the poiynucleotide encoding GFRP ma.y be administerecl to a subject to treat or prevent a disorder associated with increased expression or activity of GFRP including, but not limited to, those described above.
'S In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, accarding to conventional pharmaceutical principles. The combination of therapeutic agents ma:y act synergistically to effect the treatment or prevention of the 30 various disorders described above. Using this approach. one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of GFRP may be produced using methods which are generally known in the art. In particular. purified GFRP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind GFRP.
Antibodies to GFRP may also 35 be generated using methods that are well known in the art. Such antibodies may include, but are not limited to. polvclonal, monoclonal. chimeric. and sin~~le chain antibodies.
Fab fragments, and fragments produced by a Fab expression library. Neutralizin~~ antibodies (i.e., those which inhibit dimer formation) ;are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats. rabbits, rats. mice, humans, and others may be immunized by injection with GFRP or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species. various adjuvants may be used to increase immunological response. !inch adjuvants include, but are not limited to. Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic potyols, polyanions. peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corvnebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to GFRP have an amino acid sequence consisting of at least about ~ amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of GFRP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to GFRP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.,g., Kohter, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. ( 1983) I'roc. Natl. Acad. Sci.
USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.) In addition.. techniques developed for the production of ''chimeric antibodies," such as the ~5 splicing of mouse antibody genes to Ihuman antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison. S.L. et al. ( 1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. ( 1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies many be adapted, using methods known in the art, to produce GFRP-specific single :.0 chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R. ( 1991 )~ Proc. Natl. Acad. Sci. USA 88:101 34-10137.) Antibodies ,may also be produced by inducing in vivo production in the lymphocyte population or by screening immunaglobulin libraries or panels of highly specific binding reagents as 35 disclosed in the literature. (See, e.g., Orlandi. R. et al. ( 1989) Proc.
Natl. Acad. Sci. USA

86:3833-3837: Winter. G. et al. ( 1591 ) Nature 349:293-299.) Antibody fragments which contain specific binding sites for GFRP may also be generated.
For example, such fragments include. but are not Limited to, F(ab')= fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of s the F(ab')2 fragments. Alternatively. Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D. _ , et al. (1989} Science 246:1275-1281.) Various irnmunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols fo:r competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between GFRP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering GFRP epitopes is generally used, but a competitive binding assay may also be employed (Pound, s-,upra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for GFRP. Affinity is expressed as an association constant, K~, which is defined as the molar concentration of GFRP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
The K~ determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple GFRP epitopes, represents the average affinity, or avidity, of the antibodies for GFRP. The Ke determined for a preparation of monoclonal antibodies, which are monospecific for a particular GFRP epitope, represents a true measure of affinity. High-affinity antibody preparations with K~ ranging from about 10'to 10'' L/mole are preferred for use in immunoassays in which the GFRP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations ?5 with Ka ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of GFRP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach, IRL
Press, Washington, DC:
Liddell, J.E. and Cryer, A. (1991 ) ,A Practical Guide to Monoclonal Antibodies. John Wiiey & Sons, New York NY}.
:30 The titer and avidity of polyc:lonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least I-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of GFRP-antibody complexes. Procedures fbr evaluating antibody specificity, titer, and avidity, and guidelines ~5 for antibody quality and usage in various applications. are generally available. (See, e.g., Catty, su ra, and Coligan et al. supra.) In another embodiment of the invention, the polynucleotides encoding GFRP, or any fragment or complement thc;reot: may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding GFRP may be used in situations in which it would be desirable to block the transcription of the: mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides en~codin~~ GFRP. Thus, complementary molecules or fragments may be used to modulate GFRP activity, or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisevse oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding GFRP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids. may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding GFRP. (See, e.g., !iambrook. supra; Ausubel. 1995. supra.) Genes encoding GFRP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucieotide, or fragment thereof.
encoding GFRP. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the D1~IA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more :!0 with a non-replicating vector, and maiy last even longer if appropriate replication elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA. RNA. or PNA) to the control, 5', or regulatory regions of the gene encoding GFRP. Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +I O from the start site, may be employed.
Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors. or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E.
et al. (1994) in Huber, 3n B.E. and B.I. Carr, Molecular and Imrnunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp.
163-177.) A comple~mentarv sequence or antisense molecule may also be designed to block translation of mRNA, by preventing the transcript from binding to ribosomes.
Ribozymes. enzymatic RNA rnolecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme acaion involves sequence-specific hybridization of the ribozyme 3_'~ molecule to complementary target RNA. followed by endonucleolytic cleavage. For example, ehgineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding GFRP.
Specific ribozyme cleavage: sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites. including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 1 ~ and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site.
may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
The suitability of candidate targets rnay also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic: acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
Alternatively. RNA molecules may be generated by in vitro and in vivo transcription of DNA
sequences encoding GFRP. Such DNA sequences may be incorporated into a wide variety of vectors IS with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA,, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half life. Possible modifications include, but are not lirnited to, the addition of flanking sequences at the S' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine. as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine.
guanine, thymine. and uridine which are not as easily recognized by endogenous endonucleases.
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
Delivery by transfection, by liposomc: injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See. e.g., Goidman. C.K. et al. ( 1997) Nat.
Biotechno1.15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows. horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a phattrtaceutical or sterile composition. in conjunction with a pharmaceutically acceptable carrier. for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of GFRP.
antibodies to GFR.P, and mimetics, aeonists. antagonists. or inhibitors of GFRP. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in anv sterile. biocompatible pharmaceutical carrier including, but not limited to, saline. buffered saline, dextrose, and water. The compositions may be administered to a patient alone. or in combination with other agents. drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to. oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular. transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
Further details on techniques for formulation and administration may be found in the latest edition of Remin~ton's IS Pharmaceutics! Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
Such carriers enable the pharmaceutical compositions to be formulated as tablets. pills, dragees, capsules, liquids. gels, syrups, slurries. suspensions, and the like, for ingestion by the patient.
Pharmaceutical preparations fbr oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee .cores. Suitable auxiliaries can be added. if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose. mannitol.
and sorbitol; starch from corn, wheat, rice. potato. or other plants;
cellulose. such as methyl cellulose, :?5 hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen. If desired.
disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium aleinate.
Dragee corers may be used in conjunction with suitable coatings, such as concentrated sugar ?.0 solutions, which may also contain gum arabic, talc. polyvinylpyrrolidone.
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacqGOer solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound. i.e.. dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules made of 35 gelatin. as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.

Push-tit capsules can contain active: ingredients mired with tillers or binders. such as lactose or starches. lubricants. such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions., preferably in physiologically compatible buffers such as Hanks' solution, Ringer's _ .
solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase they viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid poiycationic amino polymers may also be used for delivery. Optionally. the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in than art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
The pharm.aceuticai composiit'ron may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and suecinic acids. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to SO mM histidine, 0.1 % to 2% sucrose, and 2%
to 7% mannitol, at a :?5 pH range of 4.5 to '~.~, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared. they can be placed in an appropriate container and labeled for treatment o:f an indicated condition. For administration of GFRP, such labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions. suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, or pigs.
An animal mode) m;ay also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for ~10 administration in humans.
A therapeutically effective dose refers to that amount of active in~~redient.
for example GFRP
or fragments thereof; antibodies of GFRP. and agonists, antagonists or inhibitors of GFRP, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals. such as by calculating the ED;° (the dose therapeutically effective in ~0% of the population) or LDS° (the dose lethal to ~0% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be e~cpiressed as the LD;°/EDS°
ratio. Pharmaceutical compositions which exhibit larger therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the EDS° with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient. and the route of administration.
The exact dosage will be determined by the practitioner. in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 :!0 days, every week, or biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 ug, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of pol~mucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind GFRP may be used for the 3~0 diagnosis of disorders characterized by expression of GFRP, or in assays to monitor patients being treated with GFRP or agonists, antagonists, or inhibitors of GFRP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for GFRP include methods which utilize the antibody and a label to detect GFRP
in human body fluids or in extracts of cells or tissues. 'The antibodies may be used with or without modification. and 3.i may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of ~l 1 reporter molecules. several of which are described above. are known in the art and may be used.
A variety of protocols for measuring GFRP. including ELISAs, RIAs. and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of GFRP expression. Normal or standard values for GFRP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects. for example, human subjects, with antibody to GFRP under conditions suitable; for complex formation. The amount of standard complex formation may be quantitated by various methods. such as photometric means. Quantities of GFRP
expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values.
Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding GFRP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RTJA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of GFRP
may be correlated with disease. The diagnostic assay may be used to determine absence. presence, and excess expression of GFRP, and to men itor regulation of GFRP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding GFRP or closely related molecules may be used to identify nucleic acid sequences which encode GFRP. The specificity of the probe. whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, arid the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occuwing sequences encoding GFRP, allelic variants, or related sequences.
Probes may also be used fc>r 'the detection of related sequences. and may have at least 50%
sequence identity to any of the GFRP encoding sequences. The hybridization probes of the subject 15 invention may be DNA or RNA and may be derived from the sequence of SEQ ID
NO:S-8 or from genomic sequences including promoters, enhancers, and introns of the GFRP
gene.
Means for producing specific hybridization probes for DNAs encoding GFRP
include the cloning of polynucleotide sequences encoding GFRP or GFRP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art. are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as '=P or'SS, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotiide sequences encoding GFRP may be used for the diagnosis of disorders 3:i associated with expression of GFRI'. Examples of such disorders include, but are not limited to, a developmental disorder such as renal tubular acidosis. anemia. Cushin~~s syndrome. achondroplastic dwarfism. Duchenne and Becker muscular dystrophy. epilepsy. ~~onadal dysgenesis. WAGR
syndrome (Wilms" tumor, aniridia, ~,enitourinary abnormalities. and mental retardation), Smith-Magenis syndrome;. myelodysplasti<; syndrome, hereditary mucoepithelial dyspiasia. hereditary keratodermas. herc;ditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, sei:~ure disorders such as Syndenham's chorea and cerebral palsy, spina bifida. anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss: a cell proliferative disorder, such as actinic keratosis, arteriosclerosis. atherosclerosis, bursitis. cirrhosis. hepatitis. mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria. polycythc:mia vera, psoriasis, primary thrombocvthemia, and a cancer including adenocarcinoma, leukemia, lymphoma. melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a~ cancer of the adrenal gland, bladder, bone, bone marrow. brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate. salivary glands, skin, spleen, testis, thymus.
thyroid. and uterus; an immune disorder, such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic derntatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, :!0 erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria.
hepatitis, hypereosinophilia, irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis. osteoarthritis, osteoporosis, pancreatitis, polycythemia vera.
polymyositis, psoriasis, Reiter's syndrome. rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative c;oiitis, uveitis, Wc:rner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, trauma, and hematopoietic cancer including lymphoma. leukemia, and myeloma: a reprodu-ctive disorder, such as a disorder of prolactin production.
infertility, including tubal disease, ovulatory defects, and endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian tumor. a uterine fibroid, autoimmune disorders. an ectopic pregnancy, and teratogenesis;
cancer of the breast. fibrocystic breast disease, and galactorrhea: a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis. cancer of the prostate, benign prostatic hyperplasia.
3:i prostatitis. Peyronie~s disease, impotence. carcinoma of the male breast, and gynecomastia: a :l3 cardiovascular disorder, such as congestive heart failure. ischemic heart disease. angina pectoris, myocardial infarction, hypertensive heart disease. degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve. mitral annular calcification, mural valve prolapse, rheumatic fever and rheumatic heart disease. infective endocarditis.
nonbacterial thrombotic endocarditis, endocarditis of systennic lupus et~.~thematosus, carcinoid heart disease. cardiomyopathy, myocarditis, peric:arditis, neoplastic heart disease. congenital heart disease, and complications of cardiac transplantation: an infection such as that caused by a viral agent classified as adenovirus, arenavirus. bunyavirus, calicivirus, coronavirus. filovirus. hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus. reovirus, retrovirus. rhabdovirus. or togavirus; an infection such as that caused by a bacterial agent classified as pneumococcus, staphylococcus, streptococcus, bacillus. corynebacterium, clostridium, meningococcus, gonococcus. listeria, moraxella, kingella. haemophilus, legionella, bordetella, gram-negative enterobacaerium including shigella. salmonella. and campylobacter, pseudomonas, vibrio, brucella. francisel,fa. yersinia, bartonella. norcardium. actinomyces.
mycobacterium. spirochaetale, rickettsia. chlamydia, or mycoplasma; an infection such as that caused by a fungal agent classified as aspergillus, blastomyces, dermatophytes, cryptococcus. coccidioides, malasezzia, histoplasma, or other fungal agents causing various mycoses: and an infection such as that caused by a parasite classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue nematodes such as trichineila. intestinal nematodes such as ascaris, lymphatic filarial nematodes, trematodes such as schistosoma, or cestrodes such as tapeworm. The polynucleotide sequences encoding GFRP may be used in Southen~ or northern analysis, dot blot, or other membrane-based technologies: in PCR
technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered GFRP expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding GFRP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding GFRP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a :30 suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding GFRP in the sample indicates thc: presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies.
in clinical trials, or to 5 monitor the treatment of an individual patient.
-t~4 In order to provide a basis for the dia;nosis of a disorder associated with expression of GFRP.
a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof. encoding GFRP, under conditions suitable for hybridization or amplification.
Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that vrhich is observed in the normal subject.
The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the: disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding GFRP
may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymaticaliy, or produced in vitro. C)ligomers will preferably contain a fragment ofa polynucleotide encoding GFRP. or a fragment of a polynucleotide complementary to the polynucleotide encoding ZS GFRP, and will be employed under crptimized conditions for identification of a specific gene or condition. Oligomc:rs may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
Methods wihich may also be used to quantify the expression of GFRP include radiolabeling or biotinylating nucleotides, coamplitic,ation of a control nucleic acid, and interpolating results from ..0 standard curves. (See, e.g.. Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C.
et al. ( 1993) Anal. Biochem. 212:229-23G.) The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer of interest is presented in various. dilutions and a spectrophotometric or colorimetric response gives rapid quantrtatron.
35 In further embodiments, oligonucleotides or lon~~er fragments derived from any of the d5 WO 00/24774. PCT/US99/25458 polynucleotide sequences described herein may be used as targets in a microarray. The microarray can be used to monitor the expression level of large numbers of ~~enes simultaneously and to identify genetic variants. rnutations. and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder. to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g.,..
Brennan, T.M. et ail. (1995) U.S. latent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiiler ct al. ( 1995) PCT application W095/2511 16; Shalon, D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. ( 1997) Proc. Natl.
Aead. Sci. USA 94:2I50-215; and Heller. 1VI.J. et al. ( 1997) U.S. Patent No. 5,605,662.) In another embodiment of the invention, nucleic acid sequences encoding GFRP
may be used to generate hybridiization probes useful in mapping the naturally occurring genomic sequence. The sequences may be mapped to a particular chromosome. to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs}, bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J. et a1. ( 1997) Nat.
Genet. I 5:345-355; Price, C.M. ( 1993) Blood Rev. 7:127-134; and Trask, B.J. ( 1991 ) Trends Genet.
7:149-154.) Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al. ( 1995) in Meyers, supra, pp. 965-968.) Examples of genetic rnap data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location ofthe gene; encoding GFRP on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA
associated with that disorder.
The nucleotide sequences of the invention may be used to detect differences in gene sequences among ;?5 normal, carrier, anf. affected individuals.
In situ hybridization of chrornosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping.
This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 I q22-33, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-X80.) The nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation.
inversion, etc.. among normal. carrier. or affected individuals.
In another embodiment of the invention. GFRP. its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between GFRP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen. et al. ( 1984) PCT
application W084/03564.) In this method. large numbers of different small test compounds are synthesized on a solid substrate. 'The test compounds are reacted with GFRP, or fragments thereof, and washed. Bound GFRP is then detected by methods well known in the art.
Purified GFRP can also be coated directly onto plates for use in the aforementioned drug screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a IS solid support.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding GFRP specifically compete with a test compound for binding GFRP. In this manner, antibodies can be used t:o detect the presence of any peptide which shares one or more antigenic determinants with GFRP.
:?0 In additional embodiments, the nucleotide sequences which encode GFRP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code .and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding ~'.5 description, utilize t:he present invention to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above and below, in particular U.S. Ser. No. [Attorney Docket No. PF-062 i P, filed October 28, 1998]. U.S. Ser. No.
30 [Attorney Docket No. PF-0644 P, fled December 1 l, 1998], and U.S. Ser. No.
[Attorney Docket No.
PF-0688 P, filed May 17, 1999], are hereby expressly incorporated by reference.
EXAMPLES
.i 7 I. Construction of eDNA Libraries The OVARTUT03 eDNA (library was constructed using RNA isolated from ovarian tumor tissue removed from the left ovary of a 52-year-old mixed ethnicity female during a total abdominal hysterectomy, bilateral salpingo-oophorectomy. peritoneal and lymphatic structure biopsy, regional lymph node excision, and peritoneal tissue destruction. Pathology indicated an invasive grade 3 (of 4).
seroanaplastic carcinoma forming a. mass in the left ovary. Multiple tumor implants were present on the surface of the ovaries and fallopian tubes, the posterior surface of the uterus, and cul-de-sac.
Multiple leiomyornata were identified. Pathology also indicated metastatic grade 3 seroanaplastic carcinoma involving the omentum. cul-de-sac peritoneum, left broad ligament peritoneum, and mesentery colon. Patient history included breast cancer, chronic peptic ulcer, and joint pain. Family history included colon cancer, cerebrovascular disease. breast cancer, type II
diabetes, esophageal cancer, and depressive disorder.
The BRST'NOT31 cDNA library was constructed using RNA isolated from right breast tissue removed from a ~ ~-year-old Caucasian female during a unilateral extended simple mastectomy.
Pathology for the associated tumor tissue indicated residual microscopic infiltrating grade 3 ductal adenocarcinoma and extensive grade: 2 intraductal carcinoma. Multiple axillary lymph nodes were positive for metast;atic adenocarcinoma with minimal extranodal extension.
Immunoperoxidase stains for estrogen and progesterone receptors were positive. Patient history included benign hypertension, hyperlipidemia, cardiac dysrhythmia., benign colon neoplasm, breast cyst, and breast neoplasm.
Family history included benign hypertension, acute leukemia, primary liver cancer, and lung cancer.
The SMCA,NOTOI library was constructed using RNA isolated from an aortic smooth muscle cell line derived from the explanted heart of a male during a heart transplant.
The BRSTtVOT32 library was constructed from RNA isolated from diseased right breast tissue removed from a 46-year-old Caucasian female during bilateral subcutaneous mammectomy.
Pathology indicateel nonproliferative fibrocystic disease bilaterally. The patient presented with fibrosclerosis of the breast. Patient history included urinary tract infection. Family history included breast cancer, benign hypertension, and atherosclerotic coronary artery disease.
For the OV.ARTUT03 and BRSTNOT3 I libraries, frozen tissue was homogenized and lysed in TRIZOL reagent (1 g tissue/10 ml TRIZOL; Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate, using a Polytron PT-3000 homogenizer (Brinkmann Instruments, Westbury NY). After brief incubation on ice, chloroform was added ( 1:~ v/v), and the mixture was centrifuged to separate the phases. The upper aqueous phase was removed to a fresh tube, and isopropanol was added to precipitate (RNA. The RNA was resuspended in RNase-free water and ~18 treated with DNase. The RNA was re-extracted once with acid phenol-chloroform and reprecipitated with sodium acetate and ethanol.
For the SMCANOTOI library. the frozen tissue was homogenized and lysed in guanidinium isothiocyanate solution using a Polvtron-PT 3000 homogenizer (Brinkrriann Instruments). RNA was isolated as per Stratagene's RNA isolation protocol (Strataeene, La Jolla CA).
RNA was extracted twice with acid phenol. precipitated with sodium acetate and ethanol.
resuspended in RNase-free water. and treated with DNase.
For the B.RSTNOT32 library, the frozen tissue was homogenized and lysed in Trizol reagent (0.8 g tissue/12 ml 'Trizol; Life Technologies), a monoplastic solution of phenol and guanidine isothiocyanate, using a Polvtron PT'-3000 homogenizer (Brinkmann Instruments).
After a brief incubation on ice. chloroform was added ( 1:5 v/v) and the lysate was centrifuged. The upper chloroform layer was removed to a fresh tube and the RNA precipitated with isopropanol, resuspended in Df:PC-treated water. and treated with DNase for 2~ min at 37°C. The mRNA was re-extracted once with acid phenol-chloroform pH 4.7 and precipitated using 0.3M
sodium acetate and 2.5 volumes ethanol.
To construct the OVARTU'f03, BRSTNOT3 l, SMCANOTOI, and BRSTNOT32 cDNA
libraries. mRNA was isolated using the QIAGEN OLIGOTEX kit (QIAGEN, Inc., Chatsworth CA).
The mRNA was handled according to the recommended protocols in the SUPERSCRIPT
plasmid system for cDNA synthesis and plasmid cloning (Life Technologies). The cDNAs were fractionated on a SEPHAROSE;CL4B column (P'harmacia), and those cDNAs exceeding 400 by were ligated into either the pINCY or pINCYI plasmi~ds. The recombinant plasmids were subsequently transformed into DHSa competent cells (Life 'ferhnologies).
II. Isolation of cDNA Clones Plasmid DIVA was released from the cells and purified using the R.E.A.L. PREP
96 plasmid kit (QIAGEN, lnc. I. This kit enabled the simultaneous purification of 96 samples in a 96-well block using multi-channel reagent dispensers. The recommended protocol was employed except for the following changes: 1 ) the bacteria were cultured in 1 ml of sterile Terrific Broth (Life Technologies) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures were incubated for 19 hours and at the end of incubation, the cells were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol. samples were transferred to a 96-well block for storage at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput t~~rmat (Rao. V.B. ( 1994) Anal. Biochem. 216: l=14). Host cell lysis and thermal cycling steps were: carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a Fluoroskan 11 t7uorescence scanner (Labsystems Oy. Helsinki. Finland).
III. Sequencing and Analysis cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the AB1 C:A.TALYST 800 (Perkin-Elmer) thermal cycler or the PTC-200 thermal cycler (M.l Research) in conjunction with the HYDRA microdispenser (Bobbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as the ABI PRISM BIfGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the IS ABI PRISM 373 or 377 sequencing system (Perkin-Elmer) in conjunction with standard ABI
protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the c:DNA sequences were selected for extension using the techniques disclosed in Example V.
:20 The polynucleotide sequences derived from cDNA sequencing were assembled and analyzed using a combination of software pro~:rams which utilize algorithms well known to those skilled in the art. Table I summarizes the tools, programs. and algorithms used and provides applicable descriptions, references, and threshold parameters. The first column of Table I shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third :!5 column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column preseints, where applicable, the scores, probability values, and other parameters used to evaluate the strenl;th of a match between two sequences (the higher the score. the greater the homology between two sequences). Sequences were analyzed using MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software 30 (DNASTAR). Polynucleotide and polypeptide sequence alignments were generated using the default parameters specified by the clustal ail;orithm as incorporated into the MEGAL(GN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.

The polynucleotide sequences were validated by removing vector. linker, and polyA
sequences and by masking ambiguous bases. using algorithms and programs based on BLAST, dynamic programing, and dinucieotiide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as the GenBank primate. rodent.
mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM. and PFAM to acquire annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred.
Phrap, and Consed, and were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
The full length pol:ynucleotide sequences were translated to derive the corresponding full length l0 amino acid sequences. and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above), SwissProt.
BLOCKS. PRINTS.
DOMO, PRODOM, I'rosite, and Hidden Markov Model (I-IMM)-based protein family databases such as PFAM. HMM is a probabilistic approach which analyzes consensus primary structures of gene families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) l5 The programs described above for the assembly and analysis of full length polynucleotide and amino acid sequences were also used to identify polynucleotide sequence fragments from SEQ ID
NO:S-8. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies were described in The Invention section above.
IV. Northern Analysis .:0 Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra. ch. 7: Ausubel, 1995, supra, ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related 25 molecules in nucleotide databases such as GenBank or LIFESEQ (Incyte Pharmaceuticals). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:
sequence identity x % maximum BLAST score 3~) 100 The product score takes into account troth the degree of'simiiarity between two sequences and the length of the sequence match. For example, with a product score of 40. the match will be exact within a I% to 2% error, and, with a product score of 70. the match will be exact.
Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding GFRP occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease. The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal. hematopoietic/immune, musculoskeletal, nervous, reproductive. and urologic. The disease/condition categories included cancer, inflammation, trauma, cell proliferation, neurological, and pooled. Far each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories.
Percentage values ~of tissue-specific and disease- or condition-specific expression are reported in the description of the invention.
V. Extension of GFRP Encoding Polynucleotides The full length nucleic acid sequences of SEQ ID NO:S-8 were produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this I S fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer, to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), ar another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68. °C to about 72 °C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction :'S mix contained DNA, template. 200 nrnol of each primer, reaction buffer containing Mg2', (NHa)ZSOa, and (3-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step I: 94°C, 3 min; Step 2:
94°C, 15 sec; Step ?~: 57°C, 1 min; Step 4: 68°C, 2 min:
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage a,t 4°C.
The concentration of DNA in each well was determined by dispensing 100 ul PICOGREEN

quantitation reagent (0.25% (v/v} PICOGREEN; Molecular Probes, Eugene OR) dissolved in I X TE
and 0.5 pl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fiuoroskan II
(Labsystems Oy. Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 ~I to 10 ~1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJl cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to relegation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised. and agar digested with Agar ACE
(Promega). Extended clones were relegated using T4 Iigase (New England Biolabs. Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site IS overhangs, and transfected into competent E. coli cells. 'Transformed cells were selected on antibiotic-containing media, individual colonies were picked and cultured overnight at 37°C in 384-well plates in LB/2;c carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following a!0 parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min;
Step S: steps 2, 3. and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries were rearnplified using the same conditions as described above.
Samples were diluted with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing 25 primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABl PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID NO:S-8 are used to obtain 5' regulatory sequences using the procedure above. oligonucleotides designed for such extension. and an appropriate genomic library.
30 VI. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID NO:S-8 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides. consisting of about 20 base pairs, is specifically described. essentially the same procedure is used with larger nucleotide fragments.

Oligonucleotides .are designed usin~l state-of the-art software such as OLIGO
4.06 software (National Biosciences) and !labeled by combining ~0 pmol of each oligomer. 250 uCi of [y-'=P] adenosine triphosphate (Amersham Pharmacia~ Biotech). and 't4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled. oligonucleotides are substantially purified usin~~ a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot containing 10' counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with ane of the following endonucleases: Ase l, Bgl II, Eco RI, Pst 1, Xba I, or Pvu II (DuPont NEN).
The DNA, from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nvtran Plus, Schleicher ~ Schuell. Durham NH). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
VII. Microarrays A chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced by hand or using :?0 available methods and machines and contain any appropriate number of elements. After hybridization, nonhybridized probes .are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
2:5 Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the :microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an 30 appropriate substrata, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et ai.
(1995) Science 270:467-470; Shalom U. et al. (1996) Genome Res. 6:639-64~.) Fluorescent probes are prepared and used for hybridization to the elements on the substrate. The substrate is analyzed by procedures described above.
VIII. Complementary Polynurleotides Sequencca complementary to the GFRP-encoding sequences, or any parts thereof.
are used to detect, decrease, or inhibit expression of naturally occurring GFRP.
Although use of oligonucleotides comprising from about I S to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucieotides are designed using OL,IGO 4.06 software (National Biosciences) and the coding sequence of GFRP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary IO oligonucleotide is designed to prevent ribosomal binding to the GFRP-encoding transcript.
IX. Expression of GFRP
Expression and purification of GFRP is achieved using bacterial or virus-based expression systems. For expression of GFRP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of eDNA
l5 transcription. Examples of such promoters include, but are not limited to, the trp-!ac (tac) hybrid promoter and the T'S or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21 (DE3).
Antibiotic resistant bacteria express GPRP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of GFRP in eukaryotic cells is achieved by infecting insect 'ZO or mammalian cell lines with recombinant Autoeraphica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding GFRF' by either homologous recombination or bacterial-mediated transposition involving transfer plasrnid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of eDNA transcription. Recombinant baculovirus is used to 35 infect Spodovtera frug_iperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.K.
et al. ( 1994) Proc. TJatl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. ( 1996) Hum. Gene Ther.
7:1937-1945.) In most expression systems. (iFRP is synthesized as a fusion protein with.
e.g., glutathione 30 S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26 kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotec:h). Following purification. the GST moiety cait be proteolvtically cleaved from GFRP at specifically engineered sites. FLAG, an 8-amino acid peptide. enables immunoaffinity purification usinc: commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN;~. Methods for protein expression and purification are discussed in Ausube) (1995, .
supra, ch. l0 and 16). Purified I:iFRP obtained by these methods can be used directly in the following activity assay.
X. Demonstration of GFRP Activity An assay for cytokine activity of GFRP measures the proliferation of leukocytes. In this assay, the amount of tritiated thymidine incorporated into newly synthesized DNA is used to estimate proliferative activity. Varying amounts of GFRP are added to cultured leukocytes, such as granulocytes, monocytes, or lymphocytes, in the presence of ['H]thymidine, a radioactive DNA
precursor. A similar assay for growth factor activity of GFRP measures the stimulation of DNA
synthesis in Swiss. mouse 3T3 cells (McKay, 1. and I. Leigh, eds. (1993) Growth Factors: A Practical Approach, Oxford University Press, New York NY). Initiation of DNA synthesis indicates the cells' entry into the mitotic cycle and their commitment to undergo later division.
3T3 cells are competent to respond to most growth factors, not only those that are mitogenic, but also those that are involved in embryonic induction. This competency is possible because the in vivo specificity demonstrated by some growth factors is not necessarily inherent but is determined by the responding tissue. Therefore, this assay is generally applicable to GFRP. In this growth factor assay, varying amounts of GFRP are added to quiescent 3T3 cultured cells in the presence of ['H]thymidine. GFRP
for these cytokine and growth factor assays can be obtained by recombinant means or from biochemical preparations.
Incorporation of ['H]thymidine into acid-precipitable DNA is measured over an appropriate time interval, and the arnount incorporated is directly proportional to the amount of newly synthesized DNA. A linear dose-response curve over at least a hundred-fold GFRP
concentration range is indicative of cytokine or growth factor activit,~, as appropriate. One unit of activity per milliliter is conventionally defined as the concentration of GFRP producing a 50% response level, where 100%
represents maximal incorporation of ['H]thymidine into acid-precipitable DNA.
An assay for chemokine activity of GFRP utilizes a Boyden micro chamber (Neuroprobe, Cabin John MD) to measure leukocyte chemotaxis (Vicari, s. upra). In this assay, about I05 migratory cells such as macrophages or monocytes are placed in cell culture media in the upper compartment of the chamber. Varying dilutions of GFRP are placed in the lower compartment.
The two compartments are separated by a ~ or 8 micron pore polycarbonate filter (Nucleopore, Pleasanton ~6 CA). After incubz~tion at 37'C for 80 to 120 minutes. the filters are fixed in methanol and stained with appropriate labeling agents. Cells which migrate to the other side of the filter are counted using standard microscopy. The chematactic index is calculated by dividing the number of migratory cells counted when GFF~P is present in the lower compartment by the number of migratory cells counted when only media is present in the lower compartment. The chemotactic index is proportional to the activity of GFRP.
The follistatin activity of GFRP is measured by its binding to activin using a sensitive and specific assay for a.ctivin-follistatin binding developed by Demurs et al. ( 1992, Biochem. Biophys.
Res. Commun. 185:1148-1 154). Activin is labelled with ''-51 and combined with a biological sample.
The mixture is treated with 50% acetonitrile in conjunction with chromatographic methods which separate free from ibound activin. The amount of GFRP bound to activin is directly proportional to its follistatin activity.
XI. Functional Assays GFRP function is assessed by expressing the sequences encoding GFRP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPC>RT (Life Technologies) and pCR3. l (Invitrogen, Carlsbad CA), both of which contain the cytomel;alovirus promoter. 5-10 ~g of recombinant vector are transiently transfected into a human cell tine, for example, an endothelial or hematopoietic cell line, using either liposome .!0 formulations or elec;troporation. 1 ~2 ug of an additional plasmid containing sequences encoding a marker protein are c:o-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include. e.g., Green Fluorescent Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify tr~ansfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide;
changes in cell size ,and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cvtometry are discussed in Orme:rod, M.G. ( 1994) Flow Cvtometrv, Oxford, New York NY.
The influence of GFRP on Gene expression can be assessed using highly purified populations of cells transfected with sequences encoding GFRP and either CD64 or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin Ci (IgG). Transfected cells are efficiently separated from nontransfected cells using _.
magnetic beads coated with either human IeG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the cells using methods well known by those of skill in the art.
Expression of mRtJA encoding GFRP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XII. Production of GFRP Specific Antibodies GFRP substantially purified using polyacrylamide gel electrophoresis (PAGE:
see, e.g., Harrington, M.G. ( 1990) Methods Ein-_rymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the GFRP amino acid sequence is analyzed using LASERGENE
software I S (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and usc;d to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art.. (See, e.g., Ausubel, 1995, supra, ch. 11.) Typically, oligopeptides of;about 15 residues in length are synthesized using an ABI 431A
:!0 peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. ( See, e.g.. Ausubel. 1995, supra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's a.djuvant. Resulting antisera are tested for antipeptide and anti GFRP activity by, fir example, binding the peptide or GFRP to a substrate, blocking with 1 % BSA, 35 reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XIII. Purification of Naturally Occurring GFRP Using Specific Antibodies Naturally occurring or recombinant GFRP is substantially purified by immunoaffinity chromatography using antibodies specifiic for GFRP. An immunoaffinity column is constructed by covalently coupling anti-GFRP antibody to an activated chromatographic resin, such as 3~D CNBr-activated SEF'HAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing GFRP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of GFRP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/GFRP binding (e.g., a buffer of pH 2 to pH ;. or a high concentration of a chaotrope. such as urea or thiocyanat:e ion), and GFRP' is collected.
XIV. Identif<c:ation of Molecules Which Interact with GFRP
GFRP, or biologically active fragments thereof. are labeled with ''-SI Bolton-Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter ( 1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a mufti-well plate are incubated with the labeled GFRP. washed, and any wells with labeled GFRP complex are assayed. Data obtained using different concentrations of GFRP are used to calculate values for the number. affinity, and association of GFRP with the candidate molecules.
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with certain embodiments, it should be understood that the: invention as claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.
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SEQUENCE LISTING
< 110 > INCYTE PFIARMACEUTICAL~~ , INC .
TANG, Y. Tom YUE, Henry HILLMAN, Jennifer L.
CORLEY, rfeil C.
GUEGLER, Karl J.
BAUGHN, N,:ariah R.
AU-YOUNG, Janice <120> GROWTH FACTOR RELATED MOLECULES
<130> PF-0627 PCT
<140> To Be Assigned <141> Herewith <150> 09/181,711; unassigned; 09/209,547; unassigned; 09/313,457;
unassigned <151> 1998-10-2B; 1998-10-28; 1998-12-11; 1998-12-11; 1999-05-17;

<160> 12 <170> PERL Program <210> 1 <211> 125 <212> PRT
<213> Homo sapiE:ns <220>
<221> misc_feature <223> Incyte ID No.: 277728201 <400> 1 Met Arg Gly Thr Arg Leu Ala Leu Leu Ala Leu Val Leu Ala Ala Cys Gly Glu Leu Ala Pro Ala heu Arg Cys Tyr Val Cys Pro Glu Pro Thr Gly Val Ser Asp Cys V'al Thr Ile A1a Thr Cys Thr Thr Asn Glu Thr Met Cys Lys Thr T'hr Leu Tyr Ser Arg Glu Ile Val Tyr Pro Phe Gln Gly Asp Ser Thr Val Thr Lys Ser Cys Ala Ser Lys Cys Lys Pro Ser Asp Val Asp Gly Ile Gly Gln Thr Leu Pro Val Ser Cys Cys Asn Thr Glu Leu Cys Asn Val Asp Gly Ala Pro Ala Leu Asn Ser Leu His Cys Gly Ala Leu Thr Leu Leu Pro Leu Leu Ser Leu Arg Leu 1/~~

<210> 2 <211> 127 <212> PRT
<213> Homo sap:iens <220>
<221> misc_feature <223> Incyte II7 No.: 4185824CD1 <400> 2 Met Gln Gln Arc_I Gly Leu Ala Ile Val Ala Leu Ala Val Cys Ala Ala Leu His Ala Ser Glu Ala IIe Leu Pro IIe Ala Ser Ser Cys Cys Thr Glu Va7. Ser His His Ile Ser Arg Arg Leu Leu Glu Arg Val Asn Met Cy~; Arg Ile Gln Arg Ala Asp Gly Asp Cys Asp Leu Ala Ala Val I1E: Leu His Val L~ys Arg Arg Arg Ile Cys Val Ser Pro His Asn His. Thr Val Lys Gln Trp Met Lys Val Gln Ala Ala Lys Lys Asn Gly Lys Gly Asn Val Cys His Arg Lys Lys His His Gly Lys Arg Asp Ser Asn Arg Ala His Gln Gly Lys His Glu Thr Tyr Gly His Lys Thr Pro Tyr <210> 3 <211> 147 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feat~ure <223> Incyte ID No.: 2484440CD1 <400> 3 Met Glu Arg Gly Ala His Gly Gly Ala Gly Gly Cys Leu Cys Leu Leu Pro Glu Gly Phe Arg Ile Leu Gly Val Lys Gly Gly Ser Trp Gly Gln Glu Pro Cys Gly Val Leu Ser Glu Met Ser Pro Glu Ala Ser Pro Gly Thr Arg Pro Ala Giu Ser Cys Glu His Val Val Cys Pro Arg Pro Gln Ser Cys Val Val Asp Gln Thr Gly Ser Ala His Cys Val Val Cys Arg Ala Ala I?ro Cys Pro Val Pro Ser Ser Pro Gly Gln Glu Leu Cys Gly Asn Asn Asn Val Thr Tyr Ile Ser Ser Cys His Met Arg Gln Ala Thr Cys Phe Leu Gly Arg Ser Ile Gly Val Arg His Ala Gly Ser Cys Ala Gly Thr Pro Glu Glu Pro Pro Gly Gly Glu Ser Ala Glu Glu Glu Glu Asn Phe Val <210> 4 <211> 345 <212> PRT
<213> Homo sapi.ens <220>
<221> misc_feat.ure <223> Incyte ID No.: 4163378CD1 <400> 4 Met Ser Leu Phe Gly Leu Leu Leu Leu Thr Ser Ala Leu Ala Gly Gln Arg Gln Gly Thr Gln Ala Glu Ser Asn Leu Ser Ser Lys Phe Gln Phe Ser Ser Asn Lys Glu Gln Tyr Gly Val Gln Asp Pro Gln His Glu Arg Ile Ile Thr Va1 Ser Thr Asn Gly Ser Ile His Ser Pro Arg Phe Pro His Thr Tyr Pro Arg Asn Thr Val Leu Val Trp Arg Leu Val Ala Val Glu Glu .Asn Val Trp I.le Gln Leu Thr Phe Asp Glu Arg Phe Gly Leu Glu .Asp Pro Glu Asp Asp Ile Cys Lys Tyr Asp Phe Val Glu Val Glu Glu Pro Ser Asp Gly Thr Ile Leu Gly Arg Trp Cys Gly Ser Gly 'Thr Val Pro Gly Lys Gln Ile Ser Lys Gly Asn Gln Ile Arg Ile Arg Phe Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro Gly Phe Cys :Ile His Tyr Asn Ile Val Met Pro Gln Phe Thr Glu Ala Val Ser 1?ro Ser Val Leu Pro Pro Ser Ala Leu Pro Leu Asp Leu Leu Asn Asn Ala Ile Thr Ala Phe Ser Thr Leu Glu Asp Leu Ile Arg Tyr Leu Glu Pro Glu Arg Trp Gln Leu Asp Leu Glu Asp Leu Tyr Arg Pro Thr Trp Gln Leu Leu Gly Lys Ala Phe Val Phe Gly Arg Lys :cer Arg Val Val Asp Leu Asn Leu Leu Thr Glu Glu Val Arg Leu Tyr Ser Cys Thr Pro Arg Asn Phe Ser Val Ser Ile Arg Glu Glu L~eu Lys Arg Thr Asp Thr Ile Phe TrpProGlyCys LeuLeuValLys ArgCysGly Gly Cys Ala Asn CysCysLeuHis AsnCysAsnGlu CysGlnCys Val Ser Lys Pro ValThrLysLy:aTyrHisGluVal LeuGlnLeu Arg Lys Thr Pro GlyValArgGly LeuHisLysSer LeuThrAsp Val Leu Glu Ala HisHisGluGlu CysAspCysVal CysArgGly Ser Gly Gly Thr <210> 5 <211> 525 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No.: 2777282CB1 <400> 5 ccagtctgtc gccacctcac ttggtgtctg ctgtccccgc caggcaagcc tggggtgaga 60 gcacagagga gtgggccggg accatgcggg ggacgcggct ggcgctcctg gcgctggtgc 120 tggctgcctg cggagagctg gcgccggccc tgcgctgcta cgtctgtccg gagcccacag 180 gagtgtcgga ctgtgtcacc atcgcc,acct gcaccaccaa cgaaaccatg tgcaagacca 240 cactctactc ccgggagata gtgtac~~cct tccaggggga ctccacggtg accaagtcct 300 gtgccagcaa gtgtaagccc tcggatgtgg atggcatcgg ccagaccctg cccgtgtcct 360 gctgcaatac tgagctgtgc aatgtagacg gggcgcccgc tctgaacagc ctccactgcg 420 gggccctcac gctcctccca ctcttgagcc tccgactgta gagtccccgc ccacccccat 480 ggccctatgc ggcccagccc cgaatgcctt gaagaagtcc ccccg 525 <210> 6 <211>-566 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No.: 4185824C'B1 <400> 6 tcgaacagcc tcact.tgtgt tgctgtcagt gccagtaggg caggcaggaa tgcagcagag 60 aggactcgcc atcgt.ggcct tggctgt.ctg tgcggcccta catgcctcag aagccatact 120 tcccattgcc tccag~ctgtt gcacggaggt ttcacatcat atttccagaa ggctcctgga 180 aagagtgaat atgtg~tcgca tccagag-agc tgatggggat tgtgacttgg ctgctgtcat 240 ccttcatgtc aagcgcagaa gaatctgtgt cagcccgcac aaccatactg ttaagcagtg 300 gatgaaagtg caagctgcca agaaaaatgg taaaggaaat gtttgccaca ggaagaaaca 360 ccatggcaag agggacagta acagggcaca tcaggggaaa cacgaaacat acggccataa 420 aactccttat taggagagtc taccggtaaa tccttccgag accattccct caagtggact 480 ttggccctgg attgggtgta agttttatca tcctgaattc tcccctaatg ttgggccacc 540 ggaccaaacc caaatatttg gttttt 566 <210> 7 <211> 2246 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feanure <223> Incyte I17 No.: 2484440CB1 <400> 7 aggggcgggg ctttgggtag cataaggggc aggagctcac gaaagaggtg tggctcctgg 60 gtacaatggg aagggcaggg cttgtgggtg gagcctgagg ggtggaatgg agaggggggc 120 tcacgggggg gcgggggggt gcttgtgtct cctaccagag ggttttcgca tcttgggggt 180 taagggtggg tctt;ggggcc aagagccctg cggggttctc agcgagatgt cccctgaagc 240 ttcccctggc acc<:ggcctg cagagr_cctg tgagcacgtg gtgtgcccgc ggccacagtc 300 gtgcgtcgtg gacc:agacgg gcagcgccca ctgcgtggtg tgtcgagcgg cgccctgccc 360 tgtgccctcc agc<:ccggcc aggagctttg cggcaacaac aacgtcacct acatctcctc 420 gtgccacatg cgcc:aggcca cctgcttcct gggccgctcc atcggcgtgc gccacgcggg 480 cagctgcgca ggcacccctg aggagc:cgcc aggtggtgag tctgcagaag aggaagagaa 540 cttcgtgtga gcct:gcagga caggcctggg cctggtgccc gaggcccccc atcatcccct 600 gttatttatt gccacagcag agtctaatt-_t atatgccacg gacactcctt agagcccgga 660 ttcggaccac ttgdggatcc cagaacctcc ctgacgatat cctggaagga ctgaggaagg 720 gaggcctggg ggcc:ggctgg tgggtc~ggat agacctgcgt tccggacact gagcgcctga 780 tttagggccc ttct:ctagga tgcr.cc:agcc cctaccctaa gacctattgc cggggaggat 840 tccacacttc cgct:cctttg gggataaacc tattaattat tgctactatc aagagggctg 900 ggcattctct gctg~gtaatt cctgaagagg catgactgct tttctcagcc ccaagcctct 960 agtctgggtg tgta.cggagg gtctadc:ctg ggtgtgtacg gagggtctag cctgggtgag 1020 tacggagggt ctag~cctggg tgagtacgga gggtctagcc tgggtgagta cggagagtct 1080 agcctgggtg tgta.tggagg atctac~cctg ggtgagtatg gagggtctag cctgggtgag 1140 tatggagggt ctag~cctggg tgtgta~tgga gggtctagcc tgggtgagta tggagggtct 1200 agcctgggtg tgta.tggagg gtctac~cctg ggtgagtatg gagggtctag cctgggtgtg 1260 tacggagggt ctagtctgag tgcgtgtggg gacctcagaa cactgtgacc ttagcccagc 1320 aagccaggcc cttcatgaag gccaagaagg ctgccaccat tccctgccag cccaagaact 1380 ccagcttccc cactgcctct gtgtgcccct ttgcgtcctg tgaaggccat tgagaaatgc 1440 ccagtgtgcc ccctgggaaa gggcacggcc tgtgctcctg acacgggctg tgcttggcca 1500 cagaaccacc cagcgtctcc cctgctgctg tccacgtcag ttcatgaggc aacgtcgcgt 1560 ggtctcagac gtggagcagc cagcggcagc tcagagcagg gcactgtgtc cggcggagcc 1620 aagtccactc tgggggagct ctggcgggga ccacgggcca ctgctcaccc actggccccg 1680 aggggggtgt agacgccaag actcacgcat gtgtgacatc cggagtcctg gagccgggtg 1740 tcccagtggc accactaggt gcctgctgcc tccacagtgg ggttcacacc cagggctcct 1800 tggtccccca caacctgccc cggccaggcc tgcagaccca gactccagcc agacctgcct 1860 cacccaccaa tgcagccggg gctggcgaca ccagccaggt gctggtcttg ggccagttct 1920 cccacgacgg ctcaccctcc cctccatctg cgttgatgct cagaatcgcc tacctgtgcc 1980 tgcgtgtaaa ccacagcctc agaccagcta tggggagagg acaacacgga ggatatccag 2040 cttccccggt ctggggtgag gagtgtgggg agcttgggca tcctcctcca gcctcctcca 2100 gcccccaggc agtgccttac ctgtggtgcc cagaaaagtg cccctaggtt ggtgggtcta 2160 caggagcctc agcc,aggcag cccaccccac cctggggccc tgcctcacca aggaaataaa 2220 gactcaaaga agcc,~aaaaa aaaaaa 2246 <210> 8 <211> 2779 <212> DNA
<213> Homo Sapiens <220>
s~l~

<221> misc_feat:ure <223> Incvte II) No.: 416337l3CB1 <400> 8 atttgtttaa acctagggaa actggta cag gtccaggttt tgctttgatc cttttcaaaa 60 actggagaca caga~agaggg ctctac~gaaa aagttttgga tgggattatg tggaaactac 120 cctgcgattc tctcrctgcca gagcac~gctc ggcgcttcca ccccagtgca gccttcccct 180 ggcggtggtg aaag~agactc gggagt:cgct gcttccaaag tgcccgccgt gagtgagctc 240 tcaccccagt cagccaaatg agcctcttcg ggcttctcct gctgacatct gccctggccg 300 gccagagaca ggggactcag gcggaa~tcca acctgagtag taaattccag ttttccagca 360 acaaggaaca gtacggagta caagat.cct.c agcatgagag aattattact gtgtctacta 420 atggaagtat tcacagccca aggtttcctc atacttatcc aagaaatacg gtcttggtat 480 ggagattagt agcagtagag gaaaatgtat ggatacaact tacgtttgat gaaagatttg 540 ggcttgaaga cccagaagat gacatatgca agtatgattt tgtagaagtt gaggaaccca 600 gtgatggaac tatattaggg cgctggtgtg gttctggtac tgtaccagga aaacagattt 660 ctaaaggaaa tcaaattagg ataagatttg tatctgatga atattttcct tctgaaccag 720 ggttctgcat ccactacaac attgtcatgc cacaattcac agaagctgtg agtccttcag 780 tgctaccccc ttca~gctttg ccactggacc tgcttaataa tgctataact gcctttagta 840 ccttggaaga cctt.attcga tatcttgaac cagagagatg gcagttggac ttagaagatc 900 tatataggcc aacttggcaa cttcttggca aggcttttgt ttttggaaga aaatccagag 960 tggtggatct gaac~~ttcta acagaggagg taagattata cagctgcaca cctcgtaact 1020 tctcagtgtc cata~agggaa gaactaaaga gaaccgatac cattttctgg ccaggttgtc 1080 tcctggttaa acgcr_gtggt gggaactgtg cctgttgtct ccacaattgc aatgaatgtc 1140 aatgtgtccc aagcaaagtt actaaa~aaat accacgaggt ccttcagttg agaccaaaga 1200 ccggtgtcag gggat~tgcac aaatcactca ccgacgtggc cctggagcac catgaggagt 1260 gtgactgtgt gtgcagaggg agcacaggag gatagccgca tcaccaccag cagctcttgc 1320 ccagagctgt gcagt:gcagt ggctgattct attagagaac gtatgcgtta tctccatcct 1380 taatctcagt tgttt:gcttc aaggac~att catcttcagg atttacagtg cattctgaaa 1440 gaggagacat caaac:agaat taggagt:t_gt gcaacagctc ttttgagagg aggcctaaag 1500 gacaggagaa aaggt:cttca atcgtgc~aaa gaaaattaaa tgttgtatta aatagatcac 1560 cagctagttt cagac~ttacc atgtacgtat tccactagct gggttctgta tttcagttct 1620 ttcgatacgg cttac~ggtaa tgtca.gt:aca ggaaaaaaac tgtgcaagtg agcacctgat 1680 tccgttgcct tgctt:aactc taaagct:cca tgtcctgggc ctaaaatcgt ataaaatctg 1740 gatttttttt ttttt.ttttg ctcatata ca catatgtaaa ccagaacatt ctatgtacta 1800 caaacctggt tttta.aaaag gaactat:gtt: gctatgaatt aaacttgtgt cgtgctgata 1860 ggacagactg gattt.ttcat atttctt:att aaaatttctg ccatttagaa gaagagaact 1920 acattcatgg tttggaagag ataaacct.ga aaagaagagt ggccttatct tcactttatc 1980 gataagtcag tttatttgtt tcattgt.gta catttttata ttctcctttt gacattataa 2040 ctgttggctt ttctaatctt gttaaat.ata tctattttta ccaaaggtat ttaatattct 2100 tttttatgac aacttagatc aactattttt agcttggtaa atttttctaa acacaattgt 2160 tatagccaga ggaacaaaga tgatata.aaa tattgttgct ctgacaaaaa tacatgtatt 2220 tcattctcgt atggtgctag agttagatta atctgcattt taaaaaactg aattggaata 2280 gaattggtaa gttgcaaaga ctttttgaaa ataattaaat tatcatatct tccattcctg 2340 ttattggaga tgaaaataaa aagcaactta tgaaagtaga cattcagatc cagccattac 2400 taacctattc cttttttggg gaaatctgag cctagctcag aaaaacataa agcaccttga 2460 aaaagacttg gcagcttcct gataaagcgt gctgtgctgt gcagtaggaa cacatcctat 2520 ttattgtgat gttgtggttt tattatctta aactctgttc catacacttg tataaataca 2580 tggatatttt tatgt~acaga agtatgtctc ttaaccagt.t cacttattgt actctggcaa 2640 tttaaaagaa aatca~~taaa atattttgct tgtaaaatgc ttaatatcgt gcctaggtta 2700 tgtggtgact atttgaatca aaaatgtatt gaatcatcaa ataaaagaat gtggctattt 2760 tggggagaaa aaaaaaaaa 2778 <210> 9 <211> 103 <212> PRT
<213> Homo sapiens <300>
<308> Genbank ID No.: 81536902 <400> 9 Met Ala Ser Arg Trp Ala Val Gln Leu Leu Leu Val Ala Ala Trp Ser Met Gly Cys Gly Glu Ala Leu Lys Cys Tyr Thr Cys Lys Glu Pro Met Thr Ser Ala Ser Cys Arg Thr Ile Thr Arg Cys Lys Pro Glu Asp Thr Ala Cys Met Thr Thr Leu Val Thr Val Glu Ala Glu Tyr Pro Phe Asn Gln Ser Pro Val Val Thr Arg Ser Cys Ser Ser Ser Cys Val Ala Thr Asp Pro Asp Ser Ile Gly Ala Ala His Leu Ile Phe Cys Cys Phe Arg Asp Leu Cys Asn Ser Glu Leu <210> 10 <211> 150 <212> PRT
<213> Homo sapi<~ns <300>
<308> Genbank II) No.: 8238862'1 <400> 10 Met Asn Leu Trp Leu Leu Ala c'ys Leu Val Ala Gly Phe Leu Gly Ala Trp Ala Pro Ala Val His '.;'hr Gln Gly Val Phe Glu Asp Cys Cys Leu Ala Tyr His Tyr Pro 7~1_e Gly Trp Ala Val Leu Arg Arg Ala Trp Thr Tyr Arg Ile Gln C~lu Val Ser Gly Ser Cys Asn Leu Pro Ala Ala Ile Phe Tyr Leu Pro Lys Arg His Arg Lys Val Cys Gly Asn Pro Lys Ser Arg Glu Val Gln Arg Ala Met Lys Leu Leu Asp Ala Arg Asn Lys Val Phe P,la Lys Leu His His Asn Met Gln Thr Phe Gln Ala Gly Pro His F,la Val Lys Lys Leu Ser Ser Gly Asn Ser Lys Leu Ser Ser Ser L,ys Phe Ser Asn Pro Ile Ser Ser 125 1.30 135 Ser Lys Arg Asn Val Ser Leu Leu Ile Ser Ala Asn Ser Gly Leu ~/ 1 ~

<210> 11 <211> 343 <212> PRT
<213> Gallus ga.llus <300>
<308> Genbank ID No.: g853834 <400> 11 Met Leu Asn Gln Arg Ile His Pro Gly Met Leu Val Leu Leu Met Phe Leu Tyr His Phe Met Glu Asp His Thr Ala Gln Ala Gly Asn Cys Trp Leu Arg Gln Ala Arg Asn Gly Arg Cys Gln Val Leu Tyr Lys Thr Asp Leu Ser Lys Glu Glu Cys Cys Lys Ser Gly Arg Leu Thr Thr Ser Trp Thr Glu Glu .Asp Val Asn Asp Asn Thr Leu Phe Lys Trp Met Ile Phe Asn Gly G:Ly Ala Pro Asn Cys Ile Pro Cys Lys Glu Thr Cys Glu Asn Val ~4sp Cys Gly Pro Gly Lys Lys Cys Lys Met Asn Lys Lys Asn Lys Pro Arg Cys Val Cys Ala Pro Asp Cys Ser Asn Ile Thr Trp Lys (31y Pro Val Cys Gly Leu Asp Gly Lys Thr Tyr Arg Asn Glu Cys Ala Leu Leu Lys Ala Arg Cys Lys Glu Gln Pro Glu Leu Glu Val G1n Tyr Gln Gly Lys Cys Lys Lys Thr Cys Arg Asp Val Leu Cys Pro Gly Ser Ser Thr Cys Val Val Asp Gln Thr Asn Asn Ala Tyr C'ys Val Thr Cys Asn Arg Ile Cys Pro Glu Pro Thr Ser Pro Glu Gln Tyr Leu Cys Gly Asn Asp Gly Ile Thr Tyr Ala Ser Ala Cys Efis Leu Arg Lys Ala Thr Cys Leu Leu Gly Arg Ser Ile Gly Leu A.la Tyr Glu Gly Lys Cys Ile Lys Ala Lys Ser Cys Glu Asp Ile Gln Cys Ser Ala Gly Lys Lys Cys Leu Trp Asp Phe Lys Val Gly Arg Gly Arg Cys Ala Leu Cys Asp Glu Leu Cys Pro Glu Ser Lys Ser Asp Glu Ala val Cys Ala Ser Asp Asn Thr Thr Tyr Pro Ser Glu Cys Ala Met Lys Glu Ala Ala Cys Ser Met Gly Val Leu Leu Glu Val Lys His Ser Gly Ser Cys Asn Ser Ile Asn GIu Asp Pro Glu Glu Glu Glu Glu Asp Glu Asp Gln Asp Tyr Ser Phe Pro Ile S~er Ser Ile Leu Glu Trp 8/1~

<210> 12 <211> 730 <212> PRT
<213> Homo Sapiens <300>
<308> Genbank I:D No.: 8179500 <400> 12 Met Pro Gly Val Ala Arg Leu Fro Leu Leu Leu Gly Leu Leu Leu Leu Pro Arg Pro Gly Arg Pro Leu Asp Leu Ala Asp Tyr Thr Tyr Asp Leu Ala Glu Glu Asp Asp Ser Glu Pro Leu Asn Tyr Lys Asp Pro Cys Lys Ala Ala Ala Phe Leu Gly Asp 21e Ala Leu Asp Glu 50 ~ 55 60 Glu Asp Leu Arg Ala Phe Gln Val Gln Gln Ala Val Asp Leu Arg Arg His Thr Ala Arg Lys Sex Ser Ile Lys Ala Ala Val Pro Gly Asn Thr Ser Thr Pro Ser Cys Gln Ser Thr Asn Gly Gln Pro Gln Arg Gly Ala Cys Gly Arg Trp ,4rg Gly Arg Ser Arg Ser Arg Arg Ala Ala Thr Ser Arg Pro Glu Arg Val Trp Pro Asp Gly Val Ile Pro Phe Val Ile Gly Gly Asn 1?he Thr Gly Ser Gln Arg Ala Val Phe Arg Gln Ala Met Arg His '.Crp Glu Lys His Thr Cys Val Thr Phe Leu Glu Arg Thr Asp Glu Asp Ser Tyr Ile Val Phe Thr Tyr Arg Pro Cys Gly Cys Cys Ser Tyr Val Gly Arg Arg Gly Gly Gly Pro Gln Ala Ile Ser Ile Gly Lys Asn Cys Asp Lys Phe Gly Ile Val Val His Glu Leu Gly His Val Val Gly Phe Trp His Glu His Thr Arg Pro Asp Arg Asp Arg His Val Ser Ile Val Arg Glu Asn Ile Gln Pro Gly Gln Glu Tyr p,sn Phe Leu Lys Met Glu Pro Gln Glu Val Glu Ser Leu Gly Glu Thr Tyr Asp Phe Asp Ser Ile Met His Tyr Ala Arg Asn Thr Phe Ser Arg Gly Ile Phe Leu Asp Thr Ile Val Pro Lys Tyr Glu Val Asn Gly Val Lys Pro Pro Ile Gly Gln Arg Thr Arg Leu Ser Lys Gly Asp Ile Ala Gln Ala Arg Lys Leu Tyr Lys Cys Pro Ala Cys Gly Glu Thr Leu Gln Asp Ser Thr Gly Asn Phe Ser Ser Pro Glu Tyr Pro Asn Gly Tyr Ser Ala His Met His Cys Val Trp Arg Ile Ser Val Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp Leu Tyr Arg Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg Asp Gly Phe Trp Arg Lys Ala Pro Leu Arg Gl~i Arg Phe Cys Gly Ser Lys Leu Pro Glu Pro Ile Val Ser Thr Asp Ser Arg Leu Trp Val Glu Phe Arg Ser Ser Ser Asn Trp Val Gly Lys Gly Phe Phe Ala Val Tyr Glu Ala Ile Cys Gly Gly Asp Va7. Lys Lys Asp Tyr Gly His Ile Gln Ser Pro Asn Tyr Pro Asp Asp Tyr Arg Pro Ser Lys Val Cys Ile Trp Arg Ile Gln Val Ser Glu Gly Phe His Val Gly Leu 'rhr Phe Gln Ser Phe Glu Ile Glu Arg' His Asp Ser Cys Ala Tyr Asp Tyr Leu Glu Val Arg Asp Gly His Ser Glu Ser Ser Thr Leu Ile Gly Arg Tyr Cys Gly Tyr Glu Lys Pro Asp Asp Ile Lys Ser Thr Ser Ser Arg Leu Trp Leu Lys Phe Val Ser Asp Gly Ser Ile Asn Lys Ala Gly Phe Ala Val Asn Phe Phe Lys Glu Val Asp Glu Cys Ser Arg Pro Asn Arg Gly Gly Cys Glu Gln Arg Cys Leu Asn Thr Leu Gly Ser Tyr Lys Cys Ser Cys Asp Pro Gly Tyr Glu Leu Ala Pro Asp Lys Arg Arg Cys Glu Ala Ala Cys Gly Gly Phe Leu Thr Lys Leu Asn Gly Ser Ile Thr Ser Pro Gly Trp Pro Lys Glu Tyr Pro Pro Asn Lys Asn Cys Ile Trp Gln Leu Val .Ala Pro Thr Gln Tyr Arg Ile Ser Leu Gln Phe Asp Phe Phe Glu 'rhr Glu Gly Asn Asp Val Cys Lys Tyr Asp Phe Val Glu Val Arg ;Ser Gly Leu Thr Ala Asp Ser Lys Leu His Gly Lys Phe Cys Gly Ser Glu Lys Pro Glu Val Ile Thr Ser Gln Tyr Asn Asn Met Arg Val Glu Phe Lys Ser Asp Asn Thr Val Ser Lys Lys Gly Phe Lys Ala His Phe Phe Ser Glu Lys Arg Pro Ala Leu Gln Pro Pro Arg Gly Arg Pro His Gln Leu Lys Phe Arg Val Gln Lys Arg Asn Arg Thr Pro Gln

Claims

What is claimed is:

1. A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4 and fragments thereof.
2. A substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of claim 1.
3. An isolated and purified polynucleotide encoding the polypeptide of claim 1.
4. An isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide of claim 3.
5. An isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide of claim 3.
6. An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide of claim 3.
7. A method for detecting a polynucleotide, the method comprising the steps of:
(a) hybridizing the polynucleotide of claim 6 to at least one nucleic acid in a sample. thereby forming a hybridization complex: and (b) detecting the hybridization complex. wherein the presence of the hybridization complex correlates with the presence of the polynucleotide in the sample.
8. The method of claim 7 further comprising amplifying the polynucleotide prior to hybridization.
9. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:5-8 and fragments thereof.
10. An isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide of claim 9.

11. An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide of claim 9.
l2. An expression vector comprising at least a fragment of the polynucleotide of claim 3 13. A host cell comprising the expression vector of claim 12.
14. A method for producing a polypeptide, the method comprising the steps of:
a) culturing the host cell of claim 13 under conditions suitable for the expression of the polypeptide: and b) recovering the polypeptide from the host cell culture.
15. A pharmaceutical composition comprising the polypeptide of claim 1 in conjunction with a suitable pharmaceutical carrier.
16. A purified antibody which specifically binds to the polypeptide of claim 1.
17. A purified agonist of the polypeptide of claim 1.
18. A purified antagonist of the polypeptide of claim 1.
19. A method for treating or preventing a disorder associated with decreased expression or activity of GFRP, the method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 15.
20. A method for treating or preventing a disorder associated with increased expression or activity of GFRP, the method comprising administering to a subject in need of such treatment an effective amount of the antagonist of claim 18.