DNA encoding a hyaluronan receptor expressed in human umbilical vein endothelial cells

The present invention provides nucleotide and amino acid sequences that identify and encode the hyaluronan receptor (hr) from human umbilical vein endothelial cells. The present invention also provides for antisense molecules to the nucleotide sequences which encode hr, expression vectors for the production of purified HR, antibodies capable of binding specifically to HR, hybridization probes or oligonucleotides for the detecting the upregulation of HR encoding nucleotide sequences, genetically engineered host cells for the expression of HR, diagnostic tests for activated, angiogenic, inflamed or metastatic cells and/or tissues based on HR-encoding nucleic acid molecules and antibodies capable of binding specifically to the receptor.

OVERVIEW 
All blood vessels are composed of three layers or tunics. The tunica intima 
consists of endothelial cells which line the vessel and rest on the basal 
lamina or middle layer. The subendothelial layer consists of loose 
connective tissue and may contain smooth muscle cells. The endothelial 
cells are generally polygonal and elongated in the direction of blood 
flow. The nucleus of the endothelial cell bulges into the capillary lumen, 
and Golgi complex is located at the nuclear poles. A few mitochondria, 
free ribosomes and rough endoplasmic reticulum are present. Endothelial 
cells are held together by zona occludentes and an occasional desmosome; 
gap junctions which offer variable permeability to macromolecules are 
present. The Weibel Palade body, a rod shaped cytoplasmic inclusion is 
characteristic of these cells. 
Vascular endothelial cells play a central role in physiological 
homeostasis, blood vessel permeability, and response to physiologic and 
pathologic stimuli. The endothelium is a primary target for cardiovascular 
risk factors such as high blood pressure, shear stress, and 
atherosclerosis. It is sensitive to endothelin, growth factors, 
interleukin 1, epinephrine, angiotensin, arginine vasopressin, heparin, 
bradykinin, acetylcholine, prostacyclin, etc. 
Hyaluronan (HA) is a negatively charged, high molecular weight, connective 
tissue polysaccharide found in the extracellular matrix of most animal 
tissues. It is synthesized in the plasma membrane of fibroblasts and other 
cells and catabolized locally as well as in the lymph nodes or liver 
sinusoids. HA is commonly isolated from the vitreous body of the eye, 
synovial fluid, umbilical cord, and skin. It has several physiological 
functions including roles in water and plasma protein homeostasis; 
mitosis, cell migration and differentiation including anglogenesis (Rooney 
P and Kumar S (1994) EXS (Switzerland) 70:179-90); and tissue remodeling, 
either as a normal or tumor-associated event. 
The matrix-induced effects on cells are directed by a wide variety of 
HA-binding proteins, such as the hyaluronan receptor (HR). The widespread 
occurrence of HRs indicate their importance in tissue organization and 
control of cellular behavior. The family is known as the hyaladherins and 
includes those HA-binding proteins which act as part of the structural 
matrix and those which interact with HA at the plasma membrane as 
cell-surface matrix receptors. With the recognition of the hypluronan 
cell-surface receptor (HR), cell biologists, pathologists, and 
immunologists have begun to investigate the importance of the HA and HR 
for their potential diagnostic and therapeutic value. 
Matrix Hyaladherins 
HRs found within the cartilage matrix have been well characterized. 
Aggrecan is the large aggregating chondroitin sulfate proteoglycan of 
cartilage which has a high affinity for HA. Link protein is a 45-48 kDa 
glycoprotein which also demonstrates strong specific binding affinity. HA 
may bind more than 100 aggrecan and link protein molecules in a 
supramolecular complex which confers the viscoelastic properties of 
cartilage. Other matrix proteins such as PG-M and type VI collagen which 
participate in assembly and integrity may also be involved. 
HA-binding proteins are also found in noncartilaginous tissues. Versican of 
fibroblasts, hyaluronectin of nervous and soft connective tissues, glial 
hyaluronan binding protein in the central nervous system, and neurocan, a 
chondroitin sulfate proteoglycan of brain also form strong structural 
complexes with HA. All matrix hyaloadherins contain tandem repeated B 
loops, a structural motif believed to contain the HA-binding domain. 
HR hyaloadherins have been detected on several cell types from a wide 
variety of tissues. Some reports suggest that HR are related to the CD44 
family of lymphocyte homing receptors which include the isoforms, Pgp-1, 
Hermes antigen, H-CAM, ECMRIII, etc. The distal extracellular domain of 
CD44 has sequence homology to one of the B loop motifs of link protein. 
The numerous isoforms suggest different cellular functions and demonstrate 
binding to other ligands such as collagens I and IV and mucosal vascular 
addressin. 
Other non-CD44 HR include cell-surface antigens termed IVd4 which block 
binding of HA, liver endothelial cell receptors (LEC) which are involved 
in the clearance of HA from the circulation, and fibroblast-produced HR 
which may be located on the cell surface where it mediates HA-induced cell 
locomotion. Its 58 kDA soluble form contains an HA-binding component 
unrelated to the B loop motif and is known as receptor for HA mediated 
motility (RHAMM). The important distinctions between cell-surface and 
matrix hyaloadherins are 1) HA hexasaccharides represent the minimum size 
molecule that interacts with these cell-surface receptors, 2) binding 
affinity increases with increasing polymer length, and 3) binding 
increases with increasing buffer ionic strength. 
Cell Migration 
Increased matrix presence of HA has been correlated with cell migration in 
embryogenesis, limb regeneration, wound healing and tumor invasion. Since 
the CD44 HR have been shown to associate with the cytoskeletal ankyrin, 
proteins of the HR complex may affect reorganization of the actin 
cytoskeleton and other activities such as cell ruffling, detachment from 
the substratum, and locomotion necessary for cell migration. RHAMM, as one 
of the HR complex proteins, binds to HA with high affinity and is 
expressed only in the leading lamellae and perinuclear regions of 
migrating fibroblasts. Since RHAMM does not include a transmembrane 
hydrophobic region, it is assumed to be a peripheral protein associated 
with intracellular, membrane-bound tyrosine kinase. In studies of timed 
administration of HA and an inhibitor of tyrosine kinase, HA stimulated 
locomotion via a rapid tyrosine kinase signal transduction pathway. 
Tumor invasion and metastasis 
Invasive or metastatic cancer cells have the capacity to exit from the 
vascular system by use of sets of molecules, at least one of which always 
has a receptor function. One series of such sets might include successive 
interactions among endothelial VLA-4 integrin and E-selectin, 
subendothelial collagen IV and .beta.-4 integrin, and soft connective 
tissue HA and CD44 or HR interactions (Zetter BR (1993) Semin Cancer Biol 
4:215-218). 
Some tumor cells also have the capacity to assemble HA-enriched 
pericellular matrices which reduce cell adhesion to the outside of the 
growing tumor and protect the tumor from immune surveillance. In addition, 
the presence of high HA attracts endothelial cells which are active in 
anglogenesis. The combination of these HA functions allows the rapid 
establishment and growth of invasive tumor cells. 
The transforming oncogene H-ras may promote cell locomotion. Hardwick et al 
(1992 J Cell Biol 117:1343-1350) reported that H-ras actually regulates 
expression of RHAMM, showed binding between HA and RHAMM, and produced an 
antibody to the protein which is capable of inhibiting HA/HR locomotion. 
SUMMARY OF THE INVENTION 
The subject invention provides nucleotide sequence which uniquely encodes a 
novel human hyaluronan receptor. The cDNA, known as hr, was fully 
contained within Incyte Clone No. 39200 and encodes a polypeptide 
designated HR. 
The invention also comprises diagnostic tests for physiologic or pathologic 
compromise which include the steps of testing a sample or an extract with 
hr nucleic acids, fragments or oligomers thereof. Aspects of the invention 
include the antisense DNA of hr; cloning or expression vectors containing 
hr; host cells or organisms transformed with expression vectors containing 
hr; a method for the production and recovery of purified HR from host 
cells; and purified protein, HR, which can be used to generate antibodies 
and other molecules for diagnosis of activated, angiogenic, inflamed or 
metastatic cells and/or tissues.

DETAILED DESCRIPTION OF THE INVENTION 
Definitions 
As used herein, HRs, refers to polypeptides, naturally occurring HRs and 
active fragments thereof, which are encoded by mRNAs transcribed from the 
cDNA of Seq ID No 1. 
"Active" refers to those forms of HR which retain the biologic and/or 
immunologic activities of any naturally occurring HR. 
"Naturally occurring HR" refers to HRs produced by human cells that have 
not been genetically engineered and specifically contemplates various HRs 
arising from post-translational modifications of the polypeptide including 
but not limited to acetylation, carboxylation, glycosylation, 
phosphorylation, lipidation and acylation. 
"Derivative" refers to HRs chemically modified by such techniques as 
ubiquitination, labeling (e.g., with radionuclides, various enzymes, 
etc.), pegylation (derivatization with polyethylene glycol), and insertion 
or substitution by chemical synthesis of aa such as ornithine, which do 
not normally occur in human proteins. 
"Recombinant variant" refers to any polypeptide differing from naturally 
occurring HRs by aa insertions, deletions, and substitutions, created 
using recombinant DNA techniques. Guidance in determining which aa 
residues may be replaced, added or deleted without abolishing activities 
of interest, such as cell adhesion and chemotaxis, may be found by 
comparing the sequence of the particular HR with that of homologous 
receptors and minimizing the number of aa sequence changes made in regions 
of high homology. 
Preferably, aa "substitutions" are the result of replacing one aa with 
another aa having similar structural and/or chemical properties, such as 
the replacement of a leucine with an isoleucine or valine, an aspartate 
with a glutamate, or a threonine with a serine, i.e., conservative aa 
replacements. "Insertions" or "deletions" are typically in the range of 
about 1 to 5 aa. The variation allowed may be experimentally determined by 
systematically making insertions, deletions, or substitutions of aa in a 
HR molecule using recombinant DNA techniques and assaying the resulting 
recombinant variants for activity. 
Where desired, a "signal or leader sequence" can direct the polypeptide 
through the membrane of a cell. Such a sequence may be naturally present 
on the polypeptides of the present invention or provided from heterologous 
protein sources by recombinant DNA techniques. 
A polypeptide "fragment," "portion," or "segment" is a stretch of aa 
residues of at least about 5 amino acids, often at least about 7 aa, 
typically at least about 9 to 13 aa, and, in various embodiments, at least 
about 17 or more aa. To be active, any HR polypeptide must have sufficient 
length to display biologic and/or immunologic activity. 
An "oligonucleotide" or polynucleotide "fragment", "portion," or "segment" 
is a stretch of nucleotide residues which is long enough to use in 
polymerase chain reaction (PCR) or various hybridization procedures to 
amplify or simply reveal related parts of mRNA or DNA molecules. 
The present invention includes purified HR polypeptide from natural or 
recombinant sources, cells transformed with recombinant nucleic acid 
molecules encoding HR. Various methods for the isolation of HR polypeptide 
may be accomplished by procedures well known in the art. For example, such 
polypeptide may be purified by immunoaffinity chromatography by employing 
the antibodies provided by the present invention. Various other methods of 
protein purification well known in the art include those described in 
Deutscher M (1990) Methods in Enzymology, Vol 182, Academic Press, San 
Diego; and Scopes R (1982) Protein Purification: Principles and Practice, 
Springer-Verlag, New York City, both incorporated herein by reference. 
"Recombinant" may also refer to a polynucleotide which encodes HR and is 
prepared using recombinant DNA techniques. The DNA which encodes HR may 
also include allelic or recombinant variants and mutants thereof. 
"Oligonucleotides" or "nucleic acid probes" are prepared based on the cDNA 
sequence which encodes HR provided by the present invention. 
Oligonucleotides comprise portions of the DNA sequence having at least 
about 15 nucleotides, usually at least about 20 nucleotides. Nucleic acid 
probes comprise portions of the sequence having fewer nucleotides than 
about 6 kb, usually fewer than about 1 kb. After appropriate testing to 
eliminate false positives, these probes may be used to determine whether 
mRNAs encoding HR are present in a cell or tissue or to isolate similar 
nucleic acid sequences from chromosomal DNA as described by Walsh PS et al 
(1992 PCR Methods Appl 1:241-250). 
Probes may be derived from naturally occurring or recombinant single- or 
double-stranded nucleic acids or be chemically synthesized. They may be 
labeled by nick translation, Klenow fill-in reaction, PCR or other methods 
well known in the art. Probes of the present invention, their preparation 
and/or labeling are elaborated in Sambrook Jet al (1989) Molecular 
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; or 
Ausubel FM et al (1989) Current Protocols in Molecular Biology, John Wiley 
& Sons, New York City, both incorporated herein by reference. 
"Activated" cells as used in this application are those which are engaged 
in migration, proliferation, vascularization or differentiation as part of 
a normal or disease process. 
Recombinant variants encoding these same or similar polypeptides may be 
synthesized or selected by making use of the "redundancy" in the genetic 
code. Various codon substitutions, such as the silent changes which 
produce various restriction sites, may be introduced to optimize cloning 
into a plasmid or viral vector or expression in a particular prokaryotic 
or eukaryotic system. Mutations may also be introduced to modify the 
properties of the polypeptide, to change ligand-binding affinities, 
interchain affinities, or polypeptide degradation or turnover rate. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides a nucleotide sequence uniquely identifying a 
novel human hyaluronan receptor, HR, which was highly expressed in the 
human umbilical vein endothelial library. Because HR is specifically 
expressed in embryonic tissue, the nucleic acid (hr), polypeptide (HR) and 
antibodies to HR are useful in diagnostic assays for invasive cancers. 
Excessive expression of HR can direct cell migration, including 
lymphocytes and/or other cells which respond to hyaluronan. Therefore, a 
diagnostic test for excess expression of HR can accelerate diagnosis and 
proper treatment of an abnormal condition caused by viral or other 
infections; anglogenesis of cancerous tissues; invasive leukemias and 
lymphomas; or other physiologic/pathologic problems which deviate from 
normal development and result in metastatic cell migration, proliferation, 
vascularization and differentiation. 
The nucleotide sequence encoding HR (or its complement) has numerous 
applications in techniques known to those skilled in the art of molecular 
biology. These techniques include use as hybridization probes, use as 
oligomers for PCR, use for chromosome and gene mapping, use in the 
recombinant production of HR, and use in generation of anti-sense DNA or 
RNA, their chemical analogs and the like. Uses of the nucleotide sequences 
encoding HR disclosed herein are exemplary of known techniques and are not 
intended to limit their use in any technique known to a person of ordinary 
skill in the art. Furthermore, the nucleotide sequences disclosed herein 
may be used in molecular biology techniques that have not yet been 
developed, provided the new techniques rely on properties of nucleotide 
sequences that are currently known, e.g., the triplet genetic code, 
specific base pair interactions, etc. 
It will be appreciated by those skilled in the art that as a result of the 
degeneracy of the genetic code, a multitude of HR-encoding nucleotide 
sequences, some bearing minimal homology to the nucleotide sequence of any 
known and naturally occurring gene may be produced. The invention has 
specifically contemplated each and every possible variation of nucleotide 
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 nucleotide sequence of naturally 
occurring HR, and all such variations are to be considered as being 
specifically disclosed. 
Although nucleotide sequences which encode HR and its variants are 
preferably capable of hybridizing to the nucleotide sequence of the 
naturally occurring HR gene under stringent conditions, it may be 
advantageous to produce nucleotide sequences encoding HR or its 
derivatives possessing a substantially different codon usage. Codons can 
be selected to increase the rate at which expression of the peptide occurs 
in a particular prokaryotic or eukaryotic expression host in accordance 
with the frequency with which particular codons are utilized by the host. 
Other reasons for substantially altering the nucleotide sequence encoding 
HR and its derivatives without altering the encoded aa sequence 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 nucleotide sequence encoding HR may be joined to a variety of other 
nucleotide sequences by means of well established recombinant DNA 
techniques (cf Sambrook J et al. (1989) Molecular Cloning: A Laboratory 
Manual, Cold Spring Harbor Laboratory, New York). Useful nucleotide 
sequences for joining to hr include an assortment of cloning vectors, 
e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the 
like, that are well known in the art. Vectors of interest include 
expression vectors, replication vectors, probe generation vectors, 
sequencing vectors, and the like. In general, vectors of interest may 
contain an origin of replication functional in at least one organism, 
convenient restriction endonuclease sensitive sites, and selectable 
markers for the host cell. 
Another aspect of the subject invention is to provide for hr-specific 
nucleic acid hybridization probes capable of hybridizing with naturally 
occurring nucleotide sequences encoding HR. Such probes may also be used 
for the detection of similar hyaluronan receptor encoding sequences and 
should preferably contain at least 50% of the nucleotides from this hr 
encoding sequence. The hybridization probes of the subject invention may 
be derived from the nucleotide sequence of the SEQ ID NO 1 or from genomic 
sequence including promoter, enhancer elements and introns of the 
respective naturally occurring hrs. Hybridization probes may be labeled by 
a variety of reporter groups, including radionuclides such as 32P or 35S, 
or enzymatic labels such as alkaline phosphatase coupled to the probe via 
avidin/biotin coupling systems, and the like. 
PCR as described in U.S. Pat. Nos. 4,683,195; 4,800,195; and 4,965,188 
provides additional uses for oligonucleotides based upon the nucleotide 
sequences which encode HR. Such probes used in PCR may be of recombinant 
origin, chemically synthesized, or a mixture of both and comprise a 
discrete nucleotide sequence for diagnostic use or a degenerate pool of 
possible sequences for identification of closely related genomic 
sequences. 
Other means of producing specific hybridization probes for hr DNAs include 
the cloning of nucleic acid sequences encoding HR or HR 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 polymerase as T7 or 
SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. 
It is now possible to produce a DNA sequence, or portions thereof, encoding 
HR and its derivatives entirely by synthetic chemistry, after which the 
gene can be inserted into any of the many available DNA using reagents, 
and cells that are known in the art at the time of the filing of this 
application. Moreover, synthetic chemistry may be used to introduce 
mutations into the hr sequences or any portion thereof. 
The nucleotide sequence can be used to construct an assay to detect 
inflammation or disease associated with abnormal levels of expression of 
HR. The nucleotide sequence can be labeled by methods known in the art and 
added to a fluid or tissue sample from a patient under hybridizing 
conditions. After an incubation period, the sample is washed with a 
compatible fluid which optionally contains a dye (or other label requiring 
a developer) if the nucleotide has been labeled with an enzyme. After the 
compatible fluid is rinsed off, the dye is quantitated and compared with a 
standard. If the amount of dye is significantly elevated, the nucleotide 
sequence has hybridized with the sample, and the assay indicates the 
presence of inflammation and/or disease. 
The nucleotide sequence for hr can be used to construct hybridization 
probes for mapping that gene. The nucleotide sequence provided herein may 
be mapped to a chromosome and specific regions of a chromosome using well 
known genetic and/or chromosomal mapping techniques. These techniques 
include in situ hybridization, linkage analysis against known chromosomal 
markers, hybridization screening with libraries or flow-sorted chromosomal 
preparations specific to known chromosomes, and the like. The technique of 
fluorescent in situ hybridization of chromosome spreads has been 
described, among other places, in Verma et al (1988) Human Chromosomes: A 
Manual of Basic Techniques, Pergamon Press, New York N.Y. 
Fluorescent in situ hybridization of chromosomal preparations and other 
physical chromosome mapping techniques may be correlated with additional 
genetic map data. Examples of genetic map data can be found in the 1994 
Genome Issue of Science (265:1981f). Correlation between the location of 
hr on a physical chromosomal map and a specific disease (or predisposition 
to a specific disease) can help delimit the region of DNA associated with 
that genetic disease. The nucleotide sequence of the subject invention may 
be used to detect differences in gene sequence between normal and carrier 
or affected individuals. 
The nucleotide sequence encoding HR may be used to produce purified HR 
using well known methods of recombinant DNA technology. Among the many 
publications that teach methods for the expression of genes after they 
have been isolated is Goeddel (1990) Gene Expression Technology, Methods 
and Enzymology, Vol 185, Academic Press, San Diego. HR may be expressed in 
a variety of host cells, either prokaryotic or eukaryotic. Host cells may 
be from the same species in which hr nucleotide sequences are endogenous 
or from a different species. Advantages of producing HR by recombinant DNA 
technology include obtaining adequate amounts of the protein for 
purification and the availability of simplified purification procedures. 
Cells transformed with DNA encoding HR may be cultured under conditions 
suitable for the expression of hyaluronan receptors and recovery of the 
protein from the cell culture. HR produced by a recombinant cell may be 
secreted or may be contained intracellularly, depending on the particular 
genetic construction used. In general, it is more convenient to prepare 
recombinant proteins in secreted form. Purification steps vary with the 
production process and the particular protein produced. 
In addition to recombinant production, fragments of HR may be produced by 
direct peptide synthesis using solid-phase techniques (cf Stewart et al 
(1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; 
Merrifield J (1963) J Am Chem Soc 85:2149-2154. In vitro protein synthesis 
may be performed using manual techniques or by automation. Automated 
synthesis may be achieved, for example, using Applied Biosystems 431A 
Peptide Synthesizer (Foster City, Calif.) in accordance with the 
instructions provided by the manufacturer. Various fragments of HR may be 
chemically synthesized separately and combined using chemical methods to 
produce the full length molecule. 
HR for antibody induction does not require biological activity; however, 
the protein must be immunogenic. Peptides used to induce specific 
antibodies may have an aa sequence consisting of at least five aa, 
preferably at least 10 aa. They should mimic a portion of the aa sequence 
of the protein and may contain the entire aa sequence of a small naturally 
occurring molecules like HR. Short stretches of HR aa may be fused with 
those of another protein such as keyhole limpet hemocyanin and antibody 
produced against the chimetic molecule. 
Antibodies specific for HR may be produced by inoculation of an appropriate 
animal with the polypeptide or an antigenic fragment. An antibody is 
specific for HR if it is produced against an epitope of the polypeptide 
and binds to at least part of the natural or recombinant protein. Antibody 
production includes not only the stimulation of an immune response by 
injection into animals, but also analogous steps in the production of 
synthetic antibodies or other specific-binding molecules such as the 
screening of recombinant immunoglobulin libraries (cf Orlandi R et al 
(1989) PNAS 86:3833-3837, or Huse WD et al (1989) Science 256:1275-1281) 
or the in vitro stimulation of lymphocyte populations. Current technology 
(Winter G and Milstein C (1991) Nature 349:293-299) provides for a number 
of highly specific binding reagents based on the principles of antibody 
formation. These techniques may be adapted to produce molecules 
specifically binding HR. 
An additional embodiment of the subject invention is the use of HR specific 
antibodies, inhibitors, or their analogs as bioactive agents to treat 
viral or other infections; anglogenesis of cancerous tissues; invasive 
leukemias and lymphomas; or other physiologic/pathologic problems which 
deviate from normal development and result in metastatic cell migration, 
proliferation, vascularization and differentiation. 
Bioactive compositions comprising agonists, antagonists, or inhibitors of 
HR may be administered in a suitable therapeutic dose determined by any of 
several methodologies including clinical studies on mammalian species to 
determine maximum tolerable dose and on normal human subjects to determine 
safe dosage. Additionally, the bioactive agent may be complexed with a 
variety of well established compounds or compositions which enhance 
stability or pharmacological properties such as half-life. It is 
contemplated that the therapeutic, bioactive composition may be delivered 
by intravenous infusion into the bloodstream or any other effective means 
which could be used for treating invasive cancers. 
The examples below are provided to illustrate the subject invention. These 
examples are provided by way of illustration and are not included for the 
purpose of limiting the invention. 
EXAMPLES 
I Isolation of mRNA and Construction of the cDNA Library 
The hyaluronan receptor cDNA sequence was identified among the sequences 
comprising the HUVEC library. The HUVEC cell line is a normal, 
homogeneous, well-characterized, early passage, endothelial cell culture 
from human umbilical vein (Cell Systems Corporation, 12815 NE 124th St, 
Kirkland, Wash. 98034). 
The HUVEC cDNA library was custom constructed by Stratagene (11099 M. 
Torrey Pines Rd., La Jolla, Calif. 92037). cDNA synthesis was primed with 
oligo dT hexamers, and synthetic adaptor oligonucleotides were ligated 
onto the cDNA ends to enable its insertion into Uni-ZAP.TM. vector system 
(Stratagene). This allowed high efficiency unidirectional (sense 
orientation) lambda library construction and the convenience of a plasmid 
system with blue/white color selection to detect clones with cDNA 
insertions. 
The quality of the cDNA library was screened using DNA probes, and then, 
the pBluescript.RTM. phagemid (Stratagene) was excised. This phagemid 
allows the use of a plasmid system for easy insert characterization, 
sequencing, site-directed mutagenesis, the creation of unidirectional 
deletions and expression of fusion polypeptides. Subsequently, the 
custom-constructed library phage particles were infected into E. coil host 
strain XL1-Blue.RTM. (Stratagene). The high transformation efficiency of 
this bacterial strain increases the probability that the cDNA library will 
contain rare, under-represented clones. Alternative unidirectional vectors 
might include, but are not limited to, pcDNAI (Invitrogen) and pSHIox-1 
(Novagen). 
II Isolation of cDNA Clones 
The phagemid forms of individual cDNA clones were obtained by the in vivo 
excision process, in which XL1-BLUE was coinfected with an f1 helper 
phage. Proteins derived from both lambda phage and f1 helper phage 
initiated new DNA synthesis from defined sequences on the lambda target 
DNA and create a smaller, single-stranded circular phagemid DNA molecule 
that includes all DNA sequences of the pBluescript plasmid and the cDNA 
insert. The phagemid DNA was released from the cells and purified, then 
used to reinfect fresh bacterial host cells (SOLR, Stratagene), where the 
double-stranded phagemid DNA was produced. Because the phagemid carries 
the gene for .beta.-lactamase, the newly transformed bacteria were 
selected on medium containing ampicillin. 
Phagemid DNA was purified using the QIAWELL-8 Plasmid Purification System 
from QIAGEN.RTM. DNA Purification System (QIAGEN Inc, 9259 Eton Ave, 
Chatsworth, Calif. 91311). This technique provides a rapid and reliable 
high-throughput method for lysing the bacterial cells and isolating highly 
purified phagemid DNA. The DNA eluted from the purification resin was 
suitable for DNA sequencing and other analytical manipulations. 
III Sequencing of cDNA Clones 
The cDNA inserts from random isolates of the HUVEC library were sequenced 
in part. Methods for DNA sequencing are well known in the art. 
Conventional enzymatic methods employed DNA polymerase Klenow fragment, 
SEQUENASE.RTM. (US Biochemical Corp, Cleveland, Ohio) or Taq polymerase to 
extend DNA chains from an oligonucleotide primer annealed to the DNA 
template of interest. Methods have been developed for the use of both 
single- and double-stranded templates. The chain termination reaction 
products were electrophoresed on urea-acrylamide gels and detected either 
by autoradiography (for radionuclide-labeled precursors) or by 
fluorescence (for fluorescent-labeled precursors). Recent improvements in 
mechanized reaction preparation, sequencing and analysis using the 
fluorescent detection method have permitted expansion in the number of 
sequences that can be determined per day (using machines such as the 
Catalyst 800 and the Applied Biosystems 373 DNA sequencer). 
IV Homology Searching of cDNA Clones and Deduced Proteins 
Each sequence so obtained was compared to sequences in GenBank using a 
search algorithm developed by Applied Biosystems Inc. and incorporated 
into the INHERIT.TM. 670 Sequence Analysis System. In this algorithm, 
Pattern Specification Language (developed by TRW Inc.) was used to 
determine regions of homology. The three parameters that determine how the 
sequence comparisons run were window size, window offset, and error 
tolerance. Using a combination of these three parameters, the DNA database 
was searched for sequences containing regions of homology to the query 
sequence, and the appropriate sequences were scored with an initial value. 
Subsequently, these homologous regions were examined using dot matrix 
homology plots to distinguish regions of homology from chance matches. 
Smith-Waterman alignments were used to display the results of the homology 
search. 
Peptide and protein sequence homologies were ascertained using the 
INHERIT.TM. 670 Sequence Analysis System in a way similar to that used in 
DNA sequence homologies. Pattern Specification Language and parameter 
windows were used to search protein databases for sequences containing 
regions of homology which were scored with an initial value. Dot-matrix 
homology plots were examined to distinguish regions of significant 
homology from chance matches 
V Identification, Full Length Sequencing and Translation of the Gene 
INHERIT.TM. analysis of the randomly picked and sequenced portions of 
clones from the HUVEC library identified the partial sequence from Incyte 
39200 as homologous to hyaluronan receptor from mouse (Hardwick et al 
(1992) J Cell Biol 117:1343 1350). The cDNA insert comprising Incyte 39200 
was fully sequenced using the same methods described above. The coding 
region of the insert (ATG-&gt;TGA) was identified and is shown as Sequence ID 
No. 1. This sequence for human hr was translated using DNASTAR software, 
the in-frame translation was identified, and is shown in Sequence ID No. 
2. When all three possible predicted translations of the sequence were 
searched against protein databases such as SwissProt and PIR, no exact 
matches were found to the possible translations of hr. FIG. 1 shows the 
degree of amino acid homology between HR and mouse RHAMM. The unmatched aa 
in the middle of the sequence may reflect the position of a mouse intron. 
The cDNA lacks the intron since it was constructed from mRNA. FIG. 2 shows 
the hydrophobicity plot for HR. 
VI Antisense analysis 
Knowledge of the correct, complete cDNA sequence of HR will enable its use 
in antisense technology in the investigation of gene function. Either 
oligonucleotides, genomic or cDNA fragments comprising the antisense 
strand of hr can be used either in vitro or in vivo to inhibit expression 
of the mRNA. Such technology is now well known in the art, and probes can 
be designed at various locations along the nucleotide sequences. By 
treatment of cells or whole test animals with such antisense sequences, 
the gene of interest can be effectively turned off. Frequently, the 
function of the gene can be ascertained by observing behavior at the 
cellular, tissue or organismal level (e.g. lethality, loss of 
differentiated function, changes in morphology, etc.). 
In addition to using sequences constructed to interrupt transcription of 
the open reading frame, modifications of gene expression can be obtained 
by designing antisense sequences to intron regions, promoter/enhancer 
elements, or even to trans-acting regulatory genes. Similarly, inhibition 
can be achieved using Hogeboom base-pairing methodology, also known as 
"triple helix" base pairing. 
VII Expression of HR 
Expression of hr may be accomplished by subcloning the cDNAs into 
appropriate expression vectors and transfecting the vectors into an 
appropriate expression hosts. In this particular case, the cloning vector 
previously used for the generation of the tissue library also provides for 
direct expression of hr sequences in E. coil. Upstream of the cloning 
site, this vector contains a promoter for .beta.-galactosidase, followed 
by sequence containing the amino-terminal Met and the subsequent 7 
residues of .beta.-galactosidase. Immediately following these eight 
residues is an engineered bacteriophage promoter useful for artificial 
priming and transcription and a number of unique restriction sites, 
including Eco RI, for cloning. 
Induction of the isolated, transfected bacterial strain with IPTG using 
standard methods will produce a fusion protein corresponding to the first 
seven residues of .beta.-galactosidase, about 15 residues of "linker", and 
the peptide encoded within the cDNA. Since cDNA clone inserts are 
generated by an essentially random process, there is one chance in three 
that the included cDNA will lie in the correct frame for proper 
translation. If the cDNA is not in the proper reading frame, it can be 
obtained by deletion or insertion of the appropriate number of bases by 
well known methods including in vitro mutagenesis, digestion with 
exonuclease III or mung bean nuclease, or oligonucleotide linker 
inclusion. 
The hr cDNA can be shuttled into other vectors known to be useful for 
expression of protein in specific hosts. Oligonuceotide amplimers 
containing cloning sites as well as a segment of DNA sufficient to 
hybridize to stretches at both ends of the target cDNA (25 bases) can be 
synthesized chemically by standard methods. These primers can then used to 
amplify the desired gene segments by PCR. The resulting new gene segments 
can be digested with appropriate restriction enzymes under standard 
conditions and isolated by gel electrophoresis. Alternately, similar gene 
segments can be produced by digestion of the cDNA with appropriate 
restriction enzymes and filling in the missing gene segments with 
chemically synthesized oligonucleotides. Segments of the coding sequence 
from more than one gene can be ligated together and cloned in appropriate 
vectors to optimize expression of recombinant sequence. 
Suitable expression hosts for such chimeric molecules include but are not 
limited to mammalian cells such as Chinese Hamster Ovary (CHO) and human 
293 cells, insect cells such as Sf9 cells, yeast cells such as 
Saccharomyces cerevisiae, and bacteria such as E. coil. For each of these 
cell systems, a useful expression vector may also include an origin of 
replication to allow propagation in bacteria and a selectable marker such 
as the .beta.-lactamase antibiotic resistance gene to allow selection in 
bacteria. In addition, the vectors may include a second selectable marker 
such as the neomycin phosphotransferase gene to allow selection in 
transfected eukaryotic host cells. Vectors for use in eukaryotic 
expression hosts may require RNA processing elements such as 3' 
polyadenylation sequences if such are not part of the cDNA of interest. 
Additionally, the vector may contain promoters or enhancers which increase 
gene expression. Such promoters are host specific and include MMTV, SV40, 
or metallothionine promoters for CHO cells; trp, lac, tac or T7 promoters 
for bacterial hosts, or alpha factor, alcohol oxidase or PGH promoters for 
yeast. Transcription enhancers, such as the rous sarcoma virus (RSV) 
enhancer, may be used in mammalian host cells. Once homogeneous cultures 
of recombinant cells are obtained through standard culture methods, large 
quantities of recombinantly produced HR can be recovered from the 
conditioned medium and analyzed using chromatographic methods known in the 
art. 
VIII Isolation of Recombinant HR 
HR may be expressed as a chimeric protein with one or more additional 
polypeptide domains added to facilitate protein purification. Such 
purification facilitating domains include, but are not limited to, metal 
chelating peptides such as histidine-tryptophan modules that allow 
purification on immobilized metals, protein A domains that allow 
purification on immobilized immunoglobulin, and the domain utilized in the 
FLAGS extension/affinity purification system (Immunex Corp., Seattle 
Wash.). The inclusion of a cleavable linker sequence such as Factor XA or 
enterokinase(Invitrogen, San Diego Calif.) between the purification domain 
and the hr sequence may be useful to facilitate expression of HR. 
IX Production of HR Specific Antibodies 
Two approaches are utilized to raise antibodies to HR, and each approach is 
useful for generating either polyclonal or monoclonal antibodies. In one 
approach, denatured protein from the reverse phase HPLC separation is 
obtained in quantities up to 75 mg. This denatured protein can be used to 
immunize mice or rabbits using standard protocols; about 100 micrograms 
are adequate for immunization of a mouse, while up to 1 mg might be used 
to immunize a rabbit. For identifying mouse hybridomas, the denatured 
protein can be radioiodinated and used to screen potential murine B-cell 
hybridomas for those which produce antibody. This procedure requires only 
small quantities of protein, such that 20 mg would be sufficient for 
labeling and screening of several thousand clones. 
In the second approach, the amino acid sequence of HR, as deduced from 
translation of the cDNA, is analyzed to determine regions of high 
immunogenicity. Oligopeptides comprising appropriate hydrophilic regions, 
as illustrated in FIG. 2, are synthesized and used in suitable 
immunization protocols to raise antibodies. Analysis to select appropriate 
epitopes is described by Ausubel FM et al (1989, Current Protocols in 
Molecular Biology, John Wiley & Sons, New York City). The optimal amino 
acid sequences for immunization are usually at the C-terminus, the 
N-terminus and those intervening, hydrophilic regions of the polypeptide 
which are likely to be exposed to the external environment when the 
protein is in its natural conformation. 
Typically, selected peptides, about 15 residues in length, are synthesized 
using an Applied Biosystems Peptide Synthesizer Model 431A using 
fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH, Sigma) by 
reaction with M-maleimidobenzoyI-N-hydroxysuccinimide ester (MBS; cf. 
Ausubel FM et al, supra). If necessary, a cysteine may be introduced at 
the N-terminus of the peptide to permit coupling to KLH. Rabbits are 
immunized with the peptide-KLH complex in complete Freund's adjuvant. The 
resulting antisera are tested for antipeptide activity by binding the 
peptide to plastic, blocking with 1% BSA, reacting with antisera, washing 
and reacting with labeled (radioactive or fluorescent), affinity purified, 
specific goat anti-rabbit IgG. 
Hybridomas may also be prepared and screened using standard techniques. 
Hybridomas of interest are detected by screening with labeled HR to 
identify those fusions producing the monoclonal antibody with the desired 
specificity. In a typical protocol, wells of plates (FAST; 
Becton-Dickinson, Palo Alto, Calif.) are coated with affinity purified, 
specific rabbit-anti-mouse (or suitable anti-species Ig) antibodies at 10 
mg/ml. The coated wells are blocked with 1% BSA, washed and exposed to 
supernatants from hybridomas. After incubation the wells are exposed to 
labeled HR at 1 mg/ml. Clones producing antibodies will bind a quantity of 
labeled HR which is detectable above background. Such clones are expanded 
and subjected to 2 cycles of cloning at limiting dilution (1 cell/3 
wells). Cloned hybridomas are injected into pristine mice to produce 
ascites, and monoclonal antibody is purified from mouse ascitic fluid by 
affinity chromatography on Protein A. Monoclonal antibodies with 
affinities of at least 10e8 Me-1, preferably 10e9 to 10e10 or stronger, 
will typically be made by standard procedures as described in Harlow and 
Lane (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory 
N.Y.; and in Goding (1986) Monoclonal Antibodies: Principles and Practice, 
Academic Press, New York N.Y. incorporated herein by reference. 
X Diagnostic Test Using HR Specific Antibodies 
Particular HR antibodies are useful for the diagnosis of prepathologic 
conditions, and chronic or acute diseases which are characterized by 
differences in the amount or distribution of HR, respectively. To date, HR 
has only been found in the HUVEC library and is thus specific for 
abnormalities or pathologies which affect embryonic, angiogenic or 
invasive cells. 
Diagnostic tests for HR include methods utilizing the antibody and a label 
to detect HR in human body fluids, tissues or extracts of such tissues. 
The polypeptides and antibodies of the present invention may be used with 
or without modification. Frequently, the polypeptides and antibodies will 
be labeled by joining them, either covalently or noncovalently, with a 
substance which provides for a detectable signal. A wide variety of labels 
and conjugation techniques are known and have been reported extensively in 
both the scientific and patent literature. Suitable labels include 
radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent 
agents, chemiluminescent agents, magnetic particles and the like. Patents 
teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 
3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. 
Also, recombinant immunoglobulins may be produced as shown in U.S. Pat. 
No. 4,816,567, incorporated herein by reference. 
A variety of protocols for measuring soluble or membrane-bound HR, using 
either polyclonal or monoclonal antibodies specific for the protein, are 
known in the art. Examples include enzyme-linked immunosorbent assay 
(ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting 
(FACS). A two-site monoclonal-based immunoassay utilizing monoclonal 
antibodies reactive to two non-interfering epitopes on HR is preferred, 
but a competitive binding assay may be employed. These assays are 
described, among other places, in Maddox, Del. et al (1983, J Exp Med 
158:1211). 
XI Purification of Native HR Using Specific Antibodies 
Native or recombinant HR can be purified by immunoaffinity chromatography 
using antibodies specific for HR. In general, an immunoaffinity column is 
constructed by covalently coupling the anti-HR antibody to an activated 
chromatographic resin. 
Polyclonal immunoglobulins are prepared from immune sera either by 
precipitation with ammonium sulfate or by purification on immobilized 
Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, 
monoclonal antibodies are prepared from mouse ascites fluid by ammonium 
sulfate precipitation or chromatography on immobilized Protein A. 
Partially purified immunoglobulin is covalently attached to a 
chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB 
Biotechnology). The antibody is coupled to the resin, the resin is 
blocked, and the derivative resin is washed according to the 
manufacturer's instructions. 
Such immunoaffinity columns were utilized in the purification of HR by 
preparing a fraction from cells containing HR in a soluble form. This 
preparation was derived by solubilization of the whole cell or of a 
subcellular fraction obtained via differential centrifugation by the 
addition of detergent or by other methods well known in the art. 
Alternatively, soluble HR containing a signal sequence may be secreted in 
useful quantity into the medium in which the cells are grown. 
A soluble HR-containing preparation was passed over the immunoaffinity 
column, and the column was washed under conditions that allow the 
preferential absorbance of HR (eg, high ionic strength buffers in the 
presence of detergent). Then, the column was eluted under conditions that 
disrupt antibody/HR binding (e.g., a buffer of pH 2-3 or a high 
concentration of a chaotrope such as urea or thiocyanate ion), and HR was 
collected. 
XII Hyaluronan Induced Cherootaxis for Cell Activation and Wound Healing 
The chemotactic interactions between HA and HR were measured in a 48-well 
microchemotaxis chambers (cf. Falk WR et al (1980) J Immunol Methods 
33:239). In each well, two compartments are separated by a filter that 
allows the passage of cells in response to a chemical gradient. Cells 
expressing HR in a culture medium such as RPMI 1640 (Sigma, St. Louis Mo.) 
are placed on one side of a filter, usually polycarbonate, and cells 
producing HA or a solution enriched with HA are placed on the opposite 
side of the filter. Sufficient incubation time is allowed for the cells to 
traverse the filter in response to the concentration gradient across the 
filter. Filters are recovered from each well, and cells adhering to the 
side of the filter facing the HA are typed and quantified. 
Those cells producing HR and migrating toward the higher end of the HA 
gradient are rated for chemotactic specificity. This assay not only 
substantiates the ability of HR-producing cells to respond to HA, but 
provides researchers using methods well known in the art with model 
systems from which to obtain and describe transcription factors and 
enhancers specific for use in regulating HR activity in native cell 
populations. The ability to artificially supply such factors dissolved in 
dimethyl sulfoxide (DMSQ) or some other carrier liquid, to upregulate 
production of HR in a localized manner, and to enhance migration 
capability provides for the use of HA as a stimulant in wound healing. HA 
could be incorporated in collagenous or other natural or artificial 
bandage materials used to treat refractory wounds. The presence of HA 
would attract activated endothelial cells, fibroblasts, etc. which would 
participate in the repair and healing process. 
XIII Drug Screening 
This invention is particularly useful for screening compounds by using HR 
or binding fragments thereof in any of a variety of drug screening 
techniques. The polypeptide or fragment employed in such a test may either 
be free in solution, affixed to a solid support, borne on a cell surface 
or located intracellularly. One method of drug screening utilizes 
eukaryotic or prokaryotic host cells which are stably transformed with 
recombinant nucleic acids expressing the polypeptide or fragment. Drugs 
are screened against such transformed cells in competitive binding assays. 
Such cells, either in viable or fixed form, can be used for standard 
binding assays. One may measure, for example, the formation of complexes 
between HR and the agent being tested. Alternatively, one can examine the 
diminution in complex formation between HR and hyaluronan caused by the 
agent being tested. 
Thus, the present invention provides methods of screening for drugs or any 
other agents which can affect cell migration, anglogenesis or infiltration 
of lymphomas or leukemias. These methods comprise contacting such an agent 
with HR polypeptide or a fragment thereof and assaying (i) for the 
presence of a complex between the agent and the HR polypeptide or 
fragment, or (ii) for the presence of a complex between the HR polypeptide 
or fragment and the cell, by methods well known in the art. In such 
competitive binding assays, the HR polypeptide or fragment is typically 
labeled. After suitable incubation, free HR polypeptide or fragment is 
separated from that present in bound form, and the amount of free or 
uncomplexed label is a measure of the ability of the particular agent to 
bind to HR or to interfere with the HR and agent complex. 
Another technique for drug screening provides high throughput screening for 
compounds having suitable binding affinity to the HR polypeptides and is 
described in detail in European Patent Application 84/03564, published on 
Sep. 13, 1984, incorporated herein by reference. Briefly stated, large 
numbers of different small peptide test compounds are synthesized on a 
solid substrate, such as plastic pins or some other surface. The peptide 
test compounds are reacted with HR polypeptide and washed. Bound HR 
polypeptide is then detected by methods well known in the art. Purified HR 
can also be coated directly onto plates for use in the aforementioned drug 
screening techniques. In addition, non-neutralizing antibodies can be used 
to capture the peptide and immobilize it on the solid support. 
This invention also contemplates the use of competitive drug screening 
assays in which neutralizing antibodies capable of binding HR specifically 
compete with a test compound for binding to HR polypeptides or fragments 
thereof. In this manner, the antibodies can be used to detect the presence 
of any peptide which shares one or more antigenic determinants with HR. 
XIV Rational Drug Design 
The goal of rational drug design is to produce structural analogs of 
biologically active polypeptides of interest or of small molecules with 
which they interact, e.g., agonists, antagonists, or inhibitors. Any of 
these examples can be used to fashion drugs which are more active or 
stable forms of the polypeptide or which enhance or interfere with the 
function of a polypeptide in vivo (cf Hodgson J. (1991) Bio/Technology 
9:19-21, incorporated herein by reference). 
In one approach, the three-dimensional structure of a protein of interest, 
or of a protein-inhibitor complex, is determined by x-ray crystallography, 
by computer modeling or, most typically, by a combination of the two 
approaches. Both the shape and charges of the polypeptide must be 
ascertained to elucidate the structure and to determine active site(s) of 
the molecule. Less often, useful information regarding the structure of a 
polypeptide may be gained by modeling based on the structure of homologous 
proteins. In both cases, relevant structural information is used to design 
efficient inhibitors. Useful examples of rational drug design may include 
molecules which have improved activity or stability as shown by Braxton S. 
and Wells J. A. (1992 Biochemistry 31:7796-7801) or which act as 
inhibitors, agonists, or antagonists of native peptides as shown by 
Athauda S. B. et al (1993 J Biochem 113:742-746), incorporated herein by 
reference. 
It is also possible to isolate a target-specific antibody, selected by 
functional assay, as described above, and then to solve its crystal 
structure. This approach, in principle, yields a pharmacore upon which 
subsequent drug design can be based. It is possible to bypass protein 
crystallography altogether by generating anti-idiotypic antibodies 
(anti-ids) to a functional, pharmacologically active antibody. As a mirror 
image of a mirror image, the binding site of the anti-ids would be 
expected to be an analog of the original receptor. The anti-id could then 
be used to identify and isolate peptides from banks of chemically or 
biologically produced peptides. The isolated peptides would then act as 
the pharmacore. 
By virtue of the present invention, sufficient amount of polypeptide may be 
made available to perform such analytical studies as X-ray 
crystallography. In addition, knowledge of the HR amino acid sequence 
provided herein will provide guidance to those employing computer modeling 
techniques in place of or in addition to x-ray crystallography. 
XV Identification of Other Members of the HR Complex 
Purified HR is useful for characterization and purification of associated 
cell surface receptors and binding molecules. Cells which respond to HA by 
chemotaxis or other specific responses are likely to express a receptor 
for HR and to interact with transmembrane signaling molecules such as 
tyrosine kinase. Radioactive labels may be incorporated into HR by various 
methods known in the art. A preferred embodiment is the labeling of 
primary amino groups in HR with .sup.125 I Bolton-Hunter reagent (Bolton, 
A. E. and Hunter, W. M. (1973) Biochem J 133: 529), which has been used to 
label other signaling molecules without concomitant loss of biological 
activity (Hebert Calif. et al (1991) J Biol Chem 266: 18989; McColl Set al 
(1993) J Immunol 150:4550-4555). Receptor-bearing cells are incubated with 
the labeled signaling molecules. The cells are then washed to removed 
unbound molecules, and receptor-bound labeled molecule is quantified. The 
data obtained using different concentrations of HR is used to calculate 
values for the number, affinity, and association of other members of the 
receptor complex. 
Labeled HR is also useful as a reagent for purification of the particular 
molecule(s) of this complex with which it interacts. In one embodiment of 
affinity purification, HR is covalently coupled to a chromatography 
column. Cells and their membranes are extracted, HA is removed and various 
HA-free subcomponents are passed over the column. HR-associated molecules 
bind to the column by virtue of their biological affinity. The HR-complex 
is recovered from the column, dissociated and the recovered molecule is 
subjected to N-terminal protein sequencing. This amino acid sequence is 
then used to identify the molecule or to design degenerate oligonucleotide 
probes for cloning the gene from an appropriate cDNA library. 
In an alternate method, mRNA is obtained from HR-complex-bearing cells and 
made into a cDNA library. The library is transfected into a population of 
cells, and cells expressing the associated molecule(s) are selected using 
fluorescently labeled HR. The molecule is identified by recovering and 
sequencing the recombinant DNA from the highly labeled cells. 
In another alternate method, antibodies are raised against HR, specifically 
monoclonal antibodies. The monoclonal antibodies are screened to identify 
those which inhibit the binding of labeled HR. These monoclonal antibodies 
are then used in affinity purification or expression cloning of the 
associated signaling molecule. 
Other soluble binding molecules are identified in a similar manner. Labeled 
HR is incubated with extracts or other appropriate materials derived from 
HUVEC cells. After incubation, HR complexes (which are larger than the 
size of purified HR molecule) are identified by a sizing technique such as 
size exclusion chromatography or density gradient centrifugation and are 
purified by methods known in the art. The soluble binding protein(s) is 
subjected to N-terminal sequencing to obtain information sufficient for 
database identification, if the soluble protein is known, or for cloning, 
if the soluble protein is unknown. 
XVI Use and Administration of HR 
Antibodies, inhibitors, recetors or antagonists of HR (or other treatments 
for excessive HR production, hereinafter abbreviated TEHR), can provide 
different effects when administered therapeutically. TEHRs will be 
formulated in a nontoxic, inert, pharmaceutically acceptable aqueous 
carrier medium preferably at a pH of about 5 to 8, more preferably 6 to 8, 
although the pH may vary according to the characteristics of the antibody, 
inhibitor, or antagonist being formulated and the condition to be treated. 
Characteristics of TEHR include solubility of the molecule, half-life and 
antigenicity/immunogenicity; these and other characteristics may aid in 
defining an effective carrier. Native human proteins are preferred as 
TEHRs, but organic or synthetic molecules resulting from drug screens may 
be equally effective in particular situations. 
TEHRs may be delivered by known routes of administration including but not 
limited to topical creams and gels; transmucosal spray and aerosol, 
transdermal patch and bandage; injectable, intravenous and lavage 
formulations; and orally administered liquids and pills particularly 
formulated to resist stomach acid and enzymes. The particular formulation, 
exact dosage, and route of administration will be determined by the 
attending physician and will vary according to each specific situation. 
Such determinations are made by considering multiple variables such as the 
condition to be treated, the TEHR to be administered, and the 
pharmacokinetic profile of the particular TEHR Additional factors which 
may be taken into account include disease state (e.g. severity) of the 
patient, age, weight, gender, diet, time of administration, drug 
combination, reaction sensitivities, and tolerance/response to therapy. 
Long acting TEHR formulations might be administered every 3 to 4 days, 
every week, or once every two weeks depending on half-life and clearance 
rate of the particular TEHR. 
Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a 
total dose of about 1 g, depending upon the route of administration. 
Guidance as to particular dosages and methods of delivery is provided in 
the literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It 
is anticipated that different formulations will be effective for different 
TEHRs and that administration targeting metastatic cancers may necessitate 
delivery in a manner different from that being delivered to vascular 
endothelial cells. 
It is contemplated that conditions or diseases which activate leukocytes 
may precipitate damage that is treatable with TEHRs. These conditions or 
diseases may be specifically diagnosed by the tests discussed above, and 
such testing should be performed in suspected cases of viral or other 
infections; anglogenesis of cancerous tissues; invasive leukemias and 
lymphomas; or other physiologic/pathologic problems which deviate from 
normal development and result in metastatic cell migration, proliferation 
and differentiation. 
All publications and patents mentioned in the above specification are 
herein incorporated by reference. The foregoing written specification is 
considered to be sufficient to enable one skilled in the art to practice 
the invention. Indeed, various modifications of the above described modes 
for carrying out the invention which are obvious to those skilled in the 
field of molecular biology or related fields are intended to be within the 
scope of the following claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 3 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1056 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Human Umbilical Vein Endothelial Cell 
(B) CLONE: 39200 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..1056 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ATGCAAAACTTAAAACAGAAGTTTATTCTTGAACAACAGGAACGTGAA48 
MetGlnAsnLeuLysGlnLysPheIleLeuGluGlnGlnGluArgGlu 
151015 
AAGCTTCAACAAAAAGAATTACAAATTGATTCACTTCTGCAACAAGAG96 
LysLeuGlnGlnLysGluLeuGlnIleAspSerLeuLeuGlnGlnGlu 
202530 
AAAGAATTATCTTCGAGTCTTCATCAGAAGCTCTGTTCTTTTCAAGAG144 
LysGluLeuSerSerSerLeuHisGlnLysLeuCysSerPheGlnGlu 
354045 
GAAATGGCTAAAGAGAAGAATCTGTTTGAGGAAGAATTAAAGCAAACA192 
GluMetAlaLysGluLysAsnLeuPheGluGluGluLeuLysGlnThr 
505560 
CTGGATGAGCTTGATAAATTACAGCAAAAGGAGGAACAAGCTGAAAGG240 
LeuAspGluLeuAspLysLeuGlnGlnLysGluGluGlnAlaGluArg 
65707580 
CTGGTCAAGCAATTGGAAGAGGAAGCAAAATCTAGAGCTGAAGAATTA288 
LeuValLysGlnLeuGluGluGluAlaLysSerArgAlaGluGluLeu 
859095 
AAACTCCTAGAAGAAAAGCTGAAAGGGAAGGAGGCTGAACTGGAGAAA336 
LysLeuLeuGluGluLysLeuLysGlyLysGluAlaGluLeuGluLys 
100105110 
AGTAGTGCTGCTCATACCCAGGCCACCCTGCTTTTGGAGGAAAAGTAT384 
SerSerAlaAlaHisThrGlnAlaThrLeuLeuLeuGluGluLysTyr 
115120125 
GACAGTATGGTGCAAAGCCTTGAAGATGTTACTGCTCAATTTGAAAGC432 
AspSerMetValGlnSerLeuGluAspValThrAlaGlnPheGluSer 
130135140 
TATAAAGCGTTAACAGCCAGTGAGATAGAAGATCTTAAGCTGGAGAAC480 
TyrLysAlaLeuThrAlaSerGluIleGluAspLeuLysLeuGluAsn 
145150155160 
TCATCATTACAGGAAAAAGTGGCCAAGGCTGGGAAAAATGCAGAGGAT528 
SerSerLeuGlnGluLysValAlaLysAlaGlyLysAsnAlaGluAsp 
165170175 
GTTCAGCATCAGATTTTGGCAACTGAGAGCTCAAATCAAGAATATGTA576 
ValGlnHisGlnIleLeuAlaThrGluSerSerAsnGlnGluTyrVal 
180185190 
AGGATGCTTCTAGATCTGCAGACCAAGTCAGCACTAAAGGAAACAGAA624 
ArgMetLeuLeuAspLeuGlnThrLysSerAlaLeuLysGluThrGlu 
195200205 
ATTAAAGAAATCACAGTTTCTTTTCTTCAAAAAATAACTGATTTGCAG672 
IleLysGluIleThrValSerPheLeuGlnLysIleThrAspLeuGln 
210215220 
AACCAACTCAAGCAACAGGAGGAAGACTTTAGAAAACAGCTGGAAGAT720 
AsnGlnLeuLysGlnGlnGluGluAspPheArgLysGlnLeuGluAsp 
225230235240 
GAAGAAGGAAGAAAAGCTGAAAAAGAAAATACAACAGCAGAATTAACT768 
GluGluGlyArgLysAlaGluLysGluAsnThrThrAlaGluLeuThr 
245250255 
GAAGAAATTAACAAGTGGCGTCTCCTCTATGAAGAACTATATAATAAA816 
GluGluIleAsnLysTrpArgLeuLeuTyrGluGluLeuTyrAsnLys 
260265270 
ACAAAACCTTTTCAGCTACAACTAGATGCTTTTGAAGTAGAAAAACAG864 
ThrLysProPheGlnLeuGlnLeuAspAlaPheGluValGluLysGln 
275280285 
GCATTGTTGAATGAACATGGTGCAGCTCAGGAACAGCTAAATAAAATA912 
AlaLeuLeuAsnGluHisGlyAlaAlaGlnGluGlnLeuAsnLysIle 
290295300 
AGAGATTCATATGCTAAATTATTGGGTCATCAGAATTTGAAACAAAAA960 
ArgAspSerTyrAlaLysLeuLeuGlyHisGlnAsnLeuLysGlnLys 
305310315320 
ATCAAGCATGTTGTGAAGTTGAAAGATGAAAATAGCCAACTCAAATCG1008 
IleLysHisValValLysLeuLysAspGluAsnSerGlnLeuLysSer 
325330335 
GAAGTATCAAAACTCCGCTGTCAGCTTGCTAAAAAAAAAACAAAGTGA1056 
GluValSerLysLeuArgCysGlnLeuAlaLysLysLysThrLys* 
340345350 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 351 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetGlnAsnLeuLysGlnLysPheIleLeuGluGlnGlnGluArgGlu 
151015 
LysLeuGlnGlnLysGluLeuGlnIleAspSerLeuLeuGlnGlnGlu 
202530 
LysGluLeuSerSerSerLeuHisGlnLysLeuCysSerPheGlnGlu 
354045 
GluMetAlaLysGluLysAsnLeuPheGluGluGluLeuLysGlnThr 
505560 
LeuAspGluLeuAspLysLeuGlnGlnLysGluGluGlnAlaGluArg 
65707580 
LeuValLysGlnLeuGluGluGluAlaLysSerArgAlaGluGluLeu 
859095 
LysLeuLeuGluGluLysLeuLysGlyLysGluAlaGluLeuGluLys 
100105110 
SerSerAlaAlaHisThrGlnAlaThrLeuLeuLeuGluGluLysTyr 
115120125 
AspSerMetValGlnSerLeuGluAspValThrAlaGlnPheGluSer 
130135140 
TyrLysAlaLeuThrAlaSerGluIleGluAspLeuLysLeuGluAsn 
145150155160 
SerSerLeuGlnGluLysValAlaLysAlaGlyLysAsnAlaGluAsp 
165170175 
ValGlnHisGlnIleLeuAlaThrGluSerSerAsnGlnGluTyrVal 
180185190 
ArgMetLeuLeuAspLeuGlnThrLysSerAlaLeuLysGluThrGlu 
195200205 
IleLysGluIleThrValSerPheLeuGlnLysIleThrAspLeuGln 
210215220 
AsnGlnLeuLysGlnGlnGluGluAspPheArgLysGlnLeuGluAsp 
225230235240 
GluGluGlyArgLysAlaGluLysGluAsnThrThrAlaGluLeuThr 
245250255 
GluGluIleAsnLysTrpArgLeuLeuTyrGluGluLeuTyrAsnLys 
260265270 
ThrLysProPheGlnLeuGlnLeuAspAlaPheGluValGluLysGln 
275280285 
AlaLeuLeuAsnGluHisGlyAlaAlaGlnGluGlnLeuAsnLysIle 
290295300 
ArgAspSerTyrAlaLysLeuLeuGlyHisGlnAsnLeuLysGlnLys 
305310315320 
IleLysHisValValLysLeuLysAspGluAsnSerGlnLeuLysSer 
325330335 
GluValSerLysLeuArgCysGlnLeuAlaLysLysLysThrLys 
340345350 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 477 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: mouse 
(B) CLONE: GI 53979 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetGlnIleLeuThrGluArgLeuAlaLeuGluArgGlnGluTyrGlu 
151015 
LysLeuGlnGlnLysGluLeuGlnSerGlnSerLeuLeuGlnGlnGlu 
202530 
LysGluLeuSerAlaArgLeuGlnGlnGlnLeuCysSerPheGlnGlu 
354045 
GluMetThrSerGluLysAsnValPheLysGluGluLeuLysLeuAla 
505560 
LeuAlaGluLeuAspAlaValGlnGlnLysGluGluGlnSerGluArg 
65707580 
LeuValLysGlnLeuGluGluGluArgLysSerThrAlaGluGlnLeu 
859095 
ThrArgLeuAspAsnLeuLeuArgGluLysGluValGluLeuGluLys 
100105110 
HisIleAlaAlaHisAlaGlnAlaIleLeuIleAlaGlnGluLysTyr 
115120125 
AsnAspThrAlaGlnSerLeuArgAspValThrAlaGlnLeuGluSer 
130135140 
ValGlnGluLysTyrAsnAspThrAlaGlnSerLeuArgAspValThr 
145150155160 
AlaGlnLeuGluSerGluGlnGluLysTyrAsnAspThrAlaGlnSer 
165170175 
LeuArgAspValThrAlaGlnLeuGluSerGluGlnGluLysTyrAsn 
180185190 
AspThrAlaGlnSerLeuArgAspValThrAlaGlnLeuGluSerVal 
195200205 
GlnGluLysTyrAsnAspThrAlaGlnSerLeuArgAspValSerAla 
210215220 
GlnLeuGluSerTyrLysSerSerThrLeuLysGluIleGluAspLeu 
225230235240 
LysLeuGluAsnLeuThrLeuGlnGluLysValAlaMetAlaGluLys 
245250255 
SerValGluAspValGlnGlnGlnIleLeuThrAlaGluSerThrAsn 
260265270 
GlnGluTyrAlaArgMetValGlnAspLeuGlnAsnArgSerThrLeu 
275280285 
LysGluGluGluIleLysGluIleThrSerSerPheLeuGluLysIle 
290295300 
ThrAspLeuLysAsnGlnLeuArgGlnGlnAspGluAspPheArgLys 
305310315320 
GlnLeuGluGluLysGlyLysArgThrAlaGluLysGluAsnValMet 
325330335 
ThrGluLeuThrMetGluIleAsnLysTrpArgLeuLeuTyrGluGlu 
340345350 
LeuTyrGluLysThrLysProPheGlnGlnGlnLeuAspAlaPheGlu 
355360365 
AlaGluLysGlnAlaLeuLeuAsnGluHisGlyAlaThrGlnGluGln 
370375380 
LeuAsnLysIleArgAspSerTyrAlaGlnLeuLeuGlyHisGlnAsn 
385390395400 
LeuLysGlnLysIleLysHisValValLysLeuLysAspGluAsnSer 
405410415 
GlnLeuLysSerGluValSerLysLeuArgSerGlnLeuValLysArg 
420425430 
LysGlnAsnGluLeuArgLeuGlnGlyGluLeuAspLysAlaLeuGly 
435440445 
IleArgHisPheAspProSerLysAlaPheCysHisAlaSerLysGlu 
450455460 
AsnPheThrProLeuLysGluGlyAsnProAsnCysCys 
465470475 
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