Note: Descriptions are shown in the official language in which they were submitted.
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fi~JiN G-PROTEIN CgB~SOICIlQS RFCBPTOR BaiGHRlO
This invention relates to newly identified
polynucleotides, polypeptides encoded by such
polynucleotides, the use of such polynucleotides and,
polypeptides, as well as the production of such
polynucleotides and polypeptides. More particularly, the
polypeptide of the present invention is a human 7-
transmembrane receptor which has been putatively identified
as a chemokiae receptor, sometimes hereinafter referred to as
"G-Protein Chemokine Receptor" or "HD~iRlO". The imrention
also relates to inhibiting the action of such polypeptides.
It is well established that many medically significant
biological processes are mediated by proteins participating
in signa7~ transduction pathways that involve G-proteins
and/or second messengers, e.g., cAMP (Lefkowitz, Nature,
351:353-354 11991)). Herein these proteins are referred to
as proteins participating in pathways with G-proteins or PPG
proteins . Some examcples of these proteins include the GPC
receptors, such as those for adrenergic agents and dopamine
(Kobilka, B.K., et al., PNA.S, 84:46-50 (1987); Kobilka, B.K.,
et al., Science. 238:650-656 11987); Bunzow, J.R., et al.,
Nature, 336:783-787 (ig88)), G-proteins themselves, effector
proteins, e.g., phospholipase C, adenyl cyclase, and
phosphodiesterase, and actuator proteins, e.g., protein
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kinase A and protein kinase C (Simon, M.I., et al., Science,
252:802-8 X1991)).
For example, in one form of signal transduction, the
effect of hormone binding is activation of an enzyme,
adenylate cyclase, inside the cell. 8azyme activation by
hormones is dependent on the presence of the nucleotide GTP,
and GTP also influences hormone binding. A G-protein
connects the hormone receptors to adenylate cyclase. G-
protein was shown to exchange GTP for bound GDP when
activated by hormone receptors. The GTP-carrying form then
binds to an activated adenylate cyclase. Hydrolysis of GTP
to GDP, catalyzed by the G-protein itself, returns-the.G-
protein to its basal, inactive form. Thus, the G-protein
serves a dual role, as an intexinediate that relays the signal
from receptor to effector, and as a clock that controls the
duration of the signal.
The ~mbrane protein gene superfamily of G-protein
coupled receptors has been characterized as having seven
putative transmembrane do~ai.ns. The domains are believed to
represent traasmembrane a-helices connected by extracellular
or cytoplasmic loops. G-protein coupled receptors include a
pride range of biologically active receptors , such as hormone ,
viral, growth factor and neuroreceptors. ,.
G-protein coupled receptors have been characterized as
including these seven consezved hydrophobic stretches of
about 20 -to 30 amino acids, connecting at least eight
divergent hydrophilic loops. The G-protein family of coupled
receptors includes dopamine receptors which bind to
neuroleptic drugs used for treating psychotic and
neurological disorders. Other examples of members of this
family include calcitonin, adrenergic, eadothelin; cAMP,
adenosine, muscarinic, acetylcholine, serotonin, histamine,
thrombin, kinin, follicle stimulating hormone, opsins,
endothelial differentiation gene-1 receptor and rhodopsins,
odorant, cytanegalovirus receptors, etc.
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G-protein coupled receptors can be intrace~lularly
coupled by heterotrimeric G-proteins to various intracellular
enzymes, ion criannels and transporters (see, Johnson et al.,
Bndoc., Rev., 10:317-331 (1989)). Different G-protein a-
subuaits preferentially stimulate particular effectors to
modulate various biological functions in a cell.
Phosphorylation of cytoplasmic residues of G-protein coupled
receptors have been identified as an important mechanism for
the regulation of G-protein coupling. of some G-protein
coupled receptors. G-protein coupled receptors are found in
numerous sites within a mammalian host.
Chemokines, also referred to as intercriae cytokines,
are a subfamily of structurally and functionally related
cytokines . These molecules are 8-10 kd in size . In general,
chemokines exhibit 20~c to 75~ homology at the amino acid
level and are characterized by four conserved cysteine~
residues that form two disulfide bonds. Based on the
arrangement of the first two cysteine residues, chemokines
have been classified into two subfamilies, alpha and beta.
In the alpha subfamily, the first two cysteines are separated
by one amino acid and hence are referred to as the "C-g-C"
subfamily. In the beta subfamily, the two cysteines are in
an adjacent position and are, therefore, referred to as the
"C-C" subfamily. Thus far, at least nine different members
of this family have been identified is humans.
The intercrine cytokines exhibit a wide variety of
functions. A hallmark feature is their ability to elicit
chemotactic migration of distinct cell types, including
monocytes, neutrophils, T lymphocytes, basophils and
fibroblaets. Many chemokines have proinflammatory activity
and are involved in multiple steps during an inflammatory
reaction. These activities include stimulation of histaiaine
release, lysosomal enzyme and leukotriene release, increased
adherence of target immune cells to endothelial cells,
enhanced binding of coatplement proteins, induced expression
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of granulocyte adhesion molecules and complement receptors,
and respiratory burst. In addition to their involvement in
inflam~aation, certain chemokines have bees shown to exhibit
other activities . For examcple, macrophage inflanrcc~aatory
protein 1 tMIP-1) is able to suppress hematopoietic stem cell
proliferation, platelet factor-4 (PF-4 ) is a potent inhibitor
of endothelial cell growth, Interleukin-8 (IL-8) promotes
proliferation of keratinocytes, and GRO is an autocrine
growth factor for melanoma cells .
In light of the diverse biological activities, it is not
surprising that che~nokiaes have been implicated in a number
of physiological and disease conditions, including lymphocyte
trafficking, wound healing, hematopoietic regulation and
immunological disorders such as allergy, asthma and
arthritis.
In accordance with one aspect of the present invention,
there are provided novel mature receptor polypeptides as well
as biologically active and diagnostically or therapeutically
useful fragments, analogs and derivatives thereof. The
receptor polypeptides of the present inveatioa'are of human
origin.
In accordance with aaother~ aspect of the present
invention, there are. provided isolated nucleic acid molecules
encoding the receptor polypeptides of the present invention,
including mRNAs, DNAs, cDNAs, genomic DNA as well as
aatisense- analogs thereof and biologically active and
diagnostically or therapeutically useful fragments thereof.
In accordance with a further aspect of the present
imreation, there are provided processes for producing such
receptor polypeptides by recombinant techniques comprising
culturing recombinant prokaryotic and/or eukaryotic host
cells, containing nucleic acid sequences encoding the
receptor polypeptides of the present invention, under
conditions promoting expression of said polypeptides and
subsequent recovery of said polypeptides.
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In accordance with yet a further aspect of the present
invention, there are provided antib.~dies against such
receptor polypeptides.
In accordance with another aspect of the present
invention there are provided methods of screening for
compounds which bind to and activate or inhibit activation of
the receptor polypeptides of the present invention.
In accordance with still another embodiment of the
present invention there are provided processes of
administering compounds to a host which bind to and activate
the receptor polypeptide of the present invention which are
useful in stinn~lating haematopoiesis, around healing,
coagulation, angiogenesis, to treat solid tumors, chronic
infections, leukemia, T-cell mediated auto-imaname diseases,
parasitic infections, psoriasis, and to stimulate growth
factor activity.
In accordance with another aspect of the present
invention there is provided a method of administering the
receptor polypeptides of the present invention via gene
therapy to treat conditions related to underexpression of the
polypeptides or underexpression of a ligand for the receptor
polypeptide_
In accordance with still another embodiment of the
present invention there are provided processes of
administering compounds to a host which bind to and inhibit
activation of the receptor polypeptides of the present
invention which are useful in the prevention and/or treatment
of allergy, atherogenesis, anaphylaxis, malignancy, chronic
and acute inflammation, histamine and Ig$-m;ediated allergic
reactions; prostaglandin-independent fever, bone marrow
failure, silicosis, sarcoidosis, rheumatoid arthritis, shock
and hyper-eosinophilic syndrome.
In accordance with yet another aspect oft. the present
invention, there are provided nucleic acid probes comprising
nucleic acid molecules of sufficient length to specifically
CA 02562162 2006-10-20
hybridize to the polynucleotide sequences of the present
invention.
Tn accordance with still another aspect of the present
invention, there are provided diagnostic assays for detecting
diseases related to mutations in the nucleic acid sequences
encoding such polypeptides and for detecting an altered level
of the soluble form of the receptor polypeptides.
In accordance with yet a further aspect of the~present
invention, there are provided processes for utilizing such
receptor polypeptides, or polynucleotides encoding such
polypepti.des, ~ for .in' vitro purposes related. ' to scientific
research, synthesis of DNA and manufacture of DNA vectors.-
These and other aspects of the present invention should
be apparent to those skilled~in the art from the teachings
herein.-
The following drawings acre illustrative of embodiments
of the invention and are not meant to limit the scope.of the
invention as encompassed by the_claims.
Figure 1 shows the cDNA sequence and the. corresponding
deduced amino acid sequence of the G-protein coupled receptor
of the present invention. the tandard one-letter
abbreviation for amino acids is used. Sequencing was
performed using a 373 Automated DNA sequ~ncer (~..pplied
Biosystems, Inc.).
Figure 2 illustrates an amino acid alignment of the G-
protein chemokine receptor~of the present invention and the
human MCP-I receptor.
In accordance with an aspect of the preser_t invention,
there is provided an isoxated nucleic acid (polyizucleotide)
which encodes for the mature polypeptide having the deduced
amino acid sequence of Figure 1 (SEQ ID N0:2) or for the
mature polypeptide encoded by the cDNA of the clone deposited
as ATCC Deposit No. 97183 with the Rmerican Type Culture
Collection, 12301 Parklawn Drive, Rockv?11e, Mary?and, 20852,
United States of America, on June ~, 1995.
The polynucleot~~.de of this invention was discovered in
a cDNA library derived from human monocytes. It is
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stiucturallv related to the G protein-coupled receptor
family. It contains an open reading frame encoding a protein
of 352 amino acid residues. The protein exhibits the highest
degree of homology to a human MCP-1 receptor with 70.1 %
identity and 82.9 % similarity over a 347 amino acid stretch.
The polynucleotide of the present invention may be is
the form of RNA or in the forca of DNA, which DNA includes
cDNA, genomic DNA, and synthetic DNA. The DN~.1 may. be double-
stranded or single-stranded, and if single stranded may be
the coding strand or non-coding (anti-sense) strand. The
coding sequence which encodes the mature polypeptide may be
identical to the coding sequence shown in Figure 1 (S$Q ID
NO:1) or that of the .deposited clone or may be a different
coding sequence which coding sequence, as a result of the
redundancy or degeneracy of the genetic code, encodes the-
same mature polypeptide as the DNA of Figure 1 (S8Q ID N0:1)~
or the deposited cDl~.
.The polynucleotide which encodes for the mature
polypeptide of Figure 1 or for the mature polypeptide encoded
by~the deposited cDNA may include: only the coding sequence
for the mature polypeptide; the coding sequence for- the
mature palypeptide and additional-coding sequence such as a
transmembraae (T1~1')- or intro-cellular domain; the coding
sequence for the . mature polypeptide (and optionally
additional coding sequence) and non-coding sequence, such as
introns or non-coding sequence 5' and/or 3' of the coding
sequence far the mature polypeptide. '
Thus, the term "polynucleotide encoding a polypeptide"
encooapasses a polynucleotide which includes only codir~g
sequence for the polypeptide as well as-.a polynucleotide
which includes additional coding and/or non-coding sequence.
The present invention further relates to variants of the
herei.nabove described polyaucleotides which encode for
fragments, analogs and derivatives of the polypeptide having
the deduced amigo acid sequence of Figure 1 or the
CA 02562162 2006-10-20
polypeptide encoded by the cDNA of the deposited clone. T~.
variant of the polynucleotide may be a naturally occurring
allelic variant of the polynucleotide or a non-naturally
occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides
encoding the same mature polypeptide as shown in Figure 1
(S$Q ID N0:2) or the same mature polypeptide encoded by the
cDNA of the deposited clone as well as .variants of such
polynucleotides which variants encode for a fragment,
derivative or analog of the polypeptide of Figure 1 (SBQ~ID
NO:~) or the polypeptide encoded by the cDI~ of the deposited
clone. Such nucleotide variants include deletion variants,
substitution variants and addition or insertion variants.
As hereinabove indicated, the.polyaucleotide may have a
coding sequence which is a naturally occurring allelic
variant of the coding sequence shown is Figure 1 (S$Q ID-
NO:~)_or of the coding sequence of the deposited clone. As
known is the art, as allelic variant is as altezaate form of
a polyaucleotide sequence which may have a substitution,
deletion or addition of one or more nucleotides, which does
not substantially alter the function of the encoded
polypeptide_ '
The polyaucieotides'may also encode for a soluble form
of the G-protein chemokine receptor polypeptide which is the
extracellular portion of the polypeptide which has been
cleaved froia the TM and intracellular domain of the full-
leagth poiypeptide of the present invention.
The polynucleotides of the present~invention may also
have the coding sequence fused~in frame to a marker sequence
which allows for purification of the polypeptide of the
present imrention_ The marker sequence may be a hexa-
histidine tag supplied by a pQB-9 vector to provide far-
purification of the mature polypeptide fused to the marker is
the case of a~bacterial host, or, for example, the marker
sequence may be a hemagglutinin~(HA) tag when'a-mammalian
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CA 02562162 2006-10-20
host, e.g. COS-7 cells, is used. The HA tag corresponds to
an epitope derived from the influenza hemagglutinin protein
(Wilson, I., et al., Cell, 37:76: (1984)y.
The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding
and following the coding region (leader and trailer) as well
as intervening sequences (introns) between individual coding
segments (axons).
Fragments of the full length gene of the present
invention may be used as a hybridization probe for a cDNA~
library to isolate the full length cDNA and to isolate other
cDNAs which have a high sequence similarity to the gene or
similar biological activity. Probes of this type preferably
have at least 30 bases and may contain, for example, 50 or
more bases . The probe may also be used to identify a cD~~iA
clone corresponding to a full length transcript and a genomic ;
clone or clones that contain the complete gene including
regulatory and pranotor regions, axons, and introns. An
example of a screen coomprises isolating the coding region of
the gene by using the laio~rn DNA sequence to synthesize an
oligonucleotide probe. Labeled oligonucleotides having a
sequence complementary to that of the gene of the present
invention are used to screen a library of human cDNA, ;.geaomic
DATA or mR~ to determine which members of the library the
probe hybridizes to.
The present invention further relates to
polynucleotides which hybridize to the hereiaabove-described
sequences if there is at least 70%, preferably at least 90%,
and more preferably at least 95% identity between the
sequences. The-present invention particularly relates to
polynucleotides which hybridize under stringent conditions to
the hereiaabove-described polynucleotides. As herein used,
the terra "stringent conditions" means hybridization will
occur only if there is at least 95% and preferably at least
97% identity between the sequences. The polynucleotides
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which hybridize to the herei.na.bave described polynucleotidE
in a preferred embodiment encode polypeptides which either
retain substantially the same biological function yr activity
as the mature polypeptide encoded by the cDl~s of Figure 1
(SBQ ID NO:1) or the deposited cDNA(s).
Alternatively, the polynucleotide may have at least 20
bases, preferably 30 bases, and awre preferaGbly at least 50
bases which hybridize to a polynucleotide'of the present
imrention and which has an identity thereto, as hereinabove
described, and which may or may not retain activity. For
example, such polynucleotides may be employed as probes for
the polynucleotide of SBQ ID I~IO:1, for ~ exan~le, for recovery
of the polyaucleotide or as a diagnostic probe or as a PQ2
primer.
Thus, the present invention is directed to
polynucleotides having at~ least a 70~ identity, preferably at
Least 90~ and more preferably at least a 95~ identity to a
polynucleotide which encodes the polypeptide of SBQ ID N0:2
as well as fraga~eats thereof, which fragments have at least
30 bases and preferably at least 50 bases and to polypeptides
encoded by such polyaucleotides.
The deposits) referred to herein will be maintained
under the terms of, the Budapest Treaty on the International
Recognition of the Deposit of Micro-organistas for purposes of
Patent Procedure. These deposits ajre provided merely as
convenience to those of skill ia-the art and are not an
adatission that a deposit is required.
The segueace of the polynucleotides contained in the
deposited materials, as well. as the axaino acid sequence of
the polypeptides encoded thereby,
are controlling in the event of nay conflict.
with~any description of~sequences herein. A license utay be
required to make, or sell the deposited materials, and
no such license is hereby granted..
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The present invention further relates to a G-protein
chemokine receptor polypeptide which has the deduced amino
acid sequence of Figure 1 (SBQ ID N0:2) or which has the
amino acid sequence encoded by the deposited'cDNA, as well as
fragments, analogs and derivatives of such polypeptide.
The terns ~fragment," "derivative" and ~anaiog~ when
referring to the polypeptide of Figure 1 or that encoded by
the deposited cDNA, means a polypeptide which either retains
substantially the same biological function or activity as
such polypeptide, i.e_ functions as a G-protein chemokine
receptor, or retains the ability to bind the ligand or the
receptor even though_the polypeptide does not function as a
G-protein chemokine receptor, for example, a soluble form of
the receptor. An analog includes a proprotein~which can be
activated by cleavage of the proprotein portion to produce an
active mature polypeptide.
The polypeptide of the present invention may be a
recombinant polypeptide, a natural polypeptide or a synthetic
polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of the polyQeptide
of Figure 1 (SNQ ID N0:2) or that encoded by the deposited
cDI~ may be (i) one in which one -or more of the am;.ao acid
residues are substituted with a conserved or non-consezved
amino acid residue (preferably a conserved amino acid
residue)~and such substituted amino acid residue may or may
not be one encoded by the genetic code, or <ii) one in which
one or more of the amino acid residues includes a substituent
group, or (iii) one in which the mature polypeptide is fused
with another compound, such as a comcpound to increase the
half-life of the- polypeptide (for example, polyethylene
glycol), or (iv) one in which the additional amino acids are
fused to the mature polypeptide for purification of the
polypeptide or (v) one in which a fragment of tie polypeptide
is soluble, i.e. not membrane bound, yet still binds ligands
to the membrane bound receptor. Such fragments, derivatives
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and analogs are deeated to be within the scope of those
skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
The polypeptides of the present invention include the
polypeptide of SBQ ID N0:2 (in particular the mature
polypeptide) as well as polypeptides which have at least 70%
similarity (preferably a 70% identity) to the polypeptide of
SBQ ID N0:2 and more preferably a 90% similarity (more .
preferably a 90% identity) to the polypeptide of SBQ ID N0:2
and still more preferably a 95% similarity (still more
preferably a 90% identity) to the polypeptide of S$Q ID N0:2
and to portions of such polypeptide with such portion of the
poiypeptide generally containing at least 30 amino acids and
more preferably at least 50 amino acids.
As known in the art "similarity" between two
polypeptides is determined by comparing the amino acid
sequence and conserved amino acid substitutes thereto of the
polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present
invention may be employed for producing the corresponding
full-length polypeptide by peptide synthesis, therefore, the
fragments may be employed as intermediates for producing the
full-length polypeptides. Fragments or portions of the
polynucleotides of the present invention may be used to
synthesize full-length polynucleotides of the present
invention.
The tezm "geese" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding
and following the coding region "leader and trailer" as well
as intervening sequences (introns) between individual coding
segments (exons)_
The term "isolated° means that the material is removed
from its original environment (e. g., the natural environment
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if it is naturally occurring). Por example, a naturally-
occurring polynucleotide or polypeptide present in a living
animal is not isolated, but the same polynucleotide or
polypeptide, separated from s~aae or all of the coexisting
~aaterials is the natural system, is isolated. Such
polynucleotides could be part of a vector and/or such
polynucleotides or polypeptides could be part of a
co~o~position, and still be isolated in that such vector or
composition is not part of its natural environment.
The polypeptides of the present invention include the
polypeptide of SSQ ID N0:2 (in particular the mature
polypeptide) as well as polypeptides which have at least 70%
similarity (preferably at least 70% identity) to the
polypeptide of S8Q ID N0:2 and more preferably at least 90%
similarity (more preferably at least 90% identity) to the
polypeptide of S$Q ID N0:2 and still more preferably at least
95% similarity (still more preferably at least 95% identity)
to the polypeptide of SBQ ID N0:2 gad also iaciude portions
of such polypeptides with such portion of the polypeptide
generally containing at least 30 amino acids and more
preferably at least 50 amigo acids.
~ ~o~ ~ the art "similarity" between two
polypeptides is determined by comparing the amino acid
sequence and its conserved amino acid substitutes of one
polypeptide to the sequence of a second po3ypeptide.
Frac~nents or portions of the polypeptides of the present
invention may be employed for producing the corresponding
full-length polypeptide by peptide synthesis; therefore, the
fragments may be employed as intermediates for producing the
full-length polypeptides: Fragments or portions of the
polynucleotides of the present invention may be used to
synthesize full-length polynucleotides of the present
invention.
The present invention also relates to vectors which
include polyaucleotides of the present invention, host cells
CA 02562162 2006-10-20
which are genetically engineered with vectors 'of the
invention and the production of polypeptides of the invention
by recombinant techniques.
Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this
invention which may be, for example, a cloning vector or an
expression vector. The vector may be, for example, in the
form of a plasmid, a viral particle, a phage, etc. The
engineered host cells can be cultured in conventional
nutrient media modified as appropriate for activating.
promoters , selecting transfo~mants or amplifying the genes of
the present invention. The culture conditions, such as
temperature, pH and the like, are those previously used with
the host cell selected for expression, and will be apparent
to the ordinarily skilled artisan.
The polynucleotides of the present invention may be
employed for producing polypeptides by recombinant
tec~iques. Thus, for example, the polyaucleotide may be
included in nay one of a variety of expression vectors for
expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences, e.g.,
derivatives of Sv40; bacterial plasmids; phage DNA;
bacuZovirus; yeast plasmids; vectors derived from
combinations of plasmids and phage DNA, viral D1~ such as
vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used as long as it is
replicabie and viable in the host.
The appropriate DNA seguence may be inserted into~the
vector by a variety of procedures. In general, the DNA
sequence is inserted into an appropriate restriction
endonuclease sites) by procedures known in the art. Such
procedures and others are deemed to be within the scope of
those skilled in the art.
The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequences)
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(promoter) to direct mRNA synthesis. As representative
examples of such pramoters, there may be mentioned: LTR or
SV4Q promoter, the E. coli. lac or t~, the phage lambda P~
promoter and other promoters known to control expression of
genes in prokaryotic or eukaryotic cells or their vizuses.
The expression vector also contains a ribosome binding site
for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for
amplifying expression.
In addition, the expression vectors preferably contain
one or more selectable marker genes to provide a phenotypic
trait for selection of transformed host cells such as
dihydrofolate reductase or neomycin resistance for eukaryotic
cell culture, or such as tetracycline or ampicillin
resistance in B. coli.
The vector containing the appropriate DID sequence as
hereinabove described, as well as an appropriate promoter or
control sequence, may be employed to traasforar an appropriate
host to permit the host to express the protein.
As representative examples of appropriate hosts, there
may be mentioned: bacterial cells, such as E. coli,
StreDtonHrces, SalaaonelZa tvnhimurium; fungal cells, such as
yeast; insect cells such as Drosonhila and cetera
animal cells such as CEO, COS or Hooves melanoma; adenovirus;
plant cells, etc. The selection of an appropriate host is
deemed to.be within the scope of those skilled in the art
from the teachings herein.
More particularly, the present invention also includes
recombinant constructs comprising one or more of the
sequences as broadly described above. The constructs
co~ac~prise a vector, such as a plasmid or viral vector, into
which a sequence of the invention has been inserted, in a
forward or reverse orientation. In a preferred aspect of this
embodiment, the construct further comprises regulatory
sequences, including, for example, a promoter, operably
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CA 02562162 2006-10-20
linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
comseercially available. The following vectors are provided
by way of example. Bactei_al: pQ$70, pQB60, pQ$-9 (Qiagen),
pbs, pDlO, phagescript, psiXi74; pbluescript SIC, pbsks,
pl~8A, pNXl6a, pN818A, pNH46A (Stratagene); ptrc99a, pKR223-
3, pKR233-3, pDR540, pRITS (Pharmacia). Bukaryotic: pWLN80,
pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3; pBPV, pMSG,
pSVL (Pharmacia). However, any other plasmid or vector may
be used as long as they are replicable aad viable in the
host.
Promoter regions can be selected from aay desired gene
using CAT (chloramphenicol transferase) vectors or other
vectors with selectable markers. Two appropriate vectors are
PKK232-8 and PCM7. Particular named bacterial promoters
include lacI, lacZ, T3, T7, gpt, lambda PR, PL aad trp.
Bukaryotic promoters include CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus,
aad mouse metallothionein-I. Selection of the appropriate
vector and promoter is well within the level. of ordinary
skill in the art.
In a further embodiment, the_present invention relates
to host cells containing the above-described constructs. The
host cell can be a higher eukaxyotic cell, such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast
cell, or the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the construct into the host
cell can be effected by calcium phosphate transfection, DSAB-
Dextran mediated transfection, or electroporation. (Davis,
L., Dibner, M., Battey, I., Basic Methods in Molecular
Biology, (198x)).
The constructs in host cells can be used in a
conventional manner to produce the gene product encoded by
the recombinant sequence. Alternatively, the polypeptides of
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CA 02562162 2006-10-20
the invention can be synthetically produced by conventional
peptide synthesizers.
Mature proteins can be expressed in mammalian cells,
yeast, bacteria, or other cells under the control of
appropriate promoters. Cell-free translation systems can
also be employed to produce such proteins using RNAs derived
from ' the Did constructs of the present invention.
Appropriate cloning and expression vectors for use with
prokaryotic and eukaryotic hosts are described by Sambrook,
et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989).
Transcription -of the DIZA encoding the polypeptides of
the present invention by higher eukaryotes is increased by
inserting an enhancer sequence into the vector. Bnhancers
are cis-acting elements of DID,, usually about from 10 to 300
by that act on apromoter to increase its~transcription.
Examples including the SV40 eahancer on the late side of the
replication origin by 100 to 270, a cytoa2egalovirus early
promoter eahancer, the polyoma exl~ancer on the late side of
the. replication origin, and adeziovirus enbancers.
Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin
resistance gene of E. coli and S. cerevisiae T1ZP3 gene, and
a promoter derived from a highly-expressed gene to direct
transcription of a downstream structural sequence. Such
promoters can be derived from operons encoding glycolytic
enzymes such as 3-phosphoglycerate kinase (PGK), a-factor,
acid phosphatase, or heat shock proteins, among others. The
heterologous structural sequence is assembled in appropriate
phase with translation initiation and ter~aination sequences,
and preferably, a leader sequence capable of directing
secretion of translated protein into the periplasmic space or
extracellular meditaa. Optionally, the heterologous sequence
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CA 02562162 2006-10-20
can encode a fusion protein including an N-terminal
identification peptide imparting desired characteristics,
e.g., stabilization~or simplified purification of expressed
. recombinant product.
Useful expression vectors for bacterial use are
constxucted by inserting a structural DNA seguence encoding
a desired protein together With suitable translation
initiation and tezmination signals in operable reading phase
with a functional promoter. The vector will.comprise one or
more phenotypic selectable markers and an origin of
replication to ensure maintenance of the vector and to, if
desirable, provide amplification within the host. Suitable
prokaryotic hosts' for transformation include B. coli,
Bacillus subtilis, Salmonella tvahimurium and various species
within the ~ genera Pseudomoaas, Streptomyces, and
.Staphylococcus, although others may also be employed as a~
matter of choice. ~ '
As a representative but noalimiting example, useful
expression vectors for bacterial use can comprise a
selectable marker and bacterial origin~of replication derived
from comsterczally available plasmids cocaprising genetic
elements of the well known cloning vector pBR322 (ATCC
37017). Such c~ercial vectors include, for eacample,
pKK223-3 (Pbar<nacia Fine Chemicals, Uppsala, Sweden) and G8M1
(Promega Biotec, Madison, WI, tTSA). These pBR322 "backbone"
sections are combined with an appropriate promoter and the
structural sequence to be expressed.
Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e. g.,
temperature shift or chemical induction) and cells are
cultured for an additional period.
Cells are typically harvested by centrifugation,
diszupted by physical or chemical means, and the resulting
crude extract retained for further purification.
_18_
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Microbial cells employed in expression of proteins can
be disrupted by any convenient method, including freeze-thaw
cycling, sonication, mechanical disruption, or use of cell
lysing agents, such methods are well know to those skilled in
the art.
various mammalian cell culture systems can also be
employed to express recombinant protein. fixamples of
mammalian expression systems include the COS-7 lines of
monkey kidney fibroblasts, described by Gluzman, Cell, 23:175
(1981), and other cell lines capable of expressing a
compatible vector, for example, the C12?, 3T3, C80, Fieha and
BHK cell lines. Mammalian expression vectors will coa~rise
an origin of replication, a suitable promoter and eahancer,
and also any necessary ribosa~ne binding sites,
polyadeaylation site, splice donor and acceptor sites,
transcriptional termination segueaces, and 5' flanking
nontranscribed sequences. D1~1 sequences derived from the
SV40 splice, and polyadeaylation sites may be used to provide
the required nontranscribed genetic elements.
The G-protein che~xaokine receptor polypeptides can be
recovered and purified from recombinant cell cultures by
methods including ammonium sulfate or ethanol precipitation,
acid extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Protein refolding
steps can be used, as necessary, in completing configuration
of the mature protein. Finally, high performance liquid
chromatography (HPLC) can be employed for final purification
steps.
The polypeptides of the present invention may be a
naturally purified product, or a product of chemical
synthetic procedures, or produced by recombinant techniques
from a prokaryotic or eukaryotic host (for example, by
bacterial, yeast, higher plant, insect and mammalian cells in
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culture). Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present
invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial
methionine amino acid residue.
The polynucleotides and polypeptides of the present
invention may be employed as research reagents and materials
for discovery of treatments and diagnostics to human disease.
The G-protein chemokine receptors of t3ie present
im~ention may be employed in a process for screening for
compounds which activate (agonists) or inhibit activation
(antagonists) of the receptor polypeptide of the present
invention .
In general, such screening procedures involve providing
appropriate cells which express the receptor polypeptide of
the present invention on the surface thereof. Such cells
include cells from mammals, yeast, drosophila or E. Cola. In
particular, a polynucleotide encoding the receptor of the
present invention is employed to transfect cells to thereby
express the G-protein chemokiae receptor. The expressed
receptor is then contacted with a test compound to obse3cve
binding, stimulation or inhibition of a functional response.
One such screening procedure involves the use of
~aelanophores which are traasfected to express the G-protein
chemokine receptor of the present invention. Such a
screening technique is described in PCT WO 92 / 01810 published
February 6, 1992.
Thus, for example, such assay may be employed for
screening for a compound which inhibits activation of the
receptor polypeptide of the present invention by contacting
the melanophore cells which encode the receptor with both the
receptor ligand and a compound to be screened. Inhibition of
the signal generated by the ligand indicates that a compound
is a potential antagonist for the receptor, i.e., inhibits
activation of the receptor.
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The screen may be employed for determining a compound
which activates the receptor by contacting such cells with
compounds to be screened and determining whether such
compound generates a signal, i.e., activates the receptor.
Other screening techniques include the use of cells
which express the G-protein chemokine receptor (for example,
transfected CHO cells) in a system which measures
extracellular pH changes caused by receptor activation, for
example, as described in Science, volume 246, pages 181-296
(October 1989). For example, compounds may be contacted with
a cell which expresses the receptor polypeptide of the
present imrention and a second messenger response, e.g.-
signal transduction or pH changes, may be measured to
determine whether the potential compound activates or
inhibits the receptor.
Another such screening technique involves introducing
RtsA encoding the G-protein chemokiae receptor into 8enopus
oocytes to transiently express the receptor.. The receptor
oocytes may then be contacted with the receptor ligand and a
cooa~pound to be screened, followed by detection of inhibition
or activation of a calcium signal in the case of screening
for caa~pounds which are thought to inhibit activation of the
receptor.
Another screening technique involves expressing the G-
protein chemokine receptor in which the receptor is linked to
a phospholipase C or D. As representative examples of such
cells, there may be mentioned endothelial cells, smooth
muscle cells, embzyonic kidney cells, etc. The screening may
be accomplished as hereinabove described by detecting
activation of the receptor or inhibition of activation of the
receptor from the phospholipase second signal.
Another method involves screening for compounds which
inhibit activation of the receptor polypeptide of the present
invention antagonists by determining inhibition binding of
labeled ligand to cells which have the receptor on the
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surface thereof. Such a method involves transfecting .a
eukaryotic cell with DNA encoding the G-protein chemokine
receptor such that the cell expresses the receptor on its
surface and contacting the cell with a compound in the
presence of a labeled form of a known ligand. The ligand can
be labeled, e.g., by radioactivity. The amount of labeled
ligand bound to the receptors is measured, e.g., by measuring
radioactivity of the receptors. If the coarpound binds to the
receptor as determined by a reduction of labeled ligand which
binds to the receptors, the binding of labeled ligand to the
receptor is inhibited.
An antibody may antagonize a G-protein chemokine
receptor of the present invention, or in so~e cases an
oligopeptide, which bind to the G-protein chemokiae receptor
but does not elicit a second messenger response such that the
activity of the G-protein chemokine receptors is prevented.
Antibodies include anti-idiotypic antibodies which recognize
unique determinants generally associated with the antigen-
binding site of as antibody. Potential antagonist compounds
also include proteins which are closely related- to the Iigaad
of the G-protein chemokine receptors, i.e. a fragment of the
ligand, which have lost biological function and when binding
to the G-protein chemokine receptor elicit no response.
An aatisease construct prepared through the use of
aatisense technology, may be used to control gene expression
through triple-helix formation or aatisense DNA or RNA, both
of which methods are based on binding of a polynucleotide to
DTiA or RIB. For example, the 5' coding portion of the
polynucleotide sequence, which encodes for the mature
polypeptides of the present invention, is used to design an
aatisease RNA oligoaucleotide of frost about 1f to 40 base
pairs in length. A DNA oligonucleotide is designed to be
coanplemeatary to a region of the gene involved in
transcription (triple helix -see Lee et al., Nucl. Acids
Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988):
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CA 02562162 2006-10-20
and Dervan et al., Science, 251: 1360 (1991)), thereby
preventing transcription and the production of G-protein
chemokine receptor. The antisense RNA oligonucleotide
hybridizes to the ~aaRNA in vivo and blocks translation of mRNA
molecules into G-protein coupled receptor (antisense - Okano,
J. Neurochem.. 56:560 (1991); Oligodeoxynucleotides as
Antisense Inhibitors of Gene Expression, CRC Press, Boca
Baton, FI: (1988)). The oligonucleotides described above can
also be delivered to cells such that the antisense RNA or DNA
may be expressed ~En vivo to inhibit production of G-protein
chemokine receptor.
A small molecule which binds to the G-protein chemokine
receptor, making it inaccessible to ligands such that nozmal
biological activity is prevented, for example small peptides
or peptide-like molecules, may also be used to inhibit
activation of the receptor polypeptide of the present
invention.
A soluble f ozln of the G-protein chemokiae receptor, a . g .
a fragment of the receptors, may be used to inhibit
activation of the receptor by binding to the ligand to a
polypeptide of the present invention and preventing the
ligand from interacting with membrane bound G-protein
chemokiae receptors.
The com~pouads which bind to and activate the G-protein
chempkine receptors of the present invention may be employed
to stite haematopoiesis, wound healing, coagulation,
aagiogenesis, to treat solid tumors, chronic infections,
leukemia, T-cell mediated auto-im~awae diseases, parasitic
affections, psoriasis, and to stimulate growth factor
activity.
The compounds which bind to and inhibit the G-protein
chemokine receptors of the present invention may be employed
to treat allergy, atherogenesis, anaphylaxis, malignancy,
chronic and acute inflammation, histamine and Ig$-mediated
allergic reactions, prostaglandin-independent fever, bone
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marrow failure, silicosis, sarcoidosis, rheumatoid arthritis
shock and hyper-eosinophilic syndrome.
The compounds may be employed in combination with a
suitable pharmaceutical carrier. Such'compositions comprise
a therapeutically effective amount of the compound and a
pharmaceutically acceptable carrier or excipient. Such a
carrier includes but is not limited to saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations
thereof. The formulation should ~ suit the mode of
administration.
The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of
the ingredients of. the pharmaceutical compositions of the
invention. Associated with such containers) can be a notice
in the fozm prescribed by a governmental agency regulating
the manufacture, use or sale. of pharmaceuticals or biological
products, which not~.ce reflects approval by the agency of
_asanufacture, use or sale for human administration_ In
additio'~n, the co~apouads. of the present invention may be
employed in conjunction with other therapeutic coxapounds.
The pharmaceutical ,cocapositions may be ac~ainistered in
a convenient manner such as by the topical, intravenous,
iatraperitoaeal, iatramuseular, subcutaneous, iatraaasal or
3atradermal (applicable) routes. The pharmaceutical
compositions are administered in as amount which is effective
for treating and/or prophylaxis of the specific indication.
In general, the phaanaceutical compositions will be
administered in an amount of at least about ZO ~eg/kg body
weight and in nmost cases they will be administered in an
amount not in excess of about 8 mg/Kg body weight per day.
In most cases, the dosage is from about 10 ,ug/kg to about 1
mg/kg body weight daily, taking into account the routes of
adnttnistratioa, symptoms, etc.,
The G-protein chemokine receptor polypeptides and
antagonists or agonists which are polypeptides, may also be
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employed in accordance with the present invention by
expression of such polypeptides in vivo, which is often
referred to as ~gene therapy.~
Thus, for example, cells from a patient may be
engineered with a polyaucleotide (DNA or RNA) encoding a
polypeptide ex vivo, with the engineered cells then being
provided to a patient to be treated with the polypeptide.
Such methods are well-known in the art. For example, cells
may be engineered by procedures known in the art by use of a
retroviral particle containing RNA encoding a polypeptide of
the present invention.
Similarly, cells may be engineered in vivo for
expression of a polypeptide in vivo by, for example,
procedures known in the art. As known in the art, a producer
cell for producing a retroviral particle containing RNA
encoding the polypeptide of the present invention may be
administered to a patient for engineering cells in vivo and
expression of the polypeptide is vivo. These and other
methods for administering a polypeptide of the present
invention by such method should be apparent to those skilled
in the art from the teachings of the preseat invention. For
example, the expression vehicle for engineering cells may be
other than a retrovirus, for example, an adenovirus which may
be used to engineer cells in vivo after combination with a
suitable delivery vehicle.
R,etraviruses frown which the retroviral plasmid vectors
hereinabove mentioned may be derived include, but are not
limited to, Moloaey Marine Leukemia Vizus, spleen necrosis
vizus, retroviruses such as Rous Sarcoma Virus, Harvey
Sarcoma Virus, avian leukosis vines, gibbon age leukemia
virus, human immunodeficiency virus, adenovirus;
Myeloproliferative Sarcoma Virus, and mammary tumor virus.
In one embodiment, the retroviral plasmid vector is derived
from Moloney Marine Leukemia Virus.
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The vector includes one or more promoters. Suitable
promoters which may be employed include, but are not limited
to, the retroviral LTR; the SV40 promoter; and the human
cytomegalovirus (HIV) promoter described in Miller, et al.,
$~otechnioues, Vol. 7, No. 9, 980-990 (1989) , or any other
promoter (e. g., cellular promoters such as eukaryotic
cellular promoters including, but not limited to, the
histone, pol III, and ~-actin promoters). Other viral
promoters which may be employed include, but are not limited
to, adenovirus promoters, thymidine kinase (TK) promoters,
and B19 parvovirus promoters. The selection of a suitable
promoter will be apparent to those skilled in the art from
the teachings contained herein.
The nucleic acid sequence encoding the polypeptide of
the present invention is under the control of a. suitable
promoter. Suitable, prou~ot~rs which may be employed include,
but are not limited to, adeaoviral promoters, such as the
adenovirai major late proafoter; or hetorologous promoters,
such as the cytomegalovirus (City) propaoter; the respiratory
syncytial virus (RSV) praanoter; inducibie~promoters, such as
the MMT promoter, the metallothionein proiaoter; heat. shock
promoters; the alhumin promoter; the ApoAI promoter; human
globin promoters; viral thymidine kinase promoters, such as
the Herpes Simplex thyad.dine kinase promoter; retroviral LTRs
(including the modified retroviral LTRs hereinabove
described).; the ~B-actin promoter; and human growth hormone
promoters . The proutoter also ~ may be the native proamoter
which controls the genes encoding the polypeptides.
The retroviral plas=aid vector is employed to transduce
packaging cell lines to foza~ producer cell lines. Bx,amples
of packaging cells which may be transfected include, but are
not limited to, the PSSOl, PA337, ~-2, ~-AM, PA12, T19-14X,
VT-39-I7-Fi2, s~CRB, ~C'RIP, GP+E-.86, GP+eavAml2, and DAN cell
lines as described in Miller, Human Gene Theratw, vol. 1,
pgs. 5-14 (1990) .
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The vector may transduce the packaging
cells through any means laiown in the art. Such means
include, but are not limited to, electroporation; the use of
liposomes, and CaP04 precipitation. In one alternative, the
retroviral 'plasmz.d vector may be encapsulated into a
liposome, or coupled to a lipid, and then administered to a
host.
The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequences)
encoding the polypeptides. Such retroviral vector particles
then may be employed, to transduce eukaryotic cells, either
za vitro or in vivo. The transduced eukaryotic cells will
express the nucleic acid sequences) encoding the
polypeptide. 8ukaryotic cells which may be transduced
include, but are not limited to, embryoaic stem cells,
embryonic carcinoma . cells, as well as hema_topo~:etic. stem
. cells, hepatocytes, fibroblasts, myoblasts, keratinocytes,
endothelial cells, aad bronchial epithelial cells.
The present invention also provides a method for
determining whether a ligand not kaown to be capable of
binding to a G-protein cheruokine receptor can bind to such
receptor which comprises contacting a mammalian cell which
expresses a, G-protein chemokine receptor-,with the ligand
under conditions percaitting binding of ligands to . the G-
protein chemokine receptor, detecting the presence of .a
ligand which binds to the receptor and thereby determining
whether the ligand binds to the G-protein chemokine receptor.
The systems hereinabove described-for determining agonists
and/or antagonists may also 'be employed for determining
ligaads which bind to the receptor.
This invention also provides a method of detecting
expression of a G-protein chemokine receptor polypeptide of
the present invention on the surface of a cell by detecting
the presence of mRNA coding for the receptor which comprises
obtaining total mRHA from the cell and contacting the mRNA so
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CA 02562162 2006-10-20
obtained with a nucleic acid probe comprising a nucleic acid
molecule of at least 10 nucleotides capable of specifically
hybridizing with a sequence included within the sequence of
a nucleic acid molecule encoding the receptor under
hybridizing conditions, detecting the presence of mRNA
hybridized to the probe, and thereby detecting the expression
of the receptor by the cell.
The present invention also provides a method for
identifying receptors related to the receptor polypeptides of
the present invention. . These related receptors .rnay be
identif led by hortnology to a G-protei a chemokine receptor
polypeptide of the present invention,-by low stringency cross
hybridization, or by identifying receptors that interact with
related natural or synthetic ligands and or elicit similar
behaviors after genetic or pharmacological blockade of the
chemokine receptor.polypeptides of the present invention.
Fragments of the genes may, be ~ used as a hybridization
probe for a cDi~.1 library to isolate other genes which have a
high sequence similarity to the genes of- the present
invention, or which have similar biological activity. Probes
of this type are at least 20 bases, preferably at least. 3.0
bases and angst preferably at least 50 bases or more. The
probe may also be used to identify a cDI~1 clone corresponding
to a full length'transcript and a genomic clone or clones
that contain the caa~plete gene of the present invention
including .regulatory and promoter regions, exons and introas .
An example of a screen of tb.is type comprises isolating the
coding region of the gene by using the known DNA sequence to
syathesize~ an oligonucleotide probe. Labeled
oligonucleotides having a sequence complementary to that of
the. genes of the present invention are used to screen a
library of human cDNA, genosaic DNA or mRNA to deter~ni.ae which
members of the library the probe hybridizes to.
The present invention also contecuplates, the use of the
genes of the present invention as a diagnostic, for example,
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CA 02562162 2006-10-20
score diseases result from inherited defective genes. .These
genes can be detected by cooaparing the sequences of the
defective gene with that of a normal one. Subsequently, one
can verify that a "mutant" gene is associated with abnormal
receptor activity. In addition, one can insert mutant
receptor genes into a suitable vector for expression in a
functional assay system (e. g., colorimetric assay, expression
on MacConkey plates, c~rplementation experiments, in a
receptor deficient strain of ~IC293 cells) as yet another
means to verify or identify mutations. Once "mutant" genes
have been identified, one can they screen population for
carriers of the "mutant" receptor gene.
Individuals carrying mutations in the gene of the
present invention may be detected at the DNA level by a
variety of techaigues. Nucleic acids used for diagnosis may
be obtained froaa a patient' s cells, including but not limited
to such as from blood, urine, saliva, tissue biopsy and
autopsy material_ The genomic DNA may be used directly for
detection or may be amplified enzymatically by using PCR
(Saiki, et al., N~.tu~, 324:163-166 1986) prior to analysis.
RNA or cDNA may also be used for the same purpose. As an
example, PCR primers como~plimentazy to the nucleic acid of the
instant invention can be used to identify and analyze
mutations in the gene of the present invention. For example,
deletions and insertions can be detected by a change in size
of the amplified product in comparison to the normal
genotype. Point mutations can be identified by hybridizing
amplified DNA to. radio labeled RNA of the invention or
alternatively, radio labeled aatisense DNA sequences of the
invention. Perfectly matched sequences can be distinguished
from mismatched duplexes by RNase A digestion or by
differences in melting te~peratures. Such a diagnostic would
be particularly useful for prenatal or eves neonatal testing.
Sequence differences between the reference gene and
"mutants" may be revealed by the direct DNA seqeiencing
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CA 02562162 2006-10-20
method. in addition, cloned DNA segments may be used as
probes to detect specific DNA segments. The sensitivity of
this method is greatly enhanced when combined with PCR. For
exa~aa~ple, a sequence primer is used with double stranded PCR
product or a single stranded template molecule generated by
a modified PCR. The sequence determination is performed by
conventional procedures with radio labeled nucleotide or h:
an autoioaatic sequencing procedure With fluorescent-tags.
Genetic testing based on DNA sequence differences may be
achieved by detection of alterations in the electrophoretic
mobility of DNA fragments in gels with or without denaturing
agents. Sequences changes at specific locations may also be
revealed by nucleus protection assays, such RNase and S1
protection or the chemical cleavage method (e.g. Cotton, et
al. , PI~~~g. USA, 85:4397-4401 1985) .
In addition, sane diseases are a result of, or are
characterized by changes in gene expression which can be
detected by changes in the mRNA. Alternatively, the genes of
the present invention can be used as a reference to identify
individuals expressing a decrease of functions associated
with receptors of this type.
The present invention also relates to a diagnostic assay
for detecting altered levels of soluble forms of the G-proem
chemokiae receptor polypeptides of the present invention in
various tissues. Assays used to detect levels of the soluble
receptor polypeptides in a sample derived from a host are
well kaoarn to those of skill in the art and include
radioimmvaoassays, competitive-binding assays, Western blot
analysis and preferably as BLISA assay.
An BLISA assay initially coa~rises preparing an antibody
specific to antigens of the G-protein chemokine receptor
polypeptides, preferably a monoclonal antibody. In addition
a reporter antibody is prepared against the monoclonal
antibody. To the reporter antibody is attached a detectable
reagent such as radioactivity, fluorescence or in this
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CA 02562162 2006-10-20
example a horseradish peroxidase enzycae. A sample is now
removed from a host and incubated on a solid support, e.g. a
polystyrene dish, that binds the proteins in the sample. Any
free protein binding sites on the dish are then covered by
incubating with a non-specific protein such as bovine serum
albumin. Next, the monoclonal antibody is incubated in the
dish during which time the monoclonal antibodies attach to
any G-protein chemokine receptor proteins attached to the
polystyrene dish. All unbound monoclonal antibody'is washed
ou~ with buffer. The reporter antibody linked to horseradish
peroxidase is now placed in the dish resulting in binding of
the reporter antibody to any monoclonal antibody bound to G-
protein chemokine receptor proteins. Unattached reporter
antibody is then washed out. Peroxidase substrates are then
added to the dish and the amount of color developed in a
given time period is a measurement of the amount of G-protein
chec~okine receptor proteins present in a given volume of
patient sample when cort~ared against a standard curve.
The seguences of the present invention are also valuable
for chroancso~ne identification. The sequence is specifically
targeted to and can hybridize with a particular location on
as individual human chromoso~ane.. Moaceover, there is a current
need for identifying particular sites on the chromosome . Few
chro~some marking reagents based on actual sequence data
(repeat polymo~cphisms) are presently available for marking
chro~nosoma,,l location. The mapping of DNAs to chromosomes
according to the present invention is an important f first step
in corzelating those sequences with genes associated with
disease.
Briefly, sequences can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp) from' the cDNA.
Computer analysis of the cDNA is used to rapidly select
primers that do not span more than one axon in the genomic
DNA, thus complicating the amplification process. These
primers are then used for PCR screening of somatic cell
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CA 02562162 2006-10-20
hybrids containing iadividtial human chro~awsomes. Oniy.those
hybrids containing the human gene corresponding to the primer
will yield an amplified fragaaent.
PCR mapping of somatic cell hybrids is a rapid procedure
for assigning a particular DNA to a particular chromosome.
Using the present invention with the same oligonucleotide
primers, sublocalization can be achieved with panels of
fragments from specific chromosomes or pools of large geaomic
clones in an analogous manner. Other mapping strategies that
can similarly be used to map to its chromosome include in
s~ttu hybridization, prescreening With labeled flow-sorted
chromosomes and preselection by hybridization to construct
chromaosome specific-cDl~ libraries.
Fluorescence ~fn situ hybridization (FISH) of a cDN~I
clone to a metaphase chromosomal spread can be used to
provide a precise chromosomal location in one step. This
technique can be used with cDNA as short as 50 or 60 bases.
For a review of this technique, see verma et al., Human
Chromosomes: a Manual of Basic Techniques, Pergamon Press,
New York ( 1988 ) .
Once a sequence has been mapped to a precise chromosomal
location, the physical position, of the sequence on the
chroaaosome can be correlated with genetic map data. Such
data are found, for example, in v. McRusick, Meadelian
Inheritance in Man (available on line through Johns Hopkins
University Welch Medical Library). The relationship between
genes and diseases that have bees mapped to the same
chromosomal region are they identified through linkage
analysis (coinheritarice of physically adjacent genes).
Next, it is necessary to determine the differences in
the cDNA or genomic sequence between affected and unaffected
individuals . If a mutation is observed in sa~ae or all of the
affected individuals but not in any normal individuals, then
the mutation is likely to be the causative agent of the
disease.
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With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a
chromosomal region associated with the disease could be one
of between 50 and 500 potential causative genes. (This
assumes z megabase mapping resolution and one gene per 20
kb) .
The polypeptides, their fragments or other derivatives,
or analogs thereof, or cells expressing them can be used as
an im~nuaogen to produce antibodies thereto. These 'antibodies
can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chimeric, single chain,
and humanized antibodies, as well as Fab fragments, or the
product of an Fab expression library. Various procedures
known in the art may be used for the production of such
antibodies and fragments.
Antibodies generated against the polypeptides
corresponding to a sequence of the present invention can be~
obtained by direct injection of the polypeptides into an
animal or by ad~ainisterinc the polvpeptides to an animal,
preferably a nonhuman . The antibody so obtained will then
bind the polypeptides itself. In this manner, even a
sequence encoding only a fragment,of the polypeptides can be
used to generate antibodies binding the whole native
polypeptides. Such antibodies can then be used to isolate
the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line
cultures can be used. Examples include the hybridoma
technique (Kohler and Milstein, 1975, Nature, 256:495-497),
the trioma technique, the human E-cell hybridama. technique
(Rozbor et al : , 1983 , Imaaunology Today 4 : 72 ) , and the BBV-
hybridoma technique to produce human monoclonal antibodies
(Cole, et al., 1985, in Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96).
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CA 02562162 2006-10-20
Techniques described for the production of single chain
antibodies tll.S. Patent 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products
of this invention. Also, tram~Anic mice may be used to
express humanized antibodies to imvanunogenic polypeptide
products of this imrention.
The present i~m~ention will be further described with
reference to the following examples; however, it is to be
understood that the present invention is not limited to such
examples. All parts or amounts, unless otherwise specified,
are by weight.
In order to facilitate understanding of the following
examples certain freguently occurring methods and/or terms
will be described.
"Plasmids~ are designated by a lower case p preceded
aad/or followed by capital letters and/or numbers. The
starting plasmids herein are either co~mmnercially available,
publicly available on an unrestricted. basis, or can be
constructed from available plasmids in accord with published
procedures. In addition, equivalent plasmids to those
described are known in the art and will be apparent to the
ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the
DNA with a restriction enzyme that acts only at certain
sequences in the DNA. The various restriction enzymes used
herein are commercially available and their reaction
conditions, cofactors and other requirements were used as
would be known to the ordinarily skilled artisan. For
analytical purposes, typically 1 ~cg of plasmid or DNA
fragment is used with about 2 units,of enzyme in about 20 ~cl
of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~cg of
DNA are digested with 20 to 250 units of enzyme in a larger
volume. Appropriate buffers and substrate amounts for
particular restriction enzyr~aes are specified by the
-34-
CA 02562162 2006-10-20
manufacturer . Incubation times of about 1 hour at 3 7~' C are
ordinarily used, but may vary in accordance with the
supplier s instructions. After digestion the reaction is
electrophoresed directly on a polyacrylamide gel to isolate
the desired fragment.
Size separation of the cleaved fragments is performed
using 8 percent polyacrylamide geI described by Goeddel, D.
et al., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be che~aically synthesized. Such synthetic
oligonucieotides have no 5' phosphate and thus will not
ligate to another oligonucleotide without adding a phosphate
with an ATP is the presence of a kinase. A synthetic
oligonucleotide will ligate to a fragment that has not been
dephosphorylated.
"Ligation" refers to the process of forming
phosphodiester bonds between two double stranded nucleic acid
fragments (Maniatis, T., et al., Id., p. 146). Unless
otherwise provided, ligation may be accomaplished using known
buffers and conditions with 10 units to T4 DNA lipase
("lipase") per 0.5 ycg of approximately equimolar amounts of
the DNA fragments to be ligated.
Unless otherwise stated, transformation was performed as
described in the method of Graham, F. and Van der Bb, A.,
Virology,.52:456-457 (1973).
Exa~le 1
Bacterial Bxflression and Purification of HDGNR10
The DNA sequence encoding for 5Z~R10, ATCC # _ is
initially amplified using PCR oligonucleotide primers
corresponding to theca 5' and sequences of the processed
~RlO protein (minus the signal peptide sequence) and the
vector sequences 3' to the F~GNR10 geese. Additional
r
nucleotides corresponding to FmGYRIO were added to the 5' and
3' sequences respectively. The 5' oligonucleotide primer has
-35-
CA 02562162 2006-10-20
the sequence S' CGGAATTCCTCCATGGATTATCAAGTGTCA 3' contains an
BcoRI restriction enzyme site followed by 18 nucleotides of
H~N'RIO coding sequence starting from the presumed terminal
amino acid of the processed protein codon. The 3' sequence
5~' CGGAAGCTTCGTCACAAGCCCACAGATAT 3' contains complementary
sequences to ~ HindIII site and-is followed by z8 nucleotides
of F~GNR10 coding sequence. The restriction enzyme sites
correspond to the restr~.ctioa enzyme sites on the bacterial
expression vector pQE-9 .(Qiagen, inc. 9259 Eton Avenue,
Chatsworth, CA, 91311). pQB-9 encodes antibiotic resistance
(A~up') , a bacterial origin of replication (ori) , as IPTG
regulatable promoter operator (P/0), a ribosome~binding site
(RBS), a 6-His tag_and restriction enzyme sites. pQE-9 Gras
then digested- with EcoRI and HindIII. The amplified
t"
sequences were ligated into pQE-9 and were inserted in frame
with the sequence encoding.for the histidine tag and the
RBS. The ligation mixture was then used to transforiu E. coli
strain M15/rep 4 (Qiagen, Inc.) by the procedure described zn
Saiabrook, J. et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Laboratory Press, (3989). M15/rep4 contains
paultiple copies of~the plasmid pRFP4, which expresses the
lacl repressor and also confers kanaa~ycin resistance (Xan°) .
Tzansfortaants are identified by their ability to grow on L8
plates and an~picillin/kanamycin resistant colonies were
selected. Plasaaid , DNA was isolated and confizzaed by
restriction analysis. - Clones containing the desired
constnzcts were grown overnight (0/N) in liquid culture in Ia8
media supple~oaented with both Amp (Z00 ug/zal) and Kan (25
ug/ZN.). The O/N culture is used to inoculate a large culture
at a ratio of 1.100 to- 1:250. The cells were grown tq an
optical density 600 (0.D.°°°) of between 0.4 and 0.6.
IPTG
("Isopropyl-B-D-thiogalacto pyranoside") was then added to a
final concentration of 1 mM. IPTG induces by inactivating
the lacI repressor, clearing. the ?/0 leading to increased
gene expression. Cells were grown an extra 3 to 4 hours .
-36=
CA 02562162 2006-10-20
Cells were then harvested by centrifugation. The cell pellet
was solubilized in the chaotropic agent 6 Molar Guanidine
HCi. After clarification, solubilized HDGNR10 was purified
from this solution by chromatography on a Nickel-Chelate~
column under conditions that allow for tight binding by
groteins containing.the 6-His tag. Hochuli, E. et al., J.
Chromatography x11:17?-I84 (198x). HDGNR10 was eluted from
the column in 6 molar guanidine HC1 pH 5.0 and for the
purpose of renaturation adjusted to 3 molar guanidine KC1,
lO.OmM.sodium phosphate, 10 mmolar glutathione (reduced) and
2 mmolar glutathione ~ (oxidized) . After incubation iri this
solution for 12 hours the protein was dialyzed to 10 mmolar
sodium phosphate.
example 2
The expression .of plasrnid, HDGNR10 Iii is :derived front a
vector pcDNAI/AmpM(Invitrogen) containing: 1) ~SV40 origin of
replication, 2) ampicillin resistance gene, 3) E_coli.
replication origin, a) CMV promoter followed by a polylinker
region, a SVaO intron and polyadenylation site. A DNA
fragment encoding the entire HDGNR10 precursor and a HA tag
fused=in frame to its 3~ end was cloned into the polylinker
region of the vector, therefore, the recombinant protein
expression is directed under the CMV promoter. The HA tag
correspond to an epitope derived from the influenza
hemagglutinin protein as previously described (I. Wilson, H.
.Niman, R. I~eighten,. A Cherenson, M. Connolly; and R. Lerner,
198x, Cell 37, 7~7) . The infusion of HA tag to the target
protein allows easy detection of the recombinant protein with
an antibody that recognizes the HA epitope.
The plasmid construction, strategy is described as
follows:
The DNA sequence encoding for HDGNR10, ATCC No. 97L83, was
constructed by PCFt using two primers: the S~ primer 5' GTCC
3T
CA 02562162 2006-10-20
AAGCTTGCCACCATGGATTATCAAGTGTCA 3' and contains a HindIIT sit~-
followed by 18 nucleotides of HDGNR10 coding sequence
starting from the initiation colon; the 3' sequence 5'
CTAGCTCGAGTCAAGCGTAGTt:TGGGACGTC~TATGGGTAGCACA.AGCCCACAGATATTTC
3' contains complementary ' sequences to an XhoI site,
translation stop colon, HA tag and the last l8 nucleotides of
the F~GNR10 coding sequence (not including the stop.codon).
Therefore, the PCR product contains a HindIII site F~GNR10
coding sequence followed by. HA tag fused in frame, a
translation termination stop colon next to the HA tag, and an
XhoI.site. The PCR amplified DNA fragment and the vector,
pcDl~tAI/Amp, were digested with HindIII and Xhol restriction
enzyme and ligated. The ligation mixture was transformed
into E. coli strain SURE (available from Stratagene Cloning'
Systems, 11099 North Torrey Pines Road, La Jolla, CA 92437)
the transformed culture was - plated on atapicillin media
plates and resistant ~ colonies were selected. Plasraid DNA was
isolated from transfozmants and PYam~ned by restriction
analysis for the presence of the correct fragment. For
expression of the recoiabinant ~GNR~.O, COS cells were
transfected 'with the expression vector by DBAE-DEXTRAN
method. (J. Sambrook, F. Pritsch, T. Maniatis, Molecular
Cloning: A Laboratory Manual, Cold Spring Laboratory press,
(3989)). The expression of the HDGNR10 HA protein was
detected by radiolabelling and iman:noprecipitation method.
(8. Harlow, D. Lane, Antibodies:. A Laboratory Manual, Cold
Spring Harbor Laboratory Press, (i988)). Cells were labelled
for 8 hours with '~S-cysteine two days past transfection.
Culture media were then collected and cells were lysed with
detergent (RIPA buffer (150 :nM NaCI, 1~ NP-40, 0.1~ SDS, i~~
NP-40, 0.5$ DOC, 50mM Tris, pH 7.5). (Wilson, I. et al " Id.
37:767 (1984)x. Both cell lysate and culture media were
precipitated with a EA specific monoclonal antibody:
Proteins precipitated were analyzed on 15~c SDS-PAGE gels.
-38-
- CA 02562162 2006-10-20
F.~s.~~.~tl a 3
n
th v'
g~grP~sion svstem
The DNA, sequence encoding the full length HDGNR10
protein, ATCC No. 97183, 'was amplified using PCR
oligonucleotide primers corresponding to the 5' .and 3'
sequences of the gene : '
The S' primer has the sequence 5' CGGGATCCCTCCATGGATTAT
CAAGTGTCA 3' and contains a BamHI restriction enzyme. site
followed by 4 nucleotides resembling-anlefficient signal for
the initiation of translation in eukaryotic cells (J. Mol.
Biol. 1987,-196, 9Q7-950, Kozak, M.), and just behind the
first 18 nucleotides of the HDGNR10 gene (the initiation
codon for translation is "ATG"). '
The 3' primer has the sequence 5' CGGGATCCCGCT
CACAAGCCCACAGATAT 3' and container the cleavage site for the
restriction endonuclease BamHI and- 18nucleotides
complementary- to the 3' non-translated sequence of the
HDGNRIO gene. The amplified 'sequences were isolated from a
I% agarose gel using a commercially available kit
( "Geneclean, " BIO 10I Inc . , La -Jolla, Ca . ) . The fragment was
then digested with the endonuclease BarnFII -and purified as
described above. This fragment is designated F2.
The vector .pRGl (modification. of pvL9~1 'vector,
discussed below) is used for the expression of the HDGNR10
protein using the baculovirus expression system (for revie~r
see: Summers, M.D, and Smith, G.E. 1987, A manual of methods
for baculovirus vectors and insect cell culture procedures,
Texas Agricultural- Experimental Statiori Bulletin No. 1555) .
This expression vector contains the stroizg polyhedrin
promoter of the Autographs californica nuclear polyhedrosis
virus (AcMNPV) -followed by the recognition sites for the
restriction endonuclease BamFiI. The polyadenylation site of
the simian virus (SV}40 is used for efficient
polyadenylation. For an easy selection of recomb~~nant
39
CA 02562162 2006-10-20
viruses the beta-galactosidase gene from E.coli is inserted
in the same orientation as the polyhedrin promoter followed
' by the polyadenylation signal of the polyhedrin gene. The
polyhedrin sequences are flanked at both sides by viral
sequences for the cell-mediated homologous recombination of
co-transfected wild-type viral DNA. Many other baculovirus
vectors could be used in place of pRGl such as pAc373, pVL941
and pAcIM1 (Luckow, V.A. aad Suaaners, M.D. , Virology, 170:31-
39) . .
The plasmid was digested With the restriction enzyme
BamHI and then dephosphorylated usiag calf intestinal
phosphatase by procedures known in the art. The DNA was then
isolated from a 1~ agarose gel as described above. This
vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 were
ligated with T4 DNA lipase. ls.coli HS101 cells were then
transformed and bacteria identified that contained the
plasmid (pBacBDGrTRIO ) with the HDC;NR10 gene using the eazyn~e
BamHI. The sequence of the cloned fragment was confirmed by
DNA sequencing.
~Cg of the plas-.nid pBac~GNRlO were co-transfected with
i.0 ~cg of a caaamercially available liaearized baculovirus
("BaculoGold" baculovirus DNA°, Phazmingen, Saa Diego, CA.)
using the lipofection method (Felgner et al. Proc. Natl.
Acad. Sci. USA, 84:7413-7417 (1987)).
leg of BaculoGold'"' virus DNA and 5 ~g of the plasmid
pBacBDGrTRIO were mixed in a sterile well of a microtiter
plate containing 50 ~ul of serum free Grace's meditma (Z.ife
Technologies Inc., Gaithersburg, MD). Afterwards 10 girl
Lipofectin plus 90 ~Cl Grace's median were added, mixed and
incubated for 15 miautes at room t~perature. Then the..
transfection mixture Was added drop wise to the Sf9 insect
cells (ATCC CRL 1711) seeded in a 35 n~ tissue culture plate
with 1 ml Grace' medium without serum. The plate was rocked
back and forth to mix the newly added solution. The plate
-40-
CA 02562162 2006-10-20
was then incubated for 5 hours at 27°C. After S hours .the
transfection solution was removed from the plate and 1 ml of
Grace's insect medium supplemented with 10% fetal calf senun
was added. The plate was put back into an incubator and
cultivation continued at 27°C for four days.
After four days the supernatant was collected and a
plaque assay performed similar as described by Summers and
Smith (supra). As a modification an agarose gel with "Blue
Gal" (Life Technologies Inc., Gaithersburg) was used which
allows as easy isolation of blue stained plaques. ~ (A
detailed description of a "plaque assay" can also be found in
the user's guide for insect cell culture and baculovirology
distributed by Life_Technologies Inc., Gaithersburg, page 9-
10) _
Four days after the serial dilution, the viruses were
added to the cells, blue stained plaques were picked with the
tip of an Eppendarf pipette. T3~e agar containing the
recombinant viruses was then resuspended in an Eppendorf tube
containing~200 ~.l of Grace's medium.. The agar was removed by
a brief centrifugation and the supernatant containing. the
recombinant baculoviruses was used to infect Sf9 cells seeded
in 35 moa dishes. Four days later the supernatants of these
culture dishes were harvested and~then stored at 4°C.
Sf9 cells were grown in Grace's anedium~supplemented~with
10% heat=inactivated FBS_ The cells were infected with the
recombinant baculovinxs V-~GNR10 at a multiplicity of
infection tMOI) of 2. Six hours later the medium Was removed
and replaced with SF900 II mediuui minus methionine and
cysteine (Life Technologies Inc., Gaithersburg). 42 hours
later S ~cCi of ~S-methionine and 5 ~.Ci 'sS cysteine (Amersham)
were added. The cells were further incubated for i6 hours
before they were harvested by centrifugation and the Zabelled
proteins visualized by SDS-PAGE and autoradiography.
Exatrmle 4
-41-
CA 02562162 2006-10-20
- ExoressiQn via Gene Therap~r
Fibroblasts are obtained from a subject by skin biopsy.
The resulting tissue is placed in tissue-culture medium and
separated into small pieces. Small chunks of the tissue are
placed on a wet surface of a tissue culture flask,
approximately ten pieces are placed in each flask. The flask
is turned upside down, closed tight and left at room
temperature over night. After 24 hours at room temperature,
the flask is inverted and the chunks of tissue remain fixed
to the bottom of the flask and fresh media (e.g., Ham's F12
media, with 10~ FBS, penicillin and streptomycin, is added.
This is then incubated at 37°C for approximately one week.
At this time, fresh media is added and subsequently changed
every several days. After an additional two weeks in
culture, a monolayer of fibroblasts emerge. The monolayex is
trypsinized and scaled into larger flasks.
pMV-7 (Rirsctmneier, P.T. et al, DNA, 7:219-25 (1988)
flanked by the long terminal repeats of the Moloaey marine
sarcoma virus, is digested with BcoRI and HiadII2 and
subsequently treated with calf intestinal phosphatase. The
linear vector is fractionated on agarose gel and purified,
using glass beads.
The cDNA encoding a polypeptide of the present invention
is amplified using PCR prisoners which correspond to the 5' and
3' end sequences respectively: The 5' primer ccntains an
$coRI sits and the 3' primer contains a HindIII site. $qual
quantities of the Moloney murinE sarcoma virus linear
backbone and the BcoRI and HindIII fragment are added
together, in the presence of T4 DNA lipase. The resulting
mixture is maintained under conditions appropriate for
ligation of the two fragments. The ligation mixture is used
to transform bacteria HB101, which are then plated onto agar-
containing kanamycin for the purpose of confirming that the
vector had the gene of interest properly inserted.
-42-
CA 02562162 2006-10-20
The amphotropic pA3l~ or GP+aml2 packaging cells are
grown in tissue culture' to confluent density in Dulbecco~s
Modified Eagles Medium (DMEM) with 10% calf serum (CS),
penicillin arid streptomycin. The.MSV vector containing the
' gene is then added to the media and the packaging cells are
transduced with the vector. The packaging cells now produce
infectious viral particles containing the gene (.the packaging
cells are now referred to as producer cells).
Fresh ~aedia is added to the transduced producer cells,
and subsequently, the media is harvested from a 10 cm plate
of confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore
filter to remove detached producer cells and this media is
then used to infect fibroblast cells. Media is removed from
a sub-confluent plate of fibroblasts and quickly replaced
with the_media from the producer cells. This media is
removed and replaced with fresh med~,a. ~ If the titer of vixus
is high, then virtually all fibroblasts will be infected and
no~selection is required. If the titer is very low, then it
is necessary to use a retroviral vector that has a selectable
marker, such as neo or hid.
The engineered fibroblasts are then infected into the
ho$t, either alone or after~~having been grows to confluence
on cytodex 3 microcarrier beads. The fibroblasts now produce
the protein product.
Numerous modifications and variations of the present
invention are possible in light of the above~teachings and,
therefore, within the scope of the appended claims, the
invention may be practiced otherwise than as particularly
described.
-43-
CA 02562162 2006-10-20
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: HUMAN GENOME SCIENCES, INC. .
9410 KEY WEST AVENUE
ROCKVILLE, MD 20850
UNITED STATES OF AMERICA
APPLICANT/INVENTOR: LI, Yi
RUBEN, Steven M.
_, (ii) TITLE OF INVENTION: IiUMAN G-PROTEIN CHEMOKINE RECEPTOR HDGNRIO
(iii) NUMBER OF SEQUENCES: 9
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
(B) STREET: 1100 NEW YORK AVE., NW, SUITE 600
(C) CITY: WASHINGTON
(D) STATE: DC
(E) COUNTRY: USA
(F) ZIP: 20005
(v) COMPtTTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible - .
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release X1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,216,912
(B) FILING DATE: 06-JUN-1995
(C) CLASSIFICATIdN:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US95/07173
(8)- FILING DATE: 06-JUN-1995
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: STEFFE, ERIC K.
(B) REGISTRATION NUMBER: 36,688
(C) REFERENCE/DOCKET NUMBER: 1488.I15CA00
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 371-2600
(B)..TELEFAX: (202) 371-2540
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1419 base pairs
(B) TYPE: nucleic acid
(C). STRANDEDNESS: double ~ .
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
CA 02562162 2006-10-20
-45-
(ix) FEATDRE:
(A) NAME/FEEY: CDS
(B) LOCATION: 259._131!
(xi) SEQUE1~TCE DESCRIPTION: SEQ ID NO:1: _
GTGAGATGGT GCTTTCATGA ATTCCCGCAA CAAGAGCCAA GCTCTCCATC TAGTGGACAG 60
GGAAGCTAGC AGCAAAGCTT CCCTTCACTA CGAAACTTCA TTGCTTGGCC CAAAAGAGAG z20
TTAATTCAAT GTAGACATCT ATGTAGGCAA TTAAAAACCT ATTGATGTAT AAAACAGTTT 180
GCATTCATGG AGGGCAAC2A'AATACFsTTCT AGGACTTTAT AAAAGATCAG TTTTTATTTA 240
TGCACAGGGT GGAACAAG ATG GAT TAT CAA GTG TCA AGT CCA ATC TAT GAC 291
Met Asp Tyr 61~x Val Ser Ser Pro Ile Tyr Asp
1 S 10
ATC AAT TAT TAT gCA TCG 6AG CCC ~'GC CCA AAA ATC AAT GTG AAG CAA . 339
Ile Asn Tyr Tyr Thr Ser Glu Pro Cys Pro hys Ile Asn Val Lys~ Gln
15 20 ~ 25
ATC GCA GCC CGC CTC CTG CCT CCG CTC TAC TCA CTG GTG TTC ATC TTT 387
IIe~Ala Ala'Arg Leu he~u Pro Pro heu Tyr Ser Leu Val Phe Ile Phe
30 35 - . 40
GGT TTT GTG GGC AAC ATG CTG GTC ATC CTC _AT~C CTG ATA AFiC TGC CAA ~ ' 435
Gly Phe Val Gly Asa Met Leu Val_ Ile ~.eu Ile Leu Ile Asa Cys Gln
45 54 ~ SS
AGG CTG GAG AGC ATG ACT GAC ATC TAC CTG CTC AAC CTG GCC.ATC TCT 9S3
Arg beu GIu Ser Met Thr Asg I1e Tyr Z.eu F.eu Asia Le3~ Ala Ile Ser
6f? 65 70 75 .
GAC CTG TTT TTC CTT CTT ACT GTC CCC TTC TGG ~GCT CAC 1'l~lT GCT GCC 53I
Asp Leu Phe Phe.heu l.eu- Thr VaI Pro Phe Trp Ala 8is Tyr Ala Ala
'. 80 85 90
GCC CAG TGG GAC TTT GGA AAT ACA. 1$TG T'GT CAA CTC T~'G' ACA. GGG CTC ' 579'
Ala Gln Trp Asp Phe Gly Asn Thr Met Cys GIr~ _ F~e~x Few TY~rr Gly heu
95 100 105
TAT TTT ATA GGC TTC TTC TCT GGA ATC TTC TTC ATC ATC GTC CTG ACA 627
Tyr Phe Ile Gly Phe Phe Ser Gly Tle Phe Phe IIe Ile Ixtz beu Thr
lla ~~.s ~ 120 . . ~ .
ATe c~T AGG~TAC eT~ GCT ATC GTC C~~ GCT.GTG TTT GCS TTA AAA cce _. s7s ..
I2e Asp Arg Tyr Lew Rla Ile Val F~is ~ Ala ~i'af Phe F~a~ ~ ~eu ~.ys P~.a
12S ~ 134 . 135
AGG ACG GTC ACC TT'F GGG GTG GTG~ACA AGT GTG ATC ACT TG6 6TG GTG 723
Arg Thr Val Thr Phe Gly Val Val Thr Ser Val Ile Thr Trp Pal Val
140 145 I50 I55
GCT GTG TTT GCG TCT CTC CCA 6GA ATC ATG TTT ACC AGA TCT CAA A13A 771
Ala Va2 Phe Ala Sec heu Pro Gly Ile fle Phe Thr Arg Ser Gln Lys
16c~ lss ~ loo
GAA~GGT CTT CAT TAC ACC TGC AGC TCT CAT TTT CCA TAC AGT CAG TAT 819
Glu Gly Leu His Tyr Thr Cys Ser Sex ~~is.Phe Pro Tyr Ser Gln Tyr
z~7s . zeo 18s
CA 02562162 2006-10-20
-q.s-
CAA TTC TGG AAG AAT TTC CAG ACA TTA AF~G ATA GTC ATC TTG GGG CTG 867
Gln Phe Trp Lys Asn Phe Gln.Thr Leu Lys Ile Val Ile heu Gly Leu
190 295 200
GTC CTG CCG CTG CTT GTC ATG GTC ATC TGC TAC TCG GGA ATC CTA AAA 915
Val Leu Pro Leu leu Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys
205 210 215
ACT"CTG CTT CGG TGT CGA AAT GAG AAG AAG AGG CAC AGG GCT GTG AGG 963
Thr Leu f~eu Arg Cys Arg Asn Glu r.ys Lys Arg His Arg~ Ala Val Arg
220 225 230 235
CTT ATC TTC ACC ATC ATG ATT GTT TAT TTT CTC TTC TGG GCT CCC TAC ' 1011
Leu Ile Phe Thr Ire Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Tyr .
24'O 245 250
AAC ATT GTC CTT CTC CTG AAC ACC TTC CAG OAA TTC TTT GGC CTG AAT 1059
Asn Ile Val Leu Leu Leu Asn Thr Phe Gln Glu Phe Phe~ Gly Leu Asn
255 260 265
AAT TGC AGT TCT AAC CAG GTG ACA GAG 1107
AGC AGG TTG
GAC CAA
GCT ATG
Asn Cys Ser Ser Asn Leu Asp Gln Ala Gln Val Thr Glu
Ser Arg Met
270 ~ 275 280
ACT CTT GGG ACG CAC TGC ATC AAC CCC ATC TAT~GCC TTT lass
ATG TGC ATC
Thr Leu Gly Thr 8is Cys Ile Asn Pro Ile Tyr AIa Phe
Met Cys Ile
285 ~ 290 295
GTC GGG GAG TTC AGA TliC CTC TTA GTC TTC CAA AAG CAC I203
AAG AAC TTC '
Val Gly Glu Phe Arg Tyr heu Leu Val Phe G,ln Lys
I:ys Asn Phe 8is-
300 305 . 310 315
ATT C,CC AAA TTC TGC TGC TGT TCT ATT CA6i CA1~~ GA6 I25I
CfC AAF1 ' TTC GCT
Ile AIa Zys Fhe Cys Cys Cys Ser Lle 61r~ Gln Glue
Arg Lys Phe Ala
324 325 330
CCC GAG C6A AGG TCA TAC ACC.CGA TCC GGG GAG CAG GAPS1299
GCA GTT ACT .
Pro Glu~Arg Ser Ser Tyr Thr Arg Ser GIg Glu GI~i
Ala Va3. Thr Glu
335 340 345 -
ATA TCT GTG GGC TTG~ TGAC~CGGAC TCAAGTGGGG TGGTGA~CC1~ GTCA6'AGTTG ~~x354
Ile Ser Val Gly Leu . '
350
TGCl~CATGGC TTAG~'TTTCA, TACACAGCCT GGGCTGGGGG TGGGGTGGAA GAGGTCTTTT 1414
(21' INP~5R2~tFtTION FCR SEø IET ~tQ:2':
( i a SEQUENCE CF~tPtCTERISTICS
(A) LENGTI#: 352 amino acid
(B~ TYPE: amino acid
(D) TOPi'~Ltt'~GY: linear
( ii ) MO1.ECDLE TYPE : protein
(xi~ SEQQE1~ICE DESCRIPTION: SEQ ID N0:2:
Met Asp Tyr Gln Va3. Sex Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thx
1 S 10 ' 15
CA 02562162 2006-10-20
-t~T_ .
Ser Glu Pro Cys Pro Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu
20 25 30
Leu Pro Pro Leu Tyr Ser Leu Val Phe~Ile Phe Gly Phe Val Gly.Asn
35 40 . 45
Met Leu Val Ile Leu Ile Leu Ile Asn Cys Gln Arg Leu GIu Ser Met
50 55 60
Thr Asp Ile Tyr Leu Leu l~sn Leu Ala Ile Ser Asp Leu Phe Phe Leu
65 70 . '- 75 80
Leu Thr Val Pro Phe Trp Ala Bis Tyr Ala Ala Ala Gln Trp Asp Phe
85 90 .95
Gly Asn Thr Met Cys Gln Leu Leu Thr Gly ?~eu Tyr Phe Ile GZy Phe
100 105 ~ 110 '
Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu
115 120 125
Ala Ile Val 8is RIa Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe
130 135 140 -
Gly Yal Val Thr Ser Val Ile Thr Txp Val Val Ala Val Phe A'La Ser
195 I50 155 160
?~eu Pro Gly Ile Ile phe Thr Arg Ser Gln Lys Glu Gly Leu 8is Tyr
165 I70 . 175
Thr Cys Sex Ser Iiis Phe Pro Tyr Ser GIn Tyr Gln Phe~Trg Lys Asr~
180 185 ' 190
Phe Gln Thr Leu T.ys IIe Val Ile Leu Gly Leu Val Leu Pra~ Leu Leu
X95 ~ 200 205
Yal Met Val Ile Cys Tys Ser GIy Ile Leu Lys Thr Leu Leu Arg Cys
210 21~ 22E? .
Arg Asn Glur ~ys Lys Arg 8is Arg Ala VaEI ArQ feu 31e Phe Thr Ile
225 230 235 244'
Met IIe Val Tyr Phe Leu Phe Trp Ala Pro Tyr Asn Ile Val Leu Leu
. 245 250 255 .
Leu .~~s~x Thr Phe Gln Glu PFse Phe Gly Leu Asn Asn Cys Ser Ser Ser
2613 265 270
Ast~ l~rg Leu asp Gln AIa Met G~.n t~'al Thr Glu Tar. Leu Gly Met Thr
2'15 . 28~T 285
ibis Cys Cys IIe Asn Pro Ile Ile Tyr Ala Phe Val fly 6Iu Lys Phe
290 2 95 300
Arg-Asn Tyr Leu Leu Val Phe Phe Gln Lys His Ile AIa I:ys Arg Phe
305 310 315 324
Cys Lys Cys Cys Ser I'le Phe Gln Gln Glu Ala Pro Glu Arg Ala Ser
325 330 335
Ser Val Tyr Thr Arg Ser Thr Gly Glu Gin Glu Ile Ser Val G1_y Leu
3~0 - . 345 350
CA 02562162 2006-10-20
(2) INFORMATION FOR SEQ ID N0:3:
'(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
CGGAATTCCT CCATGGATTA TCAAGTGTCA 30
(2) INFORMATION
FOR SEQ
ID N0:4:
(i) SEQUENCE
CHARACTERISTICS
(A) LENGTH: 29 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4.
CGGAAGCTTCGTCACAAGCC CACAGATAT 29
(2) INFORMATION FOR SEQ ID NO:S: ,
. (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 34 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) ' MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:S: .
GTCCAAGCTT GCCACCATGG ATTATCAAGT GTCA 34
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 61 BASE PAIRS ...,
(B) TYPE: NUCLEIC ACID _
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CTAGCTCGAG TCAAGCGTAG TCTGGGACGT.CGTATGGGTA GCACAAGCCC ACAGATATTT 60
C . 6I
CA 02562162 2006-10-20
-49-
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
CGGGATCCCT CCATGGATTA TCAAGTGTCA 30
(2) INFORMATION FOR SEQ ID N0:8:
(i.) SEQUENCE CHARACTERISTICS
(A) LENGTH: 29 BASE PAIRS
($) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
CGGGATCCCG CTCACAAGCC CACAGATAT 29
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 344 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single .
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Glu Glu Val Thr Thr Phe Phe Asp Tyr Asp Tyr Gly A1a Pro Cys
10 15
His Lys Phe Asp Val Lys Gln Ile Gly Ala Gln Leu Leu Pro Pro
20 25 30 ..
Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu
35 40 4S
Val Val Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys Cys Leu Thr
50 55 60
Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu
65 70 75
Ile Thr Leu Pro Leu Trp AlawHis Ser Ala Ala Asn Glu Trp Val
80 ~ 85 90
Phe Gly Asn Ala Met Cys Lys Leu Phe Thr Gly Leu Tyr His Ile
95 100 105
Gly Tyr_Phe Gly Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp
110' 115 ~ 120
Arg Tyr Leu Ala Ile Val His Ala Val Phe Ala Leu Lys Ala Arg
125 130 135
' CA 02562162 2006-10-20
-5 ~-
Thr Val ValThr SerVal IleThr TrpLeu Val
Thr Phe
Gly Va1
140 145 150
Ala Val Val ProGly IleIle PheThr LysCys Gln
Phe Ala
Ser
155 I60 ~ 165
Lys Glu SerVal Tyr ValCys GlyPro TyrPhe ProArg Gly
Asp
1~0 I75 180
Trp Asn PheHis Thr IleMet ArgAsn IleLeu GlyLeu Val
Asn
185 190 195
Leu Pro LeuIle Met ValIle CysTyr SerGly IleLeu Lys
Leu
200 ~ 205 - 210
Thr Leu ArgCys Arg AsnGlu LysLys ArgHis ArgAla Val
Leu
2I5 220 225
Arg Val PheThr Ile MetIle ValTyr PheLeu PheTrp Thr
Ile
230 235 240
Pro Tyr IleVal Ile LeuLeu AsnThr PheGln GluPhe Phe
Asn
245 250 255
Gly Leu AsnCys Glu SerThr SerGln LeuAsp GlnAla Thr
Ser
260 265 270
Gln Val GluThr Leu GlyMet ThrHis CysCys IleAsn Pro
Thr
275 280 285
rle Ile AlaPhe Val GlyGlu LysPhe ArgSer LeuPhe Eiis
Tyr
290 295 300
Ile Ala GlyCys Arg IleAla ProLeu GlnLys ProVal Cys
Leu
305 310 315
Gly Gly GlyVal Arg ProGly LysAsn ValLys ValThr Thr
Pro
320 _ 325 330
Gln Gly LeuAsp Gly ArgGly LysGly LysSer IleGly
Leu
. 340
335
