U.S. patent application number 10/818952 was filed with the patent office on 2005-10-27 for therapeutic uses for mdc and mdc antagonists.
This patent application is currently assigned to ICOS CORPORATION. Invention is credited to Chantry, David H., Deeley, Michael C., Gray, Patrick W..
Application Number | 20050238647 10/818952 |
Document ID | / |
Family ID | 32302784 |
Filed Date | 2005-10-27 |
United States Patent
Application |
20050238647 |
Kind Code |
A1 |
Gray, Patrick W. ; et
al. |
October 27, 2005 |
Therapeutic uses for MDC and MDC antagonists
Abstract
The present invention provides purified and isolated
polynucleotide sequences encoding a novel human macrophage-derived
C--C chemokine designated Macrophage Derived Chemokine (MDC), and
polypeptide analogs thereof. Also provided are materials and
methods for the recombinant production of the chemokine, and
purified and isolated chemokine protein, and polypeptide analogs
thereof. Also provided are antibodies reactive with the chemokine
and methods of making and using all of the foregoing.
Inventors: |
Gray, Patrick W.; (Seattle,
WA) ; Chantry, David H.; (Seattle, WA) ;
Deeley, Michael C.; (Edmonds, WA) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 S. WACKER DRIVE, SUITE 6300
SEARS TOWER
CHICAGO
IL
60606
US
|
Assignee: |
ICOS CORPORATION
Bothell
WA
|
Family ID: |
32302784 |
Appl. No.: |
10/818952 |
Filed: |
April 6, 2004 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10818952 |
Apr 6, 2004 |
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09067447 |
Apr 28, 1998 |
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6737513 |
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09067447 |
Apr 28, 1998 |
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08939107 |
Sep 26, 1997 |
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6498015 |
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08939107 |
Sep 26, 1997 |
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08660542 |
Jun 7, 1996 |
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5932703 |
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Current U.S.
Class: |
424/145.1 |
Current CPC
Class: |
C07K 16/24 20130101;
C07K 2319/00 20130101; A01K 2217/05 20130101; C07K 2319/02
20130101; C07K 2317/76 20130101; C07K 2317/73 20130101; C07K 14/523
20130101; A61K 39/00 20130101; C07K 14/7158 20130101; A61K 2039/505
20130101 |
Class at
Publication: |
424/145.1 |
International
Class: |
A61K 039/395 |
Claims
1-18. (canceled)
19. A method of modulating chemotaxis of C--C chemokine
receptor-four positive (CCR4.sup.+) systemic memory T cells in a
mammalian host, the method comprising: administering a CCR4
modulating agent to a mammalian host in an amount effective to
modulate chemotaxis of said cells.
20. The method of claim 19, wherein said systemic memory T cells
are further characterized as
CD4.sup.+/CD45RA.sup.-/CD45RO.sup.+.
21. The method of claim 19, wherein said CCR4.sup.+ modulating
agent comprises a Macrophage Derived Chemokine (MDC) polypeptide
that comprises the amino acid sequence of residues 1-69 of SEQ ID
NO: 2.
22. The method of claim 19, wherein the CCR4.sup.+ modulating agent
comprises a polypeptide that comprises an amino acid sequence that
is encoded by a polynucleotide that hybridizes to a DNA comprising
the non-coding strand complementary to nucleotides 92 to 298 of SEQ
ID NO: 1, under the following stringent hybridization conditions:
hybridization at 42.degree. C. in a hybridization solution
comprising 5.times.SSC, 20 mM NaPO4, pH 6.8, 50% formamide; and
washing at 42.degree. C. in a wash solution comprising
0.2.times.SSC; wherein the polypeptide binds CCR4.sup.+.
23. The method of claim 19, wherein said CCR4.sup.+ modulating
agent is a MDC antibody.
24. The method of claim 23, wherein said antibody is a monoclonal
antibody.
25. The method of claim 24, wherein said antibody is selected from
the group consisting of: 252Y, 252Z, and 272D.
26. The method of claim 19, wherein said CCR4.sup.+ modulating
agent is Thymus and Activation-Regulated Chemokine (TARC).
27. A method of inhibiting inflammation comprising: administering a
composition comprising a CCR4 modulating agent that interferes with
MDC-induced chemotaxis of CCR4.sup.+ systemic memory T cells to a
mammalian host in an amount effective to modulate MDC-induced
chemotaxis of CCR4.sup.+ systemic memory T cells.
Description
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 08/939,107, filed Sep. 26, 1997, (Attorney
docket No. 27866/34188), and is a continuation-in-part of U.S.
patent application Ser. No. 08/660,542, filed Jun. 7, 1996. These
applications are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to chemokines and
more particularly to purified and isolated polynucleotides encoding
a novel human C--C chemokine, to purified and isolated chemokine
protein encoded by the polynucleotides, to chemokine analogs, to
materials and methods for the recombinant production of the novel
chemokine protein and analogs, to antibodies reactive with the
novel chemokine, to chemokine inhibitors, and to uses of all of the
foregoing materials.
BACKGROUND
[0003] Chemokines, also known as "intercrines" and "SIS cytokines",
comprise a family of small secreted proteins (e.g., 70-100 amino
acids and about 8-10 kiloDaltons) which attract and activate
leukocytes and thereby aid in the stimulation and regulation of the
immune system. The name "chemokine" is derived from chemotactic
cytokine, and refers to the ability of these proteins to stimulate
chemotaxis of leukocytes. Indeed, chemokines may comprise the main
attractants for inflammatory cells into pathological tissues. See
generally, Baggiolini et al., Annu. Rev. Immunol, 15: 675-705
(1997); and Baggiolini et al., Advances in Immunology, 55:97-179
(1994), both of which are incorporated by reference herein. While
leukocytes comprise a rich source of chemokines, several chemokines
are expressed in a multitude of tissues. Baggiolini et al. (1994),
Table II.
[0004] Previously identified chemokines generally exhibit 20-70%
amino acid identity to each other and contain four highly-conserved
cysteine residues. Based on the relative position of the first two
of these cysteine residues, chemokines have been further classified
into two subfamilies. In the "C--X--C" or "a" subfamily, encoded by
genes localized to human chromosome 4, the first two cysteines are
separated by one amino acid. In the "C--C" or ".beta." subfamily,
encoded by genes on human chromosome 17, the first two cysteines
are adjacent. X-ray crystallography and NMR studies of several
chemokines have indicated that, in each family, the first and third
cysteines form a first disulfide bridge, and the second and fourth
cysteines form a second disulfide bridge, strongly influencing the
native conformation of the proteins. In humans alone, more than ten
distinct sequences have been described for each chemokine
subfamily. Chemokines of both subfamilies have characteristic
leader sequences of twenty to twenty-five amino acids.
[0005] The C--X--C chemokines, which include IL-8,
GRO.alpha./.beta./.gamm- a., platelet basic protein, Platelet
Factor 4 (PF4), IP-10, NAP2, and others, share approximately 25% to
60% identity when any two amino acid sequences are compared (except
for the GRO.alpha./.beta./.gamma. members, which are 84-88%
identical with each other). Most of the C--X--C chemokines
(excluding IP-10 and Platelet Factor 4) share a common E-L-R
tri-peptide motif upstream of the first two cysteine residues, and
are potent stimulants of neutrophils, causing rapid shape change,
chemotaxis, respiratory bursts, and degranulation. These effects
are mediated by seven-transmembrane-domain rhodopsin-like G
protein-coupled receptors; a receptor specific for IL-8 has been
cloned by Holmes et al., Science, 253:1278-80 (1991), while a
similar receptor (77% identity) which recognizes IL-8, GRO and NAP2
has been cloned by Murphy and Tiffany, Science, 253:1280-83 (1991).
Progressive truncation of the N-terminal amino acid sequence of
certain C--X--C chemokines, including IL-.sub.8, is associated with
marked increases in activity.
[0006] The C--C chemokines, which include Macrophage Inflammatory
Proteins MIP-1.alpha. and MIP-1.beta., Monocyte chemoattractant
proteins 1, 2, 3, and 4 (MCP-1/2/3/4), RANTES, 1-309, eotaxin,
TARC, and others, share 25% to 70% amino acid identity with each
other. Previously-identified C--C chemokines activate monocytes,
causing calcium flux and chemotaxis. More selective effects are
seen on lymphocytes, for example, T lymphocytes, which respond best
to RANTES. Several seven-transmembrane-domain G protein-coupled
receptors for C--C chemokines have been cloned to date, including a
C--C chemokine receptor-1 (CCR1) which recognizes, e.g.,
MIP-1.alpha. and RANTES (Neote et al., Cell, 72:415425 (1993)); a
CCR2 receptor which has two splice variants and which recognizes,
e.g., MCP-1 (Charo et al., Proc. Nat. Acad. Sci., 91:2752-56
(1994)); CCR3, which recognizes, e.g., eotaxin, RANTES, and MCP-3
(Combadiere, J. Biol. Chem., 270:16491 (1995)); CCR4, which
recognizes MIP-1.alpha., RANTES, and MCP-1 (Power et al., J. Biol.
Chem., 270:19495 (1995)); and CCR5, which recognizes MIP-1.alpha.,
MIP-1.beta., and RANTES (Samson et al., Biochemistry, 35:3362
(1996)). Several CC chemokines have been shown to act as
attractants for activated T lymphocytes. See Baggiolini et al.
(1997).
[0007] Truncation of the N-terminal amino acid sequence of certain
C--C chemokines also has been associated with alterations in
activity. For example, mature RANTES (1-68) is processed by CD26 (a
dipeptidyl aminopeptidase specific for the sequence
NH.sub.2--X-Pro- . . . ) to generate a RANTES (3-68) form that is
capable of interacting with and transducing a signal through CCR5
(like the RANTES (1-68) form), but is one hundred-fold reduced in
its capacity to stimulate through the receptor CCR1. See Proost et
al., J. Biol. Chem., 273(13): 7222-7227 (1998), and Oravecz et al.,
J. Exp. Med., 186: 1865-1872 (1997). U.S. Pat. No. 5,705,360 to
Rollins and Zhang purports to describe N-terminal deletions of
chemokine MCP-1 that inhibit receptor binding to the corresponding
endogenous chemokine.
[0008] The roles of a number of chemokines, particularly IL-8, have
been well documented in various pathological conditions. See
generally Baggiolini et al. (1994), supra, Table VII. Psoriasis,
for example, has been linked to over-production of IL-8, and
several studies have observed high levels of IL-8 in the synovial
fluid of inflamed joints of patients suffering from rheumatic
diseases, osteoarthritis, and gout.
[0009] The role of C--C chemokines in pathological conditions also
has been documented, albeit less comprehensively than the role of
IL-8. For example, the concentration of MCP-1 is higher in the
synovial fluid of patients suffering from rheumatoid arthritis than
that of patients suffering from other arthritic diseases. The MCP-1
dependent influx of mononuclear phagocytes may be an important
event in the development of idiopathic pulmonary fibrosis. The role
of C--C chemokines in the recruitment of monocytes into
atherosclerotic areas is currently of intense interest, with
enhanced MCP-1 expression having been detected in macrophage-rich
arterial wall areas but not in normal arterial tissue. Expression
of MCP-1 in malignant cells has been shown to suppress the ability
of such cells to form tumors in vivo. (See U.S. Pat. No. 5,179,078,
incorporated herein by reference.) A need therefore exists for the
identification and characterization of additional C--C chemokines,
to further elucidate the role of this important family of molecules
in pathological conditions, and to develop improved treatments for
such conditions utilizing chemokine-derived products.
[0010] Chemokines of the C--C subfamily have been shown to possess
utility in medical imaging, e.g., for imaging sites of infection,
inflammation, and other sites having C--C chemokine receptor
molecules. See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778,
incorporated herein by reference. Such methods involve chemical
attachment of a labeling agent (e.g., a radioactive isotope) to the
C--C chemokine using art recognized techniques (see, e.g., U.S.
Pat. Nos. 4,965,392 and 5,037,630, incorporated herein by
reference), administration of the labeled chemokine to a subject in
a pharmaceutically acceptable carrier, allowing the labeled
chemokine to accumulate at a target site, and imaging the labeled
chemokine in vivo at the target site. A need in the art exists for
additional new C--C chemokines to increase the available arsenal of
medical imaging tools.
[0011] The C--C chemokines RANTES, MIP-.alpha., and MIP-1.beta.
also have been shown to be the primary mediators of the suppressive
effect of human T cells on the human immunodeficiency virus (HIV),
the agent responsible for causing human Acquired Immune Deficiency
Syndrome (AIDS). These chemokines show a dose-dependent ability, to
inhibit specific strains of HIV from infecting cultured T cell
lines [Cocchi et al., Science, 270:1811 (1995)]. In addition,
International patent publication number WO 97/44462, filed by
Institut Pasteur, describes the use of fragments and analogs of the
chemokine RANTES as antagonists, to block RANTES interaction with
its receptors, for the purpose of suppressing HIV. The C--X--C
chemokine stromal derived factor-1 (SDF-1) also is capable of
blocking infection by T-tropic HIV-1 strains. See Winkler et al.,
Science, 279:389-393 (1998). However, the processes through which
chemokines exert their protective effects have not been fully
elucidated, and these chemokines in fact may stimulate HIV
replication in cells exposed to the chemokines before HIV
infection. See Kelly et al., J. Immunol, 160:3091-3095 (1998).
Moreover, not all tested strains of the virus are equally
susceptible to the inhibitory effects of chemokines; therefore, a
need exists for additional C--C chemokines for use as inhibitors of
strains of HIV.
[0012] Similarly, it has been established that certain chemokine
receptors such as CCR5 [International Patent Publication No. WO
97/44055, published 27 Nov. 1997], CCR8, CCR2, and CXCR4) are
essential co-receptors (with the CD4 receptor) for HIV-1 entry into
susceptible cells, and that progression to AIDS is delayed in
patients having certain variant alleles of these receptors. A need
exists for additional therapeutics to inhibit HIV-1 infection
and/or proliferation by interfering with HIV-1 entry and/or
proliferation in susceptible cells.
[0013] More generally, due to the importance of chemokines as
mediators of chemotaxis and inflammation, a need exists for the
identification and isolation of new members of the chemokine family
to facilitate modulation of inflammatory and immune responses.
[0014] For example, substances that promote inflammation may
promote the healing of wounds or the speed of recovery from
conditions such as pneumonia, where inflammation is important to
eradication of infection. Modulation of inflammation is similarly
important in pathological conditions manifested by inflammation.
Crohn's disease, manifested by chronic inflammation of all layers
of the bowel, pain, and diarrhea, is one such pathological
condition. The failure rate of drug therapy for Crohn's disease is
relatively high, and the disease is often recurrent even in
patients receiving surgical intervention. The identification,
isolation, and characterization of novel chemokines facilitates
modulation of inflammation.
[0015] Similarly, substances that induce an immune response may
promote palliation or healing of any number of pathological
conditions. Due to the important role of leukocytes (e.g.,
neutrophils and monocytes) in cell-mediated immune responses, and
due to the established role of chemokines in leukocyte chemotaxis,
a need exists for the identification and isolation of new
chemokines to facilitate modulation of immune responses.
[0016] Additionally, the established correlation between chemokine
expression and inflammatory conditions and disease states provides
diagnostic and prognostic indications for the use of chemokines, as
well as for antibody substances that are specifically
immunoreactive with chemokines; a need exists for the
identification and isolation of new chemokines to facilitate such
diagnostic and prognostic indications.
[0017] In addition to their ability to attract and activate
leukocytes, some chemokines, such as IL-8, have been shown to be
capable of affecting the proliferation of non-leukocytic cells. See
Tuschil, J. Invest. Dermatol, 99:294-298 (1992). A need exists for
the identification and isolation of new chemokines to facilitate
modulation of such cell proliferation.
[0018] It will also be apparent from the foregoing discussion of
chemokine activities that a need exists for modulators of chemokine
activities, to inhibit the effects of endogenously-produced
chemokines and/or to promote the activities of
endogenously-produced or exogenously administered chemokines. Such
modulators typically include small molecules, peptides, chemokine
fragments and analogs, and/or antibody substances. Chemokine
inhibitors interfere with chemokine signal transduction, i.e., by
binding chemokine molecules, by competitively or non-competitively
binding chemokine receptors, and/or by interfering, with signal
transduction downstream from the chemokine receptors. A need exists
in the art for effective assays to rapidly screen putative
chemokine modulators for modulating activity.
[0019] For all of the aforementioned reasons, a need exists for
recombinant methods of production of newly discovered chemokines,
which methods facilitate clinical applications involving the
chemokines and chemokine inhibitors.
SUMMARY OF THE INVENTION
[0020] The present invention provides novel purified and isolated
polynucleotides and polypeptides, antibodies, and methods and
assays that fulfill one or more of the needs outlined above.
[0021] For example, the invention provides purified and isolated
polynucleotides (i.e., DNA and RNA, both sense and antisense
strands) encoding a novel human chemokine of the C--C subfamily,
herein designated "Macrophage Derived Chemokine" or "MDC".
Preferred DNA sequences of the invention include genomic and cDNA
sequences and chemically synthesized DNA sequences. Polynucleotides
encoding non-human vertebrate forms of MDC, especially mammalian
and avian forms of MDC, also are intended as aspects of the
invention.
[0022] The nucleotide sequence of a cDNA, designated MDC cDNA,
encoding this chemokine, is set forth in SEQ ID NO: 1, which
sequence includes 5' and 3' non-coding sequences. A preferred DNA
of the present invention comprises nucleotides 20 to 298 of SEQ ID
NO. 1, which nucleotides comprise the MDC coding sequence.
[0023] The human MDC protein comprises a putative twenty-four amino
acid signal sequence at its amino terminus. Another preferred DNA
of the present invention comprises nucleotides 92 to 298 of SEQ ID
NO. 1, which nucleotides comprise the putative coding sequence of
the mature (secreted) MDC protein, without the signal sequence.
[0024] The amino acid sequence of human chemokine MDC is set forth
in SEQ ID NO: 2. Preferred polynucleotides of the present invention
include, in addition to those polynucleotides described above,
polynucleotides that encode the amino acid sequence set forth in
SEQ ID NO: 2, and that differ from the polynucleotides described in
the preceding paragraphs only due to the well-known degeneracy of
the genetic code.
[0025] Similarly, since twenty-four amino acids (positions -24 to
-1) of SEQ ID NO: 2 comprise a putative signal peptide that is
cleaved to yield the mature MDC chemokine, preferred
polynucleotides include those which encode amino acids 1 to 69 of
SEQ ID NO: 2. Thus, a preferred polynucleotide is a purified
polynucleotide encoding a polypeptide having an amino acid sequence
comprising amino acids 1-69 of SEQ ID NO: 2.
[0026] Among the uses for the polynucleotides of the present
invention is the use as a hybridization probe, to identify and
isolate genomic DNA encoding human MDC, which gene is likely to
have a three exon/two intron structure characteristic of C--C
chemokines genes. (See Baggiolini et al. (1994), supra); to
identify and isolate DNAs having sequences encoding non-human
proteins homologous to MDC; to identify human and non-human
chemokine genes having similarity to the MDC gene; and to identify
those cells which express MDC and the conditions under which this
protein is expressed. Polynucleotides encoding human MDC have been
employed to successfully isolate polynucleotides encoding at least
two exemplary non-human embodiments of MDC (rat and mouse). (See
SEQ ID NOs: 35-38.)
[0027] Hybridization probes of the invention also have diagnostic
utility, e.g., for screening for inflammation in human tissue, such
as colon tissue. More particularly, hybridization studies using an
MDC polynucleotide hybridization probe distinguished colon tissue
of patients with Crohn's disease (MDC hybridization detected in
epithelium, lamina propria, Payer's patches, and smooth muscle)
from normal human colon tissue (no hybridization above
background).
[0028] Generally speaking, a continuous portion of the MDC cDNA of
the invention that is at least about 14 nucleotides, and preferably
about 18 nucleotides, is useful as a hybridization probe of the
invention. Thus, in one embodiment, the invention includes a DNA
comprising a continuous portion of the nucleotide sequence of SEQ
ID NO: 1 or of the non-coding strand complementary thereto, the
continuous portion comprising at least 18 nucleotides, the DNA
being capable of hybridizing under stringent conditions to a coding
or non-coding strand of a human MDC gene. For diagnostic utilities,
hybridization probes of the invention preferably show hybridization
specificity for MDC gene sequences. Thus, in a preferred
embodiment, hybridization probe DNAs of the invention fail to
hybridize under the stringent conditions to other human chemokine
genes (e.g., MCP-1 genes, MCP-2 genes, MCP-3 genes, RANTES genes,
MIP-1.alpha. genes, MIP-1.beta. genes, and 1-309 genes, etc.).
[0029] In another aspect, the invention provides a purified
polynucleotide which hybridizes under stringent conditions to the
non-coding strand of the DNA of SEQ ID NO: 1. Similarly, the
invention provides a purified polynucleotide which, but for the
redundancy of the genetic code, would hybridize under stringent
conditions to the non-coding strand of the DNA of SEQ ID NO: 1.
Exemplary stringent hybridization conditions are as follows:
hybridization at 42.degree. C. in 5.times.SSC, 20 mM NaPO.sub.4, pH
6.8, 50% formamide; and washing at 42.degree. C. in 0.2.times.SSC.
Those skilled in the art understand that it is desirable to vary
these conditions empirically based on the length and the GC
nucleotide base content of the sequences to by hybridized, and that
formulas for determining such variation exist. [See, e.g., Sambrook
et al., Molecular Cloning: a Laboratory Manual. Second Edition,
Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
(1989).]
[0030] In another aspect, the invention includes plasmid and viral
DNA vectors incorporating DNAs of the invention, including any of
the DNAs described above or elsewhere herein. Preferred vectors
include expression vectors in which the incorporated MDC-encoding
cDNA is operatively linked to an endogenous or heterologous
expression control sequence. Such expression vectors may further
include polypeptide-encoding DNA sequences operably linked to the
MDC-encoding DNA sequences, which vectors may be expressed to yield
a fusion protein comprising the MDC polypeptide of interest.
[0031] In another aspect, the invention includes a prokaryotic or
eukaryotic host cell stably transfected or transformed with a DNA
or vector of the present invention. In preferred host cells, the
mature MDC polypeptide encoded by the DNA or vector of the
invention is expressed. The DNAs, vectors, and host cells of the
present invention are useful, e.g., in methods for the recombinant
production of large quantities of MDC polypeptides of the present
invention. Such methods are themselves aspects of the invention.
For example, the invention includes a method for producing MDC
wherein a host cell of the invention is grown in a suitable
nutrient medium and MDC protein is isolated from the cell or the
medium.
[0032] Knowledge of DNA sequences encoding MDC makes possible
determination of the chromosomal location of MDC coding sequences,
as well as identification and isolation by DNA/DNA hybridization of
genomic DNA sequences encoding the MDC expression control
regulatory sequences such as promoters, operators, and the
like.
[0033] According to another aspect of the invention, host cells may
be modified by activating an endogenous MDC gene that is not
normally expressed in the host cells or that is expressed at a
lower level than is desired. Such host cells are modified (e.g., by
homologous recombination) to express MDC by replacing, in whole or
in part, the naturally-occurring MDC promoter with part or all of a
heterologous promoter so that the host cells express MDC. In such
host cells, the heterologous promoter DNA is operatively linked to
the MDC coding sequences, i.e., controls transcription of the MDC
coding sequences. See, for example, PCT International Publication
No. WO 94/12650; PCT International Publication No. WO 92/20808; and
PCT International Publication No. WO 91/09955. The invention also
contemplates that, in addition to heterologous promoter DNA,
amplifiable marker DNA (e.g., ada, dhfr, and the multi-functional
CAD gene which encodes carbamyl phosphate synthase, aspartate
transcarbamylase, and dihydro-orotase) and/or intron DNA may be
recombined along with the heterologous promoter DNA into the host
cells. If linked to the MDC coding sequences, amplification of the
marker DNA by standard selection methods results in
co-amplification of the MDC coding sequences in such host
cells.
[0034] The DNA sequence information provided by the present
invention also makes possible the development, by homologous
recombination or "knockout" strategies [see, Capecchi, Science.,
244: 1288-1292 (1989)], of rodents that fail to express functional
MDC or that express a variant of MDC. Such rodents are useful as
models for studying the activities of MDC, MDC variants, and MDC
modulators in vivo. Rodents having a humanized immune system are
useful as models for studying the activities of MDC and MDC
modulators toward HIV infection and proliferation.
[0035] In yet another aspect, the invention includes purified and
isolated MDC polypeptides. Mammalian and avian MDC polypeptides are
specifically contemplated. A preferred peptide is a purified
chemokine polypeptide having an amino acid sequence comprising
amino acids 1 to 69 of SEQ ID NO: 2 (human mature MDC). Throughout
the application, human mature MDC usually will be referred to
simply as "MDC" or as "mature MDC". In instances where context
warrants, such as certain descriptions of experiments that involve
both human and non-human mature MDCs and/or that involve MDC
fragments and analogs, human mature MDC will sometimes be
specifically referred to as "human" and will sometimes be referred
to as "MDC(1-69)."
[0036] Mouse and Rat MDC polypeptides of the invention are taught
in SEQ ID NOs: 36 and 38. The sequence in SEQ ID NO: 36 depicts a
complete murine MDC, consisting of a 24 residue leader peptide
(residues -24 to -1 of SEQ ID NO: 36) and a 68 residue murine
mature MDC. The sequence in SEQ ID NO: 38 depicts a partial rat
MDC, consisting of 13 residues of the leader peptide (residues -13
to -1) and the complete 68 residue rat mature MDC.
[0037] The polypeptides of the present invention may be purified
from natural sources, but are preferably produced by recombinant
procedures, using the DNAs, vectors, and/or host cells of the
present invention, or are chemically synthesized. Purified
polypeptides of the invention may be glycosylated or
non-glyclosylated, water soluble or insoluble, oxidized, reduced,
etc., depending on the host cell selected, recombinant production
method, isolation method, processing, storage buffer, and the
like.
[0038] Moreover, an aspect of the invention includes MDC
polypeptide analogs wherein one or more amino acid residues is
added, deleted, or replaced from the MDC polypeptides of the
present invention, which analogs retain one or more of the
biological activities characteristic of the C--C chemokines,
especially of MDC. The small size of MDC facilitates chemical
synthesis of such polypeptide analogs, which may be screened for
MDC biological activities (e.g., the ability to induce macrophage
chemotaxis, or inhibit monocyte chemotaxis) using the many activity
assays described herein. Alternatively, such polypeptide analogs
may be produced recombinantly using well-known procedures, such as
site-directed mutagenesis of MDC-encoding DNAs of the invention,
followed by recombinant expression of the resultant DNAs.
[0039] In a related aspect, the invention includes polypeptide
analogs wherein one or more amino acid residues is added, deleted,
or replaced from the MDC polypeptides of the present invention,
which analogs lack the biological activities of C--C chemokines or
MDC, but which are capable of competitively or non-competitively
inhibiting the binding of MDC polypeptides with a C--C chemokine
receptor. Such polypeptides are useful, e.g., for modulating the
biological activity of endogenous MDC in a host, as well as useful
for medical imaging methods described above.
[0040] Certain specific analogs of MDC are contemplated to modulate
the structure, intermolecular binding characteristics, and
biological activities of MDC. For example, amino-terminal
(N-terminal) and carboxy-terminal (C-terminal) deletion analogs
(truncations) are specifically contemplated to change MDC structure
and function. Among the amino terminal deletion analogs that are
specifically contemplated are analogs wherein 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, or 11 amino terminal residues have been deleted (i.e.,
deletions up to the conserved cysteine pair at positions 12 and 13
of human, murine, and rat mature MDC). As set forth in detail
below, experimental data indicates that most or all of these
analogs will possess reduced MDC biological activities and, in
fact, will act as inhibitors of one or more biological activities
of mature MDC.
[0041] Additionally, the following single-amino acid alterations
(alone or in combination) are specifically contemplated: (1)
substitution of a non-basic amino acid for the basic arginine
and/or lysine amino acids at positions 24 and 27, respectively, of
SEQ ID NO: 2; (2) substitution of a charged or polar amino acid
(e.g., serine, lysine, arginine, histidine, aspartate, glutamate,
asparagine, glutamine or cysteine) for the tyrosine amino acid at
position 30 of SEQ ID NO: 2, the tryptophan amino acid at position
59 of SEQ ID NO: 2, and/or the valine amino acid at position 60 of
SEQ ID NO: 2; and (3) substitution of a basic or small, non-charged
amino acid (e.g., lysine, arginine, histidine, glycine, alanine)
for the glutamic acid amino acid at position 50 of SEQ ID NO: 2.
Specific analogs having these amino acid alterations are
encompassed by the following formula (SEQ ID NO: 25):
1 Met Ala Arg Leu Gln Thr Ala Leu Leu Val Val Leu Val Leu Leu Ala
-24 -20 -15 -10 Val Ala Leu Gln Ala Thr Glu Ala Gly Pro Tyr Gly Ala
Asn Met Glu -5 1 5 Asp Ser Val Cys Cys Arg Asp Tyr Val Arg Tyr Arg
Leu Pro Leu Xaa 10 15 20 Val Val Xaa His Phe Xaa Trp Thr Ser Asp
Ser Cys Pro Arg Pro Gly 25 30 35 40 Val Val Leu Leu Thr Phe Arg Asp
Lys Xaa Ile Cys Ala Asp Pro Arg 45 50 55 Val Pro Xaa Xaa Lys Met
Ile Leu Asn Lys Leu Ser Gln 60 65
[0042] wherein the amino acid at position 24 is selected from the
group consisting of arginine, glycine, alanine, valine, leucine,
isoleucine, proline, serine, threonine, phenylalanine, tyrosine,
tryptophan, aspartate, glutamate, asparagine, glutamine, cysteine,
and methionine, wherein the amino acid at position 27 is
independently selected from the group consisting of lysine,
glycine, alanine, valine, leucine, isoleucine, proline, serine,
threonine, phenylalanine, tyrosine, tryptophan, aspartate,
glutamate, asparagine, glutamine, cysteine, and methionine, wherein
the amino acid at position 30 is independently selected from the
group consisting of tyrosine, serine, lysine, arginine, histidine,
aspartate, glutamate, asparagine, glutamine, and cysteine; wherein
the amino acid at position 50 is independently selected from the
group consisting of glutamic acid, lysine, arginine, histidine,
glycine, and alanine; wherein the amino acid at position 59 is
independently selected from the group consisting of tryptophan,
serine, lysine, arginine, histidine, aspartate, glutamate,
asparagine, glutamine, and cysteine; and wherein the amino acid at
position 50 is independently selected from the group consisting of
valine, serine, lysine, arginine, histidine, aspartate, glutamate,
asparagine, glutamine, and cysteine. Such MDC polypeptide analogs
are specifically contemplated to modulate the binding
characteristics of MDC to chemokine receptors and/or other
molecules (e.g., heparin, glycosaminoglycans, erythrocyte chemokine
receptors) that are considered to be important in presenting MDC to
its receptor. In one preferred embodiment, MDC polypeptide analogs
of the invention comprise amino acids 1 to 69 of SEQ ID NO: 25.
[0043] The following additional analogs have been synthesized and
also are intended as aspects of the invention: (a) a polypeptide
comprising a sequence of amino acids identified by positions 1 to
70 of SEQ ID NO: 30; (b) a polypeptide comprising a sequence of
amino acids identified by positions 9 to 69 of SEQ ID NO: 2; (c) a
polypeptide comprising a sequence of amino acids identified by
positions 1 to 69 of SEQ ID NO: 31; and (d) a polypeptide
comprising a sequence of amino acids identified by positions 1 to
69 of SEQ ID NO: 32.
[0044] As set forth in detail below, experimental data indicates
that the addition of as few as one additional amino acid at the
amino terminus of human mature MDC is sufficient to confer useful
MDC inhibitory properties to the resultant analog. Thus, all amino
terminal addition analogs are contemplated as an aspect of the
invention. Such addition analogs include the addition of one or a
few randomly selected amino acids; the addition of common tag
sequences (e.g., polyhistidine sequences, hemagglutinin sequences,
or other sequences commonly used to facilitate purification); and
chemical additions to the amino terminus (e.g., the addition of an
amino terminal aminooxypentane moiety). See Proudfoot et al., J.
Biol. Chem., 271:2599-2603 (1996); Simmons et al, Science, 276
(5310): 276-279 (1997).
[0045] Also as set forth in detail below, evidence exists that
mature MDC is cleaved in vivo by a dipeptidyl amino peptidase,
resulting in an MDC(3-69) form that exhibits at least some
activities antagonistic to MDC. An additional aspect of the
invention includes analogs wherein the proline at position 2 of a
mature MDC (e.g., human, murine, and rat MDC) is deleted or changed
to an amino acid other than proline. Such analogs are collectively
referred to as "MDC.DELTA.Pro.sub.2 polypeptides. Those
MDC.DELTA.Pro.sub.2 polypeptides that retain MDC biological
activities are contemplated as useful in all indications wherein
mature MDC is useful; and are expected to be less susceptible to
activity-destroying depeptidyl amino peptidases that recognize and
cleave the sequence NH2-Xaa-Pro- (e.g., CD26). Those
MDC.DELTA.Pro.sub.2 polypeptides that lack MDC biological
activities are contemplated as being used as MDC inhibitors.
[0046] It will be appreciated that, while the foregoing analogs
were often described with reference to human mature MDC, similar
analogs of other vertebrate MDC's, especially mammalian MDC's, also
are contemplated as aspects of the invention.
[0047] It also will be appreciated that it may be advantageous to
express MDC or MDC analogs as fusions with immunoglobulin
sequences, human serum albumin sequences, or other sequences, or to
perform other standard chemical modifications, for the purpose of
extending the serum half-life of the MDC or MDC analog. See, e.g.,
Yeh et al., Proc. Nat'l. Acad. Sci. U.S.A., 89(5): 1904-1908
(1992); Sambrook et al., supra. The definition of polypeptides of
the invention is intended to encompass such modifications.
[0048] In related aspects, the invention provides purified and
isolated polynucleotides encoding such MDC polypeptide analogs,
which polynucleotides are useful for, e.g., recombinantly producing
the MDC polypeptide analogs; plasmid and viral vectors
incorporating such polynucleotides, and prokaryotic and eukaryotic
host cells stably transformed with such DNAs or vectors.
[0049] In another aspect, the invention includes antibody
substances (e.g., monoclonal and polyclonal antibodies, single
chain antibodies, chimeric or humanized antibodies, antigen-binding
fragments of antibodies, and the like) which are immunoreactive
with MDC polypeptides and polypeptide analogs of the invention.
Such antibodies are useful, e.g., for purifying polypeptides of the
present invention, for quantitative measurement of endogenous MDC
in a host, e.g., using well-known ELISA techniques, and for
modulating binding of MDC to its receptor(s), The invention further
includes hybridoma cell lines that produce antibody substances of
the invention. Exemplary antibodies of the invention include
monoclonal antibodies 252Y and 252Z, which are produced by
hybridoma cell line 252Y and hybridoma cell line 252Z,
respectively. The hybridoma cell lines are themselves aspects of
the invention, and have been deposited with the American Type
Culture Collection (ATCC Accession Nos. HB-12433 and HB-12434,
respectively). Another exemplary antibody of the invention is
monoclonal antibody 272D, which is produced by hybridoma cell line
272D (itself an aspect of the invention and deposited with the
American Type Culture Collection (ATCC Accession No. HB-12498).
[0050] Recombinant MDC polypeptides and polypeptide analogs of the
invention may be utilized in a like manner to antibodies in binding
reactions, to identify cells expressing receptor(s) of MDC and in
standard expression cloning techniques to isolate polynucleotides
encoding the receptor(s). Such MDC polypeptides, MDC polypeptide
analogs, and MDC receptor polypeptides are useful for modulation of
MDC chemokine activity, and for identification of polypeptide and
chemical (e.g., small molecule) MDC agonists and antagonists.
[0051] Additional aspects of the invention relate to pharmaceutical
utilities of MDC polypeptides and polypeptide analogs of the
invention. For example, MDC has been shown to modulate leukocyte
chemotaxis. In particular, MDC has been shown to induce macrophage
chemotaxis and to inhibit monocyte chemotaxis. Thus, in one aspect,
the invention includes a method for modulating (e.g., up-regulating
or down-regulating) leukocyte chemotaxis in a mammalian host
comprising the step of administering to the mammalian host an MDC
polypeptide or polypeptide analog of the invention, wherein the MDC
polypeptide or MDC polypeptide analog modulates leukocyte
chemotaxis in the host. In preferred methods, the leukocytes are
monocytes and/or macrophages. For example, empirically determined
quantities of MDC are administered (e.g., in a pharmaceutically
acceptable carrier) to induce macrophage chemotaxis or to inhibit
monocyte chemotaxis, whereas inhibitory MDC polypeptide analogs are
employed to achieve the opposite effect.
[0052] In another aspect, the invention provides a method for
palliating an inflammatory or other pathological condition in a
patient, the condition characterized by at least one of (i)
monocyte chemotaxis toward a site of inflammation in said patient
or (ii) fibroblast cell proliferation, the method comprising the
step of administering to the patient a therapeutically effective
amount of MDC. In one embodiment, a therapeutically effective
amount of MDC is an amount capable of inhibiting monocyte
chemotaxis. In another embodiment, a therapeutically effective
amount of MDC is an amount capable of inhibiting fibroblast cell
proliferation. Such therapeutically effective amounts are
empirically determined using art-recognized dose-response
assays.
[0053] As an additional aspect, the invention provides a
pharmaceutical composition comprising an MDC polypeptide or
polypeptide analog of the invention in a pharmaceutically
acceptable carrier. Similarly, the invention relates to the use of
a composition according to the invention for the treatment of
disease states, e.g., inflammatory disease states. In one
embodiment, the inflammatory disease state is characterized by
monocyte chemotaxis toward a site of inflammation in a patient
having the disease state. In another embodiment, the disease state
is characterized by fibroblast cell proliferation in a patient
having the disease state.
[0054] MDC induced chemotaxis of natural killer cells (NK) can lead
to enhanced cytotoxicity of targeted NK cells against carious forms
of cancers. These forms of cancers include all solid tumor and
cancerous cells found in various organs and skin (e.g., breast,
ovarian, prostate, kidney, lung, pancreas, liver and bone cancers).
NK cells also play an important role in antibody-dependent
cell-mediated cytotoxicity. Stimulation of this process with MDC or
MDC agonists would lead to improved immune response to tumors. [See
generally Immunology (Ed. Kuby, J.) pp 304-6, W.H. Freeman and Co.,
New York, N.Y. (1992)]. Similarly, NK cells lead to viral immunity.
MDC may be used to potentiate resistance to common viral diseases
(e.g., influenza and rhinoviruses) by stimulating NK conferred
viral immunity by stimulating antigen-specific TH memory cells.
[Immunology Ed. Kuby J pp 420-425, W.H. Freeman and Co. New York,
N.Y. (1992)]. "Treatment" as used herein includes both prophylactic
and therapeutic treatment.
[0055] The apparent optimal concentration of mature MDC in receptor
binding and chemotaxis experiments is about 10 ng/ml. Thus, for
therapeutic methods involving the systemic administration of MDC
(or MDC analogs retaining a desired MDC biological activity), doses
and dosing schedules are preferably selected to maintain
circulating concentrations in blood of about 0.1-10 ng/ml.
Preferred approaches for preparing a dose and maintaining such
levels in the bloods include administration of MDC in a bolus
fashion, so as to administer approximately 0.1-10 mg of MDC. This
administration is repeated in order to maintain the stated blood
concentration. For example, MDC is stable at 1 mg/ml in
phosphate-buffered saline (PBS) and is administered to experimental
animals using this formulation. This formulation, either liquid or
lyophilized and reconstituted, is suitable for human parenteral
use, e.g., via intravenous injection. Other formulations can be
devised to concentrate the protein drug and stabilize it for use
years after its preparation. [See, erg., Stability and
Characterization of Protein and Peptide Drugs, Cbase Histories,
Wang Y J and Pearlman R. (Eds.), Plenum Press, New York (1993)
(describing methods for the preparation of cytokines and other
similar protein drug formulations by the inclusion of a variety of
excipients to maintain solubility and stability and minimize
aggregation)]. Exemplary excipients include citrate, EDTA,
detergents of the Tween family, zwittergent family, or pluronic
family, and amino acids such as cysteine to maintain the proper
oxido-reductant state.
[0056] In a second preferred approach, MDC is administered using
any of a number of drug delivery methods that are known in the art
to facilitate slow-release of the bioactive product. This can be
accomplished as easily as employing intramusculature administration
[see for example M. Groves in Parental Technology Manual, Second
edition, M. J. Groves (Ed.), Interpharm Press, Inc., Prairie View,
Ill., pp. 6-7 (1988)] to cause the MDC to be adsorbed into the
blood stream over a delayed period of time. Alternatively, the MDC
product can be delivered using a number of drug delivery methods
[see for a general review L M Sanders, in Peptide aid Protein Drug
Delivery, V. H. L. Lee (Ed.), Marcel Dekker, Inc., New York, pp.
785-806 (1991)]. For example, MDC is incorporated into
biodegradable microspheres, such as poly(lactic-co-glycolic acid of
PLGA) microspheres as shown using Human Growth Hormone, [Tracy,
Biotechnol. Progress, 14: 108-115 (1988)], or leuprolide acetate
microspheres [Okada et al., Pharm. Res, 8: 787-791 (1991)] which
can permit administrations as infrequently as once monthly. A
variety of other drug delivery approaches will be apparent to those
in the art, including dry powder formulations suitable for
inhalation made available by Inhale Corporation, Palo Alto, Calif.,
and transdermal delivery made available by Alza Corporation, Palo
Alto, Calif.
[0057] It will also be apparent from the teachings herein relating
to the various activities of MDC that modulators of MDC activities,
to inhibit the effects of endogenously-produced MDC and/or to
promote the activities of endogenously-produced or exogenously
administered MDC, have therapeutic utility. Such modulators
typically include small molecules, peptides, chemokine fragments
and analogs, and/or antibody substances. MDC inhibitors interfere
with MDC signal transduction, e.g., by binding MDC molecules, by
competitively or non-competitively binding MDC receptors on target
cells, and/or by interfering with signal transduction in the target
cells downstream from the chemokine receptors. Thus, in another
aspect, the invention provides assays to screen putative chemokine
modulators for modulating activity. Modulators identified by
methods of the invention also are considered aspects of the
invention.
[0058] In one embodiment, the invention provides a method for
identifying a chemical compound having MDC modulating activity
comprising the steps of: (a) providing first and second receptor
compositions comprising MDC receptors; (b) providing a control
composition comprising detectably-labeled MDC, (c) providing a test
composition comprising detectably-labeled MDC and further
comprising the chemical compound; (d) contacting the first receptor
composition with the control composition under conditions wherein
MDC is capable of binding to MDC receptors; (e) contacting the
second receptor composition with the test composition under
conditions wherein MDC is capable of binding to MDC receptors; (f)
washing the first and second receptor compositions to remove
detectably-labeled MDC that is unbound to MDC receptors; (g)
measuring detectably-labeled MDC in the first and second receptor
compositions; and (h) identifying a chemical compound having MDC
modulating activity, wherein MDC modulating activity is correlated
with a difference in detectably-labeled MDC between the first
second receptor compositions.
[0059] As reported herein, the chemokine receptor CCR4 has been
demonstrated to be a high affinity receptor for MDC. Thus, in a
preferred embodiment of the foregoing method, the first and second
receptor compositions comprise the MDC receptor that is CCR4. Since
CCR4 is a membrane protein, a preferred embodiment for practicing
the method is one wherein the first and second receptor
compositions comprise CCR4-containing cell membranes derived from
cells that express CCR4 on their surface. The cell membranes may be
on intact cells, or may constitute an isolated fraction of cells
that express CCR4. Cells that naturally express CCR4 and cells that
have been transformed or transfected to express CCR4 recombinantly
are contemplated.
[0060] In a related aspect, the invention provides a method for
identifying a modulator of binding between MDC and CCR4, comprising
the steps of (a) contacting MDC and CCR4 both in the presence of,
and in the absence of, a putative modulator compound; (b) detecting
binding between MDC and CCR4; and (c) identifying a putative
modulator compound in view of decreased or increased binding
between. MDC and CCR4 in the presence of the putative modulator, as
compared to binding in the absence of the putative modulator. The
contacting is performed, for example, by combining MDC with cell
membranes that contain CCR4, in a buffered aqueous suspension.
[0061] In one embodiment, the method is performed with labeled MDC.
In step (b), binding between MDC and CCR4 is detected by detecting
labeled MDC bound to CCR4. In a preferred embodiment, the
contacting step comprises contacting a suspension of cell membranes
comprising CCR4 with a solution containing MDC. In a highly
preferred embodiment, the method further comprises the steps of
recovering the cell membranes from the suspension after the
contacting step (e.g., via filtration of the suspension); and
washing the cell membranes prior to the detecting step to remove
unbound MDC.
[0062] In an alternative embodiment, the method is performed with a
host cell expressing CCR4 on its surface. In step (b), binding
between MDC and CCR4 is detected by measuring the conversion of GTP
to GDP in the host cell.
[0063] In yet another alternative embodiment, the method is
performed with a host cell that expresses CCR4 on its surface, and
binding between MDC and CCR4 expressed in the host cell is detected
by measuring cAMP levels in the host cell.
[0064] It will be appreciated that assays for modulators such as
those described above are often performed by immobilizing (e.g., on
a solid support) one of the binding partners (e.g., MDC or a
fragment thereof that is capable of binding CCR4, or CCR4 or a
fragment thereof that is capable of binding MDC). In a preferred
variation, the non-immobilized binding, partner is labeled with a
detectable agent. The immobilized binding partner is contacted with
the labeled binding partner in the presence and in the absence of a
putative modulator compound capable of specifically reacting with
MDC or CCR4; binding between the immobilized binding partner and
the labeled binding partner is detected; and modulating compounds
are identified as those compounds that affect binding between the
immobilized binding partner and the labeled binding partner.
[0065] In yet another embodiment, the invention provides a method
for identifying a chemical compound having MDC modulating activity,
comprising the steps of (a) providing first and second receptor
compositions comprising MDC receptors; (b) contacting the first
receptor composition with a control composition comprising
detectably-labeled MDC; (c) contacting the second receptor
composition with a test composition comprising detectably-labeled
MDC and further comprising the chemical compound; (d) washing the
first and second receptor compositions to remove detectably-labeled
MDC that is unbound to MDC receptors; (e) measuring
detectably-labeled MDC in the first and second receptor
compositions after the washing; and (f) identifying a chemical
compound having MDC modulating activity, wherein MDC modulating
activity is correlated with a difference in detectably-labeled MDC
between the first and the second receptor compositions.
[0066] In yet another embodiment, MDC binding to its receptor is
measured by measurement of the activation of a reporter gene that
has been coupled to the receptor using procedures that have been
reported in the art for other receptors. See, e.g., Himmler et al.,
Journal of Receptor Research, 13:79-94 (1993).
[0067] MDC-binding fragments of high affinity receptors of MDC are
specifically contemplated as inhibitor compounds of the invention;
antibodies to such receptors also are contemplated as inhibitor
compounds of the invention.
[0068] MDC's involvement in various aspects of immune responses is
described in detail below. Based on the involvement of MDC in
immune response, the administration of MDC antagonists is
indicated, for example, in the treatment anaphylaxis [Brown, A. F.,
J. Accid. Emerg. Med. 12(2):89-100 (1995)], shock [Brown (1995)
supra], ischemia, reperfusion injury and central ischemia
[Lindsberg et al., Ann. Neurol., 30(2):117-129 (1991)],
atherogenesis [Handley et al., Drug Dev. Res., 7:361-375 (1986)],
Crohn's disease [Denizot et al., Digestive Diseases and Sciences,
37(3):432-437 (1992)], ischemic bowel necrosis/necrotizing
enterocolitis [Denizot et al. (1992), supra, and Caplan et al.,
Acta Pediat. Suppl., 396:11-17 (1994)], ulcerative colitis (Denziot
et al. (1992), supra), ischemic stroke [Satoh et al., Stroke,
23:1090-1092 (1992)]" ischemic brain injury [Lindsberg et al.,
Stroke, 21:1452-1457 (1990) and Lindsberg et al. (1991), supra],
systemic lupus erythematosus [Matsuzaki et al., Clinica Chimica
Acta, 210: 139-144 (1992)], acute pancreatitis [Kald et al.,
Pancreas, 8(4):440-442 (1993)], septicemia (Kald et al. (1993),
supra), acute post-streptococcal glomerulonephritis [Mezzano et
al., J. Am. Soc. Nephrol., 4:235-242 (1993)], pulmonary edema
resulting from IL-2 therapy [Rabinovichi et al., J. Clin. Invest.,
89:1669-1673 (1992)], ischemic renal failure [Grino et al., Animals
of Internal Medicine, 121(5):345-347 (1994)]; pre-term labor
[Hoffman et al., Am. Soc. Nephrol., 162(2):525-528 (1990) and Maki
et al., Proc. Natl. Acad. Sci. USA, 85:728-732 (1988)], adult
respiratory distress syndrome [Rabinovichi et al., J. Appl.
Physiol., 74(4):1791-1802 (1993); Matsumoto et al., Clin. Exp.
Pharmacol. Physiol., 19:509-515 (1992); and Rodriguez-Roisin et
al., J. Clin. Invest., 93:188-194 (1994)]. "Treatment" as used
herein includes both prophylactic and therapeutic treatment.
[0069] MDC acts as a chemoattractant for T.sub.H2 differentiated
memory cells, which produce the cytokines IL-4, IL-5 IL-10 and
others. It is expected that, in some instances, MDC leads to an
immune state in which T.sub.111 cytokine driven responses are
reduced. In such instances, antagonism of MDC would lead to a state
in which T.sub.H1 cytokine driven responses are enhanced.
Modulation of the T.sub.H1-T.sub.H2 balance may lead to enhanced
"immune surveillance," and improved eradication of viral and
parasitic infections. Administration of MDC antagonists of the
invention to mammalian subjects, especially humans, for the
purposes of ameliorating pathological conditions associated with
undesirable or excessive T.sub.H2 responses and/or
less-than-desirable T.sub.H1 responses are contemplated as
additional aspects of the invention. Administration of sufficient
MDC antagonists to substantially reduce endogenous IL-10, a
T.sub.H1 immune suppressing cytokine, would lead to enhanced
cytotoxic T-lymphocyte mediated immunity and immune surveillance
[see Muller et al., J. Infect, 177: 586-94 (1998); Kenney et al.,
J. Infect. Dis., 177: 815-9 (1998)]. In these situations an
effective dose and dosing schedule can be determined by monitoring
circulating IL-10 levels and increasing the dose and frequency of
administration to reduce IL-10 levels to near normal levels.
Treatment of chronic or persistent viral infections and parasitic
infections is specifically contemplated, especially in combination
with other antiviral or anti-parasitic infection therapeutics.
Similarly, treatment or prevention of graft failure or graft
rejection with MDC antagonists is contemplated. The administration
of MDC antagonists is indicated, for example, in Leishmaniasis [Li
et al., Infect. Immunol., 64:5248-5254 (1996); Krishnan et al., J.
Immunol., 156(2):653-62 (1996)], opportunistic lung infections in
cystic fibrosis patients [Moser et al., APMIS, 105(11):838-42
(1997)], to delay HIV-1 induced immunodeficiency [Berger et al.,
Rev. Virol., 147(2-3):103-108 (1996); Barker et al., Proc. Nail.
Acad. Sci. (USA, 9,2(24): 11135-9 (1995); Jason et al., J. Acquir.
Immune. Defic. Syndrome Retrovirol, 10(4): 471-6 (1995); Maggi et
al., J. Biol. Regul. Homeost. Agents, 9(3): 78-81 (0.1995)],
chronic interstitial lung disease [Kunkel et al., Sarcoidosis Vasc.
Diffuse Lung Dis., 13: 120-128 (1996)], in neurological disorders
associated with a T.sub.H2 response [Windhagen et al., Chem.
Immunol., 63: 171-86 (1996); Bai et al., Clin. Immunol.
Immunopathol., 83(2): 117-26 (1997)], colorectal cancer [Pellegrini
et al., Cancer Immunol Immunother. 42(1): 1-8 (1996)], viral
infection, for example various species of herpes and hepatitis
[Spruance et al., Antiviral Rev., 28(1): 39-55 (1995); Pope et al.,
J. Immunol, 156(9): 3342-9 (1996); Bartoletti et al.,
Gastroenterol., 112(1): 193-199 (1997)], candidiasis and other
fungal infections [Spaccapelo et al., J. Immunol., 155(3): 1349-60
(1995); Fidel et al., J. Infect. Dis., 176(3): 728-39 (1995); Cenci
et al., J. Infect Dis., 171 (5): 1.279-88 (1995)], chronic
pneumonia [Johansen et al., Behring Inst. Mitt., 98: 269-73
(1997)], solid tumor cancer [Khar e al., Cytokines Mol. Ther.,
2(1): 39-46 (1996)], Bordella pertussis respiratory infection [Ryan
et al., J. Infect Dis., 175(5): 1246-50 (1997)], systemic lupus
erythrematosus [Segal et al., J. Immunol., 158(6): 2648-53 (1997)],
Bullous pemphigoidd pathogenesis [Deptia et al., Arch. Dermatol
Res., 289(12): 667-70 (1997)], glomerulonephritis [Kitching et al.,
Kidney Int., 53(1): 112-8 (1998); Huang et al., J. Am Soc. Neprol.,
8(7): 1101-8 (1997); Tipping et al., Eur. J. Immunol., 27(2):
515-21 (1997)], pulmonary respiratory syncytial virus infection
[Hussell et al, Eur. J. Immunol., 27(12): 3341-9 (1997)],
complications of trauma associated with surgical stress [Decker et
al., Surgery 119(3): 316-25 (1996)], celiac disease [Karban et al.,
Isr. J. Med. Sci., 33(3): 209-14 (1997)], Gulf War syndrome [Rook
et al., Lancet, 349 (9068): 1831-3 (1997)], ameobocyte infection,
for example Plasmodium falciparum [Eighazali et al., Clin. Exp.
Immunol., 109(1): 84-9 (1997)] and schistosoma mansoni [Wolowczuk
et al., Immunol., 91(1): 35-44 (1997)], and B-cell lymphoma,
especially mucosa-associated lymphoid tissue type [Greiner et al.,
Am J. Pathol., 150(5): 1583-93 (1997)]. "Treatment" as used herein
includes both prophylactic and therapeutic treatment.
[0070] With respect to any of the conditions, disorders, and
disease states identified in the preceding paragraphs, an exemplary
method of treatment comprises the steps of identifying a human
subject in need of therapeutic or prophylactic treatment for one of
the above-identified conditions, disorders, or disease states; and
administering to the human subject a therapeutically or
prophylactically effective amount of an N/DC antagonist compound.
By "therapeutically effective amount" is meant a dose and dosing
schedule that is sufficient to cure the disease state, or to reduce
the symptoms or severity of the disease state. By "prophylactically
effective amount" is meant a dose and dosing schedule that is
sufficient to reduce the likelihood of occurrence of a disease
state, or delay its onset, relative to human subjects that are
considered to have equivalent risk of developing the disease state
but whom are not treated with an MDC antagonist. Therapeutically
effective amounts are readily determined by dose-response studies
that are conventionally performed in the art.
[0071] In one highly preferred embodiment, the invention includes a
method of inhibiting proliferation of a mammalian immunodeficiency
virus comprising the step of contacting mammalian cells that are
infected with a mammalian immunodeficiency virus with a composition
comprising an MDC-IV antagonist compound or TARC-IV antagonist
compound, in an amount effective to inhibit proliferation of said
virus in said cells. The family of mammalian immunodeficiency
viruses is intended to include human immunodeficiency viruses, such
as strains of HIV-1 and HIV-2, and analogous viruses known to
infect other mammalian species, including but not limited to simian
and feline immunodeficiency viruses. The method can be performed in
vitro (e.g., in cell culture), but preferably is performed in vivo
by administering the antagonist to an infected subject, e.g., an
HIV-infected human subject. (In yet another embodiment, the method
is performed prophylacticly on a subject at risk of developing an
HIV infection, e.g., due to the subject's likelihood of exposure to
contaminated blood samples, contaminated needles, or intimate
exposure to an HIV-infected person.)
[0072] The term "MDC-IV antagonist compound" refers to compounds
that antagonize the apparent Immunodeficiency Virus-proliferative
effects of MDC in infected cells. Thus, the term "MDC-IV antagonist
compound" is meant to include any compound that is capable of
inhibiting proliferation of the immunodeficiency virus in a manner
analogous to either the inhibition reported herein for MDC
neutralizing antibodies or the inhibition reported herein for
certain MDC analogs (e.g., analogs having amino terminal additions
or truncations). For example, anti-MDC antibodies are highly
preferred MDC-IV antagonist compounds. For treatment of humans
infected with an HIV virus, humanized antibodies are highly
preferred. Similarly, polypeptides that comprise an antigen-binding
fragment of an anti-MDC antibody and that are capable of binding to
MDC are preferred MDC-IV antagonist compounds.
[0073] As described elsewhere herein in greater detail,
amino-terminal truncations of mature human MDC(1-69) possess
antiproliferative activity against HIV-1. Thus, another set of
preferred MDC-IV antagonist compounds are polypeptides whose amino
acid sequence consists of a portion of the amino acid sequence set
forth in SEQ ID NO: 2 sufficient to bind to the chemokine receptor
CCR4, said portion having an amino-terminus between residues 3 and
12 of SEQ ID NO: 2 (i.e., analogs lacking at least three amino
acids from the amino terminus of MDC(1-69). Amino terminal deletion
analogs that have been further modified. e.g., by including an
oligopeptide tag to facilitate purification, or by including, an
initiator methionine for bacterial expression, are also
contemplated.
[0074] Amino-terminal additions to mature MDC also result in
analogs possessing antiproliferative activity against HIV-1. Thus,
another set of preferred MDC-IV antagonist compounds are
polypeptides that comprise a mature MDC sequence (e.g., amino acids
1-69 of SEQ ID NO: 1), and that further comprise a chemical
addition to the amino terminus of the mature MDC sequence to render
said polypeptide antagonistic to MDC. Additions of additional amino
acids and other chemical moieties are contemplated.
[0075] It will further be appreciated that substitution of amino
acids in a mature MDC sequence (especially substitutions in the
amino terminus of mature MDC) may be expected, in some instances,
to result in analogs possessing antiproliferative activity against
HIV-1. Such analogs also are intended MDC-IV antagonist compounds,
and are identifiable using HIV proliferation assays described
herein.
[0076] It is postulated that MDC's HIV-proliferative effects are
mediated, at least in part, through the chemokine receptor CCR4.
Thus, the family of MDC-IV antagonist compounds includes
polypeptides that comprise the C--C chemokine receptor 4 (CCR4)
amino acid sequence set forth in SEQ ID NO: 34 or that comprise a
continuous fragment thereof that is capable of binding to MDC or
TARC. Such polypeptides are expected to bind endogenous MDC and
thereby inhibit HIV proliferation in a manner analogous to anti-MDC
antibodies. Also contemplated are anti-CCR4 antibodies, which are
expected to block MDC-CCR4 interactions, thereby inhibiting
MDC-induced HIV proliferation.
[0077] As described herein in detail, the chemokine TARC possesses
sequence similarity to MDC possesses various overlapping biological
activities, and, like MDC, binds to the chemokine receptor CCR4.
These similarities suggest that compounds that inhibit TARC-CCR4
interactions will also be useful for inhibiting proliferation of
immunodeficiency viruses. Compounds that inhibit TARC-induced
proliferation of such viruses are collectively referred to as
"TARC-IV antagonist compounds." Such compounds include anti-CCR4
antibodies, anti-TARC antibodies (especially humanized versions),
and polypeptides that are capable of binding to TARC and that
comprise an antigen-binding fragment of an anti-TARC antibody.
[0078] It is also contemplated that modifications to the amino
terminus of mature TARC polypeptides will result in TARC-IV
antagonist compounds, in a manner analogous to what has been
reported herein for MDC analogs. Thus, TARC-IV antagonist compounds
for use in methods of the invention include polypeptides that have
an amino acid sequence consisting of a portion of the amino acid
sequence set forth in SEQ ID NO: 43 that is sufficient to bind to
the chemokine receptor CCR4, said portion having an amino-terminus
between residues 1 and 10 of SEQ ID NO: 43. Polypeptide comprising
mature TARC sequences, and further comprising chemical additions to
the amino terminus to render the polypeptide antagonistic to TARC
also are contemplated. Polypeptides comprising the mature TARC
amino acid sequence, into which substitutions have been introduced
to confer HIV antiproliferative activity, also are contemplated as
TARC-IV antagonist compounds.
[0079] In another highly preferred embodiment, the invention
includes a method of inhibiting platelet aggregation in a mammalian
subject (especially a human subject) comprising the step of
administering to a mammalian subject a composition comprising an
MDC-PA antagonist compound or TARC-PA antagonist compound, in an
amount effective to inhibit platelet aggregation in the subject.
Such methods may be performed for therapeutic purposes, e.g., in
patients suffering from undesirable blood clotting, or for
prophylactic purposes on a subject at risk of developing
undesirable blood clotting or coagulation. Such patients would
include, e.g., patients who have previously suffered myocardial
infarction or stroke or other clotting disorders, or who are deemed
to be at high risk for developing such conditions.
[0080] The term "MDC-PA antagonist compound" refers to compounds
that antagonize the apparent Platelet Aggregating effects of MDC.
Thus, the term "MDC-PA antagonist compound" is meant to include any
compound that is capable of inhibiting platelet aggregation that is
observable after administration of MDC to a mammalian subject
(e.g., to a mouse or rat). Those compounds described above as
MDC-IV antagonist compounds are specifically contemplated as MDC-PA
antagonist compounds as well. For example, anti-MDC antibodies are
highly preferred MDC-PA antagonist compounds. For treatment of
humans, humanized antibodies are highly preferred. Similarly,
polypeptides that comprise an antigen-binding fragment of an
anti-MDC antibody and that are capable of binding to MDC are
preferred MDC-PA antagonist compounds. All MDC analogs that inhibit
the platelet aggregating effects of MDC also are preferred. Analogs
having additions, deletions, and/or substitutions in the amino
terminus are specifically contemplated.
[0081] The structural and functional similarities between MDC and
TARC reported herein indicate that compounds that inhibit TARC-CCR4
interactions will be useful for inhibiting platelet aggregation.
Compounds that inhibit TARC-induced platelet aggregation are
collectively referred to as "TARC-PA antagonist compounds." Such
compounds include anti-CCR4 antibodies, anti-TARC antibodies
(especially humanized versions); various TARC analogs described
elsewhere herein, and polypeptides that are capable of binding to
TARC and that comprise an antigen-binding fragment of an anti-TARC
antibody.
[0082] As described herein in detail, the expression patterns of
MDC and its receptor, CCR4, provide an indication for the use of
MDC as an adjuvant in a vaccine. Thus, in another aspect, the
invention includes a vaccine composition comprising an antigen of
interest in a suitable pharmaceutical carrier, improved by the
inclusion of MDC in the vaccine composition. The antigen of
interest may be any composition intended to (generate a desirable
immune response in a human or other animal. Such compositions would
include, for example, killed or attenuated pathogens or antigenic
portions thereof. In a related aspect, the invention includes a
method of immunizing a human or animal, wherein the improvement
comprises administering MDC to the human or animal, either
concurrently or before or after administering an antigen of
interest.
[0083] The foregoing, aspects and numerous additional aspects will
be apparent from the drawing and detailed description which
follow.
BRIEF DESCRIPTION OF THE DRAWING
[0084] FIG. 1 is a comparison of the amino acid sequence of human
MDC (SEQ ID NO: 2) with the amino acid sequences of other,
previously characterized human C--C chemokines: MCP-3 [Van Damme et
al., J. Exp. Med., 176:59 (1992)] (SEQ ID NO: 18); MCP-1[Matsushima
et al., J. Med., 169:1485 (1989)] (SEQ ID NO: 19); MCP-2 (mature
form) [Van Damme et al., supra; Chang et al., Int. Immunol., 1:388
(1989)] (SEQ ID NO: 20); RANTES [Schall et al., J. Immunol.,
141:1018 (1988)] (SEQ ID NO: 21); MIP-1.beta. [Brown et al., J.
Immunol., 142:679 (1989)] (SEQ ID NO: 22); MIP-1.alpha. [Nakao et
al., Mol. Cell Biol., 10:3646 (1990)] (SEQ ID NO: 23); and I-309
[Miller et al., J. Immunol., 143:2907 (1989)] (SEQ ID NO: 24). A
slash "/" marks the site at which putative signal peptides are
cleaved. Dashes are inserted to optimize alignment of the
sequences.
[0085] FIG. 2 is a graph depicting the chemotactic effect (measured
in fluorescence units) of increasing concentrations of MDC on human
mononuclear cell migration in a chemotaxis assay. Closed circles
show the response of human mononuclear cells derived from the cell
line THP-1. The open diamond shows the response to the positive
control, zymosan activated serum (ZAS).
[0086] FIG. 3 is a graph depicting the chemotactic effect (measured
in fluorescence units) of increasing concentrations of MDC on human
polymorphonuclear (pmn) leukocyte migration. Closed circles show
response to MDC, and an open diamond shows the response to the
positive control, IL-8.
[0087] FIG. 4 is a graph depicting the chemotactic effect (measured
in fluorescence units) of increasing concentrations of MDC on
macrophage and monocyte migration. Closed circles show the response
to MDC of macrophages derived from the cell line THP-1. Open
circles show the response to MDC of monocytes derived from the cell
line THP-1.
[0088] FIG. 5 is a graph depicting the chemotactic effect (measured
in fluorescence units) of increasing concentrations of MDC on
guinea pig peritoneal macrophage migration. Closed circles show the
response of macrophages to MDC. An open triangle shows the response
to the positive control, zymosan activated serum (ZAS).
[0089] FIG. 6 is a graph depicting the chemotactic-inhibitory
effect (measured in fluorescence units) of increasing
concentrations of MDC on THP-1 monocyte migration induced by MCP-1.
Closed circles depict the chemotactic-inhibitory effects of MDC
where chemotaxis has been induced by MCP-1. Open circles depict the
chemotactic-inhibitory effects of MDC in a control experiment
wherein only the basal medium (RPMI with 0.2% BSA (RBSA), no MCP-1)
was employed. The zero point on the x axis corresponds to the
response of cells to MCP-1 and RBSA in the absence of any MDC.
[0090] FIG. 7 is a graph depicting the effect (measured in counts
per minute (cpm)) of increasing concentrations of MDC on fibroblast
proliferation. Closed circles depict the proliferative response
with purified MDC that was recombinantly produced in CHO cells
(Example 10F). Open circles depict the response with chemically
synthesized MDC (Example 11).
[0091] FIG. 8 schematically depicts the construction of mammalian
expression vector pDC1.
[0092] FIG. 9 depicts the nucleotide and deduced amino acid
sequence (SEQ ID NOs: 39 and 40) of a S. cerevisiae alpha factor
pre-pro/human MDC cDNA chimeric construct used to express human MDC
in yeast.
[0093] FIG. 10 depicts the structure of plasmid pYGL/preproMDC,
used to express human MDC in yeast.
[0094] FIG. 11 depicts the inhibitory effects of the anti-MDC
antibodies 252Y and 252Z on the binding of the fusion protein
MDC-SEAP to the MDC receptor designated CCR4. Binding (depicted as
percent of maximal binding) is plotted as a function of increased
concentrations of antibody
[0095] FIG. 12 depicts the inhibitory effects of the anti-MDC
antibodies 252Y and 252Z on the MDC-induced chemotaxis of
CCR4-transfected L1.2 cells. The number of cells observed migrating
toward MDC in a standard chemotaxis assay are plotted as a function
of increased concentrations of antibody.
DETAILED DESCRIPTION
[0096] The present invention is illustrated by the following
examples related to a human cDNA, designated MDC cDNA, encoding a
novel C--C chemokine designated MDC (for "macrophage-derived
chemokine"). More particularly, Example 1 describes the isolation
of a partial MDC cDNA from a human macrophage cDNA library. Example
2 describes the isolation of additional cDNAs from the cDNA library
using the cDNA from Example 1 as a probe, one of these additional
cDNAs containing the entire MDC coding sequence. Additionally,
Example 2 presents a composite MDC cDNA nucleotide sequence and
presents a characterization of the deduced amino acid sequence of
the chemokine (MDC) encoded thereby. In Example 3, experiments are
described which reveal the level of MDC gene expression in various
human tissues. The greatest MDC gene expression was observed in the
thymus, with much weaker expression detectable in spleen and lung
tissues. Example 4 describes more particularly the expression of
the MDC gene during monocyte maturation into macrophages and during
inducement of HL60 cell differentiation to a macrophage-like cell
type.
[0097] Since MDC gene expression was detected in thymus and spleen
in Example 3, in situ hybridization studies were conducted to
localize further the MDC gene expression in these tissues.
Moreover, iii site hybridization revealed a correlation between
elevated MDC gene expression in inflamed tissues, as exemplified
using intestinal tissue from Crohn's diseased patients. These in
situ hybridization experiments are described in Example 5.
[0098] Example 6 describes the recombinant production of MDC as a
GST fusion protein in prokaryotic cells, as well as the cleavage of
the fusion protein and purification of the recombinant MDC. Example
7 describes alternative DNA constructs useful for expression of
recombinant MDC protein, and describes the production of MDC by a
bacterial host transformed with such a construct.
[0099] Example 8 provides experimental protocols for purification
of recombinant MDC produced, e.g., as described in Example 7.
Examples 9 and 10 provide protocols for the recombinant production
of MDC in yeast and mammalian cells, respectively. In addition,
Example 10 provides additional protocols for purification of
recombinant MDC, and describes the determination of the amino
terminus of MDC recombinantly produced in mammalian cells. Example
11 describes production of MDC and MDC polypeptide analogs by
peptide synthesis. Certain preferred analogs are specifically
described in Example 11.
[0100] Examples 12-17 provide protocols for the determination of
MDC biological activities. For instance. Example 12 provides an
assay of MDC effects upon basophils, mast cells, and eosinophils.
MDC-induced chemotaxis of eosinophils is specifically demonstrated.
Example 13 describes assays of chemoattractant and cell-activation
properties of MDC on monocytes/macrophages, neutrophils, and
granulocytes. MDC induced macrophage chemotaxis, but inhibited
monocyte chemotaxis.
[0101] Examples 14-17 provide protocols for the determination of
MDC biological activities in vivo. Example 14 provides an MDC tumor
growth-inhibition assay. Examples 15 and 16 provide protocols for
assaying MDC activity via intraperitoneal and subcutaneous
injection, respectively. Example 17 provides protocols for
determining the myelosuppressive activity of MDC.
[0102] Example 18 provides protocols for generating antibodies that
are specifically immunoreactive with MDC, including polyclonal,
monoclonal, and humanized antibodies. Uses of the antibodies also
are described.
[0103] Example 19 provides a calcium flux assay for determining the
ability of MDC to induce cellular activation.
[0104] Example 20 provides assays and experimental results relating
to the HIV proliferative and anti-proliferative effects of human
mature MDC and MDC antagonists.
[0105] Example 21 demonstrates the anti-proliferative effects of
MDC on fibroblasts. Example 22 provides in vitro assays for the
effects of MDC upon the proliferation of additional cell types.
Example 23 provides an in vivo assay for determining the
anti-proliferative effects of MDC on fibroblasts.
[0106] Example 24 describes the chromosomal localization of the
human MDC gene.
[0107] Example 25 describes procedures which identified the CC
chemokine receptor "CCR4" as a high affinity binding partner of
MDC. Examples 26 and 27 provide assays for identifying MDC
modulators.
[0108] Example 28 describes the isolation of cDNAs encoding rat and
mouse MDC, and characterizes the MDC proteins encoded thereby.
Example 29 further characterizes selected MDC analogs.
[0109] Example 30 describes experiments that demonstrate that
anti-MDC monoclonal antibodies are effective for neutralizing
biological activities of MDC that were elucidated in other
examples.
[0110] Example 31 describes experiments that demonstrate that MDC
induces chemotaxis of T.sub.H2 helper cells, a discovery with
therapeutic implications as discussed in Example 31 and elsewhere
herein.
[0111] Example 32 describes platelet-aggregating activities of MDC,
and describes the use of MDC and MDC antagonists to modulate
platelet aggregation.
EXAMPLE 1
Isolation of a cDNA Encoding MDC
[0112] A partial cDNA for a new C--C chemokine, designated pMP390,
was isolated from a macrophage cDNA library as described in U.S.
patent application Ser. No. 08/939,107, filed Sep. 26, 1997, and in
related international publication number WO 96/40923, both of which
are incorporated herein by reference. Sequence comparisons were
performed on Dec. 14, 1994, by the BLAST Network Service of the
National Center for Biotechnology Information (e-mail:
"blast@ncbi.nlm.nih.gov"), using the alignment algorithm of
Altschul et al., J. Mol. Biol., 215: 403-410 (1990). The sequence
analysis revealed that a portion of the isolated macrophage cDNA
clone designated pMP390 contained a gene sequence having
approximately 60-70% identity with previously-identified chemokine
genes, including the human MCP-3 gene and rat MIP-1.beta. gene.
[0113] The 2.85 kb cDNA insert of pMP390 was subcloned into the
vector pBluescript SK.sup.- (Stratagene, La Jolla Calif.) to
facilitate complete sequencing. The complete sequence of this
pMP390 cDNA corresponds to nucleotides 73 to 2923 of SEQ ID NO: 1
(and to deduced amino acids -6 to 69 of SEQ ID NO 2). The sequence
that was originally compared to database sequences corresponds to
nucleotides 73 to 610 of SEQ ID NO: 1.
EXAMPLE 2
Isolation of Additional cDNA Clones Having the Complete MDC Coding
Sequence
[0114] Using the pMP390 cDNA clone isolated in Example 1,
additional cDNA clones were isolated from the same human macrophage
cDNA library, these additional cDNAs containing additional 5'
sequence and encoding the complete amino acid sequence of a
macrophage derived chemokine. The additional cloning and sequencing
is described in detail in U.S. Ser. No. 08/939,107 and WO 96/40923,
incorporated herein by reference.
[0115] Of the additional clones, clones designated pMP390-12 and
pMP390B contained the largest additional 5' coding sequence, each
extending an additional 72 nucleotides upstream of the sequence
previously obtained from the cDNA clone pMP390. A composite DNA
sequence, herein designated MDC cDNA, was generated by alignment of
the pMP390 and pMP390-12 cDNA sequences. This 2923 base pair
composite cDNA sequence, and the deduced amino acid sequence of the
chemokine MDC, are set forth in SEQ ID NOs: 1 and 2,
respectively.
[0116] Manual comparison of the deduced MDC amino acid sequence
with sequences of known chemokines indicates that the MDC cDNA
sequence encodes a novel C--C chemokine ninety-three amino acids in
length, sharing 28-34% amino acid identity with other C--C
chemokines (FIG. 1). As aligned in FIG. 1, MDC shares 29% amino
acid identity with MCP-1 and MIP-1.alpha., 28% identity with MCP-2,
32% identity with I-309, 33% identity with MCP-3 and MIP-1.beta.,
and 34 % identity with RANTES. Importantly, the four cysteine
residues characteristic of the chemokines are conserved in MDC.
Five additional residues also are completely conserved in the eight
sequences presented in FIG. 1.
[0117] The first 24 amino acids of the 93 amino acid MDC sequence
are predominantly hydrophobic and are consistent with von Heijne's
rules [Nucleic Acids Rev., 14: 4683-90 (1986)] governing signal
cleavage. These features and the polypeptide comparison in FIG. 1
collectively suggest that the MDC cDNA encodes a twenty-four amino
acid signal peptide that is cleaved to produce a mature form of MDC
beginning with the glycine residue at position 1 of SEQ ID NO: 2.
This prediction was confirmed by direct sequencing of MDC protein
produced recombinantly in mammalian cells, as described below in
Example 10. The MDC composite cDNA sequence shown in SEQ ID NO: 1
extends nineteen nucleotides upstream of the predicted initiating
methionine codon, and 2.6 kb downstream of the termination
codon.
EXAMPLE 3
Determination of MDC Gene Expression in Human Tissues
[0118] Northern blot analysis were conducted to determine the
tissues in which the MDC gene is expressed.
[0119] A radiolabeled pMP390 5' fragment which corresponds to the
region of the MDC cDNA encoding the putative mature form of MDC
plus 163 bases of the adjacent 3' noncoding region was used to
probe Multiple Tissue Northern blots (Clontech, Palo Alto, Calif.)
containing RNA from various normal human tissues. The probe was
denatured by boiling prior to use, and the hybridizations were
conducted according to the manufacturer's specifications.
Autoradiographs were exposed 5 days at -80.degree. C. with 2
intensifying screens.
[0120] The greatest MDC gene expression was observed in the thymus,
with much weaker expression detectable in spleen and lung tissues.
Expression of MDC in tissue from the small intestine was at even
lower levels, and no expression was detected in brain, colon,
heart, kidney, liver, ovary, pancreas, placenta, prostate, skeletal
muscle, testis, or peripheral blood leukocytes.
[0121] As discussed in detail below in Example 25, MDC is a ligand
for the CC chemokine receptor CCR4, which receptor also has been
reported to be a ligand for the chemokine TARC. See Imai et al., J.
Biol. Chem., 272: 15036-15042 (1997). Like MDC, TARC is abundantly
expressed in the thymus, with little expression observed in other
tissues. More particularly. CCR4 is expressed on T cells,
especially CD4.sup.+ T cells [See Imai et al. (1997), and Power et
al., J. Biol. Chem., 270: 19495-19500 (1995)], while MDC and TARC
are expressed by cells of the dendritic lineage which form a major
component of the thymic architecture. See Godiska et al., J. Exp.
Med., 185: 1595-1604 (1997), incorporated herein by reference; and
Imai et al., J. Biol. Chem., 271: 21514-21521 (1996). These
expression patterns suggest a biological activity of MDC, CCR4, and
TARC in T cell development, since immature progenitor cells undergo
differentiation and expansion (leading to the establishment of the
major T cell lineages and the elimination of potentially
autoreactive T cells) within the highly specialized
microenvironment of the thymus. See von Boehmer, Current Biology,
7: 308-310 (1997). The fact that MDC also is expressed at high
levels in cultured macrophages suggests an MDC activity in the
initiation and/or triggering of the immune response, by
facilitating the interaction of T cells with antigen-presenting
cells at sites of inflammation.
[0122] These expression pattern data suggest therapeutic utilities
of MDC (or MDC mimetics or agonists) to stimulate beneficial immune
responses. For example, MDC, MDC agonists, or MDC mimetics may be
administered to augment/enhance T cell activation where T cell
activation may be beneficial. The use of MDC as an adjuvant in
vaccine development or in tumor immunotherapy is specifically
contemplated.
[0123] Conversely, the expression pattern data also indicates a
therapeutic utility for modulators of MDC's interaction with CCR4
in T cell-mediated autoimmune diseases, including but not limited
to psoriasis, graft versus host disease, and allograft rejection,
and in T cell and/or B cell mediated allergic responses.
EXAMPLE 4
MDC Gene Expression During Macrophage Maturation
[0124] Because the cDNAs encoding MDC were isolated from a human
macrophage cDNA library, MDC gene expression during differentiation
of monocytes into macrophages was examined.
A
[0125] Human monocytes from a single donor were cultured on a
series of tissue culture plates, and cells from one plate were
harvested after 0, 2, 4 or 6 days. See generally Elstad et al., J.
Immunol. 140:1618-1624; Tjoelker et al, Nature, 374:549-552 (1995).
Under these conditions, the monocytes differentiated into
macrophages by days 4-6 [Stafforini et al., J. Biol Chem., 265:
9682-9687 (1990)].
[0126] A Northern blot of RNA (10 .mu.g per lane) isolated from the
cells harvested at each time point was prepared and probed, using a
radiaolabeled pMP390 fragment. No signal was detectable in RNA from
freshly isolated monocytes, whereas a very strong signal was
generated from cells that had differentiated into macrophages after
six days of culture. Cells cultured for four days produced a much
weaker signal, whereas the signal generated from cells cultured for
two days could be seen only after prolonged exposure of the
filter.
B
[0127] To confirm the expression of MDC in differentiated human
macrophages, culture supernatants were analyzed by western blotting
with anti-MDC monoclonal antibodies produced as described below in
Example 18. Several plates of human macrophages were differentiated
by growth on plastic for eight days in the presence of macrophage
colony stimulating factor (0.5 ng/ml, R&D Systems, Minneapolis,
Minn.).
[0128] The medium from the differentiated macrophage cell cultures
was removed and replaced with similar medium or with medium
containing low density lipoprotein (LDL, Sigma), oxidized LDL
(oxidized by incubation in 5 .mu.M CuSO.sub.4.smallcircle.5H.sub.2O
according to the method of Maiden et al., J. Biol. Chem., 266:13901
(1991)), or dexamethazone (6 nM, Sigma Chemical Co.). Following 3
days of each treatment, the culture medium was removed, brought to
pH 6.8 by the addition of HCl, and passed over a Heparin-Sepharose
CL-6B column (Pharmacia, Piscataway, N.J.). The column was washed
with 0.2 M NaCl in 20 mM Tris, pH 8, and eluted with 0.6 M NaCl in
20 mM Tris, pH 8. The eluted material was fractionated on an 18%
acrylamide SDS-PAGE gel (NOVEX) and electroblotted to PVDF membrane
(Millipore, Bedford Mass.). The filter was blocked, washed, and
reacted with monoclonal antibodies against MDC using standard
techniques (Sambrook et al.). In each of the culture media
analyzed, MDC protein was detected at a concentration of
approximately 0.5 .mu.g/ml, thus confirming expression of MDC in
differentiated human macrophages.
[0129] Expression of MDC also was analyzed in human epithelial cell
lines. The colon epithelial cell line T84 (ATCC #CCL-248) was grown
in DMEM/F12 medium (GIBCO, Gaithersburg Md.), and the lung
epithelial cell line A549 (ATCC #CCL-185) was grown in F12 medium.
Screening for the presence of MDC mRNA in the cells and MDC protein
in the culture medium was performed as described above for
macrophages. No evidence of MDC expression was detectable by either
method in these cell lines.
[0130] In addition, samples of the T84 cell line were treated for 1
day with TNF.alpha. (5 ng/ml, Pepro Tech, Rocky Hill, N.J.),
TGF-.beta. (1 ng/ml, R&D Systems), or interferon-.gamma. (200
U/ml, Pepro Tech), each with or without addition of recombinant MDC
at 100 ng/ml (derived from CHO cell transfectants; see Ex. 10).
Samples of the A549 cell line were treated with 50 ng/ml PMA (Sigma
Chemical Co.) for 0, 1, 3, 5, or 7 days. None of these treatments
resulted in detectable expression of MDC mRNA in the T84 or A549
cells when screened by Northern blotting as described above.
C
[0131] Further examination of MDC gene expression--in macrophages
was conducted by treating the human cell line HL60 with either 1%
DMSO (Sigma Chemical Co.) or 50 ng/ml PMA (Sigma). Treatment with
DMSO induces differentiation of HL60 cells into a granulocytic cell
type, whereas PMA induces their differentiation into a macrophage
lineage [Perussia et al., Blood, 58: 836-843 (1981)]. RNA was
isolated from untreated cells and from cells treated for one or
three days with DMSO or PMA, electrophoresed (10 .mu.g/lane), and
blotted. The Northern blot of the RNA was probed with the
radiolabeled pMP390 5' fragment described in Example 3.
[0132] After three days of PMA treatment, the HL-60 cells clearly
expressed MDC mRNA, although the level of expression was apparently
less than that of macrophages after six days of culture (see
above). No expression was seen after one day of treatment or in
untreated cells. Further, no detectable expression of MDC was
induced by treatment with DMSO for one or three days.
EXAMPLE 5
In Situ Hybridization
[0133] Because MDC gene expression was detected in the thymus and
spleen, in situ hybridization was carried out to localize the
source of the message in these tissues. Further, in situ
hybridization was used to correlate MDC gene expression with tissue
inflammation, using intestinal tissue from Crohn's diseased
patients as an example. The procedures used for these experiments
are described in detail in U.S. Ser. No. 08/939,107 and WO
96/40923, both of which are incorporated by reference.
[0134] Observed hybridization of the anti-sense strand indicated
that the MDC gene was expressed in cells throughout the cortex of
normal human thymus, with weak signal in the follicles. Expression
of MDC in the thymus may indicate a T lymphocyte developmental role
of MDC. Expression in normal human spleen was localized to cells of
the red pulp, whereas little signal was detected in the white pulp.
A high level of expression in inflamed tonsil was localized to the
epithelial region, although inflammatory cells appeared to have
infiltrated the entire tissue sample.
[0135] Colon samples from patients with Crohn's disease exhibited
hybridization in cells of the epithelium, lamina propria, Payer's
patches, and smooth muscle. In contrast, normal human colon showed
no hybridization above background. The observed pattern of MDC
expression in the colons of Crohn's disease patients closely
correlates with the expression of a macrophage-specific gene,
Platelet Activating Factor Acetylhydrolase (PAF-AH) [Tjoelker et
al., supra]. This result, together with the data presented in
Example 4, suggest that macrophages express MDC cDNA int vivo
during pathogenic inflammation. Moreover, the identification of MDC
in Crohn's disease colon tissue samples suggest diagnostic
relevance of MDC levels (e.g., in a patient's blood, stool sample,
and/or intestinal lesions) to a patient's disease state or clinical
prognosis.
EXAMPLE 6
Production of Recombinant MDC
[0136] To produce recombinant MDC protein, the sequence encoding
the putative mature form of the protein was amplified by PCR and
cloned into the vector pGEX-3 .times. (Pharmacia, Piscataway,
N.J.). The pGEX vector is designed to produce a fusion protein
comprising glutathione-S-transferase (GST), encoded by the vector,
and a protein encoded by a DNA fragment inserted into the vector's
cloning site.
[0137] An MDC cDNA fragment was amplified by PCR using the primers
390-2R (SEQ ID NO: 8) and 390-FX2 (SEQ ID NO: 11). Primer 390-FX2
contains a BamHI restriction site, followed by a sequence encoding
a thrombin cleavage site [Chang et al., Eur. J. Biochem., 151:217
(1985)] followed by bases 92-115 of SEQ ID NO: 1. The thrombin
cleavage site is as follows:
leucine-valine-proline-arginine-glycine-proline, in which glycine
and proline are the first two residues of the mature form of MDC.
Treatment of the recombinant fusion protein with thrombin is
expected to cleave the arginine-glycine bond of the fusion protein,
releasing the mature chemokine from the GST fusion.
[0138] The PCR product was purified by agarose gel electrophoresis,
digested with BamHI endonuclease, and cloned into the BamHI site of
pGEX-3.times.. This pGEX-3.times./MDC construct was transformed
into E. coli XL-1 Blue cells (Stratagene, La Jolla Calif.), and
individual transformants were isolated and grown. Plasmid DNA from
individual transformants was purified and partially sequenced using
an automated sequencer and primer GEX5 (SEQ ID NO: 12), which
hybridizes to the pGEX-3.times. vector near the BamHI cloning site.
The sequence obtained with this primer confirmed the presence of
the desired MDC insert in the proper orientation.
[0139] Induction of the GST-MDC fusion protein was achieved by
growing the transformed XL-1 Blue culture at 37.degree. C. in LB
medium (supplemented with carbenicillin) to an optical density at
wavelength 600 nm of 0.4, followed by further incubation for 4
hours in the presence of 0.25 to 1.0 mM Isopropyl
.beta.-D-Thiogalactopyranoside (Sigma Chemical Co., St. Louis
Mo.).
[0140] The fusion protein, produced as an insoluble inclusion body
in the bacteria, was purified as follows. Cells were harvested by
centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA,
and treated with 0.1 mg/ml lysozyme (Sigma Chemical Co.) for 15
minutes at room temperature. The lysate was cleared by sonication,
and cell debris was pelleted by centrifugation for 10 minutes at
12,000.times.g. The fusion protein-containing pellet was
resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered over 50%
glycerol, and centrifuged for 30 min. at 6000.times.g. The pellet
was resuspended in standard phosphate buffered saline solution
(PBS) free of Mg.sup.++ and Ca.sup.++. The fusion protein, which
remained insoluble, was approximately 80-90% of the protein mass
and migrated in denaturing SDS-polyacrylamide gels with a relative
molecular weight of 33 kD. The protein yield, as judged by
Coomassie staining, was approximately 100 mg/l of E. coli
culture.
[0141] The fusion protein was subjected to thrombin digestion to
cleave the GST from the mature MDC protein. The digestion reaction
(20-40 ug fusion protein, 20-30 units human thrombin (4000 U/mg
(Sigma) in 0.5 ml PBS) was incubated 16-48 hrs. at room temperature
and loaded on a denaturing SDS-PAGE gel to fractionate the reaction
products. The gel was soaked in 0.4 M KCl to visualize the GST and
MDC protein bands, which migrated as fragments of approximately 26
kD and 7 kD, respectively.
[0142] The identity of the 7 kD SDS-PAGE fragment was confirmed by
partial amino acid sequence analysis. First, the protein was
excised from the gel, electroeluted in 25 mM Tris base and 20 mM
glycine, and collected onto a PVDF membrane in a ProSpin column
(Applied Biosystems, Foster City, Calif.). Subjecting the sample to
automated sequencing (Applied Biosystems Model 473A, Foster City,
Calif.) yielded 15 residues of sequence information, which
corresponded exactly to the expected N-terminus of the predicted
mature form of MDC (SEQ ID NO: 2, amino acid residues 1 to 15).
EXAMPLE 7
Production of Recombinant MDC in Bacteria
[0143] MDC peptides and analogs can be expressed using a variety of
bacterial expression systems including E. coli, Bacillis subtilis,
streptomyces lividans, and many others. [For a general review see
"Gene Expression Technology" in Methods in Enzymology, Vol. 185:
pp. 1-283. Ed. D. V. Goeddel, Academic Press, San Diego, Calif.
(1990).] In general, an expression cassette comprised of a
transcription element (a promoter), a translation element, a coding
region to be expressed (for example MDC), and a transcription
termination element is developed and optimized to effect
significant gene expression. This cassette is incorporated into
either episomal plasmids, which confer stable propagation, or into
integration vectors to mediate the insertion or creation (via
homologous recombination) of an expression cassette within the host
genome. The gene can be expressed directly or can be fused to
signal sequences (e.g., pelB, ompA, est2) to direct secretion of
the gene product out of the cytoplasm into either the periplasmic
space or media, or to other leader sequences (e.g., ubiquitin) to
enhance the folding or otherwise stabilize the recombinantly
expressed coding region. The gene product, either properly folded
or not, can be recovered in a crude state or as inclusion bodies
from the cells following a fermentation phase and either directly
purified or refolded prior to purification.
[0144] A. Construction and Testing of Bacterial MDC Expression
Vector P2-390
[0145] The portion of the MDC cDNA encoding the predicted mature
MDC protein was cloned into a plasmid containing the arabinose
promoter (araB) and the pelB leader sequence [see Better et al.,
Science, 240:1041-43 (1988)].
[0146] More particularly, an MDC cDNA was amplified by PCR using
approximately 0.11 .mu.g of pMP390-12 as template and synthetic
oligonucleotide primers 390-2R (SEQ ID NO:8) and 390-Pel (SEQ ID
NO: 13). Primer 390-Pel contains an Nco I restriction site,
followed by two cytosine residues, followed by bases 92 to 115 of
SEQ ID NO: 1.
[0147] The expected PCR product of 232 bp was purified by agarose
gel electrophoresis, digested with Nco I and BamHI, and cloned
along with a portion of the arabinose operon and pelB leader
sequence (Better et al., supra) into the vector pUC19 (New England
Biolabs, Beverly. MA). The resultant construct, designated P2-390,
encodes a fusion of the pelB leader (encoded by the vector) to the
mature MDC protein. The sequence of this construct was confirmed by
automated sequencing using the primers Ara1 (SEQ ID NO:28) and Ara2
(SEQ ID NO:29), which anneal to the vector adjacent to the cloning
site. The plasmid P2-390 was transformed into the E. coli strain
MCI 061 using standard procedures, and an ampicillin resistant
clone was selected for MDC production. The clone was grown in a 3
liter fermenter (Applikon, Foster City, Calif.) and MDC production
was induced by the addition of 50% arabinose to a final
concentration of 0.1%. After one day of cultivation in the presence
of arabinose, the cells were harvested. Western blotting revealed
that MDC was present within the cells at a level of approximately 4
.mu.g/g of cell paste and was secreted into the culture medium to a
level of approximately 1 .mu.g/ml.
[0148] B. Protocol for Bacterial Expression of MDC Using Plasmid
P2-390
[0149] The plasmid P2-390 was transformed into E. coli strain
SB7219 (Sheppard and Englesberg, J. Molec. Biol., 25:443-454 (1967)
and Wilcox et al., J. Biol. Chem., 249:2946-2952 (1974)). SB7219 is
a prototrophic strain incapable of degrading arabinose, the inducer
of the araB promoter used to transcribe the pelB-MDC coding region.
The genotype of SB7219 is E. coli K12 F.sup.- del(codb-lac).sub.3
del(ara735) rpsL150(str.sup.R).lambda..sup.-. The production strain
SB7219:P2-390 was grown in the fermenter (run FC563) in a fed batch
format. A frozen aliquot of the seed is inoculated into 250 ml of
fermentation basal medium in the shake flask. The composition of
the basal medium is as follows:
2 Component Quantity per L Basal Medium Na.sub.3citrate 1 g 5.4%
FeCl.sub.3.6H.sub.2O 2 ml glucose 2 g NaH.sub.2PO.sub.4.H.sub.2O 3
g K.sub.2HPO.sub.4 6 g (NH.sub.4).sub.2SO.sub.4 5 g 20% yeast
extract solution 5 ml 1 M CaCl.sub.2 0.5 ml 1 M MgCl.sub.2 2.0 ml
trace elements 4 ml trace vitamins 2 ml 1% thiamine 1 ml
tetracycline 5 mg pH is set to 7.0 Trace Elements Solution Boric
Acid 5.0 g Copper Sulfate .multidot. 5H.sub.2O 2.0 g Potassium
Iodide 1.0 g Manganese sulfate 10 g Molybdic acid 0.5 g ZnCl.sub.2
(Anhydrous) 5.2 g Cobalt chloride 0.5 g Trace Vitamin Solution
Sodium Hydroxide, 50% 1.3 ml Riboflavin 0.42 g Folic Acid 0.04 g
D-Pantothenic Acid (hemicalcium salt) 5.4 g Nicotiinic Acid
(niacin) 6.1 g Pyridoxine HCl 1.4 g Biotin 0.06 g
[0150] The shake flask culture is grown at 37 C and 220 RPM to an
optical density corresponding to mid-exponential growth
(approximately OD.sub.600.apprxeq.0.7). The inoculum is added to
the fermentor containing 1.5 L of basal media and grown at
30.degree. C. for 5 hours. A feed is then initiated at 3.6 ml/hr
and exponentially increased to effect a doubling time of 5 hr until
a maximum of 18 ml/hr of feed is achieved.
3 Feed Medium Component Quantity per L Na.sub.3citrate 5 g 5.4%
FeCl.sub.3.6H.sub.20 10 ml glycerol 500 g (NH.sub.4).sub.2SO.sub.4
5 g 1 M CaCl.sub.2 4 ml 1 M MgCl.sub.2 100 ml 1 M MnCl.sub.2 0.4 ml
trace elements 10 ml
[0151] When the wet cell mass is approximately 100 g/L, 20 ml of
50% arabinose solution is added to induce expression of MDC. The
temperature is raised to 37.degree. C. and the feed rate is
decreased to 12 ml/hr. The fermentation is allowed to continue for
approximately 20 more hours, at which time the cell paste is
harvested from the tank and stored frozen at -70.degree. C. The MDC
contained in the cell paste is suitable for recovery by mechanical
lysis, re-folding, and purification as described below in Example
8.
[0152] C. Direct Expression of MDC in E. coli
[0153] In a similar way, MDC that is directly expressed (i.e.,
without a fused in-frame leader sequence) is engineered into the
same vector. The plasmid pBAR5/MDC/RC is a plasmid identical to
P2-90 except for the elimination of the pelB leader sequence. In
addition, the first fourteen percent of the MDC(1-69) coding
sequence (amino acid codons 1-6 and 8-10) have been modified to
change cytosine residues at codon position three to either an
adenosine or thymidine nucleotide (while preserving the encoded
amino acid). Additionally, a translation initiation codon was
added. Thus, the coding sequence in pBAR5/MDC/RC begins:
4 5' ATG GGA CCA TAT GGA GCA AAT ATG GAA GAT AGT . . . (SEQ ID NO:
44)
[0154] E. coli strain SB7219 harboring this plasmid is grown in a
fermentor essentially as described above and the MDC that is
produced is similarly recovered.
[0155] D. P2-390 Variant Expression Vector
[0156] In addition, a derivative of P2-390 pBAR5/PelB/MDC/RC in
which the amino acid codons described above in part C were
substituted for the wild-type sequence was created. E. coli SB7219
harboring this plasmid is grown in a fermentor in a comparable
fashion and the MDC produced is similarly recovered.
EXAMPLE 8
Purification of Recombinant MDC from Bacteria and Culture
Medium
[0157] The following are experimental protocols for purification of
the recombinant MDC produced as described in Example 7.
[0158] A. Recovery and Purification of Secreted Recombinant
MDC.
[0159] The secreted recombinant MDC protein is purified from the
bacterial culture media by, e.g., adapting methods previously
described for the purification of recombinantly produced RANTES
chemokine [Kuna et al., J. Immunol., 149:636-642 (1992)], MGSA
chemokine [Horuk et al, J. Biol. Chem. 268:541-46 (1993)], and
IP-10 chemokine (expressed in insect cells) [Sarris el al., J. Exp.
Med., 178:1127-1132 (1993)].
[0160] B. Recovery, and Re-Folding of MDC Bound in Inclusion
Bodies
[0161] Methods for recovery of inclusion bodies from E. coli paste
has been well described [see Lin et al., Biotechniques, 11(6):
748-52 (1991); Myers et al., Prot. Express. Purif., 2: 136-143
(1991); Krueger et al., BioPharm., pp. 40-45 (March, 1989); Marston
et al., "Solubilization of Protein Aggregates," Methods in
Enzymology, M P Deutcher (Ed.), Academic Press, New York, 182:
264-276 (1990)]. Briefly, MDC is released from intact cells using a
mechanical lysis device (e.g., Mauton-Gaulin). The cell paste is
resuspended (20-30% w/v) in buffer [for example, containing 50 mM
Tris HCl, pH 8.0, 1 mM EDTA, 50 mM NaCl, 0.2 mg/ml lysozyme, and
0.5% (v/v) Triton X-100] and passed through the machine at a
constant pressure of 8-12,000 PSI for one to two passes at
4-15.degree. C. The soluble components of the cell are separated
from MDC and the other cellular-derived insoluble components by
applying a centrifugal force of approximately 12,000.times.g for a
period of about 5-10 minutes. The insoluble pelleted material is
then re-suspended and re-centrifuged using dilute solutions of
detergent [for example, 0.5% (v/v) Triton X-100 and 10 mM EDTA, pH
8.0]. Other wash steps can be used, including 0.5% (v/v)
Zwittergent 3-14 (Calbiochem, Inc.), as well as treatments to
minimize viscosity including lysozyme, DNase, Nonidet and EDTA [see
Bartholome-DeBelder et al., Mol., Microbiol., 2:519 (1988)].
[0162] To achieve proper folding of MDC contained in exclusion
bodies, inclusion body preparations are reduced at a protein
concentration of 5-10 mg/ml in 6 M guanidine-HCl containing 0.1 M
Tris HCl, pH 8.6, 20% .beta.-mercaptoethanol, for 1 hour at
37.degree. C. Complete reduction results in a completely clear
solution. Confirmation of complete reduction is obtained using an
analytical reverse phase (rp) HPLC procedure. For example, a Vydac
C4 analytical column (e.g., 214 nm) is equilibrated in 5%
acetonitrile/water/0.1% trifluoroacetic acid. The sample is
injected and a linear gradient with increasing acetylnitrile
content is run at a rate of 2% increase per minute. A single peak
indicates that complete reduction of the MDC protein has been
achieved.
[0163] The pH of the solution containing the fully reduced MDC is
gradually lowered to 4.0 with 10% HCl. The MDC is then recovered
from the reduction solution using preparative rpHPLC [e.g., a Vydak
C4 preparative column with the gradient as described above] to
remove HCl salts and denaturant. The recovered MDC is then diluted
into 2 M guanidine-HCl, 0.1 M Tris HCl, pH 8.6, 8 mM cysteine, 1 mM
cystine to a protein concentration of 2 g/L. The solution is
stirred slowly at room temperature for 4-8 hours and shielded from
light. The concentration of properly refolded MDC is monitored
using the analytical rpHPLC method described above and is
distinguished from reduced MDC by a 2-4 minute reduction in
retention time on the HPLC column, relative to the reduced MDC.
Confirmation of disulfide bond formation in refolded MDC is
confirmed using mass spectrometry [i.e., MALDI MS].
[0164] C. Purification of Refolded MDC
[0165] MDC is purified using a two column procedure as follows:
SP-Sepharose-fast flow (Pharmacia) resin is packed for column
purification and equilibrated in loading buffer (0.2 M NaCl, 20 mM
Tris base, pH 7.5). The recovered, refolded MDC solution is diluted
with buffer until the conductivity of the supernatant equals 18-19
mS, and the pH is adjusted to 7.5. The solution is filtered to
remove insoluble materials and applied to the column to a capacity
of 0.5 mg MDC/ml of resin. Loading buffer is then used until the
OD.sub.280) returns to baseline. MDC is eluted using a higher salt
buffer (0.6 M NaCl, 20 mM Tris, pH 7.5).
[0166] The SP-Sephadex elution peak is then chromatographed on an
WP Hi-Propyl (C3) hydrophobic interaction column (JT Baker
#7585-O.sub.2). The column is equilibrated with 2.4 M NaCl, 20 mM
Tris, pH 7.5. The 0.6 M NaCl containing S--P eluate is then
adjusted with the appropriate amount of 5 M NaCl to bring the salt
concentration of the eluate to 2.4M NaCl. The adjusted eluate is
loaded onto the propyl column at 2 mg of MDC/ml and washed with 2.4
M NaCl, 20 mM Tris, pH 7.5, until the OD.sub.280 returns to
baseline. The column is then washed with two column volumes of 2.0
M NaCl, 20 mM NaCl. The purified MDC is eluted from the column with
0.8 M NaCl, 20 mM Tris, pH 7.5. Purified MDC is then filter
sterilized and stored at -70.degree. C.
EXAMPLE 9
Recombinant Production of MDC in Yeast
[0167] Following are protocols for the recombinant expression of
MDC in yeast and for the purification of the recombinant MDC.
Heterologous expression of human genes using microbial hosts can be
an effective method to produce therapeutic proteins both for
research and commercial manufacture. Secretion from yeast hosts
(see recent review by Romanos, Yeast, 8: 423-488 (1992)) such as
Saccharomyces cerevisiae (Price et al., Gene, 55:287 (1987))
Kluyvreomyces lactis (Fleer et al., Bio/Technology, 9: 968-975
(1991)), Pichia pastoris (Cregg et al., Bio/Technology, 11: 905-910
(1993)), Schizosaccharomyces pombe(Broker et al., FEBS Lett., 248:
105-110 (1989)), and related organisms provide a particularly
useful approach to obtain both high titer production of crude bulk
product and rapid recovery and purification. These expression
systems typically are comprised of an expression cassette
containing a strong transcriptional segment of DNA or promoter to
effect high levels of mRNA expression in the host. The mRNA
typically encodes a coding region of interest preceded by an
in-frame leader sequence, e.g., S. cerevisiae pre-pro alpha factor
(Brake et al., Proc. Nat. Acad. Sci., 81: 4642-4646 (1984)) or
equivalent signal, which directs the mature gene product to the
culture medium. As taught below, MDC can be expressed in such a
manner.
[0168] In one exemplary protocol, the coding region of the MDC cDNA
is amplified from pMP390-12 by PCR, using as primers synthetic
oligonucleotides containing the MDC cDNA sequences present in
primers 390-IF (SEQ ID NO: 7) and 390-2R (SEQ ID NO: 8). A DNA
encoding the yeast pre-pro-alpha leader sequence is amplified from
yeast genomic DNA in a PCR reaction using one primer containing
bases 1-20 of the alpha mating factor gene and another primer
complimentary to bases 2-55-235 of this gene [Kurjan and
Herskowitz, Cell, 30: 933-943 (1982)]. The pre-pro-alpha leader
coding sequence and MDC coding sequence fragments are ligated into
a plasmid containing the yeast alcohol dehydrogenase (ADH2)
promoter, such that the promoter directs expression of a fusion
protein consisting of the pre-pro-alpha factor fused to the mature
MDC polypeptide. As taught by Rose and Broach, Meth. Enz., 185:
234-279, D. Goeddel, ed., Academic Press, Inc., San Diego, Calif.
(1990), the vector further includes an ADH2 transcription
terminator downstream of the cloning site, the yeast "2-micron"
replication origin, the yeast leu-2d gene, the yeast REP1 and REP2
genes, the E. coli beta-lactamase gene, and an E. coli origin of
replication. The beta-lactamase and leu-2d genes provide for
selection in bacteria and yeast, respectively. The leu-2d gene also
facilitates increased copy number of the plasmid in yeast to induce
higher levels of expression. The REP1 and REP2, genes encode
proteins involved in regulation of the plasmid copy number.
[0169] The DNA construct described in the preceding paragraph is
transformed into yeast cells using a known method, e.g., lithium
acetate treatment [Stearns et al., Meth. Enz., supra pp. 280-297].
The ADH2 promoter is induced upon exhaustion of glucose in the
growth media [Price et al., Gene, 55:287 (1987)]. The pre-pro-alpha
sequence effects secretion of the fusion protein from the cells.
Concomitantly, the yeast KEX2 protein cleaves the pre-pro sequence
from the mature MDC chemokine [Bitter et. al., Proc. Natl. Acad.
Sci. USA, 81:5330-5334 (1984)].
[0170] Alternatively, MDC is recombinantly expressed in yeast using
a commercially available expression system e.g., the Pichia
Expression System (Invitrogen, San Diego, Calif.), following the
manufacturer's instructions. This system also relies on the
pre-pro-alpha sequence to direct secretion, but transcription of
the insert is driven by the alcohol oxidase (AOX1) promoter upon
induction by methanol.
[0171] The secreted MDC is purified from the yeast growth medium
by, e.g., the methods used to purify MDC from bacterial and
mammalian cell supernatants (see Examples 8 and 10).
[0172] MDC was expressed in yeast as follows. Using standard
molecular biological methods (Sambrook et al., Molecular Cloning: a
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989)) such as those described
above, the S. cerevisiae alpha factor pre-pro sequence (codons 1-85
in FIG. 9) was fused to the presumptive mature form of MDC (SEQ ID
NO: 1, positions 1-69; codons 86-155 in FIG. 9). Expression of the
resultant coding region is under control of the K. lactis LAC4
promoter present in the plasmid pYGL/preproMDC (see FIG. 10). This
plasmid is a derivative of the K. lactis expression plasmid
developed by Fleer et al., (supra) and used to secrete high titers
of human serum albumin. This vector class is derived from the
plasmid pKD1, a 2.mu. like plasmid from in K. drosophilarium (Chen
et al., Nucleic Acids Research, 14: 447-81 (1986)). These vectors
are autonomously replicated and maintained at high copy number and
have been shown to confer high levels of protein production when K.
lactis strains containing these plasmids are grown in either
galactose or lactose as "inducing" agents and as the sole carbon
source. The construct pYGL/preproMDC confers to the host both
resistance to G418 (200 mg/L) and the glycolytic enzyme
phosphoglucokinase (PGK). Efficient selection for transformed cells
containing the plasmid is effected by providing a sole carbon
source that requires processing via the glycolytic pathway of
intermediary metabolism.
[0173] Plasmid pYGL/preproMDC was transformed into the pgko
deficient host strain FBO5 (Delta Biotechnology Limited) by
selecting for G418 resistance in YEPPglycerol/ethanol medium (0.5%
yeast extract, 1% peptone, 1 M KPO.sub.4, pH 7.0, containing 3%
glycerol and 2% ethanol). Following clonal isolation, the
transformed seed was grown in shake flask production medium YEPPgal
(0.5% yeast extract, 1% peptone, 1 M KPO.sub.4, pH 7.0, containing
2% galactose as sole carbon source). SDS-PAGE analysis of the
culture medium indicated that a protein species of the molecular
weight expected of that for mature MDC was present. This protein
migrated comparably to synthetic MDC (Gryphon Sciences
Corporation). Titration data using dilutions of purified synthetic
MDC and culture supernatants in Coomassie blue stained SDS-PAGE
gels suggested that MDC was present in the range of 4-10 mg/L.
[0174] Western analyses using an anti-MDC monoclonal antibody did
not reveal the presence of MDC-related degradation products, even
after further culturing of the seed 24 hours past the completion of
growth. This observation suggested that the seed is capable of
producing and stably accumulating MDC, indicating that high cell
fermentation methods would be effective to increase titer.
[0175] The MDC production seed was used to inoculate a fermentor
maintained at 26.degree. C. containing a batch medium. The
composition of the batch medium (1200 ml) was as follows: 7.5 g
Yeast extract; 0.6 g MgSO.sub.4; 6.0 g NH.sub.4SO.sub.4; 9.6 g
KH.sub.2PO.sub.4; 26.4 g K.sub.2HPO.sub.4; 11 mg CaCl.sub.2 5.0 ml
1000.times. vitamins [Bitter et al., J. Med. Virol., 25(2): 123-140
(1988)]; 2.5 ml 1000.times. trace elements [Bitter et al. (1988)];
and 1.2 g 30% galactose.
[0176] One hour following inoculation, a feed was initiated at a
rate of 12 ml/hour and maintained for four days. The feed medium
composition (1500 ml) was as follows: Galactose, 600 g; yeast
extract, 50 g; MgSO.sub.4, 4 g; NH.sub.4SO.sub.4, 40 g;
KH.sub.2PO.sub.4, 60 g; K.sub.2 HPO.sub.4, 165 g; 1000.times.trace
elements, 15 ml; 100.times. vitamins, 30 ml; 4% CaCl.sub.2
solution, 20 ml.
[0177] Samples were collected and analyzed throughout the run. MDC
accumulated during the first three days of the fermentation to a
final titer of approximately 50 mg/L as determined from
purification recovery experiments. The primary protein species
present is MDC. Significant levels of degradation were not observed
by SDS-PAGE analysis. A sample of the harvest supernatant was
partially purified using ion exchange chromatography. Following
dialysis into phosphate buffered saline, the yeast-produced MDC
exhibited a single molecular mass of 8088 daltons, as compared with
the theoretical value of 8086, well within the expected error of
the measurement.
[0178] Yeast-produced MDC was further analyzed for biological
activity by calcium flux assay and found to exhibit activity
comparable to the activity of synthetic MDC and CHO-produced MDC.
Using the assay described below in Example 25, yeast-produced MDC
was also successful in competing with synthetic MDC-SEAP for
binding to CCR4 recombinantly expressed on a mammalian cell
surface.
EXAMPLE 10
Recombinant Production of MDC in Mammalian Cells
[0179] MDC was recombinantly produced in mammalian cells according
to the following procedures.
[0180] A. Synthesis of Expression Vector 390HXE
[0181] A truncated version of the MDC cDNA was synthesized by PCR
using pMP390-12 as template and the synthetic oligonucleotides
390RcH (SEQ ID NO: 14) and 390RcX (SEQ ID NO: 15) as primers.
Primer 390RcH contains a Hind III restriction site followed by
bases 1 to 20 of SEQ ID NO: 1 primer 390RcX contains an Xba I
restriction site followed by the sequence complimentary to bases
403 to 385 of SEQ ID NO: 1.
[0182] The expected 423 bp PCR product was purified by agarose gel
electrophoresis and cloned into Hind III/Xha I-digested pRc/CMV
((InVitrogen, San Diego Calif.) a vector which allows for direct
expression in mammalian cells). The resulting plasmid, designated
390HXE, contained bases 1 to 403 of SEQ ID NO: 1. The sequence of
the insert was confirmed by automated sequencing using the primers
DC03 (SEQ ID NO: 16) and JHSP6 (SEQ ID NO: 3). Primer DC03 anneals
to the pRc/CMV vector sequence adjacent to the cloning site.
[0183] B. Synthesis of Expression Vector 390HmX
[0184] Another MDC cDNA construct was generated by PCR, using
pMP390-12 as template and the primers 390RcH (SEQ ID NO: 14) and
390mycRX (SEQ ID NO: 17). Primer 390mycRX contains an Xba I
restriction site, a sequence complementary to the sequence encoding
a "myc" epitope [Fowlkes et al., BioTechniques, 13:422-427 (1992)],
and a sequence complementary to bases 298 to 278 of SEQ ID NO: 1.
This reaction amplified the expected 354 bp fragment containing
bases 1 to 298 of SEQ ID NO: 1 fused to a "myc" epitope at the MDC
carboxy-terminus. This epitope can be used to facilitate
immunoprecipitation, affinity purification, and detection of the
MDC-myc fusion protein by Western blotting. The fragment was cloned
into pRc/CMV to generate the plasmid 390HmX. The sequence of the
insert was confirmed by automated sequencing using the primer DC03
(SEQ ID NO: 16).
[0185] C. Expression of MDC in 293T and NS0 Cells
[0186] Two transfection protocols were used to express the two MDC
cDNA constructs described above in subparts A and B transient
transfection into the human embryonic kidney cell line 293T and
stable transfection into the mouse myeloma cell line NS0 (ECACC
85110503).
[0187] Transient transfection of 293T cells was carried out by the
calcium phosphate precipitation protocol of Chen and Okayama,
BioTechniques, 6:632-638 (1988) and Mol. Cel. Biol., 87:2745-2752
(1987). Cells and supernatants were harvested four days after
transfection. A Northern blot was prepared from 4 .mu.g of total
RNA from each cell lysate and probed with a radiolabeled MDC
fragment prepared by PCR. The template for the labeling reaction
was a PCR fragment previously generated by amplifying pMP390 with
the primers 390-IF (SEQ ID NO: 17) and 390-4R (SEQ ID NO: 9).
Approximately 30 ng of this fragment was employed in a PCR reaction
containing the following: 1.5 mM MgCl.sub.2, 50 mM KCl, 10 mM Tris,
pH 8.4, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dGTP, 1 .mu.M dCTP, 50
.mu.Ci .alpha..sup.32P-dCTP (DuPont/New England Nuclear, Boston
Mass.), 2.5 U Taq polymerase, and 10 .beta.g/ml each of primers
390-1F and 390-2R. The reaction was denatured by heating for 4
minutes at 94.degree. C., followed by 15 cycles of amplification
(denaturation for 15 seconds at 94.degree. C., annealing for 15
seconds at 60.degree. C., and extension for 30 seconds at
72.degree. C.). The probe was purified by passage over a G-25 Quick
Spin column (BMB). Conditions for hybridization were as follows:
The filters were incubated at 42.degree. C. for 16 hours with
5.times.10.sup.7 counts per minute (cpm) of the probe, in 40-50 ml
of a solution containing 50% formamide, 5.times. Denhardt's
solution, 5.times.SSC (1.times.SSC is 0.15 M NaCl, 15 mM sodium
citrate), 50 mM sodium phosphate, pH 6.5, and 0.1 mg/ml sheared
salmon sperm DNA (Sigma, St. Louis Mo.).
[0188] Filters were subsequently washed in 0.5.times.SSC and 0.2%
SDS at 42.degree. C. for 30 minutes. Autoradiography was carried
out at -80.degree. C. with one intensifying screen for sixteen
hours. The MDC DNA constructs were very highly expressed in the
transfected cells and not detectable in the non-transfected
cells.
[0189] For stable transfections, NS0 cells were grown to 80%
confluency in D-MEM (Gibco), collected by centrifugation, and
washed with PBS. Twenty .mu.g of plasmid DNA was linearized with
Sca I restriction endonuclease (BMB), added to the cells, and
incubated on ice for 15 minutes in a 0.4 cm gap cuvette (BioRad,
Hercules Calif.). The cells were electroporated with two pulses of
3 microfarad at 1.5 kilovolts. Cells were diluted into 20 ml D-MEM,
incubated at 37.degree. C. in 5% CO.sub.2 for 24 hours, and
selected by plating into 96-well plates at various dilutions in
D-MEM containing 800 .mu.g/ml geneticin. Wells containing single
drug-resistant colonies were expanded in selective media. Total RNA
was analyzed by Northern blotting as described in the preceding
paragraph. Message for MDC was seen only in transfected cell
lines.
[0190] MDC is purified from mammalian culture supernatants by,
e.g., adapting methods described for the purification of
recombinant TCA3 chemokine [Wilson et al., J. Immunol.,
145:2745-2750 (1990], or as described below in subpart F.
[0191] D. Expression of MDC in CHO Cells
[0192] PCR was used to amplify bases 1 to 403 of the MDC cDNA clone
(SEQ ID NO: 1) using primers 390RcH and 390RcX (SEQ. ID NOs: 14 and
15), as described above in subpart A. The fragment was cloned into
the HindIII and XbaI sites of the expression vector pDC1, a pUC19
derivative that contains the cytomegalovirus (CMV) promoter to
drive expression of the insert. More specifically, vector pDC1,
depicted in FIG. 8, was derived from pRc/CMV and pSV2-dhfr (ATCC
vector #37146). Vector pDC1 is similar to the mammalian expression
vector pRc/CMV (Invitrogen, San Diego) except that pDC1 carries the
mouse dihydrofolate reductase (dhfr) gene as a selectable marker,
in place of the neomycin phosphotransferase gene. Transcription of
the target gene in pDC1 is under the control of the strong CMV
promoter. See Stenberg et al, J. Virology, 49:190-199 (1984).
Additionally, a polyadenylation sequence from the bovine growth
hormone gene [Goodwin and Rottman, J. Biol. Chem., 267:166330-16334
(1992)] is provided on the 3' side of the target gene. The dhfr
expression cassette [Subramani et al., Mol. Cell. Biol. 1:854-864
(1981)] allows selection for pDC1 in cells lacking a functional
dhfr gene.
[0193] XL-1 Blue bacteria (Stratagene) were transformed with the
pDC1/MDC plasmid using standard techniques of CaCl.sub.2 incubation
and heat shock (Sambrook et al). Transformants were grown in LB
medium containing 100 .mu.g/ml carbenicillin. Plasmid DNA from
individual transformed clones was isolated using the Promega Wizard
Maxiprep system (Madison, Wis.) and its sequence was confirmed by
automated sequencing using the primers 390-IF and 390-2R (SEQ ID
NOs: 7 & 8). The plasmid was linearized by restriction
digestion with Pvu I endonuclease (Boehringer Mannheim), which cuts
once within the vector sequence.
[0194] The Chinese hamster ovary (CHO) cell line used for
production of MDC was DG-44, which was derived by deleting the dhfr
gene. See Urlaub et al., Cell, 33:405 (1983). For electroporation,
10' of these CHO cells were washed in PBS, resuspended in 1 ml PBS,
mixed with 25 .mu.g of linearized plasmid, and transferred to a 0.4
cm cuvette. The suspension was electroporated with a Biorad Gene
Pulser (Richmond, Calif.) at 290 volts, 960 .mu.Farad.
Transfectants were selected by growth in a medium (Cat. No. 12000,
Gibco, Gaithersburg, Md.) containing 10% dialyzed fetal bovine
serum (FBS) (Hyclone, Logan, Utah) and lacking hypoxanthine and
thymidine. Cells from several hundred transfected colonies were
pooled and re-plated in a medium containing 20 nM methotrexate
(Sigma, St. Louis, Mo.). Colonies surviving this round of selection
were isolated and expanded in a medium containing 20 nM
methotrexate.
[0195] E. Purification of MDC for Protein Sequencing
[0196] Transfected CHO clones were grown on plastic tissue culture
dishes to approximately 90% confluence in a medium, at which time
the medium was replaced with P5 medium containing 0.2% to 1.0% FBS.
P5 medium consists of the components listed in Table 2, below
(purchased as a premixed powder form Hyclone, Logan Utah),
supplemented with the following additional components: (1) 3 g/l
sodium bicarbonate (Sigma, St. Louis, Mo.); (2) 2 .mu.g/l sodium
selenite (Sigma); (3) 1% soy bean hydrolysate (Quest International,
Naarden, The Netherlands); (4) 1.times. ferrous sulfate/EDTA
solution (Sigma); (5) 1.45 ml/1 EX-CYTE VLE solution (Bayer,
Kankakee, Ill.); (6) 10 .mu.g/ml recombinant insulin (Nucellin, Eli
Lily, Indianapolis, Ind.); (7) 0.1% pluronic F-68 (Sigma); (8) 30
.mu.g/ml glycine (Sigma); (9) 50 .mu.M ethanolamine (Sigma); and
(10) 1 mM sodium pyruvate (Sigma).
5 TABLE 2 Powder Component #5 gm/L INORGANIC Sodium Chloride 4.0
SALTS Potassium Chloride 0.4 Sodium Phosphate Dibasic, Anhydrous
0.07102 Sodium Phosphate Monobasic H.sub.20 0.0625 Magnesium
Sulfate, Anhydrous 0.1 Cupric sulfate 5 H.sub.20 0.00000125 Ferrous
Sulfate 7 H.sub.20 0.000417 Zinc Sulfate 7 H.sub.20 0.0004315
Ferric Nitrate 9 H.sub.20 0.00005 Calcium Chloride, Anhydrous
0.11661 Magnesium Chloride, Anhydrous 0 AMINO L-Alanine 0 ACIDS
L-Arginine HCl 0.15 L-Asparagine H.sub.20 0.075 L-Aspartic Acid
0.04 L-Cysteine HCl H.sub.20 0.035 L-Cystine 2 HCl 0.12 L-Glutamic
Acid 0.02 L-Glutamine 0.5846 Glycine 0.02 L-Histidine HCl H.sub.20
0.04 L-Isoleucine 0.15 L-Leucine 0.15 L-Lysine HCl 0.1 L-Methionine
0.05 L-Proline 0.05 L-Phenylainine 0.05 L-Serine 0.075 L-Threonine
0.075 L-Tryptophan 0.02 L-Tyrosine 2 Na 2 H.sub.20 0.075 L-Valine
0.125 VITAMINS Biotin 0.001 D-Calcium Pantothenate 0.0025 Choline
Chloride 0.015 Folic Acid 0.005 i-Inositol 0.175 Nicotinamide 0.005
Pyridoxal HCl 0.005 Pyrdoxine HCl 0.005 Riboflavin 0.001 Thiamine
HCl 0.005 Cyanocobalamine 0.001 OTHER D-Glucose 1.0 Hypoxanthine,
Na 0.005 Thymidine 0.005 Putrescine 2HCl 0.000081 Sodium Pyruvate
0.11004 Linoleic Acid 0.0001 DL-Alpha-Lipoic Acid 0.0002 Phenol
Red, Na Salt 0.0086022
[0197] After two additional days in culture an aliquot of each
supernatant was mixed with an equal volume of acetone. The
precipitated proteins were pelleted by centrifugation, fractionated
on an 18% Tris Glycine gel (NOVEX), and blotted to a PVDF membrane
(Millipore, Bedford, Mass.).
[0198] MDC bound to the membrane was detected by a crude
preparation of monoclonal antibody to MDC (prepared as described in
Example 18). Cells from the clone secreting the highest level of
MDC protein (approx. 1 .mu.g/ml) were removed from the plate by
treatment with a solution of 0.5 % trypsin and 5.3 mM EDTA (GIBCO)
and used to start a suspension culture in a medium plus 10% fetal
bovine serum (FBS). Over the course of 8 days, 5 volumes of P5
medium were added to the culture. Proteins were precipitated from
the culture supernatant by addition of polyethylene glycol (MW
8000, Union Carbide, Danbury, Conn.) to 20% (weight/volume),
fractionated on an 18% Tris glycine gel, and electroblotted to a
PVDF membrane (Millipore, Bedford, Mass.) in CAPS buffer
(3-[Cyclohexylamino]-1-propanesulfonic acid, pH 10.4) (Sigma, St.
Louis, Mo.). A strip of the filter was removed for detection of MDC
by western blotting with the supernatant from a hybridoma cell line
producing anti-MDC monoclonal antibodies (See Example 18). The
reactive band, which migrated with an apparent molecular weight of
6.4 kD, was excised from the remaining portion of the filter.
[0199] Using an automated sequencer (Applied Biosystems, Model 473
A, Foster City, Calif.), the sequence of the N-terminus of the
protein was determined to be: GPYGANMEDS. This sequence is
identical to that of residues 1 to 10 of SEQ ID NO. 2,
corresponding to the N-terminus of the predicted mature form of
MDC.
[0200] F. Purification of MDC for Biological Assays
[0201] For growth of larger cultures, MDC-expressing CHO cells were
grown to 80% confluence on tissue culture plates in .alpha. medium.
The cells were removed from the plates by treatment with trypsin
and EDTA and resuspended at a density of 3.times.10.sup.5 Cells/ml
in P5 medium plus 1% FBS in a spinner flask at 37.degree. C.
Additional P5/1% FBS medium was added as needed to keep the cell
density in the range of 1.times.10.sup.6 to 3.times.10.sup.6.
[0202] After 11 days in culture, the cells were removed from the
medium by filtration. The pH of the culture medium was adjusted to
6.8, and it was passed over a heparin-Sepaharose CL-6B column
(Pharmacia, Piscataway, N.J.). After washing with 0.2 M NaCl in
potassium phosphate buffer, pH 7, the column was eluted with a
linear gradient of 0.2 to 0.7 M NaCl. Fractions were analyzed by
SDS-PAGE and Coomassie stained to determine which of them contained
MDC. MDC eluted from the column at approximately 0.6 M NaCl.
[0203] The fractions containing MDC were pooled and concentrated by
ultrafiltration in stirred-cell chamber (Amicon, Beverly, Mass.)
using a filter with a MW cutoff of 3 kD. Octylglucoside (10 mM
final concentration, Boehringer Mannheim Biochemicals) was added to
the concentrated MDC, which subsequently was passed through a
Sephacryl HR100 column (Pharmacia, Piscataway, N.J.). Fractions
were analyzed by SDS-PAGE for the presence of MDC. The final yield
of MDC protein was approximately 0.1 mg/liter of culture
supernatant, and the purity was estimated to be greater than 95%,
as judged by Coomassie staining.
EXAMPLE 11
Production of MDC and MDC Analogs by Peptide Synthesis
[0204] MDC and MDC polypeptide analogs are prepared by chemical
peptide synthesis using techniques that have been used successfully
for the production of other chemokines such as IL-8 [Clark-Lewis et
al. J. Biol. Chem., 266:23128-34 (1991)] and MCP-1. Such methods
are advantageous because they are rapid, reliable for short
sequences such as chemokines, and enable the selective introduction
of novel, unnatural amino acids and other chemical
modifications.
[0205] For example, MDC and MDC analogs were chemically synthesized
using optimized stepwise solid-phase methods [Schnolzer et al.,
Int. J. Pept. Protein Res., 10:180 (1992)] based on
1-butyloxycarbonyl (Boc) chemistries of Merrifield [J. Am. Chem.
Soc., 85:2149-2154 (1963)] on an Applied Biosystems 430A Peptide
Synthesizer (Foster City, Calif.). The proteins were purified by
reverse-phase HPLC and characterized by standard methods, including
electrospray mass spectrometry and nuclear magnetic resonance.
[0206] The chemically synthesized MDC corresponded to the mature
form of recombinant MDC, consisting of residues 1 to 69 of SEQ ID
NO. 2. Several methods were used to compare the chemically
synthesized MDC to the recombinant MDC produced by CHO cell
transfectants as described in Example 10. The migration of
chemically synthesized MDC was identical to that of the recombinant
MDC in denaturing SDS-PAGE (18% Tris glycine gel, NOVEX). In
addition, the proteins reacted similarly in western blot analysis
using monoclonal and polyclonal antibodies raised against
bacterially produced MDC as described below in Example 18. The
chemically synthesized MDC also appeared to behave in the same
manner as the recombinant MDC in immunoprecipitation assays with
the anti-MDC monoclonal antibodies. These studies indicate that the
denatured and the non-denatured structures of chemically
synthesized MDC are similar to those of recombinant MDC.
[0207] The following MDC analogs also have been chemically
synthesized:
[0208] 1. "MDC (n+1)" (SEQ ID NO: 30) consists of Leucine followed
by residues 1 to 69 of SEQ ID NO. 2. This analog has alternatively
been referred to herein as "MDC(0-69)."
[0209] 2. "MDC (9-69)" consists of residues 9 to 69 of SEQ ID NO.
2.
[0210] 3. "MDC-yl" (SEQ ID NO: 31) consists of residues 1 to 69 of
SEQ ID NO. 2, with the following substitution: Residues 59-60
(Trp-Val) were replaced with the sequence Tyr-Leu. A related analog
"MDC-wvas" consists of residues 1 to 69 of SEQ ID NO. 2, with the
following substitution: Residues 59-60 (Trp-Val) were replaced with
the sequence Ala-Ser.
[0211] 4. "MDC-eyfy" (SEQ ID NO: 32) consists of residues 1 to 69
of SEQ ID NO. 2, with the following substitution: Residues 28-31
(His-Phe-Tyr-Trp) were replaced with the sequence Glu-Tyr-Phe-Tyr,
derived from the amino acid sequence of the chemokine RANTES
(residues 26-29 of SEQ ID NO: 21).
[0212] The analogs "MDC (n+1)", "MDC (9-69)", and "MDC-yl" are
expected to be antagonists of MDC activity, inhibiting MDC activity
by competitively binding to the same receptor that recognizes MDC.
Alternatively, they may effect inhibition by forming inactive
heterodimers with the native MDC. Possible activities of the analog
"MDC-eyfy" include inhibition of MDC as described for the previous
analogs. Alternatively, "MDC-eyfy" may confer some of the
activities typical of the chemokine RANTES, such as chemotaxis of T
lymphocytes, monocytes, or eosinophils.
[0213] Additionally, the following single-amino acid alterations
(alone or in combination) are specifically contemplated: (1)
substitution of a non-basic amino acid for the basic arginine
and/or lysine amino acids at positions 24 and 27, respectively, of
SEQ ID NO: 2; (2) substitution of a charged or polar amino acid
(e.g., serine, lysine, arginine, histidine, aspartate, glutamate,
asparagine, glutamine or cysteine) for the tyrosine amino acid at
position 30 of SEQ ID NO: 2, the tryptophan amino acid at position
59 of SEQ ID NO: 2, and/or the valine amino acid at position 60 of
SEQ ID NO: 2, and (3) substitution of a basic or small, non-charged
amino acid (e.g., lysine, arginine, histidine, glycine, alanine)
for the glutamic acid amino acid at position 50 of SEQ ID NO: 2.
Specific analogs having these amino acid alterations are
encompassed by the following formula (SEQ ID NO: 25):
6 Met Ala Arg Leu Gln Thr Ala Leu Leu Val Val Leu Val Leu Leu Ala
-24 -20 -15 -10 Val Ala Leu Gln Ala Thr Glu Ala Gly Pro Tyr Gly Ala
Asn Met Glu -5 1 5 Asp Ser Val Cys Cys Arg Asp Tyr Val Arg Tyr Arg
Leu Pro Leu Xaa 10 15 20 Val Val Xaa His Phe Xaa Trp Thr Ser Asp
Ser Cys Pro Arg Pro Gly 25 30 35 40 Val Val Leu Leu Thr Phe Arg Asp
Lys Xaa Ile Cys Ala Asp Pro Arg 45 50 55 Val Pro Xaa Xaa Lys Met
Ile Leu Asn Lys Leu Ser Gln 60 65
[0214] wherein the amino acid at position 24 is selected from the
group consisting of arginine, glycine, alanine, valine, leucine,
isoleucine, proline, serine, threonine, phenylalanine, tyrosine,
tryptophan, aspartate, glutamate, asparagine, glutamine, cysteine,
and methionine; wherein the amino acid at position 27 is
independently selected from the group consisting of lysine,
glycine, alanine, valine, leucine, isoleucine, proline, serine,
threonine, phenylalanine, tyrosine, tryptophan, aspartate,
glutamate, asparagine, glutamine, cysteine, and methionine; wherein
the amino acid at position 30 is independently selected from the
group consisting of tyrosine, serine, lysine, arginine, histidine,
aspartate, glutamate, asparagine, glutamine, and cysteine; wherein
the amino acid at position 50 is independently selected from the
group consisting of glutamic acid, lysine, arginine, histidine,
glycine, and alanine; wherein the amino acid at position 59 is
independently selected from the group consisting of tryptophan,
serine, lysine, arginine, histidine, aspartate, glutamate,
asparagine, glutamine, and cysteine; and wherein the amino acid at
position 60 is independently selected from the group consisting of
valine, serine, lysine, arginine, histidine, aspartate, glutamate,
asparagine, glutamine, and cysteine. Such MDC polypeptide analogs
are specifically contemplated to modulate the binding
characteristics of MDC to chemokine receptors and/or other
molecules (e.g., heparin, glycosaminoglycans, erythrocyte chemokine
receptors) that are considered to be important in presenting MDC to
its receptor.
[0215] Additionally, analogs wherein the proline at position 2 of
SEQ ID NO: 1 is deleted or substituted for by another amino acid
are specifically contemplated. Such mutants will collectively be
referred to as "MDC.DELTA.Pro.sub.2 polypeptides." As described
below in Example 20, MDC (3-69) derived from an HIV-infected T cell
line displays properties that are, at least in some respects,
opposite or antagonistic from properties observed for mature MDC
(1-69). It is hypothesized that a dipeptidyl amino peptidase such
as CD26 [Oravecz et al., J. Exper. Med., 186:1865 (1997)] possesses
a specificity for the sequence NH.sub.2--X-Pro (wherein X is any
amino acid), and that the dipeptidase therefore is capable of
converting mature MDC (1-69) (having the amino terminus
NH.sub.2-Gly-Pro-Tyr) to the MDC (3-69) form in vivo. It is
expected that the dipeptidase CD26 will not cleave the amino
terminus from MDC.DELTA.Pro.sub.2 polypeptides, rendering such
mutants more stable than MDC(1-69) in vivo. MDC.DELTA.Pro.sub.2
polypeptides that retain the biological activities of mature MDC
(1-69) are useful in all therapeutic indications wherein MDC (1-69)
is useful as a therapeutic, whereas MDC.DELTA.Pro.sub.2
polypeptides that antagonize the activity of mature MDC (1-69)
(e.g., by competitively binding but failing to signal through CCR4)
are useful as MDC antagonists. In preferred embodiments,
substitution of the proline with a glycine, alanine, valine,
leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, or
tryptophan is contemplated. Introducing the MDC.DELTA.Pro.sub.2
mutation into any of the analogs described above is also
specifically contemplated.
[0216] After synthesis, synthetic MDC or MDC analogs may be reduced
and refolded substantially as described in Example 8 for
bacterially-produced MDC bound in inclusion bodies, or using
procedures that are well-known in the art. See, e.g., Protein
Folding, T. E. Creighton (Ed.), W.H. Freeman & Co., New York,
N.Y. (1992): van Kimmenade et al., Eur. J. Biochem., 173: 109-114
(1988); and PCT publication no. WO 89/01046.
[0217] Recombinant techniques such as those described in the
preceding examples also are contemplated for preparing MDC
polypeptide analogs. More particularly, polynucleotides encoding
MDC are modified to encode polypeptide analogs of interest using
well-known techniques, e.g., site-directed mutagenesis and the
polymerase chain reaction. See generally Sambrook et al, supra,
Chapter 15. The modified polynucleotides are expressed
recombinantly, and the recombinant MDC polypeptide analogs are
purified, as described in the preceding examples.
[0218] The chemoattractant and/or cell-activation properties of MDC
or MDC polypeptide analogs on one or more types of cells involved
in the inflammatory process (e.g., T lymphocytes, monocytes,
macrophages, basophils, eosinophils, neutrophils, mast cells, and
natural killer cells), on endothelial cells, epithelial cells,
fibroblasts, or others are assayed by art-recognized techniques
that have been used for numerous other chemokines. Native MDC,
recombinant MDC or MDC polypeptide analogs, or synthetic MDC or MDC
polypeptide analogs purified and isolated as described in one or
more of the preceding examples are assayed for activity as
described in the following examples with respect to MDC.
EXAMPLE 12
Assay of MDC Effects upon Basophils, Mast Cells, and
Eosinophils
[0219] The effect of MDC upon basophils, mast cells, and
eosinophils is assayed, e.g., by methods described by Weber et al.,
J. Immunol., 154:4166-4172 (1995) for the assay of MCP-1/2/3
activities. In these methods, changes in free cytosolic calcium and
release of proinflammatory mediators (such as histamine and
leukotriene) are measured. Blocking chemokine-mediated activation
of these cell types has implications in the treatment of late-phase
allergic reactions, in which secretion of proinflammatory mediators
plays a significant role [Weber et al., supra].
[0220] In one signaling assay synthetic MDC (0.01-10 nM) caused
dose-dependent chemotaxis of purified human eosinophils (maximum
chemotaxis approximately four-fold greater than in controls). The
relative chemotactic activity of MDC, in relation to other known
chemotactic factors of eosinophils, was as follows:
MDC.apprxeq.eotaxin<RANTES<MCP-4.ltoreq.eotaxin-2. In
contrast, the MDC analog MDC(9-69) displayed no chemotactic
activity in the same assay. This data demonstrates a biological
activity and utility for MDC in stimulating the chemotaxis of
eosinophils, and further demonstrates a utility of MDC modulators
for modulating this chemotactic activity.
[0221] It was further determined that the eosinophil-chemotactic
activity of MDC appears to operate in a manner independent of the
chemokine receptor CCR3. CCR3-transfected HEK cells labeled with
Fura-2 demonstrated a rapid rise in intracellular free calcium
following stimulation with 10-50 nM eotaxin, eotaxin-2, or MCP-4,
but not with 10-100 nM MDC. Similarly, purified eosinophils
cultured for 72 hours in 10 ng/ml IL-5 and labeled with Fura-2
demonstrated a rapid rise in intracellular free calcium following
stimulation with 10-50 nM eotaxin, eotaxin-2, or MCP-4, whereas no
such rise was observed following stimulation with MDC (up to 100
nM).
EXAMPLE 13
Assay of Chemoattractant and Cell-Activation Properties of MDC upon
Human Monocytes/Macrophages and Human Neutrophils
[0222] The effects of MDC upon human monocytes/macrophages or human
neutrophils is evaluated, e.g. by methods described by Devi et al.,
J. Immunol., 153:5376-5383 (1995) for evaluating murine
TCA3-induced activation of neutrophils and macrophages. Indices of
activation measured in such studies include increased adhesion to
fibrinogen due to integrin activation, chemotaxis, induction of
reactive nitrogen intermediates, respiratory burst (superoxide and
hydrogen peroxide production), and exocytosis of lysozyme and
elastase in the presence of cytochalasin B. As discussed by Devi et
al., these activities correlate to several stages of the leukocyte
response to inflammation. This leukocyte response, reviewed by
Springer, Cell, 76:301-314 (1994), involves adherence of leukocytes
to endothelial cells of blood vessels, migration through the
endothelial layer, chemotaxis toward a source of chemokines, and
site-specific release of inflammatory mediators. The involvement of
MDC at any one of these stages provides an important target for
clinical intervention, for modulating the inflammatory
response.
[0223] In one art-recognized chemotaxis assay, a modified Boyden
chamber assay, leukocytes to be tested are fluorescently labeled
with calcein by incubating for 20 minutes at room temperature. The
labeled cells are washed twice with serum-free RPMI, resuspended in
RPMI containing 2 mg/ml of BSA, and then added quantitatively to
the upper wells of the chambers, which are separated from the lower
wells by a polycarbonate filter (Neuroprobe Inc. Cabin John, Md.).
MDC diluted in the same medium as the leukocytes is added to the
lower wells at various concentrations. Chambers are incubated for 2
hours at 37.degree. C. At the end of the assay, cells that have not
migrated through the membrane are removed by rinsing the filter
with PBS and scraping with a rubber policeman. Cells that have
migrated through the filter are quantitated by reading fluorescence
per well in a fluorescent plate reader (Cytofluor, Millipore Inc.,
Boston, Mass.).
[0224] A series of experiments were performed using art-recognized
procedures to determine the chemotactic properties of MDC.
Initially, the response of human mononuclear cells to MDC was
determined. The effect of MDC on the chemotactic response of
polymorphonuclear leukocytes (granulocytes) also was examined.
[0225] It has been established that MCP-1, which is a C--C
chemokine, causes both recruitment and activation of monocytes but
appears to have limited ability to induce the migration of
macrophages. The failure of MCP-1 to attract macrophages appears to
be correlated to the differentiation process: as monocytic cells
differentiate, there is a progressive decrease in cell response to
MCP-1[Denholm and Stankus, Cytokine, 7: 436-440 (1995)]. The
biological activities of MCP-1 appear to correlate with the
expression of this chemokine, with MCP-1 mRNA being found in
monocytes but decreasing as these cells differentiate.
[0226] The pattern of expression of MDC appears to be the reverse
of that described for MCP-1, with the amount of mRNA for MDC
increasing as monocytes differentiate to macrophages. To determine
whether this expression pattern correlates to the biological
response to MDC, the effects of MDC on the migration of monocytes
and macrophages were compared.
[0227] A number of different leukocyte cells types were analyzed in
chemotaxis and chemotaxis inhibition assays. Human mononuclear and
polymorphonuclear leukocytes were isolated from peripheral blood
using methods known in the art [Denholm et al., Amer. J. Pathol.,
135:571-580 (1989)]. Second, the human monocytic cell line, THP-1
(obtained from the ATCC, Rockville, Md., and maintained in culture
in RPMI with 10% FBS and with pennicillin/steptomycin) was
employed. THP-1 cells can be cultured as monocytes or can be
induced to differentiate to macrophages by treatment with phorbol
myristate acetate (PMA) [Denholm and Stankus, Cytokine, 7:436-440
(1995)]. In some experiments monocytic THP-1 cells were employed,
and in others monocytic THP-1 cells were differentiated to
macrophages by incubation with phorbol myristate acetate (PMA).
Third, guinea pig peritoneal macrophages were obtained essentially
as described in Yoshimura, J. Immunol, 150(5025-5032 (1993).
Briefly, animals were given an intraperitoneal injection of 3%
sterile thioglycollate (DIFCO) two days prior to cell harvest.
Macrophages were obtained from the peritoneal cavity by lavage with
phosphate buffered saline (PBS) with 1 mM EDTA and 0.1% glucose.
Cells were washed once by centrifugation and then utilized in
chemotaxis assays as described below.
[0228] Assays of chemotactic activity were carried out, using the
cell preparations described above, essentially as described by
Denholm and Stankus, Cytometry, 19:366-369 (1995), using 96-well
chambers (Neuroprobe Inc., Cabin John, Md.) and cells labeled with
the fluorescent dye, calcein (Molecular Probes, Eugene, Oreg.).
Polycarbonate filters used in this assay were PVP-free (Neuroprobe
Inc.); filter pore sizes used for different cell types were: 5
.mu.m for monocytes and THP-1 cells, 3 .mu.m for polymorphonuclear
leukocytes, and 8 .mu.m for guinea pig macrophages.
[0229] Fifty thousand calcein labelled cells were resuspended in
RPMI medium containing 2 mg/ml BSA and placed in the upper wells.
MDC or other test substances were diluted in RPMI with BSA (e.g.,
final MDC concentrations of 25, 50, 100, 250 ng/ml) and placed in
the lower wells. Following incubation at 37.degree. C. for 2 hours,
unmigrated cells remaining above the filter were removed by wiping;
the filter was then air-dried. Controls in these assays were: RPMI
with BSA as the negative control, and 50 ng/ml of MCP-1 and 1%
zymosan activated serum (ZAS, prepared as described [Denholm and
Lewis, Amer. J Pathol., 126:464-474, (1987)]) were used as positive
controls. Migration of cells was quantitated on a fluorescent plate
reader (Cytofluor, Millipore Inc. Bedford, Mass.) and the number of
cells migrated expressed as fluorescent units.
[0230] In assays of inhibitory activity, cells in the upper wells
of the chambers were suspended in varying concentrations (0.005,
0.05, 0.5, 5.0, and 50 ng/ml) of MDC. The lower wells of the
chamber were filled with either medium alone or the chemotactic
factors, MCP-1 or zymosan activated serum (ZAS). Inhibition was
assessed by comparing the number of cells that migrated to MCP-1 or
ZAS, in the absence of MDC, to the number of cells that migrated
with increasing concentrations of MDC. Preparation of cells and
quantitation of assays was performed exactly as described above for
the chemotaxis assays. The number of cells migrated was expressed
as fluorescent units.
[0231] As indicated in FIG. 2, MDC did not induce THP-1-derived
mononuclear cell migration, but rather appeared to inhibit
mononuclear cell migration, at concentrations between 10 and 100
ng/ml. Other C--C chemokines, such as MCP-1 and RANTES, typically
induce maximal monocyte chemotaxis within this concentration
range.
[0232] As shown in FIG. 3, MDC, at concentrations of 001 to 100
ng/ml had no net effect on granulocyte migration. In respect to
this lack of effect on granulocyte chemotaxis, MDC is similar to
other previously described C--C chemokines.
[0233] The response of both macrophage and monocyte THP-1 cells to
MDC is shown in FIG. 4. Macrophages (closed circles) migrated to
MDC in a dose dependent manner, with optimal activity at 50 ng/ml.
The decrease in macrophage chemotactic response to MDC at higher
concentrations (100 ng/ml) reflects a desensitization of cells
which is typical of most chemotactic factors at high concentrations
[Falk and Leonard, Infect. Immunol., 32:464-468 (1981)]. Monocytic
THP-1 cells (open circles) however, did not migrate to MDC.
[0234] The chemotactic activity of MDC for macrophages was further
verified in experiments utilizing elicited guinea pig peritoneal
macrophages. MDC induced a dose dependent migration of guinea pig
macrophages (FIG. 5), at concentrations between 100 and 500 ng/ml.
The concentrations necessary to induce the migration of guinea pig
macrophages was approximately ten-fold of that for human cells
(FIG. 4). Similar differences in concentrations necessary for peak
biological activity of human chemokines in other species have been
reported for MCP-1 by Yashimura, J. Immunol., 150:5025-5032
(1993).
[0235] The results of these experiments suggest that the biological
activities of MDC are linked to the differentiation of monocytes to
macrophages. In contrast to MCP-1 [Yoshimura, J. Immunol.,
150:5025-5032 (1993)], MDC induces macrophage but not monocyte
chemotaxis.
[0236] The ability of MDC to attract macrophages indicates that
this chemokine might act to induce the focal accumulation of tissue
macrophages. The accumulation of tissue macrophages in specific
areas is important in the formation of granulomas, in which lung
macrophages act to surround and enclose foreign particulates or
relatively nondestructible bacterial pathogens such as
Mycobacterium sp. [Adams, Am. J. Pathol., 84:164-191 (1976)].
[0237] In certain conditions such as arthritis, the accumulation of
macrophages is understood to be detrimental and destructive. The
ability of MDC to promote macrophage chemotaxis indicates a
therapeutic utility for MDC inhibitors of the invention, to
prevent, reduce, or eliminate macrophage accumulation in
tissues.
[0238] The results of the chemotaxis assays with human mononuclear
cells, presented in FIG. 2, suggested that MDC might inhibit cell
migration. In the absence of MDC, monocytic THP-1 cells migrate to
MCP-1, as shown in FIG. 6 (MDC of 0 ng/ml). However, when cells are
exposed to MDC, the chemotactic response to MCP-1 (closed circles)
is decreased. MDC, at concentrations of 0.005-0.5 ng/ml, inhibited
monocyte chemotactic response to MCP-1. Although MDC inhibited the
chemotactic response of monocytes to MCP-1, there was no
significant effect of MDC on chemokinesis, or random migration, as
reflected by the numbers of cells migrating to medium alone (open
circles, RPMI with BSA), either in the presence of absence of
MDC.
[0239] The inhibitory activity of MDC on monocyte chemotaxis
indicates therapeutic utility for MDC in the treatment of several
chronic inflammatory conditions (atherosclerosis, arthritis,
pulmonary fibrosis) in which monocyte chemotaxis appears to play an
important pathogenic role. Enhancing the activity of MDC in such
diseases might result in the decreased migration of monocytes into
tissues, thereby lessening the severity of disease symptoms.
EXAMPLE 14
MDC In Vivo Tumor Growth Inhibition Assay
[0240] Tumor growth-inhibition properties of MDC are assayed, e.g.,
by modifying the protocol described by Laning et al, J. Immunol.,
153:4625-4635 (1994) for assaying the tumor growth-inhibitory
properties of murine TCA3. An MDC-encoding cDNA is transfected by
electroporation into the myeloma-derived cell line J558 (American
Type Culture Collection, Rockville, Md.). Transfectants are
screened for MDC production by standard techniques such as ELISA
(enzyme-linked immunoadsorbant assay) using a monoclonal antibody
generated against MDC as detailed in Example 18. A bolus of 10
million cells from an MDC-producing clone is injected
subcutaneously into the lower right quadrant of BALB/c mice. For
comparison, 10 million non-transfected cells are injected into
control mice. The rate and frequency of tumor formation in the two
groups is compared to determine efficacy of MDC in inhibiting tumor
growth. The nature of the cellular infiltrate subsequently
associated with the tumor cells is identified by histologic means.
In addition, recombinant MDC (20 ng) is mixed with non-transfected
J558 cells and injected (20 ng/day) into tumors derived from such
cells, to assay the effect of MDC administered exogenously to tumor
cells.
EXAMPLE 15
Intraperitoneal Injection Assay
[0241] The cells which respond to MDC in vivo are determined
through injection of 1-1000 ng of purified MDC into the
intraperitoneal cavity of mice or other mammals (e.g., rabbits or
guinea pigs), as described by Luo et al., J. Immunol.,
153:4616-4624 (1994). Following injection, leukocytes are isolated
from peripheral blood and from the peritoneal cavity and identified
by staining with the Diff Quick kit (Baxter, McGraw, Ill.). The
profile of leukocytes is measured at various times to assess the
kinetics of appearance of different cell types. In separate
experiments, neutralizing antibodies directed against MDC (Example
18) are injected along with MDC to confirm that the infiltration of
leukocytes is due to the activity of MDC.
EXAMPLE 16
In vivo Activity Assay--Subcutaneous Injection
[0242] The chemoattractant properties of MDC are assayed in vivo by
adapting the protocol described by Meurer et al., J. Exp. Med.,
178:1913-1921 (1993). Recombinant MDC (10-500 pmol/site) is
injected intradermally into a suitable mammal, e.g., dogs or
rabbits. At times of 4 to 24 hours, cell infiltration at the site
of injection is assessed by histologic methods. The presence of MDC
is confirmed by immunocytochemistry using antibodies directed
against MDC. The nature of the cellular infiltrate is identified by
staining with Baxter's Diff Quick kit.
EXAMPLE 17
Myelosuppression Activity Assays
[0243] The myelosuppressive activity of MDC is assayed by injection
of MDC into mice or another mammal (e.g. rabbits, guinea pigs),
e.g., as described by Maze et al., J. Immunol., 149:1004-1009
(1992) for the measurement of the myelosuppressive action of
MIP-1.alpha.. A single dose of 0.2 to 10 ug of recombinant MDC is
intravenously injected into C3H/HeJ mice (Jackson Laboratories, Bar
Harbor Me.). The myelosuppressive effect of the chemokine is
determined by measuring the cycling rates of myeloid progenitor
cells in the femoral bone marrow and spleen. The suppression of
growth and division of progenitor cells has clinical implications
in the treatment of patients receiving chemotherapy or radiation
therapy. The myeloprotective effect of such chemokine treatment has
been demonstrated in pre-clinical models by Dunlop et al., Blood,
79:2221 (1992).
[0244] An in vitro assay also is employed to measure the effect of
MDC on myelosuppression, in the same manner as described previously
for derivatives of the chemokines interleukin-8 (IL-8) and platelet
factor 4 (PF-4). See Daly et al., J. Biol. Chem., 270:23282 (1995).
Briefly, low density (less than 1.077 g/cm) normal human bone
marrow cells are plated in 0.3% agar culture medium with 16% fetal
bovine serum (HyClone, Logan, Utah) with 100 units/ml recombinant
human GM-CSF (R&D Systems, Minneapolis, Minn.) plus 50 ng/ml
recombinant human Steel factor (Immunex Corp., Seattle, Wash.) in
the absence (control) and presence of MDC for assessment of
granulocyte-macrophage precursors. For assessment of granulocyte
erythroid myeloid megakaryocyte colony forming units (CFU-GEMM) and
erythroid burst forming units (BFU-E), cells are grown in 0.9%
methylcellulose culture medium in the presence of recombinant human
erythropoietin (1-2 units/ml) in combination with 50 ng/ml Steel
factor. Plates are scored for colonies after incubation at
37.degree. C. in lowered (5%) O.sub.2 for 14 days. The combination
of GM-CSF and Steel factor or erythropoietin and Steel factor allow
detection of large colonies (usually >1000 cells/colony) which
come from early, more immature subsets of granulocyte myeloid
colony forming units (CFU-GM), CFU-GEMM, and BFU-E.
EXAMPLE 18
Antibodies to Human MDC
[0245] A. Monoclonal Antibodies
[0246] Recombinant MDC, produced by cleavage of a GST-MDC fusion
protein as described in Example 6, was used to immunize a mouse for
generation of monoclonal antibodies. In addition, a separate mouse
was immunized with a chemically synthesized peptide corresponding
to the N-termunus of the mature form of MDC (residues 1 to 12 of
SEQ ID NO. 2). The peptide was synthesized on an Applied Biosystem
Model 473A Peptide Synthesizer (Foster City, Calif.), and
conjugated to Keyhole Lympet Hemocyanine (Pierce), according to the
manufacturer's recommendations. For the initial injection to
produce "Fusion 191" hybridomas, approximately 10 .mu.g of MDC
protein or conjugated peptide was emulsified with Freund's Complete
Adjuvant and injected subcutaneously. At intervals of two to three
weeks, additional aliquots of MDC protein were emulsified with
Freund's Incomplete Adjuvant and injected subcutaneously. Prior to
the final prefusion boost, a sample of serum was taken from the
immunized mice. These sera were assayed by western blot to confirm
their reactivity with MDC protein. For a prefusion boost, the mouse
was injected with MDC in PBS, and four days later the mouse was
sacrificed and its spleen removed.
[0247] For the production of "Fusion 252" hybridomas, a mouse was
immunized with the MDC(0-0.69) chemically synthesized peptide (See
Example 11). On Day 0, the mouse was pre-bled and injected
subcutaneously at two sites with 10 ug of MDC(0-69) in 200 ul
complete Freund's adjuvant. On Day 22, the mouse was boosted with
30 ug of MDC(0-69) in 150 ul of incomplete Freund's adjuvant. On
Day 40, the mouse was boosted with 20 ug MDC(0-69) in 100 ul of
incomplete Freund's adjuvant. On day 54, blood was drawn and
screened for anti-MDC antibodies via western blot, and reactivity
was observed against MDC. On days 127 through 130, the mouse was
injected on each of four consecutive days with 10 ug of MDC(0-69)
in a volume of 200 ul PBS. On day 131, the mouse was sacrificed and
the spleen was removed for a fusion.
[0248] For the production of "Fusion 272" hybridomens, a mouse was
treated in a similar fashion as the mouse for fusion 252, except,
on day 356, the mouse was boosted with MDC(0-69) in incomplete
Freund's adjuvant. Test bleeds were taken on day 367 and screened
by ELISA. On days 385, 386, 387, and 388, the mouse was boosted
with 5 .mu.g injections of MDC(0-69). On day 389 the spleen was
removed for a fusion.
[0249] The spleens were placed in 10 ml serum-free RPMI 1640, and
single cell suspensions were formed by grinding the spleens between
the frosted ends of two glass microscope slides submerged in
serum-free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM
sodium pyruvate, 100 units/ml penicillin, and 100 .mu.g/ml
streptomycin (RPMI) (Gibco, Canada). The cell suspensions were
filtered through a sterile 70-mesh Nitex cell strainer (Becton
Dickinson, Parsippany, N.J.), and were washed twice by centrifuging
at 200 g for 5 minutes and resuspending the pellet in 10 ml
serum-free RPMI. Thymocytes taken from three naive Balb/c mice were
prepared in a similar manner and used as a Feeder Layer. NS-1
myeloma cells, kept in log phase in RPMI with 10% fetal bovine
serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three
days prior to fusion, were centrifuged at 200 g for 5 minutes, and
the pellet was washed twice as described above.
[0250] Spleen cells (2.times.10.sup.8) were combined with
4.times.10.sup.7NS-1 cells and centrifuged, and the supernatant was
aspirated. The cell pellet was dislodged by tapping the tube, and 2
ml of 37.degree. C. PEG 1500 (50% in 75 mM Hepes, pH 8.0)
(Boehringer Mannheim) was added with stirring over the course of 1
minute, followed by the addition of 14 ml of serum-free RPMI over 7
minutes. An additional 16 ml RPMI was added and the cells were
centrifuged at 200 g for 10 minutes. After discarding the
supernatant, the pellet was resuspended in 200 ml RPMI containing
15% FBS, 100 .mu.M sodium hypoxanthine, 0.4 .mu.M aminopterin, 16
.mu.M thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer
Mannheim) and 1.5.times.10.sup.6 thymocytes/ml and plated into 10
Corning flat-bottom 96-well tissue culture plates (Corning, Corning
N.Y.).
[0251] On days 2, 4, and 6, after the fusion, 100 .mu.l of medium
was removed from the wells of the fusion plates and replaced with
fresh medium. On day 8, Fusion 191 was screened by ELISA, testing
for the presence of mouse IgG binding to MDC as follows. Immulon 4
plates (Dynatech, Cambridge, Mass.) were coated for 2 hours at
37.degree. C. with 100 ng/well of MDC diluted in 25 mM Tris, pH
7.5. The coating solution was aspirated and 200 .mu.l/well of
blocking solution [0.5% fish skin gelatin (Sigma) diluted in
CMF-PBS] was added and incubated for 30 min. at 37.degree. C. The
blocking solution was aspirated and 50 .mu.l culture supernatant
was added. After incubation at 37.degree. C. for 30 minutes, and
washing three times with PBS containing 0.05% Tween 20 (PBST), 50
.mu.l of horseradish peroxidase conjugated goat anti-mouse IgG(fc)
(Jackson ImmunoResearch, West Grove, Pa.) diluted 1:7000 in PBST
was added. Plates were incubated as above, washed four times with
PB ST, and 100 .mu.L substrate, consisting of 1 mg/ml o-phenylene
diamine (Sigma) and 0.1 .mu.l/ml 30% H.sub.2O.sub.2 in 100 mM
Citrate, pH 4.5, was added. The color reaction was stopped after 5
minutes with the addition of 50 .mu.l of 15% H.sub.2SO.sub.4.
A.sub.490 was read on a plate reader (Dynatech). Fusions 252 and
272 were screened in a similar manner, except ELISA plates were
coated with 50 ng/well of MDC.
[0252] Selected fusion wells were cloned twice by dilution into
96-well plates and visually scored for the number of colonies/well
after 5 days. The monoclonal antibodies produced by hybridomas were
isotyped using the Isostrip system (Boehringer Mannheim,
Indianapolis, Ind.).
[0253] Anti-MDC antibodies were characterized further by western
blotting against recombinant MDC produced as described above in E.
coli or mammalian CHO cells. To prepare the blot, approximately 3
.mu.l of sedimented cells (transformed E. coli producing MDC;
transfected CHO cells producing MDC; untransformed E. coli
(control); and untransfected CHO cells (control)) were dissolved in
standard sample preparation buffer containing SDS (sodium dodecyl
sulfate), and DTT (dithiolthreitol) (Sambrook el al.). After
boiling, the lysates were fractionated via denaturing SDS-PAGE (18%
acrylamide, Tris Glycine gel. NOVEX) and electroblotted to PVDF
membranes (Millipore, Bedford, Mass.). MDC monoclonal antibodies
were diluted to 0.7 .mu.g/ml in PBS for use in the western
blotting, following standard techniques (Sambrook et al.). As an
additional control, the monoclonal antibodies were further tested
for cross-reactivity on western blots of whole tissue lysates of
human skin, tonsil, and thymus.
[0254] One anti-MDC monoclonal antibody, designated monoclonal
antibody 191 D, reacted strongly with recombinant MDC produced by
both bacteria and mammalian cells. Further, this antibody displayed
very little background reactivity in preliminary screening against
bacteria, the CHO mammalian cell line, or the whole human tissues
tested. In addition, this antibody showed the ability to
immunoprecipitate recombinant CHO-derived MDC, following standard
immunoprecipitation protocols (Sambrook et al.).
[0255] Some background reactivity was observed in subsequent
western analyses using the anti-MDC monoclonal antibody 191D.
Further anti-MDC monoclonal antibodies designated 252Y and 252Z
(derived from Fusion 252), used at a concentration of 4 ug/ml,
showed less background and strong reactivity with synthetic MDC at
a concentration of 0.5 ng. No band was seen on the western blot
with human tissue lysates of either colon, skin or tonsil, and
background reactivity was minimal. The hybridomas that produce
monoclonals 252Y and 252Z have been designated "hybridoma 252Y" and
"hybridoma 252Z," respectively.
[0256] Monoclonal antibody 272D, at 1 .mu.g/ml, recognized 200 ng
of wild type MDC by western blot, although less strongly than
antibody 252Y. Antibody 272D showed no background reactivity
against lanes loaded with human thymus whole cell lysate or human
skeletal muscle lysate.
[0257] The hybridoma cell line which produces monoclonal antibody
191 D (designated hybridoma 191D) has been deposited with the
American Type Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville Md. 20852 (USA) pursuant to the provisions of the
Budapest Treaty (ATCC Deposit date: Jun. 4, 1996, ATCC Accession
No. HB-12122). The hybridoma cell lines that produce monoclonal
antibodies 252Y and 252Z (designated "hybridoma 252Y" and
"hybridoma 252Z") were also deposited with the ATCC pursuant to the
provisions of the Budapest Treaty (ATCC Deposit date: Nov. 19,
1997. ATCC Accession Nos. HB-12433 and HB-12434, respectively). The
hybridoma cell line that produces monoclonal antibody 272D was
deposited with the ATCC pursuant to the provisions of the Budapest
Treaty on Mar. 27, 1998 (ATCC Accession No. HB-12498). Availability
of the deposited materials is not to be construed as a license to
practice the invention in contravention of the rights granted under
the authority of any government in accordance with its patent
laws.
[0258] B. Polyclonal Antibodies.
[0259] Polyclonal antibodies against MDC were raised in rabbits
following standard protocols (Sambrook et. al.). Recombinant MDC
produced as a GST fusion protein as described above was diluted in
PBS, emulsified with Freund's Complete Adjuvant, and injected
subcutaneously into rabbits. At intervals of three and six weeks,
additional MDC diluted in PBS was emulsified with Freund's
Incomplete Adjuvant and injected subcutaneously into the same
rabbits. Ten days after the third immunization, serum was withdrawn
from the rabbits and diluted ten-fold in Tris-buffered saline with
0.5% Tween 20 (TBS-T, Sambrook et al.) for characterization via
western blotting against recombinant MDC as described above.
[0260] In a similar set of experiments, polyclonal antisera was
generated in a rabbit against a 12-mer peptide corresponding to the
amino-terminus of mature MDC (SEQ ID NO: 2, positions 1-12). The
resultant antiserum was characterized in Western blot experiments
using synthetic MDC (mature form, residues 1-69); MDC(0-69);
MDC(9-69); MDC-eyfy; and MDC-wvas (see Example 11). The antiserum
recognized all forms but the MDC(9-69) peptide.
[0261] C. MDC Detection Assay
[0262] Monoclonal antibodies 252Y and 252Z were employed in an MDC
detection assay as follows: Aliquots of the antibodies 252Y and
252Z were biotinylated using NHS-LC-Biotin (Pierce) according to
manufacturer's instructions. Immulon 4 ELISA plates were coated
with one monoclonal antibody (252Y or 252Z, unbiotinylated)
overnight at 4.degree. C. The next day, the plates were blocked
with 0.5% fish skin for 30 minutes at 37.degree. C. Known
quantities of MDC were loaded onto the plate for 30 minutes at
37.degree. C. The plates were washed and coated with the other
monoclonal antibody (biotinylated) for 30 minutes at 37.degree. C.
The plates were washed and loaded with streptavidin-HRP for 30
minutes at 37.degree. C. The plates were then developed and read on
a Dynatech MR5000 plate reader. Preliminary results indicate that,
by using the antibody pair 252Y and 252Z. MDC is detectable in the
concentration range of low nonograms to high picograms per
milliliter.
[0263] In a related set of experiments, an ELISA format was
employed to examine the relative affinity of antibodies 191D, 252Y,
and 252Z for antigen. Antibodies were produced as ascites and
purified over a protein A matrix (Prosep-A, Bioprocessing, LTD,
Durham, England) according to manufacturer's instructions. Eluted
antibody was dialyzed against PBS and antibody concentration was
assessed by A.sub.280 measurements. MDC was coated onto Immulon 4
plates in four-fold dilutions ranging from 2000 to 0.4 ng/ml. After
blocking and washing the plates as described above, each antibody
was added at a constant concentration of 250 ng/ml, and A.sub.280
measurements were taken to quantify antibody bound to the plates.
The absorbance values for antibodies 252Y and 252Z were more than
five-fold higher than those of antibody 191 D (1.86 and 1.90 versus
0.34) at 2000 ng/ml MDC; more than seven-fold higher (1.22, 1.29,
and 0.16, respectively) at 500 ng/ml, and more than three-fold
higher (0.47, 0.47, and 0.13) at 125 ng/ml MDC. At 31 ng/ml MDC,
the A.sub.280 measurements were at background levels for all three
antibodies.
[0264] D. Characterization of Epitopes Recognized by Antibodies
252Y and 252Z
[0265] The ability of monoclonal antibodies 252Y and 252Z to
recognize synthetic MDC (mature form, residues 1-69) and MDC
variants (MDC(0-69); MDC(9-69); MDC-eyfy; and MDC-wvas (see Example
11)) was analyzed via Western blot. One hundred to 500 nanograms of
each synthetic peptide was electrophoresed on a denaturing
polyacrylamide gel, transferred, and probed with antibody 252Y or
antibody 252Z at a concentration of 1 .mu.g/ml. Immunoreactivity
was visualized by incubating the probed blot with horseradish
peroxidase-conjugated goat anti-mouse immunoglobulin G
(Transduction Laboratories #M 15345) at a concentration of 0.2
.mu.g/ml or 1:5000 dilution in TRIS buffered saline with 0.1% Tween
20 (TBS Tw20) and 1% bovine serum albumin for 30 minutes at room
temperature. The blot was washed three times in the TRIS buffered
saline/0.1% Tween 20 solution and detection of antibody binding was
measured by autoradiography (Kodak Hyperfilm) using
electro-chemiluminescence (NEN Renaissance ECL # NEL 102). Both
monoclonal antibodies were observed to recognize wildtype MDC and
the analogs MDC(0-69), MDC(9-69), and MDC-eyfy. However, antibody
252Y and antibody 252Z both failed to recognize MDC-wvas,
suggesting that the epitope(s) recognized by these antibodies
include(s) the wv motif near the carboxyl-terminus of MDC. This
motif tends to be highly conserved in all CC chemokines (see FIG.
1).
[0266] To further characterize the epitope(s) recognized by
antibodies 252Y and 252Z, an Immulon 4 plate was coated with MDC at
1.0 .mu.g/ml. After blocking the plate with fish skin as described
above in part C, unlabeled antibody 252Y, 252Z, or an
isotype-matched control was added at 5 .mu.g/ml and incubated for
30 minutes at 37.degree. C. Without washing, either biotinlyated
antibody 252Y or 252Z was added at a concentration of 0.25
.mu.g/ml, and the plate was incubated an additional 30 minutes at
37.degree. C. Thereafter, the plate was washed and developed with
streptavidin-HRP. The results showed that either 252Y or 252Z was
capable of reducing the signal of either biotinlyated antibody
ten-fold, as compared with the signal of either biotynilated
antibody blocked with the control antibody. These results further
indicate that antibodies 252Y and 252Z recognize similar or
overlapping epitopes.
[0267] In contrast, unpurified supernatant from hydriboma 272D was
tested in a similar experiment for its ability to compete with
biotynilated 252Y or biotynilated 252Z, but was unable to reduce
the signal of either antibody. Thus, monoclonal antibody 272D
recognizes an epitope different from that recognized by monoclonals
252Y and 252Z.
[0268] E. Antibodies 252Y and 252Z are useful for
Immunoprecipitating MDC
[0269] The following experiments were conducted which demonstrate a
utility for antibodies 252Y and 252Z for immunoprecipitation of
MDC. Antibodies 252Y, 252Z, and an irrelevant isotype-matched
control were added separately at a concentration of 10 .mu.g/ml to
an extraction buffer (1% triton X-100, 10 mM Tris base, 5 mM EDTA,
10 mM NaCl, 30 mM Na pyrophospate, 50 mM NaF, 100 .mu.M Na
Orthovanadate, pH 7.6) containing 100 ng/ml MDC. These samples were
incubated on ice for 1 hour. To precipitate the immune complexes,
15 .mu.l of protein G sepharose (Pharmacia Biotech # 17-0618-01)
were added to each sample and incubated on a rotation apparatus at
4.degree. C. for 30 minutes. The samples were then centrifuged to
collect the protein G sepharose/immune complexes, washed three
times (1 ml each) in extraction buffer, boiled/solubilized in
2.times.SDS-PAGE buffer, electrophoresed on an 18% SDS-PAGE gel,
and western blotted to PVDF membrane (Novex # LC2002). Nonspecific
binding sites on the PVDF membrane were blocked with TBS Tw20/1%
BSA for 30 minutes at room temperature. The blot was then probed
with 1 .mu.g/ml of antibody 252Y in TBS Tw20/1% BSA for 1 hour,
washed three times with TBS Tw20, probed with horseradish
peroxidase-conjugated goat anti-mouse IgG in TBS Tw20/1% BSA for 30
minutes at room temperature, washed three times with TBS Tw20, and
detected by autoradiography using ECL. Bands at approximately 8 kD
were detected in the 252Y and 252Z lanes but not in the negative
isotype-matched control lane. Additionally, MDC was
immunoprecipitated from cell culture supernatants containing RPMI
(Rosell Park Memorial Institute--Gibco) medium with 10% fetal
bovine serum spiked with 25 ng/ml MDC using the same conditions
stated above.
[0270] F. Humanization of Anti-MDC Monoclonal Antibodies
[0271] The activities of MDC as reported herein suggest numerous
therapeutic indications for MDC inhibitors (antagonists).
MDC-neutralizing antibodies (see Example 30) comprise one class of
therapeutics useful as MDC antagonists. Following are protocols to
improve the utility of anti-MDC monoclonal antibodies as
therapeutics in humans, by "humanizing" the monoclonal antibodies
to improve their serum half-life and render them less immunogenic
in human hosts (i.e., to prevent human antibody response to
non-human anti-MDC antibodies).
[0272] The principles of humanization have been described in the
literature and are facilitated by the modular arrangement of
antibody proteins. To minimize the possibility of binding
complement, a humanized antibody of the IgG4 isotype is
preferred.
[0273] For example, a level of humanization is achieved by
generating chimeric antibodies comprising the variable domains of
non-human antibody proteins of interest with the constant domains
of human antibody molecules. (See, e.g., Morrison and Oi, Adv.
Immunol., 44:65-92 (1989). The variable domains of MDC neutralizing
anti-MDC antibodies are cloned from the genomic DNA of a B-cell
hybridoma or from cDNA generated from mRNA isolated from the
hybridoma of interest. The V region gene fragments are linked to
exons encoding human antibody constant domains, and the resultant
construct is expressed in suitable mammalian host cells (e.g.,
myeloma or CHO cells).
[0274] To achieve an even greater level of humanization, only those
portions of the variable region gene fragments that encode
antigen-binding complementarity determining regions ("CDR") of the
non-human monoclonal antibody genes are cloned into human antibody
sequences. [See, e.g., Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-36 (1988); and Tempest et al., Bio/Technology,
9:266-71 (1991). If necessary, the .beta.-sheet framework of the
human antibody surrounding the CDR3 regions also is modified to
more closely mirror the three dimensional structure of the
antigen-binding domain of the original monoclonal antibody. (See
Kettleborough et al., Protein Engin., 4:773-783 (1991) and Foote et
al., J. Mol. Biol., 224:487-499 (1992).)
[0275] In an alternative approach, the surface of a non-human
monoclonal antibody of interest is humanized by altering selected
surface residues of the non-human antibody, e.g., by site-directed
mutagenesis, while retaining all of the interior and contacting
residues of the non-human antibody. See Padlan, Molecular Immunol,
28(4/5):489-98 (1991).
[0276] The foregoing approaches are employed using MDC-neutralizing
anti-MDC monoclonal antibodies and the hybridomas that produce
them, such as antibodies 252Y and 252Z, to generate humanized
MDC-neutralizing antibodies useful as therapeutics to treat or
palliate conditions wherein MDC-expression is detrimental.
[0277] G. Human MDC-Neutralizing Antibodies from Phage Display
[0278] Human MDC-neutralizing antibodies are generated by phage
display techniques such as those described in Aujame et al., Human
Antibodies, 8(4):155-168 (1997); Hoogenboom, TIBTECH. 15:62-70
(1997); and Rader et al., Curr. Opin. Biotechnol., 8:503-508
(1997), all of which are incorporated by reference. For example,
antibody variable regions in the form of Fab fragments or linked
single chain Fv fragments are fused to the amino terminus of
filamentous phage minor coat protein pill. Expression of the fusion
protein and incorporation thereof into the mature phage coat
results in phage particles that present an antibody on their
surface and contain the genetic material encoding the antibody. A
phage library comprising such constructs is expressed in bacteria,
and the library is panned (screened) for MDC-specific
phage-antibodies using labelled or immobilized MDC as
antigen-probe.
[0279] H. Human MDC-Neutralizing Antibodies from Transgenic
Mice
[0280] Human MDC-neutralizing antibodies are generated in
transgenic mice essentially as described in Bruggemann and
Neuberger, sImmunol. Today, 17(8):391-97 (1996) and Bruggemann and
Taussig, Curr. Opin. Biotechnol., 8:455-58 (1997). Transgenic mice
carrying human V-gene segments in germline configuration and that
express these transgenes in their lymphoid tissue are immunized
with an MDC composition using conventional immunization protocols.
Hybridomas are generated using B cells from the immunized mice
using conventional protocols and screened to identify hybridomas
secreting anti-MDC human antibodies (e.g., as described above).
[0281] 1. ELISA for Detecting and Monitoring Serum Concentrations
of MDC
[0282] The measurement of endogenous levels of MDC is useful to
monitor the immune state of a patient, especially a patient who is
immunocompromized, in a hyperimmune state, or undergoing treatment
with MDC neutralizing antibodies or other MDC antagonists.
[0283] A sensitive ELISA to measure MDC in biological fluids, for
example serum, can be established using, monoclonal antibodies,
polyclonal antibodies, immuno-conjugates containing MDC ligands
(for example heparin conjugates), or combinations thereof. For
example, monoclonal antibodies 272D, 252Y and 252Z were employed in
an MDC detection assay as described below.
[0284] Aliquots of the antibodies 252Y and 252Z were biotinylated
using NHS-LC-Biotin (Pierce) according to manufacturer's
instructions. Immulon 4 ELISA plates were coated with antibody 272D
overnight at 4.degree. C. The next day, the plates were blocked
with 0.5% fish skin for 30 minutes at 37.degree. C. Known
quantities of MDC(1-69) were loaded onto the plate for 30 minutes
at 37.degree. C. The plates were washed and coated with either 252Y
or 252Z (biotinylated) for 30 minutes at 37.degree. C. The plates
were washed and loaded with streptavidin-HRP for 30 minutes at
37.degree. C. The plates were then developed and read on a Dynatech
MR5000 plate reader. Preliminary results indicate that MDC is
detectable in the concentration range of low nanograms per
milliliter in this ELISA format. It is expected that use of
polyclonal antibodies for the capture antibody will lead to a still
more sensitive ELISA assay.
EXAMPLE 19
Calcium Flux Assay
[0285] Changes in intracellular calcium concentrations, indicative
of cellular activation by chemokines, were monitored in several
cell lines by an art-recognized calcium flux assay. Cells were
incubated in 1 ml complete media containing 1 .mu.M Fura-2/AM
(Molecular Probes, Eugene, Oreg.) for 30 minutes at room
temperature, washed once, and resuspended in D-PBS at
.about.10.sup.6 cells/ml.
[0286] Two ml of suspended cells were placed in a continuously
stirred cuvette at 37.degree. C. in a fluorimeter (AMINCO-Bowman
Series 2, Rochester, N.Y.). The concentration of intracellular
calcium was indicated by fluorescence, which was monitored at 510
nm emission wavelength while switching between excitation
wavelengths of 340 nm and 380 nm every 0.5 seconds. The ratio of
the emissions from the 340 nm relative to the 380 nm excitation
wavelengths corresponds to the level of intracellular calcium.
[0287] Cell lines measured by this assay included the following:
the human embryonic kidney cell line HEK-293 stably transfected
with the putative chemokine receptor gene V28 [Raport et al., Gene,
163:295-299 (1995)]; HEK-293 cells stably transfected with the
chemokine receptor gene CCR5 [Samson et al., Biochemistry,
35:3362-3367 (1996); see also co-owned, co-pending U.S. patent
application Ser. No. 08/575,967, filed Dec. 20, 1995, incorporated
herein by reference, disclosing chemokine receptor materials and
methods, including CCR5 (identified therein as "88C")], the human
monocytic cell line THP-1, the human lung epithelial cell line
A-549; and the human fibroblast cell line IMR-90. None of these
cell lines fluxed calcium in response to the recombinant MDC
protein. As positive controls, the HEK-293 transfectants responded
strongly to thrombin, indicating that the assay was valid. In
addition, the THP-1 cells responded strongly to the commercially
available chemokines MCP-1 and MCP-3 (Peprotech, Rocky Hill, N.J.)
at a final concentration of 25 ng/ml. No additional stimuli were
tested on the A-549 or IMR-90 cell lines.
EXAMPLE 20
Inhibition of HIV Proliferation
[0288] Several CC chemokines have been implicated in suppressing
the proliferation of Human Immunodeficiency Virus (HIV), the
causative agent of human Acquired Immune Deficiency Syndrome
(AIDS). See Cocchi et al, Science, 270:1811 (1995), Winkler et al.,
Science, 279:389-393 (1998). The HIV antiproliferative activity of
MDC is measured by means such as those described by Cocchi et al.,
in which a CD4.sup.+ T cell line is acutely infected with an HIV
strain and cultured in the presence of various concentrations of
MDC. After three days, a fresh dilution of MDC in the culture
medium is added to the cells. At 5 to 7 days following infection,
the level of HIV is measured by testing the culture supernatants
for the presence of HIV p24 antigen by a commercial ELISA test
(Coulter, Miami, Fla.).
[0289] One technical report teaches that MDC possesses an HIV
antiproliferative activity. See Pal et al., Science, 278: 695-698
(1997). The agent used in the study consisted of purified
polypeptides that had been secreted from an immortalized cell line
derived from CD8 T cells from an HIV-1-infected individual. Pal et
al. reported that the purified "native MDC" from this cell line
possessed an NH.sub.2-terminus corresponding to the tyrosine at
position 3 of SEQ ID NO: 1. A "minor" sequence beginning with the
proline at position 2 of SEQ ID NO: 1 also was detected. The
authors did not detect a peptide beginning with the glycine at
position 1 of SEQ ID NO: 1 in their "native MDC" composition.
According to Pal et al., a reversed-phase HPLC fraction containing
the "native MDC" suppressed the acute infection of CD8.sup.+
cell-depleted PBMCs by HIV-1.sub.IIIB and various NSI HIV isolates
in a concentration-dependent fashion. Similar HIV suppressor
activity was not observed in supernatants from other cell lines
that appeared (from Northern blot studies) to demonstrate
equivalent MDC gene expression.
A. Use of MDC Antagonists to Inhibit HIV Proliferation
[0290] An acute HIV-1.sub.BAl infectivity assay reported in Pal et
al. was repeated (100 TCID.sub.50 units/well) using the macrophage
cell line PM-1 (1.times.10.sup.5 cells/well) and using purified
mature MDC recombinantly expressed in CHO cells and having an amino
acid sequence beginning at position 1 of SEQ ID NO: 1 (see Example
10). Interestingly, mature MDC was found to have no HIV suppressive
activity. The same assay was performed with MDC(0-69) (See Example
11), an analog that exhibits properties of a partial MDC antagonist
(see Example 19) in that it binds CCR4 with wild-type affinity, but
exhibits substantially reduced capacity to induce a calcium flux or
induce chemotaxis. At a concentration of 1 .mu.g/ml, MDC(0-69)
conferred a 58% and 67% reduction in the production of infectious
particles (TCID.sub.50 units measured on days 5 and 7). The
positive control RANTES produced greater than 95% inhibition at 5
ng/ml. Without intending to be limited to a particular theory, one
explanation for these results is that mature MDC (1-69) induces HIV
proliferation, and that the anti-proliferative effects of MDC(0-69)
results from this species competitively inhibiting the capacity of
endogenous mature MDC (1-69) to stimulate HIV-1 production.
[0291] The effects of mature MDC and of MDC-neutralizing antibodies
were analyzed in Pal et al.'s acute HIV-1.sub.Bal. (0.01 MOI/well)
infectivity assay using peripheral blood mononuclear cells (PBMC,
1.times.10.sup.6 cells/well) depleted of CD8.sup.+ cells. The
mature MDC (1-69) failed to inhibit p24 production, as compared to
a control murine IgG1 antibody. However, the murine monoclonal
anti-MDC neutralizing antibodies 252Y (IgG1) and 252Z each
inhibited p24 production when tested separately at a concentration
of 2 ng/ml (37% and 28% inhibition, respectively). Again, one
explanation for these data is that PBMC contain and produce
endogenous MDC (1-69) that acts to stimulate HIV-1 functions, and
that MDC antagonists inhibit this effect.
[0292] To confirm the apparent role of MDC as an HIV-1 agonist, an
infectivity assay (such as that described in Pal et al.) is
repeated using MDC neutralizing antibody and titrating exogenous
mature MDC(1-69) into the assay wells. If native MDC(1-69) exerts
an agonistic effect on HIV-1 infectivity and/or proliferation, then
it is expected that the antiviral effect of the neutralizing
antibody will be reduced with increasing amounts of mature MDC, and
will be overwhelmed with the addition of a molar excess of MDC.
[0293] Collectively, these results provide a therapeutic indication
for MDC antagonists for inhibiting proliferation of infectious
retroviruses, especially HIV retroviruses. Such therapeutic methods
and uses are intended as an aspect of the invention. For use in
this context, the term "MDC antagonist" includes any compound
capable of inhibiting HIV-1 proliferation in a manner analogous to
MDC neutralizing antibodies, or MDC(0-69), or MDC(3-69). For
example, anti-MDC antibodies (especially neutralizing antibodies,
and preferably humanized antibodies) are highly preferred MDC
antagonists. Similarly, polypeptides that are capable of binding to
MDC that comprise an antigen-binding fragment of an anti-MDC
antibody are contemplated. Effective MDC analogs also are
contemplated as MDC antagonists. For example, N-terminal deletion
analogs of MDC are contemplated, especially deletion analogs having
an amino acid sequence consisting of a portion of the amino acid
sequence set forth in SEQ ID NO: 2 that is sufficient to bind to
the chemokine receptor CCR4, the portion having an amino-terminus
between residues Land 12 of SEQ ID NO: 2. Likewise, analogs
comprising chemical addition to the amino terminus to render said
polypeptide antagonistic to MDC are contemplated. The chemical
addition may be added to the amino terminus of MDC(1-69) to form
the analog, or to the amino terminus of an MDC analog that has had
amino acids deleted from its amino terminus (e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, or 11 residues deleted).
[0294] Additional classes of MDC antagonists useful in anti-HIV
therapeutic methods include antagonists derived from CCR4 or from
other MDC receptors. For example, a solubilized, MDC-binding
version of CCR4 or CCR4 fragment is contemplated. Similarly,
humanized antibodies that block but do not signal through CCR4 are
contemplated as useful as anti-HIV therapeutics. Such antibodies
are made using techniques described herein for making anti-MDC
antibodies and/or techniques that have been described in the art
for generating antibodies to other seven transmembrane receptor
proteins (e.g., using as an antigen CCR4-transfected cells that
express CCR4 on their surface). See Wu et al., J. Exp. Med., 185:
1681-1691 (1997).
[0295] Yet another class of MDC antagonists useful in anti-HIV
therapeutic methods of the invention include agents that have the
effect of transforming mature MDC(1-69) to antagonist forms in
vivo, e.g., by modifying the amino terminus of MDC. For example,
administration of a therapeutically effective amount of the
dipeptidyl aminopeptidase CD26 is contemplated.
[0296] Therapeutically effective amounts of MDC antagonists (i.e.,
for inhibiting HIV infectivity and/or proliferation) are readily
determined using standard dose-response studies. Moreover,
determination of proper dose and dosing is facilitated by anti-MDC
antibodies of the invention (Example 18), which can be used in an
ELISA or other standard assays to monitor serum MDC levels in
subjects receiving treatment. A therapeutic MDC neutralizing
antibody should be administered in sufficient quantity and with
sufficient frequency so as to maintain serum concentrations of MDC
below detectable levels. Doses of an MDC neutralizing antibody on
the order of 0.1 to 100 mg antibody per kilogram body weight, and
more preferably 1 to 10 mg/kg, are specifically contemplated. For
humanized antibodies, which typically exhibit a long circulating
half-life, dosing at intervals ranging from daily to every other
month, and more preferably every week, or every other week, or
every third week, are specifically contemplated. Use of an IgG4
type humanized MDC-neutralizing antibody is highly preferred, to
minimize or eliminate the possibility of inducing a complement
reaction.
[0297] Moreover, determination of therapeutically effective MDC
antagonists, doses, and dosing schedules is facilitated by
dose-response studies in art-recognized in vivo models for HIV
infection and proliferation, such as studies in appropriate mice
[Pettoello-Mantovani et al., J. Infect. 177:337 (1998); J. M.
McCune et al., "The Hematophtology of HIV-1 Disease: Experimental
Analysis in vivo," in Human Hematopoiesis in SCID Mice. M.
Roncarolo et al. (eds.), Landes Publishing Co., New York, N.Y., pp.
129-156 (1995); and McCune et al., "The SCID-hu mouse: a small
animal model for HIV infection and antiviral testing," in Progress
in Immunol., Vol. VII, Melchers et al. (eds.), Springer-Verlag
Berlin-Heidelberg, pp. 1046-1049 (1989)] or primate models.
[0298] B. Use of TARC Antagonists to Inhibit HIV Proliferation
[0299] The foregoing experiments also suggest further analysis
wherein an HIV-1 infectivity assay is repeated using neutralizing
antibodies directed against other beta chemokines. For those
.beta.-chemokines lacking an activity towards T.sub.H2 cells
(analogous to MDC's activity toward such cells), it is expected
that chemokine-specific neutralizing antibodies will behave much
like the murine control IgG1 antibody above. However, for those
.beta. chemokines that possess an activity toward T.sub.H2 cells
that is comparable to that of MDC (i.e., TARC), it is expected that
chemokine-specific neutralizing antibodies will behave much like
MDC-neutralizing antibodies and inhibit HIV-1 infectivity and/or
proliferation. The use of TARC-neutralizing antibodies and/or other
TARC inhibitors to suppress the infectivity and/or proliferation of
immunodeficiency viruses is specifically contemplated as an aspect
of the invention.
[0300] The nucleotide and deduced amino acid sequences of TARC have
been reported in the literature and are set forth herein in SEQ ID
NOs: 42 and 43. See Imai et al., J. Biol. Chem. 271: 21514-21521
(1996); GENBANK ACCESSION NO. D43767. TARC polypeptides and
anti-TARC antibodies are synthesized using procedures essentially
as described herein for making MDC and anti-MDC antibodies, or
using procedures described in the literature for TARC. [See Imai et
al., J. Biol. Chem., 272: 15036-15042 (1997); and Imai et al., J.
Biol. Chem., 271: 21514-21521 (1996).] The
HIV-proliferative/anti-proliferative effects of TARC polypeptides
(e.g., native mature TARC and TARC analogs, especially
amino-terminal deletion and addition analogs) and TARC-neutralizing
antibodies are assayed essentially as described in Pal et al. or
Cocchi et al.
[0301] Based on the theory that the HIV antiproliferative efficacy
of MDC antagonists is mediated by blocking the signaling of MDC
through CCR4 in target cells that express CCR4, it is further
contemplated that antibodies to any other chemokine that known or
is discovered to signal through CCR4 will be useful as anti-HIV
therapeutics of the invention.
EXAMPLE 21
Effects of MDC on Fibroblast Proliferation
[0302] In addition to their ability to attract and activate
leukocytes, some chemokines, such as IL-8, have been shown to be
capable of affecting the proliferation of non-leukocytic cells [see
Tuschil, J. Invest. Dermatol., 99:294-298 (1992)]. Fibroblasts
throughout the body are important to the structural integrity of
most tissues. The proliferation of fibroblasts is essential to
wound healing and response to injury but can be deleterious as
well, as in the case of chronic inflammatory diseases, such as
pulmonary fibrosis [Phan, in: Immunology of Inflammation, Elsevier
(1983), pp. 121-162].
[0303] In Vitro cell proliferation assays were utilized to assess
the effects of MDC on the proliferation of fibroblasts. Human
fibroblasts (CRL-1635) were obtained from ATCC and maintained in
culture in DMEM with 10% FBS and 1% antibiotics. Proliferation
assays were performed and quantitated as previously described in
the art by Denholm and Phan, Amer. J Pathol., 134:355-363 (1989).
Briefly, on day 1, 2.5.times.10.sup.3 cells/well were plated into
96 well plates in DMEM with 10% FBS. Day 2: twenty-four hours after
plating, medium on cells was changed to serum-free DMEM. Day 3:
medium was removed from cells and replaced with MDC diluted in DMEM
containing 0.4% FBS. Day 5: one microCurie of .sup.3H-thymidine was
added per well and incubation continued for an additional 5 hours.
Cells were harvested onto glass fiber filters. Cell proliferation
was expressed as cpm of .sup.3H-thymidine incorporated into
fibroblasts. Controls for this assay included the basal medium for
this assay, DMEM with 0.4% FBS as the negative control, and DMEM
with 10% FBS as the positive control.
[0304] As shown in FIG. 7, MDC treatment decreased the
proliferation of fibroblasts in a dose dependent manner. Similar
inhibition of fibroblast proliferation was observed with both MDC
purified from CHO cells (closed circles) and chemically synthesized
MDC (open circles). The fibroblast-antiproliferative effect of MDC
indicates a therapeutic utility for MDC in the treatment of
diseases such as pulmonary fibrosis and tumors, in which enhanced
or uncontrolled cell proliferation is a major feature.
EXAMPLE 22
Cell Proliferation Assays
[0305] The effects of MDC upon the proliferation of epithelial
cells, T cells, fibroblasts, endothelial cells, macrophages, and
tumor cells are assayed by methods known in the art, such as those
described in Denholm et al., Amer. J. Pathol, 134:355-363 (1989),
and "In Vitro Assays of Lymphocyte Functions," in: Current
Protocols Immunology, Sections 3-4, Wiley and Sons (1992), for the
assay of growth factor activities. In these methods, enhancement or
inhibition of cell growth and the release of growth factors are
measured.
[0306] MDC effects on the proliferation of epithelial cells and
endothelial cells are assayed using the same procedures as those
described above for fibroblasts (Example 21).
[0307] The effects on the proliferation of T cells are determined
using peripheral blood lymphocytes. Mononuclear cells are isolated
from peripheral blood as described in Denholm et al., Amer., J.
Pathol., 135:571-580 (1989); cells are resuspended in RPMI with 10%
FBS and incubated overnight in plastic tissue culture flasks.
Lymphocytes remain in suspension in these cultures and are obtained
by centrifugation of culture medium. One hundred thousand
lymphocytes are plated into each well of a 96 well plate and
incubated for three days in medium (RPMI plus 10% FBS) containing 1
.mu.g/ml PHA with or without 50, 125, 250 or 500 ng/ml of MDC. One
microCurie of .sup.3H-thymidine is added during the last 18 hours
of incubation. Cells are harvested and proliferations expressed as
described for fibroblasts in Example 21.
[0308] The effects of MDC on macrophage proliferation are
determined using elicited guinea pig peritoneal macrophages,
obtained as described above in Example 13. Macrophages are plated
into 96 well plates at a density of one hundred thousand cells per
well in RPMI with 10% FBS, and incubated 2 hours to allow cells to
adhere. Medium is then removed and replaced with fresh medium with
or without 50, 125, 250 or 500 ng/ml of MDC. Cells with MDC are
incubated three days, and proliferation is determined as described
above for lymphocytes.
[0309] Chemokine-mediated control of the proliferation of these
cell types has therapeutic implications in enhancing tissue repair
following injury, and in limiting the proliferation of these cells
in chronic inflammatory reactions such as psoriasis, fibrosis, and
atherosclerosis, and in neoplastic conditions.
EXAMPLE 23
In Vivo Fibroblast Proliferation Assay
[0310] The anti-proliferative effects of MDC upon fibroblasts are
determined in vivo by the methods known in the art, such as those
reported by Phan and Fantone, Amer. J. Pathol., 50:587-591 (1984),
which utilize a rat model of pulmonary fibrosis in which the
disease is induced by bleomycin. This model is well-characterized
and allows for the assessment of fibroblast proliferation and
collagen synthesis during all stages of this disease.
[0311] Briefly, rats are divided into four treatment groups: 1)
controls, given intratracheal injections of normal saline; 2)
saline-injected rats which also receive a daily intraperitoneal
injection of 500 ng of MDC in saline; 3) bleomycin-treated, given
an intratracheal injection of 1.5 mg/kg bleomycin (Calbiochem, Palo
Alto, Calif.); and 4) bleomycin-treated rats which also are given a
daily intraperitoneal injection of 500 ng of MDC.
[0312] Three rats per group are sacrificed at 4, 7, 14, 21, and 28
days after the initial intratracheal injections. Lungs are removed
and samples of each lobe taken for histological examination and
assays of collagen content.
EXAMPLE 24
MDC Chromosomal Localization
[0313] A 20 kb genomic fragment containing the human MDC gene was
labelled with digoxigenin by nick translation and used as a probe
for fluorescence in situ hybridization of human chromosomes (Genome
Systems, Inc., St. Louis, Mo.). The labelled probe was hybridized
to normal metaphase chromosomes derived from PHA-stimulated
peripheral blood lymphocytes. Reactions were carried out in the
presence of sheared human DNA in 50% formamide, 10% dextran
sulfate, 30 mM sodium chloride, 3 mM sodium citrate, and 0.1%
sodium dodecyl sulphate. Hybridization signals were detected by
treating slides with fluoresceinated anti-digoxigenin antibodies,
followed by counter-staining with 4,6-diamidino-2-phenylindol- e.
An initial hybridization experiment localized the gene to the q
terminus of a group E chromosome.
[0314] A genomic probe that specifically hybridizes to the short
arm of chromosome 16 was used to demonstrate co hybridization of
chromosome 16 with the MDC probe. A total of 80 metaphase cells
were analyzed with 61 exhibiting specific labeling. The MDC probe
hybridized to a region immediately adjacent to the
heterochromatic/euchromatic boundary, corresponding to band 16q 13.
The gene encoding TARC also is localized in this region. See
Nomiyama et al., Genomics, 40: 211-213 (1997).
[0315] These chromosomal mapping data indicate a utility of
MDC-encoding polynucleotides as a chromosomal marker. Contiguous
fragments of SEQ ID NO: 1 of at least 15 nucleotides, and more
preferably at least 20, 25, 50, 75, 100, 150, 200, 500, or more
nucleotides, and the complements of such fragments, are
specifically contemplated as probes of the invention. Moreover,
probes having partial degeneracy from SEQ ID NO: 1 are contemplated
as being useful as well. Probes having preferably at least 90%, and
more preferably 95%, 96%, 97%, 98%, 99%, or more similarity to SEQ
ID NO: 1 are preferred as probes of the invention.
EXAMPLE 25
MDC is a High-Affinity Ligand for CCR4
[0316] The chemokine receptor designated CCR4 has been
characterized previously [Power et al., J. Biol. Chem., 270:
19495-19500 (1995)], and shown to bind the CC chemokine TARC
(Thymus and Activation-Regulated Chemokine, Genbank Accession No.
D43767). See Imai et al., J. Biol. Chem., 272: 15036-15042 (1997);
and Imai et al., J. Biol. Chem., 271: 21514-21521 (1996). The cDNA
and deduced amino acid sequences of human CCR4 are set forth in SEQ
ID NOs: 33 and 34, and are deposited with Genbank (Accession No.
X85740). The following experiments were performed that demonstrate
that MDC is a high affinity ligand for CCR4.
[0317] A. Preparation of CCR4-Transfected Cells
[0318] The murine pre-B cell line L 1.2 [See, e.g., Gallatin et
al., Nature, 304:30-34 (1983)] maintained in RPMI 1640 media
supplemented with 10% fetal calf serum, was selected for
transformation with the CCR4 expression vector described in Imai et
al., J. Biol. Chem., 272: 15036-15042 (1997), incorporated herein
by reference. L1.2 cells were stably transfected as described
previously by electroporation with 10 .mu.g linearized plasmid at
260 V, 960 microfarads using a Gene Pulser (BioRad). See Imai et
al., J. Biol. Chem., 272: 15036-15042 (1997). It will be apparent
that other cell lines in the art are suitable for CCR4 transfection
for the following assays. For example, 293 cell lines have been
transfected with CCR4 cDNA and employed effectively in calcium Flux
assays.
[0319] B. Preparation of Recombinant Chemokines
[0320] The mature sequences of both MDC and TARC were chemically
synthesized by Gryphon Sciences (South San Francisco Calif.) using
t-butyl-oxycarbonyl chemistries on a peptide synthesizer (430A,
Applied Biosystems). Lyophilized protein was dissolved at 10 mg/ml
in 4 mM HCl and immediately diluted to 0.1 mg/ml in
phosphate-buffered saline plus 0.1% bovine-serum albumin (BSA) for
storage at -80.degree. C.
[0321] Recombinant MDC also was expressed as a fusion protein with
the secreted form of placental alkaline phosphatase (SEAP) in the
expression vector pcDNA3 (Clontech, Palo Alto Calif.). A similar
TARC-SEAP fusion protein is described in Imai et al. (1997).
Briefly, the coding region of MDC, followed by a sequence encoding
a five amino acid linker (Ser-Arg-Ser-Ser-Gly) was fused in-frame
to a sequence encoding mature SEAP, followed by a sequence encoding
a (His).sub.6 tag. The MDC-SEAP expression plasmid was transfected
into COS cells by the DEAE Dextran method. See Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1989). The transfected cells
were cultured in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum. Twenty-four hours after transfection,
the serum levels were reduced from 10% to 10%. After 3-4 days, the
culture supernatants were collected, centrifuged, filtered through
a 0.45 micron membrane, and stored at 4.degree. C. The
concentration of MDC-SEAP in the filtered supernatant was
determined by comparison with the reported specific activity of
secreted placental alkaline phosphatase [Berger et al., Gene, 66:
1-10 (1988)], and confirmed using known concentrations of TARC-SEAP
[Imai et al., (1997)] as an internal reference standard.
[0322] C. CCR4 Binding Assays
[0323] The MDC-SEAP was used as a probe to examine MDC binding to
CCR4-transfected L1.2 cells. For displacement and saturation
experiments, transfected L1.2 cells (approx. 3.times.10.sup.5) were
incubated for one hour at 16.degree. C. in the presence of 0.5 nM
MDC-SEAP in the presence or absence of various concentrations of
unlabeled chemokines in 200 .mu.l binding buffer (RPMI 1640 media
containing 25 mM HEPES, pH 7.4, 1% BSA, and 0.02% sodium azide).
Following incubation, the cells were washed four times in binding
buffer and lysed in 50 .mu.l of 10 mM Tris-HCl, pH 8.0, and 1%
Triton X-100. Samples were heated at 65.degree. C. for 15 minutes
to inactivate cellular phosphatases, centrifuged, and stored at
-20.degree. C. until assayed.
[0324] Alkaline phosphatase activity in 10 .mu.l of sample was
determined by a chemiluminescence assay using the Great Escape
Detection kit (Clontech, Palo Alto, Calif.) according to the
manufacturer's instructions. The saturation binding curve was
fitted (Table Curve.TM.) using the Hill equation
y=a(x.sub.c)/(x.sub.c+b.sub.c), where y is the amount of ligand
bound, a is the maximum amount of ligand bound, x is the
concentration of ligand, b is the concentration of ligand at which
50% of receptor sites are occupied (K.sub.D), and c is the Hill
coefficient. Binding competition curves were fitted
(TableCurve.TM.) using a three-parameter logistic model described
by the equation y=a/[1+(x/b).sup.c], where y is the amount of
labelled ligand bound, a is the maximum amount of labelled ligand
bound, x is the concentration of the competitive chemokine, b is
the IC.sub.50, and c is a parameter that determines the slope of
the curve at the IC.sub.50.
[0325] These binding assays demonstrated that MDC-SEAP bound to
CCR4-expressing cells. This binding was to a single high affinity
site with a K.sub.d of 0.18 nM, as demonstrated by Scatchard
analysis. Binding of MDC-SEAP was competitively inhibited with
increasing concentrations of unlabeled MDC or TARC. The IC.sub.50
for MDC was 0.65 nM, while the IC.sub.50 for TARC was 2.1 nM. These
data suggest that both MDC and TARC recognize a common binding,
site on CCR4, and that MDC has more than three-fold higher affinity
than TARC for CCR4.
[0326] To examine the specificity of MDC binding to CCR4, six
additional chemokines (MCP-1, MCP-3, MCP-4, RANTES, MIP-1.alpha.,
and MIP-1.beta.) were tested for competition of MDC-SEAP binding. A
200-fold molar excess of each chemokine was tested for competition
with a constant quantity of MDC-SEAP (0.5 nM). The additional
chemokines did not compete for binding of MDC-SEAP to CCR4. In
contrast, unlabeled MDC and TARC both blocked binding of MDC-SEAP
to CCR4 transfectants.
[0327] D. Calcium Mobilization Assay
[0328] Imai et al. (1997) showed that TARC signals through CCR4 by
inducing calcium mobilization. To determine the ability of MDC to
cause signaling through chemokine receptors, we examined calcium
mobilization in L1.2 cells recombinantly expressing CCR1, CCR2B,
CCR3, CCR4, CCR5, CCR6, or CCR7.
[0329] Transfected L1.2 cells were suspended at a concentration of
3.times.10.sup.6 cells/ml in Hank's balanced salt solution
supplemented with 1 mg/ml BSA and 10 mM HEPES, pH 7.4. Cells were
incubated with 1 .mu.M fura-PE3-AM (Texas Fluorescence Labs) at
room temperature for 1 hour in the dark. After washing twice, cells
were resuspended at a concentration of 2.5.times.10.sup.6 cells/ml.
To measure intracellular calcium, 2 ml of cells were placed in a
quartz cuvette in a Perkin-Elmer LS 50B spectrofluorimeter.
Fluorescence was monitored at 340 nm (excitation wavelength 1), 380
nm (excitation wavelength 2), and 510 nm (emission wavelength)
every 200 ms.
[0330] In these experiments, MDC did not cause calcium flux in L1.2
cells transfected with CCR1, CCR2B. CCR3, CCR5, CCR6, or CCR7,
whereas each of these transfected cell lines responded to its known
cognate ligand. In contrast, L1.2 cells transfected with CCR4
produced a strong calcium flux when stimulated with 10 nM MDC.
Similar to other G protein-coupled receptors, CCR4 was refractory
to subsequent stimulation with the same concentration of MDC. Ten
nanomolar MDC also completely desensitized CCR4 transfectants to
subsequent 10 nM TARC treatment. However, pre-treatment of
CCR4-transfected L1.2 cells with TARC did not desensitize the
receptor to subsequent stimulation with MDC. The signal produced by
initial TARC stimulation was of lower intensity than both the
primary MDC signal and the MDC signal secondary to TARC
stimulation. These results further confirm that MDC is a ligand for
CCR4.
[0331] E. Chemotaxis Assay
[0332] We next examined the ability of MDC and TARC to induce
migration of CCR4-transfected L1.2 cells. Approximately 10.sup.6
CCR4-transfected L1.2 cells, resuspended in 0.1 ml RPMI 1640 media
with 0.5% BSA, were loaded in the upper wells of a transwell
chamber (3 .mu.m pore size, Costar). Untransfected L1.2 cells were
used as a control. Test chemokines were added to the lower wells at
a concentration of 0-100 nM in a volume of 0.6 ml. After 4 hours at
37.degree. C.,-cells in the lower chamber were collected and
counted by FACS.
[0333] Both MDC and TARC induced migration of CCR4-transfected L1.2
cells. Both chemokines produced classic bell-shaped migration
responses with maximal migration at about 10 nM. The migration
observed with MDC was significantly higher than that for TARC, with
MDC inducing migration of greater than 7% of input cells versus
less than 3% migration for TARC. Untransfected L1.2 cells failed to
migrate when treated with MDC. These chemotaxis results further
confirm that both MDC and TARC are functional ligands for CCR4.
[0334] F. Conclusion
[0335] Collectively, the foregoing experiments provide compelling
evidence that MDC acts as a high affinity ligand for the chemokine
receptor CCR4.
[0336] As described below in Example 32, CCR4 has been found to be
abundantly and nearly exclusively expressed on antigen-specific
T.sub.H2 helper T cells. Such cells are particularly susceptible to
HIV-1 infection. (See Maggi et al., Science, 265:244-252 (1994).)
The identification herein of a high affinity MDC receptor on
HIV-susceptible T cells indicates a putative mechanism/pathway
through which MDC(1-69) exerts its agonistic activity relating to
enhanced HIV-1 infectivity and or viral production in infected
cells (see Example 20), and likewise indicates a target for
therapeutic intervention. Without intending to be limited to a
particular theory, MDC-mediated activation of T.sub.H2 cells,
through the CCR4 receptor, is postulated to enhance infectivity
and/or production of HIV-1 virus, in a manner analogous to the
increased infectivity that has previously been observed for
activated target cells. See Woods et al., Blood, 89: 1635-1641
(1997); and Roederer et al., J. Clin. Invest., 99(7): 1555-1564
(1997).
EXAMPLE 26
MDC Modulator Assays
[0337] Modulators of MDC activity may be useful for the treatment
of diseases or symptoms of diseases wherein MDC plays a role. Such
modulators may be either agonists or antagonists of MDC binding.
The following receptor binding assays provide procedures for
identifying such MDC modulators.
[0338] MDC is labelled with a detectable label such as .sup.125I,
.sup.3H, .sup.14C, biotin, or Europium. A preparation of cell
membranes containing MDC receptors is prepared from natural cells
that respond to MDC, such as human macrophages, phorbol
ester-stimulated THP-1 cells, human fibroblasts, human fibroblast
cell lines, or guinea pig macrophages. (Alternatively, a
recombinant receptor preparation is made from cells transfected
with an MDC receptor cDNA, such as a mammalian cell line
transfected with a cDNA encoding CCR4 and expressing CCR4 on its
surface.) The membrane preparation is exposed to .sup.125I-labelled
MDC, for example and incubated under suitable conditions (e.g., ten
minutes at 37.degree. C.). The membranes, with any bound
.sup.125I-MDC, are then collected on a filter by vacuum filtration
and washed to remove unbound .sup.125I-MDC. The radioactivity
associated with the bound MDC is then quantitated by subjecting the
filters to liquid scintillation spectrophotometry.
[0339] The specificity of MDC binding may be confirmed by repeating
the foregoing assay in the presence of increasing quantities of
unlabeled MDC, and measuring the level of competition for binding
to the receptor. These binding assays also can be employed to
identify modulators of MDC receptor binding.
[0340] The foregoing receptor binding assay also may be performed
with the following modification: in addition to labelled MDC, a
potential MDC modulator is exposed to the membrane preparation. In
this assay variation, an increased level (quantity) of
membrane-associated label indicates the potential modulator is an
activator of MDC binding; a decreased level (quantity) of
membrane-associated label indicates the potential modulator is an
inhibitor of MDC receptor binding. This assay can be utilized to
identify specific activators and inhibitors of MDC binding from
large libraries of chemical compounds or natural products. Rapid
screening of multiple modulator candidate compounds simultaneously
is specifically contemplated.
EXAMPLE 27
Assay to Identify Modulators of the MDC/CCR4 Interaction
[0341] The discovery that CCR4 acts as an MDC receptor prompted the
development of the following additional assays to identify
modulators of the interaction between MDC and CCR4. Such assays are
intended as aspects of the present invention.
[0342] A. Direct Assay
[0343] In one embodiment, the invention comprehends a direct assay
for modulation (potentiation or inhibition) of MDC-receptor
binding. In one direct assay, membrane preparations presenting the
chemokine receptor CCR4 in a functional conformation are exposed to
either MDC alone or MDC in combination with potential
modulators.
[0344] For suitable membrane preparations, tissue culture cells,
such as 293 or K-562 cells (ATCC CRL-1573 and CCL-243,
respectively), are transfected with an expression vehicle encoding
the MDC receptor CCR4. Cells that express the receptor are selected
and cultured, and a membrane preparation is made from the
transfected cells expressing the chemokine receptor. By way of
example, suitable membrane preparations are made by homogenizing
cells in TEM buffer (25 mM Tris-HCl, pH 7.4, 1 mM EDTA, 6 mM
MgCl.sub.2, 10 .mu.M PMSF, 1 .mu.g/ml leupeptin). The homogenate is
centrifuged at 800.times.g for 10 minutes. The resulting pellet is
homogenized again in TEM and re-pelleted. The combined supernatants
are then centrifuged at 100,000.times.g for one hour. The pellets
containing the membrane preparations are resuspended in TEM at 1.5
mg/ml.
[0345] Membrane preparations are exposed to labelled MDC (e.g., MDC
labelled with I.sup.125 or other isotope, MDC prepared as an
MDC-secreted alkaline phosphatase fusion protein, or MDC labelled
in some other manner) either in the presence (experimental) or
absence (control) of one or more compounds to be tested for the
ability to modulate MDC-receptor binding activity. To practice the
assay in standard 96-well plates, an exemplary reaction would
include 2 .mu.g of the membrane preparation, 0.06 nM of
radio-labelled MDC, and 0.01 to 100 .mu.M of one or more test
compounds, in a reaction buffer comprising 50 mM HEPES, pH 7.4, 1
mM CaCl.sub.2, 5 mM MgCl.sub.2, and 0.1% BSA. The reactions are
then incubated under suitable conditions (e.g., for 1-120 minutes,
or more preferably 10-60 minutes, at a temperature from about room
temperature to about 37 .degree. C.).
[0346] After incubation, the membranes, with any bound MDC and test
compounds, are collected on a filter by vacuum filtration and
washed to remove any unbound ligand and test compound. Thereafter,
the amount of labelled MDC associated with the washed membrane
preparation is quantified. In an embodiment wherein the label is a
radioisotope, then bound MDC preferably is quantified by subjecting
the filters to liquid scintillation spectrophotometry. In an
embodiment wherein an MDC-alkaline phosphatase fusion protein is
employed, alkaline phosphatase activity is measured using, for
example, the "Great Escape" detection kit (Clontech, Palo Alto,
Calif.) according to the manufacturer's instructions. The amount of
label (e.g., scintillation counts or alkaline phosphatase activity)
associated with the membranes is proportional to the amount of
labelled MDC bound thereto. If the quantity of bound, labelled MDC
observed in an experimental reaction is greater than the amount
observed in the corresponding control, then the experimental
reaction is scored as containing one or more putative agonists
(i.e., activators, potentiators) of MDC receptor binding. If the
quantity of bound, labelled MDC observed in an experimental
reaction is less than the amount observed in the corresponding
control, then the experimental reaction is scored as containing one
or more putative antagonists (inhibitors) of MDC receptor
binding.
[0347] The specificity of modulator binding may be confirmed by
repeating the foregoing assay in the presence of increasing
quantities of unlabeled test compound and noting the level of
competition for binding to the receptor. The assay may also be
repeated using labelled modulator compounds, to determine whether
the modulator compound operates by binding with the MDC
receptor.
[0348] B. Indirect GDP Assay
[0349] In another embodiment, the invention comprehends indirect
assays for identifying modulations of MDC receptor binding that
exploit the coupling of chemokine receptors to G proteins. As
reviewed in Linder et al., Sci. Am., 267: 56-65 (1992), during
signal transduction, an activated receptor interacts with and
activates a G protein. The G protein is activated by exchanging GDP
for GTP. Subsequent hydrolysis of the G protein-bound GTP
deactivates the G protein. Therefore, one can indirectly assay for
G protein activity by monitoring the release Of .sup.32P.sub.i from
[.gamma.-.sup.32P]-GTP.
[0350] For example, approximately 5.times.10.sup.7 HEK-293 cells
that have been transformed or transfected (e.g., with a CCR4
expression vector) to express CCR4 are grown in MEM+10% fetal calf
serum (FCS). The growth medium is supplemented with mCi/ml
[.sup.32P]-sodium phosphate for 2 hours to uniformly label
nucleotide pools. The cells are subsequently washed in a
low-phosphate isotonic buffer.
[0351] An experimental aliquot of washed cells is exposed to MDC in
the presence of one or more test compounds, while a control aliquot
of cells is exposed to MDC, but without exposure to the test
compound. Following an incubation period (e.g., minutes, 37.degree.
C.), cells are pelleted and lysed and nucleotide compounds are
fractionated using, e.g., thin layer chromatography (TLC) developed
with 1 M LiCl. Labelled GTP and GDP are identified in the TLC by
developing known GTP and GDP standards in parallel. The labelled
GTP and GDP are then quantified by autoradiographic techniques that
are standard in the art.
[0352] In this assay, the extent of MDC interaction with its
receptor is proportional to the levels of .sup.32P-labelled GDP
that are observed, thereby permitting the identification of
modulators of MDC-CCR4 binding. An intensified signal resulting
from a relative increase in GTP hydrolysis, producing
.sup.32P-labelled GDP, indicates a relative increase in receptor
activity. The intensified signal therefore identifies the potential
modulator as an activator of MDC-CCR4 activity, or possibly as an
MDC mimetic. Conversely, a diminished relative signal for
.sup.32P-labelled GDP, indicative of decreased receptor activity,
identifies the potential modulator as an inhibitor of MDC receptor
binding or an inhibitor of MDC-induced CCR4 signal
transduction.
[0353] C. cAMP Assay
[0354] The activities of G protein effector molecules (e.g.,
adenylyl cyclase, phospholipase C, ion channels, and
phosphodiesterases) are also amenable to assay. Assays for the
activities of these effector molecules have been previously
described. For example, adenylyl cyclase, which catalyzes the
synthesis of cyclic adenosine monophosphate (cAMP), is activated by
G proteins. Therefore, MDC binding and activation of CCR4 that
activates a G protein, which in turn activates adenylyl cyclase,
can be detected by monitoring cAMP levels in a host cell that
recombinantly expresses CCR4.
[0355] Host cells that recombinantly express CCR4 are preferred for
use in the assay. The host cells are incubated in the presence of
either MDC alone or MDC plus one or more test compounds as
described above. The cells are lysed, and the concentration of cAMP
is measured by a suitable assay, such as a commercial enzyme
immunoassay. For example, the BioTrak Kit (Amersham, Inc.,
Arlington Heights, Ill.) provides reagents for a suitable
competitive immunoassay for cAMP.
[0356] An elevated level of intracellular cAMP in a test reaction
relative to a control reaction is attributed to the presence of one
or more test compounds that increase or mimic MDC-induced CCR4
activity, thereby identifying a potential activator compound. A
relative reduction in the concentration of cAMP would indirectly
identify an inhibitor of MDC-induced CCR4 activity.
[0357] It will be apparent to those in the art that the foregoing
assays may be performed using MDC analogs described herein.
Moreover, variations of the foregoing assays will be apparent to
those in the art. Any variations that utilize both MDC and CCR4,
and especially those variations which utilize MDC and cells that
recombinantly express CCR4, are intended as aspects of the
invention.
[0358] While the use of human MDC and CCR4 comprises a highly
preferred embodiment, it will be apparent that the source organism
for MDC and CCR4 is not a limiting factor, and the foregoing assays
may be practiced effectively with MDC and/or with CCR4 that are
derived from non-human organisms. By way of example, rat and mouse
MDC are taught herein, and a Mus musculus chemokine receptor 4
sequence has been reported in the art. See Hoogewerf et al.,
Biochem. Biophys. Res. Comm., 218(1): 337-343, and GenBank
Accession No. X90862. Moreover, the methods used herein to obtain
rat and mouse MDC are employable to obtain MDC or CCR4 from other
organisms.
[0359] Moreover, evidence exists that there is at least one
additional receptor that recognizes MDC. For example, MDC
stimulates migration of dendritic cells and IL-2 activated natural
killer cells. Godiska et al., J. Exp. Med., 185: 1595-1604 (1997),
incorporated herein by reference. This migration is not likely to
be mediated by CCR4, since CCR4 appears to be expressed primarily
on T cells, but not on monocytes or NK cells. See Imai et al.
(1997). Consistent with this, CCR4 clones were represented very
rarely in a human macrophage cDNA library (less than one in a
million clones). Variations of the assays reported herein that
utilize MDC with other MDC receptors also are intended as aspects
of the invention.
[0360] Additionally, it will be apparent that the protocols
described in preceding examples for assaying MDC biological
activities (in vivo or with respect to specific cell types in
vitro) are useful as assays for MDC modulators. In a highly
preferred embodiment, a compound is first identified as a candidate
MDC modulator using any of the assays described in Examples 26 and
27. Compounds that modulate MDC-receptor activity in one or more of
these initial assays are further screened in any of the protocols
described in preceding examples, to determine the ability of the
compounds to modulate the MDC biological activities to which those
examples specifically relate.
EXAMPLE 28
Isolation of cDNA Encoding Rat and Mouse MDC Proteins
[0361] Knowledge of the human MDC gene sequence described herein
was used as described below to isolate and clone putative rat and
mouse MDC cDNAs, which are intended as aspects of the
invention.
[0362] To clone a rat MDC cDNA, a labelled probe was prepared using
standard random primer extension techniques. A fragment of the
human MDC cDNA was generated by PCR, which fragment includes the
MDC coding region plus approximately 300 bases of 3' untranslated
sequence. This fragment was labelled with
.sup.32P-deoxyribonucleotides using the Random Primed DNA Labeling
kit (Boehringer Mannhein, Indianapolis, Ind.). The labelled MDC
probe was used to screen approximately 106 bacteriophage lambda
clones from a commercially-available rat thymus cDNA library
(Stratagene, La Jolla, Calif., Cat. No. 936502). Three positive
clones were obtained. Sequencing of one of the positive clones,
designated RT3, provided an approximately 958 base pair sequence
(SEQ ID NO: 37) that included an MDC open reading frame (SEQ ID NO:
38) and about 0.5 kb of 3' untranslated sequence. The open reading
frame included sequence encoding the putative mature MDC protein
(SEQ ID NO: 38, residues 1 to 69) plus 13 amino acids of the
putative signal peptide sequence; it lacked the initiator
methionine codon and sequence encoding the amino terminus of the
signal peptide. A complete rat MDC cDNA or genomic clone is
obtainable using all or a portion of the RT3 sequence as a labelled
probe to re-probe the Stratagene rat cDNA library, and/or other rat
cDNA libraries, and/or a rat genomic DNA library.
[0363] To clone a mouse MDC cDNA, approximately 10.sup.6
bacteriophage lambda clones of a commercially-available mouse
thymus cDNA library (Stratagene, Cat. No. 935303) were screened
with a radiolabeled fragment of the above-described rat MDC cDNA.
The probe was generated using overlapping primers in a primer
extension reaction. The primer extension reaction comprised:
partially overlapping primers corresponding to nucleotides 41 to
164 of SEQ ID NO: 37 (and to nucleotides 92-215 of SEQ ID NO: 1);
.sup.32P-labelled deoxyribonucleotides; and the Klenow fragment of
E. coli DNA polymerase. Twelve positive clones were isolated.
[0364] One positive clone, designated MT3, was sequenced and found
to contain a 1.8 kb cDNA insert that included the entire putative
murine MDC coding region and about 1507 bases of 3' untranslated
sequence. The cDNA and deduced amino acid sequences for this murine
MDC clone are set forth in SEQ ID NOs: 35 and 36, respectively. The
mouse MDC has a putative 24 amino acid signal sequence followed by
a 68 amino acid MDC sequence.
[0365] Comparisons of the human, rat, and mouse MDC protein and DNA
(coding region) sequences reveal the following levels of
similarity:
7 Human vs. rat protein: 65% identity; Human vs. rat DNA: 74%
identity; Human vs. mouse protein: 64% identity; Human vs. mouse
DNA: 72% identity; Rat vs. mouse protein: 88% identity; Rat vs.
mouse DNA: 92% identity.
[0366] The four cysteines characteristic of C--C chemokines are
conserved in all three MDC proteins.
[0367] It is contemplated that the encoded rat and mouse MDC
polypeptides corresponding to SEQ ID NOs: 38 and 36 are processed
into mature mouse MDC proteins, in a manner analogous to the
processing of the human MDC precursor, by cleavage of a signal
peptide. The signal peptides for both human and murine MDC are 24
amino acids. The exact length of the rat MDC signal peptide will be
readily apparent upon isolation of a full length rat MDC cDNA. It
will be appreciated that these proteins can be synthesized
recombinantly or synthetically and assayed for MDC biological
activities as described herein for human MDC. Likewise, it will be
appreciated that any analogs described herein for human MDC can be
similarly prepared for these other mammalian MDC proteins.
[0368] The foregoing results demonstrate the utility of
polynucleotides of the invention for identifying and isolating
polynucleotides encoding other vertebrate MDC proteins, especially
other mammalian or avian MDC proteins. Such identified and isolated
polynucleotides, in turn, can be expressed (using procedures
similar to those described in preceding examples) to produce
recombinant polypeptides corresponding to other vertebrate forms of
MDC, which proteins would be useful in the same manners that human
MDC is useful, including therapeutic veterinary applications.
Polynucleotides encoding vertebrate (and especially mammalian or
avian) MDC proteins, the proteins themselves, and analogs thereof
are all contemplated to be aspects of the present invention.
EXAMPLE 29
Receptor Binding and Stimulation Assays
[0369] Using procedures essentially as described in Example 25,
selected MDC analogs described in Example 11 were screened for the
ability to bind CCR4 and/or induce calcium (Ca.sup.++) flux and
chemotaxis in L1.2 cells transfected with CCR4.
[0370] The analog MDC(n+1) bound CCR4 with similar affinity to MDC,
but induced calcium flux and chemotaxis in L1.2/CCR4 cells with a
slightly lower potency than MDC. For example, in chemotaxis, the
peak activity for MDC(n+1) was observed at 100 ng/ml rather than 10
ng/ml, and the maximum number of cells migrating was 5000, compared
to 9000 for MDC.
[0371] MDC(9-69) bound CCR4 with reduced affinity relative to that
of MDC (0-69). MDC(9-69) did not induce calcium flux in L1.2/CCR4
cells, and it was much less potent in chemotaxis. The fact that
MDC(9-69) binds CCR4 but does not signal through CCR4 indicates a
utility of MDC(9-69) as an MDC inhibitor.
[0372] Collectively, the activities of MDC (n+1) and MDC (9-69)
indicate that amino-terminal additions and deletions and other
modifications may result in useful MDC inhibitors.
[0373] The analog "MDC-wvas" bound CCR4 with 500-fold less affinity
than MDC, induced only a very small calcium flux, and did not
induce any chemotaxis. The analog "MDC-eyfy" acted similar to MDC
in CCR4-binding, chemotaxis, and calcium flux assays.
EXAMPLE 30
Monoclonal Antibodies 252Y & 252Z Inhibit CCR4-Mediated
Cellular Responses to MDC
[0374] Using procedures similar to those described in Example 25,
the monoclonal antibodies 252Y and 252Z described in Example 18
were screened for the ability to modulate MDC-CCR4 binding and
modulate the CCR4-mediated biological activities of MDC.
[0375] A. Antibodies 252Y and 252Z Inhibit MDC Binding to CCR4
[0376] The fusion protein MDC-SEAP (Example 25) was employed to
evaluate the ability of the antibodies to inhibit MDC binding to
its receptor CCR4. MDC-SEAP at a concentration of 0.5 nM was
incubated for fifteen minutes at room temperature with varying
concentrations (0.01-10 .mu.g/ml, shown in FIG. 1) of antibody
252Y, antibody 252Z, or an isotype control (final reaction volume
100 .mu.l). Thereafter, the mixtures were added to CCR4-expressing
L1.2 cells (100 .mu.l, 4000 cells per .mu.l), and incubated at
4.degree. C. for an additional 60 minutes. The extent of MDC-SEAP
binding to the CCR4-expressing cells was determined by alkaline
phosphatase chemiluminescent assay as described in Example 25. A
baseline level of non-specific binding (defined as the amount of
binding that could not be competed by a 200-fold molar excess of
native MDC) was determined and subtracted from experimental
measurements. FIG. 11 presents the experimental results in
graphical form, wherein each data point represents a percentage of
maximum binding. (Maximum binding was defined as the amount of
MDC-SEAP bound to the cells in the absence of antibody, minus
non-specific binding.) As shown in FIG. 11, both antibody 252Y and
antibody 252Z (but not the isotype control) inhibited MDC-SEAP
binding to CCR4-infected cells in a dose-dependent manner. Fifty
percent inhibition of binding was observed for both antibodies at
an antibody concentration of about 2 .mu.g/ml.
[0377] B. Antibodies 252Y and 252Z Inhibit MDC-Induced
Chemotaxis
[0378] To confirm that antibodies 252Y and 252Z also were capable
of inhibiting CCR4-mediated cellular responses to MDC, both calcium
flux and chemotaxis assays were performed using the
CCR4-transfected L1.2 cells.
[0379] For the calcium flux assay, the transfected L1.2 cells were
labelled with Fura-2/AM (see Example 19) and monitored for
Ca.sup.++-induced fluorescence changes using an AMINCO-Bowman
Series 2 fluorimeter. Addition of 75 nM MDC to the cells induced a
rapid, transient increase in intracellular Ca.sup.++ levels. This
Ca.sup.++ flux response was completely inhibited when either
antibody 252Y or antibody 252Z were added to the cells at a
concentration of 10 .mu.g/ml one minute before contacting the cells
with the MDC solution. An isotype-matched control antibody had no
effect on the MDC-induced Ca.sup.++ flux. Thus, both antibodies
blocked the calcium flux response to MDC in CCR4-transfected L1.2
cells.
[0380] For the chemotaxis assay, CCR4-transfected L1.2 cells
(approx. 10 million cells/ml in a volume of 0.1 ml) were
preincubated with antibody 252Y, antibody 252Z, or an
isotype-matched control in RPMI-1640 media (Gibco) at various
concentrations ranging from 0.5 to 50 g/ml for 30 minutes at room
temperature. Thereafter, the cells were exposed to 100 ng/ml MDC
(i.e., the peak concentration for maximum chemotaxis) for 4 hours
in a Costar Transwell apparatus. The number of cells migrating
toward MDC was counted using a Becton-Dickinson FACScan apparatus.
As shown in FIG. 12, MDC-induced chemotaxis of these cells was
totally inhibited by either antibody 252Y or antibody 252Z at
concentrations of 2-5 .mu.g/ml, but not by the isotype-matched
control. The IC.sub.50 antibody concentration (required to inhibit
50% migration) was 1 .mu.g/ml. The same antibodies did not inhibit
chemotaxis of the CCR4/L1.2 cells toward the C--C chemokine TARC,
indicating that the inhibitory effect was specific for MDC.
[0381] In a similar set of experiments, antibody 272D was screened
for its ability to inhibit MDC stimulated chemotaxis. Ten .mu.g/ml
of antibody 272D was required to inhibit chemotaxis toward
recombinant MDC (30 ng/ml) by greater than 90%. Only 2 .mu.g/ml of
antibody 252Z was required to achieve a similar level of
inhibition, indicating that antibody 252Z is a more potent
inhibitor of MDC induced chemotaxis.
EXAMPLE 31
MDC Induces Chemotaxis of T.sub.H2 Helper T Cells
[0382] A transendothelial migration assay was performed essentially
as described in the art [Ponath, et al., J. Clin. Invest., 97:
604-612 (1996); Ponath et al., J. Exp. Med., 183: 2437-2448 (1996);
and Imai, et al., Cell, 91: 521-530 (1997)] to determine the
presence and the phenotype of T cells that migrate toward the
chemokines TARC and MDC. Briefly, about 2.times.10.sup.5 cells of
the endothelial cell line ECV304 (ATCC CRL-1998 or European Cell
Culture Collection, Portions Down, UK) were added to Transwell
inserts (Coaster) with a 5 .mu.m pore size and cultured at
37.degree. C. for 48-96 hours in M199 medium (GIBCO/BRL)
supplemented with 10% FCS. Chemokines were diluted (serial
dilutions of 0.1 to 100 nM) in a migration medium (a 1:1 mixture of
RPMI-1640: M 99, supplemented with 0.5% BSA, 20 mM HEPES, pH 7.4)
and added to 24-well tissue culture plates in a final volume of 600
.mu.l. Endothelial cell-coated inserts were placed in each well and
106 peripheral blood mononuclear cells (PBMC) or T cell lines in
100 .mu.l were added to the upper chambers. The cells were allowed
to migrate through the endothelial cells into the lower chambers at
37.degree. C. for 4 hours (PBMC) or 90 minutes (T cell lines). The
migrated cells in the lower chambers were stained with FITC- or
PE-conjugated monoclonal antibodies (mAb) for indicated cell
surface makers and counted by flow cytometry.
[0383] In the transendothelial cell migration assay, both TARC and
MDC induced dose-dependent vigorous migration of CD14.sup.-
lymphocytes but not of CD14.sup.+ monocytes, with MDC consistently
inducing cell migration about 2 times more efficiently than TARC.
Migration activity was detected with chemokine concentrations as
low as 1 nM. Significant migration occurred with 10 nM TARC and 10
nM MDC. Analysis of the migrating lymphocytes revealed that 10 nM
of either TARC or MDC attracted predominantly CD4.sup.- T cells.
Neither TARC nor MDC induced migration of CD19.sup.+ B cells or
CD16.sup.+ NK cells. Furthermore, TARC and MDC attracted almost
exclusively CD45RA.sup.-/CD45RO.sup.- effector/memory T cells. This
observation was consistent with the observation that a murine (IgG)
monoclonal antibody to CCR4 stained highly selectively a fraction
(20%) of CD45RO.sup.-CD4.sup.- memory helper T cells.
[0384] Effector/memory helper T cells represent a population of
cells that have encountered cognate antigens in vivo and have
differentiated into T.sub.H1 or T.sub.H2 cells. Since CCR4 is
expressed on about 200/0 of effector/memory helper T cells,
additional experiments were conducted to determine whether CCR4 is
selectively expressed on certain subsets of helper T cells.
[0385] First, CD4.sup.-CD45RO.sup.-T cells (obtained from PBMC by
negative selection with Dynabeads (Dynal) after incubation with
anti-CD16, anti-CD14, anti-CD20, anti-CD 8, and anti-CD45RA
antibodies) were fractionated into CCR4.sup.+ and CCR4.sup.-
-subpopulations by staining with the anti-CCR4 mAb and cell
sorting. The cell subpopulations were expanded as polyclonal cell
lines by culturing for 9-14 days at 37.degree. C. in RPMI medium
supplemented with PHA (diluted 1:100) and 100 U/ml IL-2. Expanded
cells were subjected to a second round of enrichment by staining
with anti-CCR4 monoclonal antibody and sorting. Sorted cells were
immediately activated with 50 ng/ml PMA (Sigma) and 1000 ng/ml
ionomycin (Sigma) for 24 hours, at which time the culture medium
was analyzed by ELISA (R&D) to determine each population's
pattern of cytokine production. Since helper T cells are classified
into T.sub.H1 and T.sub.H2 subsets based on their profiles of
cytokine production [Mosmann et al., Immunol. Today, 17: 138-146
(1996)], this analysis permitted determination of whether CCR4 is
selectively expressed in one or the other subpopulation.
[0386] Analysis of the culture medium revealed that the CCR4.sup.+
T cells produced significantly larger amounts of IL-4 and IL-5 than
the cultured CCR4.sup.-T cells (>12 ng/ml for CCR4.sup.- T cells
versus<2.5 ng/ml for CCR4.sup.- T cells for each cytokine).
Conversely, CCR4.sup.- T cells produced IFN-.gamma. at levels much
higher than CCR4.sup.+ T cells (>300 ng/ml vs. <25 ng/ml).
These cytokine expression patterns indicate that the CCR4.sup.+
population of cells contained almost exclusively T.sub.H2 cells,
whereas CCR4.sup.- cells were enriched for T.sub.H1 cells.
[0387] To support the conclusion that CCR4.sup.+T cells are
predominantly T.sub.H.sup.2 cells, the CD4.sup.+CD45RO.sup.- T
cells that had been attracted by TARC or MDC in the
transendothelial migration assay were expanded by culturing in PHA
and IL-2 and then examined for their pattern of cytokine production
as described above. Compared to total CD4.sup.+CD45RO.sup.+T cells,
the cells attracted by TARC or MDC were enriched for producers of
IL-4 and IL-5 and depleted of producers of IFN-.gamma..
[0388] To further confirm the observed selective expression of CCR4
on T.sub.H2 cells, experiments were performed to polarize CD4.sup.+
CD45RA.sup.+ naive T cells in vitro, and the artificially polarized
cell populations were examined for CCR4 expression. The naive T
cells (obtained from PBMC by negative selection with Dynabeads
after incubation with anti-CD16, anti-CD14, anti-CD20, anti-CD8,
and anti-CD45RO antibodies) were polarized into T.sub.H1 cells by
culturing in the presence of PHA (1:100), 2 ng/ml IL-12, and 200
ng/ml anti-IL-4 monoclonal antibodies (Pharmingen); or into
T.sub.H2 cells by culturing with PHA (1:100), 10 ng/ml IL-4, and 2
.mu.g/ml anti-IL-12 monoclonal antibodies. After 3-4 days, 100 U/ml
IL-2 was added to the cultures. CCR4 expression and transmigration
were analyzed at day 9-14.
[0389] Analysis of the cultured cells with an anti-CCR4 monoclonal
antibody revealed that 60% of cells polarized into T.sub.H2 cells
expressed CCR4, compared to only 4% of cells polarized into
T.sub.H1 cells. Northern blot analysis of the RNA isolated from
these cell populations also demonstrated that T.sub.H2 cells
expressed CCR4 mRNA at levels much higher than T.sub.H1 cells. As
controls, CCR7 mRNA was expressed in both types of cells whereas
CCR3 mRNA was not detected in either type of cell.
[0390] In the endothelial transmigration assay, the artificially
polarized T.sub.H2 cells, but not those polarized into T.sub.H1,
migrated vigorously toward TARC and MDC, whereas both types of
cells migrated toward SLC. (See Nagira, et al., "Molecular cloning
of a novel human CC chemokine secondary lymphoid-tissue chemokine
that is a potent chemoattractant for lymphocytes and mapped to
chromosome 9p 13," J. Biol. Chem., 272: 19518-19524 (1997).)
Neither population of cells migrated toward eotaxin, a ligand for
CCR3.
[0391] Collectively, the foregoing experiments demonstrate that a
significant population of T.sub.H2 cells express the chemokine
receptor CCR4, and that the chemokines TARC and MDC represent
selective chemoattractants of T.sub.H2 cells, an effect that
presumably is mediated at least in part through CCR4. Tissues of
allergic inflammation are infiltrated by T.sub.H2 cells, as well as
by eosinophils, another cell type selectively attracted by MDC (see
Example 12). Furthermore, T cells migrating into tissues after
antigen challenge have been reported to be involved in localized
production of the T.sub.H.sup.2 cytokines, IL-4 and IL-5, and in
accumulation of eosinophils. (See Garlisi et al., Clin. Immunol.
Immunopathol., 75: 75-83 (1995).) Additionally, TARC and MDC are
abundantly produced by dendritic cells whose close interactions
with migrating lymphocytes constitute essential parts in initiation
and promotion of immune responses. (See Steinman, R. M., Annu. Rev.
Immunol., 9: 271-296 (1991).) Enhanced TARC and MDC production from
antigen presenting cells in T.sub.H2 responses would be expected to
lead to further recruitment of T.sub.H2 cells via CCR4. Thus, the
discoveries herein relating to the biological effects of MDC
indicate that the effects may be deeply intertwined and involved in
multiple aspects of an immunological or allergic cascade, a factor
of direct clinical importance. For example, agents that interfere
with the interactions of TARC or MDC with the receptor CCR4 (and/or
that interfere with the interactions of TARC or MDC with T.sub.H2
cells or eosinophils in cell-based assays) have therapeutic
indications for reducing allergic inflammatory responses. The use
of such agents in the treatment of asthma, a conditions
characterized by eosinophilic infiltration and probable involvement
of presentation of sensitizing antigen by mucosal dendritic cells
to T.sub.H2 T cells, is specifically contemplated.
EXAMPLE 32
Use of MDC and MDC Antagonists to Modulate Platelet Aggregation
[0392] The following experimental data indicates that MDC promotes
platelet aggregation, and suggests a therapeutic indication for MDC
and MDC antagonists to modulate platelet aggregation.
[0393] Female Lewis rats, six to eight weeks old, were administered
0.5 .mu.g of synthetic mature human MDC(1-69) intravenously in a
saline solution, via the tail vein. At various time points, the
animals (4) were anesthetized with 100 .mu.l ACE cocktail
(Ketamine, ACE promazine and Rompon) and blood samples were
collected into Microcontainers containing EDTA (Beckton Dickinson).
Samples (300-400 .mu.l) were stored overnight at 4-8.degree. C. A
CBC with Differential analysis was conducted to identify changes in
cell number in the rats compared to control rats that had been
administered only phosphate-buffered saline. In all four animals
treated, marked platelet aggregation was observed. This aggregation
was most pronounced at the time of MDC administration and
dissipated with time after the bolus. A similar phenomenon was
observed in mice using an analogous protocol.
[0394] Receptor analyses have indicated that platelets express
detectable levels of the MDC receptor CCR4. These experiments
suggest a receptor through which MDC may exert its
platelet-aggregating effects.
[0395] The foregoing observations suggest that mature MDC
stimulates platelet aggregation, and suggests that MDC antagonists
are useful for inhibiting coagulation. Such use is indicated, e.g.,
in myocardial infarction patients to prevent further inappropriate
blood clotting, and in patients for the therapeutic or prophylactic
treatment of stroke.
[0396] The concentrations at which MDC induces platelet aggregation
and at which MDC antagonists prevent platelet aggregation are
determined in vivo using purified platelets and serial dilutions of
MDC and MDC antagonists and procedures that are well known in the
art. See, e.g., Jeske et al., Thromb. Res., 88(3):271-281 (1997);
Herault et al., Thromb. Haemost., 79(2):383-388 (1998); and
Furakawa et al., Jpn. J. Pharmacol, 75(3):295-298 (1997). Putative
MDC antagonists for screening in such assays include all of the
putative MDC antagonists identified above, e.g., in Example 20.
Those MDC analogs that inhibit platelet aggregation and those that
promote aggregation are determined by such dose response studies
and/or by mouse studies as described above.
[0397] Similarly, since TARC also signals through CCR4, the use of
TARC and TARC antagonists to modulate platelet aggregation also is
intended as an aspect of the invention.
[0398] The biological functions of MDC, elucidated as described
above, suggest several clinical applications.
[0399] Chemokines in general attract and activate monocytes and
macrophages (Baggiolini et al., supra), and MDC in particular
attracts macrophages and inhibits monocyte chemotaxis. Thus, MDC
expression in a pathogenic inflammatory setting may exacerbate
disease states by recruiting additional macrophages or other
leukocytes to the disease site, by activating the leukocytes that
are already there, or by inducing leukocytes to remain at the site.
Thus, inhibiting the chemoattractant activity of MDC may be
expected to alleviate deleterious inflammatory processes.
Significantly, the potential benefits of such an approach have been
directly demonstrated in experiments involving IL-8, a C--X--C
chemokine that attracts and activates neutrophils. Antibodies
directed against IL-8 have a profound ability to inhibit
inflammatory disease mediated by neutrophils [Harada et al., J.
Leukoc. Biol., 56:559 (1994)]. Inhibition of MDC is expected to
have a similar effect in diseases in which macrophages are presumed
to play a role, e.g., Crohn's disease, rheumatoid arthritis, or
atherosclerosis.
[0400] Alternatively, augmenting the effect of MDC may have a
beneficial role in such diseases, as chemokines have also been
shown to have a positive effect in wound healing and angiogenesis.
Thus, exogenous MDC or MDC agonists may be beneficial in promoting
recovery from such diseases.
[0401] In addition, the myelosuppressive effect demonstrated for
the C--C chemokine MIP-1.alpha. (Maze et al., supra) suggests that
MDC may have a similar activity. Such activity, provided by MDC or
MDC agonists, may yield substantial benefits for patients receiving
chemotherapy or radiation therapy, reducing the deleterious effects
of the therapy on the patient's myeloid progenitor cells.
[0402] MDC or MDC agonists may also prove to be clinically
important in the treatment of tumors, as suggested by the ability
of the C--C chemokine TCA3 to inhibit tumor formation in mice (see
Laning et al., supra). MDC may act directly or indirectly to
inhibit tumor formation, e.g., by attracting and activating various
non-specific effector cells to the tumor site or by stimulating a
specific anti-tumor immunity. The fibroblast-antiproliferative
effect of MDC indicates a therapeutic utility for MDC in the
treatment of diseases such as pulmonary fibrosis and tumors, in
which enhanced or uncontrolled cell proliferation is a major
feature.
[0403] While the present invention has been described in terms of
specific embodiments, it is understood that variations and
modifications will occur to those skilled in the art. Accordingly,
only such limitations as appear in the appended claims should be
placed on the invention.
Sequence CWU 1
1
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