U.S. patent application number 10/804289 was filed with the patent office on 2004-10-14 for 7 transmembrane receptor family member blrx.
Invention is credited to Hedrick, Joseph A., Homey, Bernhard, Vicari, Alain, Zepeda, Monica L., Zlotnik, Albert.
Application Number | 20040203080 10/804289 |
Document ID | / |
Family ID | 46280023 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040203080 |
Kind Code |
A1 |
Hedrick, Joseph A. ; et
al. |
October 14, 2004 |
7 transmembrane receptor family member BLRx
Abstract
Novel chemokines and 7 transmembrane receptors from mammals,
reagents related thereto, including purified proteins, specific
antibodies, and nucleic acids encoding the chemokines and receptors
are disclosed. Methods of using the chemokines, receptors, reagents
and diagnostic kits are also provided.
Inventors: |
Hedrick, Joseph A.; (South
River, NJ) ; Homey, Bernhard; (Dusseldorf, DE)
; Vicari, Alain; (Lyon, FR) ; Zepeda, Monica
L.; (San Diego, CA) ; Zlotnik, Albert; (San
Diego, CA) |
Correspondence
Address: |
SCHERING-PLOUGH CORPORATION
PATENT DEPARTMENT (K-6-1, 1990)
2000 GALLOPING HILL ROAD
KENILWORTH
NJ
07033-0530
US
|
Family ID: |
46280023 |
Appl. No.: |
10/804289 |
Filed: |
March 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10804289 |
Mar 19, 2004 |
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09910695 |
Jul 20, 2001 |
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6737252 |
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09910695 |
Jul 20, 2001 |
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09122585 |
Jul 24, 1998 |
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60053693 |
Jul 25, 1997 |
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Current U.S.
Class: |
435/7.9 ;
530/350 |
Current CPC
Class: |
C07K 14/705 20130101;
C07K 14/521 20130101; A61K 39/00 20130101; C07K 14/522 20130101;
C07K 14/7158 20130101 |
Class at
Publication: |
435/007.9 ;
530/350 |
International
Class: |
G01N 033/53; G01N
033/542; C07K 014/715 |
Claims
What is claimed is:
1. A substantially pure polypeptide comprising at least 26
contiguous amino acids of the amino acid sequence set forth in SEQ
ID NO: 8.
2. A kit comprising said polypeptide of claim 1 and instructions
for use or disposal of reagents in said kit.
3. A substantially pure polypeptide comprising the amino acid
sequence set forth in SEQ ID NO: 8.
4. A kit comprising said polypeptide of claim 3 and instructions
for use or disposal of reagents in said kit.
5. The polypeptide of claim 3 wherein the polypeptide is detectably
labeled.
6. The polypeptide of claim 3 wherein the polypeptide is attached
to a solid substrate.
7. The polypeptide of claim 3 wherein the polypeptide is a fusion
protein.
8. A substantially pure polypeptide comprising a polypeptide that
is a 3-fold or less substituted form of the polypeptide defined by
the amino acid sequence set forth in SEQ ID NO: 8.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 09/910,695, filed Jul. 20, 2001, now allowed,
which is a continuation-in-part of U.S. application Ser. No.
09/122,585, filed Jul. 24, 1998, from which priority is claimed
pursuant to 35 USC .sctn.120, which application is related to U.S.
Provisional Application Serial No. 60/053,693, filed Jul. 25, 1997,
from which priority is claimed pursuant to 35 USC .sctn.119(e)(1),
which applications are incorporated herein by reference in their
entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions related to
proteins which function in controlling physiology, development,
and/or differentiation of mammalian cells. In particular, it
provides proteins which are implicated in the regulation of
physiology, development, differentiation, or function of various
cell types, e.g., chemokines, 7 transmembrane receptors, reagents
related to each, e.g., antibodies or nucleic acids encoding them,
and uses thereof.
BACKGROUND OF THE INVENTION
[0003] The circulating component of the mammalian circulatory
system comprises various cell types, including red and white blood
cells of the erythroid and myeloid cell lineages. See, e.g.,
Rapaport (1987) Introduction to Hematology (2d ed.) Lippincott,
Philadelphia, Pa.; Jandl (1987) Blood: Textbook of Hematology,
Little, Brown and Co., Boston, Mass.; and Paul (ed.) (1993)
Fundamental Immunology (3d ed.) Raven Press, N.Y.
[0004] For some time, it has been known that the mammalian immune
response is based on a series of complex cellular interactions,
called the "immune network." Recent research has provided new
insights into the inner workings of this network. While it remains
clear that much of the response does, in fact, revolve around the
network-like interactions of lymphocytes, macrophages,
granulocytes, and other cells, immunologists now generally hold the
opinion that soluble proteins, known as lymphokines, cytokines, or
monokines, play a critical role in controlling these cellular
interactions. Thus, there is considerable interest in the
isolation, characterization, and mechanisms of action of cell
modulatory factors, an understanding of which should lead to
significant advancements in the diagnosis and therapy of numerous
medical abnormalities, e.g., immune system and other disorders.
[0005] Lymphokines apparently mediate cellular activities in a
variety of ways. They have been shown to support the proliferation,
growth, and differentiation of the pluripotential hematopoietic
stem cells into vast numbers of progenitors comprising diverse
cellular lineages making up a complex immune system. These
interactions between the cellular components are necessary for a
healthy immune response. These different cellular lineages often
respond in a different manner when lymphokines are administered in
conjunction with other agents.
[0006] The chemokines are a large and diverse superfamily of
proteins. The superfamily is subdivided into two classical
branches, based upon whether the first two cysteines in the
chemokine motif are adjacent (termed the "C-C" branch), or spaced
by an intervening residue ("C-X-C"). A more recently identified
branch of chemokines lacks two cysteines in the corresponding
motif, and is represented by the chemokines known as lymphotactins.
Another recently identified branch has three intervening residues
between the two cysteines, e.g., CX3C chemokines. See, e.g., Schall
and Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon
and Schall (1996) Int. Arch. Allergy & Immunol. 109:97-109.
[0007] The chemokine receptors are typically members of the
superfamily of G-protein coupled (or linked) receptors (GPCR, or
GPLR). As a class, these receptors are integral membrane proteins
characterized by amino acid sequences which contain seven
hydrophobic domains. See, e.g., Ruffolo and Hollinger (eds. 1995)
G-Protein Coupled Transmembrane Signaling Mechanisms CRC Press,
Boca Raton, Fla.; Watson and Arkinstall (1994) The G-Protein Linked
Receptor FactsBook Academic Press, San Diego, Calif.; Peroutka (ed.
1994) G Protein-Coupled Receptors CRC Press, Boca Raton, Fla.;
Houslay and Milligan (1990) G-Proteins as Mediators of Cellular
Signaling Processes Wiley and Sons, New York, N.Y.; and Dohlman, et
al. (1991) Ann. Rev. Biochem. 60:653-688. These hydrophobic domains
are predicted to represent transmembrane spanning regions of the
proteins. These GPCRs are found in a wide range of organisms and
are typically involved in the transmission of signals to the
interior of the cell, e.g., through interaction, e.g., with
heterotrimeric G-proteins. They respond to a wide and diverse range
of agents including lipid analogs, amino acid derivatives, small
peptides, and other molecules.
[0008] The presumed transmembrane segments are typically 20-25
amino acids in length. Based upon models and data on
bacteriorhodopsin, these regions are predicted to be
.alpha.-helices and be oriented to form a ligand binding pocket.
See, e.g., Findley, et al. (1990) Trends Pharmacol. Sci.
11:492-499. Other data suggest that the amino termini of the
proteins are extracellular, and the carboxy termini are
intracellular. See, e.g., Lodish, et al. (1995) Molecular Cell
Biology 3d ed., Scientific American, New York; and Watson and
Arkinstall (1994) The G-Protein Linked Receptor FactsBook Academic
Press, San Diego, Calif. Phosphorylation cascades have been
implicated in the signal transduction pathway of these
receptors.
[0009] Although the full spectrum of biological activities mediated
by these 7 transmembrane receptors has not been fully determined,
chemoattractant effects are recognized. Chemokine receptors are
notable members of the GPCR family. See, e.g., Samson, et al.
(1996) Biochemistry 35:3362-3367; and Rapport, et al. (1996) J.
Leukocyte Biology 59:18-23. The best known biological functions of
these chemokine molecules relate to chemoattraction of leukocytes.
However, new chemokines and receptors are being discovered, and
their biological effects on the various cells responsible for
immunological responses are topics of continued study.
[0010] Many factors have been identified which influence the
differentiation process of precursor cells, or regulate the
physiology or migration properties of specific cell types. These
observations indicate that other factors exist whose functions in
immune function were heretofore unrecognized. These factors provide
for biological activities whose spectra of effects may be distinct
from known differentiation or activation factors. The absence of
knowledge about the structural, biological, and physiological
properties of the regulatory factors which regulate cell physiology
in vivo prevents the modulation of the effects of such factors.
[0011] In addition, other factors exist whose functions in
hematopoiesis, neural function, immune development, and leukocyte
trafficking were heretofore unrecognized. These receptors mediate
biological activities whose spectra of effects are distinct from
known differentiation, activation, or other signaling factors. The
absence of knowledge about the structural, biological, and
physiological properties of the receptors which regulate cell
physiology, development, or function prevents the modification of
the effects of such factors.
[0012] Thus, medical conditions where regulation of the development
or physiology of relevant cells is required remain
unmanageable.
SUMMARY OF THE INVENTION
[0013] The present invention is based, in part, upon the discovery
of new genes encoding various chemokines, e.g., those designated
CXC N4; or 7 transmembrane receptors, e.g., those designated
DNAXCCR10, which encode rodent receptors; and BLRx, which encode
primate receptors. Each GPCR gene encodes a polypeptide exhibiting
structural and/or sequence homology to 7 transmembrane receptors.
Such receptors are typically G-protein coupled (or linked)
receptors (GPCR or GPLR), though the complete set of ligands for
each has not yet been identified.
[0014] The invention also provides mutations (muteins) of the
respective natural sequences, fusion proteins, chemical mimetics,
antibodies, and other structural or functional analogs. It is also
directed to isolated nucleic acids, e.g., genes encoding respective
proteins of the invention. Various uses of these different protein,
antibody, or nucleic acid compositions are also provided.
[0015] The present invention provides a composition selected from
the group of: a substantially pure antigenic polypeptide comprising
sequence from a CXC N4, a DNAXCCR10, or BLRX; a binding composition
comprising an antigen binding portion of an antibody specific for
binding to such an antigenic polypeptide; a nucleic acid encoding
such an antigenic polypeptide; and a fusion protein comprising at
least two non-overlapping segments of at least 10 amino acids of
such an antigenic polypeptide.
[0016] In certain embodiments of the antigenic polypeptide, it is
from a warm blooded animal, e.g., a rodent or primate; it comprises
a sequence of FIGS. 1-5; it exhibits a post-translational
modification pattern distinct from a natural form of said
polypeptide; it is detectably labeled; or it is made by expression
of a recombinant nucleic acid. In other embodiments, a sterile form
is provided, including, e.g., composition comprising the
polypeptide and an acceptable carrier. A detection kit comprising a
compartment or container holding such an antigenic polypeptide is
also provided.
[0017] In other binding composition forms, e.g., antibody
embodiments, the polypeptide is a mouse or human protein; the
antibody is raised against a peptide sequence of FIGS. 1-5; the
antibody is a monoclonal antibody; the binding composition is fused
to a heterologous protein, or is detectably labeled. An alternative
embodiment is a binding compound comprising an antigen binding
fragment of the antibody described. Also provided is a detection
kit comprising such a binding compound. With the antibodies are
provided methods of purifying a polypeptide using the binding
compound or antibody to specifically separate the polypeptides from
others, or for detection, e.g., immunohistochemistry or
immunoprecipitation.
[0018] Nucleic acid embodiments are provided, e.g., where the
nucleic acid is in an expression vector and: encodes a polypeptide
from a mouse or human; comprises a sequence of a mature protein of
FIGS. 1-5; or comprises a deoxyribonucleic acid nucleotide. The
invention also provides a kit with such nucleic acids, e.g., which
include PCR primers for amplifying such sequences.
[0019] With nucleic acids are provided fusion proteins, comprising:
a sequence of FIGS. 1-5; and/or sequence of another chemokine or 7
transmembrane receptor, as appropriate. Also provided is a cell
comprising a recombinant nucleic acid, as described, and methods of
producing a polypeptide comprising expressing the nucleic acid in
an expression system.
[0020] Other embodiments include methods of modulating physiology
or development of a cell, or treating a disorder, with a step of
contacting that cell with a composition comprising an agonist or
antagonist of the chemokine or receptor. Ordinarily, the cell is a
neuron, macrophage, a lymphocyte, or a skin cell, such as found in
the epidermis or dermis. Various physiological effects to be
modulated include a cellular calcium flux, a chemoattractant
response, cellular morphology modification responses,
phosphoinositide lipid turnover, an antiviral response, or a
proliferative response.
[0021] Yet further embodiments are directed to a method comprising
administering to a subject in need thereof an effective amount of
an agent that modulates the expression or activity of BLRx or
fragments thereof. In a further embodiment, the method of treatment
or prevention comprises administering to a subject in need thereof
an effective amount of an agent that enhances or decreases the
activity or expression of BLRx or fragments thereof. The agent can
be, for example, a polynucleotide encoding BLRX, a polynucleotide
that includes a transcriptional or translational regulator, an
antibody that specifically binds to BLRx, an antisense
oligonucleotide having a sequence that binds to a sequence encoding
BLRx, or a small molecule inhibitor.
[0022] The present methods can be directed to the treatment of
various disorders, including, for example, wound healing,
proliferative disorders, fibrotic disorders, sclerotic disorders,
cancer, and angiogenesis.
[0023] In another aspect, the present invention encompasses
pharmaceutical compositions comprising an effective amount of an
agent that modulates the expression or activity of BLRx and a
suitable carrier. The agent can be a BLRx agonist or antagonist,
for example, or can comprise BLRx and a suitable carrier.
[0024] These and other embodiments of the present invention will
readily occur to those of ordinary skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 (SEQ ID NOS:1 and 2) depicts the partial nucleotide
sequence (5' to 3') and corresponding amino acid sequence of a
rodent embodiment of a chemokine designated CXC N4 (SEQ ID NO:1).
The predicted signal cleavage site is at approximately the
gly19-gln20 peptide bond. The CXC motif corresponds to residues
cys27 through cys29.
[0026] FIGS. 2A-2B (SEQ ID NOS:3 and 4) show the nucleotide
sequence (5' to 3') and corresponding amino acid sequence of a
human embodiment of a chemokine receptor similar to one designated
GPCR W. Nucleotides 1106 and 1139 differ from the previously
reported human and canine sequences.
[0027] FIGS. 3A-3C (SEQ ID NOS:7 and 8) show the nucleotide
sequence (5' to 3') and corresponding amino acid sequence of a
human embodiment of a primate 7 transmembrane receptor family
member, designated BLRx. Ambiguities in the sequence are as
follows: residue 1462 may be G/T; 1473 may be A/C; 1490 may be
A/C/G/T; and 1495 may be A/T. Only the first is in the coding
sequence, thus residue 193 may be gly or arg.
[0028] FIGS. 4A-4B (SEQ ID NOS:9 and 10) show a mouse DNAXCCR10
nucleotide sequence segment and the corresponding amino acid
sequence.
[0029] FIG. 5 (SEQ ID NOS:5 and 6) shows a partial mouse DNAXCCR10
nucleotide sequence segment and corresponding amino acid
sequence.
[0030] FIG. 6 shows expression levels of BLRx in various tissues
and cell types. Expression levels were normalized and expressed as
femtograms mRNA per 50 ng total cDNA. Acronyms are as follows:
DMEC=dermal microvascular endothelial cell; DMEC N=untreated dermal
microvascular endothelial cell; FB=fibroblast; FB N=untreated
fibroblast; MC=melanocyte; MC N=untreated melanocyte; and
KC=keratinocyte.
[0031] FIG. 7 shows BLRx expression during various phases of wound
healing.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] All references cited herein are incorporated herein by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference.
[0033] I. General
[0034] The present invention provides DNA sequences encoding
various mammalian proteins, including chemokines, or which exhibit
structural properties characteristic of a 7 transmembrane receptor.
See, e.g., Ruffolo and Hollinger (eds. 1995) G-Protein Coupled
Transmembrane Signaling Mechanisms CRC Press, Boca Raton, Fla.;
Watson and Arkinstall (1994) The G-Protein Linked Receptor
FactsBook Academic Press, San Diego, Calif.; Peroutka (ed. 1994) G
Protein-Coupled Receptors CRC Press, Boca Raton, Fla.; Houslay and
Milligan (1990) G-Proteins as Mediators of Cellular Signaling
Processes Wiley and Sons, New York, N.Y. Certain mouse and human
embodiments are described herein.
[0035] Among the many types of ligands which mediate biology via
these receptors are chemokines and certain proteases. Chemokines
play an important role in immune and inflammatory responses by
inducing migration and adhesion of leukocytes. See, e.g., Schall
(1991) Cytokine 3:165-183; and Thomson (ed.) The Cytokine Handbook
Academic Press, NY. Chemokines are secreted by activated leukocytes
and act as a chemoattractant for a variety of cells which are
involved in inflammation. Besides chemoattractant properties,
chemokines have been shown to induce other biological responses,
e.g., modulation of second messenger levels such as Ca.sup.++;
inositol phosphate pool changes (see, e.g., Berridge (1993) Nature
361:315-325 or Billah and Anthes (1990) Biochem. J. 269:281-291);
cellular morphology modification responses; phosphoinositide lipid
turnover; possible antiviral responses; and others. Thus, the
chemokines provided herein may, alone or in combination with other
therapeutic reagents, have advantageous combination effects.
[0036] Moreover, there are reasons to suggest that chemokines may
have effects on other cell types, e.g., attraction or activation of
monocytes, dendritic cells, T cells, eosinophils, and/or perhaps on
basophils and/or neutrophils. They may also have chemoattractive
effects on various neural cells including, e.g., dorsal root
ganglia neurons in the peripheral nervous system and/or central
nervous system neurons.
[0037] G-protein coupled receptors, e.g., chemokine receptors, are
important in the signal transduction mechanisms mediated by their
ligands. They are useful markers for distinguishing cell
populations, and have been implicated as specific receptors for
retroviral infections.
[0038] The chemokine superfamily was classically divided into two
groups exhibiting characteristic structural motifs, the Cys-X-Cys
(C-X-C) and Cys-Cys (C-C) families. These were distinguished on the
basis of a single amino acid insertion between the NH-proximal pair
of cysteine residues and sequence similarity. Typically, the C-X-C
chemokines, i.e., IL-8 and MGSA/Gro-.alpha. act on neutrophils but
not on monocytes, whereas the C-C chemokines, i.e., MIP-1.alpha.
and RANTES, are potent chemoattractants for monocytes and
lymphocytes but not neutrophils. See, e.g., Miller, et al. (1992)
Crit. Rev. Immunol. 12:17-46. A recently isolated chemokine,
lymphotactin, does not belong to either group and may constitute a
first member of a third chemokine family, the C family.
Lymphotactin does not have a characteristic CC or CXC motif, and
acts on lymphocytes but not neutrophils and monocytes. See, e.g.,
Kelner et al. (1994) Science 266:1395-1399. This chemokine defines
a new C-C chemokine family. Even more recently, another chemokine
exhibiting a CX3C motif has been identified, which establishes a
fourth structural class.
[0039] The present invention provides additional chemokine
reagents, e.g., nucleic acids, proteins and peptides, antibodies,
etc., related to the newly discovered chemokine designated rodent
CXC N4.
[0040] In other embodiments, the invention provides genes encoding
novel G-protein coupled receptors, designated mouse DNAXCCR10 and
primate BLRX. Their ligands have not yet specifically been
identified, but the DNAXCCR10 responds to binding to the chemokine
MIP-3.alpha.. See Hieshima, et al. (1997) J. Biol. Chem.
272:5846-5853; Hromas, et al. (1997) Blood 89:3315-3322; and Baba,
et al. (1997) J. Biol. Chem. 272:14893-14898. The receptors exhibit
structural features typical of known 7 transmembrane spanning
receptors, which receptors include chemokine receptors. The
receptors may exhibit properties of binding many different
cytokines at varying specificities (shared or promiscuous binding
specificity) or may exhibit high affinity for one (specific) or a
subset (shared) of chemokines. Alternatively, the ligands may be
other molecules, including molecules such as epinephrine,
serotonin, or glucagon.
[0041] The described chemokines or receptors should be important
for mediating various aspects of cellular, organ, tissue, or
organismal physiology or development.
[0042] II. Purified Chemokines; Receptors
[0043] Nucleotide and derived amino acid sequences of a novel
rodent CXC chemokine, e.g., from mouse, designated CXC N4 are shown
in FIG. 1 (SEQ ID NOS:1 and 2). The gene encodes a novel protein
exhibiting structure and motifs characteristic of a chemokine. Its
closest reported chemokines are the mouse SDF-1, IP-10, and MIG
chemokines. See, e.g., Aiuti, et al. (1997) J. Exp. Med.
185:111-120; Loetscher, et al. (1996) J. Exp. Med. 184:963-969; and
Sgadari, et al. (1997) Blood 89:2635-2643.
[0044] Nucleotide and derived amino acid sequences of a novel
primate GPCR, e.g., from human, designated DNAXCCR10, are shown in
FIGS. 2A-2B (SEQ ID NOS:3 and 4). Sequence analysis shows closest
sequence homology to a human GPCR designated GPCR W (accession
number L42324) or to a canine GPCR W (accession number L42326); and
to a mouse sequence (accession number AA423677). As indicated
above, the receptor responds to binding with chemokine ligand
MIP-3.alpha., which immediately allows for screening for ligands
since a positive control is available.
[0045] Nucleotide and derived amino acid sequences of a rodent
GPCR, e.g., from mouse, DNAXCCR10, are shown in FIGS. 4A-4B (SEQ ID
NOS:9 and 10). A partial mouse sequence is shown in FIG. 5 (SEQ ID
NOS:5 and 6)
[0046] Nucleotide and derived amino acid sequences of a novel
primate, e.g., from human, 7 transmembrane receptor family member,
designated BLRX herein, are shown in FIGS. 3A-3C (SEQ ID NOS:7 and
8). Sequence analysis shows sequence homology to various GPCR
family members, particularly the bovine gustative receptor. See
Forster, et al. (1996) Cell 13:1037-1047.
[0047] Certain general descriptions of physical properties of
polypeptides, nucleic acids, and antibodies, where directed to one
embodiment clearly are usually applicable to other chemokines or
receptors described herein.
[0048] These amino acid sequences, provided amino to carboxy, are
important in providing sequence information on the chemokine ligand
or receptor, allowing for distinguishing the protein from other
proteins, particularly naturally occurring versions. Moreover, the
sequences allow preparation of peptides to generate antibodies to
recognize and distinguish such segments, and allow preparation of
oligonucleotide probes, both of which are strategies for isolation,
e.g., cloning, of genes encoding such sequences, or related
sequences, e.g., natural polymorphic or other variants, including
fusion proteins. Similarities of the chemokines have been observed
with other cytokines. See, e.g., Bosenberg, et al. (1992) Cell
71:1157-1165; Huang, et. al. (1992) Molecular Biology of the Cell
3:349-362; and Pandiella, et al. (1992) J. Biol. Chem.
267:24028-24033. Likewise for the GPC receptors.
[0049] As used herein, the term "CXC N4" shall encompass, when used
in a protein context, a protein having an amino acid sequence as
shown in FIG. 1. Similarly, by the term "BLRx" is meant a protein
with an amino acid sequence as depicted in FIGS. 3A-3C herein.
Likewise, the term "DNAXCCR10" refers to a protein having the amino
acid sequence depicted in FIGS. 2A-2B. Preferably, such sequences
will be the native or natural sequence, e.g., a sequence
corresponding to the native sequence from a mammal, such as a
primate, rodent, etc. The invention also embraces a polypeptide
comprising a significant fragment of such protein. The invention
also encompasses a counterpart polypeptide from another mammalian
species, e.g., which exhibits similar sequence, and is more
homologous in the native coding sequence than other genes from the
species. Typically, in the case of homologs of the reference
sequence, the molecule will interact with its specific binding
components, such as antibodies which bind the sequence, its
receptor if the molecule is a chemokine, or the appropriate ligand,
if the molecule is a receptor. These binding components, e.g.,
antibodies, typically bind to the chemokine with high affinity,
e.g., at least about 100 nM, usually better than about 30 nM,
preferably better than about 10 nM, and more preferably at better
than about 3 nM. Similar concepts apply to the binding components
which recognize mammalian embodiments for the GPCRs DNAXCCR10 and
BLRx.
[0050] The term "polypeptide" as used herein includes a significant
fragment or segment, and encompasses a stretch of amino acid
residues of at least about 8 amino acids, generally at least 10
amino acids, more generally at least 12 amino acids, often at least
14 amino acids, more often at least 16 amino acids, typically at
least 18 amino acids, more typically at least 20 amino acids,
usually at least 22 amino acids, more usually at least 24 amino
acids, preferably at least 26 amino acids, more preferably at least
28 amino acids, and, in particularly preferred embodiments, at
least about 30 or more amino acids, e.g., about 35, 40, 45, 50,
etc. Similar proteins will likely comprise a plurality of such
segments. Such fragments may have ends which begin and/or end at
virtually all positions, e.g., beginning at residues 1, 2, 3, etc.,
and ending at, e.g., 54, 53, 52, etc., in all combinatorial pairs
in the coding segment. Typically, the plurality will be at least
two, more usually at least three, and preferably 5, 7, or even
more. While the length minima are provided, longer lengths, of
various sizes, may be appropriate, e.g., one of length 7, and two
of length 12.
[0051] Particularly interesting peptides have ends corresponding to
structural domain boundaries, e.g., intracellular or extracellular
loops of the receptor embodiments. Such peptides will typically be
immunogenic peptides, e.g., including peptides of at least 12, 14,
16, etc., residues or may be concatenated to generate larger
polypeptides. Short peptides may be attached or coupled to a larger
carrier.
[0052] The term "binding composition" refers to molecules that bind
with specificity to the respective chemokine or receptor, e.g., in
antibody-antigen interaction, or alternatively in a ligand-receptor
type fashion. These compositions may be compounds, e.g., proteins,
which specifically associate with the chemokine or receptor,
including natural physiologically relevant protein-protein
interactions, either covalent or non-covalent. The binding
composition may be a polymer, or another chemical reagent. No
implication as to whether the chemokine presents a concave or
convex shape in its ligand-receptor interaction is necessarily
represented, other than the interaction exhibit similar
specificity, e.g., specific affinity. A functional analog may be a
ligand with structural modifications, or may be a wholly unrelated
molecule, e.g., which has a molecular shape which interacts with
the appropriate ligand binding determinants. The ligands may serve
as agonists or antagonists of a physiological or natural receptor,
see, e.g., Goodman, et al. (eds.) (1990) Goodman & Gilman's:
The Pharmacological Bases of Therapeutics (8th ed.), Pergamon
Press. The term expressly includes compounds comprising antigen
binding portions of antibodies, polyclonal or monoclonal, e.g.,
which specifically bind to the respective antigen.
[0053] Substantially pure means that the protein is free from other
contaminating proteins, nucleic acids, and/or other biologicals
typically derived from the original source organism. Purity may be
assayed by standard methods, and will ordinarily be at least about
40% pure, more ordinarily at least about 50% pure, generally at
least about 60% pure, more generally at least about 70% pure, often
at least about 75% pure, more often at least about 80% pure,
typically at least about 85% pure, more typically at least about
90% pure, preferably at least about 95% pure, more preferably at
least about 98% pure, and in most preferred embodiments, at least
99% pure. Analyses will typically be by weight, but may be by molar
amounts.
[0054] Solubility of a polypeptide or fragment depends upon the
environment and the polypeptide. Many parameters affect polypeptide
solubility, including temperature, electrolyte environment, size
and molecular characteristics of the polypeptide, and nature of the
solvent. Typically, the temperature at which the polypeptide is
used ranges from about 4.degree. C. to about 65.degree. C. Usually
the temperature at use is greater than about 18.degree. C. and more
usually greater than about 22.degree. C. For diagnostic purposes,
the temperature will usually be about room temperature or warmer,
but less than the denaturation temperature of components in the
assay. For therapeutic purposes, the temperature will usually be
body temperature, typically about 37.degree. C. for humans, though
under certain situations the temperature may be raised or lowered
in situ or in vitro.
[0055] The electrolytes will usually approximate in situ
physiological conditions, but may be modified to higher or lower
ionic strength where advantageous. The actual ions may be modified,
e.g., to conform to standard buffers used in physiological or
analytical contexts.
[0056] The size and structure of the polypeptide should generally
be in a substantially stable state, and usually not in a denatured
state, though in certain circumstances denatured protein will be
important. The polypeptide may be associated with other
polypeptides in a quaternary structure, e.g., to confer solubility,
or associated with lipids or detergents in a manner which
approximates natural lipid bilayer interactions.
[0057] The solvent will usually be a biologically compatible
buffer, of a type used for preservation of biological activities,
and will usually approximate a physiological solvent. Usually the
solvent will have a neutral pH, typically at least about 5,
preferably at least 6, and typically less than 10, preferably less
than 9, and more preferably about 7.5. On some occasions, a
detergent will be added, typically a mild non-denaturing one, e.g.,
CHS (cholesteryl hemisuccinate) or CHAPS
(3-([3-cholamido-propyl]dimethylammonio)-1-propane sulfonate), or a
low enough concentration as to avoid significant disruption of
structural or physiological properties of the protein.
[0058] Solubility is reflected by sedimentation measured in
Svedberg units, which are a measure of the sedimentation velocity
of a molecule under particular conditions. The determination of the
sedimentation velocity was classically performed in an analytical
ultracentrifuge, but is typically now performed in a standard
ultracentrifuge. See, Freifelder (1982) Physical Biochemistry (2d
ed.), W. H. Freeman; and Cantor and Schimmel (1980) Biophysical
Chemistry, parts 1-3, W. H. Freeman & Co., San Francisco. As a
crude determination, a sample containing a putatively soluble
polypeptide is spun in a standard full sized ultracentrifuge at
about 50 K rpm for about 10 minutes, and soluble molecules will
remain in the supernatant. A soluble particle or polypeptide will
typically be less than about 30 S, more typically less than about
15 S, usually less than about 10 S, more usually less than about 6
S, and, in particular embodiments, preferably less than about 4 S,
and more preferably less than about 3 S.
[0059] III. Physical Variants
[0060] This invention also encompasses proteins or peptides having
substantial amino acid sequence homology with the amino acid
sequence of each respective chemokine or chemokine receptor. The
variants include species or polymorphic variants.
[0061] Amino acid sequence homology, or sequence identity, is
determined by optimizing residue matches, if necessary, by
introducing gaps as required. This changes when considering
conservative substitutions as matches. Conservative substitutions
typically include substitutions within the following groups:
glycine, alanine; valine, isoleucine, leucine; aspartic acid,
glutamic acid; asparagine, glutamine; serine, threonine; lysine,
arginine; and phenylalanine, tyrosine. Homologous amino acid
sequences are typically intended to include natural allelic and
interspecies variations in each respective protein sequence.
Typical homologous proteins or peptides will have from 25-100%
homology (if gaps can be introduced), to 50-100% homology (if
conservative substitutions are included) with the amino acid
sequence of the appropriate chemokine or receptor, or any
percentage between these stated ranges. Homology measures will be
at least about 35%, generally at least 40%, more generally at least
45%, often at least 50%, more often at least 55%, typically at
least 60%, more typically at least 65%, usually at least 70%, more
usually at least 75%, preferably at least 80%, and in particularly
preferred embodiments, at least 85%, 90%, 95% or more. See also
Needleham, et al. (1970) J. Mol. Biol. 48:443-453; Sankoff, et al.
(1983) Chapter One in Time Warps, String Edits, and Macromolecules:
The Theory and Practice of Sequence Comparison Addison-Wesley,
Reading, Mass.; and software packages from IntelliGenetics,
Mountain View, Calif.; and the University of Wisconsin Genetics
Computer Group, Madison, Wis.
[0062] Each of the isolated chemokine or GPC receptor DNAs can be
readily modified by nucleotide substitutions, nucleotide deletions,
nucleotide insertions, and inversions of nucleotide stretches.
These modifications may result in novel DNA sequences which encode
these proteins, their derivatives, or proteins having similar
physiological, immunogenic, or antigenic activity. These modified
sequences can be used to produce mutant proteins or to enhance
expression, or to introduce convenient enzyme recognition sites
into the nucleotide sequence without significantly affecting the
encoded protein sequence. Enhanced expression may involve gene
amplification, increased transcription, increased translation, and
other mechanisms. Enhanced expression may be useful in the context
of wound healing, as described further below. Such mutant receptor
derivatives include predetermined or site-specific mutations of the
respective protein or its fragments. "Mutant chemokine" or "mutant
chemokine receptor" encompasses a polypeptide otherwise falling
within the homology definition of the chemokine or receptor as set
forth above, but having an amino acid sequence which differs from
that of the chemokine or receptor as found in nature, whether by
way of deletion, substitution, or insertion. These include amino
acid residue substitution levels from none, one, two, three, five,
seven, ten, twelve, fifteen . . . 75 etc., and any number within
this range. In particular, "site specific mutant" generally
includes proteins having significant homology with a protein having
sequences of FIGS. 1-5, and as sharing various biological
activities, e.g., antigenic or immunogenic, ligand binding, with
those sequences, and in preferred embodiments contain a plurality,
or most, of disclosed sequences, particularly those found in
various related groups of animals. As stated before, it is
emphasized that descriptions are generally meant to encompass the
various chemokine or receptor proteins from other members of
related groups, not limited to the mouse or human embodiments
specifically discussed.
[0063] Although site specific mutation sites are often
predetermined, mutants need not be site specific. Chemokine or
receptor mutagenesis can be conducted by making amino acid
insertions or deletions, including with PCR mutagenesis.
Substitutions, deletions, insertions, or combinations may be
generated to arrive at a final construct. Insertions include amino-
or carboxy-terminal fusions. Random mutagenesis can be conducted at
a target codon and the expressed mutants can then be screened for
the desired activity. Methods for making substitution mutations at
predetermined sites in DNA having a known sequence are well known
in the art, e.g., by M13 primer mutagenesis or polymerase chain
reaction (PCR) techniques. See also Sambrook, et al. (1989) and
Ausubel, et al. (1987 and Supplements). Many structural features
are known about the chemokines and GPCRs which allow determination
of whether specific residues are embedded into the core of the
secondary or tertiary structures, or whether the residues will have
relatively little effect on protein folding. Preferred positions
for mutagenesis are those which do not prevent functional folding
of the resulting protein.
[0064] The mutations in the DNA normally should not place coding
sequences out of reading frames and preferably will not create
complementary regions that could hybridize to produce secondary
mRNA structure such as loops or hairpins. But certain situations
exist where such problems are compensated. See, e.g., Gesteland and
Atkins (1996) Ann. Rev. Biochem. 65:741-768.
[0065] The present invention also provides recombinant proteins,
e.g., heterologous fusion proteins using segments from these
proteins, or antibodies. A heterologous fusion protein is a fusion
of proteins or segments which are naturally not normally fused in
the same manner. Thus, the fusion product of an immunoglobulin with
a receptor polypeptide is a continuous protein molecule having
sequences fused in a typical peptide linkage, typically made as a
single translation product and exhibiting properties derived from
each source peptide. A similar chimeric concept applies to
heterologous nucleic acid sequences.
[0066] In addition, new constructs may be made from combining
similar functional or structural domains from other proteins. For
example, ligand-binding or other segments may be "swapped" between
different new fusion polypeptides or fragments. See, e.g.,
Cunningham, et al. (1989) Science 243:1330-1336; and O'Dowd, et al.
(1988) J. Biol. Chem. 263:15985-15992. Thus, new chimeric
polypeptides exhibiting new combinations of specificities will
result from the functional linkage of ligand-binding specificities
and other functional domains. Such may be chimeric molecules with
mixing or matching of the various structural segments, e.g., the
.beta.-sheet or .alpha.-helix structural domains for the chemokine,
or receptor segments corresponding to each of the transmembrane
segments (TM1-TM7), or the intracellular (cytosolic, C1-C4) or
extracellular (E1-E4) loops from the various receptor types. The C3
loop is particularly important.
[0067] The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable
synthetic DNA fragments. A double stranded fragment will often be
obtained either by synthesizing the complementary strand and
annealing the strand together under appropriate conditions or by
adding the complementary strand using DNA polymerase with an
appropriate primer sequence, e.g., PCR techniques.
[0068] IV. Functional Variants
[0069] The blocking of physiological response to various
embodiments of these chemokines or GPCRs may result from the
inhibition of binding of the ligand to its receptor, likely through
competitive inhibition. Thus, in vitro assays of the present
invention will often use isolated protein, membranes from cells
expressing a recombinant membrane associated receptor, e.g., ligand
binding segments, or fragments attached to solid phase substrates.
These assays will also allow for the diagnostic determination of
the effects of either binding segment mutations and modifications,
or ligand mutations and modifications, e.g., ligand analogs.
[0070] This invention also contemplates the use of competitive drug
screening assays, e.g., where neutralizing binding compositions,
e.g., antibodies, to antigen or receptor fragments compete with a
test compound for binding to the protein. In this manner, the
antibodies can be used to detect the presence of polypeptides which
share one or more antigenic binding sites of the ligand and can
also be used to occupy binding sites on the protein that might
otherwise interact with a receptor.
[0071] Additionally, neutralizing antibodies against a specific
chemokine embodiment and soluble fragments of the chemokine which
contain a high affinity receptor binding site, can be used to
inhibit chemokine activity in tissues, e.g., tissues experiencing
abnormal physiology.
[0072] "Derivatives" of chemokine proteins include amino acid
sequence mutants, glycosylation variants, and covalent or aggregate
conjugates with other chemical moieties. Covalent derivatives can
be prepared by linkage of functionalities to groups which are found
in chemokine amino acid side chains or at the N- or C-termini, by
means which are well known in the art. These derivatives can
include, without limitation, aliphatic esters or amides of the
carboxyl terminus, or of residues containing carboxyl side chains,
O-acyl derivatives of hydroxyl group-containing residues, and
N-acyl derivatives of the amino terminal amino acid or amino-group
containing residues, e.g., lysine or arginine. Acyl groups are
selected from the group of alkyl-moieties including C3 to C18
normal alkyl, thereby forming alkanoyl aroyl species. Covalent
attachment to carrier proteins may be important when immunogenic
moieties are haptens.
[0073] In particular, glycosylation alterations are included, e.g.,
made by modifying the glycosylation patterns of a polypeptide
during its synthesis and processing, or in further processing
steps. Particularly preferred means for accomplishing this are by
exposing the polypeptide to glycosylating enzymes derived from
cells which normally provide such processing, e.g., mammalian
glycosylation enzymes. Deglycosylation enzymes are also
contemplated. Also embraced are versions of the same primary amino
acid sequence which have other minor modifications, including
phosphorylated amino acid residues, e.g., phosphotyrosine,
phosphoserine, or phosphothreonine, or nucleoside or nucleotide
derivatives, e.g., guanyl derivatized.
[0074] A major group of derivatives are covalent conjugates of the
respective chemokine or receptor or fragments thereof with other
proteins or polypeptides. These derivatives can be synthesized in
recombinant culture such as - or C-terminal fusions or by the use
of agents known in the art for their usefulness in cross-linking
proteins through reactive side groups. Preferred chemokine
derivatization sites with cross-linking agents are at free amino
groups, carbohydrate moieties, and cysteine residues.
[0075] Fusion polypeptides between these chemokines or receptors
and other homologous or heterologous proteins, e.g., other
chemokines or receptors, are also provided. Many growth factors and
cytokines are homodimeric entities, and a repeat construct may have
various advantages, including lessened susceptibility to
proteolytic cleavage. Moreover, many cytokine receptors require
dimerization to transduce a signal, and various dimeric ligands or
domain repeats can be desirable. Homologous polypeptides may be
fusions between different surface markers, resulting in, e.g., a
hybrid protein exhibiting receptor binding specificity. Likewise,
heterologous fusions may be constructed which would exhibit a
combination of properties or activities of the derivative proteins.
Typical examples are fusions of a reporter polypeptide, e.g.,
luciferase, with a segment or domain of a ligand, e.g., a
receptor-binding segment, so that the presence or location of the
fused ligand, or a binding composition, may be easily determined.
See, e.g., Dull, et al., U.S. Pat. No. 4,859,609. Other gene fusion
partners include bacterial .beta.-galactosidase, trpE, Protein A,
.beta.-lactamase, alpha amylase, alcohol dehydrogenase, a FLAG
fusion, and yeast alpha mating factor. See, e.g., Godowski, et al.
(1988) Science 241:812-816.
[0076] The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable
synthetic DNA fragments. A double stranded fragment will often be
obtained either by synthesizing the complementary strand and
annealing the strand together under appropriate conditions or by
adding the complementary strand using DNA polymerase with an
appropriate primer sequence.
[0077] Such polypeptides may also have amino acid residues which
have been chemically modified by phosphorylation, guanylation,
sulfonation, biotinylation, or the addition or removal of other
moieties, particularly those which have molecular shapes similar to
phosphate or guanyl groups. In some embodiments, the modifications
will be useful labeling reagents, or serve as purification targets,
e.g., affinity tags as FLAG.
[0078] Fusion proteins will typically be made by either recombinant
nucleic acid methods or by synthetic polypeptide methods.
Techniques for nucleic acid manipulation and expression are
described generally, for example, in Sambrook, et al. (1989)
Molecular Cloning: A Laboratory Manual (2d ed.), Vols. 1-3, Cold
Spring Harbor Laboratory. Techniques for synthesis of polypeptides
are described, for example, in Merrifield (1963) J. Amer. Chem.
Soc. 85:2149-2156; Merrifield (1986) Science 232: 341-347; and
Atherton, et al. (1989) Solid Phase Peptide Synthesis: A Practical
Approach, IRL Press, Oxford; and chemical ligation, e.g., Dawson,
et al. (1994) Science 266:776-779, a method of linking long
synthetic peptides by a peptide bond.
[0079] This invention also contemplates the use of derivatives of
these chemokines or receptors other than variations in amino acid
sequence or glycosylation. Such derivatives may involve covalent or
aggregative association with chemical moieties. These derivatives
generally include: (1) salts, (2) side chain and terminal residue
covalent modifications, and (3) adsorption complexes, for example
with cell membranes. Such covalent or aggregative derivatives are
useful as immunogens, as reagents in immunoassays, or in
purification methods such as for affinity purification of ligands
or other binding ligands. For example, a chemokine antigen can be
immobilized by covalent bonding to a solid support such as cyanogen
bromide-activated Sepharose, by methods which are well known in the
art, or adsorbed onto polyolefin surfaces, with or without
glutaraldehyde cross-linking, for use in the assay or purification
of anti-chemokine antibodies or its receptor. These chemokines can
also be labeled with a detectable group, for example radioiodinated
by the chloramine T procedure, covalently bound to rare earth
chelates, or conjugated to a fluorescent moiety for use in
diagnostic assays. Purification of chemokine, receptor, or binding
compositions may be effected by immobilized antibodies or
receptor.
[0080] Other modifications may be introduced with the goal of
modifying the therapeutic pharmacokinetics or pharmacodynamics of a
target chemokine. For example, certain means to minimize the size
of the entity may improve its pharmacoaccessibility; other means to
maximize size may affect pharmacodynamics. Similarly, changes in
ligand binding kinetics or equilibrium of a receptor may be
engineered.
[0081] A solubilized chemokine or receptor or appropriate fragment
of this invention can be used as an immunogen for the production of
antisera or antibodies specific for the ligand, receptor, or
fragments thereof. The purified proteins can be used to screen
monoclonal antibodies or chemokine-binding fragments prepared by
immunization with various forms of impure preparations containing
the protein. In particular, antibody equivalents include antigen
binding fragments of natural antibodies, e.g., Fv, Fab, or
F(ab).sub.2. Purified chemokines can also be used as a reagent to
detect antibodies generated in response to the presence of elevated
levels of the protein or cell fragments containing the protein,
both of which may be diagnostic of an abnormal or specific
physiological or disease condition. Additionally, chemokine protein
fragments, or their concatenates, may also serve as immunogens to
produce binding compositions, e.g., antibodies of the present
invention, as described immediately below. For example, this
invention contemplates antibodies raised against certain amino acid
sequences, e.g., in FIGS. 1-5, or proteins containing them. In
particular, this invention contemplates antibodies having binding
affinity to or being raised against specific fragments, e.g., those
which are predicted to lie on the outside surfaces of protein
tertiary structure. Similar concepts apply to antibodies specific
for receptors of the invention.
[0082] The present invention contemplates the isolation of
additional closely related species variants. Southern and Northern
blot analysis should establish that similar genetic entities exist
in other related mammals, and establish the stringency of
hybridization conditions to isolate such. It is likely that these
chemokines and receptors are widespread in species variants, e.g.,
among the rodents and the primates.
[0083] The invention also provides means to isolate a group of
related chemokines or receptors displaying both distinctness and
similarities in structure, expression, and function. Elucidation of
many of the physiological effects of the proteins will be greatly
accelerated by the isolation and characterization of distinct
species variants of the ligands. Related genes found, e.g., in
various computer databases will also be useful, in many instances,
for similar purposes with structurally related proteins. In
particular, the present invention provides useful probes or search
features for identifying additional homologous genetic entities in
different species.
[0084] The isolated genes will allow transformation of cells
lacking expression of a corresponding chemokine or receptor, e.g.,
either species types or cells which lack corresponding antigens and
exhibit negative background activity. Expression of transformed
genes will allow isolation of antigenically pure cell lines, with
defined or single specie variants. This approach will allow for
more sensitive detection and discrimination of the physiological
effects of chemokine or receptor proteins. Subcellular fragments,
e.g., cytoplasts or membrane fragments, can be isolated and
used.
[0085] Dissection of critical structural elements which effect the
various differentiation functions provided by ligands is possible
using standard techniques of modern molecular biology, particularly
in comparing members of the related class. See, e.g., the
homolog-scanning mutagenesis technique described in Cunningham, et
al. (1989) Science 243:1339-1336; and approaches used in O'Dowd, et
al. (1988) J. Biol. Chem. 263:15985-15992; and Lechleiter, et al.
(1990) EMBO J. 9:4381-4390.
[0086] In addition, various segments can be substituted between
species variants to determine what structural features are
important in both receptor binding affinity and specificity, as
well as signal transduction. An array of different chemokine or
receptor variants will be used to screen for variants exhibiting
combined properties of interaction with different species
variants.
[0087] Intracellular functions would probably involve segments of
the receptor which are normally accessible to the cytosol. However,
ligand internalization may occur under certain circumstances, and
interaction between intracellular components and "extracellular"
segments may occur. The specific segments of interaction of a
particular chemokine with other intracellular components may be
identified by mutagenesis or direct biochemical means, e.g.,
cross-linking or affinity methods. Structural analysis by
crystallographic or other physical methods will also be applicable.
Further investigation of the mechanism of signal transduction will
include study of associated components which may be isolatable by
affinity methods or by genetic means, e.g., complementation
analysis of mutants.
[0088] Further study of the expression and control of the various
chemokines or receptors will be pursued. The controlling elements
associated with the proteins may exhibit differential
developmental, tissue specific, or other expression patterns.
Upstream or downstream genetic regions, e.g., control elements, are
of interest. Differential splicing of message may lead to membrane
bound forms, soluble forms, and modified versions of ligand.
[0089] Structural studies of the proteins will lead to design of
new ligands or receptors, particularly analogs exhibiting agonist
or antagonist properties on the receptor. This can be combined with
previously described screening methods to isolate ligands
exhibiting desired spectra of activities.
[0090] Expression in other cell types will often result in
glycosylation differences in a particular chemokine or receptor.
Various species variants may exhibit distinct functions based upon
structural differences other than amino acid sequence. Differential
modifications may be responsible for differential function, and
elucidation of the effects are now made possible.
[0091] Thus, the present invention provides important reagents
related to a physiological ligand-receptor interaction. Although
the foregoing description has focused primarily upon the mouse and
human embodiments of the chemokines or receptors specifically
described, those of skill in the art will immediately recognize
that the invention provides other counterparts, e.g., from related
species, rodents or primates.
[0092] V. Antibodies
[0093] Antibodies can be raised to these chemokines or receptors,
including species or polymorphic variants, and fragments thereof,
both in their naturally occurring forms and in their recombinant
forms. Additionally, antibodies can be raised to chemokines or
receptors in either their active or inactive forms, or in their
native or denatured forms. Anti-idiotypic antibodies are also
contemplated.
[0094] Antibodies, including binding fragments and single chain
versions, against predetermined fragments of the ligands can be
raised by immunization of animals with concatemers or conjugates of
the fragments with immunogenic proteins. Monoclonal antibodies are
prepared from cells secreting the desired antibody. These
antibodies can be screened for binding to normal or defective
chemokines or receptors, or screened for agonistic or antagonistic
activity. These monoclonal antibodies will usually bind with at
least a K.sub.D of about 1 mM, more usually at least about 300
.mu.M, typically at least about 10 .mu.M, more typically at least
about 30 .mu.M, preferably at least about 10 .mu.M, and more
preferably at least about 3 .mu.M or better.
[0095] The antibodies, including antigen binding fragments, of this
invention can have significant preparative, diagnostic, or
therapeutic value. They can be useful to purify or label the
desired antigen in a sample, or may be potent antagonists that bind
to ligand and inhibit binding to receptor or inhibit the ability of
a ligand to elicit a biological response. They also can be useful
as non-neutralizing antibodies and can be coupled to, or as fusion
proteins with, toxins or radionuclides so that when the antibody
binds to antigen, a cell expressing it, e.g., on its surface via
receptor, is killed. Further, these antibodies can be conjugated to
drugs or other therapeutic agents, either directly or indirectly by
means of a linker, and may effect drug targeting. Antibodies to
receptors may be more easily used to block ligand binding and/or
signal transduction.
[0096] The antibodies of this invention can also be useful in
diagnostic or reagent purification applications. As capture or
non-neutralizing antibodies, they can be screened for ability to
bind to the chemokines or receptors without inhibiting
ligand-receptor binding. As neutralizing antibodies, they can be
useful in competitive binding assays. They will also be useful in
detecting or quantifying chemokine or receptors, e.g., in
immunoassays. They may be used as purification reagents in
immunoaffinity columns or as immunohistochemistry reagents.
[0097] Ligand or receptor fragments may be concatenated or joined
to other materials, particularly polypeptides, as fused or
covalently joined polypeptides to be used as immunogens. Short
peptides will preferably be made as repeat structures to increase
size. A ligand and its fragments may be fused or covalently linked
to a variety of immunogens, such as keyhole limpet hemocyanin,
bovine serum albumin, tetanus toxoid, etc. See Microbiology, Hoeber
Medical Division, Harper and Row, 1969; Landsteiner (1962)
Specificity of Serological Reactions, Dover Publications, New York,
and Williams, et al. (1967) Methods in Immunology and
Immunochemistry, Vol. 1, Academic Press, New York, for descriptions
of methods of preparing polyclonal antisera. A typical method
involves hyperimmunization of an animal with an antigen. The blood
of the animal is then collected shortly after the repeated
immunizations and the gamma globulin fraction is isolated.
[0098] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies may be found in, e.g., Stites, et al. (eds.)
Basic and Clinical Immunology (4th ed.), Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.) Academic Press, New York; and particularly in Kohler and
Milstein (1975) in Nature 256:495-497, which discusses one method
of generating monoclonal antibodies. Summarized briefly, this
method involves injecting an animal with an immunogen. The animal
is then sacrificed and cells taken, e.g., from its spleen, which
are then fused with myeloma cells. The result is a hybrid cell or
"hybridoma" that is capable of reproducing in vitro. The population
of hybridomas is then screened to isolate individual clones, each
of which secrete a single antibody species to the immunogen. In
this manner, the individual antibody species obtained are the
products of immortalized and cloned single B cells from the immune
animal generated in response to a specific site recognized on the
immunogenic substance. Large amounts of antibody may be derived
from ascites fluid from an animal.
[0099] Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively to
selection of libraries of antibodies in phage or similar vectors.
See, Huse, et al. (1989) "Generation of a Large Combinatorial
Library of the Immunoglobulin Repertoire in Phage Lambda," Science
246:1275-1281; and Ward, et al. (1989) Nature 341:544-546. The
polypeptides and antibodies of the present invention may be used
with or without modification, including chimeric or humanized
antibodies. Frequently, the polypeptides and antibodies will be
labeled by joining, either covalently or non-covalently, a
substance which provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and are reported
extensively in both the scientific and patent literature. Suitable
labels include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent moieties, chemiluminescent moieties,
magnetic particles, and the like. Patents, teaching the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced, see Cabilly, U.S. Pat. No.
4,816,567; and Queen et al. (1989) Proc. Nat'l. Acad. Sci.
86:10029-10033.
[0100] The antibodies of this invention can also be used for
affinity chromatography in isolating the protein. Columns can be
prepared where the antibodies are linked to a solid support, e.g.,
particles, such as agarose, Sephadex, or the like, where a cell
lysate may be passed through the column, the column washed,
followed by increasing concentrations of a mild denaturant, whereby
the purified chemokine protein will be released.
[0101] The antibodies may also be used to screen expression
libraries for particular expression products. Usually the
antibodies used in such a procedure will be labeled with a moiety
allowing easy detection of presence of antigen by antibody
binding.
[0102] Antibodies raised against these chemokines or receptors will
also be useful to raise anti-idiotypic antibodies. These will be
useful in detecting or diagnosing various immunological conditions
related to expression of the respective antigens.
[0103] VI. Nucleic Acids
[0104] The described peptide sequences and the related reagents are
useful in isolating a DNA clone encoding these chemokines or
receptors, e.g., from a natural source. Typically, it will be
useful in isolating a gene from another individual, and similar
procedures will be applied to isolate genes from related species,
e.g., rodents or primates. Cross hybridization will allow isolation
of ligand from other closely related species. A number of different
approaches should be available to successfully isolate a suitable
nucleic acid clone. Similar concepts apply to the receptor
embodiments.
[0105] The purified protein or defined peptides are useful for
generating antibodies by standard methods, as described above.
Synthetic peptides or purified protein can be presented to an
immune system to generate monoclonal or polyclonal antibodies. See,
e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene;
and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold
Spring Harbor Press. Alternatively, a chemokine or receptor may be
used as a specific binding reagent, and advantage can be taken of
its specificity of binding, much like an antibody would be used.
The chemokine receptors are typically 7 transmembrane proteins,
which could be sensitive to appropriate interaction with lipid or
membrane. The signal transduction typically is mediated through a
G-protein, through interaction with a G-protein coupled
receptor.
[0106] For example, the specific binding composition could be used
for screening of an expression library made from a cell line which
expresses a particular chemokine. The screening can be standard
staining of surface expressed ligand, or by panning. Screening of
intracellular expression can also be performed by various staining
or immunofluorescence procedures. The binding compositions could be
used to affinity purify or sort out cells expressing the
ligand.
[0107] The peptide segments can also be used to predict appropriate
oligonucleotides to screen a library, e.g., to isolate species
variants. The genetic code can be used to select appropriate
oligonucleotides useful as probes for screening. See, e.g., FIGS.
1-5. In combination with polymerase chain reaction (PCR)
techniques, synthetic oligonucleotides will be useful in selecting
correct clones from a library. Complementary sequences will also-be
used as probes or primers. Anchored vector or poly-A complementary
PCR techniques or complementary DNA of other peptides may be
useful. Complementary nucleic acid sequences may also be used as
mutagenesis primers.
[0108] This invention contemplates use of isolated DNA or fragments
to encode a biologically active corresponding chemokine
polypeptide. In addition, this invention covers isolated or
recombinant DNA which encodes a biologically active protein or
polypeptide which is capable of hybridizing under appropriate
conditions with the DNA sequences described herein. Said
biologically active protein or polypeptide can be an intact ligand.
receptor, or fragment, and have an amino acid sequence as disclosed
in FIGS. 1-5. Further, this invention covers the use of isolated or
recombinant DNA, or fragments thereof, which encode proteins which
are homologous to a chemokine or receptor or which was isolated
using such a cDNA encoding a chemokine or receptor as a probe. The
isolated DNA can have the respective regulatory sequences in the 5'
and 3' flanks, e.g., promoters, enhancers, poly-A addition signals,
and others.
[0109] An "isolated" nucleic acid is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially separated from
other components which naturally accompany a native sequence, e.g.,
ribosomes, polymerases, and flanking genomic sequences from the
originating species. The term embraces a nucleic acid sequence
which has been removed from its naturally occurring environment,
and includes recombinant or cloned DNA isolates and chemically
synthesized analogs or analogs biologically synthesized by
heterologous systems. A substantially pure molecule includes
isolated forms of the molecule.
[0110] An isolated nucleic acid will generally be a homogeneous
composition of molecules, but will, in some embodiments, contain
minor heterogeneity. This heterogeneity is typically found at the
polymer ends or portions not critical to a desired biological
function or activity.
[0111] A "recombinant" nucleic acid is defined either by its method
of production or its structure. In reference to its method of
production, e.g., a product made by a process, the process is use
of recombinant nucleic acid techniques, e.g., involving human
intervention in the nucleotide sequence, typically selection or
production. Alternatively, it can be a nucleic acid made by
generating a sequence comprising fusion of two fragments which are
not naturally contiguous to each other, but is meant to exclude
products of nature, e.g., naturally occurring purified forms. Thus,
for example, products made by transforming cells with any
unnaturally occurring vector is encompassed, as are nucleic acids
comprising sequence derived using a synthetic oligonucleotide
process. Such is often done to replace a codon with a redundant
codon encoding the same or a conservative amino acid, while
typically introducing or removing a sequence recognition site.
Alternatively, it is performed to join together nucleic acid
segments of desired functions to generate a single genetic entity
comprising a desired combination of functions not found in the
commonly available natural forms. Restriction enzyme recognition
sites are often the target of such artificial manipulations, but
other site specific targets, e.g., promoters, DNA replication
sites, regulation sequences, control sequences, or other useful
features may be incorporated by design. A similar concept is
intended for a recombinant, e.g., fusion, polypeptide. Specifically
included are synthetic nucleic acids which, by genetic code
redundancy, encode polypeptides similar to fragments of these
antigens, and fusions of sequences from various different species
variants.
[0112] A significant "fragment" in a nucleic acid context is a
contiguous segment of at least about 17 nucleotides, generally at
least about 20 nucleotides, more generally at least about 23
nucleotides, ordinarily at least about 26 nucleotides, more
ordinarily at least about 29 nucleotides, often at least about 32
nucleotides, more often at least about 35 nucleotides, typically at
least about 38 nucleotides, more typically at least about 41
nucleotides, usually at least about 44 nucleotides, more usually at
least about 47 nucleotides, preferably at least about 50
nucleotides, more preferably at least about 53 nucleotides, and in
particularly preferred embodiments will be at least about 56 or
more nucleotides, e.g., 60, 65, 75, 85, 100, 120, 150, 200, 250,
300, 400, etc. Such fragments may have ends which begin and/or end
at virtually all positions, e.g., beginning at nucleotides 1, 2, 3,
etc., and ending at, e.g., 300, 299, 298, 287, etc., in
combinatorial pairs. Particularly interesting polynucleotides have
ends corresponding to structural domain boundaries.
[0113] A DNA which codes for a particular chemokine or receptor
protein or peptide will be very useful to identify genes, mRNA, and
cDNA species which code for related or homologous ligands or
receptors, as well as DNAs which code for homologous proteins from
different species. There are likely homologs in closely related
species, e.g., rodents or primates. Various chemokine proteins
should be homologous and are encompassed herein, as would be
receptors. However, proteins can readily be isolated under
appropriate conditions using these sequences if they are
sufficiently homologous. Typically, primate chemokines or receptors
are of particular interest.
[0114] This invention further covers recombinant DNA molecules and
fragments having a DNA sequence identical to or highly homologous
to the isolated DNAs set forth herein. In particular, the sequences
will often be operably linked to DNA segments which control
transcription, translation, and DNA replication. Alternatively,
recombinant clones derived from the genomic sequences, e.g.,
containing introns, will be useful for transgenic studies,
including, e.g., transgenic cells and organisms, and for gene
therapy. See, e.g., Goodnow (1992) "Transgenic Animals" in Roitt
(ed.) Encyclopedia of Immunology Academic Press, San Diego, pp.
1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al. (1991)
Science 254:707-710; Capecchi (1989) Science 244:1288; Robertson
(1987) (ed.) Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach IRL Press, Oxford; and Rosenberg (1992) J. Clinical
Oncology 10:180-199.
[0115] Homologous nucleic acid sequences, when compared, exhibit
significant similarity, or identity. The standards for homology in
nucleic acids are either measures for homology generally used in
the art by sequence comparison or based upon hybridization
conditions. The hybridization conditions are described in greater
detail below.
[0116] Substantial homology in the nucleic acid sequence comparison
context means either that the segments, or their complementary
strands, when compared, are identical when optimally aligned, with
appropriate nucleotide insertions or deletions, in at least about
50% of the nucleotides, generally at least about 56%, more
generally at least about 59%, ordinarily at least about 62%, more
ordinarily at least about 65%, often at least about 68%, more often
at least about 71%, typically at least about 74%, more typically at
least about 77%, usually at least about 80%, more usually at least
about 85%, preferably at least about 90%, more preferably at least
about 95 to 98% or more, and in particular embodiments, as high at
about 99% or more of the nucleotides. Alternatively, substantial
homology exists when the segments will hybridize under selective
hybridization conditions, to a strand, or its complement, typically
using a sequence derived from FIGS. 1-5. Typically, selective
hybridization will occur when there is at least about 55% homology
over a stretch of at least about 30 nucleotides, preferably at
least about 65% over a stretch of at least about 25 nucleotides,
more preferably at least about 75%, and most preferably at least
about 90% over about 20 nucleotides. See, Kanehisa (1984) Nuc.
Acids Res. 12:203-213. The length of homology comparison, as
described, may be over longer stretches, and in certain embodiments
will be over a stretch of at least about 17 nucleotides, usually at
least about 20 nucleotides, more usually at least about 24
nucleotides, typically at least about 28 nucleotides, more
typically at least about 40 nucleotides, preferably at least about
50 nucleotides, and more preferably at least about 75 to 100 or
more nucleotides. PCR primers will generally have high levels of
matches over potentially shorter lengths.
[0117] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0118] Optical alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment
algorithm of Needlman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity method of Pearson and Lipman (1988) Proc.
Nat'l Acad. Sci. USA 85:2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by visual inspection (see generally Ausubel
et al., supra).
[0119] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng and Doolittle (1987) J. Mol. Evol. 35:351-360. The method used
is similar to the method described by Higgins and Sharp (1989)
CABIOS 5:151-153. The program can align up to 300 sequences, each
of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. For example, a reference sequence can be
compared to other test sequences to determine the percent sequence
identity relationship using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps.
[0120] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described Altschul, et al. (1990) J.
Mol. Biol. 215:403-410. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http:www.ncbi.nlm.nih.gov/- ). This algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match
or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to
as the neighborhood word score threshold (Altschul, et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
then extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Extension of the
word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLAST program uses as defaults a
wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and
Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915) alignments (B)
of 50, expectation (E) of 10, M=5, N=4, and a comparison of both
strands.
[0121] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and Altschul
(1993) Proc. Nat'l Acad. Sci. USA 90:5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example., a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0122] A further indication that two nucleic acid sequences of
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the polypeptide encoded by the second nucleic acid, as
described below. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules hybridize to each other under
stringent conditions, as described below.
[0123] Stringent conditions, in referring to homology in the
hybridization context, will be stringent combined conditions of
salt, temperature, organic solvents, and other parameters,
typically those controlled in hybridization reactions. Stringent
temperature conditions will usually include temperatures in excess
of about 30.degree. C., more usually in excess of about 37.degree.
C., typically in excess of about 45.degree. C., more typically in
excess of about 55.degree. C., preferably in excess of about
65.degree. C., and more preferably in excess of about 70.degree. C.
Stringent salt conditions will ordinarily be less than about 1000
mM, usually less than about 500 mM, more usually less than about
400 mM, typically less than about 300 mM, preferably less than
about 200 mM, and more preferably less than about 150 mM, e.g.,
20-50 mM. However, the combination of parameters is much more
important than the measure of any single parameter. See, e.g.,
Wetmur and Davidson (1968). J. Mol. Biol. 31:349-370. Hybridization
under stringent conditions should give a background of at least
2-fold over background, preferably at least 3-5 or more.
[0124] Corresponding chemokines or receptors from other closely
related species can be cloned and isolated by cross-species
hybridization. Alternatively, sequences from a sequence data base
may be recognized as having similarity. Homology may be very low
between distantly related species, and thus hybridization of
relatively closely related species is advisable. Alternatively,
preparation of an antibody preparation which exhibits less species
specificity may be useful in expression cloning approaches. PCR
approaches using segments of conserved sequences will also be
used.
[0125] VII. Making Chemokines or Receptors; Mimetics
[0126] DNA which encodes each respective chemokine, receptor, or
fragments thereof can be obtained by chemical synthesis, screening
cDNA libraries, or by screening genomic libraries prepared from a
wide variety of cell lines or tissue samples. A "coding sequence"
or a sequence which "encodes" a particular protein, is a nucleic
acid sequence which is transcribed (in the case of DNA) and
translated (in the case of mRNA) into a polypeptide in vitro or in
vivo when placed under the control of appropriate regulatory
sequences. The boundaries of the coding sequence are determined by
a start codon at the 5' (amino) terminus and a translation stop
codon at the 3' (carboxy) terminus. A transcription termination
sequence will usually be located 3' to the coding sequence.
[0127] This DNA can be expressed in a wide variety of host cells
for the synthesis of a full-length ligand or fragments which can in
turn, for example, be used to generate polyclonal or monoclonal
antibodies; for binding studies; for construction and expression of
modified molecules; for expression cloning or purification; and for
structure/function studies. Each protein or its fragments can be
expressed in host cells that are transformed with appropriate
expression vectors. These molecules can be substantially purified
to be free of protein or cellular contaminants, other than those
derived from the recombinant host, and therefore are particularly
useful in pharmaceutical compositions when combined with a
pharmaceutically acceptable carrier and/or diluent. The antigens or
antibodies, or portions thereof, may be expressed as fusions with
other proteins.
[0128] Expression vectors are typically self-replicating DNA or RNA
constructs containing the desired protein gene or its fragments,
usually operably linked to suitable genetic control elements that
are recognized in a suitable host cell. These control elements are
capable of effecting expression within a suitable host. The
specific type of control elements necessary to effect expression
will depend upon the eventual host cell used. Generally, the
genetic control elements can include a prokaryotic promoter system
or a eukaryotic promoter expression control system, and typically
include a transcriptional promoter, an optional operator to control
the onset of transcription, transcription enhancers to elevate the
level of mRNA expression, a sequence that encodes a suitable
ribosome binding site, and sequences that terminate transcription
and translation. Thus, the term "control sequences" or "control
elements" refers collectively to promoter sequences,
polyadenylation signals, transcription termination sequences,
upstream regulatory domains, origins of replication, internal
ribosome entry sites ("IRES"), enhancers, and the like, which
collectively provide for the replication, transcription and
translation of a coding sequence in a recipient cell. Not all of
these control sequences need always be present so long as the
selected coding sequence is capable of being replicated,
transcribed and translated in an appropriate host cell. Expression
vectors also usually contain an origin of replication that allows
the vector to replicate independently of the host cell.
[0129] The vectors of this invention contain DNA which encode
embodiments of a chemokine, receptor, or a fragment thereof,
typically encoding a biologically active polypeptide. The DNA can
be under the control of a promoter, such as a mammalian or viral
promoter and can encode a selection marker. This invention further
contemplates use of such expression vectors which are capable of
expressing eukaryotic cDNA coding for each chemokine or receptor in
a prokaryotic or eukaryotic host, where the vector is compatible
with the host and where the eukaryotic cDNA coding for the protein
is inserted into the vector such that growth of the host containing
the vector expresses the cDNA in question. Usually, expression
vectors are designed for stable replication in their host cells or
for amplification to greatly increase the total number of copies of
the desirable gene per cell. It is not always necessary to require
that an expression vector replicate in a host cell, e.g., it is
possible to effect transient expression of the ligand or its
fragments in various hosts using vectors that do not contain a
replication origin that is recognized by the host cell. It is also
possible to use vectors that cause integration of a chemokine or
receptor gene or its fragments into the host DNA by recombination,
or to integrate a promoter which controls expression of an
endogenous gene. Thus, as used herein, a cell has been
"transformed" by exogenous DNA when such exogenous DNA has been
introduced inside the cell membrane. Exogenous DNA may or may not
be integrated (covalently linked) into chromosomal DNA making up
the genome of the cell. A stably transformed cell is one in which
the exogenous DNA has become integrated into the chromosome so that
it is inherited by daughter cells through chromosome replication.
This stability is demonstrated by the ability of the eucaryotic
cell to establish cell lines or clones comprised of a population of
daughter cells containing the exogenous DNA.
[0130] Vectors, as used herein, comprise plasmids, viruses,
bacteriophage, integratable DNA fragments, and other vehicles,
including those which enable the integration of DNA fragments into
the genome of the host. Expression vectors are specialized vectors
which contain genetic control elements that effect expression of
operably linked genes. Plasmids are the most commonly used form of
vector but many other forms of vectors which serve an equivalent
function and which are, or become, known in the art are suitable
for use herein. See, e.g., Pouwels, et al. (1985 and Supplements)
Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., and
Rodriquez, et al. (1988)(eds.) Vectors: A Survey of Molecular
Cloning Vectors and Their Uses, Buttersworth, Boston, Mass.
[0131] Transformed cells include cells, preferably mammalian, that
have been transformed with a chemokine or receptor gene containing
vector constructed using recombinant DNA techniques. Transformed
host cells usually express the ligand, receptor, or its fragments,
but for purposes of cloning, amplifying, and manipulating its DNA,
do not need to express the protein. This invention further
contemplates culturing transformed cells in a nutrient medium, thus
permitting the protein to accumulate in the culture. The protein
can be recovered, from the culture or from the culture medium, or
from cell membranes.
[0132] For purposes of this invention, DNA sequences are operably
linked when they are functionally related to each other. For
example, DNA for a presequence or secretory signal is operably
linked to a polypeptide if it is expressed as a preprotein or
participates in directing the polypeptide to the cell membrane or
in secretion of the polypeptide. A promoter is operably linked to a
coding sequence if it controls the transcription of the
polypeptide; a ribosome binding site is operably linked to a coding
sequence if it is positioned to permit translation. Usually,
operably linked means contiguous and in reading frame, however,
certain genetic elements such as repressor genes are not
contiguously linked but still bind to operator sequences that in
turn control expression.
[0133] Suitable host cells include prokaryotes, lower eukaryotes,
and higher eukaryotes. Prokaryotes include both gram negative and
gram positive organisms, e.g., E. coli and B. subtilis. Lower
eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and
species of the genus Dictyostelium. Higher eukaryotes include
established tissue culture cell lines from animal cells, both of
non-mammalian origin, e.g., insect cells, and birds, and of
mammalian origin, e.g., human, primates, and rodents.
[0134] Prokaryotic host-vector systems include a wide variety of
vectors for many different species. As used herein, E. coli and its
vectors will be used generically to include equivalent vectors used
in other prokaryotes. A representative vector for amplifying DNA is
pBR322 or many of its derivatives. Vectors that can be used to
express these chemokines or their fragments include, but are not
limited to, such vectors as those containing the lac promoter
(pUC-series); trp promoter (pBR322-trp); Ipp promoter (the
pIN-series); lambda-pP or pR promoters (pOTS); or hybrid promoters
such as ptac (pDR540). See Brosius, et al. (1988) "Expression
Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters",
in Rodriguez and Denhardt (eds.) Vectors: A Survey of Molecular
Cloning Vectors and Their Uses, Buttersworth, Boston, Chapter 10,
pp. 205-236.
[0135] Lower eukaryotes, e.g., yeasts and Dictyostelium, may be
transformed with chemokine or receptor sequence containing nucleic
acids. For purposes of this invention, the most common lower
eukaryotic host is the baker's yeast, Saccharomyces cerevisiae. It
will be used to generically represent lower eukaryotes although a
number of other strains and species are also available. Yeast
vectors typically consist of a replication origin (unless of the
integrating type), a selection gene, a promoter, DNA encoding the
desired protein or its fragments, and sequences for translation
termination, polyadenylation, and transcription termination.
Suitable expression vectors for yeast include such constitutive
promoters as 3-phosphoglycerate kinase and various other glycolytic
enzyme gene promoters or such inducible promoters as the alcohol
dehydrogenase 2 promoter or metallothionine promoter. Suitable
vectors include derivatives of the following types:
self-replicating low copy number (such as the YRp-series),
self-replicating high copy number (such as the YEp-series);
integrating types (such as the YIp-series), or mini-chromosomes
(such as the YCp-series).
[0136] Higher eukaryotic tissue culture cells are the preferred
host cells for expression of the functionally active chemokine or
receptor proteins. In principle, most any higher eukaryotic tissue
culture cell line is workable, e.g., insect baculovirus expression
systems, whether from an invertebrate or vertebrate source.
However, mammalian cells are preferred, in that the processing,
both cotranslationally and posttranslationally, will be typically
most like natural. Transformation or transfection and propagation
of such cells has become a routine procedure. Examples of useful
cell lines include HeLa cells, Chinese hamster ovary (CHO) cell
lines, baby rat kidney (BRK) cell lines, insect cell lines, bird
cell lines, and monkey (COS) cell lines. Expression vectors for
such cell lines usually include an origin of replication, a
promoter, a translation initiation site, RNA splice sites (if
genomic DNA is used), a polyadenylation site, and a transcription
termination site. These vectors also usually contain a selection
gene or amplification gene. Suitable expression vectors may be
plasmids, viruses, or retroviruses carrying promoters derived,
e.g., from such sources as from adenovirus, SV40, parvoviruses,
vaccinia virus, or cytomegalovirus. Representative examples of
suitable expression vectors include pcDNA1; pCD, see Okayama, et
al. (1985) Mol. Cell Biol. 5:1136-1142; pMClneo Poly-A, see Thomas,
et al. (1987) Cell 51:503-512; and a baculovirus vector such as pAC
373 or pAC 610.
[0137] It will often be desired to express a chemokine or receptor
polypeptide in a system which provides a specific or defined
glycosylation pattern. In this case, the usual pattern will be that
provided naturally by the expression system. However, the pattern
will be modifiable by exposing the polypeptide, e.g., an
unglycosylated form, to appropriate glycosylating proteins
introduced into a heterologous expression system. For example, a
chemokine or receptor gene may be co-transformed with one or more
genes encoding mammalian or other glycosylating enzymes. Using this
approach, certain mammalian glycosylation patterns will be
achievable or approximated in prokaryote or other cells.
[0138] A chemokine, receptor, or a fragment thereof, may be
engineered to be phosphatidyl inositol (PI) linked to a cell
membrane, but can be removed from membranes by treatment with a
phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl inositol
phospholipase-C. This releases the antigen in a biologically active
form, and allows purification by standard procedures of protein
chemistry. See, e.g., Low (1989) Biochim. Biophys. Acta
988:427-454; Tse, et al. (1985) Science 230:1003-1008; and Brunner,
et al. (1991) J. Cell Biol. 114:1275-1283.
[0139] Now that these chemokines and receptors have been
characterized, fragments or derivatives thereof can be prepared by
conventional processes for synthesizing peptides. These include
processes such as are described in Stewart and Young (1984) Solid
Phase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill.;
Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis,
Springer-Verlag, New York; and Bodanszky (1984) The Principles of
Peptide Synthesis, Springer-Verlag, New York. For example, an azide
process, an acid chloride process, an acid anhydride process, a
mixed anhydride process, an active ester process (for example,
p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl
ester), a carbodiimidazole process, an oxidative-reductive process,
or a dicyclohexyl-carbodiimide (DCCD)/additive process can be used.
Solid phase and solution phase syntheses are both applicable to the
foregoing processes.
[0140] These chemokines, receptors, fragments, or derivatives are
suitably prepared in accordance with the above processes as
typically employed in peptide synthesis, generally either by a
so-called stepwise process which comprises condensing an amino acid
to the terminal amino acid, one by one in sequence, or by coupling
peptide fragments to the terminal amino acid. Amino groups that are
not being used in the coupling reaction are typically protected to
prevent coupling at an incorrect location.
[0141] If a solid phase synthesis is adopted, the C-terminal amino
acid is typically bound to an insoluble carrier or support through
its carboxyl group. The insoluble carrier is not particularly
limited as long as it has a binding capability to a reactive
carboxyl group. Examples of such insoluble carriers include
halomethyl resins, such as chloromethyl resin or bromomethyl resin,
hydroxymethyl resins, phenol resins,
tert-alkyloxycarbonyl-hydrazidated resins, and the like.
[0142] An amino group-protected amino acid is bound in sequence
through condensation of its activated carboxyl group and the
reactive amino group of the previously formed peptide or chain, to
synthesize the peptide step by step. After synthesizing the
complete sequence, the peptide is split off from the insoluble
carrier to produce the peptide. This solid-phase approach is
generally described, e.g., by Merrifield, et al. (1963) in J. Am.
Chem. Soc. 85:2149-2156.
[0143] The prepared ligand and fragments thereof can be isolated
and purified from the reaction mixture by means of peptide
separation, e.g., by extraction, precipitation, electrophoresis,
and various forms of chromatography, and the like. The various
chemokines or receptors of this invention can be obtained in
varying degrees of purity depending upon its desired use.
Purification can be accomplished by use of the protein purification
techniques disclosed herein or by the use of the antibodies herein
described, e.g., in immunoabsorbant affinity chromatography. This
immunoabsorbant affinity chromatography is typically carried out,
e.g., by first linking the antibodies to a solid support and then
contacting the linked antibodies with solubilized lysates of
appropriate source cells, lysates of other cells expressing the
ligand or receptor, or lysates or supernatants of cells producing
the desired proteins as a result of DNA techniques, see below.
[0144] VIII. Uses
[0145] The present invention provides reagents which will find use
in diagnostic applications as described elsewhere herein, e.g., in
the general description for developmental abnormalities, or below
in the description of kits for diagnosis.
[0146] This invention also provides reagents with significant
therapeutic potential. These chemokines and receptors (naturally
occurring or recombinant), fragments thereof, and binding
compositions, e.g., antibodies thereto, along with compounds
identified as having binding affinity to them, should be useful in
the treatment of conditions associated with abnormal physiology or
development, including inflammatory conditions, e.g., asthma, and
epithelial conditions, for example, improper wound healing. In
particular, modulation of trafficking of leukocytes is one likely
biological activity, but a wider tissue distribution might suggest
broader biological activity, including, e.g., antiviral effects.
Abnormal proliferation, regeneration, degeneration, and atrophy may
be modulated by appropriate therapeutic treatment using the
compositions provided herein. For example, a disease or disorder
associated with abnormal expression or abnormal signaling by a
chemokine or ligand for a receptor should be a likely target for an
agonist or antagonist of the ligand.
[0147] Various abnormal physiological or developmental conditions
are known in cell types shown to possess the chemokine or receptor
mRNAs by Northern blot analysis. See Berkow (ed.) The Merck Manual
of Diagnosis and Therapy, Merck & Co., Rahway, N.J.; and Thorn,
et al. Harrison's Principles of Internal Medicine, McGraw-Hill,
N.Y. Developmental or functional abnormalities, e.g., of the immune
system, cause significant medical abnormalities and conditions
which may be susceptible to prevention or treatment using
compositions provided herein.
[0148] Antibodies to the chemokines or receptors, including
recombinant forms, can be purified and then used diagnostically or
therapeutically, alone or in combination with other chemokines,
cytokines, or antagonists thereof. These reagents can be combined
for therapeutic use with additional active or inert ingredients,
e.g., in conventional pharmaceutically acceptable carriers or
diluents, e.g., immunogenic adjuvants, along with physiologically
innocuous stabilizers and excipients. These combinations can be
sterile filtered and placed into dosage forms as by lyophilization
in dosage vials or storage in stabilized aqueous preparations. This
invention also contemplates use of antibodies or binding fragments
thereof, including forms which are not complement binding.
Moreover, modifications to the antibody molecules or antigen
binding fragments thereof, may be adopted which affect the
pharmacokinetics or pharmacodynamics of the therapeutic entity.
[0149] Drug screening using antibodies or receptor or fragments
thereof can be performed to identify compounds having binding
affinity to each chemokine or receptor, including isolation of
associated components. Subsequent biological assays can then be
utilized to determine if the compound has intrinsic stimulating
activity and is therefore a blocker or antagonist in that it blocks
the activity of the ligand. Likewise, a compound having intrinsic
stimulating activity can activate the receptor and is thus an
agonist in that it simulates the activity of a ligand. This
invention further contemplates the therapeutic use of antibodies to
these chemokines as antagonists, or to the receptors as antagonists
or agonists. This approach should be particularly useful with other
chemokine or receptor species variants.
[0150] Moreover, the novel chemokines and receptors described
herein can be used to effect wound healing, as well as to treat a
wide variety of disorders associated with proliferative responses.
For example, tissue fibrosis is the formation of excessive amounts
of fibrotic or scar tissue. Major organs such as the heart, kidney,
liver, eye, and skin are prone to chronic scarring. Hypertrophic
scars (nonmalignant tissue bulk) are a common form of fibrosis
caused by burns and other trauma. In addition, there are a number
of other fibroproliferative disorders, including scleroderma,
keloids, and atherosclerosis, which are associated with general
tissue scarring, tumor-like growths in the skin, or sustained
scarring of blood vessels. Fibrosis can follow surgery in the form
of adhesions, keloid tumors or hypertrophic scarring. Fibrosis
causes contractures and joint dislocation following severe burns,
wounds or orthopaedic injuries; it can occur in any organ and
accompanies many disease states, such as hepatitis (liver
cirrhosis), hypertension (heart failure), tuberculosis (pulmonary
fibrosis) and diabetes (nephropathy).
[0151] Fibrosis, despite the cause, activates a proliferative
response related to the events that occur in the healing of skin
wounds. Specifically, acute physical damage causes the death of
cells, the clotting of blood, an inflammatory response
characterized by the migration of inflammatory cells into the
lesion, the proliferation of fibroblastic cells in the lesion and
the production and deposition of collagen fibers and scars.
[0152] As demonstrated herein, BLRx, a representative chemokine
receptor, is expressed by fibroblasts, melanocytes and
keratinocytes and is involved in wound healing. In particular,
enhanced expression of BLRx is associated with the onset of the
wound healing process. Thus, the compositions and methods described
herein may be used for treating wounds, such as burns and lesions,
as well as for treating or preventing any of the various forms of
fibrosis described above as the processes involved in fibrosis are
the same as those involved in wound healing. Moreover, the
compositions and methods may be used in the treatment of
hyperproliferative diseases of the epidermis, such as psoriasis and
basal cell carcinoma. The methods and compositions are also useful
for stimulation of transplanted corneal tissue.
[0153] For example, the compositions and methods described herein
can be used to enhance wound healing, block the fibrotic process
and/or control hyperproliferative disorders using agents that
modulate (i.e., either inhibit or enhance) endogenous BLRx
production. In treating these disorders, an increase in the
concentration of endogenous BLRx may be desired, such as in the
case of wound healing. Thus, one method of treatment includes
administering a composition-that enhances BLRX activity, for
example, the ability of BLRx to bind to its ligand, or a
composition that enhances endogenous BLRX expression. For example,
target cells can be-transformed with DNA constructs that either
encode BLRx or that include transcriptional and/or translational
regulatory elements that enhance expression of endogenous BLRx. The
exogenous polynucleotides need not code for exact copies of the
endogenous BLRx proteins. Modified protein chains can also be
readily designed utilizing various recombinant DNA techniques well
known to those skilled in the art and described for instance, in
Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2d
ed.), Vols. 1-3, Cold Spring Harbor Laboratory. For example,
hydroxylamine can also be used to introduce single base mutations
into the coding region of the gene (Sikorski et al. (1991) Meth.
Enzymol. 194: 302-318). Additionally, an agonist, i.e., a molecule
that increases or prolongs the duration of the effect of BLRx, can
be administered. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules that bind to and modulate the
effects of the target molecule.
[0154] Conversely, a decrease in the concentration or expression of
endogenous BLRx may be desired, such as when treating a
BLRx-associated proliferative disorder, for example fibrotic or
sclerotic disease such as liver sclerosis, lung fibrosis and
local/systemic sclerosis, cancer, angiogenesis, and
atherosclerosis. In this context, one method of treatment includes
the administration of a composition that decreases BLRx activity,
or causes a decreased production of endogenous BLRx. Thus, an
antagonist, i.e., a molecule which, when bound to BLRx, decreases
the extent or duration of the effect of the biological activity,
can be administered. Antagonists may include proteins, nucleic
acids, carbohydrates, antibodies, or any other molecules which
decrease the effect of the target molecule. Thus, one method of
treatment includes transforming a target cell with a polynucleotide
that includes transcriptional and/or translational regulatory
elements that decrease or suppress expression of endogenous BLRx.
Another method of treatment includes administering a composition
that interferes with the binding of BLRx to its ligand, such as by
competitively binding to BLRX or by otherwise decreasing the
ability of BLRx to bind to or process its ligand, or by effecting
the ability of BLRx to participate in downstream signaling. Thus,
in one embodiment of the present invention, methods for treatment
of a proliferative disorder involve the administration of a
therapeutically effective amount of an antibody which specifically
reacts with BLRX. Such antibodies are described in detail
above.
[0155] In another embodiment, a method of the present invention
involves the administration of a therapeutically effective amount
of an antisense oligonucleotide having a sequence capable of
binding specifically with any sequences of genomic DNA or an mRNA
molecule which encodes BLRx, so as to prevent transcripton or
translation of BLRX mRNA. By "antisense" is meant a composition
containing a nucleic acid sequence which is complementary to the
"sense" strand of a specific nucleic acid sequence. Once introduced
into a cell, the complementary nucleotides combine with endogenous
sequences produced by the cell to form duplexes and to block either
transcription or translation. See, e.g., Agrawal, S., ed. (1996)
Antisense Therapeutics, Humana Press Inc., Totawa N.J.; Alama et
al. (1997) Pharmacol. Res. 36:171-178; Crooke, S. T. (1997) Adv.
Pharmacol. 40:1-49; and Lavrosky et al. (1997) Biochem. Mol. Med.
62(1):11-22. Antisense sequences can be any nucleic acid material,
including DNA, RNA, or any nucleic acid mimics or analogs. See,
e.g., Rossi et al. (1991) Antisense Res. Dev. 1:285-288; Pardridge
et al. (1995) Proc. Nat. Acad. Sci. 92:5592-5596; Nielsen and
Haaima (1997) Chem. Soc. Rev. 96:73-78; and Lee et al. (1998)
Biochemistry 37:900-1010. Delivery of antisense sequences can be
accomplished in a variety of ways, such as through intracellular
delivery using a recombinant vector.
[0156] Antisense oligonucleotides of about 15 to 25 nucleic acid
bases are typically preferred as such are easily synthesized and
are capable of producing the desired inhibitory effect. Molecular
analogs of antisense oligonucleotides may also be used for this
purpose and can have added advantages such as stability,
distribution, or limited toxicity advantageous in a pharmaceutical
product. In addition, chemically reactive groups, such as
iron-linked ethylenediamine-tetraacetic acid (EDTA-Fe), can be
attached to antisense oligonucleotides, causing cleavage of the RNA
at the site of hybridization. These and other uses of antisense
methods to inhibit the in vitro translation of genes are well known
in the art. See, e.g., Marcus-Sakura (1988) Anal. Biochem.
172:289.
[0157] The delivery of polynucleotides, e.g., for delivering BLRX
genes or antisense oligonucleotides, can be achieved using
recombinant expression vectors, with or without carrier viruses or
particles. Such methods are well known in the art. See, e.g., U.S.
Pat. Nos. 6,214,804; 6,147,055; 5,703,055; 5,589,466; 5,580,859;
Slater et al. (1998) J. Allergy Clin. Immunol. 102:469-475. For
example, delivery of polynucleotide sequences can be achieved using
various viral vectors, including retrovirus and adeno-associated
virus vectors. See, e.g., Miller A. D. (1990) Blood 76:271; and
Uckert and Walther (1994) Pharmacol. Ther. 63:323-347. Vectors
which can be utilized for antisense gene therapy include, but are
not limited to, adenoviruses, herpes viruses, vaccinia, or,
preferably, RNA viruses such as retroviruses. Other gene delivery
mechanisms that can be used for delivery of polynucleotide
sequences to target cells include colloidal dispersion and
liposome-derived systems, artificial viral envelopes, and other
systems available to one of skill in the art. See, e.g., Rossi, J.
J. (1995) Br. Med. Bull. 51:217-225; Morris et al. (1997) Nucl.
Acids Res. 25:2730-2736; and Boado et al. (1998) J. Pharm. Sci.
87:1308-1315. For example, delivery systems can make use of
macromolecule complexes, nanocapsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes.
[0158] Site-specific delivery of exogenous genes is also
contemplated, such as techniques in which cells are first
transfected in culture and stable transfectants are subsequently
delivered to the target site. Moreover, delivery can be to the
region of the target cell or tissue, i.e., in an area proximal to
the tissue to be treated, for example, to the region of the
wound.
[0159] The quantities of reagents necessary for the various
therapies described above will depend upon many different factors,
including means of administration, target site, physiological state
of the patient, and other medicants administered. Thus, treatment
dosages should be titrated to optimize safety and efficacy in
various populations, including racial subgroups, age, gender, etc.
Typically, dosages used in vitro may provide useful guidance in the
amounts useful for in situ administration of these reagents. Animal
testing of effective doses for treatment of particular disorders
will provide further predictive indication of human dosage. Various
considerations are described, e.g., in Gilman, et al. (eds.) (1990)
Goodman and Gilman's: The Pharmacological Bases of Therapeutics,
8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences,
17th ed. (1990), Mack Publishing Co., Easton, Penn. Methods for
administration are discussed therein and below, e.g., for oral,
intravenous, intraperitoneal, or intramuscular administration,
transdermal diffusion, and others. Pharmaceutically acceptable
carriers typically include water, saline, buffers, and other
compounds described, e.g., in the Merck Index, Merck & Co.,
Rahway, N.J. Dosage ranges would ordinarily be expected to be in
amounts lower than 1 mM concentrations, typically less than about
10 .mu.M concentrations, usually less than about 100 nM, preferably
less than about 10 pM (picomolar), and most preferably less than
about 1 fM (femtomolar), with an appropriate carrier. Slow release
formulations, or a slow release apparatus will often be utilized
for continuous administration.
[0160] A chemokine, fragments thereof, or antibodies to it or its
fragments, antagonists, and agonists, may be administered directly
to the host to be treated or, depending on the size of the
compounds, it may be desirable to conjugate them to carrier
proteins such as ovalbumin or serum albumin prior to their
administration. Therapeutic formulations may be administered in
many conventional dosage formulations. While it is possible for the
active ingredient to be administered alone, it is often preferable
to present it as a pharmaceutical formulation. Formulations
typically comprise at least one active ingredient, as defined
above, together with one or more acceptable carriers thereof. Each
carrier should be both pharmaceutically and physiologically
acceptable in the sense of being compatible with the other
ingredients and not injurious to the patient. Carriers may improve
storage life, stability, etc. Formulations include those suitable
for oral, rectal, nasal, or parenteral (including subcutaneous,
intramuscular, intravenous and intradermal) administration. The
formulations may conveniently be presented in unit dosage form and
may be prepared by any methods well known in the art of pharmacy.
See, e.g., Gilman, et al. (eds.) (1990) Goodman and Gilman's: The
Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and
Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack
Publishing Co., Easton, Penn.; Avis, et al. (eds.) (1993)
Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New
York; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Tablets Dekker, New York; and Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York. The
therapy of this invention may be combined with or used in
association with other therapeutic agents. Similar considerations
will often apply to receptor based reagents.
[0161] Both the naturally occurring and the recombinant forms of
the chemokines or receptors of this invention are particularly
useful in kits and assay methods which are capable of screening
compounds for binding activity to the proteins. Several methods of
automating assays have been developed in recent years so as to
permit screening of tens of thousands of compounds in a short
period. See, e.g., Fodor, et al. (1991) Science 251:767-773, which
describes means for testing of binding affinity by a plurality of
defined polymers synthesized on a solid substrate. The development
of suitable assays can be greatly facilitated by the availability
of large amounts of purified, soluble chemokine as provided by this
invention.
[0162] For example, antagonists can normally be found once a ligand
has been structurally defined. Testing of potential ligand analogs
is now possible upon the development of highly automated assay
methods using physiologically responsive cells. In particular, new
agonists and antagonists will be discovered by using screening
techniques described herein.
[0163] Viable cells could also be used to screen for the effects of
drugs on respective chemokine or G-protein coupled receptor
mediated functions, e.g., second messenger levels, i.e., Ca.sup.++;
inositol phosphate pool changes (see, e.g., Berridge (1993) Nature
361:315-325 or Billah and Anthes (1990) Biochem. J. 269:281-291);
cellular morphology modification responses; phosphoinositide lipid
turnover; an antiviral response. and others. Some detection methods
allow for elimination of a separation step, e.g., a proximity
sensitive detection system. Calcium sensitive dyes will be useful
for detecting Ca.sup.++ levels, with a fluorimeter or a
fluorescence cell sorting apparatus.
[0164] Rational drug design may also be based upon structural
studies of the molecular shapes of the chemokines, other effectors
or analogs, or the receptors. Effectors may be other proteins which
mediate other functions in response to ligand binding, or other
proteins which normally interact with the receptor. One means for
determining which sites interact with specific other proteins is a
physical structure determination, e.g., x-ray crystallography or 2
dimensional NMR techniques. These will provide guidance as to which
amino acid residues form molecular contact regions. For a detailed
description of protein structural determination, see, e.g.,
Blundell and Johnson (1976) Protein Crystallography, Academic
Press, New York.
[0165] Purified chemokine or receptor can be coated directly onto
plates for use in the aforementioned drug screening techniques, and
may be associated with detergents or lipids. However,
non-neutralizing antibodies, e.g., to the chemokines or receptors
can be used as capture antibodies to immobilize the respective
protein on the solid phase.
[0166] Similar concepts also apply to the chemokine receptor
embodiments of the invention.
[0167] IX. Kits
[0168] This invention also contemplates use of chemokine or
receptor proteins, fragments thereof, peptides, binding
compositions, and their fusion products in a variety of diagnostic
kits and methods for detecting the presence of ligand, antibodies,
or receptors. Typically the kit will have a compartment containing
a defined chemokine or receptor peptide or gene segment or a
reagent which recognizes one or the other, e.g., binding
reagents.
[0169] A kit for determining the binding affinity of a test
compound to a chemokine or receptor would typically comprise a test
compound; a labeled compound, for example an antibody having known
binding affinity for the protein; a source of chemokine or receptor
(naturally occurring or recombinant); and a means for separating
bound from free labeled compound, such as a solid phase for
immobilizing the ligand or receptor. Once compounds are screened,
those having suitable binding affinity to the ligand or receptor
can be evaluated in suitable biological assays, as are well known
in the art, to determine whether they act as agonists or
antagonists to the receptor. The availability of recombinant
chemokine or receptor polypeptides also provide well defined
standards for calibrating such assays or as positive control
samples.
[0170] A preferred kit for determining the concentration of, for
example, a chemokine or receptor in a sample would typically
comprise a labeled compound, e.g., antibody, having known binding
affinity for the target, a source of ligand or receptor (naturally
occurring or recombinant) and a means for separating the bound from
free labeled compound, for example, a solid phase for immobilizing
the chemokine or receptor. Compartments containing reagents, and
instructions for use or disposal, will normally be provided.
[0171] Antibodies, including antigen binding fragments, specific
for the chemokine or receptor, or fragments are useful in
diagnostic applications to detect the presence of elevated levels
of chemokine, receptor, and/or its fragments. Such diagnostic
assays can employ lysates, live cells, fixed cells,
immunofluorescence, cell cultures, body fluids, and further can
involve the detection of antigens related to the ligand or receptor
in serum, or the like. Diagnostic assays may be homogeneous
(without a separation step between free reagent and antigen
complex) or heterogeneous (with a separation step). Various
commercial assays exist, such as radioimmunoassay (RIA),
enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay
(EIA), enzyme-multiplied immunoassay technique (EMIT),
substrate-labeled fluorescent immunoassay (SLFIA), and the like.
For example, unlabeled antibodies can be employed by using a second
antibody which is labeled and which recognizes the primary antibody
to a chemokine or receptor or to a particular fragment thereof.
Similar assays have also been extensively discussed in the
literature. See, e.g., Harlow and Lane (1988) Antibodies: A
Laboratory Manual, CSH.
[0172] Anti-idiotypic antibodies may have similar uses to diagnose
presence of antibodies against a chemokine or receptor, as such may
be diagnostic of various abnormal states. For example,
overproduction of a chemokine or receptor may result in production
of various immunological reactions which may be diagnostic of
abnormal physiological states, particularly in various inflammatory
or asthma conditions.
[0173] Frequently, the reagents for diagnostic assays are supplied
in kits, so as to optimize the sensitivity of the assay. For the
subject invention, depending upon the nature of the assay, the
protocol, and the label, either labeled or unlabeled antibody or
labeled chemokine or receptor is provided. This is usually in
conjunction with other additives, such as buffers, stabilizers,
materials necessary for signal production such as substrates for
enzymes, and the like. Preferably, the kit will also contain
instructions for proper use and disposal of the contents after use.
Typically the kit has compartments or containers for each useful
reagent. Desirably, the reagents are provided as a dry lyophilized
powder, where the reagents may be reconstituted in an aqueous
medium providing appropriate concentrations of reagents for
performing the assay.
[0174] The aforementioned constituents of the drug screening and
the diagnostic assays may be used without modification or may be
modified in a variety of ways. For example, labeling may be
achieved by covalently or non-covalently joining a moiety which
directly or indirectly provides a detectable signal. In any of
these assays, the ligand, test compound, chemokine, receptor, or
antibodies thereto can be labeled either directly or indirectly.
Possibilities for direct labeling include label groups: radiolabels
such as .sup.125I, enzymes (U.S. Pat. No. 3,645,090) such as
peroxidase and alkaline phosphatase, and fluorescent labels (U.S.
Pat. No. 3,940,475) capable of monitoring the change in
fluorescence intensity, wavelength shift, or fluorescence
polarization. Possibilities for indirect labeling include
biotinylation of one constituent followed by binding to avidin
coupled to one of the above label groups.
[0175] There are also numerous methods of separating bound from the
free ligand, or alternatively bound from free test compound. The
chemokine or receptor can be immobilized on various matrixes,
perhaps with detergents or associated lipids, followed by washing.
Suitable matrixes include plastic such as an ELISA plate, filters,
and beads. Methods of immobilizing the chemokine or receptor to a
matrix include, without limitation, direct adhesion to plastic, use
of a capture antibody, chemical coupling, and biotin-avidin. The
last step in this approach may involve the precipitation of
antigen/antibody complex by any of several methods including those
utilizing, e.g., an organic solvent such as polyethylene glycol or
a salt such as ammonium sulfate. Other suitable separation
techniques include, without limitation, the fluorescein antibody
magnetizable particle method described in Rattle, et al. (1984)
Clin. Chem. 30:1457-1461, and the double antibody magnetic particle
separation as described in U.S. Pat. No. 4,659,678.
[0176] Methods for linking proteins or their fragments to the
various labels have been extensively reported in the literature and
do not require detailed discussion here. Many of the techniques
involve the use of activated carboxyl groups either through the use
of carbodiimide or active esters to form peptide bonds, the
formation of thioethers by reaction of a mercapto group with an
activated halogen such as chloroacetyl, or an activated olefin such
as maleimide, for linkage, or the like. Fusion proteins will also
find use in these applications.
[0177] Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence
of the chemokine or receptor. These sequences can be used as probes
for detecting levels of the ligand message in samples from patients
suspected of having an abnormal condition, e.g., an inflammatory,
physiological, or developmental problem. The preparation of both
RNA and DNA nucleotide sequences, the labeling of the sequences,
and the preferred size of the sequences has received ample
description and discussion in the literature. Normally an
oligonucleotide probe should have at least about 14 nucleotides,
usually at least about 18 nucleotides, and the polynucleotide
probes may be up to several kilobases. Various labels may be
employed, most commonly radionuclides, particularly .sup.32P.
However, other techniques may also be employed, such as using
biotin modified nucleotides for introduction into a polynucleotide.
The biotin then serves as the site for binding to avidin or
antibodies, which may be labeled with a wide variety of labels,
such as radionuclides, fluorescers, enzymes, or the like.
Alternatively, antibodies may be employed which can recognize
specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA
hybrid duplexes, or DNA-protein duplexes. The antibodies in turn
may be labeled and the assay carried out where the duplex is bound
to a surface, so that upon the formation of duplex on the surface,
the presence of antibody bound to the duplex can be detected. The
use of probes to the novel anti-sense RNA may be carried out in
conventional techniques such as nucleic acid hybridization, plus
and minus screening, recombinational probing, hybrid released
translation (HRT), and hybrid arrested translation (HART). This
also includes amplification techniques such as polymerase chain
reaction (PCR).
[0178] Diagnostic kits which also test for the qualitative or
quantitative presence of other markers are also contemplated.
Diagnosis or prognosis may depend on the combination of multiple
indications used as markers. Thus, kits may test for combinations
of markers. See, e.g., Viallet, et al. (1989) Progress in Growth
Factor Res. 1:89-97.
[0179] X. Receptor for Chemokine; Ligands for Receptors
[0180] Having isolated a ligand binding partner of a specific
interaction, methods exist for isolating the counter-partner. See,
Gearing, et al EMBO J. 8:3667-4676 or McMahan, et al. (1991) EMBO
J. 10:2821-2832. For example, means to label a chemokine without
interfering with the binding to its receptor can be determined. For
example, an affinity label can be fused to either the amino- or
carboxy-terminus of the ligand. An expression library can be
screened for specific binding of chemokine, e.g., by cell sorting,
or other screening to detect subpopulations which express such a
binding component. See, e.g., Ho, et al. (1993) Proc. Nat'l Acad.
Sci. 90:11267-11271. Alternatively, a panning method may be used.
See, e.g., Seed and Aruffo (1987) Proc. Nat'l. Acad. Sci.
84:3365-3369.
[0181] With a receptor, means to identify the ligand exist. Methods
for using the receptor, e.g., on the cell membrane, can be used to
screen for ligand by, e.g., assaying for a common G-protein linked
signal such as Ca++ flux. See Lerner (1994) Trends in Neurosciences
17:142-146. It is likely that the ligands for these receptors are
chemokines.
[0182] Protein cross-linking techniques with label can be applied
to a isolate binding partners of a chemokine. This would allow
identification of protein which specifically interacts with a
chemokine, e.g., in a ligand-receptor like manner.
[0183] The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the invention to specific embodiments.
EXAMPLES
[0184] I. General Methods
[0185] Many of the standard methods below are described or
referenced, e.g., in Maniatis, et al. (1982) Molecular Cloning, A
Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor
Press, NY; Sambrook, et al. (1989) Molecular Cloning: A Laboratory
Manual (2d ed.) Vols. 1-3, CSH Press, NY; Ausubel, et al., Biology
Greene Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al.
(1987 and Supplements) Current Protocols in Molecular Biology
Wiley/Greene, NY; Innis, et al. (eds.) (1990) PCR Protocols: A
Guide to Methods and Applications Academic Press, NY. Methods for
protein purification include such methods as ammonium sulfate
precipitation, column chromatography, electrophoresis,
centrifugation, crystallization, and others. See, e.g., Ausubel, et
al. (1987 and periodic supplements); Deutscher (1990) "Guide to
Protein Purification," Methods in Enzymology vol. 182, and other
volumes in this series; Coligan, et al. (1995 and supplements)
Current Protocols in Protein Science John Wiley and Sons, New York,
N.Y.; P. Matsudaira (ed.) (1993) A Practical Guide to Protein and
Peptide Purification for Microsequencing, Academic Press, San
Diego, Calif.; and manufacturer's literature on use of protein
purification products, e.g., Pharmacia, Piscataway, N.J., or
Bio-Rad, Richmond, Calif. Combination with recombinant techniques
allow fusion to appropriate segments (epitope tags), e.g., to a
FLAG sequence or an equivalent which can be fused, e.g., via a
protease-removable sequence. See, e.g., Hochuli (1989) Chemische
Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant
Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic
Engineering, Principle and Methods 12:87-98, Plenum Press, NY; and
Crowe, et al. (1992) QIAexpress: The High Level Expression &
Protein Purification System QUIAGEN, Inc., Chatsworth, Calif.
[0186] Standard immunological techniques are described, e.g., in
Hertzenberg, et al. (eds. 1996) Weir's Hanbook of Experimental
Immunology vols 1-4, Blackwell Science; Coligan (1991) Current
Protocols in Immunology Wiley/Greene, NY; and Methods in Enzymology
volumes. 70, 73, 74, 84, 92, 93, 108, L16, 121, 132, 150, 162, and
163. Assays for neural cell biological activities are described,
e.g., in Wouterlood (ed. 1995) Neuroscience Protocols modules 10,
Elsevier; Methods in Neurosciences Academic Press; and Neuromethods
Humana Press, Totowa, N.J. Methodology of developmental systems is
described, e.g., in Meisami (ed.) Handbook of Human Growth and
Developmental Biology CRC Press; and Chrispeels (ed.) Molecular
Techniques and Approaches in Developmental Biology
Interscience.
[0187] FACS analyses are described in Melamed, et al. (1990) Flow
Cytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro
(1988) Practical Flow Cytometry Liss, New York, N.Y.; and Robinson,
et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New
York, N.Y.
[0188] II. Isolation and Characterization of Rodent CXC N4
cDNAs
[0189] The rodent CXC N4 was identified from a mouse cDNA library.
Individual cDNA clones are sequenced using standard methods, e.g.,
the Taq DyeDeoxy Terminator Cycle Sequencing kit (Applied
Biosystems, Foster City, Calif.), and the sequence is further
characterized.
[0190] The predicted signal sequence corresponds to amino acids
met1 to about gly19, so the mature form should begin with gln20 and
run about the standard chemokine length, e.g., about 90 to 110
residues. Additional processing may occur in a physiological
system.
[0191] Computer analysis and alignments for related genes indicates
the closest match is to the mouse chemokines SDF-1, IP-10, and MIG.
This similarity in sequence may well correlate with similarity in
regulation, which suggests related functions. The absence of the
ELR motif in the CXC chemokine suggests that it will not bind to
the IL-8 receptors, and is probably not angiogenic. It may still be
angiostatic, suggesting some possible use in tumor or related
therapies. Conversely, an antagonist is likely to block its
activity, which suggests therapeutic or research use where
angiostasis is undesirable, e.g., wound healing, etc.
[0192] Other rodent counterparts should be isolatable using the
entire coding portion of this clone as a hybridization probe. A
Southern blot or PCR analysis may indicate the extent of homology
across species, and either a cDNA library or mRNA can be screened
to identify an appropriate cell source. The physiological state of
many different cell types may also be evaluated for increased
expression of the gene.
[0193] III. Isolation and Characterization of GPCR cDNAs
[0194] A. Rodent DNAXCCR10
[0195] The partial rodent DNAXCCR10 clone was derived from mouse
cDNA library. The nucleotide sequence is provided in FIG. 5 (SEQ ID
NOS:5 and 6), encoding a polypeptide of about 75 amino acids, at
the carboxy terminus of the natural gene.
[0196] Computer analysis suggests that the closest related genes
are various G-protein coupled receptors. These include the
chemokine receptors, and protease, e.g., thrombin, receptors.
Structural motifs suggest that the receptor contains motifs
characteristic of the chemokine receptor family, and of the
protease receptor family. The transmembrane segments, based upon
hydrophobicity plots and comparisons with other similar GPCRs,
particularly the human and canine GPCR W genes, should be about as
follows: TM6 ends at gln2; and TM7 from phe14 to gln36. See, e.g.,
Loetscher, et al. (1996) J. Expt'l Med. 184:963-969. The amino
terminal segment is probably an extracellular segment (E1), and the
others would be E2 between TM2 and TM3; E3 between TM4 and TM5; and
E4 between TM6 and TM7. The intracellular segments should then run
I1 between TM1 and TM2; I2 between TM3 and TM4, I3 between TM5 and
TM6, and I4 the carboxy terminus from the end of TM7. Additional
processing may occur in a physiological system. A computer analysis
of GPCR sequences will indicate residues characteristic of the
family members.
[0197] Other rodent counterparts should be isolatable using the
entire coding portion of this mouse clone as a hybridization probe.
A Southern blot may indicate the extent of homology across species,
and either a cDNA library or mRNA can be screened to identify an
appropriate cell source. The physiological state of many different
cell types may also be evaluated for increased expression of the
gene.
[0198] Screening for response to various chemokine ligands
indicates that the receptor responds to the presence of the
MIP-3.alpha. chemokine. See Hieshima, et al. (1997) J. Biol. Chem.
272:5846-5853; Hromas, et al. (1997) Blood 89:3315-3322; and Baba,
et al. (1997) J. Biol. Chem. 272:14893-14898. The receptor induced
a Ca++ flux upon transfection into various cell types and
contacting with the MIP-3.alpha.. This receptor is distributed on T
cells, as discussed below.
[0199] This suggests that the MIP-3.alpha. chemokine ligand, or its
antagonist, should have activity in recruiting resting T cells
and/or NK cells to sites of inflammation. It may also be useful in
vaccines, exhibiting certain adjuvant effects. Thus, these reagents
may be useful in regulation of inflammation, or initiation of an
immune response.
[0200] B. Primate BLRx
[0201] The primate BLRX clone was derived from a cDNA library made
from human sequence, including both olfactory epithelium and tonsil
cells.
[0202] Computer analysis suggests that the closest related genes
are various G-protein coupled receptors. Structural analysis
indicates that the receptor contains motifs characteristic of the
chemokine receptor family, and exhibits similarity to a bovine
gustative receptor. The transmembrane segments, based upon
hydrophobicity plots and comparisons with other similar GPCRs,
should be about as follows: TM1 from val42 to tyr69; TM2 from val78
to val102; TM3 from ile114 to ile135; TM4 from trp155 to val174;
TM5 from ala198 to tyr220; TM6 from lys240 to asn257; TME7 from
gln286 to phe305.
[0203] Other primate counterparts should be isolatable using the
entire coding portion of this human clone as a hybridization probe.
A Southern blot may indicate the extent of homology across species,
and either a cDNA library or mRNA can be screened to identify an
appropriate cell source. The physiological state of many different
cell types may also be evaluated for increased expression of the
gene.
[0204] IV. Preparation of Antibodies
[0205] Many standard methods are available for preparation of
antibodies. For example, synthetic peptides may be prepared to be
used as antigen, administered to an appropriate animal, and either
polyclonal or monoclonal antibodies prepared. Short peptides, e.g.,
less than about 10 amino acids may be expressed as repeated
sequences, while longer peptides may be used alone or conjugated to
a carrier. For example, with the GPCRs, animals are immunized with
peptides or complete proteins from apprpriate portions of FIGS.
1-5. Highest specificity will result when the polypeptides are
selected from portions which are most unique, e.g., not from
conserved sequence regions. The animals may be used to collect
antiserum, or may be used to generate monoclonal antibodies.
[0206] Antiserum is evaluated for use, e.g., in an ELISA, and will
be evaluated for utility in immunoprecipitation, e.g., typically
native, or Western blot, e.g., denatured antigen, analysis.
Monoclonal antibodies will also be evaluated for those same
uses.
[0207] The antibodies provided will be useful as immunoaffinity
reagents, as detection reagents, for immunohistochemistry, and as
potential therapeutic reagents, either as agonist or antagonist
reagents.
[0208] V. Assays for Chemotactic Activity of Chemokines
[0209] Chemokine proteins are produced, e.g., in COS cells
transfected with a plasmid carrying the chemokine cDNA by
electroporation. See, Hara, et al. (1992) EMBO J. 10:1875-1884.
Physical analytical methods may be applied, e.g., CD analysis, to
compare tertiary structure to other chemokines to evaluate whether
the protein has likely folded into an active conformation. After
transfection, a culture supernatant is collected and subjected to
bioassays. A mock control, e.g., a plasmid carrying the luciferase
cDNA, is used. See, de Wet, et al. (1987) Mol. Cell. Biol.
7:725-757. A positive control, e.g., recombinant murine
MIP-1.alpha. from R&D Systems (Minneapolis, Minn.), is
typically used. Likewise, antibodies may be used to block the
biological activities, e.g., as a control.
[0210] Lymphocyte migration assays are performed as previously
described, e.g., in Bacon, et al. (1988) Br. J. Pharmacol.
95:966-974. Murine Th2 T cell clones, CDC-25 (see Tony, et al.
(1985) J. Exp. Med. 161:223-241) and HDK-1 (see Cherwinski, et al.
(1987) J. Exp. Med. 166:1229-1244), made available from R. Coffman
and A. O'Garra (DNAX, Palo Alto, Calif.), respectively, are used as
controls.
[0211] Ca2+ flux upon chemokine stimulation is measured, e.g.,
according to the published procedure described in Bacon, et al.
(1995) J. Immunol. 154:3654-3666.
[0212] Maximal numbers of migrating cells in response to the
chemokine being tested are measured. See Schall (1993) J. Exp. Med.
177:1821-1826. A dose-response curve is determined, preferably
giving a characteristic bell shaped dose-response curve.
[0213] After stimulation with various chemokines, lymphocytes often
exhibit a measurable intracellular Ca2+ flux. MIP-1.alpha., e.g.,
is capable of inducing immediate transients of calcium
mobilization. Typically, the levels of chemokine used in these
assays will be comparable to those used for the chemotaxis assays
({fraction (1/1000)} dilution of conditioned supernatants).
[0214] Retroviral infection assays have also been described, and
recent description of certain chemokine receptors in retroviral
infection processes may indicate that similar roles may apply these
receptors. See, e.g., Balter (1996) Science 272:1740 (describing
evidence for chemokine receptors as coreceptors for HIV); and Deng,
et al. (1996) Nature 381:661-666.
[0215] For receptors, biological activity may be measured in
response to an appropriate ligand. The receptors are transfected
into an assortment of cell types, each of which is likely to
possess the intracellular signaling components compatible with the
expressed receptor. Various ligand sources are tested to find a
source of ligand which results in a G-protein coupled response.
Alternatively, the cells are tested for Ca++ flux in response to
such ligands. Flux may be conveniently measured by
electrophysiological means, or by Ca++ sensitive dyes.
[0216] VI. Analysis of Individual Variation
[0217] From the distribution data, an abundant easily accessible
cell type is selected for sampling from individuals. Using PCR
techniques, a large population of individuals are analysed for this
gene. cDNA or other PCR methods are used to sequence the
corresponding gene in the different individuals, and their
sequences are compared. This indicates both the extent of
divergence among racial or other populations, as well as
determining which residues are likely to be modifiable without
dramatic effects on function.
[0218] VII. Biological Activities, Direct and Indirect
[0219] A robust and sensitive assay is selected as described above,
e.g., on immune cells, neuronal cells, or stem cells. Chemokine is
added to the assay in increasing doses to see if a dose response is
detected. For example, in a proliferation assay, cells are plated
out in plates. Appropriate culture medium is provided, and
chemokine is added to the cells in varying amounts. Growth is
monitored over a period of time which will detect either a direct
effect on the cells, or an indirect effect of the chemokine.
[0220] Alternatively, an activation assay or attraction assay is
used. An appropriate cell type is selected, e.g, hematopoietic
cells, myeloid (macrophages, neutrophils, polymorphonuclear cells,
etc.) or lymphoid (T cell, B cell, or NK cells), neural cells
(neurons, neuroglia, oligodendrocytes, astrocytes, etc.), or stem
cells, e.g., progenitor cells which differentiate to other cell
types, e.g., gut crypt cells and undifferentiated cell types.
[0221] Other assays will be those which have been demonstrated with
other chemokines. See, e.g., Schall and Bacon (1994) Current
Opinion in Immunology 6:865-873; and Bacon and Schall (1996) Int.
Arch. Allergy & Immunol. 109:97-109.
[0222] VIII. Structure Activity Relationship
[0223] Information on the criticality of particular residues is
determined using standard procedures and analysis. Standard
mutagenesis analysis is performed, e.g., by generating many
different variants at determined positions, e.g., at the structural
positions identified above, and evaluating biological activities of
the variants. This may be performed to the extent of determining
positions which modify activity, or to focus on specific positions
to determine the residues which can be substituted to either
retain, block, or modulate biological activity.
[0224] Alternatively, analysis of natural variants can indicate
what positions tolerate natural mutations. This may result from
populational analysis of variation among individuals, or across
strains or species. Samples from selected individuals are analyzed,
e.g., by PCR analysis and sequencing. This allows evaluation of
population polymorphisms.
[0225] IX. Chromosomal Localization
[0226] The cDNA is labeled, e.g., nick-translated with biotin-14
dATP and hybridized in situ at a final concentration of 5 ng/.mu.l
to metaphases from two normal males. Fluorescence in situ
hybridization (FISH) method may be modified from that described by
Callen, et al. (1990). Ann. Genet. 33:219-221, in that chromosomes
are stained before analysis with both prodidium iodide (as counter
stain) and DAPI (for chromosome identification). Images of
metaphase preparations are captured by a CCD camera and computer
enhanced. Identification of the appropriate labeled chromosomes is
determined. Localization to the standard locations for such
molecule, or different location may also provide information as to
function.
[0227] X. Expression Analysis of Chemokine/Receptor Genes
[0228] RNA blot and hybridization are performed according to the
standard methods in Maniatis, et al. (1982) Molecular Cloning: A
Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. An appropriate fragment or the whole coding sequence
of a cDNA fragment is selected for use as a probe. To verify the
amount of RNA loaded in each lane, the substrate membrane is
reprobed with a control cDNA, e.g., glyceraldehyde 3-phosphate
dehydrogenase (G3PDH) cDNA (Clontech, Palo Alto Calif.).
[0229] Analysis of mRNA from the appropriate cell source using the
probe will determine the natural size of message. It will also
indicate whether different sized messages exist. The messages will
be subject to analysis after isolation, e.g., by PCR or
hybridization techniques.
[0230] Northern blot analysis may be performed on many different
mRNA sources, e.g., different tissues, different species, or cells
exhibiting defined physiological responses, e.g., activation
conditions or developmental conditions. However, in certain cases,
cDNA libraries may be used to evaluate sources which are difficult
to prepare. A "reverse Northern" uses cDNA inserts removed from
vector, but multiplicity of bands may reflect either different
sized messages, or may be artifact due to incomplete reverse
transcription in the preparation of the cDNA library. In such
instances, verification may be appropriate by standard Northern
analysis.
[0231] Similarly, Southern blots may be used to evaluate species
distribution of a gene. The stringency of washes of the blot will
also provide information as to the extent of homology of various
species counterparts.
[0232] Tissue distribution, and cell distribution, may be evaluated
by immunohistochemistry using antibodies. Alternatively, in situ
nucleic acid hybridization may also be used in such analysis.
Certain distribution data may be ascertained by the frequency and
tissue types where messages have been found and collected in
sequence databases, e.g., GenBank or proprietary collections.
[0233] A. Rodent CXC N4
[0234] The mouse CXC N4 sequence was identified from a mouse
sequence data base. There is little distribution data generated at
this time.
[0235] B. Rodent DNAXCCR10
[0236] The rodent DNAXCCR10 was isolated from a mouse cDNA library.
A human counterpart nucleic acid is expressed in activate
monocytes, and in spleen, lymph node, and appendix, with lesser
signals detected in thymus and testis. The signal in testis may be
artifact, and should probably be retested under more stringent
conditions of isolated tissue. The message appears to be
down-regulated in activated PBMC, in activated splenocytes, and
activated T cell clones. The message appears to be up-regulated in
activated NK cells. It appears up-regulated in activated monocytes,
but down-regulated in IL-10 treated monocytes.
[0237] The distribution of the receptor points more to the function
of the ligand, MIP-3.alpha., as discussed above.
[0238] C. Primate BLRx
[0239] The BLRx GPCR was identified from primate cDNA library from
Weizman Olfactory Epithelium and human tonsilar cells enriched for
germinal center B cells. A full length clone was isolated from
human spleen, and fragments have been identified in cDNA libraries
derived from endometrium stromal cells and synovial
fibroblasts.
[0240] The message has been detected in cDNA libraries from liver,
brain, gall bladder, small intestine, ovary, uterus, spleen, and
tonsil. A weak signal was detected in activated monocytes, but this
needs to be confirmed. Message has also been detected in various T
cell lines and a CHA cell line, but not in the tested Th1, Th2, or
Th0 T cell clones. A signal was not detected in PBMC. There appear
to be short sequences available from mouse sequences which
correspond to a rodent counterpart for this gene.
[0241] XI. Biological Activity of BLRx
[0242] A. Expression Analysis
[0243] In order to identify cells types that express BLRx, a large
panel of cDNA libraries was analyzed for the expression of BLRX
using quantitative PCR. In particular, cDNA libraries from various
cellular sources (indicated in FIG. 6) were prepared as described
previously (Bolin, et al. (1997) J. Neurosci. 17:5493) and used as
templates for Taqman-PCR analyses. Cells were stimulated with
various cytokines as indicated in FIG. 6. The cDNAs (50 ng per
reaction) were analyzed for the expression of BLRx genes by the
Fluorogenic 5'-nuclease PCR assay (Holland, et al. (1991) Proc.
Natl. Acad. Sci. USA 88:7276), using an ABI Prism 7700 Sequence
Detection System (Perkin Elmer, Foster City, Calif.). Reactions
were incubated for 2 min at 50.degree. C., denatured for 10 min at
95.degree. C. and subjected to 40 two-step amplification cycles
with annealing/extension at 60.degree. C. for 1 min followed by
denaturation at 95.degree. C. for 15 sec. The PCR products were
analyzed with FAM-labeled probes. Values were expressed as fg/50 ng
total cDNA.
[0244] Results of the expression analysis are presented in FIG. 6.
In particular, expression analysis indicated that BLRx was
constitutively expressed in dermal fibroblasts (FB), with
expression significantly down-regulated by TNF-.alpha. and
IL-1.beta.. IFN-.gamma. slightly down-regulated expression by
fibroblasts, while IL-4 and IL-10 had no significant effects on
expression levels.
[0245] BLRx was expressed at lower levels in keratinocytes (KC),
melanocytes (MC) and dermal microvascular endothelial cells
(DMEC).
[0246] These results indicate that BLRx may play a role in wound
healing, sclerotic processes, keloid formation, collagen synthesis,
scleroderma, systemic sclerosis or in other biological process
where these cell types are implicated.
[0247] B. In Vivo Wound Healing Studies
[0248] In order to test the hypothesis that BLRx is involved in the
wound healing process, the following experiment was conducted.
Groups of female BALB/c mice (3 animals per group) were
anesthetized with an intraperitoneal injection of Ketamine and
Zylazine. The dorsal region was shaved and the surgical area
disinfected. A 2 cm dorsal incisional wound was made through the
epidermis and dermis leaving the subcutaneous muscle layer
undisturbed. The wound was closed and the wound and surrounding
area covered. Animals were sacrificed at 12 hours, 1, 2, 3, 5, 7
and 10 days following incision.
[0249] Wounds were harvested and RNA was extracted using the RNA
STAT 60 method (Tel-Test, New Jersey) and BLRx expression analyzed
using Taqman-PCR analysis, as described above. Results are
presented in FIG. 7. Values are expressed as fg/25 ng total cDNA.
As can be seen, BLRx expression is up-regulated during early wound
healing, peaking at 12 hours. The results evidence that BLRx plays
a role in wound healing.
[0250] XII. Screening for Receptor/Ligand
[0251] Labeled reagent is useful for screening of an expression
library made from a cell line which expresses a chemokine or
receptor, as appropriate. Standard staining techniques are used to
detect or sort intracellular or surface expressed ligand, or
surface expressing transformed cells are screened by panning.
Screening of intracellular expression is performed by various
staining or immunofluorescence procedures. See also, e.g., McMahan,
et al. (1991) EMBO J. 10:2821-2832.
[0252] For example, on day 0, precoat 2-chamber permanox slides
with 1 ml per chamber of fibronectin, 10 ng/ml in PBS, for 30 min
at room temperature. Rinse once with PBS. Then plate COS cells at
2-3.times.10.sup.5 cells per chamber in 1.5 ml of growth media.
Incubate overnight at 37.degree. C.
[0253] On day 1 for each sample, prepare 0.5 ml of a solution of 66
.mu.g/ml DEAE-dextran, 66 .mu.M chloroquine, and 4 .mu.g DNA in
serum free DME. For each set, a positive control is prepared, e.g.,
of mMIG-FLAG cDNA at 1 and 1/200 dilution, and a negative mock.
Rinse cells with serum free DME. Add the DNA solution and incubate
5 hr at 37.degree. C. Remove the medium and add 0.5 ml 10% DMSO in
DME for 2.5 min. Remove and wash once with DME. Add 1.5 ml growth
medium and incubate overnight.
[0254] On day 2, change the medium. On days 3 or 4, the cells are
fixed and stained. Rinse the cells twice with Hank's Buffered
Saline Solution (HBSS) and fix in 4% paraformaldehyde (PFA)/glucose
for 5 min. Wash 3.times. with HBSS. The slides may be stored at
-80.degree. C. after all liquid is removed. For each chamber, 0.5
ml incubations are performed as follows. Add HBSS/saponin (0.1%)
with 32 .mu.l/ml of 1M NaN.sub.3 for 20 min. Cells are then washed
with HBSS/saponin 1.times.. Add antibody complex to cells and
incubate for 30 min. Wash cells twice with HBSS/saponin. Add second
antibody, e.g., Vector anti-mouse antibody, at 1/200 dilution, and
incubate for 30 min. Prepare ELISA solution, e.g., Vector Elite ABC
horseradish peroxidase solution, and preincubate for 30 min. Use,
e.g., 1 drop of solution A (avidin) and 1 drop solution B (biotin)
per 2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add
ABC HRP solution and incubate for 30 min. Wash cells twice with
HBSS, second wash for 2 min, which closes cells. Then add Vector
diaminobenzoic acid (DAB) for 5 to 10 min. Use 2 drops of buffer
plus 4 drops DAB plus 2 drops of H.sub.2O.sub.2 per 5 ml of glass
distilled water. Carefully remove chamber and rinse slide in water.
Air dry for a few minutes, then add 1 drop of Crystal Mount and a
cover slip. Bake for 5 min at 85-90.degree. C.
[0255] Alternatively, the binding compositions are used to affinity
purify or sort out cells expressing the ligand or receptor. See,
e.g., Sambrook, et al. or Ausubel et al.
[0256] Many modification an variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of the equivalents to which such
claims are entitled.
Sequence CWU 1
1
* * * * *