U.S. patent application number 10/106623 was filed with the patent office on 2002-10-17 for methods of screening for modulators of hiv infection.
Invention is credited to Gray, Patrick W., Raport, Carol J., Schweickart, Vicki L..
Application Number | 20020150888 10/106623 |
Document ID | / |
Family ID | 24302427 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020150888 |
Kind Code |
A1 |
Gray, Patrick W. ; et
al. |
October 17, 2002 |
Methods of screening for modulators of HIV infection
Abstract
The present invention provides polynucleotides that encode the
chemokine receptors 88-2B or 88C and materials and methods for the
recombinant production of these two chemokine receptors. Also
provided are assays utilizing the polynucleotides which facilitate
the identification of ligands and modulators of the chemokine
receptors. Receptor fragments, ligands, modulators, and antibodies
are useful in the detection and treatment of disease states
associated with the chemokine receptors such as atherosclerosis,
rheumatoid arthritis, tumor growth suppression, asthma, viral
infection, AIDS, and other inflammatory conditions.
Inventors: |
Gray, Patrick W.; (Seattle,
WA) ; Schweickart, Vicki L.; (Seattle, WA) ;
Raport, Carol J.; (Bothell, WA) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN
6300 SEARS TOWER
233 SOUTH WACKER
CHICAGO
IL
60606-6357
US
|
Family ID: |
24302427 |
Appl. No.: |
10/106623 |
Filed: |
March 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10106623 |
Mar 26, 2002 |
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08771276 |
Dec 20, 1996 |
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08771276 |
Dec 20, 1996 |
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08661393 |
Jun 7, 1996 |
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6268477 |
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08661393 |
Jun 7, 1996 |
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08575967 |
Dec 20, 1995 |
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6265184 |
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Current U.S.
Class: |
435/5 |
Current CPC
Class: |
A61P 31/12 20180101;
C07K 16/18 20130101; C07K 16/2866 20130101; A61P 17/06 20180101;
A61P 7/02 20180101; A61P 19/02 20180101; A61P 29/00 20180101; C07K
2319/43 20130101; C07K 14/7158 20130101; A61P 31/18 20180101 |
Class at
Publication: |
435/5 |
International
Class: |
C12Q 001/70 |
Claims
We claim:
1. A method of screening for a modulator of human immunodeficiency
virus (HIV) or simian immunodeficiency virus (SIV) infection,
comprising steps of: (a) contacting a first composition comprising
an 88C receptor polypeptide with a second composition comprising a
HIV or SIV envelope protein in the presence and absence of a
compound, wherein the 88C receptor polypeptide comprises an amino
acid sequence encoded by a nucleotide sequence that hybridizes to
the complement of nucleotides 55-1110 of SEQ ID NO: 1 or the
complement of nucleotides 1-1056 of SEQ ID NO: 19 under the
following stringent conditions: 42.degree. C. in 50% formamide, 5X
SSC, 20 mM sodium phosphate, pH 6.8 and washing in 0.2X SSC at
55.degree. C.; (b) measuring the interaction of the 88C receptor
polypeptide with the envelope protein in the presence and absence
of the compound; and (c) screening for a modulator of HIV or SIV
infection, wherein reduced or increased interaction of the 88C
receptor polypeptide with the envelope protein in the presence of
the compound versus in the absence of the compound is indicative of
the compound being a modulator of HIV or SIV infection.
2. A method according to claim 1, wherein the 88C receptor
polypeptide comprises the amino acid sequence set forth in SEQ ID
NO: 2.
3. A method according to claim 2 wherein the envelope protein
comprises an HIV envelope protein.
4. A method according to claim 1, wherein the 88C receptor
polypeptide comprises the amino acid sequence set forth in SEQ ID
NO: 20.
5. A method according to claim 4, wherein the envelope protein
comprises an SIV envelope protein.
6. A method according to claim 1, wherein the first composition
comprises a cell that has been recombinantly modified to express
the 88C receptor polypeptide on its surface.
7. A method according to claim 6, wherein the second composition
comprises HIV which comprises HIV envelope protein.
8. A method according to claim 7, wherein the measuring step
comprises measuring HIV infection of the cell.
9. A method according to claim 1, further comprising a step of
forming a modulator composition by mixing a modulator identified
according to step (c) with a pharmaceutically acceptable diluent or
carrier.
10. A method according to claim 9, further comprising a step of
administering the modulator composition to a mammalian subject, and
screening for modulation of HIV or SIV infection in said
subject.
11. A method of screening for a modulator of human immunodeficiency
virus (HIV) infection, comprising steps of: (a) contacting a first
composition comprising an 88-2B receptor polypeptide with a second
composition comprising a HIV envelope protein, in the presence and
absence of a compound, wherein the 88-2B receptor polypeptide
comprises an amino acid sequence encoded by a nucleotide sequence
that hybridizes to the complement of nucleotides 362-1426 of SEQ ID
NO:3 under the following stringent conditions: 42.degree. C. in 50%
formamide, 5X SSC, 20 mM sodium phosphate, pH 6.8 and washing in
0.2X SSC at 55.degree. C.; (b) measuring the interaction of the
88-2B receptor polypeptide with the HIV envelope protein in the
presence and absence of the compound; and (c) screening for a
modulator of HIV infection, wherein reduced or increased
interaction of the 88-2B receptor polypeptide with the HIV envelope
protein in the presence of the compound versus in the absence of
the compound is indicative of the compound being a modulator of HIV
infection.
12. A method according to claim 11, wherein the 88-2B receptor
polypeptide comprises the amino acid sequence set forth in SEQ ID
NO: 4.
13. A method according to claim 12, wherein the first composition
comprises a cell that has been recombinantly modified to express
the 88-2B receptor polypeptide on its surface.
14. A method according to claim 13, wherein the second composition
comprises a human immunodeficiency virus which comprises HIV
envelope protein.
15. A method according to claim 14, wherein the measuring step
comprises measuring HIV infection of the cell.
16. A method according to claim 12, further comprising a step of
forming a modulator composition by mixing a modulator identified
according to step (c) with a pharmaceutically acceptable diluent or
carrier.
17. A method according to claim 16, further comprising a step of
administering the modulator composition to a mammalian subject, and
screening for modulation of HIV infection in said subject.
18. A method for detecting human immunodeficiency virus (HIV)
infection of cells, comprising steps of: (a) contacting a cell that
has been recombinantly modified to express at least one of human
chemokine receptors 88C and 88-2B with HIV; and (b) detecting HIV
infection of the cell.
19. A method for inhibiting human immunodeficiency virus (HIV)
infection of cells, comprising steps of: (a) contacting cells with
an antibody to at least one of human chemokine receptors 88C and
88-2B with HIV; and (b) detecting HIV infection of the cell after
said contacting step.
Description
[0001] This is a continuation application of U.S. patent
application Ser. No. 08/771,276 filed Dec. 20, 1996, which is a
continuation-in-part of U.S. patent application Ser. No. 08/661,393
filed Jun. 7, 1996 (issued as U.S. Pat. No. 6,268,477 on Jul. 31,
2001), which was in turn a continuation-in-part of U.S. patent
application No. 08/575,967 filed Dec. 20, 1995 (issued as U.S. Pat.
No. 6,265,184 on Jul. 24, 2001). All of these priority applications
are incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to signal
transduction pathways. More particularly, the present invention
relates to chemokine receptors, nucleic acids encoding chemokine
receptors, chemokine receptor ligands, modulators of chemokine
receptor activity, antibodies recognizing chemokines and chemokine
receptors, methods for identifying chemokine receptor ligands and
modulators, methods for producing chemokine receptors, and methods
for producing antibodies recognizing chemokine receptors.
BACKGROUND OF THE INVENTION
[0003] Recent advances in molecular biology have led to an
appreciation of the central role of signal transduction pathways in
biological processes. These pathways comprise a central means by
which individual cells in a multicellular organism communicate,
thereby coordinating biological processes. See Springer, Cell
76:301-314 (1994), Table I, for a model. One branch of signal
transduction pathways, defined by the intracellular participation
of guanine nucleotide binding proteins (G-proteins), affects a
broad range of biological processes.
[0004] Lewin, GENES V 319-348 (1994) generally discusses G-protein
signal transduction pathways which involve, at a minimum, the
following components: an extracellular signal (e.g.,
neurotransmitters, peptide hormones, organic molecules, light, or
odorants), a signal-recognizing receptor [G-protein-coupled
receptor, reviewed in Probst et al., DNA and Cell Biology 11:1-20
(1992) and also known as GPR or GPCR], and an intracellular,
heterotrimeric GTP-binding protein, or G protein. In particular,
these pathways have attracted interest because of their role in
regulating white blood cell or leukocyte trafficking.
[0005] Leukocytes comprise a group of mobile blood cell types
including granulocytes (i.e., neutrophils, basophils, and
eosinophils), lymphocytes, and monocytes. When mobilized and
activated, these cells are primarily involved in the body's defense
against foreign matter. This task is complicated by the diversity
of normal and pathological processes in which leukocytes
participate. For example, leukocytes function in the normal
inflammatory response to infection. Leukocytes are also involved in
a variety of pathological inflammations. For a summary, see Schall
et al., Curr. Opin. Immunol. 6:865-873 (1994). Moreover, each of
these processes can involve unique contributions, in degree, kind,
and duration, from each of the leukocyte cell types.
[0006] In studying these immune reactions, researchers initially
concentrated on the signals acting upon leukocytes, reasoning that
a signal would be required to elicit any form of response. Murphy,
Ann. Rev. Immunol. 12:593-633 (1994) has reviewed members of an
important group of leukocyte signals, the peptide signals. One type
of peptide signal comprises the chemokines (chemoattractant
cytokines), termed intercrines in Oppenheim et al., Ann. Rev.
Immunol. 9:617-648 (1991). In addition to Oppenheim et al.,
Baggiolini et al., Advances in Immunol. 55:97-179 (1994), documents
the growing number of chemokines that have been identified and
subjected to genetic and biochemical analyses.
[0007] Comparisons of the amino acid sequences of the known
chemokines have led to a classification scheme which divides
chemokines into two groups: the a group characterized by a single
amino acid separating the first two cysteines (CXC; N-terminus as
referent), and the .beta.group, where these cysteines are adjacent
(CC). See Baggiolini et al., supra. Correlations have been found
between the chemokines and the particular leukocyte cell types
responding to those signals. Schall et al., supra, has reported
that the CXC chemokines generally affect neutrophils; the CC
chemokines tend to affect monocytes, lymphocytes, basophils and
eosinophils. For example, Baggiolini et al., supra, recited that
RANTES, a CC chemokine, functions as a chemoattractant for
monocytes, lymphocytes (i.e., memory T cells), basophils, and
eosinophils, but not for neutrophils, while inducing the release of
histamine from basophils.
[0008] Chemokines were recently shown by Cocchi et. al., Science,
270:1811-1815 (1995) to be suppressors of HIV proliferation. Cocchi
et al. (supra) demonstrated that RANTES, MIP-1.alpha., and
MIP-1.beta. suppressed HIV-1, HIV-2 and SIV infection of a
CD4.sub.+cell line designated PM1 and of primary human peripheral
blood mononuclear cells.
[0009] Recently, however, attention has turned to the cellular
receptors that bind the chemokines, because the extracellular
chemokines seem to contact cells indiscriminately, and therefore
lack the specificity needed to regulate the individual leukocyte
cell types.
[0010] Murphy (supra) reported that the GPCR superfamily of
receptors includes the chemokine receptor family. The typical
chemokine receptor structure includes an extracellular
chemokine-binding domain located near the N-terminus, followed by
seven spaced regions of predominantly hydrophobic amino acids
capable of forming membrane-spanning .alpha.-helices. Between each
of the a-helical domains are hydrophilic domains localized,
alternately, in the intra- or extra-cellular spaces. These features
impart a serpentine conformation to the membrane-embedded chemokine
receptor. The third intracellular loop typically interacts with
G-proteins. In addition, Murphy (supra) noted that the
intracellular carboxyl terminus is also capable of interacting with
G-proteins.
[0011] The first chemokine receptors to be analyzed by molecular
cloning techniques were the two neutrophil receptors for human IL8,
a CXC chemokine. Holmes et al., Science 253:178-1280 (1991) and
Murphy et al., Science 253:1280-1283 (1991), reported the cloning
of these two receptors for IL8. Lee et al., J. Biol. Chem.
267:16283-16287 (1992), analyzed the cDNAs encoding these receptors
and found 77% amino acid identity between the encoded receptors,
with each receptor exhibiting features of the G protein coupled
receptor family. One of these receptors is specific for IL-8, while
the other binds and signals in response to IL-8, gro/MGSA, and
NAP-2. Genetic manipulation of the genes encoding IL-8 receptors
has contributed to our understanding of the biological roles
occupied by these receptors. For example, Cacalano et al., Science
265:682-684 (1994) reported that deletion of the IL-8 receptor
homolog in the mouse resulted in a pleiotropic phenotype involving
lymphadenopathy and splenomegaly. In addition, a study of missense
mutations described in Leong et al., J. Biol. Chem. 269:19343-19348
(1994) revealed amino acids in the IL-8 receptor that were critical
for IL-8 binding. Domain swapping experiments discussed in Murphy
(supra) implicated the amino terminal extracellular domain as a
determinant of binding specificity.
[0012] Several receptors for CC chemokines have also been
identified and cloned. CCCKR1 binds both MIP-1.alpha. and RANTES
and causes intracellular calcium ion flux in response to both
ligands. Charo et al., Proc Natl. Acad. Sci. (USA) 91:2752-2756
(1994) reported that another CC chemokine receptor, MCP-R1
(CCCKR2), is encoded by a single gene that produces two splice
variants which differ in their carboxyl terminal domains. This
receptor binds and responds to MCP-3 in addition to MCP-1.
[0013] A promiscuous receptor that binds both CXC and CC chemokines
has also been identified. This receptor was originally identified
on red blood cells and Horuk et al., Science 261:1182-1184 (1993)
reports that it binds IL-8, NAP-2, GRO.alpha., RANTES, and MCP-1.
The erythrocyte chemokine receptor shares about 25% identity with
other chemokine receptors and may help to regulate circulating
levels of chemokines or aid in the presentation of chemokines to
their targets. In addition to binding chemokines, the erythrocyte
chemokine receptor has also been shown to be the receptor for
plasmodium vivax, a major cause of malaria (id.) Another G-protein
coupled receptor which is closely related to chemokine receptors,
the platelet activating factor receptor, has also been shown to be
the receptor for a human pathogen, the bacterium Streptococcus
pneumoniae [Cundell et al., Nature 377:435-438 (1995)].
[0014] In addition to the mammalian chemokine receptors, two viral
chemokine receptor homologs have been identified. Ahuja et al., J.
Biol. Chem. 268:20691-20694 (1993) describes a gene product from
Herpesvirus saimiri that shares about 30% identity with the IL-8
receptors and binds CXC chemokines. Neote et al., Cell, 72:415-425
(1993) reports that human cytomegalovirus contains a gene encoding
a receptor sharing about 30% identity with the CC chemokine
receptors which binds MIP-1.alpha., MIP-1.beta., MCP-1, and RANTES.
These viral receptors may affect the normal role of chemokines and
provide a selective pathological advantage for the virus.
[0015] Because of the broad diversity of chemokines and their
activities, there are numerous receptors for the chemokines. The
receptors which have been characterized represent only a fraction
of the total complement of chemokine receptors. There thus remains
a need in the art for the identification of additional chemokine
receptors. The availability of these novel receptors will provide
tools for the development of therapeutic modulators of chemokine or
chemokine receptor function. It is contemplated by the present
invention that such modulators are useful as therapeutics for the
treatment of atherosclerosis, rheumatoid arthritis, tumor growth
suppression, asthma, viral infections, and other inflammatory
conditions. Alternatively, fragments or variants of the chemokine
receptors, or antibodies recognizing those receptors, are
contemplated as therapeutics.
SUMMARY OF THE INVENTION
[0016] The present invention provides purified and isolated nucleic
acids encoding chemokine receptors involved in leukocyte
trafficking. Polynucleotides of the invention (both sense and
anti-sense strands thereof) include genomic DNAs, cDNAs, and RNAs,
as well as completely or partially synthetic nucleic acids.
Preferred polynucleotides of the invention include the DNA encoding
the chemokine receptor 88-2B that is set out in SEQ ID NO:3, the
DNA encoding the chemokine receptor 88C that is set out in SEQ ID
NO: 1, and DNAs which hybridize to those DNAs under standard
stringent hybridization conditions, or which would hybridize but
for the redundancy of the genetic code. Exemplary stringent
hybridization conditions are as follows: hybridization at
42.degree. C. in 50% formamide, 5X SSC, 20 mM sodium phosphate, pH
6.8 and washing in 0.2X SSC at 55.degree. C.
[0017] It is understood by those of skill in the art that variation
in these conditions occurs based on the length and GC nucleotide
content of the sequences to be hybridized. Formulas standard in the
art are appropriate for determining exact hybridization conditions.
See Sambrook et al., .sctn..sctn. 9.47-9.51 in Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y (1989). Also contemplated by the invention are
polynucleotides encoding domains of 88-2B or 88C, for example,
polynucleotides encoding one or more extracellular domains of
either protein or other biologically active fragments thereof.
88-2B extracellular domains correspond to SEQ ID NO:3 and SEQ ID
NO:4 at amino acid residues 1-36, 93-107, 171-196, and 263-284. The
extracellular domains of 88-2B are encoded by polynucleotide
sequences corresponding to SEQ ID NO:3 at nucleotides 362-469,
638-682, 872-949, and 1148-1213. Extracellular domains of 88C
correspond to SEQ ID NO: 1 and SEQ ID NO:2 at amino acid residues
1-32, 89-112, 166-191, and 259-280. The 88C extracellular domains
are encoded by polynucleotide sequences that correspond to SEQ ID
NO: 1 at nucleotides 55-150, 319-390, 550-627, and 829-894. The
invention also comprehends polynucleotides encoding intracellular
domains of these chemokine receptors. The intracellular domains of
88-2B include amino acids 60-71, 131-151, 219-240, and 306-355 of
SEQ ID NO:3 and SEQ ID NO:4. Those domains are encoded by
polynucleotide sequences corresponding to SEQ ID NO:3 at
nucleotides 539-574, 752-814, 1016-1081, and 1277-1426,
respectively. The 88C intracellular domains include amino acid
residues 56-67, 125-145, 213-235, and 301-352 of SEQ ID NO: 1 and
SEQ ID NO:2. The intracellular domains of 88C are encoded by
polynucleotide sequences corresponding to SEQ ID NO: 1 at
nucleotides 220-255, 427-489, 691-759, and 955-1110. Peptides
corresponding to one or more of the extracellular or intracellular
domains, or antibodies raised against those peptides, are
contemplated as modulators of receptor activities, especially
ligand and G protein binding activities of the receptors.
[0018] The nucleotide sequences of the invention may also be used
to design oligonucleotides for use as labeled probes to isolate
genomic DNAs encoding 88-2B or 88C under stringent hybridization
conditions (i.e., by Southern analyses and polymerase chain
reaction methodologies). Moreover, these oligonucleotide probes can
be used to detect particular alleles of the genes encoding 88-2B or
88C, facilitating both diagnosis and gene therapy treatments of
disease states associated with particular alleles. In addition,
these oligonucleotides can be used to alter chemokine receptor
genetics to facilitate identification of chemokine receptor
modulators. Also, the nucleotide sequences can be used to design
antisense genetic elements of use in exploring or altering the
genetics and expression of 88-2B or 88C. The invention also
comprehends biological replicas (i.e., copies of isolated DNAs made
in vivo or in vitro) and RNA transcripts of DNAs of the
invention.
[0019] Autonomously replicating recombinant constructions such as
plasmid, viral, and chromosomal (e.g., YAC) nucleic acid vectors
effectively incorporating 88-2B or 88C polynucleotides, and,
particularly, vectors wherein DNA effectively encoding 88-2B or 88C
is operatively linked to one or more endogenous or heterologous
expression control sequences are also provided.
[0020] The 88-2B and 88C receptors may be produced naturally,
recombinantly or synthetically. Host cells (prokaryotic or
eukaryotic) transformed or transfected with polynucleotides of the
invention by standard methods may be used to express the 88-2B and
88C chemokine receptors. Beyond the intact 88-2B or 88C gene
products, biologically active fragments of 88-2B or 88C, analogs of
88-2B or 88C, and synthetic peptides derived from the amino acid
sequences of 88-2B, set out in SEQ ID NO:4, or 88C, set out in SEQ
ID NO:2, are contemplated by the invention. Moreover, the 88-2B or
88C gene product, or a biologically active fragment of either gene
product, when produced in a eukaryotic cell, may be
post-translationally modified (e.g., via disulfide bond formation,
glycosylation, phosphorylation, myristoylation, palmitoylation,
acetylation, etc.) The invention further contemplates the 88-2B and
88C gene products, or biologically active fragments thereof, in
monomeric, homomultimeric, or heteromultimeric conformations.
[0021] In particular, one aspect of the invention involves antibody
products capable of specifically binding to the 88-2B or 88C
chemokine receptors. The antibody products are generated by methods
standard in the art using recombinant 88-2B or 88C receptors,
synthetic peptides or peptide fragments of 88-2B or 88C receptors,
host cells expressing 88-2B or 88C on their surfaces, or 88-2B or
88C receptors purified from natural sources as immunogens. The
antibody products may include monoclonal antibodies or polyclonal
antibodies of any source or sub-type. Moreover, monomeric,
homomultimeric, and heteromultimeric antibodies, and fragments
thereof, are contemplated by the invention. Further, the invention
comprehends CDR-grafted antibodies, "humanized" antibodies, and
other modified antibody products retaining the ability to
specifically bind a chemokine receptor.
[0022] The invention also contemplates the use of antibody products
for detection of the 88-2B or 88C gene products, their analogs, or
biologically active fragments thereof. For example, antibody
products may be used in diagnostic procedures designed to reveal
correlations between the expression of 88-2B, or 88C, and various
normal or pathological states. In addition, antibody products can
be used to diagnose tissue-specific variations in expression of
88-2B or 88C, their analogs, or biologically active fragments
thereof.
[0023] Antibody products specific for the 88-2B and 88C chemokine
receptors may also act as modulators of receptor activities. In
another aspect, antibodies to 88-2B or 88C receptors are useful for
therapeutic purposes.
[0024] Assays for ligands capable of interacting with the chemokine
receptors of the invention are also provided. These assays may
involve direct detection of chemokine receptor activity, for
example, by monitoring the binding of a labeled ligand to the
receptor. In addition, these assays may be used to indirectly
assess ligand interaction with the chemokine receptor. As used
herein the term "ligand" comprises molecules which are agonists and
antagonists of 88-2B or 88C, and other molecules which bind to the
receptors.
[0025] Direct detection of ligand binding to a chemokine receptor
may be achieved using the following assay. Test compounds (i.e.,
putative ligands) are detectably labeled (e.g., radioiodinated).
The detectably labeled test compounds are then contacted with
membrane preparations containing a chemokine receptor of the
invention. Preferably, the membranes are prepared from host cells
expressing chemokine receptors of the invention from recombinant
vectors. Following an incubation period to facilitate contact
between the membrane-embedded chemokine receptors and the
detectably labeled test compounds, the membrane material is
collected on filters using vacuum filtration. The detectable label
associated with the filters is then quantitated. For example,
radiolabels are quantitated using liquid scintillation
spectrophotometry. Using this technique, ligands binding to
chemokine receptors are identified. To confirm the identification
of a ligand, a detectably labeled test compound is exposed to a
membrane preparation displaying a chemokine receptor in the
presence of increasing quantities of the test compound in an
unlabeled state. A progressive reduction in the level of
filter-associated label as one adds increasing quantities of
unlabeled test compound confirms the identification of that
ligand.
[0026] Agonists are ligands which bind to the receptor and elicit
intracellular signal transduction and antagonists are ligands which
bind to the receptor but do not elicit intracellular signal
transduction. The determination of whether a particular ligand is
an agonist or an antagonist can be determined, for example, by
assaying G protein-coupled signal transduction pathways. Activation
of these pathways can be determined by measuring intracellular ca++
flux, phospholipase C activity or adenylyl cyclase activity, in
addition to other assays (see examples 5 and 6).
[0027] As discussed in detail in the Examples herein, chemokines
that bind to the 88C receptor include RANTES, MIP-1.alpha., and
MIP-1.beta., and chemokines that bind to the 88-2B receptor include
RANTES.
[0028] In another aspect, modulators of the interaction between the
88C and 88-2B receptors and their ligands are specifically
contemplated by the invention. Modulators of chemokine receptor
function may be identified using assays similar to those used for
identifying ligands. The membrane preparation displaying a
chemokine receptor is exposed to a constant and known quantity of a
detectably labeled functional ligand. In addition, the
membrane-bound chemokine receptor is also exposed to an increasing
quantity of a test compound suspected of modulating the activity of
that chemokine receptor. If the levels of filter-associated label
correlate with the quantity of test compound, that compound is a
modulator of the activity of the chemokine receptor. If the level
of filter-associated label increases with increasing quantities of
the test compound, an activator has been identified.
[0029] In contrast, if the level of filter-associated label varies
inversely with the quantity of test compound, an inhibitor of
chemokine receptor activity has been identified. Testing for
modulators of receptor binding in this way allows for the rapid
screening of many putative modulators, as pools containing many
potential modulators can be tested simultaneously in the same
reaction.
[0030] The indirect assays for receptor binding involve
measurements of the concentration or level of activity of any of
the components found in the relevant signal transduction pathway.
Chemokine receptor activation often is associated with an
intracellular Ca++ flux. Cells expressing chemokine receptors may
be loaded with a calcium-sensitive dye. Upon activation of the
expressed receptor, a Ca++ flux would be rendered
spectrophotometrically detectable by the dye. Alternatively, the
Ca++ flux could be detected microscopically. Parallel assays, using
either technique, may be performed in the presence and absence of
putative ligands. For example, using the microscopic assay for Ca++
flux, RANTES, a CC chemokine, was identified as a ligand of the
88-2B chemokine receptor. Those skilled in the art will recognize
that these assays are also useful for identifying and monitoring
the purification of modulators of receptor activity. Receptor
activators and inhibitors will activate or inhibit, respectively,
the interaction of the receptors with their ligands in these
assays.
[0031] Alternatively, the association of chemokine receptors with G
proteins affords the opportunity of assessing receptor activity by
monitoring G protein activities. A characteristic activity of G
proteins, GTP hydrolysis, may be monitored using, for example,
.sup.32P-labeled GTP.
[0032] G proteins also affect a variety of other molecules through
their participation in signal transduction pathways. For example, G
protein effector molecules include adenylyl cyclase, phospholipase
C, ion channels, and phosphodiesterases. Assays focused on any of
these effectors may be used to monitor chemokine receptor activity
induced by ligand binding in a host cell that is both expressing
the chemokine receptor of interest and contacted with an
appropriate ligand. For example, one method by which the activity
of chemokine receptors may be detected involves measuring
phospholipase C activity. In this assay, the production of
radiolabeled inositol phosphates by host cells expressing a
chemokine receptor in the presence of an agonist is detected. The
detection of phospholipase activity may require cotransfection with
DNA encoding an exogenous G protein. When cotransfection is
required, this assay can be performed by cotransfection of chimeric
G protein DNA, for example, Gqi5 [Conklin et al., Nature
363:274-276 (1993)], with 88-2B or 88C DNA and detecting
phosphoinositol production when the cotransfected cell is exposed
to an agonist of the 88-2B or 88C receptor. Those skilled in the
art will recognize that assays focused on G-protein effector
molecules are also useful for identifying and monitoring the
purification of modulators of receptor activity. Receptor
activators and inhibitors will activate or inhibit, respectively,
the interaction of the receptors with their ligands in these
assays.
[0033] Chemokines have been linked to many inflammatory diseases,
such as psoriasis, arthritis, pulmonary fibrosis and
atherosclerosis. See Baggiolini et al. (supra).
[0034] Inhibitors of chemokine action may be useful in treating
these conditions. In one example, Broaddus et al., J. of Immunol.
152:2960-2967 (1994), describes an antibody to IL-8 which can
inhibit neutrophil recruitment in endotoxin-induced pleurisy, a
model of acute inflammation in rabbit lung. It is also contemplated
that ligand or modulator binding to, or the activation of, the 88C
receptor may be useful in treatment of HIV infection and HIV
related disease states. Modulators of chemokine binding to specific
receptors contemplated by the invention may include antibodies
directed toward a chemokine or a receptor, biological or chemical
small molecules, or synthetic peptides corresponding to fragments
of the chemokine or receptor.
[0035] Administration of compositions containing 88-2B or 88C
modulators to mammalian subjects, for the purpose of monitoring or
remediating normal or pathological immune reactions And viral
infections including infection by retroviruses such as HIV-1, HIV-2
and SIV is contemplated by the invention. In particular, the
invention comprehends the mitigation of inflammatory responses,
abnormal hematopoietic processes, and viral infections by delivery
of a pharmaceutically acceptable quantity of 88-2B or 88C chemokine
receptor modulators. The invention further comprehends delivery of
these active substances in pharmaceutically acceptable compositions
comprising carriers, diluents, or medicaments. The invention also
contemplates a variety of administration routes. For example, the
active substances may be administered by the following routes:
intravenous, subcutaneous, intraperitoneal, intramuscular, oral,
anal (i.e., via suppository formulations), or pulmonary (i.e., via
inhalers, atomizers, nebulizers, etc.)
[0036] In another aspect, the DNA sequence information provided by
the present invention makes possible the development, by homologous
recombination or "knockout" strategies [see, e.g. Kapecchi,
Science, 244:1288-1292 (1989)], of rodents that fail to express a
functional 88C or 88-2B chemokine receptor or that express a
variant of the receptor. Alternatively, transgenic mice which
express a cloned 88-2B or 88C receptor can be prepared by well
known laboratory techniques [Manipulating the Mouse Embryo: A
Laboratory Manual, Brigid Hohan, Frank Costantini and Elizabeth
Lacy, eds. (1986) Cold Spring Harbor Laboratory ISBN
0-87969-175-I]. Such rodents are useful as models for studying the
activities of 88C or 88-2B receptors in vivo.
[0037] Other aspects and advantages of the present invention will
become apparent to one skilled in the art upon consideration of the
following examples.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The following examples illustrate the invention. Example 1
describes the isolation of genomic DNAs encoding the 88-2B and 88C
chemokine receptors. Example 2 presents the isolation and
sequencing of cDNAs encoding human 88-2B and 88C and macaque 88C.
Example 3 provides a description of Northern analyses revealing the
expression patterns of the 88-2B and 88C receptors in a variety of
tissues. Example 4 details the recombinant expression of the 88-2B
and 88C receptors. Example 5 describes Ca++ flux assays,
phosphoinositol hydrolysis assays, and binding assays for 88-2B and
88C receptor activity in response to a variety of potential
ligands. Experiments describing the role of 88C and 882B as
co-receptors for HIV is presented in Examples 6 and 7. The
preparation and characterization of monoclonal and polyclonal
antibodies immunoreactive with 88C is described in Example 8.
Example 9 describes additional assays designed to identify 88-2B or
88C ligands or modulators.
Example 1
[0039] Partial genomic clones encoding the novel chemokine receptor
genes of this invention were isolated by PCR based on conserved
sequences found in previously identified genes and based on a
clustering of these chemokine receptor genes within the human
genome. The genomic DNA was amplified by standard PCR methods using
degenerate oligonucleotide primers.
[0040] Templates for PCR amplifications were members of a
commercially available source of recombinant human genomic DNA
cloned into Yeast Artificial Chromosomes (i.e., YACs) (Research
Genetics, Inc., Huntsville, Ala., YAC Library Pools, catalog no.
95011 B). A YAC vector can accommodate inserts of 500-1000 kilobase
pairs. Initially, pools of YAC clone DNAs were screened by PCR
using primers specific for the gene encoding CCCKR1. In particular,
CCCKR(2)-5', the sense strand primer (corresponding to the sense
strand of CCCKR1), is presented in SEQ ID NO: 15. Primer
CCCKR(2)-5' consisted of the sequence 5'-CGTAAGCTTAGAGAAGCCGGG-
ATGGGAA-3', wherein the underlined nucleotides are the translation
start codon for CCCKR1. The anti-sense strand primer was CCCKR-3'
(corresponding to the anti-sense strand of CCCKR1) and its sequence
is presented in SEQ ID NO:16. The sequence of CCCKR-3',
5'-GCCTCTAGAGTCAGAGACCAGCAGA-3', contains the reverse complement of
the CCCKR1 translation stop codon (underlined).
[0041] Pools of YAC clone DNAs yielding detectable PCR products
(i.e., DNA bands upon gel electrophoresis) identified appropriate
sub-pools of YAC clones, based on a proprietary identification
scheme (Research Genetics, Inc., Huntsville, Ala.). PCR reactions
were initiated with an incubation at 94.degree. C. for four
minutes. Sequence amplifications were achieved using 33 cycles of
denaturation at 94.degree. C. for one minute, annealing at
55.degree. C. for one minute, and extension at 72.degree. C. for
two minutes.
[0042] The sub-pools of YAC clone DNAs were then subjected to a
second round of PCR reactions using the conditions, and primers,
that were used in the first round of PCR. Results from sub-pool
screenings identified individual clones capable of supporting PCR
reactions with the CCCKR-specific primers. One clone, 881F10,
contained 640 kb of human genomic DNA from chromosome 3p21
including the genes for CCCKR1 and CCCKR2, as determined by PCR and
hybridization. An overlapping YAC clone, 941A7, contained 700 kb of
human genomic DNA and also contained the genes for CCCKR1 and
CCCKR2. Consequently, further mapping studies were undertaken using
these two YAC clones. Southern analyses revealed that CCCKR1 and
CCCKR2 were located within approximately 100 kb of one another.
[0043] The close proximity of the CCCKR1 and CCCKR2 genes suggested
that novel related genes might be linked to CCCKR1 and CCCKR2.
Using DNA from yeast containing YAC clones 881F10 and 941A7 as
templates, PCR reactions were performed to amplify any linked
receptor genes. Degenerate oligodeoxyribonucleotides were designed
as PCR primers. These oligonucleotides corresponded to regions
encoding the second intracellular loop and the sixth transmembrane
domain of CC chemokine receptors, as deduced from aligned sequence
comparisons of CCCKR1, CCCKR2, and V28. V28 was used because it is
an orphan receptor that exhibits the characteristics of a chemokine
receptor; V28 has also been mapped to human chromosome 3 [Raport et
al., Gene 163:295-299 (1995)]. Of further note, the two splice
variants of CCCKR2, CCCKR2A and CCCKR2B, are identical in the
second intracellular loop and sixth transmembrane domain regions
used in the analysis. The 5' primer, designated V28degf2, contains
an internal BamHI site (see below); its sequence is presented in
SEQ ID NO:5. The sequence of primer V28degf2 corresponds to DNA
encoding the second intracellular loop region of the canonical
receptor structure. See Probst et al., supra. The 3' primer,
designated V28degr2, contains an internal HindIII site (see below);
its sequence is presented in SEQ ID NO:6. The sequence of primer
V28degr2 corresponds to DNA encoding the sixth transmembrane domain
of the canonical receptor structure.
[0044] Amplified PCR DNA was subsequently digested with BamHI and
HindIII to generate fragments of approximately 390 bp, consistent
with the fragment size predicted from inspection of the canonical
sequence. Following endonuclease digestion, these PCR fragments
were cloned into pBluescript (Stratagene Inc., LaJolla, Calif.). A
total of 54 cloned fragments were subjected to automated nucleotide
sequence analyses. In addition to sequences from CCCKR1 and CCCKR2,
sequences from the two novel chemokine receptor genes of the
invention were identified. These two novel chemokine receptor genes
were designated 88-2B and 88C.
[0045] Restriction endonuclease mapping and hybridization were
utilized to map the relative positions of genes encoding the
receptors 88C, 88-2B, CCCKR1, and CCCKR2. These four genes are
closely linked, as the gene for 88C is approximately 18 KBP from
the CCCKR2 gene on human chromosome 3p21.
EXAMPLE 2
[0046] Full-length 88-2B and 88C cDNAs were isolated from a
macrophage cDNA library by the following procedure. Initially, a
cDNA library, described in Tjoelker et al., Nature 374:549-553
(1995), was constructed in pRc/CMV (Invitrogen Corp., San Diego,
Calif.) from human macrophage MRNA. The cDNA library was screened
for the presence of 88-2B and 88C cDNA clones by PCR using unique
primer pairs corresponding to 88-2B or 88C. The PCR protocol
involved an initial denaturation at 94.degree. C. for four minutes.
Polynucleotides were then amplified using 33 cycles of PCR under
the following conditions: Denaturation at 94.degree. C. for one
minute, annealing at 55.degree. C. for one minute, and extension at
72.degree. C. for two minutes. The first primer specific for 88-2B
was primer 88-2B-f1, presented in SEQ ID NO:11. It corresponds to
the sense strand of SEQ ID NO:3 at nucleotides 844-863. The second
PCR primer specific for the gene encoding 88-2B was primer
88-2B-r1, presented in SEQ ID NO: 12; the 88-2B-r1 sequence
corresponds to the anti-sense strand of SEQ ID NO:3 at nucleotides
1023-1042. Similarly, the sequence of the first primer specific for
the gene encoding 88C, primer 88C-f1, is presented in SEQ ID NO: 13
and corresponds to the sense strand of SEQ ID NO: 1 at nucleotides
453-471. The second primer specific for the gene encoding 88C is
primer 88C-r3, presented in SEQ ID NO: 14; the sequence of 88C-r3
corresponds to the anti-sense strand of SEQ ID NO: 1 at nucleotides
744-763.
[0047] The screening identified clone 777, a cDNA clone of 88-2B.
Clone 777 contained a DNA insert of 1915 bp including the full
length coding sequence of 88-2B as determined by the following
criteria: the clone contained a long open reading frame beginning
with an ATG codon, exhibited a Kozak sequence, and had an in-frame
stop codon upstream. The DNA and deduced amino acid sequences of
the insert of clone 777 are presented in SEQ ID NO:3 and SEQ ID
NO:4, respectively. The 88-2B transcript was relatively rare in the
macrophage cDNA library. During the library screen, only three
88-2B clones were identified from an estimated total of three
million clones.
[0048] Screening for cDNA clones encoding the 88C chemokine
receptor identified clones 101 and 134 which appeared to contain
the entire 88C coding region, including a putative initiation
codon. However, these clones lacked the additional 5' sequence
needed to confirm the identity of the initiation codon. The 88C
transcript was relatively abundant in the macrophage cDNA Library.
During the library screen, it was estimated that 88C was present at
one per 3000 transcripts (in a total of approximately three million
clones in the library).
[0049] RACE PCR (Rapid Amplification of cDNA Ends) was performed to
extend existing 88C clone sequences, thereby facilitating the
accurate characterization of the 5' end of the 88C cDNA. Human
spleen 5'-RACE-ready cDNA was purchased from Clontech Laboratories,
Inc., Palo Alto, Calif., and used according to the manufacturer's
recommendations. The cDNA had been made "5' -RACE-ready" by
ligating an anchor sequence to the 5' ends of the cDNA fragments.
The anchor sequence is complementary to an anchor primer supplied
by Clontech Laboratories, Inc., Palo Alto, Calif. The anchor
sequence-anchor primer duplex polynucleotide contains an EcoRI
site. Human spleen cDNA was chosen as template DNA because Northern
blots had revealed that 88C was expressed in this tissue. The PCR
reactions were initiated by denaturing samples at 94.degree. C. for
four minutes. Subsequently, sequences were amplified using 35
cycles involving denaturation at 94.degree. C. for one minute,
annealing at 60.degree. C. for 45 seconds, and extension at
72.degree. C. for two minutes. The first round of PCR was performed
on reaction mixtures containing 2 .mu.l of the 5'-RACE-ready spleen
cDNA, 1 .mu.l of the anchor primer, and 1 .mu.l of primer 88c-r4
(100 ng/.mu.l) in a total reaction volume of 50 .mu.l. The
88C-specific primer, primer 88c-r4 (5'-GATAAGCCTCACAGCCCTGTG-3'),
is presented in SEQ ID NO:7. The sequence of primer 88c-r4
corresponds to the anti-sense strand of SEQ ID NO: 1 at nucleotides
745-765. A second round of PCR was performed on reaction mixtures
including 1 .mu.l of the first PCR reaction with 1 .mu.l of anchor
primer and 1 .mu.l of primer 88C-rlb (100 ng/.mu.l) containing the
following sequence (5'-GCTAAGCTTGATGACTATCTTTAATGTC-3') and
presented in SEQ ID NO:8. The sequence of primer 88C-rlb contains
an internal HindIII cloning site (underlined). The sequence 3' of
the HindIII site corresponds to the anti-sense strand of SEQ ID
NO:1 at nucleotides 636-654. The resulting PCR product was digested
with EcoRI and HindIII and fractionated on a 1% agarose gel. The
approximately 700 bp fragment was isolated and cloned into
pBluescript. Clones with the largest inserts were sequenced.
Alternatively, the intact PCR product was ligated into vector pCR
using a commercial TA cloning kit (Invitrogen Corp., San Diego,
Calif.) for subsequent nucleotide sequence determinations.
[0050] The 88-2B and 88C cDNAs were sequenced using the PRISM.TM.
Ready Reaction DyeDeoxy.TM. Terminator Cycle Sequencing Kit (Perkin
Elmer Corp., Foster City, Calif.) and an Applied Biosystems 373A
DNA Sequencer. The insert of clone 777 provided the double-stranded
template for sequencing reactions used to determine the 88-2B cDNA
sequence. The sequence of the entire insert of clone 777 was
determined and is presented as the 88-2B cDNA sequence and deduced
amino acid sequence in SEQ ID NO:3. The sequence is 1915 bp in
length, including 361 bp of 5' untranslated DNA (corresponding to
SEQ ID NO:3 at nucleotides 1-361), a coding region of 1065 bp
(corresponding to SEQ ID NO:3 at nucleotides 362-1426), and 489 bp
of 3' untranslated DNA (corresponding to SEQ ID NO:3 at nucleotides
1427-1915). The 88-2B genomic DNA, described in Example 1 above,
corresponds to SEQ ID NO:3 at nucleotides 746-1128. The 88C cDNA
sequence, and deduced amino acid sequence, is presented in SEQ ID
NO: 1. The 88C cDNA sequence is a composite of sequences obtained
from RACE-PCR cDNA, clone 134, and clone 101. The RACE-PCR cDNA was
used as a sequencing template to determine nucleotides 1-654 in SEQ
ID NO: 1, including the unique identification of 9 bp of 5'
untranslated cDNA sequence in SEQ ID NO: 1 at nucleotides 1-9. The
sequence obtained from the RACE PCR cDNA confirmed the position of
the first methionine codon at nucleotides 55-57 in SEQ ID NO: 1,
and supported the conclusion that clone 134 and clone 101 contained
full-length copies of the 88C coding region. Clone 134 contained 45
bp of 5' untranslated cDNA (corresponding to SEQ ID NO: 1 at
nucleotides 10-54), the 1056 bp 88C coding region (corresponding to
SEQ ID NO:1 at nucleotides 55-1110), and 492 bp of 3' untranslated
cDNA (corresponding to SEQ ID NO:1 at nucleotides 1111-1602). Clone
101 contained 25 bp of 5' untranslated cDNA (corresponding to SEQ
ID NO: 1 at nucleotides 30-54), the 1056 bp 88C coding region
(corresponding to SEQ ID NO: 1 at nucleotides 55-1110), and 2273 bp
of 3' untranslated cDNA (corresponding to SEQ ID NO: 1 at
nucleotides 1111-3383). The 88C genomic DNA described in Example 1
above, corresponds to SEQ ID NO: 1 at nucleotides 424-809.
[0051] The deduced amino acid sequences of 88-2B and 88C revealed
hydrophobicity profiles characteristic of GPCRs, including seven
hydrophobic domains corresponding to GPCR transmembrane domains.
Sequence comparisons with other GPCRs also revealed a degree of
identity. Significantly, the deduced amino acid sequences of both
88-2B and 88C had highest identity with the sequences of the
chemokine receptors.
[0052] Table 1 presents the results of these amino acid sequence
comparisons.
1 TABLE 1 Chemokine Receptors 88-2B 88C IL-8RA 30% 30% IL-8RB 31%
30% CCCKR1 62% 54% CCCKR2A 46% 66% CCCKR2B 50% 72% 88-2B 100% 50%
88-C 50% 100%
[0053] Table 1 shows that 88-2B is most similar to CCCKR1 (62%
identical at the amino acid level) and 88C is most similar to
CCCKR2 (72% identical at the amino acid level).
[0054] The deduced amino acid sequences of 88-2B and 88C also
reveal the intracellular and extracellular domains characteristic
of GPCRs. The 88-2B extracellular domains correspond to the amino
acid sequence provided in SEQ ID NO:3, and SEQ ID NO:4, at amino
acid residues 1-36, 93-107, 171-196, and 263-284. The extracellular
domains of 88-2B are encoded by polynucleotide sequences
corresponding to SEQ ID NO:3 at nucleotides 362-469, 638-682,
872-949, and 1148-1213. Extracellular domains of 88C include amino
acid residues 1-32, 89-112, 166-191, and 259-280 in SEQ ID NO: 1
and SEQ ID NO:2. The 88C extracellular domains are encoded by
polynucleotide sequences that correspond to SEQ ID NO: 1 at
nucleotides 55-150, 319-390, 550-627, and 829-894. The
intracellular domains of 88-2B include amino acids 60-71, 131-151,
219-240, and 306-355 of SEQ ID NO:3 and SEQ ID NO:4. Those domains
are encoded by polynucleotide sequences corresponding to SEQ ID
NO:3 at nucleotides 539-574, 752-814, 1016-1081, and 1277-1426,
respectively. The 88C intracellular domains include amino acid
residues 56-67, 125-145, 213-235, and 301-352 of SEQ ID NO:1 and
SEQ ID NO:2. The intracellular domains of 88C are encoded by
polynucleotide sequences corresponding to SEQ ID NO:1 at
nucleotides 220-255, 427-489, 691-759, and 955-1110.
[0055] In addition, a macaque 88C DNA was amplified by PCR from
macaque genomic DNA using primers corresponding to 5' and 3'
flanking regions of the human 88C cDNA. The 5' primer corresponded
to the region immediately upstream of and including the initiating
Met codon. The 3' primer was complementary to the region
immediately downstream of the termination codon. The primers
included restriction sites for cloning into expression vectors. The
sequence of the 5' primer was GACAAGCTTCACAGGGTGGAACAAGATG (with
the HindIII site underlined) (SEQ ID NO: 17) and the sequence of
the 3' primer was GTCTCTAGACCACTTGAGTCCGTGTCA (with the XbaI site
underlined) (SEQ ID NO: 18). The conditions of the PCR
amplification were 94.degree. C. for eight minutes, then 40 cycles
of 94.degree. C. for one minute, 55.degree. C. for 45 seconds, and
72.degree. C. one minute. The amplified products were cloned into
the HindIII and XbaI sites of pcDNA3 and a clone was obtained and
sequenced. The full length macaque cDNA and deduced amino acid
sequences are presented in SEQ ID NOs:19 and 20, respectively. The
nucleotide sequence of macaque 88C is 98% identical to the human
88C sequence. The deduced amino acid sequences are 97%
identical.
Example 3
[0056] The mRNA expression patterns of 88-2B and 88C were
determined by Northern blot analyses.
[0057] Northern blots containing immobilized poly A++ RNA from a
variety of human tissues were purchased from Clontech Laboratories,
Inc., Palo Alto, Calif. In particular, the following tissues were
examined: heart, brain, placenta, lung, liver, skeletal muscle,
kidney, pancreas, spleen, thymus, prostate, testis, ovary, small
intestine, colon and peripheral blood leukocytes.
[0058] A probe specific for 88-2B nucleotide sequences was
generated from cDNA clone 478. The cDNA insert in clone 478
contains sequence corresponding to SEQ ID NO: 3 at nucleotides
641-1915. To generate a probe, clone 478 was digested and the
insert DNA fragment was isolated following gel electrophoresis. The
isolated insert fragment was then radiolabeled with
.sup.32P-labeled nucleotides, using techniques known in the
art.
[0059] A probe specific for 88C nucleotide sequences was generated
by isolating and radiolabeling the insert DNA fragment found in
clone 493. The insert fragment from clone 493 contains sequence
corresponding to SEQ ID NO: 1 at nucleotides 421-1359. Again,
conventional techniques involving .sup.32P-labeled nucleotides were
used to generate the probe.
[0060] Northern blots probed with 88-2B revealed an approximately
1.8 kb mRNA in peripheral blood leukocytes. The 88C Northerns
showed an approximately 4 kb MRNA in several human tissues,
including a strong signal when probing spleen or thymus tissue and
less intense signals when analyzing MRNA from peripheral blood
leukocytes and small intestine. A relatively weak signal for 88C
was detected in lung tissue and in ovarian tissue.
[0061] The expression of 88C in human T-cells and in hematopoietic
cell lines was also determined by Northern blot analysis. Levels of
88C in CD4.sup.+ and CD8.sup.+T-cells were very high. The
transcript was present at relatively high levels in myeloid cell
lines THP1 and HL-60 and also found in the B cell line Jijoye. In
addition, the cDNA was a relatively abundant transcript in a human
macrophage cDNA library based on PCR amplification of library
subfractions.
Example 4
[0062] The 88-2B and 88C cDNAs were expressed by recombinant
methods in mammalian cells.
[0063] For transient transfection experiments, 88C was subcloned
into the mammalian cell expression vector pBJ1 [Ishi et al., J.
Biol. Chem 270:16435-16440 (1995)]. The construct included
sequences encoding a prolactin signal sequence for efficient cell
surface expression and a FLAG epitope at the amino terminus of 88C
to facilitate detection of the expressed protein. The FLAG epitope
consists of the sequence "DYKDDDD. "COS-7 cells were transiently
transfected with the 88C expression plasmid using Lipofectamine
(Life Technology, Inc., Grand Island, N.Y.) following the
manufacturer's instructions. Briefly, cells were seeded in 24-well
plates at a density of 4.times.10.sup.4 cells per well and grown
overnight. The cells were then washed with PBS, and 0.3 mg of DNA
mixed with 1.5 .mu.l of lipofectamine in 0.25 ml of Opti-MEM was
added to each well. After 5 hours at 37.degree. C., the medium was
replaced with medium containing 10% FCS. quantitative ELISA
confirmed that 88C was expressed at the cell surface in transiently
transfected COS-7 cells using the M1 antibody specific for the FLAG
epitope (Eastman Co., New Haven, Conn.).
[0064] The FLAG-tagged 88C receptor was also stably transfected
into HEK-293 cells, a human embryonic kidney cell line, using
transfection reagent DOTAP
(N-[1-[(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammoniummet-
hylsulfate, Boehringer-Mannheim, Inc., Indianapolis, Ind.)
according to the manufacturer's recommendations. Stable lines were
selected in the presence of the drug G418. The transfected HEK-293
cells were evaluated for expression of 88C at the cell surface by
ELISA, using the Ml antibody to the FLAG epitope. ELISA showed that
88C tagged with the FLAG epitope was expressed at the cell surface
of stably transformed HEK-293 Cells.
[0065] The 88-2B and 88C cDNAs were used to make stable HEK-293
transfectants. The 88-2B receptor cDNA was cloned behind the
cytomegalovirus promoter in pRc/CMV (Invitrogen Corp., San Diego,
Calif.) using a PCR-based strategy. The template for the PCR
reaction was the cDNA insert in clone 777. The PCR primers were
88-2B-3 (containing an internal XbaI site) and 88-2B-5 (containing
an internal HindIII site).
[0066] The nucleotide sequence of primer 88-2B-3 is presented in
SEQ ID NO:9; the nucleotide sequence of primer 88-2B-5 is presented
in SEQ ID NO: 10. An 1104 bp region of cDNA was amplified.
Following amplification, the DNA was digested with XbaI and HindIII
and cloned into similarly digested pRc/CMV. The resulting plasmid
was named 777XP2, which contains 18 bp of 5' untranslated sequence,
the entire coding region of 88-2B, and 3 bp of 3' untranslated
sequence. For the 88C sequence, the full-length cDNA insert in
clone 134 was not further modified before transfecting HEK-293
cells.
[0067] To create stably transformed cell lines, the pRc/CMV
recombinant clones were transfected using transfection reagent
DOTAP
(N-[1-[(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammoniummethylsulfate,
Boehringer-Mannheim, Inc., Indianapolis, Ind.) according to the
manufacturer's recommendations, into HEK-293 cells, a human
embryonic kidney cell line. Stable lines were selected in the
presence of the drug G418. Standard screening procedures (i.e.,
Northern blot analyses) were performed to identify stable cell
lines expressing the highest levels of 88-2B and 88C mRNA.
EXAMPLE 5
[0068] A. Ca++ Flux Assays
[0069] To analyze polypeptide expression, a functional assay for
chemokine receptor activity was employed. A common feature of
signaling through the known chemokine receptors is that signal
transduction is associated with the release of intracellular
calcium cations. Therefore, intracellular Ca++ concentration in the
transfected HEK-293 cells was assayed to determine whether the
88-2B or 88C receptors responded to any of the known
chemokines.
[0070] HEK-293 cells, stably transfected with 88-2B, 88C (without
the FLAG epitope sequence), or a control coding region (encoding
IL8R or CCCKR2, see below) as described above, were grown in T75
flasks to approximately 90% confluence in MEM+10% serum. Cells were
then washed, harvested with versene (0.6 mM EDTA, 10 mM
Na.sub.2HPO.sub.4, 0.14 M NaCl, 3 mM KCl, and 1 mM glucose), and
incubated in MEM+10% serum+1 .mu.M Fura-2 AM (Molecular Probes,
Inc., Eugene, Oreg.) for 30 minutes at room temperature. Fura-2 AM
is a Ca++-sensitive dye. The cells were resuspended in Dulbecco's
phosphate-buffered saline containing 0.9 mM CaCl.sub.2 and 0.5 mM
MgCl.sub.2 (D-PBS) to a concentration of approximately 10.sup.7
cells/ml and changes in fluorescence were monitored using a
fluorescence spectrophotometer (Hitachi Model F-4010).
Approximately 10.sup.6 cells were suspended in 1.8 ml D-PBS in a
cuvette maintained at 37.degree. C. Excitation wavelengths
alternated between 340 and 380 nm at 4 second intervals; the
emission wavelength was 510 nm. Test compositions were added to the
cuvette via an injection port; maximal Ca++ flux was measured upon
the addition of ionomycin.
[0071] Positive responses were observed in cells expressing IL-8RA
when stimulated with IL-8 and also when CCCKR2 was stimulated with
MCP-1 or MCP-3. However, HEK-293 cells expressing either 88-2B or
88C failed to show a flux in intracellular Ca++ concentration when
exposed to any of the following chemokines: MCP-1, MCP-2, MCP-3,
MIP-1.alpha., MIP-1.mu., IL8, NAP-2, gro/MGSA, IP-10, ENA-78, or
PF-4. (Peprotech, Inc., Rocky Hill, N.J.).
[0072] Using a more sensitive assay, a Ca++ flux response to RANTES
was observed microscopically in Fura-2 AM-loaded cells expressing
88-2B. The assay involved cells and reagents prepared as described
above. RANTES (Regulated on Activation, Normal T Expressed and
Secreted) is a CC chemokine that has been identified as a
chemoattractant and activator of eosinophils. See Neote et al.,
supra. This chemokine also mediates the release of histamine by
basophils and has been shown to function as a chemoattractant for
memory T cells in vitro. Modulation of 88-2B receptor activities is
therefore contemplated to be useful in modulating leukocyte
activation.
[0073] FLAG tagged 88C receptor was expressed in HEK-293 cells and
tested for chemokine interactions in the CA++ flux assay. Cell
surface expression of 88C was confirmed by ELISA and by FACScan
analysis using the M1 antibody. The chemokines RANTES,
MIP-1.alpha., and MIP-1.beta. all induced a Ca++ flux in
88C-transfected cells when added at a concentration of 100 nM.
[0074] Ca++ flux assays can also be designed to identify modulators
of chemokine receptor binding. The preceding fluorimetric or
microscopic assays are carried out in the presence of test
compounds. If Ca++ flux is increased in the presence of a test
compound, that compound is an activator of chemokine receptor
binding. In contrast, a diminished Ca++ flux identifies the test
compound as an inhibitor of chemokine receptor binding.
[0075] B. Phosphoinositol Hydrolysis
[0076] Another assay for ligands or modulators involves monitoring
phospholipase C activity, as described in Hung et al., J. Biol.
Chem. 116:827-832 (1992). Initially, host cells expressing a
chemokine receptor are loaded with .sup.3H-inositol for 24 hours.
Test compounds (i.e., potential ligands) are then added to the
cells and incubated at 37.degree. C. for 15 minutes. The cells are
then exposed to 20 mM formic acid to solubilize and extract
hydrolyzed metabolites of phosphoinositol metabolism (i.e., the
products of phospholipase C-mediated hydrolysis). The extract is
subjected to anion exchange chromatography using an AG1X8 anion
exchange column (formate form). Inositol phosphates are eluted with
2 M ammonium formate/0. 1 M formic acid and the .sup.3H associated
with the compounds is determined using liquid scintillation
spectrophotometry. The phospholipase C assay can also be exploited
to identify modulators of chemokine receptor activity. The
aforementioned assay is performed as described, but with the
addition of a potential modulator. Elevated levels of detectable
label would indicate the modulator is an activator;
[0077] depressed levels of the label would indicate the modulator
is an inhibitor of chemokine receptor activity.
[0078] The phospholipase C assay was performed to identify
chemokine ligands of the FLAG-tagged 88C receptor. Approximately 24
hours after transfection, COS-7 cells expressing 88C were labeled
for 20-24 hours with myo-[2-.sup.3H]inositol (1 .mu.Ci/ml) in
inositol-free medium containing 10% dialyzed FCS. Labeled cells
were washed with inositol-free DMEM containing 10 mM LiCl and
incubated at 37.degree. C. for 1 hour with inositol-free DMEM
containing 10 mM LiCl and one of the following chemokines: RANTES,
MIP-1.beta., MIP-1.alpha., MCP-1, IL-8, or the murine MCP-1 homolog
JE. Inositol phosphate (IP) formation was assayed as described in
the previous paragraph. After incubation with chemokines, the
medium was aspirated and cells were lysed by addition of 0.75 ml of
ice-cold 20 mM formic acid (30 min). Supernatant fractions were
loaded onto AG1-X8 Dowex columns (Biorad, Hercules, Calif.),
followed by immediate addition of 3 ml of 50 mM NH.sub.4OH. The
columns were then washed with 4 ml of 40 mM ammonium formate,
followed by elution with 2 M ammonium formate. Total inositol
phosphates were quantitated by counting beta-emissions.
[0079] Because it has been shown that some chemokine receptors,
such as IL8RA AND IL8RB, require contransfection with an exogenous
G protein before signaling can be detected in COS-7 cells, the 88C
receptor was co-expressed with the chimeric G protein Gqi5
(Conklin, et al., Nature 363:274-276, (1993). Gqi5 ia a G protein
which has the carboxyl terminal five amino acids of Gi (which bind
to the receptor) spliced onto G.alpha.q. Co-transfection with Gqi5
significantly potentiates signaling by CCCKR1 and CCKR2B.
Co-transfection with Gqi5 revealed that 88C signaled well in
response to RANTES, MIP-1.beta., and MIP-1.alpha., but not in
response to MCP-1, IL-8 or the murine MCP-1 homologue JE.
Dose-response curves revealed EC.sub.50 values of 1 nM for RANTES,
6 nM for MIP-1.beta., and 22 nM for MIP-1 .alpha..
[0080] 88C is the first cloned human receptor with a signaling
response to MIP-1.beta.. Compared with other CC chemokines,
MIP-1.beta.clearly has a unique cellular activation pattern. It
appears to activate T cells but not monocytes (Baggiolini et al.,
supra) which is consistent with receptor stimulation studies. For
example, while MIP-1.beta.binds to CCCKR1, it does not induce
calcium flux (Neote et al., supra). In contrast, MIP-1.alpha. and
RANTES bind to and causes signaling in CCCKR1 and CCCKR5 (RANTES
also causes activation of CCCKR3). MIP-1p thus appears to be much
more selective than other chemokines of the CC chemokine family.
Such selectivity is of therapeutic significance because a specific
beneficial activity can be stimulated (such as suppression of HIV
infection) without stimulating multiple leukocyte populations which
results in general pro-inflammatory activities.
[0081] C. Binding Assays
[0082] Another assay for receptor interaction with chemokines was a
modification of the binding assay described by Ernst et al., J.
Immunol. 152:3541-3549 (1994). MIP-1.beta. as labeled using the
Bolton and Hunter reagent (di-iodide, NEN, Wilmington, Del.),
according to the manufacturer's instructions. Unconjugated iodide
was separated from labeled protein by elution using a PD-10 column
(Pharmacia) equilibrated with PBS and BSA (1% w/v). The specific
activity was typically 2200 Ci/mmole. Equilibrium binding was
performed by adding .sup.125I-labeled ligand with or without a
100-fold excess of unlabeled ligand, to 5.times.10.sup.5 HEK-293
cells transfected with 88C tagged with the FLAG epitope in
polypropylene tubes in a total volume of 300 .mu.l(50 mM HEPES pH
7.4, 1 mM CaCl.sub.2, MgCl.sub.2, 0.5% BSA) and incubating for 90
minutes at 27.degree. C. with shaking at 150 rpm. The cells were
collected, using a Skatron cell harvester (Skatron Instruments
Inc., Sterling, Va.), on glass fiber filters presoaked in 0.3%
polyethyleneimine and 0.2% BSA. After washing, the filters were
removed and bound ligand was quantitated by counting gamma
emissions. Ligand binding by competition with unlabeled ligand was
determined by incubation of 5.times.10.sup.5 transfected cells (as
above) with 1.5 nM of radiolabeled ligand and the indicated
concentrations of unlabeled ligand. The samples were collected,
washed and counted as above. The data was analyzed using the
curve-fitting program Prism (GraphPad Inc., San Diego, Calif.) and
the iterative non-linear regression program, LIGAND (PM220).
[0083] In equilibrium binding assays, 88C receptor bound
radiolabeled MIP-1.beta. in a specific and saturable manner.
Analysis of this binding data by the method of Scatchard revealed a
dissociation constant (Kd) of 1.6 nM. Competition binding assays
using labeled MIP-1.beta. revealed high-affmity binding of
MIP-1.beta.(IC.sub.50=7.4 nM), RANTES (IC.sub.50=6.9 nM), and
MIP-1.alpha.(IC.sub.50=7.4 nM), consistent with the signaling data
obtained in transiently transfected COS-7 cells as discussed in
section B above.
[0084] Example 6
[0085] The chemokines MIP-1.alpha., MIP-1.beta. and RANTES have
been shown to inhibit replication of HIV-1 and HIV-2 in human
peripheral blood mononuclear cells and PM1 cells (Cocchi et al.,
supra). In view of this finding and in view of the results
described in Example 5, the present invention contemplates that
activation of or ligand binding to the 88C receptor may provide a
protective role in HIV infection.
[0086] Recently, it has been reported that the orphan G
protein-coupled receptor, fusin, can act as a co-receptor for HIV
entry. Fusin/CXCR4 in combination with CD4, the primary HIV
receptor, apparently facilitates HIV infection of cultured T cells
([Feng et al., Science 272:872-877 (1996)]. Based upon the homology
of fusin to chemokine receptors and the chemokine binding profile
of 88C, and because 88C is constitutively expressed in T cells and
abundantly expressed in macrophages, 88C is likely to be involved
in viral and HIV infection.
[0087] The function of 88C and 88-2B as co-receptors for HIV was
determined by transfecting cells which express CD4 with 88C or
88-2B and challenging the co-transfected cells with HIV. Only cells
expressing both CD4 and a functional co-receptor for HIV become
infected. HIV infection can be determined by several methods.
ELISAs which test for expression of HIV antigens are commercially
available, for example Coulter HIV-1 .sub.p24 antigen assay (U.S.
Pat. No. 4,886,742), Coulter Corp., 11800 SW 147th Ave., Miami,
Fla. 33196. Alternatively, the test cells can be engineered to
express a reporter gene such as LACZ attached to the HIV LTR
promoter [Kimpton et al., J. Virol. 66:2232-2239 (1992)]. In this
method, cells that are infected with HIV are detected by a
colorimetric assay.
[0088] 88C was transiently transfected into a cat cell line, CCC
[Clapham, et al., 181:703-715 (1991)], which had been stably
tranformed to express human CD4 (CCC-CD4). These cells are normally
resistant to infection by any strain of HIV-1 because they do not
endogenously express 88C. In these experiments, CCC/CD4 cells were
transiently transfected with 88C cloned into the expression vector
pcDNA3.1 (Invitrogen Corp., San Diego, Calif.) using lipofectamine
(Gibco BRL, Gaithersburg, Md.). Two days after transfection, cells
were challenged with HIV. After 4 days of incubation, cells were
fixed and stained for p24 antigen as a measure of HIV infection.
88C expression by these cells rendered them susceptible to
infection by several strains of HIV-1. These strains included four
primary non-syncytium-inducing HIV-1 isolates (M23, E80, SL-2 and
SF-162) which were shown to use only 88C as a co-receptor but not
fusin. Several primary syncytium-inducing strains of HIV-1 (2006,
M13, 2028 and 2076) used either 88C or fusin as a co-receptor.
Also, two established clonal HIV-1 viruses (GUN-1 and 89.6) used
either 88C or fusin as a co-receptor.
[0089] It has been reported that some strains of HIV-2 can infect
certain CD4-negative cell lines, thus implying a direct interaction
of HIV-2 with a receptor other than CD4 [Clapham et al., J. Virol.
66:3531-3537 (1992)] For some strains of HIV-2, this infection is
facilitated by the presence of soluble CD4 (sCD4). Since 88-2B
shares high sequence similarity with other chemokine receptors that
act as HIV co-receptors (namely 88C and fusin), 88-2B was
considered to be a likely HIV-2 co-receptor. The role of 88-2B as
an HIV-2 co-receptor was demonstrated using HIV-2 strain ROD/B. Cat
CCC cells which do not endogenously express CD4 were transfected
with 88-2B. In these experiments, cells were transfected with
pcDNA3.1 containing 88-2B using lipofectamine and infected with
HIV-2 48 hours later. Three days after infection, cells were
immunostained for the presence of HIV-2 envelope glycoproteins. The
presence of sCD4 during HIV-2.sub.ROD/B challenge increased the
infection of these cells by 10-fold. The entry of HIV-2 into the
88-2B transfected cells could be blocked by the presence of 400-800
ng/ml eotaxin, one of the ligands for 88-2B. The baseline
infectivity levels of CCC/88-2B (with no soluble CD4) were
equivalent to CCC cells which were not transfected with 88-2B.
[0090] The role of 88-2B and 88C as co-receptors for HIV was
confirmed by preparing and challenging cell lines stably
transformed to express 88C or 88-2B with various strains of HIV and
SIV. These results are described in Example 7.
[0091] Alternatively, the co-receptor role of 88C and 88-2B can be
demonstrated by an experimental method which does not require the
use of live virus. In this method, cell lines co-expressing 88C or
88-2B, CD4 and a LACZ reporter gene are mixed with a cell line
co-expressing the HIV envelope glycoprotein (ENV) and a
transcription factor for the reporter gene construct [Nussbaum et
al., J. Virol. 68:5411 (1994)]. Cells expressing a functional
co-receptor for HIV will fuse with the ENV expressing cells and
thereby allow expression of the reporter gene. In this method,
detection of reporter gene product by colorimetric assay indicates
that 88C or 88-2B function as a co-receptor for HIV.
[0092] The mechanism by which chemokines inhibit viral infection
has not yet been elucidated. One possible mechanism involves
activation of the receptor by binding of a chemokine. The binding
of the chemokine leads to signal transduction events in the cell
that renders the cell resistant to viral infection and/or prevents
replication of the virus in the cell. Similar to interferon
induction, the cell may differentiate such that it is resistant to
viral infection, or an antiviral state is established.
Alternatively, a second mechanism involves direct interference with
viral entry into cells by blocking access of viral envelope
glycoproteins to the co-receptor by chemokine binding. In this
mechanism, G-protein signaling is not required for chemokine
suppression of HIV infection.
[0093] To distinguish between two mechanisms by which 88C or 88-2B
may function as co-receptors for viral or HIV infection, chemokine
binding to the receptor is uncoupled from signal transduction and
the effect of the chemokine on suppression of viral infection is
determined.
[0094] Ligand binding can be uncoupled from signal transduction by
the addition of compounds which inhibit G-protein mediated
signaling. These compounds include, for example, pertussis toxin
and cholera toxin. In addition, downstream effector polypeptides
can be inhibited by other compounds such as wortmannin. If
G-protein signaling is involved in suppression of viral infection,
the addition of such compounds would prevent suppression of viral
infection by the chemokine. Alternatively, key residues or receptor
domains of 88C or 88-2B receptor required for G-protein coupling
can be altered or deleted such that G-protein coupling is altered
or destroyed but chemokine binding is not affected.
[0095] Under these conditions, if chemokines are unable to suppress
viral or HIV infection, then signaling through a G-protein is
required for suppression of viral or HIV infection. If however,
chemokines are able to suppress viral infection, then G-protein
signaling is not required for chemokine suppression of viral
infection and the protective effects of chemokines may be due to
the chemokine blocking the availability of the receptor for the
virus.
[0096] Another approach involves the use of antibodies directed
against 88C or 88-2B. Antibodies which bind to 88C or 88-2B which
can be shown not to elicit G-protein signaling may block access to
the chemokine or viral binding site of the receptor. If in the
presence of antibodies to 88C or 88-2B, viral infection is
suppressed, then the mechanism of the protective effects of
chemokines is blocking viral access to its receptor. Feng et al.
(1996) reported that antibodies to the amino terminus of the fusin
receptor suppressed HIV infection.
[0097] Example 7
[0098] Cell lines were stably transformed with 88C or 88-2B to
further delineate the role of 88C and 88-2B in HIV infection.
Kimpton and Emerman ["Detection of Replication-Competent and
Pseudotyped Human Immunodeficiency Virus with a Sensitive Cell Line
on the Basis of Activation of an Integrated Beta-Galactosidase
Gene," J. Virol, 66(4):2232-2239 (1992)] previously described an
indicator cell line, herein identified as HeLa-MAGI cells.
HeLa-MAGI cells are HeLa cells that have been stably transformed to
express CD4 as well as integrated HIV-1 LTR which drives expression
of a nuclear localized .beta.-galactosidase gene. Integration of an
HIV provirus in the cells leads to production of the viral
transactivator, Tat, which then turns on expression of the
.beta.-galactosidase gene. The number of cells that stain positive
with X-gal for .beta.-galactosidase activity in situ is directly
proportional to the number of infected cells.
[0099] These HeLa-MAGI cells can detect lab-adapted isolates of
HIV-1 but only a minority of primary isolates [Kimpton and Emerman,
supra], and cannot detect most SIV isolates [Chackerian et al.,
"Characterization of a CD4-Expressing Macaque Cell Line that can
Detect Virus After A Single Replication Cycle and can be infected
by Diverse Simian Immunodeficiency Virus Isolates," Virology,
213(2):6499-6505 (1995)].
[0100] In addition, Harrington and Geballe ["Co-Factor Requirement
for Human Immunodeficiency Virus Type 1 Entry into a CD4-Expressing
Human Cell Line, J. Virol., 67:5939-5947 (1993)] described a cell
line based on U373 cells that had been engineered to express CD4
and the same LTR-.beta.-galactosidase construct. It was previously
shown that this cell line, herein identified as U373-MAGI, could
not be infected with any HIV (M or T-tropic) strain of HIV, but
could be rendered susceptible to infection by fusion with HeLa
cells (Harrington and Geballe, supra).
[0101] In order to construct indicator cell lines that could detect
either macrophage or T cell tropic viruses, epitope-tagged 88C or
88-2B encoding DNA was transfected into HeLa-MAGI or U373-MAGI
cells by infection with a retroviral vector to generate
HeLa-MAGI-88C or U373-MAGI-88C cell lines, respectively. Expression
of the co-receptors on the cell surface was demonstrated by
immunostaining live cells using the anti-FLAG M1 antibody and by
RT-PCR.
[0102] The 88C and 88-2B genes utilized to construct HeLa-MAGI-88C
and U373-MAGI-88C included sequences encoding the prolactin signal
peptide followed by a FLAG epitope as described in Example 4. This
gene was inserted into the retroviral vector pBabe-Puro
[Morgenstern and Land, Nucleic Acids Research, 18(12):3587-3596
(1990)].
[0103] High titer retroviral vector stocks pseudotyped with the
VSV-G protein were made by transient transfection as described in
Bartx et al., J. Virol. 70:2324-2331 (1996), and used to infect
HeLa-MAGI and U373-MAGI cells. Cells resistant to 0.6 .mu./ml
puromycin (HeLa) or 1 .mu./ml puromycin (U373) were pooled. Each
pool contained at least 1000 independent transduction events. An
early passage (passage 2) stock of the original HeLa-MAGI cells
(Kimpton and Emerman, supra) was used to create HeLa-MAGI-88C
cells.
[0104] Infections of the indicator cell lines with HIV were
performed in 12-well plates with 10-fold serial dilutions of 300
.mu.l of virus in the presence of 30 .mu./ml DEAE-Dextran as
described (Kimpton and Emerman, supra).
[0105] All HIV-1 strains and SIV.sub.mac 239 were all obtained from
the NIH AIDS Reference and Reagent Program. Molecular clones of
primary HIV-2.sub.7312A [Gao et al., "Genetic Diversity of Human
Immunodeficiency Virus Type 2: Evidence for Distinct Sequence
Subtypes with Differences in Virus Biology," J. Virol.,
68(11):7433-7447 (1992)] and SIVsmPbj1.9 [Dewhurst et al.,
"Sequence Analysis and Acute Pathogenicity of Molecularly Cloned
SIV.sub.smm-PBj14,"Nature, 345:636-640 (1990)] were obtained from
B.
[0106] Hahn (UAB). All other SIV.sub.mneisolates were obtained from
Julie Overbaugh (U.
[0107] Washington, Seattle). Stocks from cloned proviruses were
made by transient transfection of 293 cells. Other viral stocks
were made by passage of virus in human peripheral blood mononuclear
cells or in CEMx174 cells (for SIV stocks.) Viral stocks were
normalized by ELISA or p24.sup.gag (Coulter Immunology) or
p27.sup.gag (Coulter Immunology) for HIV-1 and HIV-2/SIV,
respectively, using standards provided by the manufacturer.
[0108] U373-MAGI-88C cells and U373-MAGI cells (controls) and were
infected with limiting dilutions of a T-tropic strain of HIV-1
(HIV.sub.LAI), an M-tropic strain (HIV.sub.YU-2), and an SIV
isolate, SIV.sub.MAC239.Infectivity was measured by counting the
number of blue cells per well per volume of virus (Table 2).
2 TABLE 2 titer on cell line (IU/ml).sup.b virus strain.sup.a
U373-MAGI U373-MAGI-88C HIV-1.sub.LAI <100 <100
HIV-1.sub.YU-2 <100 2.2 .times. 10.sup.6 SIV.sub.MAC239 1.2
.times. 10.sup.3 4 .times. 10.sup.5 .sup.aViruses derived by
transfection of molecular clones into 293 cells. .sup.bInfectious
units (IU) per ml is the number of blue cells per well multiplied
by the dilution of virus supernatant and normalized to 1 ml final
volume.
[0109] Two days after infection, cells were fixed and stained for
.beta.-galactosidase activity with X-gal. The U373-derived MAGI
cells were stained for 120 minutes at 370.degree. C. and the
HeLa-derived MAGI cells were stained for 50 minutes at 37.degree.
C. Background staining of non-infected cells never exceeded more
than approximately three blue cells per well. Only dark blue cells
were counted, and syncytium with multiple nuclei were counted as a
single infected cell. The infectious titer is the number of blue
cells per well multiplied by the dilution of virus and normalized
to 1 ml. The titer of HIV.sub.YU-2 on U373-MAGI-88C cells was
2.times.10.sup.6. In contrast, the titer of HIV-1.sub.LA1, was less
than 100 on U373-MAGI-88C. Thus, the specificity of a particular
HIV strain for 88C varied by four orders of magnitude.
[0110] Although SIV.sub.MAC239 infection was increased to
4.times.10.sup.5 in U373-MAGI-88C it also clearly infected
U373-MAGI cells (Table 2).
[0111] Next, a series of primary uncloned HIV strains and cloned
M-tropic strains of HIV-1 were analyzed for their ability to infect
indicator cell lines that express 88C.
[0112] As described above, HeLa-MAGI and HeLa-MAGI-88C cells were
infected with limiting dilutions of various HIV strains. The two
cloned M-tropic viruses, HIV.sub.JR-CSF and HIV.sub.YU-2, both
infected HeLa-MAGI-88C, but not HeLa-MAGI cells, showing that both
strains use 88C as a co-receptor (Table 3, See note c). However, a
great disparity in the ability of each of these two viral strains
to infect HeLa-MAGI-88C cells was observed, 6.2.times.10.sup.5
IU/ml for HIV.sub.YU-2 and 1.2.times.10.sup.4 for HIV.sub.JR-CSF.
The infectivity of virus stock (Table 3) is the number of
infectious units per physical particle (represented here by the
amount of viral core protein). In addition, it was observed that
the infectivity of these two cloned viral strains differed by over
50-fold in viral stocks that were independently prepared.
[0113] The variability of infectivity of primary viral isolates was
further examined by analyzing a collection of twelve different
uncloned virus stocks from three different clades (Table 3). Three
clade A primary isolates, three clade E isolates, and three
additional clade B isolates from geographically diverse origins
were used. With all nine strains, the primary strains of HIV could
be detected on HeLa-MAGI-88C cells, but not on HeLa-MAGI cells
(Table 3). However, the efficacy of infection varied from five
infectious units per ng p24.sup.gag to over 100 infectious units
per ng p24.sup.gag (table 3). These results indicate that absolute
infectivity of M-tropic strains varies considerably and is
independent of clade. A hypothesis that may explain this
discrepancy may involve the affinity of the V3 loop of each viral
strain for 88C after CD4 binding [Trkola et al., Nature,
384(6605):184-187 (1996); Wu et al., Nature, 384(6605):179-183
(1996)].
[0114] Table 3
3TABLE 3 viral sub-type titer (IU/ml) (country of on HeLa-
P24.sup.gag virus strain.sup.a origin).sup.b MAGI-88C.sup.c ng/ml
Infectivity.sup.d HIV-1.sub.YU-2 B (USA) 6.2 .times. 10.sup.5 2200
281 HIV-1.sub.JR-CSF B (USA) 12000 2800 4.2 HIV-1.sub.TH020 E
(Thailand) 4133 93 44 HIV-1.sub.TH021 E (Thailand) 4967 52 96
HIV-1.sub.TH022 E (Thailand) 200 15 13 HIV-1.sub.US660 B (USA) 2367
127 19 HIV-1.sub.UG031 A (Uganda) 1633 71 23 HIV-1.sub.RW009 A
(Rwanda) 3333 158 21 HIV-1.sub.RW026 A (Rwanda) 739 143 5.2
HIV-1.sub.US727 B (USA) 14,067 289 49 HIV-1.sub.US056 B (USA) 5833
284 21 HIV-1.sub.LAI B (France) 2.8 .times. 10.sup.5 167 1600
.sup.aHIV-1.sub.YU-2 and HIV-1.sub.JR-CSF were derived by
transfection of molecular clones. All others were tested as crude
supernatants of uncloned viral stocks derived from infection of
heterologous peripheral blood mononuclear cells. .sup.bClade
designation according to Myers et al., 1995 for the env gene;
country of origin refers to the country of residence of the
HIV-positive individual from whom blood was obtained for viral
isolation (World Health Organization Viral Isolate Program).
.sup.cInfectious units (IU) per ml is the number of blue cells per
well multiplied by the dilution of virus supernatant and normalized
to 1 ml final volume. All viruses, except HIV-1.sub.LAI, had less
than 10 IU/ml when tested on HeLa-MAGI cells without 88C.
HIV.sub.LAI, a T-tropic strain, has a titer of 2.8 .times. 10.sup.5
on HeLa-MAGI cells with or without 88C. .sup.dInfectivity is the
infectious units per ng P24.sup.gag (column four divided by column
five).
[0115] The ability of the HeLa-MAGI-88C cells to detect HIV-2 and
other SIV strains was also determined. HIV-2.sub.Rod has been
reported to use fusin as a receptor even in the absence of CD4
[Endres et al., Cell, 87(4):745-756 (1996)]. HIV-2.sub.Rod is able
to infect HeLa-MAGI cells, however its infectivity is enhanced at
least 10-fold in HeLa-MagI-88C (Table 4). HeLa cells endogenously
express fusin. Thus, the molecular clone of HIV-2.sub.Rod is dual
tropic, and is able to use 88C as one of its co-receptors in
addition to CXCR4. Similarly, a primary strain of HIV-2.sub.7312A
infected HeLa-MAGI-88C cells and not the HeLa-MAGI cells,
indicating that like primary strain of HIV-1, it uses 88C as a
receptor.
4TABLE 4 titer (IU/ml) titer (IU/ml) Infectivity on HeLa- on HeLa-
on HeLa- virus strain.sup.a reference MAGI.sup.b MAGI-88C
MAGI-88C.sup.c HIV-2.sub.ROD9 (Guyader et al., 967 5900 13 1987)
HIV-2.sub.7312A (Gao et al., <30 6500 17 1994) SIV.sub.MAC239
(Naidu et al., <30 20900 90 1988) SIV.sub.MNEc18 (Overbaugh et
<30 15700 19 al., 1991) SIV.sub.MNE170 (Rudensey et <30 10700
27 al., 1995) SIV.sub.SMPbj1.9 (Dewhurst et <30 776 ND.sup.d
al., 1990) SIV.sub.AGM9063 (Hirsch et al., <30 50 <1 1995)
.sup.aHIV-2 stocks, SIV.sub.SMPbj1.9, and SIV.sub.AGM9063 were
tested directly after by transfection of molecular clones in 293
cells. All others were derived from transfection of molecular
clones and subsequently amplified in CEMx174 cells.
.sup.bInfectious units (IU) per ml is the number of blue cells per
well multiplied by the dilution of virus supernatant and normalized
to 1 ml final volume. All viruses in this panel were also negative
on HeLa-MAGI-88-2B. .sup.cInfectivity is the infectious units (on
88C expressing cells) per ng P27.sup.gag determined by ELISA.
.sup.dND, not determined.
[0116] None of the SIV strains tested infected the HeLa-MAGI cells
(Table 4), and none infected HeLa-MAGI cells that expresses 88-2B.
This indicates that an alternative co-receptor used by SIV in U373
cells is not expressed in HeLa cells, and is not 88-2B. All SIV
strains tested infected the HeLa-MAGI-88C cells to some extent
(Table 3) indicating that all of the tested SIV strains use at
least 88C as one of their co-receptors.
[0117] The classification of M-tropic and T-tropic strains of HIV
in the past has often been correlated with another designation
"non-syncytium inducing" (NSI), and "syncytium inducing" (SI),
respectively. Assays based on the cell lines described herein are
sensitive to syncytium formation. The infected cells can form large
and small foci of infection containing multiple nuclei (Kimpton and
Emerman, supra).
[0118] Experiments using multiple different viral strains and
U373-MAGI-88C or HeLa-MAGI-88C indicate that SI/NSI designation is
not meaningful because all viral strains formed syncytia if the
correct co-receptor was present. These experiments show that
syncytium formation is more likely a marker for the presence of an
appropriate co-receptor on the infected cell, rather than an
indication of tropism. Infection of the HeLa-MAGI-88C cells with
SIV strains reported in the literature to be non-syncytium forming
strains, in particular, SIV.sub.MAC239, SIV.sub.MNEc18, and
SIV.sub.MNE170, was remarkable because the size of the syncytia
induced in the monolayer was much larger than those induced by any
other the HIV strains.
[0119] EXAMPLE 8
[0120] Mouse monoclonal antibodies which specifically recognize 88C
were prepared. The antibodies were produced by immunizing mice with
a peptide corresponding to the amino terminal twenty amino acids of
88C. The peptide was conjugated to Keyhole Limpet Cyanin (KLH)
according to the manufacturer's directions (Pierce, Imject
maleimide activated KLH), emulsified in complete Freund's adjuvant
and injected into five mice. Two additional injections of
conjugated peptide in incomplete Freund's adjuvant occurred at
three week intervals. Ten days after the final injection, serum
from each of the five mice was tested for immunoreactivity with the
twenty amino acid peptide by ELISA. In addition, the
immunoreactivity of the sera were tested against intact 88C
receptor expressed on the surface of 293 cells by fluorescence
activated cell sorting (FACS). The mouse with the best anti-88C
activity was chosen for spleen cell fusion and production of
monoclonal antibodies by standard laboratory methods. Five
monoclonal cell lines (227K, 227M, 227N, 227P, and 227R) were
established which produced antibodies that recognized the peptide
by ELISA and the 88C protein on 293 cells by FACS. Each antibody
was shown to react only with 88C-expressing 293 cells, but not with
293 cells expressing the closely related MCP receptor (CCCKR-2).
Each antibody was also shown to recognize 88C expressed transiently
in COS cells.
[0121] Rabbit polyclonal antibodies were also generated against
88C. Two rabbits were injected with conjugated amino-terminal
peptide as described above. The rabbits were further immunized by
four additional injections of the conjugated amino-terminal
peptide. Serum from each of the rabbits (2337J and 2470J) was
tested by FACS of 293 cells expressing 88C. The sera specifically
recognized 88C on the surface of 293 cells.
[0122] The five anti-88C monoclonal antibodies were tested for
their ability to block infection of cells by SIV, the simian
immunodeficiency virus closely related to HIV [Lehner et al.,
Nature Medicine, 2:767 (1996)]. Simian CD4.sup.+T cells, which are
normally susceptible to infection by SIV, were incubated with the
SIV.sub.mac32HJ5 clone in the presence of the anti-88C monoclonal
antibody supernatants diluted 1:5. SIV infection was measured by
determining reverse transcriptase (RT) activity on day nine using
the RT detection and quantification method (Quan-T-RT assay kit,
Amersham, Arlington Heights, Ill.). Four of the antibodies were
able to block SIV infection: antibody 227K blocked by 53%, 227M by
59%, 227N by 47% and 227P by 81%. Antibody 227R did not block SIV
infection.
[0123] The five monoclonal antibodies raised against human 88C
amino-terminal peptide were also tested for reactivity against
macaque 88C (SEQ ID NO: 20) (which has two amino acid differences
from human 88C within the amino-terminal peptide region).
[0124] The coding regions of human 88C and macaque 88C were cloned
into the expression vector pcDNA3 (Invitrogen). These expression
plasmids were used to transfect COS cells using DEAE. The empty
vector was used as a negative control. Three days after
transfection, cells were harvested and incubated with the five
anti-88C monoclonal antibodies and prepared for FACS. The results
showed that four of the five antibodies (227K, 227M, 227N, and
227P) recognized macaque 88C while one (227R) did not. All five
antibodies recognized the transfected human 88C, and none
cross-reacted with cells transfected with vector alone. On Feb. 4,
1997, the Applicants deposited hybridoma cell lines 227P, 227R, and
227M with the American Type Culture Collection (ATCC), which is
located at 10801 University Blvd., Manassas, Va. 20110-2209, USA,
pursuant to the provisions of the Budapest Treaty. These hybridoma
cell lines were accorded ATCC designations HB-12281, HB-12282, and
HB-12283, respectively.
EXAMPLE 9
[0125] Additional methods may be used to identify ligands and
modulators of the chemokine receptors of the invention.
[0126] In one embodiment, the invention comprehends a direct assay
for ligands.
[0127] Detectably labeled test compounds are exposed to membrane
preparations presenting chemokine receptors in a functional
conformation. For example, HEK-293 cells, or tissue culture cells,
are transfected with an expression vehicle encoding a chemokine
receptor. A membrane preparation is then made from the transfected
cells expressing the chemokine receptor. The membrane preparation
is exposed to .sup.125I-labeled test compounds (e.g., chemokines)
and incubated under suitable conditions (e.g., 10 minutes at
37.degree. C.). The membranes, with any bound test compounds, are
then collected on a filter by vacuum filtration and washed to
remove unbound test compounds. The radioactivity associated with
the bound test compound is then quantitated by subjecting the
filters to liquid scintillation spectrophotometry. The specificity
of test compound binding may be confirmed by repeating the assay in
the presence of increasing quantities of unlabeled test compound
and noting the level of competition for binding to the receptor.
These binding assays can also identify modulators of chemokine
receptor binding. The previously described binding assay may be
performed with the following modifications. In addition to
detectably labeled test compound, a potential modulator is exposed
to the membrane preparation. An increased level of
membrane-associated label indicates the potential modulator is an
activator; a decreased level of membrane-associated label indicates
the potential modulator is an inhibitor of chemokine receptor
binding.
[0128] In another embodiment, the invention comprehends indirect
assays for identifying receptor ligands that exploit the coupling
of chemokine receptors to G proteins. As reviewed in Linder et al.,
Sci. Am., 267:56-65 (1992), during signal transduction, an
activated receptor interacts with a G protein, in turn activating
the G protein. The G protein is activated by exchanging GDP for
GTP. Subsequent hydrolysis of the G protein-bound GTP deactivates
the G protein. One assay for G protein activity therefore monitors
the release of .sup.32p.sub.i from [.gamma.-.sup.32P]-GTP. For
example, approximately 5.times.10.sup.7 HEK-293 cells harboring
plasmids of the invention are grown in MEM+10% FCS. The growth
medium is supplemented with 5 mCi/ml [.sup.32P]-sodium phosphate
for 2 hours to uniformly label nucleotide pools. The cells are
subsequently washed in a low-phosphate isotonic buffer. One aliquot
of washed cells is then exposed to a test compound while a second
aliquot of cells is treated similarly, but without exposure to the
test compound. Following an incubation period (e.g., 10 minutes),
cells are pelleted, lysed and nucleotide compounds fractionated
using thin layer chromatography developed with 1 M LiCl. Labeled
GTP and GDP are identified by co-developing known standards. The
labeled GTP and GDP are then quantitated by autoradiographic
techniques that are standard in the art. Relatively high levels of
.sup.32P-labeled GDP identify test compounds as ligands. This type
of GTP hydrolysis assay is also useful for the identification of
modulators of chemokine receptor binding.
[0129] The aforementioned assay is performed in the presence of a
potential modulator. An intensified signal resulting from a
relative increase in GTP hydrolysis, producing 32P-labeled GDP,
indicates a relative increase in receptor activity. The intensified
signal therefore identifies the potential modulator as an
activator. Conversely, a diminished relative signal for
.sup.32P-labeled GDP, indicative of decreased receptor activity,
identifies the potential modulator as an inhibitor of chemokine
receptor binding.
[0130] The activities of G protein effector molecules (e.g.,
adenylyl cyclase, phospholipase C, ion channels, and
phosphodiesterases) are also amenable to assay.
[0131] Assays for the activities of these effector molecules have
been previously described. For example, adenylyl cyclase, which
catalyzes the synthesis of cyclic adenosine monophosphate (cAMP),
is activated by G proteins. Therefore, ligand binding to a
chemokine receptor that activates a G protein, which in turn
activates adenylyl cyclase, can be detected by monitoring cAMP
levels in a recombinant host cell of the invention. Implementing
appropriate controls understood in the art, an elevated level of
intracellular cAMP can be attributed to a ligand-induced increase
in receptor activity, thereby identifying a ligand. Again using
controls understood in the art, a relative reduction in the
concentration of cAMP would indirectly identify an inhibitor of
receptor activity. The concentration of cAMP can be measured by a
commercial enzyme immunoassay. For example, the BioTrak Kit
provides reagents for a competitive immunoassay (Amersham, Inc.,
Arlington Heights, Ill.). Using this kit according to the
manufacturer's recommendations, a reaction is designed that
involves competing unlabeled cAMP with cAMP conjugated to
horseradish peroxidase. The unlabeled cAMP may be obtained, for
example, from activated cells expressing the chemokine receptors of
the invention. The two compounds compete for binding to an
immobilized anti-cAMP antibody. After the competition reaction, the
immobilized horseradish peroxidase-cAMP conjugate is quantitated by
enzyme assay using a tetramethylbenzidine/H.sub.2O.sub.2 single-pot
substrate with detection of colored reaction products occurring at
450 nM. The results provide a basis for calculating the level of
unlabeled cAMP, using techniques that are standard in the art. In
addition to identifying ligands binding to chemokine receptors, the
cAMP assay can also be used to identify modulators of chemokine
receptor binding. Using recombinant host cells of the invention,
the assay is performed as previously described, with the addition
of a potential modulator of chemokine receptor activity. By using
controls that are understood in the art, a relative increase or
decrease in intracellular cAMP levels reflects the activation or
inhibition of adenylyl cyclase activity. The level of adenylyl
cyclase activity, in turn, reflects the relative activity of the
chemokine receptor of interest. A relatively elevated level of
chemokine receptor activity identifies an activator; a relatively
reduced level of receptor activity identifies an inhibitor of
chemokine receptor activity.
[0132] While the present invention has been described in terms of
specific embodiments, it is understood that variations and
modifications will occur to those skilled in the art. Accordingly,
only such limitations as appear in the appended claims should be
placed on the invention.
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