Polynucleotide encoding novel chemokine expressed in inflamed adenoid

The present invention provides nucleotide and amino acid sequences that identify and encode a novel expressed chemokine (ADEC) from inflamed adenoid tissue. The present invention also provides for antisense molecules to the nucleotide sequences which encode ADEC, expression vectors for the production of purified ADEC, antibodies capable of binding specifically to ADEC, hybridization probes or oligonucleotides for the detection of ADEC-encoding nucleotide sequences, genetically engineered host cells for the expression of ADEC, diagnostic tests for inflammation or disease based on ADEC-encoding nucleic acid molecules or antibodies capable of binding specifically to ADEC.

CROSS-REFERENCE TO RELATED APPLICATIONS 
This application is related to co-pending U.S. patent application Ser. No. 
08/194,317 (Novel Human Adenoid Cell-Derived Polypeptides, Their 
Production and Uses) filed Feb. 4, 1994. 
BACKGROUND OF THE INVENTION 
Leukocytes including monocytes, macrophages, basophils, and eosinophils 
play important roles in the pathological mechanisms initiated by T and/or 
B lymphocytes. Macrophages, in particular, produce powerful oxidants and 
proteases which contribute to tissue destruction and secrete a range of 
cytokines which recruit and activate other inflammatory cells. 
The investigation of the critical, regulatory processes by which white 
cells proceed to their appropriate destination and interact with other 
cells is underway. The current model of leukocyte movement or trafficking 
from the blood to injured or inflamed tissues comprises the following 
steps. The first step is the rolling adhesion of the leukocyte along the 
endothelial cells of the blood vessel wall. This movement is mediated by 
transient interactions between selectins and their ligands. A second step 
involves cell activation which promotes a more stable 
leukocyte-endothelial cell interaction mediated by the integrins and their 
ligands. This stronger, more stable adhesion precipitates the final 
steps-leukocyte diapedesis and extravasation into the tissues. 
The chemokine family of polypeptide cytokines, also known as intercrines, 
possesses the cellular specificity required to explain leukocyte 
trafficking in different inflammatory situations. First, chemokines 
mediate the expression of particular adhesion molecules on endothelial 
cells; and second, they generate gradients of chemoattractant factors 
which activate specific cell types. In addition, the chemokines stimulate 
the proliferation of specific cell types and regulate the activation of 
cells which bear specific receptors. Both of these activities demonstrate 
a high degree of target cell specificity. 
The chemokines are small polypeptides, generally about 70-100 amino acids 
(aa) in length, 8-11 kD in molecular weight and active over a 1-100 ng/ml 
concentration range. Initially, they were isolated and purified from 
inflamed tissues and characterized relative to their bioactivity. More 
recently, chemokines have been discovered through molecular cloning 
techniques and characterized by structural as well as functional analysis. 
The chemokines are related through a four cysteine motif which is based 
primarily on the spacing of the first two cysteine residues in the mature 
molecule. Currently the chemokines are assigned to one of two families, 
the C-X-C chemokines (.alpha.) and the C-C chemokines (.beta.). Although 
exceptions exist, the C-X-C chemokines generally activate neutrophils and 
fibroblasts while the C-C chemokines act on a more diverse group of target 
cells which include monocytes/macrophages, basophils, eosinophils, T 
lymphocytes and others. The known chemokines of both families are 
synthesized by many diverse cell types as reviewed in Thomson A. (1994) 
The Cytokine Handbook, 2d Ed. Academic Press, New York. The two groups of 
chemokines will be described in turn. 
The archetypal and most extensively studied C-X-C chemokine is platelet 
factor 4 (PF4). This 70 aa protein displays the definitive four cysteines 
and is released along with platelet derived growth factor (PDGF), 
transforming growth factor .beta. (TGF-.beta.) and .beta.-thromboglobulin 
(.beta.-TG) from the granules of stimulated platelets. This homotetrameric 
molecule shares structural similarity with interleukin-8 (IL-8), induces 
the migration of fibroblasts, neutrophils and monocytes, and binds 
heparin. PF4 provides the biological model for a link among thrombosis, 
inflammation, and wound healing. 
Other chemokines found in the platelet a granule include .beta.-TG, 
connective tissue activating protein III (CTAP-III) and neutrophil 
activating peptide 2 (NAP-2). All three peptides are derived from the 
differential processing of a precursor molecule, platelet basic protein 
(PBP). .beta.-TG is an 81 aa, highly basic protein which influences the 
migration of fibroblasts but has no effect on neutrophils or monocytes. 
CTAP-III is 85 aa long, and aa 4-85 are identical to .beta.-TG. Since 
CTAP-III is the primary protein in the a granule and its role as a 
purified protein has not elucidated, it may be a secondary precursor, 
inactive until further processed. NAP-2 appears to attract neutrophils but 
not monocytes. 
Nonplatelet C-X-C chemokines include IL-8, .gamma. interferon inducible 
protein (IP-10), melanocyte growth stimulatory activity (MGSA or gro) 
proteins, epithelial derived neutrophil attractant-78 (ENA-78), 
granulocyte chemotactic protein-2 (GPC-2) and stromal cell-derived 
factors-1.alpha. and 1.beta. (SDF-1.alpha. and -1.beta.). IL-8 (also know 
as NAP-1) is secreted by monocytes/macrophages, neutrophils, fibroblasts, 
endothelial cells, keratinocytes and T lymphocytes in response to 
proinflammatory cytokines, IL-1 and 3, IFN-.gamma. and TNF, as well as 
endotoxin, mitogens, parciculates, bacteria and viruses. IL-8 stimulates 
acute inflammation including the upregulation of both neutrophil adhesion 
and keratinocyte growth and the downregulation of histamine production by 
basophils. 
IP-10 is a 10 kD protein of undefined function whose mRNA has been found in 
monocytes, fibroblasts and endothelial cells. Monocytes, keratinocytes and 
activated T cells secrete IP-10 protein which has been localized to sites 
of delayed hypersensitivity reactions. The cDNA of MGSA/gro .alpha. 
produces a 15 kD protein which appears in fibroblasts. Its transcription 
is growth related, and it functions as an autocrine growth factor. The 
distinct and non-allelic forms, gro .beta. and gro .gamma. are 90% and 86% 
identical to gro .alpha., respectively. Recombinant gro .alpha. proteins 
attract and activate neutrophils. ENA-78 was purified from supernatants of 
lung alveolar cells. Like gro .alpha., it attracts and activates 
neutrophils in vitro. 
GCP-2 is a 6 kD protein isolated from the supernatants of osteosarcoma 
cells. GCP-2 exists in various N-terminally truncated forms, and it 
attracts and activates neutrophils in vitro and causes granulocyte 
accumulation in vivo. SDF-1.alpha. and -1.beta. are newly isolated cDNAs 
which encode secreted molecules and type I membrane proteins. 
Current techniques for diagnosis of abnormalities in the inflamed or 
diseased tissues mainly rely on observation of clinical symptoms or 
serological analyses of body tissues or fluids for hormones, polypeptides 
or various metabolites. Patients often manifest no clinical symptoms at 
early stages of disease or tumor development. Furthermore, serological 
analyses do not always differentiate between invasive diseases and genetic 
syndromes which have overlapping or very similar ranges. Thus, development 
of new diagnostic techniques comprising small molecules such as the 
expressed chemokines are important to provide for early and accurate 
diagnoses, to give a better understanding of molecular pathogenesis, and 
to use in the development of effective therapies. 
The chemokine molecules were reviewed in Schall T. J. (1994) Chemotactic 
Cytokines: Targets for Therapeutic Development. International Business 
Communications, Southborough, Mass., pp 180-270; and in Paul W. E. (1993) 
Fundamental Immunology, 3rd Ed. Raven Press, New York City, pp 822-826. 
SUMMARY OF THE INVENTION 
The subject invention provides a nucleotide sequence which uniquely encodes 
a novel human protein from inflamed adenoid. The new gene, which is known 
as adenoid expressed chemokine, or adec (Incyte Clone No. 20293), encodes 
a polypeptide designated ADEC SEQ ID NO:2, of the C-X-C chemokine family. 
The invention also comprises diagnostic tests for inflammatory conditions 
which include the steps of testing a sample or an extract thereof with 
adec DNA, fragments or oligomers thereof. Aspects of the invention include 
the antisense DNAs of adec; cloning or expression vectors containing adec; 
host cells or organisms transformed with expression vectors containing 
adec; a method for the production and recovery of purified ADEC from host 
cells; and purified ADEC.

DETAILED DESCRIPTION OF THE INVENTION 
Definitions 
As used herein, "adenoid expressed chemokine" or ADEC, refers to a 
polypeptide, a naturally occurring ADEC or active fragments thereof, which 
is encoded by an mRNA transcribed from ADEC cDNA of a particular SEQ ID 
NO:1. 
"Active" refers to those forms of ADEC which retain the biologic and/or 
immunologic activities of naturally occurring ADEC. 
"Naturally occurring ADEC" refers to ADEC produced by human cells that have 
not been genetically engineered and specifically contemplates various ADEC 
forms arising from post-translational modifications of the polypeptide 
including but not limited to acetylation, carboxylation, glycosylation, 
phosphorylation, lipidation and acylation. 
"Derivative" refers to polypeptides derived from naturally occurring ADEC 
by chemical modifications such as ubiquitination, labeling (e.g., with 
radionuclides, various enzymatic modifications), pegylation 
(derivatization with polyethylene glycol) or by 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 ADEC 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, cell adhesion and chemotaxis, may be found by comparing the 
sequence of the particular ADEC with that of homologous cytokines 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 
ADEC 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, ADEC 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 ADEC polypeptides from natural or 
recombinant sources, cells transformed with recombinant nucleic acid 
molecules encoding ADEC. Various methods for the isolation of the ADEC 
polypeptides may be accomplished by procedures well known in the art. For 
example, such polypeptides 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 ADEC and is 
prepared using recombinant DNA techniques. The DNA which encodes ADEC may 
also include allelic or recombinant variants and routants thereof. 
"Oligonucleotides" or "nucleic acid probes" are prepared based on the cDNA 
sequence which encodes ADEC 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 
mRNA encoding ADEC is present in a cell or tissue or to isolate similar 
nucleic acid sequences from chromosomal DNA as described by Walsh P. S. 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 J. et al (1989) Molecular 
Cloning: A Laboratory Manual, 2d Ed, Cold Spring Harbor; or Ausubel F. M. 
et al (1989) Current Protocols in Molecular Biology, Vol 2, John Wiley & 
Sons, both incorporated herein by reference. 
Alternatively, 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 vital 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 chemokine of the C-X-C family, ADEC, which is highly expressed in 
inflamed adenoid. Because ADEC is specifically expressed in the tissue 
from which it was identified and has not been found in other tissues, the 
nucleic acid (adec), polypeptide (ADEC) and antibodies to ADEC are useful 
in diagnostic tests for inflamed or diseased adenoid. Excessive expression 
of ADEC leads to activation of neutrophils and fibroblasts which respond 
by producing abundant proteases and other molecules which can lead to 
tissue damage or destruction. Therefore, a diagnostic test for excess 
expression of ADEC can accelerate diagnosis and proper treatment of the 
inflammation before extensive tissue damage or destruction occurs. 
The nucleotide sequences encoding ADEC (or their complement) have 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 ADEC, and use in generation of anti-sense DNA or 
RNA, their chemical analogs and the like. Uses of nucleotides encoding 
ADEC 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 ADEC-encoding nucleotide 
sequences, some bearing minimal nucleotide sequence 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 ADEC, and all such variations 
are to be considered as being specifically disclosed. 
Although nucleotide sequences which encode ADEC and/or ADEC variants are 
preferably capable of hybridizing to the nucleotide sequence of the 
naturally occurring ADEC gene under stringent conditions, it may be 
advantageous to produce nucleotide sequences encoding ADEC or ADEC 
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 
ADEC and/or ADEC derivatives without altering the encoded aa sequence 
include the production of RNA transcripts having more desirable 
properties, e.g., a greater half-life, than transcripts produced from the 
naturally occurring nucleotide sequence. 
Nucleotide sequences encoding ADEC 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, 2d Ed, Cold Spring Harbor). 
Useful nucleotide sequences for joining to adec include an assortment of 
cloning vectors, e.g., plasmids, cosmids, lambda phage derivatives, 
phagemids, and the like, that are 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 adec-specific 
nucleic acid hybridization probes capable of hybridizing with naturally 
occurring nucleotide sequences encoding ADEC. Such probes for the 
detection of similar chemokine encoding sequences should preferably 
contain at least 50% of the nucleotides from a C-X-C encoding sequence. 
The hybridization probes of the subject invention may be derived from the 
nucleotide sequences of the SEQ ID NO 1 or from genomic sequences 
including promoters, enhancer elements and introns of naturally occurring 
adec. Hybridization probes may be labeled by a variety of reporter groups, 
including rad. ionuclides such as .sup.32 P or .sup.32 S, or enzymatic 
labels such as alkaline phosphatase, coupled to the probe via 
avidin/biotin coupling systems, and the like. 
PCR as described U.S. Pat. Nos. 4,965,188 and 4,683,195 and 4,800,195 
provides additional uses for oligonucleotides based upon the nucleotide 
sequences which encode ADEC. Such probes used in PCR may be of recombinant 
origin, may be 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 adec-specific hybridization probes include the 
cloning of nucleic acid sequences encoding ADECs and ADEC derivatives into 
vectors for the production of mRNA probes. Such vectors are known in the 
art and 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 
ADEC and ADEC derivatives entirely by synthetic chemistry, after which the 
gene can be inserted into any of the many available DNA vectors using 
reagents, vectors 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 adec sequence or any portion thereof. 
The nucleotide sequence can be used to construct an assay to detect 
inflammation and disease associated with abnormal levels of expression of 
ADEC. 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. If adec is present at an abnormal 
level, the assay indicates the presence of inflammation and/or disease. 
The nucleotide sequence for adec 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 City. 
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 O'Brien 
(1990) Genetic Maps: Locus Maps of Complex Genomes, Book 5: Human Maps, 
Cold Spring Harbor Laboratory Press. Correlation between the location of 
adec 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, carrier and affected individuals. 
Nucleotide sequences encoding ADEC may be used to produce purified ADEC 
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. ADEC may be expressed 
in a variety of host cells, either prokaryotic or eukaryotic. Host cells 
may be from species either the same or different from the species in which 
adec nucleotide sequences are endogenous. Advantages of producing ADEC by 
recombinant DNA technology include obtaining highly enriched sources of 
the proteins for purification and the availability of simplified 
purification procedures. 
Cells transformed with DNA encoding ADEC may be cultured under conditions 
suitable for the expression of the ADEC and the recovery of the protein 
from the cell culture. ADEC 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 depend on the 
nature of the production process used and the particular ADEC produced. 
In addition to recombinant production, ADEC fragments may be produced by 
direct peptide synthesis using solid-phase techniques (cf Stewart et al 
(1969) Solid-Phase Peptide Synthesis, W. H. 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 ADEC may be chemically synthesized separately and combined 
using chemical methods to produce the full length molecule. 
ADEC for antibody induction does not need to have biological activity; 
however, it must be immunogenic. Peptides used to induce ADEC 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 ADEC and may contain the entire aa sequence of the naturally occurring 
molecule. Short stretches of ADEC aa may be fused with those of another 
protein such as keyhole limpet hemocyanin and the chimeric molecule used 
for antibody production. 
Antibodies specific for ADEC may be produced by inoculation of an 
appropriate animal with the polypeptide or an antigenic fragment. An 
antibody is specific for ADEC if it is produced against an epitope of the 
polypeptide and binds to at least part of the natural or recombinant 
protein. Induction of antibodies 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 W. D. et al (1989) Science 
256:1275-1281) or the in vitro stimulation of lymphocyte populations. 
Current technology (Winter G., 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 ADEC. 
An additional embodiment of the subject invention is the use of 
ADEC-specific antibodies, inhibitors, receptors or their analogs as 
bioactive agents to treat inflammation or disease of the adenoid 
including, but not limited to, tonsilitis, Epstein-Barr virus, Hodgkin's 
disease, various neoplasms or nonspecific pharyngitis. Compositions 
comprising the above mentioned molecules may be administered in a suitable 
therapeutic dose determined by any of several methodologies including 
clinical studies on mammalian species to determine maximal tolerable dose 
and on normal human subjects to determine safe dose. 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 agent may be delivered orally via lozenges, syrups, sprays or 
topical application, by subcutaneous injection, airgun, etc. 
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 cDNA libraries 
The adec cDNA sequence was identified among the sequences comprising the 
inflamed adenoid library. This library was constructed from mixed adenoid 
and tonsil lymphoid tissue surgically removed from a child during a 
tonsilectomy. The adenoid tissue was obtained from University of 
California at Los Angeles and frozen for future use. The frozen tissue was 
ground in a mortar and pestle and lysed immediately in buffer containing 
guanidinium isothiocyanate (cf Chirgwin J. M. et al (1979) Biochemistry 
18:5294). Lysis was followed by several phenol-chloroform extractions and 
ethanol precipitations. Poly-A+ mRNA was isolated using biotinylated oligo 
d(T) and streptavidin coupled to paramagnetic particles (Promega, Poly(A) 
Tract Isolation System). 
The poly A mRNA from the inflamed adenoid tissue was used by Stratagene 
Inc. (11011 N. Torrey Pines Rd., La Jolla, Calif. 92037) to construct a 
cDNA library. cDNA synthesis was primed using oligo dT and/or random 
hexamers. Synthetic adapter oligonucleotides were ligated onto cDNA ends 
enabling its insertion into the UNI-ZAP.TM. vector system (Stratagene 
Inc.). This allows 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 each cDNA library was screened using either DNA probes 
or antibody probes, and then the pBluescript.RTM. phagemid (Stratagene 
Inc.) was rapidly excised in living cells. The 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 proteins. Phage particles from each library were 
infected into the E. coil host strain XL1-BLUE.RTM. (Stratagene Inc.). The 
high transformation efficiency of XL1-BLUE increases the probability of 
obtaining rare, under-represented clones from the cDNA library. 
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 re-infect fresh bacterial host cells (SOLR, Stratagene Inc.), 
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 Seguencing of cDNA Clones 
The cDNA inserts from random isolates of the inflamed adenoid 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. (U.S. 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 Peduced Protein 
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 
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. 
The nucleotide and amino acid sequences for the adenoid expressed 
chemokine, ADEC are shown in FIG. 1 (SEQ ID NO:1 and SEQ ID NO:2). 
V Identification and Full Length Sequencing of the Gene 
From all of the randomly picked and sequenced clones of the inflamed 
adenoid library, adec sequences were homologous to but clearly different 
from any known C-X-C chemokine molecule. The nucleotide sequence for adec 
was found within Incyte clone 20293. 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 adec. FIG. 2 shows the comparison of ADEC with other 
.alpha. chemokine molecules; substantial regions of homology including the 
C-X-C motif are shaded. The phylogenetic analysis, however, (FIG. 3) shows 
that adec is not very closely related to other well characterized human 
C-X-C chemokines. The most related of these molecules cluster together at 
the right hand side of the figure. It appears that adec may represent a 
new subfamily or variant of the C-X-C chemokines. 
VI Antisense analysis 
Knowledge of the correct, complete cDNA sequences of the novel expressed 
chemokine genes will enable their use in antisense technology in the 
investigation of gene function. Either oligonucleotides, genomic or cDNA 
fragments comprising the antisense strand of adec can be used either in 
vitro or in vivo to inhibit expression of the specific protein. Such 
technology is now well known in the art, and probes can be designed at 
various locations along the nucleotide sequence. 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 opening 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 ADEC 
Expression of adec may be accomplished by subcloning the cDNA into an 
appropriate expression vector and transfecting this vector into an 
appropriate expression host. In this particular case, the cloning vector 
previously used for the generation of the tissue library also provides for 
direct expression of the included sequence in E. coli. 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 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 mug bean 
nuclease, or oligonucleotide linker inclusion. 
Adec cDNA can be shuttled into other vectors known to be useful for 
expression of protein in specific hosts. Oligonucleotide amplifiers 
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 Hamstar Ovary 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 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 ADEC can be recovered from the conditioned medium 
and analyzed using chromatographic methods known in the arc. 
VIII Isolation of Recombinant ADEC 
ADEC 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) between the purification domain and the ADEC-encoding 
sequence may be useful to facilitate production of ADEC. 
IX Production of ADEC-Specific Antibodies 
Two approaches are utilized to raise antibodies to ADEC, and each approach 
is useful for generating either polyclonal or monoclonal antibodies. In 
one approach, denatured ADEC 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 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 ADEC, as deduced from 
translation of the cDNA, is analyzed to determine regions of high 
immunogenicity. Oligopeptides comprising hydrophilic regions, as shown in 
FIG. 4, are synthesized and used in suitable immunization protocols to 
raise antibodies. Analysis to select appropriate epitopes is described by 
Ausubel F. M. et al (1989, Current Protocols in Molecular Biology, Vol 2, 
John Wiley & Sons). 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-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; cf. 
Ausubel F. M. 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 ADEC 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 
supematants from hybridomas. After incubation the wells are exposed to 
labeled ADEC, 1 mg/ml. Clones producing antibodies will bind a quantity of 
labeled ADEC 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 10.sup.8 M.sup.-1, preferably 10.sup.9 to 10.sup.10 
or stronger, will typically be made by standard procedures as described in 
Harlow and Lane (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor 
Laboratory; and in Goding (1986) Monoclonal Antibodies: Principles and 
Practice, 2d Ed, Academic Press New York City, both incorporated herein by 
reference. 
X Diagnostic Test Using ADEC-Specific Antibodies 
Particular ADEC 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 ADEC. To date, ADEC has only 
been found in inflamed adenoid and is thus specific for abnormalities or 
pathologies of that tissue. 
Diagnostic tests for ADEC include methods utilizing the antibody and a 
label to detect ADEC 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 radionudides, 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; 3,996,346; 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 ADEC, using 
either polyclonal or monoclonal antibodies specific for that ADEC are 
known in the arc. 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 ADEC is preferred, 
but a competitive binding assay may be employed. These assays are 
described, among other places, in Maddox, D. E. et al (1983) J Exp Med 
158:1211. 
XI Purification of Native ADEC Using Specific Antibodies 
Native or recombinant ADEC was purified by immunoaffinity chromatography 
using ADEC-specific antibodies. In general, an immunoaffinity column is 
constructed by covalently coupling the anti-ADEC 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 immunoglobolin is covalently attached to a 
chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB 
Biotechnology, Piscataway, N.J.). The antibody is coupled to the resin, 
the resin is blocked, and the derivative resin is washed according to the 
manufacturer's instructions. 
Such an immunoaffinity column was utilized in the purification of ADEC by 
preparing a fraction from cells containing ADEC 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 ADEC containing a signal sequence may be secreted 
in useful quantity into the medium in which the cells are grown. 
A soluble ADEC-containing preparation was passed over the immunoaffinity 
column, and the column was washed under conditions that allow the 
preferential absorbance of ADEC (e.g., high ionic strength buffers in the 
presence of detergent). Then, the column was eluted under conditions that 
disrupt antibody/ADEC binding (e.g., a buffer of pH 2-3 or a high 
concentration of a chaotrope such as urea or thiocyanate ion), and the 
ADEC was collected. 
XII Determination of ADEC-Induced Chemotaxis or Cell Activation 
The chemotactic activity of ADEC is measured in a 48-well microchemotaxis 
chamber (Falk W. R. 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. Cell culture medium such as RPMI 
1640 containing ADEC is placed on one side of a filter, usually 
polycarbonate, and cells suspended in the same media 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 ADEC are typed and quantified. 
The specificity of the chemoattraction is determined by performing the 
chemotaxis assay on specific populations of cells. First, blood cells 
obtained from venipuncture are fractionareal by density gradient 
centrifugation and the chemotactic activity of ADEC is tested on enriched 
populations of neutrophils, peripheral blood monohuclear cells, monocytes 
and lymphocytes. Optionally, such enriched cell populations are further 
fractionareal using CD8.sup.+ and CD4.sup.+ specific antibodies for 
negative selection of CD4.sup.+ and CD8.sup.+ enriched T-cell populations, 
respectively. 
Another assay elucidates the chemotactic effect of ADEC on activated 
T-cells. There, unfractionated T-cells or fractionareal T-cell subsets are 
cultured for 6 to 8 hours in tissue culture vessels coated with CD-3 
antibody. After this CD-3 activation, the chemotactic activity of ADEC is 
tested as described above. Many other methods for obtaining enriched cell 
populations are known in the art. 
Some chemokines also produce a non-chemotactic cell activation of 
neutrophils and monocytes. This is tested via standard measures of 
neutrophil activation such as actin polymerization, increase in 
respiratory burst activity, alegranulation of the azurophilic granule and 
mobilization of Ca.sup.++ as part of the signal transduction pathway. The 
assay for mobilization of Ca.sup.++ involves preloading neutrophils with a 
fluorescent probe whose emission characteristics have been altered by 
Ca.sup.++ binding. When the cells are exposed to an activating stimulus, 
Ca.sup.++ flux is determined by observation of the cells in a fluorometer. 
The measurement of Ca.sup.++ mobilization has been described in 
Gpynkievicz G. et al. (1985) J Biol Chem 260:3440, and McColl S. et al. 
(1993) J Immunol 150:4550-4555, incorporated herein by reference. 
Degranulation and respiratory burst responses are also measured in 
monocytes (Zachariae C. O. C. et al. (1990) J Exp Med 171:2177-82). 
Further measures of monocyte activation are regulation of adhesion 
molecule expression and cytokjne production (Jiang Y. et al. (1992) J 
Immunol 148: 2423-8). Expression of adhesion molecules also varies with 
lymphocyte activation (Taub. D. et al. (1993) Science 260: 355-358). 
XIII Drug Screening 
This invention is particularly useful for screening compounds by using ADEC 
polypeptide or binding fragment thereof in any of a variety of drug 
screening techniques. The ADEC 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 ADEC or fragment and the agent being tested or examine 
the diminution in complex formation between ADEC and a neutrophil or 
fibroblast caused by the agent being tested. 
Thus, the present invention provides methods of screening for drugs or any 
other agents which can affect inflammation and disease. These methods 
comprise contacting such an agent with an ADEC polypeptide or fragment 
thereof and assaying (i) for the presence of a complex between the agent 
and the ADEC polypeptide or fragment, or (ii) for the presence of a 
complex between the ADEC polypeptide or fragment and the cell, by methods 
well known in the art. In such competitive binding assays, the ADEC 
polypeptide or fragment is typically labeled. After suitable incubation, 
free ADEC 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 ADEC or to interfere with the 
ADEC/cell complex. 
Another technique for drug screening provides high throughput screening for 
compounds having suitable binding affinity to the ADEC 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 ADEC polypeptide and washed. Bound ADEC 
polypeptide is then detected by methods well known in the art. Purified 
ADEC 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 ADEC 
specifically compete with a test compound for binding to ADEC polypeptide 
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 ADEC. 
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 
analogous chemokine-like molecules or to identify 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 
(antiids) 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 ADEC 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 ADEC Receptors 
Purified ADEC is useful for characterization and purification of specific 
cell surface receptors and other binding molecules. Cells which respond to 
ADEC by cherootaxis or other specific responses are likely to express a 
receptor for ADEC. Radioactive labels may be incorporated into ADEC by 
various methods known in the art. A preferred embodiment is the labeling 
of primary amino groups in ADEC 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 chemokines without concomitant loss of biological 
activity (Hebert C. A. et al (1991) J Biol Chem 266: 18989; McColl S. et 
al (1993) J Immunol 150:4550-4555). Receptor-bearing cells are incubated 
with labeled ADEC. The cells are then washed to removed unbound ADEC, and 
receptor-bound ADEC is quantified. The data obtained using different 
concentrations of ADEC are used to calculate values for the number and 
affinity of receptors. 
Labeled ADEC is useful as a reagent for purification of its specific 
receptor. In one embodiment of affinity purification, ADEC is covalently 
coupled to a chromatography column. Receptor-bearing cells are extracted, 
and the extract is passed over the column. The receptor binds to the 
column by virtue of its biological affinity for ADEC. The receptor is 
recovered from the column and subjected to N-terminal protein sequencing. 
This amino acid sequence is then used to design degenerate oligonucleotide 
probes for cloning the receptor gene. 
In an alternate method, expression cloning, mRNA is obtained from 
receptor-bearing cells and made into a cDNA expression library. The 
library is transfected into a population of cells, and those cells in the 
population which express the receptor are selected using fluorescently 
labeled ADEC. The receptor is identified by recovering and sequencing 
recombinant DNA from highly labeled cells. 
In another alternate method, antibodies are raised against the surface of 
receptor-bearing cells, specifically monoclonal antibodies. The monoclonal 
antibodies are screened to identify those which inhibit the binding of 
labeled ADEC. These monoclonal antibodies are then used in affinity 
purification or expression cloning of the receptor. 
Soluble receptors or other soluble binding molecules are identified in a 
similar manner. Labeled ADEC is incubated with extracts or other 
appropriate materials derived from inflamed adenoid. After incubation, 
ADEC complexes larger than the size of purified ADEC 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 
receptors or binding protein(s) are subjected to N-terminal sequencing to 
obtain information sufficient for database identification, if the soluble 
protein is known, or cloning, if the soluble protein is unknown. 
XVI USE AND ADMINISTRATION OF ADEC 
Antibodies, inhibitors, receptors or analogs of ADEC (treatments for 
excessive ADEC production, hereafter abbreviated TEC), can provide 
different effects when administered therapeutically. TECs 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, receptor or analog being formulated and the condition to be 
treated. Characteristics of the TEC include solubility of the molecule, 
half-life and antigenicity/immunogenicity and may aid in defining an 
effective carrier. Native human proteins are preferred as TECs, but 
organic molecules resulting from drug screens may be equally effective in 
particular situations. 
TECs may be delivered by known routes of administration including but not 
limited to topical creams or gels; transmucosal spray or aerosol, 
transdermal patch or bandage; injectable, intravenous or lavage 
formulations; or orally administered liquids or pills. 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 TEC to be administered, and the 
pharmacokinetic profile of the particular TEC. 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 TEC 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 TEC. 
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 
TECs and that administration targeting the adenoid may necessitate 
delivery in a manner different from that for delivery targeted to a more 
internal tissue. 
It is contemplated that conditions or diseases of the adenoids which 
activate, fibroblasts, neutrophils or other leukocytes may precipitate 
permanent damage that is treatable with TECs. These conditions or diseases 
may be specifically diagnosed by the tests discussed above, and such 
testing should be performed in suspected cases of Epstein-Barr virus, 
Hodgkin's disease, various neoplasms or nonspecific pharyngitis. 
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 arc 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: 9 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 330 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Inflamed Adenoid 
(B) CLONE: 20293 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ATGAAGTTCATCTCGACATCTCTGCTTCTCATGCTGCTGGTCAGCAGCCTCTCTCCAGTC60 
CAAGGTGTTCTGGAGGTCTATTACACAAGCTTGAGGTGTAGATGTGTCCAAGAGAGCTCA120 
GTCTTTATCCCTAGACGCTTCATTGATCGAATTCAAATCTTGCCCCGTGGGAATGGTTGT180 
CCAAGAAAAGAAATCATAGTCTGGAAGAAGAACAAGTCAATTGTGTGTGTGGACCCTCAA240 
GCTGAATGGATACAAAGAATGATGGAAGTATTGAGAAAAAGAAGTTCTTCAACTCTACCA300 
GTTCCAGTGTTTAAGAGAAAGATTCCCTGA330 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 109 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: polypeptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Inflamed Adenoid 
(B) CLONE: 20293 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetLysPheIleSerThrSerLeuLeuLeuMetLeuLeuValSerSer 
151015 
LeuSerProValGlnGlyValLeuGluValTyrTyrThrSerLeuArg 
202530 
CysArgCysValGlnGluSerSerValPheIleProArgArgPheIle 
354045 
AspArgIleGlnIleLeuProArgGlyAsnGlyCysProArgLysGlu 
505560 
IleIleValTrpLysLysAsnLysSerIleValCysValAspProGln 
65707580 
AlaGluTrpIleGlnArgMetMetGluValLeuArgLysArgSerSer 
859095 
SerThrLeuProValProValPheLysArgLysIlePro 
100105 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 114 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetSerLeuLeuSerSerArgAlaAlaArgValProGlyProSerSer 
151015 
SerLeuCysAlaLeuLeuValLeuLeuLeuLeuLeuThrGlnProGly 
202530 
ProIleAlaSerAlaGlyProAlaAlaAlaValLeuArgGluLeuArg 
354045 
CysValCysLeuGlnThrThrGlnGlyValHisProLysMetIleSer 
505560 
AsnLeuGlnValPheAlaIleGlyProGlnCysSerLysValGluVal 
65707580 
ValAlaSerLeuLysAsnGlyLysGluIleCysLeuAspProGluAla 
859095 
ProPheLeuLysLysValIleGlnLysIleLeuAspGlyGlyAsnLys 
100105110 
GluAsn 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 107 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetAlaArgAlaThrLeuSerAlaAlaProSerAsnProArgLeuLeu 
151015 
ArgValAlaLeuLeuLeuLeuLeuLeuValAlaAlaSerArgArgAla 
202530 
AlaGlyAlaProLeuAlaThrGluLeuArgCysGlnCysLeuGlnThr 
354045 
LeuGlnGlyIleHisLeuLysAsnIleGlnSerValLysValLysSer 
505560 
ProGlyProHisCysAlaGlnThrGluValIleAlaThrLeuLysAsn 
65707580 
GlyGlnLysAlaCysLeuAsnProAlaSerProMetValLysLysIle 
859095 
IleGluLysMetLeuLysAsnGlyLysSerAsn 
100105 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 106 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
MetAlaHisAlaThrLeuSerAlaAlaProSerAsnProArgLeuLeu 
151015 
ArgValAlaLeuLeuLeuLeuLeuLeuValGlySerArgArgAlaAla 
202530 
GlyAlaSerValValThrGluLeuArgCysGlnCysLeuGlnThrLeu 
354045 
GlnGlyIleHisLeuLysAsnIleGlnSerValAsnValArgSerPro 
505560 
GlyProHisCysAlaGlnThrGluValIleAlaThrLeuLysAsnGly 
65707580 
LysLysAlaCysLeuAsnProAlaSerProMetValGlnLysIleIle 
859095 
GluLysIleLeuAsnLysGlySerThrAsn 
100105 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 99 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
MetThrSerLysLeuAlaValAlaLeuLeuAlaAlaPheLeuIleSer 
151015 
AlaAlaLeuCysGluGlyAlaValLeuProArgSerAlaLysGluLeu 
202530 
ArgCysGlnCysIleLysThrTyrSerLysProPheHisProLysPhe 
354045 
IleLysGluLeuArgValIleGluSerGlyProHisCysAlaAsnThr 
505560 
GluIleIleValLysLeuSerAspGlyArgGluLeuCysLeuAspPro 
65707580 
LysGluAsnTrpValGlnArgValValGluLysPheLeuLysArgAla 
859095 
GluAsnSer 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 107 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
MetAlaArgAlaAlaLeuSerAlaAlaProSerAsnProArgLeuLeu 
151015 
ArgValAlaLeuLeuLeuLeuLeuLeuValAlaAlaGlyArgArgAla 
202530 
AlaGlyAlaSerValAlaThrGluLeuArgCysGlnCysLeuGlnThr 
354045 
LeuGlnGlyIleHisProLysAsnIleGlnSerValAsnValLysSer 
505560 
ProGlyProHisCysAlaGlnThrGluValIleAlaThrLeuLysAsn 
65707580 
GlyArgLysAlaCysLeuAsnProAlaSerProIleValLysLysIle 
859095 
IleGluLysMetLeuAsnSerAspLysSerAsn 
100105 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 101 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
MetSerSerAlaAlaGlyPheCysAlaSerArgProGlyLeuLeuPhe 
151015 
LeuGlyLeuLeuLeuLeuProLeuValValAlaPheAlaSerAlaGlu 
202530 
AlaGluGluAspGlyAspLeuGlnCysLeuCysValLysThrThrSer 
354045 
GlnValArgProArgHisIleThrSerLeuGluValIleLysAlaGly 
505560 
ProHisCysProThrAlaGlnLeuIleAlaThrLeuLysAsnGlyArg 
65707580 
LysIleCysLeuAspLeuGlnAlaProLeuTyrLysLysIleIleLys 
859095 
LysLeuLeuGluSer 
100 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 109 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
MetLysPheIleSerThrSerLeuLeuLeuMetLeuLeuValSerSer 
151015 
LeuSerProValGlnGlyValLeuGluValTyrTyrThrSerLeuArg 
202530 
CysArgCysValGlnGluSerSerValPheIleProArgArgPheIle 
354045 
AspArgIleGlnIleLeuProArgGlyAsnGlyCysProArgLysGlu 
505560 
IleIleValTrpLysLysAsnLysSerIleValCysValAspProGln 
65707580 
AlaGluTrpIleGlnArgMetMetGluValLeuArgLysArgSerSer 
859095 
SerThrLeuProValProValPheLysArgLysIlePro 
100105 
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