Substantially pure heparin-binding growth factor polypeptides (HBGFs), nucleic acids encoding the HBGFs and antibodies which bind to the HBGFs of the invention are provided. The HBGF polypeptides are useful in methods for the induction of bone, cartilage and tissue formation, growth and development of the endometrium and in the acceleration of wound healing. HBGF is related to Connective Tissue Growth Factor (CTGF).

FIELD OF THE INVENTION 
This invention relates generally to the field of growth factors, more 
specifically to heparin-binding growth factors (HBGF). 
BACKGROUND OF THE INVENTION 
Growth factors are a class of polypeptides that stimulate target cells to 
proliferate, differentiate and organize in developing tissues. The action 
of growth factors is dependent on their binding to specific receptors 
which stimulates a signaling event within the cell. Examples of growth 
factors include platelet-derived growth factor (PDGF), insulin-like growth 
factor (IGF-I, IGF-II), transforming growth factor beta (TGF-.beta.), 
transforming growth factor alpha (TGF-.alpha.), epidermal growth factor 
(EGF), acidic and basic fibroblast growth factors (aFGF, bFGF) and 
connective tissue growth factor (CTGF) which are known to stimulate cells 
to proliferate. 
PDGF is a cationic, heat stable protein found in the alpha granules of 
circulating platelets and is known to be a mitogen and a chemotactic agent 
for connective tissue cells such as fibroblasts and smooth muscle cells. 
Because of the activities of this molecule, PDGF is believed to be a major 
factor involved in the normal healing of wounds and pathologically 
contributing to such conditions as atherosclerosis and fibrotic 
conditions. PDGF is a dimeric molecule consisting of combinations of 
.alpha. and/or .beta. chains. The chains form heterodimers or homodimers 
and all combinations isolated to date are biologically active. 
Studies on the role of various growth factors in tissue regeneration and 
repair have led to the discovery of PDGF-like proteins. These proteins 
share both immunological and biological activities with PDGF and can be 
blocked with antibodies specific to PDGF. 
Polypeptide growth factors and cytokines are emerging as an important class 
of uterine proteins that may form growth signaling pathways between the 
maternal uterus and developing embryo or fetus. Studies in a variety of 
species have suggested that EGF, heparin-binding EGF-like growth factor 
(HB-EGF), IGF-I, IGF-II, aFGF, bFGF, pleitrophin (PTN), leukemia 
inhibitory factor, colony-stimulating factor-1 (CSF-1), and TGF-.alpha. 
may be among the uterine growth-regulatory molecules involved in these 
processes. 
CTGF is a cysteine-rich monomeric peptide of M.sub.r 38,000, which is a 
growth factor having mitogenic and chemotactic activities for connective 
tissue cells. CTGF is secreted by cells and is active upon interaction 
with a specific cell-surface receptor. CTGF is the product of a gene 
unrelated to the .alpha. or .beta. chain genes of PDGF. It is a member of 
a family of growth regulators which includes the mouse (also know as 
fisp-12 or .beta.IG-M2) and human CTGF, Cyr61 (mouse), Cef10 (chicken), 
and Nov (chicken). Based on sequence comparisons, it has been suggested 
that the members of this family all have a modular structure, consisting 
of (1) an insulin-like growth factor domain responsible for binding, (2) a 
von Willebrand factor domain responsible for complex formation, (3) a 
thrombospondin type I repeat, possibly responsible for binding matrix 
molecules, and (4) a C-terminal module found in matrix proteins, 
postulated to be responsible for receptor binding. 
The sequence of the cDNA for human CTGF (hCTGF) contains an open reading 
frame of 1047 nucleotides with an initiation site at position 130 and a 
TGA termination site at position 1177 and encodes a peptide of 349 amino 
acids. There is only a 40% sequence homology between the CTGF cDNA and the 
cDNA for either the .alpha. or .beta. chains of PDGF. 
The hCTGF open reading frame encodes a polypeptide which contains 39 
cysteine residues, indicating a protein with multiple intramolecular 
disulfide bonds. The amino terminus of the peptide contains a hydrophobic 
signal sequence indicative of a secreted protein and there are two 
N-linked glycosylation sites at asparagine residues 28 and 225 in the 
amino acid sequence. There is a 45% overall sequence homology between the 
CTGF polypeptide and the polypeptide encoded by the CEF-10 mRNA 
transcript; the homology reaches 52% when a putative alternative splicing 
region is deleted. 
CTGF is antigenically related to PDGF although there is little if any 
peptide sequence homology. Anti-PDGF antibody has high affinity to the 
non-reduced forms of PDGF or CTGF, and ten-fold less affinity to the 
reduced forms of these peptides, which lack biological activity. This 
suggests that there are regions of shared tertiary structure between the 
PDGF isomers and the CTGF molecule, resulting in common antigenic 
epitopes. 
The synthesis and secretion of CTGF are selectively induced by TGF-.beta., 
BMP-2 and possibly other members of the TGF-.beta. superfamily of 
proteins. Although TGF-.beta. can stimulate the growth of normal 
fibroblasts in soft agar, CTGF alone cannot induce this property in 
fibroblasts. However, it has been shown that the synthesis and action of 
CTGF are essential for the TGF-.beta. to stimulate anchorage independent 
fibroblast growth. 
It is probable that CTGF functions as a growth factor in wound healing. 
Pathologically, CTGF has been postulated to be involved in conditions in 
which there is an overgrowth of connective tissue cells, such as systemic 
sclerosis, cancer, fibrotic conditions, and atherosclerosis. 
The primary biological activity of CTGF polypeptide is its mitogenicity, or 
ability to stimulate target cells to proliferate. The ultimate result of 
this mitogenic activity in vivo, is the growth of targeted tissue. CTGF 
also possesses chemotactic activity, which is the chemically induced 
movement of cells as a result of interaction with particular molecules. 
SUMMARY OF THE INVENTION 
The present invention is based on the discovery, purification and 
characterization of heparin-binding growth factors (HBGFs) in uterine 
secretory fluids. These growth factor polypeptides bind heparin and 
exhibit many of the functional characteristics of full length CTGF. 
In a first aspect, the present invention provides heparin-binding 
polypeptides (HBGF polypeptides) that have been identified as having 
mitogenic activity and nucleic acids encoding such polypeptides. 
In yet a further aspect of the present invention, there are provided 
antibodies which bind to HBGFs. 
In yet a further aspect of the present invention, there are also provided 
nucleic acid probes comprising nucleic acid molecules of sufficient length 
to specifically hybridize to a nucleic acid sequence encoding HBGFs. 
In accordance with yet a further aspect of the invention, there is provided 
a method for using HBGFs, the nucleic acid molecules encoding HBGFs, or 
antisense sequences to nucleic acid molecules encoding HBGFs for affecting 
wound healing, tissue formation, sclerotic or cell proliferative 
disorders, atherosclerosis or fibrotic disease.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Before the present methods, apparatus, compositions and formulations are 
described, it is to be understood that this invention is not limited to 
the particular methods, apparatus, compositions and formulations described 
herein, as such methods, apparatus, compositions and formulations may, of 
course, vary. It is also to be understood that the terminology used herein 
is for the purpose of describing 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 referents unless the 
context clearly dictates otherwise. Thus, for example, reference to "an 
organism" includes one or more different organisms, reference to "an amino 
acid" includes one or more of such amino acids, and reference to "a 
method" include reference to equivalent steps and methods known to those 
skilled in the art, and so forth. 
Unless defined otherwise, all technical and scientific terms used herein 
have the same meaning as commonly understood by one of ordinary skill in 
the art to which this invention belongs. Although any methods and 
materials similar or equivalent to those described herein can be used in 
the practice or testing of the invention, the preferred methods and 
materials are now described. The publications discussed above are provided 
solely for their disclosure prior to the filing date of the present 
application. Nothing herein is to be construed as an admission that the 
invention is not entitled to antedate such disclosure by virtue of prior 
invention. 
The present invention provides heparin-binding growth factors (HBGF 
polypeptides or HBGFs), which are mitogenic for fibroblasts and smooth 
muscle cells in vitro. HBGFs are heat- and acid-labile, and exist in two 
forms, HBGF-0.8-P1, and HBGF-0.8-P2, each of which has different heparin 
binding properties, and each of which has a M.sub.r of about 10-kDa under 
reducing conditions by SDS-PAGE. HBGFs are related structurally and 
functionally to CTGF. Both HBGF-0.8-P1 and HBGF-0.8-P2 require the 
presence of 0.8M NaCl for elution from a heparin affinity column. 
Sequencing revealed that the N-terminal sequence of HBGF-0.8-P1 
corresponded to amino acid residues 247-262 of the 349-residue predicted 
primary translation product of porcine connective tissue growth factor 
(CTGF) while the N-terminal sequence of HBGF-0.8-P2 corresponded to amino 
acid residues 248-259 of CTGF. Thus, HBGFs correspond to two 
microheterogenous, highly truncated N-terminal forms of the translation 
product of CTGF, both of which are biologically active. HBGF-0.8-P2 is 
identical to HBGF-0.8-P1 except for the presence of an additional Glu 
residue at the N-terminus of HBGF-0.8-P1. 
The HBGFs of the invention are highly N-terminally truncated forms of CTGF, 
however, there is no intron/exon boundary that could directly give rise to 
the N terminus of the two proteins. HBGFs do not align with the proposed 
modular components of CTGF; the proteins of the invention contain none of 
the sulfated glycoconjugate binding motif of CTGF, termed a thrombospondin 
type I repeat, which is postulated to be responsible for binding matrix 
molecules. A C-terminal module of CTGF found in matrix proteins, which is 
postulated to be involved in receptor binding, is entirely present in the 
HBGFs. The proposed binding motif for sulfated glycoconjugates between 
amino acid residues 206 and 214 of CTGF is absent from HBGFs, yet HBGFs 
bind heparin, and the heparin interactions are functionally significant. 
The N terminus of HBGF-0.8-P1 and HBGF-0.8-P2 may be involved in heparin 
binding, as the two proteins of the invention differ by only a single 
N-terminal Glu, yet display differential binding to heparin. 
The HBGFs of the invention are secreted from both cultured human and mouse 
fibroblasts. Production of HBGFs is not limited to a particular species or 
biological system. Preferably, the HBGFs of the invention are mitogenic 
and chemotactic for mesenchymally derived cells (e.g., fibroblasts, 
chondrocytes, osteoclasts, osteoblasts, and astroglial), however, other 
cell types (e.g., muscle cells, connective tissue cells, epithelial cells 
and secretory cells) are responsive to HBGFs as well. HBGFs can play a 
significant role in the normal development, growth and repair of human 
tissue. HBGFs are present in uterine flushings, and may play an additional 
role in the growth and remodeling of the endometrium, and, during 
pregnancy, may affect the growth and development of the extra-embryonic or 
placental membranes. 
Therapeutic agents derived from HBGFs can be useful in augmenting normal or 
impaired growth processes involving connective tissues in certain clinical 
states (e.g., wound healing). When these HBGFs are involved in 
pathological conditions, therapeutic developments from these proteins can 
be used to control or modulate uncontrolled tissue growth. 
The term "substantially pure" as used herein refers to HBGFs which are 
substantially free of other proteins, lipids, carbohydrates or other 
materials with which they are naturally associated. A substantially pure 
HBGF polypeptide will yield a single major band on a non-reducing 
polyacrylamide gel. The purity of HBGFs can also be determined by 
amino-terminal amino acid sequence analysis. HBGFs, as defined herein, 
include functional fragments of the polypeptide, so long as HBGF 
biological activity is retained (e.g., inducing a biologic response in 
fibroblasts as determined using standard assays common in the art and as 
taught herein). Smaller polypeptides containing HBGF biological activity 
are included in the invention. Additionally, more effective HBGFs 
produced, for example, through site directed mutagenesis of HBGF 
polypeptide cDNA are included. "Recombinant" HBGFs refer to HBGF 
polypeptides produced by recombinant DNA techniques; i.e., produced from 
cells transformed by an exogenous DNA construct encoding the desired HBGF 
polypeptide. "Synthetic" HBGFs are those prepared by chemical synthesis. A 
DNA "coding sequence of" or a "nucleotide sequence encoding" a particular 
HBGF polypeptide, is a DNA sequence which is transcribed and translated 
into an HBGF polypeptide when placed under the control of appropriate 
regulatory sequences. 
The invention provides nucleic acids encoding HBGF polypeptides. These 
nucleic acids include DNA, cDNA and RNA sequences which encode for HBGFs. 
It is understood that all nucleic acids encoding all or a portion of HBGF 
polypeptides are also included herein, so long as they encode a 
polypeptide with HBGF biological activity. Such nucleic acids include both 
naturally occurring and intentionally manipulated nucleic acids. For 
example, HBGF polypeptides may be subjected to site-directed mutagenesis. 
The nucleic acids of the invention include sequences that are degenerate as 
a result of the genetic code. There are only 20 natural amino acids, most 
of which are specified by more than one codon. Therefore, as long as the 
amino acid sequence of an HBGF polypeptide is functionally unchanged, all 
degenerate nucleotide sequences are included in the invention. The 
fragment, derivative or analog of the HBGF polypeptides may be (i) one in 
which one or more of the amino acid residues are substituted with a 
conserved or non-conserved amino acid residue (preferably a conserved 
amino acid residue) and such substituted amino acid residue may or may not 
be one encoded by the genetic code, or among preferred variants are those 
that vary from a reference by conservative amino acid substitutions, (such 
substitutions are those that substitute a given amino acid in a 
polypeptide by another amino acid of like characteristics. Typically, 
conservative substitutions are the replacements, one for another, among 
the aliphatic amino acids Ala, Val, Leu and Ile; interchange of the 
hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and 
Glu, substitution between the amide residues Asn and Gln, exchange of the 
basic residues Lys and Arg and replacements among the aromatic residues 
Phe, Tyr); (ii) one in which one or more of the amino acid residues 
includes a substituent group; (iii) one in which an HBGF polypeptide is 
fused with another compound, such as a compound to increase the half-life 
of the HBGF polypeptides (for example, polyethylene glycol); or (iv) one 
in which additional amino acids are fused to HBGF polypeptides, such as a 
leader or secretory sequence or a sequence which is employed for 
purification of HBGF polypeptides or a pro-protein sequence. Such 
fragments, derivatives and analogs are deemed to be within the scope of 
those skilled in the art from the teachings herein. The HBGFs of the 
present invention and nucleic acids coding for them are preferably 
provided in an isolated form, and preferably are purified to homogeneity. 
DNA sequences encoding the HBGF polypeptides of the invention can be 
obtained by several methods. For example, the DNA can be isolated using 
well known hybridization procedures. These include, but are not limited 
to: 1) hybridization of probes to genomic or cDNA libraries to detect 
shared nucleotide sequences (see, for example: Current Protocols in 
Molecular Biology, Ausubel F. M. et al. (EDS.) Green Publishing Company 
Assoc. and John Wiley Interscience, New York, Current Edition) and 2) 
antibody screening of expression libraries to detect shared structural 
features. It is appreciated by one skilled in the art that the nucleic 
acids (comprising at least 12 contiguous nucleotides) encoding the HBGFs, 
are particularly useful as probes. 
"Selective hybridization" as used herein refers to hybridization under 
moderately stringent or highly stringent physiological conditions (See, J. 
Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring 
Harbor Laboratory (Current Edition) which is hereby incorporated by 
reference in its entirety) that distinguish related from unrelated HBGF 
based upon the degree of identity between nucleotide sequences in 
proximity for hybridization to occur. Also, it is understood that a 
fragment of a 100 bps sequence that is 95 bps in length has 95% identity 
with the 100 bps sequence from which it is obtained. As used herein, a 
first DNA (RNA) sequence is at least 70% and preferably at least 80% 
identical to another DNA (RNA) sequence if there is at least 70% and 
preferably at least a 80% or 90% identity, respectively, between the bases 
of the first sequence and the bases of another sequence, when properly 
aligned with each other, for example, when aligned by BLASTN. 
"Identity" as the term is used herein, refers to a polynucleotide sequence 
which comprises a percentage of the same bases as a reference 
polynucleotide. For example, a polynucleotide which is at least 90% 
identical to a reference polynucleotide, has polynucleotide bases that are 
identical in 90% of the bases which make up the reference polynucleotide 
(i.e., when the sequences are properly aligned with each other using 
standard alignment and homology adjustments common to those in the art 
(e.g., NetBlast or GRAIL)) and may have different bases in 10% of the 
bases which comprise that polynucleotide sequence. 
Screening procedures which rely on nucleic acid hybridization make it 
possible to isolate any gene sequence from any organism, provided the 
appropriate probe is available. For example, oligonucleotide probes, which 
correspond to a part of the sequence encoding the protein in question, can 
be synthesized chemically. This requires that short, oligopeptide 
stretches of amino acid sequence must be known. The DNA sequence encoding 
the protein can be deduced from the genetic code, however, the degeneracy 
of the code must be taken into account. It is possible to perform a mixed 
addition reaction when the sequence is degenerate. This includes a 
heterogeneous mixture of denatured double-stranded DNA. For such 
screening, hybridization is preferably performed on either single-stranded 
DNA or denatured double-stranded DNA. Hybridization is particularly useful 
in the detection of cDNA clones derived from sources where an extremely 
low amount of mRNA sequences relating to the polypeptide of interest is 
present. In other words, by using selective hybridization conditions 
directed to avoid non-specific binding, it is possible, for example, to 
allow the autoradiographic visualization of a specific cDNA clone by the 
hybridization of the target DNA to that single probe in the mixture which 
is its complete complement (Wallace, et al, Nucleic Acid Research, 9:879, 
1981). It is also appreciated that such selective hybridization probes can 
be and are preferably labeled with an analytically detectable reagent to 
facilitate identification of the probe. Useful reagents include but are 
not limited to radioactivity, fluorescent dyes or enzymes capable of 
catalyzing the formation of a detectable product. The selective 
hybridization probes are thus useful to isolate complementary copies of 
DNA from other sources or to screen such sources for related sequences. 
A cDNA expression library, such as lambda gt11, can be screened indirectly 
for HBGFs having at least one epitope, using antibodies specific for HBGF 
polypeptides or antibodies to CTGF which cross react with HBGF 
polypeptides, or antibodies to PDGF which cross react with HBGF 
polypeptides. Such antibodies can be either polyclonally or monoclonally 
derived and used to detect expression products indicative of the presence 
of HBGF polypeptide cDNA. 
DNA sequences encoding HBGF polypeptides can be expressed in vitro by DNA 
transfer into a suitable host cell. "Host cells" are genetically 
engineered cells (transduced or transformed or transfected) with the 
vectors of this invention which may be, for example, a cloning vector or 
an expression vector. The vector may be, for example, in the form of a 
plasmid, a viral particle, a phage, etc. The engineered host cells can be 
cultured in conventional nutrient media modified as appropriate for 
activating promoters, selecting transformants or amplifying the genes of 
the present invention. The culture conditions, such as temperature, pH and 
the like, are those previously used with the host cell selected for 
expression, and will be apparent to the ordinarily skilled artisan. The 
term also includes any progeny of the subject host cell. It is understood 
that all progeny may not be identical to the parental cell since there may 
be mutations that occur during replication. However, such progeny are 
included when the term "host cell" is used. Introduction of the construct 
into the host cell can be effected by calcium phosphate transfection, 
DEAE-Dextran mediated transfection, electroporation or any other method of 
the art (Davis, L. et al., Basic Methods in Molecular Biology, (Current 
Edition)). 
The nucleic acids of the present invention may be employed for producing 
HBGFs by recombinant techniques. Thus, for example, the polynucleotide may 
be included in any one of a variety of expression vectors for expressing 
HBGF polypeptides. Such vectors include chromosomal, nonchromosomal and 
synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; 
phage DNA; baculovirus; yeast plasmids; vectors derived from combinations 
of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl 
pox virus, and pseudorabies. However, any other vector may be used as long 
as it is replicable and viable in the host. 
The appropriate DNA sequence may be inserted into the vector by a variety 
of procedures. In general, the DNA sequence is inserted into an 
appropriate restriction endonuclease site(s) by procedures known in the 
art. Such procedures and others are deemed to be within the scope of those 
skilled in the art. DNA sequences encoding HBGFs can be expressed in vivo 
in either prokaryotes or eukaryotes. Methods of expressing DNA sequences 
having eukaryotic coding sequences in prokaryotes are well known in the 
art. Hosts include microbial, yeast and mammalian organisms. 
Biologically functional viral and plasmid DNA vectors capable of expression 
and replication in a host are known in the art. Such vectors are used to 
incorporate DNA sequences of the invention. In general, expression vectors 
containing promotor sequences which facilitate the efficient transcription 
of the inserted eukaryotic genetic sequence are used in connection with 
the host. The expression vector typically contains an origin of 
replication, a promoter, and a terminator, as well as specific genes 
capable of providing phenotypic selection of the transformed cells. 
In addition to expression vectors known in the art such as bacterial, yeast 
and mammalian expression systems, baculovirus vectors may also be used. 
One advantage to expression of foreign genes in this invertebrate virus 
expression vector is that it is capable of expression of high levels of 
recombinant proteins, which are antigenically and functionally similar to 
their natural counterparts. Baculovirus vectors and the appropriate insect 
host cells used in conjunction with the vectors are known to those skilled 
in the art. The isolation and purification of host cell expressed 
polypeptides of the invention may be by any conventional means such as, 
for example, preparative chromatographic separations and immunological 
separations such as those involving the use of monoclonal or polyclonal 
antibodies. 
The invention provides antibodies which are specifically reactive with HBGF 
polypeptides or fragments thereof Although this polypeptide may be cross 
reactive with antibodies to PDGF or CTGF, not all antibodies to HBGFs will 
also be reactive with PDGF, and not all antibodies to CTGF will be 
reactive to HBGFs. Antibody which consists essentially of pooled 
monoclonal antibodies with different epitopic specificities, as well as 
distinct monoclonal antibody preparations are provided. Monoclonal 
antibodies are made from antigen containing fragments of the protein by 
methods well known in the art (Kohler, et al., Nature 256:495, 1975; 
Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989). 
Polyclonal antibodies to the HBGFs of the invention are also included 
using methods common to those in the art (see Harlow and Lane, 1988, 
Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 
Current Edition). Monoclonal antibodies specific for HBGFs can be 
selected, for example, by screening for hybridoma culture supernatants 
which react with HBGF polypeptides, but do not react with PDGF. Antibodies 
generated against HBGFs corresponding to the present invention can be 
obtained by direct injection of the polypeptides into an animal or by 
administering the polypeptides to an animal, preferably a nonhuman. The 
antibody so obtained will then bind the polypeptide itself. In this 
manner, even a sequence encoding only a fragment of the polypeptides can 
be used to generate antibodies binding the original polypeptides. Such 
antibodies can then be used to isolate the polypeptides from cells 
expressing that polypeptide. 
For preparation of monoclonal antibodies, any technique which provides 
antibodies produced by continuous cell line cultures can be used. Examples 
include the hybridoma technique (Kohler, et al., Nature 256:495, 1975), 
the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 
1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce 
human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies 
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). 
Techniques described for the production of single chain antibodies (U.S. 
Pat. No. 4,946,778) can be adapted to produce single chain antibodies to 
immunogenic peptide products of this invention. Additionally included 
within the bounds of the invention, are the production and use for 
diagnostic and therapeutic applications of both "human" and "humanized" 
antibodies directed to HBGF polypeptides or fragments thereof. Humanized 
antibodies are antibodies, or antibody fragments, that have the same 
binding specificity as a parent antibody (i.e., typically of mouse 
origin), but which have increased human characteristics. Humanized 
antibodies may be obtained by chain shuffling, or using phage display 
technology. For example, a polypeptide comprising a heavy or light chain 
variable domain of a non-human antibody specific for a HBGF is combined 
with a repertoire of human complementary (light or heavy) chain variable 
domains. Hybrid pairings which are specific for the antigen of interest 
are selected. Human chains from the selected pairings may then be combined 
with a repertoire of human complementary variable domains (heavy or light) 
and humanized antibody polypeptide dimers can then be selected for binding 
specificity for an antigen. Such techniques are described in U.S. Pat. No. 
5,565,332 or can be obtained commercially (Scotgene, Scotland or Oxford 
Molecular, Palo Alto, Calif., USA). Furthermore, techniques described for 
the production of "human" antibodies (i.e., de novo antibodies with human 
constant region sequences) in transgenic mice (U.S. Pat. No. 5,545,806 and 
U.S. Pat. No. 5,569,825) can also be adapted to produce "human" HBGF 
antibodies or antibody fragments or may also be commercially contracted 
(GenPharm International, Inc., Mountain View, Calif., USA). 
Antibodies generated against the polypeptides of the present invention may 
be used in screening for similar HBGF polypeptides from other organisms 
and samples. Such screening techniques are known in the art. 
The invention provides a method for accelerating wound healing in a 
subject, e.g., human, by applying to the wound an therapeutically 
effective amount of a composition which contains purified HBGF 
polypeptides, PDGF, PDGF-related molecule or combinations thereof. The 
HBGF polypeptides of this invention are valuable as a therapeutic in cases 
in which there is impaired healing of skin wounds or there is a need to 
augment normal healing mechanisms. HBGF polypeptides, or functional 
fragments thereof, are more stable and less susceptible to protease 
degradation than PDGF and other growth factors known to be involved in 
wound healing. In addition, HBGF polypeptides may have a higher specific 
biologic activity than CTGF. 
HBGF polypeptides are derived from fibroblastic cells, which are present at 
a wound site. Therefore, agents which stimulate the production of HBGF 
polypeptides can be added to a composition that is used to accelerate 
wound healing. Preferably, the agent is a member of the family of growth 
factors such as insulin-like growth factor (IGF-I), platlet-derived growth 
factor (PGF), epidermal growth factor (EGF), transforming growth factor 
beta (TGF-.beta.) and basic fibroblast growth factor (bFGF). More 
preferably, the agent is transforming growth factor beta (TGF-.beta.) or 
other member of the TGF-.beta. superfamily. Additionally, the biologic 
effect of HBGF can be modulated by the addition of heparin in a 
concentration in the range of about 1 .mu.g/ml to 100 .mu.g/ml. The HBGF 
compositions of the invention aid in healing the wound, in part, by 
promoting the growth of connective tissue. The HBGF compositions are 
prepared by combining, in any pharmaceutically acceptable carrier 
substance, e.g., inert gels or liquids, the purified HBGF polypeptides of 
the invention. Other modulating compositions such as heparin, or growth 
factors such as TGF-.beta. can be included in the HBGF compositions. 
The term "cell proliferative disorder" refers to a condition characterized 
by an abnormal number of cells. The condition can include both 
hypertrophic (the continual multiplication of cells resulting in an 
overgrowth of a cell population within a tissue) and hypotrophic (a lack 
or deficiency of cells within a tissue) cell growth or an excessive influx 
or migration of cells into an area of a body. The cell populations are not 
necessarily transformed, tumorigenic or malignant cells, but can include 
normal cells as well. For example, HBGFs may be involved in a pathological 
condition by inducing a proliferative lesion in the intimal layer of an 
arterial wall, resulting in atherosclerosis. Instead of trying to reduce 
risk factors for the condition, e.g., lowering blood pressure or reducing 
elevated cholesterol levels, HBGF polypeptide inhibitors or antagonists of 
the invention would be useful in interfering with the in vivo activity of 
HBGFs associated with atherosclerosis. HBGF polypeptide antagonists are 
also useful in treating other disorders associated with an overgrowth of 
connective tissues, such as various fibrotic conditions, including 
scleroderma, arthritis and liver cirrhosis. 
These diseases, disorders or ailments modulated by HBGF include tissue 
repair subsequent to traumatic injuries or conditions including arthritis, 
osteoporosis and other skeletal disorders, and burns. Because these 
problems are due to a poor growth response of the fibroblasts, stem cells, 
chondrocytes, osteoblasts or fibroblasts at the site of injury, the 
addition of an active biologic agent that stimulates or induces growth of 
these cells is beneficial. The term "induce" or "induction" as used 
herein, refers to the activation, stimulation, enhancement, initiation and 
or maintenance of the cellular mechanisms or processes necessary for the 
formation of any of the tissue, repair process or development as described 
herein 
The present invention further provides a method for modulating female 
reproductive tract function. Growth factors have been shown to play a role 
in cyclic mitosis and differentiation of endometrial cellular components, 
recruitment of macrophages in decidualizing the endometrium, 
endometrial-trophoblast interactions, early pregnancy maintenance, and 
endometrial functional regeneration. The term "modulate" as used herein, 
denotes a modification of an existing condition or biologic state. 
Modulation of a condition as defined herein, encompasses both an increase 
or a decrease in the determinants affecting the existing condition. For 
example, administration of HBGFs could be used to augment uterine 
functions in a condition where the promotion of growth is desired. For 
example, the uterus may be treated with HBGFs to promote the growth and 
development of placental membranes or endometrial growth. Furthermore, 
treatment with HBGFs may be used to promote and maintain a pregnancy by 
facilitating endometrial-trophoblast interaction. Alternatively, 
antagonists to HBGFs are administered to modulate conditions of excessive 
endometrial growth in which the level of HBGF is excessive in comparison 
to a normal biologic condition. 
The invention also discloses a method for treating conditions characterized 
by a cell proliferative disorder by treating the condition using an 
therapeutically effective amount of a HBGF reactive agent. The term 
"treat" denotes a lessening of the detrimental effect of the condition in 
the subject receiving the reactive agent. Where the condition is due to an 
overgrowth of cells, an antagonist of HBGF is therapeutically effective in 
decreasing the amount of growth factor that can bind to an HBGF specific 
receptor on a cell. Such an antagonist may be a HBGF specific antibody or 
functional fragments thereof (e.g., Fab, F(ab)). The treatment requires 
contacting or delivering to the site of the condition with the antagonist 
of the HBGF polypeptide. Where the cell proliferative disorder is due to a 
diminished amount of growth of cells, a HBGF reactive agent which is 
stimulatory is contacted with, or delivered to the site of the condition. 
For example, TGF-.beta. (or another member of the TGF-.beta. superfamily) 
can be such a reactive agent. Other biologic agents will be known to those 
skilled in the art. 
When a cell proliferative disorder is associated with the expression of 
HBGFs, a therapeutic approach which directly interferes with the 
transcription of HBGF into mRNA or the translation of HBGF mRNA into 
protein is possible. For example, antisense nucleic acid or ribozymes that 
bind to the HBGF mRNA or cleave it are also included within the invention. 
Antisense RNA or DNA molecules bind specifically with a targeted gene's 
RNA message, interrupting the expression of that gene's protein product. 
The antisense binds to the mRNA forming a double stranded molecule which 
cannot be translated by the cell. Antisense oligonucleotides of about 
15-25 nucleotides are preferred since they are easily synthesized and have 
an inhibitory effect just like antisense RNA molecules. In addition, 
chemically reactive groups, such as iron-linked ethylenediaminetetraacetic 
acid (EDTA-F.sub.c) can be attached to an antisense oligonucleotide, 
causing cleavage of the RNA at the site of hybridization. These and other 
uses of antisense methods to inhibit the in vivo translation of genes are 
well known in the art (e.g., De Mesmaeker, et al., 1995. Backbone 
modifications in oligonucleotides and peptide nucleic acid systems. Curr. 
Opin. Struct. Biol. 5:343-355; Gewirtz, A. M., et al., 1996b. Facilitating 
delivery of antisense oligodeoxynucleotides: Helping antisense deliver on 
its promise; Proc. Natl. Acad. Sci. U.S.A. 93:3161-3163; Stein, C. A. A 
discussion of G-tetrads 1996. Exploiting the potential of antisense: 
beyond phosphorothioate oligodeoxynucleotides. Chem. and Biol. 3:319-323). 
Another therapeutic approach included within the invention involves direct 
administration of reagents or compositions including the HBGFs of the 
invention by any conventional administration technique (for example, but 
not restricted to, local injection, inhalation, or systemic 
administration), to a subject with a fibrotic, a scelortic, or a cell 
proliferative disorder, atherosclerosis. Administration of HBGFs, as 
described above, accelerate wound healing, can induce the formation of 
tissue repair or regeneration, or the growth and development of the 
endometrium. The reagent, formulation or composition may also be targeted 
to specific cells or receptors by any method described herein or by any 
method known in the art of delivering, targeting and expressing genes 
encoding HBFG. The actual dosage of reagent, formulation or composition 
that modulates a fibrotic disorder, a scelortic disorder, a cell 
proliferative disorder, atherosclerosis or wound healing depends on many 
factors, including the size and health of an organism. However, one of 
ordinary skill in the art can use the following teachings describing the 
methods and techniques for determining clinical dosages (Spilker B., Guide 
to Clinical Studies and Developing Protocols, Raven Press Books, Ltd., New 
York, 1984, pp. 7-13, 54-60; Spilker B., Guide to Clinical Trials, Raven 
Press, Ltd., New York, 1991, pp. 93-101; Craig C., and R. Stitzel, eds., 
Modern Pharmacology, 2d ed., Little, Brown and Co., Boston, 1986, pp. 
127-33; T. Speight, ed., Avery's Drug Treatment: Principles and Practice 
of Clinical Pharmacology and Therapeutics, 3d ed., Williams and Wilkins, 
Baltimore, 1987, pp. 50-56; R. Tallarida, R. Raffa and P. McGonigle, 
Principles in General Pharmacology, Springer-Verlag, New York, 1988, pp. 
18-20) or to determine the appropriate dosage to use; but, generally, in 
the range of about between 0.5 .mu.g/ml and 500 .mu.g/ml inclusive final 
concentration are administered per day to an adult in any 
pharmaceutically-acceptable carrier. 
The present invention also provides a method to detect the presence of 
abnormal levels of HBGFs in a subject to be used diagnostically to 
determine the presence of conditions or pathologies associated with 
abnormal levels of HBGFs. Such conditions include but are not restricted 
to cell proliferative disorders, various fibrotic conditions including 
scleroderma, arthritis, liver cirrhosis, and uterine fibroids. For 
example, a sample suspected of containing HBGFs is obtained from a 
subject, the level of HBGF polypeptide is determined and compared with the 
level of HBGF polypeptide in a normal tissue sample. The level of HBGFs 
can be determined by immunoassays using anti-HBGF polypeptide antibodies, 
for example. Other variations of such assays include radioimmunoassay 
(RIA), ELISA and immunofluorescence. Alternatively, nucleic acid probes 
can be used to detect and quantitate HBGF polypeptide mRNA for the same 
purpose. 
The following examples are put forth so as to provide those of ordinary 
skill in the art with a complete disclosure and description of how to make 
and use the HBGFs of the present invention, and are not intended, nor 
should they be construed, to limit the scope of what the inventors regard 
as their invention. Efforts have been made to ensure accuracy with respect 
to numbers used (e.g., amounts, time, temperature, etc.) but some 
experimental errors and deviations should be accounted for. Unless 
indicated otherwise, parts are parts by weight, molecular weight is weight 
average molecular weight, temperature is in degrees Centigrade, and 
pressure is at or near atmospheric. 
EXAMPLE 1 
Characterization and Purification of HBGF Polypeptides 
Uteri were collected at random from slaughterhouse pigs that were 
approximately 8 months or less in age. Each uterine horn was flushed with 
cold (4.degree. C.) phosphate-buffered saline (PBS) to collect uterine 
luminal components. Growth factor purification was performed on 4-liter 
pools of ULF obtained from up to 120 animals. Uterine luminal flushings 
(ULF) were clarified by centrifugation at 13,500.times.g for 30 minutes at 
4.degree. C., and the supernatant was passed through glass wool. 
Four liter samples of clarified ULF supernatant were applied at 4.degree. 
C. to a BioRex 70 cation exchange column (5.times.6 cm; Bio-Rad) that had 
previously been equilibrated in PBS, 0.2M NaCl. After sample application, 
the column was washed with 500 ml of PBS, 0.2M NaCl, and bound proteins 
were eluted using a 500 ml gradient of 0.2-2M NaCl in PBS. The flow rate 
was 3.5 ml/min throughout, and fractions of 10 ml were collected during 
treatment of the column with the NaCl gradient. Fractions demonstrating 
mitogenic activity for Balb/c 3T3 fibroblasts were selected for further 
use. All subsequent chromatographic steps were performed at room 
temperature. 
The ion exchange chromatograph of ULF showed the presence of cationic 
growth factor activity for Balb/c 3T3 cells eluted from BioRex 70 columns 
by 0.3-0.6M NaCl. Heparin affinity chromatography revealed the presence of 
an additional unidentified HBGF polypeptide that required 0.8M NaCl for 
elution from an EconoPac heparin column. In terms of the amount of 
bioactivity recovered from the column, the fraction requiring 0.8M NaCl 
for elution appeared to be a principal cationic heparin-binding growth 
factor for 3T3 cells. The elution position of HBGF polypeptides from 
heparin affinity columns was clearly distinct from PDGF, HB-EGF, PTN, 
aFGF, bFGF, and amphiregulin. HBGF mitogenic activity was destroyed by 
exposure to heat (100.degree. C. for 2 mins or 56.degree. C. for 30 mins) 
or acid (pH2.0 for 2 mins). 
Gel filtration chromatography was used to show that HBGFs had an apparent 
relative molecular mass of approximately 10,000 daltons. For these 
studies, 0.5 ml of a fraction containing the 0.8M NaCl eluate from 
EconoPac heparin affinity FPLC of ULF from 30 animals was applied at 0.5 
ml/min to a TSK G2000 SW FPLC column (30 cm.times.8 mm, 10-.mu.m particle 
size, M, 500-100,000 fractionation range; TosoHaas) equipped with a SW 
guard column (4 cm.times.8 mm, 10-.mu.m; TosoHaas). Proteins were eluted 
with PBS containing 0.3M NaCl. Fractions of 200 .mu.l were collected and 
tested for their ability to stimulate DNA synthesis in 3T3 cells. Column 
calibration was performed using EGF (6,000 MW), lactalbumin (14,200 MW), 
trypsin inhibitor (20,100 MW), and ovalbumin (45,000 MW). Fractions were 
tested for their ability to stimulate DNA synthesis in 3T3 cells at 40 
.mu.l/ml, as described above. 
Fractions that contained HBGF activity (fractions 16-19 collected after the 
cation exchange chromatography and heparin affinity chromatography) were 
pooled, diluted, and subjected to a second cycle of heparin affinity FPLC 
using a TSK heparin 5PW column. To perform the second heparin affinity 
purification step, biologically active HBGF fractions containing the 0.8M 
NaCl eluate from the EconoPac heparin purification step were pooled, 
diluted 3-fold with 20 mM Tris-HCl (pH 7.4), and clarified by passage 
through a 0.2-.mu.m filter. The sample was applied at 2 ml/min to a TSK 
heparin 5PW column (0.8.times.7.5 cm; TosoHaas, Philadelphia, Pa.), that 
was washed and eluted as described above, except that CHAPS was omitted 
from the buffers and fractions of 0.5 ml were collected. Fractions 
containing proteins that were eluted by 0.8M NaCl and which demonstrated 
mitogenic activity of 3T3 cells were divided into two pools consisting of 
fractions 31-34 (peak 1) and fractions 35 and 36 (peak 2). HBGF 
polypeptide was again eluted by 0.8M NaCl (fractions 31-36), but was 
resolved as two peaks of mitogenic activity which had distinct heparin 
binding properties. The activity peaks were termed HGBF-0.8-P1 for 
fractions 31-34 and HGBF-0.8-P2 for fractions 35 and 36. 
HBGF-0.8-P1 and -P2 were adjusted to 10% acetonitrile, 0.1% trifluroacetic 
acid, and individually subjected to C.sub.8 reverse-phase HPLC. 
Reverse-phase HPLC was performed on a Hitachi HPLC system (Hitachi 
Instruments Inc., Danbury, Conn.) using a C.sub.8 column (0.46.times.25 
cm, 5-.mu.m particle size; Rainin Instrument Co., Woburn, Mass.) that was 
equilibrated with water containing 10% (v/v) acetonitrile and 0.1% (v/v) 
trifluoroacetic acid. Pooled fractions containing peaks 1 and 2 from the 
TSK heparin purification step were individually adjusted so that they 
contained 10% acetonitrile, 0.1% trifluoroacetic acid and were clarified 
by passage through a 0.2-.mu.m filter. Conditions for the elution of bound 
proteins were 10% acetonitrile from zero to 10 min. after sample injection 
and 10-90% from 10 min. to 146 min. The flow rate was 1 ml/min throughout, 
and the chromatogram (A.sub.214) was archived as described (Bray, and 
Brigstock, (1994) Amer. Lab. 26, 38). The eluate was collected as 0.5 ml 
fractions in siliconized tubes containing 50 .mu.l of 125 mM NaOH to 
immediately neutralize the trifluoroacetic acid. The 80 .mu.l aliquots of 
selected fractions were evaporated to dryness in a SpeedVac concentrator 
(Savant Instruments, Farmingdale, N.Y.) and reconstituted in 25 .mu.l of 
10 mM Tris-HCl (pH 7.4). 10 .mu.l of this concentrate were assayed for 
their stimulation of 3T3 cell DNA synthesis, and 10 .mu.l were used for 
analytical SDS-PAGE. For the second step C.sub.8 HPLC purification, two 
active fractions from the first HPLC step were pooled (1 ml total volume), 
diluted 5-fold with water, 0.1% trifluoroacetic acid, and subjected to the 
same chromatographic elution conditions as described herein. The elution 
positions of HGBF-0.8-P1 and -P2 were determined by bioassay of aliquots 
of fractions containing the column eluate after they had been evaporated 
and reconstituted in PBS, demonstrating that there was sufficient activity 
in the purified HGBF samples to permit their detection and further 
characterization despite prolonged (approximately 30 to 40 minute) 
exposure to pH=2 during the HPLC step. 
Following HPLC, silver-stained SDS-PAGE analysis of the fractions 
containing either HBGF-0.8-P1 or -P2 was performed under reducing 
conditions using 18% polyacrylamide mini-gels as described (Kim, G. Y., et 
al., (1995) Biol. Reprod. 52, 561-571). Subsequently, silver staining of 
proteins was performed as described (Wray, W., et al., (1981) Anal 
Biochem. 118, 197-203). SDS-PAGE was performed on (i) HPLC-purified growth 
factors, (ii) 8 .mu.l of unfractionated ULF, or (iii) 100 .mu.l of ULF 
after passage through 20-.mu.l beds of heparin-Sepharose in the presence 
of 10 mM Tris-HCl, 0.5M NaCl (pH 7.4) and subsequent extraction of the 
heparin beads with SDS-PAGE sample buffer. Gels were then prepared as 
described (Kim, G. Y., et al., (1995) Biol. Reprod. 52, 561-571). 
Subsequent analysis revealed the presence of a single 10-kDa protein that 
co-purified with Balb/c 3T3 mitogenic activity. Levels of mitogenic 
activity were directly correlated with those of the 10-kDa protein, which 
was completely pure as shown by silver staining. The results from 18 
individual HPLC purifications confirmed a direct, causative relationship 
between the 10-kDa protein(s) and the mitogenic activity of HBGF-0.8-P1 
and -P2. 
Analysis of the individual purification steps showed that 0.5-1.1 .mu.g of 
HBGF-0.8-P1 or -P2 were each purified from 342 mg of crude ULF protein and 
that 10-22 activity units for HBGF-0.8-P1 or P2 were recovered after the 
first HPLC step as compared with 66,666 units in 1 liter of starting 
material (Table 1). It should be noted that the apparent low recovery of 
HBGF peptide-0.8 activity was attributable to (i) a major contribution by 
IGF, EGF, PDGF, bFGF, HB-EGF, and PTN to the overall 3T3 cell mitogenic 
activity of the crude and partially purified samples (2, 8, 9, 12, 25-27) 
and (ii) acid lability of HBGF peptide-0.8 mitogenic activity during the 
HPLC separation step(s). Although alternative strategies were attempted to 
recover purified growth factors of higher specific activity, it was not 
possible to avoid the use of either reverse-phase HPLC or trifluoroacetic 
acid for ion pairing without compromising the purity of the final product. 
While, in terms of their biological activity, recovery of HBGF-0.8-P1 and 
-P2 was somewhat compromised, structural characterization of the proteins 
was readily achieved, since they retained sufficient activity to be 
unequivocally attributable to a single, homogenous 10-kDa band in 
SDS-polyacrylamide gels, and sufficient quantities of each protein were 
isolated from several liters of ULF (Table 1). 
TABLE 1 
__________________________________________________________________________ 
Protein Total 
Purification 
recovered 
ED.sub.50.sup.a 
activity 
Activity 
Purification 
step ng ng/ml units recovered 
factor 
__________________________________________________________________________ 
Crude ULF 
3.4 .times. 10.sup.8 
25,650 
66,666 
100 
BioRex 70 
2.2 .times. 10.sup.7 
3825 28,649 
43 7 
EconoPac Hep 
4.8 .times. 10.sup.5 
3225 744 1.1 8 
HBGF-0.8-P1 
TSK-Hep 2.1 .times. 10.sup.5 
2230 473 0.7 11 
C.sub.8 HPLC, step 1.sup.d 
1100 250 22 0.03 103 
C.sub.8 HPLC, step 2.sup.d 
100 25 20 0.03 1026 
HBGF-0.8-P2 
TSK-Hep 2.9 .times. 10.sup.4 
417 347 0.5 62 
C.sub.8 HPLC.sup.d 
500 250 10 0.015 103 
__________________________________________________________________________ 
.sup.a Concentration of HBFG0.8 preparation required to give 50% maximal 
DNA synthesis. 
.sup.b 1 unit of activity is the quantity of HBGF0.8 required to give the 
ED.sub.50. 
.sup.c Compared with crude ULF. 
.sup.d Bioactivity diminished due to acid exposure. 
EXAMPLE 2 
HBGF Polypeptide Sequencing 
Fractions containing the HPLC purified growth factors were pooled, dried, 
and subjected to preparative SDS-PAGE. Proteins in the gel were 
transferred for 90 min. at 300 mA to a polvinylidene difluoride membrane 
using 10 mM CAPS buffer (pH 11). The location of the proteins of interest 
was determined by staining the blots with 0.1% Coomassie R250 in 50% 
methanol for 2 min, followed by destaining with 50% methanol, 10% acetic 
acid. Half of each 10-kDa protein band was excised and submitted for 
N-terminal amino acid sequencing on a model 470A gas phase sequenator 
(Applied BioSystems, Foster City, Calif.). Phenylthiohydantoin-derivatives 
were identified by C.sub.18 reverse phase HPLC. A 16-residue sequence was 
obtained for HBGF-0.8-P1 with an undetermined residue at position 10, and 
a 12-residue sequence was obtained for HBGF-0.8-P2 with an undetermined 
residue at position 9 (Table 2). These data showed that HBGF-0.8-P1 and 
-P2 were N-terminally identical except for the presence of an additional 
Glu residue at the N terminus of HBGF-0.8-P1. A search of GenBank.TM. 
revealed that these sequences aligned perfectly with predicted internal 
sequences of hCTGF and mouse fisp-12 (also termed .beta.IG-M2), the murine 
homologue of CTGF (Bradham, D. M., et al., (1991) J. Cell Biol. 114, 
1285-1294; Ryseck, R-P., et al., (1991) Cell Growth Differ. 2, 225-233; 
Brunner, A., et al., (1991) DNA Cell Biol. 10, 293-300). The unassigned 
residue in cycle 10 of HBGF-0.8-P1 and cycle 9 of HBGF-0.8-P2 corresponded 
to Cys.sup.256 of hCTGF and Cys.sup.255 of fisp-12 (Table 2). 
TABLE 2 
__________________________________________________________________________ 
HBGF-0.8-P1.sup.a (SEQ ID NO:1) 
Glu--Glu--Asn--Ile--Lys--Lys--Gly--Lys--Lys--Xaa--Ile--Arg-- 
Thr--Pro--Lys--Ile 
HBGF-0.8-P2.sup.b (SEQ ID NO:2) 
Glu--Asn--Ile--Lys--Lys--Gly--Lys--Lys--Xaa--Ile--Arg--Thr 
Human CTGF-(247-262).sup.c 
Glu--Glu--Asn--Ile--Lys--Lys--Gly--Lys--Lys--Cys--Ile--Arg-- 
Thr--Pro--Lys--Ile 
fisp-12-(246-261).sup.d 
Glu--Glu--Asn--Ile--Lys--Lys--Gly--Lys--Lys--Cys--Ile--Arg-- 
Thr--Pro--Lys--Ile 
Porcine CTGF-(247-262).sup.e 
Glu--Glu--Asn--Ile--Lys--Lys--Gly--Lys--Lys--Cys--Ile--Arg-- 
Thr--Pro--Lys--Ile 
__________________________________________________________________________ 
.sup.a Repetitive yield = 88%; initial yield 7 pmol. 
.sup.b Repetitive yield = 90%; initial yield 3 pmol. 
.sup.c See Bradham et al. J. Cell Biol. 114:1285-1294, 1991. 
.sup.d See Ryseck et al. Cell Growth Differ. 2:225-233, 1991. 
.sup.e From cDNA analysis in this study. 
To verify that the partial sequences of HBGF-0.8-P1 and -P2 were actually 
present in the porcine CTGF (pCTGF) molecule, a full-length pCTGF cDNA was 
isolated by hybridization screening of a pig endometrial cDNA library 
using a .sup.32 P-labeled hCTGF probe. For these studies, total pig 
endometrial RNA was obtained as described (Kim, G. Y., et al., (Biol. 
Reprod. 52, 561-571 (1995)). A poly(A)Tract mRNA isolation system 
(Promega, Madison, Wis.) was used to isolate poly(A.sup.+) RNA, 5 .mu.g of 
which was subjected to first strand cDNA synthesis using Moloney murine 
leukemia virus reverse transcriptase and oligo(dT) linker-primer 
containing XhoI. Second strand synthesis was primed by treating the 
mRNA-cDNA complex with RNase. Double-stranded cDNA was blunted using 
Klenow fragment and ligated to EcoRI adaptors that were subsequently 
phosphorylated with T4 polynucleotide kinase. 100 ng of XhoI-digested 
cDNA, purified on a Sephacryl S-400 column, were ligated into 1 .mu.g of 
Uni-ZAP XR vector arms at the XhoI-EcoRI multiple cloning site, and the 
product was packaged using Gigapack II packaging extract (Stratagene, La 
Jolla, Calif.). The primary library was amplified in XL1-Blue MRF' cells 
to a titer of 1.4.times.10.sup.10 plaque-forming units/ml. 
A verified .sup.32 P-labeled CTGF probe, corresponding to the 3' end of the 
predicted hCTGF primary translational product, was obtained by reverse 
transcriptase-polymerase chain reaction of RNA from human foreskin 
fibroblasts using the forward and reverse primers, 
5'-GCCGTCTAGAGCGGCCGCATGGAAGAGAACATTAAGAAGGG-3' (SEQ ID NO:3) and 
3'-CCTCTGTACCGTACTTAAGCGCCGGCGACC-5' (SEQ ID NO:4), respectively. The 
probe was used to screen 10.sup.6 plaques, two of which showed 
reproducible hybridization and were isolated using a Rapid Excision Kit 
(Stratagene). Two .about.5.0-kilo-basepair pBluescript SK pig CTGF clones, 
termed pBSK-pBSK-pCTGF1 and pBSK-p-pCTGF2, were obtained and used for 
initial sequencing reactions. pBSK-pCTGF1 was then fully sequenced by a 
combination of manual and automated dideoxy terminator sequencing (Sanger, 
F., et al., Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467 (1977)). Sequence 
data were obtained from both strands of DNA. Sequences of HBGF0.8-P1 and 
-P2 are listed in Table 2. 
The cloned pig CTGF cDNA was determined to be 1.51 kilobase pairs, with an 
open reading frame of 1,047 base pairs. The primary translational product 
of pCTGF is predicted to comprise 349 amino acids and contains HBGF 
peptide-0.8 sequence between residues 247 and 262 (Table 2). At the amino 
acid level, pCTGF is approximately .about.92% identical to fisp-12 and 
hCTGF. After cleavage of its presumptive 26-residue signal peptide, pCTGF 
is predicted to comprise 323 amino acids and to contain 38 Cys residues 
that are fully conserved in hCTGF and fisp-12. 
EXAMPLE 3 
HBGF Antibody Production 
Since HBGFs represent microheterogenous forms of truncated CTGF, the 
relationship of HBGF to CTGF was investigated. The presence of the 10-kDa 
protein in the starting material was confirmed by Western blotting of 
unfractionated ULF samples using a CTGF antibody that reacted with 
HPLC-purified HBGF polypeptides. 
To produce the antibody, a four-branched multiple antigenic CTGF-(247-260) 
peptide comprising the sequence EENIKKGKKCIRTP (residues 247-260) (SEQ ID 
NO:5) was produced on a Synergy 432A peptide synthesizer (Applied 
BioSystems) and purified by reverse-phase HPLC using a C.sub.18 column 
(0.46.times.36 cm; Rainin Instruments) that was developed with a 90-min 
5-95% acetonitrile gradient in water, 0.1% trifluoroacetic acid. Fractions 
containing the purified polypeptides were pooled, evaporated to dryness, 
and reconstituted in sterile water. Two New Zealand White rabbits (rabbits 
A and B), which had been bled to collect preimmune serum, were injected 
subcutaneously with 1 mg of polypeptide in Freund's complete adjuvant, 
followed 3 weeks later by an intramuscular injection of 250 .mu.g of 
polypeptide in Freund's incomplete adjuvant. Animals were bled 7 days 
later for collection of antiserum. Reactivity of the antisera was 
validated by Western blotting and immunoprecipitation. Pre-immune serum 
and antiserum from rabbit A were used in these experiments. 
EXAMPLE 4 
Generation of the 10-kDa HBGF Polypeptides 
Western blotting was performed as has been previously described (Harlow and 
Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor 
Laboratory, New York, Current Edition). Briefly, SDS-PAGE was performed 
under reducing conditions using 18% polyacrylamide mini-gels as described 
(Kim, G. Y., et al., Biol. Reprod. 52, 561-571 (1995)). Silver staining of 
proteins was performed as described (Wray, W., et al., Anal. Biochem. 118, 
197-203 (1981)). Western blotting was performed on (i) HPLC-purified 
growth factors, (ii) 8 .mu.l of unfractionated ULF, or (iii) 100 .mu.l of 
ULF after passage through 20-.mu.l beds of heparin-Sepharose in the 
presence of 10 mM Tris-HCl, 0.5M NaCl (pH 7.4) and subsequent extraction 
of the heparin beads with SDS-PAGE sample buffer. Gels were blotted and 
blocked as described (Kim, G. Y.,et al., Biol. Reprod. 52, 561-571 (1995)) 
and incubated with a 1:1,000 dilution of rabbit preimmune serum or a 
1:1,00 dilution of rabbit anti-pCTGF-(247-260) peptide antiserum (rabbit 
A). Immunoreactive bands were visualized using alkaline 
phosphatase-conjugated goat anti-rabbit IgG followed by nitro blue 
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate chromogenic substrates. 
In addition to the 10-kDa protein, two additional mass forms of CTGF (16 
and 20-kDa) were also present in ULF, but convincing evidence for the 
38-kDa CTGF was not obtained. The Western blot further verified that HPLC 
purified HBGF comprised a single immunoreactive 10-kDa protein. Comparison 
of the staining intensity of HBGF from defined volumes of undiluted 
uterine fluid (i.e. 0.7-2.3 .mu.l) with the staining intensity of 
mitogenic amounts of purified HGBF indicated that mitogenic concentrations 
of HBGFs exist in uterine fluid in vivo. Taken together, the data showing 
that ULF did not contain detectable levels of 38-kDa CTGF but did contain 
HBGFs in amounts likely to be mitogenic, demonstrate that HBGFs occur 
naturally in vivo and is not the result of a breakdown of 38 kDa CTGF 
during their purification. 
EXAMPLE 5 
Heparin Binding Properties of HBGF Polypeptides 
The presence of an additional acidic Glu residue at the N-terminus of HBGF 
polypeptide 0.8-P1 was correlated with the lower heparin affinity of this 
molecule as compared with HBGF-0.8-P2, suggesting that the N-terminus of 
HBGF peptide-0.8 may be part of a heparin-binding domain. To test the 
heparin-binding properties of the N-terminal region as well as other 
portions of the CTGF molecule, the ability of 18 polypeptides spanning the 
entire C-terminal 103 residues of hCTGF to bind .sup.3 H!heparin was 
investigated. 
Eighteen synthetic polypeptides spanning the entire 103 C-terminal residues 
of CTGF were synthesized and received as a cleaved PepSet.TM. from Chiron 
Mimotopes (Clayton, Victoria, Australia). All polypeptides were 
synthesized with acetylated N-termini and amidated C-termini except 
CTGF-(247-255) and CTGF-(247-260), which were synthesized with free 
N-terminal amines, and CTGF-(326-349) and CTGF-(339-349), which were 
synthesized with acid C-termini (Table 3). 
All polypeptides contained one or no Cys residues; Cys.sup.292 in 
CTGF(285-292) and Cys.sup.325 in CTGF-(318-328) were replaced with Ser to 
prevent intra-chain disulfide bridging to Cys.sup.287 or Cys.sup.323 
within the respective polypeptides. Heparin-binding properties were 
determined using an adaptation of the method of Baird et al. (Baird, A., 
et al. Proc. Natl. Acad. Sci. U.S.A. 85, 2324-2328 (1988)). Briefly, 37.5 
nmol of each polypeptide were absorbed in duplicate to nitrocellulose 
using a dot-blot apparatus. The blot was blocked for 30 min. with 100 mM 
Tris-HCl, 0.15M NaCl, 0.1% bovine serum albunin (pH 7.4) and then 
incubated for 3 hr. at room temperature in this solution containing 10 
.mu.Ci/ml .sup.3 H!heparin (NEN Life Science Products). The blot was 
washed four times with 10 mM Tris-HCl, 0.15M NaCl, and individual dots 
were mixed with scintillation fluid for counting of .sup.3 H!. 
Table 3 summarizes the results obtained with the synthetic polypeptides. 
The highest level of heparin binding was obtained for polypeptides 
containing residues 247-260, 274-286, and 305-328. It should be noted that 
none of these polypeptides had HBGF polypeptide agonist or antagonist 
activity in a 3T3 cell DNA synthesis assay. 
TABLE 3 
__________________________________________________________________________ 
.sup.3 H! Heparin bound 
(mean .+-. S.D.) 
Peptide domain 
Sequence cpm/.mu.g 
__________________________________________________________________________ 
None 11 .+-. 0.2 
CTGF-(247-255) 
EENIKKGKK.sup.a 10 .+-. 0.3 
CTGF-(247-260) 
EENIKKGKKCIRTP.sup.a 
836 .+-. 1 
CTGF-(257-272) 
IRTPKISKPIKFELSG 70 .+-. 13 
CTGF-(259-275) 
TPKISKPIKFELSGCTS 124 .+-. 3 
CTGF-(274-283) 
TSMKTYRAKF 388 .+-. 12 
CTGF-(274-286) 
TSMKTYRAKFCGV 1108 .+-. 119 
CTGF-(285-291) 
GVCTDGR 7 .+-. 0.3 
Ser.sup.292 CTGF-(285-292) 
GVCTDGRS 8 .+-. 0.4 
CTGF-(293-306) 
CTPHRTTTLPVEFK 9 .+-. 1.1 
CTGF-(294-306) 
TPHRTTTLPVEFK 11 .+-. 0.4 
CTGF-(305-322) 
FKCPDGEVMKKNMMFIKT 
237 .+-. 22 
CTGF-(308-322) 
PDGEVMKKNMMFIKT 71 .+-. 2 
CTGF-(318-324) 
MFIKTCA 475 .+-. 116 
Ser.sup.325 CTGF-(318-328) 
MFIKTCASHYN 601 .+-. 40 
CTGF-(324-328) 
ACHYN 9 .+-. 1 
CTGF-(326-349) 
HYNCPGDNDIFESLYYRKMYGDMA.sup.b 
10 .+-. 1 
CTGF-(330-340) 
PGDNDIFESLY 10 .+-. 0.5 
CTGF-(339-349) 
LYYRKMYGDMA.sup.b 9 .+-. 0.5 
__________________________________________________________________________ 
.sup.a Free Nterminal amine. 
.sup.b Acid C teminus 
Previous studies have shown that heparin modulates receptor binding and 
biological activity of several HBGF polypeptides including bFGF, HB-EGF, 
and amphiregulin (Besner, G. E., et al., Growth Factors 7, 289-296 (1992); 
Higashiyama, S., et al., J. Cell Biol., 122, 933-940 (1993); Rapraeger, A. 
C., et al., Science 252, 1705-1708 (1991); Olwin, B. B., et al. J. Cell 
Biol. 118, 631-639 (1992); Cook, P., et al. J. Cell Physio. 163, 418-429 
(1995); Yayon, A., et al., Cell 64, 841-848 (1991); Aviezer, D., et al. 
Proc. Natl. Acad. Sci. U.S.A. 91, 12173-12177 (1994)). Since HBGF 
peptide-0.8 exhibited strong affinity for heparin, we examined the effect 
of this glycosaminoglycan on the mitogenic activity of HBGF peptide-0.8. 
The activity of a high stimulatory dose of HBGF peptide-0.8 was 
significantly potentiated by 1-3 .mu.g/ml heparin but was inhibited by 
30-100 .mu.g/ml heparin. The same heparin dosages had no effect on basal 
or calf serum-stimulated DNA synthesis in 3T3 cells. 
EXAMPLE 6 
HBGF Mitogenic Assay 
To assess the relative mitogenic capability of HBGFs with IGF-1, EGF, bFGF, 
and PDGF-AB, DNA synthesis assays on 3T3 cells were performed (Table 4). 
Biologically active fractions containing the 0.3-0.6M NaCl eluate from the 
Bio-Rex column were pooled, diluted 3-fold with 20 mM Tris-HCL (pH 7.4) 
containing 0.1% CHAPS, passed through a 0.45-.mu.m membrane filter, placed 
in a siliconized polypropylene vessel, and applied with a peristaltic pump 
to an EconoPac heparin column (0.7.times.3.6 cm; Bio-Rad) at 2 ml/min. The 
heparin column was then washed with 50 ml of 20 mM Tris-HCl buffer, 0.2M 
NaCl, 0.1% CHAPS and developed at 1 ml/min with a 40 ml gradient of 
0.1-2.0M NaCl in 20 mM Tris-HCl, 0.1% CHAPS (pH 7.4) using a fast protein 
liquid chromatography (FPLC) system (Pharmacia Biotech Inc.). Fractions (1 
ml) were collected into siliconized tubes during NaCl gradient elution and 
tested for 3T3 cell mitogenic activity. 
Column fractions were tested for their ability to stimulate DNA synthesis 
as measured by .sup.3 H!thymidine incorporation into the DNA of confluent 
quiescent Balb/c 3T3 cells grown in 200 .mu.l of Dulbecco's modified 
Eagle's medium, 10% bovine calf serum in 96-well culture plates as 
described (Kim, G. Y., et al., Biol. Reprod. 52, 561-571 (1995)). 
Dose-response curves to purified growth factors from ULF were established 
by assaying each dose in triplicate, with data computed as mean.+-.S.D. 
Statistical significance of the effects of 1-100 .mu.g/ml porcine heparin 
(Sigma) on growth factor activity was determined by Students' t test. 
.sup.3 H! thymidine incorporation by HBGF peptide was comparable with that 
of calf serum or purified PDGF or bFGF rather than that of weaker mitogens 
such as IGF or EGF. Further, it was found that the 3T3 mitogenic and 
biologic activity of HBGFs was synergistically potentiated by 10 ng/ml 
IGF-I, 10 ng/ml PDGF, 3 ng/ml EGF, or 0.3 ng/ml bFGF. 
Target cell specificity was studied using Balb/c 3T3 cells, bovine 
capillary endothelial cells (BCECs), and vascular smooth muscle cells. 3T3 
cells were utilized as described above. 
BCECs were obtained from Dr. J. Folkman (Children's Hospital, Boston, 
Mass.) and were maintained in gelatinized culture flasks in Dulbecco's 
modified Eagle's medium containing 3 ng/ml bFGF and 10% heat-inactivated 
bovine calf serum. Smooth muscle cells were isolated from a 2-3-cm length 
of pig thoracic aorta using established procedures (Weich, H. A., et al., 
Growth Factors 2, 313-320 (1990)) and maintained in 10% Dulbecco's 
modified Eagle's medium, 10% fetal bovine serum. BCEC and smooth muscle 
cell DNA synthesis assays were performed in 48- or 96-well plates 
essentially as described (Besner, G. E., Higashiyama, S., and Kagsbrun, M. 
Cell Regul. 1, 811-819 (1990)). BCEC DNA synthesis assays were also 
performed in the presence of 100 .mu.g/ml porcine heparin. HGBF was found 
to be mitogenic for smooth muscle cells and produced a level of 
stimulation that exceeded that of a maximal amount of EGF but was less 
than that of bFGF. HBGFs lacked mitogenic activity for endothelial cells 
when tested alone or in the presence of 100 .mu.g of heparin (see Table 
4). 
TABLE 4 
______________________________________ 
.sup.3 H! Thymidine 
incorporation 
(mean .+-. S.D.) 
Cell Type Treatment Cpm/well 
______________________________________ 
Balb/c 3T3 fibroblasts 
None 428 .+-. 18 
20% calf serum 
123,820 .+-. 7,470 
30 ng/ml IGF-1 
4,412 .+-. 170 
30 ng/ml EGF 11,550 .+-. 101 
10 ng/ml bFGF 73,853 .+-. 3,122 
30 ng/ml PDGF-AB 
110,110 .+-. 7,077 
20 .mu.l/ml HBGF-0.8 
114,730 .+-. 3,200 
Vascular smooth muscle 
None 680 .+-. 341 
cells 
3 ng/ml EGF 1,343 .+-. 378 
3 ng/ml bFGF 3,082 .+-. 374 
15 .mu.l/ml HBGF-0.8 
1,709 .+-. 403 
Capillary endothelial cells 
None 316 .+-. 84 
100 .mu.g/ml heparin 
240 .+-. 52 
3 ng/ml bFGF 2,865 .+-. 276 
3 ng/ml bFGF + 100 
1,840 .+-. 4 
.mu.g/ml heparin 
3 ng/ml aFGF 603 .+-. 46 
3 ng/ml aFGF + 100 
2,232 .+-. 236 
.mu.g/ml heparin 
20 .mu.l/ml HBGF-0.8 
243 .+-. 4 
20 .mu.l/ml HBGF-0.8 + 
195 .+-. 12 
100 .mu.g/ml heparin 
______________________________________ 
Although the invention has been described with reference to the presently 
preferred embodiment, it should be understood that various modifications 
can be made without departing from the spirit of the invention. 
Accordingly, the invention is limited only by the following claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 22 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GluGluAsnIleLysLysGlyLysLysXaaIleArgThrProLysIle 
151015 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GluAsnIleLysLysGlyLysLysXaaIleArgThr 
1510 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GCCGTCTAGAGCGGCCGCATGGAAGAGAACATTAAGAAGGG41 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CCTCTGTACCGTACTTAAGCGCCGGCGACC30 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GluGluAsnIleLysLysGlyLysLysCysIleArgThrPro 
1510 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GluGluAsnIleLysLysGlyLysLys 
15 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
IleArgThrProLysIleSerLysProIleLysPheGluLeuSerGly 
151015 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
ThrProLysIleSerLysProIleLysPheGluLeuSerGlyCysThr 
151015 
Ser 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
ThrSerMetLysThrTyrArgAlaLysPhe 
1510 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
ThrSerMetLysThrTyrArgAlaLysPheCysGlyVal 
1510 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GlyValCysThrAspGlyArg 
15 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
GlyValCysThrAspGlyArgSer 
15 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CysThrProHisArgThrThrThrLeuProValGluPheLys 
1510 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
ThrProHisArgThrThrThrLeuProValGluPheLys 
1510 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
PheLysCysProAspGlyGluValMetLysLysAsnMetMetPheIle 
151015 
LysThr 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
ProAspGlyGluValMetLysLysAsnMetMetPheIleLysThr 
151015 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
MetPheIleLysThrCysAla 
15 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
MetLysIleLysThrCysAlaSerHisTyrAsn 
1510 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
AlaCysHisTyrAsn 
15 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
HisTyrAsnCysProGlyAspAsnAspIlePheGluSerLeuTyrTyr 
151015 
ArgLysMetTyrGlyAspMetAla 
20 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
ProGlyAspAsnAspIlePheGluSerLeuTyr 
1510 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
LeuTyrTyrArgLysMetTyrGlyAspMetAla 
1510 
__________________________________________________________________________