Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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MACROPHAGE DERIVED CHEMOIaNE (MDC), MDC ANALOGS,
MDC ll~IHIBITOR SUBSTANCES. AND USES THEREOF
This application is a continuation-in-part of U.S. Patent Application Serial
No.
09/067,447, filed April 28, 1998, and a continuation-in-part of U. S. Patent
Application Serial No.
08/939,107, fill September 26, 1997, (Attorney docket No. 27866/34188), and a
continuation-
in-part of U. S. Patent Application Serial No. 08/660, 542, filed June 7,
1996, and a continuation-
in-part of U.S. Patent Application Serial No. 08/558,658, filed November 16,
1995, and a
continuation-in part ofU.S. Patent Application Serial No. 08/479,620, filed
June 7, 1995. These
applications are hereby incorporated by reference in their entirety.
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. Of particular interest is the use of chemokine inhibitor
substances to treat
allergic conditions such as asthma.
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
kiloDaitons) which attract
and activate leukocytes and thereby aid in the stimulation and regulation of
the immune system.
2 5 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., Armu.
Rev Immunol, I5: 675-705 (1997); and Baggiolini et al., Advances in
Immunolog)r, 55:97-179
(1994), both of which are incorporated by reference herein. While leukocytes
comprise a rich
3 0 source of chemokines, several chemokines are expressed in a multitude of
tissues. Baggiolini et
al. (1994), Table II.
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Preciously 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
"~i" 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 subfiunily.
Chemokines of both
subfi~cnities have characteristic leader sequences of twenty to twenty-five
amino acids.
The C-X-C chemokines, which include IL-8, GROaJ~i/y, platelet basic protein,
Platelet Factor 4 (PF4), IP-10, NAP2, and others, share approximately 25% to
60% identity when
airy two amino acid sequences are compared (except for the GROal~3/y members,
which are 84-
88% idemical with each other). Most of the C-X-C chemokines (excluding IP-10
and Platelet
Factor 4) share a common E-I,-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 rexeptors; a receptor specific for IIr8 has been cloned by
Holmes et al.,
2 0 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 IG8, is associated with marked increases in activity.
The C-C cheanokines, which include Macrophage Inflammatory Proteins MIP-la
and MIP-1(3, Monocyte cheanoattractant profane 1, 2, 3, and 4 (MCP-1/2/3/4),
RANTES, I-309,
eotaxin, TARC, and others, share 25% to 70% amino acid identity with each
other. Previously-
identified C-C chemokines activate monoc5rtes, 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
3 0 chemokines have been cloned to date, including a C-C chemokine receptor-1
(CCRl) which
recognizes, e.g., MIP-la andRANTES (Neoteetal., Cell, 72:415-425 (1993)); a
CCR2 receptor
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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. Bio~ Chem., 270:16491 (1995)); CCR4, which recognizes MIP-1 a,
P;ANTES,
and MCP-1 (Power et a1, J. Bio~ Chem., 270:19495 (1995)); and CCRS, which
recognizes MIP-
la, MIP-lei, and RANTES (Samson ~t aL, Biochemstry, 35:3362 (1996)). Several
CC
chemokines have been shown to act as attractants for activated T lymphocytes.
See Baggiolini
et al. (1997).
Truncation of the N-terminal amino acid sequ~ce 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 NIi2-
X-Pro- . . .) to
generate a RANTES (3-68) form that is capable of interacting with and
transducing a signal
through CCRS (like the RANTES (1-68) form), but is one hundred-fold reduced in
its capacity
to stimulate through the receptor CCRl. See Proost et al., J. Biol. Chem.,
273(13): 7222-7227
(1998); and Oravecz et al., J. Fxp. Med, 186: 1865-1872 (1997). United States
Patent Nos.
5;459,128, 5,705,360, and 5,739,103 to Rollins and Zhang purport to describe N-
terminal
deletions of chemokine MCP-1 that inhibit receptor binding to the
corresponding endogenous
chemokine.
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,
2 0 Table VII. Psoriasis, for example, has been linked to over-production of
IL,-8, and several studies
have observed high levels of I>i8 in the synovial fluid of inflamed joints of
patients suffering from
rheumatic diseases, osteoarthritis, and gout.
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
2 5 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
3 0 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. Patent
No. 5,179,078,
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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.
With respect to the involvement of chemolcines in allergic diseases, interest
has
focused on chemokines belonging to the CC family, such as RATTTES, eotaxin,
eotaxin-2, MCP-3
and MCP-4, because of their ability to cause migration of human eosinophils in
vitro and in vivo.
The ability ofthese chemokines to selectively activate human eosinophil
migration appears to be
due primarily to their activation of chemokine receptor CCR3. A need exists to
elucidate the
involvement of these and other chemokines in eosinophil stimulation and
activation, to facilitate
better treatments for late-phase allergic reactions, such as asthma [see
Aalbers et a~,
Eur.Respir.J., 6:840(1993); and Frigas et al., J. Allergy Clip. Immunol.,
77:527(1986)], in which
eosinophil activation and migration have been implicated.
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. Patent No. 5,413,778,
incorporated herein by
reference. Such methods involve chemical attachment of a labeling agent (eg.,
a radioactive
isotope) to the C-C chemokine using art recognized techniques (see, e.g., U.S.
Patent Nos.
4,965,392 and 5,037,630, incorporated herein by reference), administration of
the labeled
2 0 chemokine to a subject in a pharmaceutically acceptable carrier, allowing
the labeled chemokine
to accumulate at a target site, and imaging the labeled chemolcine 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.
The C-C che<nokines RANTES, M~-a, and MIP-1(3 also have been shown to be
2 5 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 a~, Science, 270:1811 (1995)]. In
addition, International
patent publication number WO 97/44462, filed by Institut Pasteur, describes
the use of fragments
3 0 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
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(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 a~, J. Irrtmunol.,
160:3091-3095 (1998). Moreover, not all~ested strains of the virus are equally
susceptible to the
inlu'bitory effects of chemokines; therefore, a need exists for additional C-C
chemokines for use
as inhibitors of strains of HIV.
Similarly, it has been established that certain chemokine receptors such as
CCRS
[International Patent Publication No. WO 97/44055, published 27 November
1997], CCR8,
CCR2, and CXCR4) are essential co-receptors (with the CD4 receptor) for HIV-1
entry into
susceptible cells, and that progression to AI17S 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.
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.
For example, substances that promote inflamtnation 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
2 0 pathological conditions manifested by inflanunation. 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
characterizaxion of novel chemokines facilitates modulation of inflammation.
2 5 Similarly, substances that induce an immune response may promote
palliation or
healing of arty number of pathological conditions. Due to the important rote
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 imriiune responses.
3 0 Additionally, the established correlation between chemokine expression and
inflammatory conditions and disease states provides diagnostic and prognostic
indications for the
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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.
In addition to their ability to attract and activate leukocytes, some
chemokines,
such as IIr-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.
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 fi-agments and analogs, andJor antibody substances. Chemokine
inhibitors interfere
with chemokine signal transduction, i.e., by binding chemokine molecules, by
competitively or
non-competitively binding chemokine receptors, andlor by interfering with
signal transduction
downstream finm the chemokine n~eptors. A need exists in the art for egective
assays to rapidly
screen putative chemokine modulators for modulating activity.
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.
2a
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.
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. The cDNA and deduced amino acid sequence of human MDC has been
3 0 published. See, e.g., International Patent Publication No. WO 96/40923,
published 19 December
1996; and Godiska et al., J. F..,xp. Med, 185(9): 1595-1604 (199'x. Compare
International
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Publication No. WO 96/39521 (12 December 1996); and Chang et al., J. Biol.
Chem., 272(40):
25229-25237 (1997).
Polynucleotides encoding non-human vertebrate forms of MDC, especially
mammalian and avian forms of N1DC, also are intended as aspects of the
invention.
The nucleotide sequence of a cDNA, designated MDC cDNA, encoding this
chemokine, is set forth in SEQ D7 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
ff~ NO. 1, which nucleotides comprise the MDC coding sequence.
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 1D NO. 1, which nucleotides comprise the putative
coding
sequence of the mature (secreted) 1V1DC protein, without the signal sequence.
The amino acid sequence of human chemokine 1V1DC is set forth in SEQ >D NO:
2. Pry polynucleotides of the present invention include, in addition to those
polynucleotides
described above, polynucleotides that encode the amino acid sequence set forth
in SEQ m NO:
2, and that differ from the polynucleotides described in the preceding
paragraphs only due to the
well-known degeneracy of the genetic code.
Similarly, since twenty-four amino acids (positions -24 to -1) of SEQ D7 NO: 2
comprise a putative signal peptide that is cleaved to yield the mature MDC
chemokine, preferred
2 0 polynucleotides include those which encode amino acids 1 to 69 of SEQ m
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 m NO: 2.
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
2 5 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 lVmC and
the conditions
under which this protein is expressed. Polynucleotides encoding human MDC have
been
3 0 employed to successfi~lly isolate polynucleotides encoding at least three
exemplary non-human
embodiments ofMDC (rat, mouse, macaque). (See SEQ 1D NOs: 35-38 & 45-46.)
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Hybridization probes of the invention also have diagnostic utility, e.g., for
screening for inflanunation 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).
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 D7 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,
M1P-la genes, NEP-1[i genes, and I-309 genes, etc.).
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
2 0 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°C in SX SSC, 20 rnM NaP04, pH 6.8, 50% formamide; and washing at
42°C in 0.2X 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, New York: Cold Spring
Harbor
Laboratory (1989).]
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
3 0 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
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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 NIDC polypeptide of interest.
In another aspect, the invention includes a prokaryotic or eukaryotic host
cell
stably transfected or transformed with a~NA or vector of the present
invention. In preferred
host cells, the mature lViDC polypeptide encoded by the DNA or vector of the
imrention is
expressed. The DNAs, vectors, and host cells of the present invention are
useful, e.g., in methods
for the re<:ombinant 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 ivmC protein is isolated from the cell or the medium.
Knowledge of DNA sequences encoding 1VI,DC makes possible determination of
the chromosomal location of Ivl,DC 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.
According to another aspect of the invention, host cells may be modified by
activating an endogenous 1VI,DC 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 lvIDC by replacing, in whole or in part, the
naturally-occurring lvIDC
2 0 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 lVmC
coding sequences,
i. e., controls transcription of the 1VIDC coding sequences. See, for example,
PCT International
Publication No. WO 94/12650; PCT International Publication No. WO 92120808;
and PCT
International Publication No. WO 91/09955. The invention also contemplates
that, in addition
to heterologous promoter DNA, amplifiable marker DNA (e.g., ado, dhfr, and the
multi-functional
CAD gene which encodes carbamyl phosphate synthase, aspartate
transcarbamylase, and dihydro-
orotase) and/or iron DNA may be recombined along with the heterologous
promoter DNA into
the host cells. If linked to the lvIDC coding sequences, amplification of the
marker DNA by
standard selection methods results in co-amplification of the NiDC coding
sequences in such host
3 o cells.
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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.
In yet another aspect, the invention includes purified and isolated MDC
polypeptides. Mammalian and avian MDC polypeptides are specifically
contemplated. A
prefer peptide is a purified chemolcine 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-lmrnan 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)."
Mouse and Rat MDC polypeptides of the invention are taught in SEQ ID NOs:
36 and 38. The sequence in SEQ ff~ 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.
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
2 5 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.
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
3 0 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
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of such polyp~tide analogs, which may be scxeened 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.
In a related aspect, the invention includes polypeptide analogs wherein one or
more
amino acid residues is added, deleted, or replaced from the 1V>DC polypepddes
of the present
invention, which analogs lank 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 IVjDC in a host, as well as useful for medical imaging
methods described
above.
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 tenxdnal residues have been deleted (i.e., deletions up to the
conserved cysteine pair
at positions 12 and 13 of human, marine, and rat mature 1W7C). As set forth in
detail below,
2 0 a~perimental data indicates that most or all of these analogs will possess
reduced 11~C biological
activities and, in fact, will act as inhibitors of one or more biological
activities of mature MDC.
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 1D NO: 2,
the tryptophan amino acid at position 59 of SEQ 1D NO: 2, and/or the valine
amino acid at
position 60 of SEQ m NO: 2; and (3) substitution of a basic or small, non-
charged amino acid
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(e.g., lysine, arginine, histidine, glycine, alanine) for the glutamic acid
amino acid at position 50
of SEQ m NO: 2. Specific analogs having these amino acid alterations are
encompassed by the
following formula (SEQ m NO: 25):
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 G~rs 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 G~rs Pro Arg Pro Gly
25 30 35 40
Val Val Leu Leu Thr Phe Arg Asp Lys Xaa Ile CSrs Ala Asp Pro Arg
45 50 55
Val Pro Xaa Xaa Lys Met Ile Leu Asn Lys Leu Ser Gln
2 0 60 65
wherein the amino acid at position 24 is selected from the group consisting of
arginine, glycine,
alanine, valine, leucine, isolauxne, proline, serine, threonine,
phenylalanine, tyrosine, tryptophan,
aspartate, glutamate, asparagine, glutamine, cysteine, and methionine; wherein
the amino acid at
2 5 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
3 0 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, argirune, 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, argmine,
histidine, aspartate,
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glutamate, aspaiagine, 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 lVmC to its receptor. In one
preferred embodiment,
1VIDC polypeptide analogs of the invention comprise amino acids 1 to 69 of SEQ
ID NO: 25.
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 )D NO: 2; (c) a polypeptide comprising
a sequence of
amino aads 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.
As set forth in detail below, experimental data indicates that the addition of
as few
as one additional amino aad at the amino terminus of human mature lvmC 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
2 0 (1996); Simmons et al., Science, 276 (5310): 276-279 (1997).
Also as set forth in detail below, evidence exists that mature NiDC is cleaved
in
vii by a dipeptidyl amino peptidase, resulting in an lViDC(3-69) forn 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 NJDC (e.g., human, marine, and rat NJDC)
is deleted or
2 5 changed to an amino acid other than proline. Such analogs are collectively
referred to as
"MDC~Pro2 polypeptides." Those IvmCAPro2 polypeptides that retain 11~C
biological
activities are contemplated as useful in all indications wherein mature 11~C
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 MDCOProZ polypeptides
that lack
3 0 MDC biological activities are contemplated as being used as lvmC
inhibitors.
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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.
It also will be appreciated that it may be advantageous to express MDC or MDC
analogs as fusions with immunoglobuliri sequences, human serum albumin
sequences, yr other
sequences, or to perform other standard chemical modifications, for the
purpose of extending the
serum half life of the MDC or 1VIDC 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.
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 IVIDC polypeptide analogs; plasmid and viral
vectors incorporating
such polynucleotides, and prokaryotic and eukaryotic host cells stably
transformed with such
DNAs or vectors.
In another aspect, the imrention includes antibody substances (eg., 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
2 0 well-known ELISA techniques, and for modulating binding of MDC to its
receptor(s). The
invention fixrrl~r includes hybridoma cell lines that produce antibody
substances of the invention.
Exemplary antt'bodies of the invention include monoclonal antibodies 252Y and
2522, which are
produced by hybridoma cell line 252Y and hybridoma cdt line 2522,
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).
Recombinant MDC polypeptides and polypeptide analogs of the invention may be
3 0 utilized in a like manner to antibodies in binding reactions, to identify
cells expressing receptors)
of NIDC and in standard expression cloning techniques to isolate
poiynucleotides encoding the
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receptor(s). Such NN1DC 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 agorusts and antagonists.
Additional aspects of the invention relate to pharmaceutical utilities of NiDC
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
IviDC polypeptide
or polypeptide analog of the imrention, wherein the IvIDC polypeptide or IVIDC
polypeptide
analog modulates leukocyte chemotaxis in the host. In preferred methods, the
leukocytes are
monocytes and/or macrophages. For example, empirically determined quantities
of IvIDC are
administered (e.g., in a pharmaceutically acceptable carrier) to induce
macrophage chemotaxis or
to inhibit monocyte chemotaxis, whereas inhibitory NIDC polypeptide analogs
are employed to
achieve the opposite effect.
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
2 0 eive 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 1VIDC is an amount capable of inhibiting fibroblast cell
proliferation. Such
therapeutically effective amounts are empirically determined using art-
recognized dose-response
assays.
As an additional aspect, the invention provides a pharmaceutical composition
comprising an 1VIDC polypeptide or polypeptide analog of the invention in a
pharmaceutically
acceptable carries. 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
3 0 site of inflaimrnation in a patiem having the disease state. In another
embodiment, the disease state
is characterized by fibroblast cell proliferation in a patient having the
disease state.
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NB7C induced chemotaxis of natural killer cells (NK) can lead to enhanced
cytotoxieity 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 N)DC or MDC
agonists would lead to improved immune response to tumors. [See generally
~nunolo~r (Ed.
Kuby, J.} pp 304-6, W.H. Freeman and Co., New York, New York (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. Ed. Kuby J. pp 420-425, W.H. Freeman and Co. New
York, New York (1992}]. "Treatmeraa' as used herein includes both prophylactic
and therapeutic
treatment.
The apparent optimal concentration of mature MDC in receptor binding and
chemotaxis eacp~imems is about 10 ng/ml. Thus, for therapeutic methods
involving the systemic
administration of MDC (or MDC analogs retain;ng a desired NiDC biological
activity), doses and
dosing schedules are preferably selected to maintain circulating
concentrations in blood of about
0.1-10 nglml. Preferred approaches for preparing a dose and maintaining such
levels in the bloods
include administration oflVmC in a bolus fashion, so as to administer
approximately 0.1-10 mg
of ~. 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, e.g., Stability and Characterization of Protein and Peptide
Drugs; Case
Histories, Wang YJ and Pearhnan 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
fiimily, zwittergent
family, or pluronic fi~mily, and amino acids such as cysteine to maintain the
proper oxido-
3 0 reductant state.
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'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, IL, 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 LM Sanders, in
Peptide and Protein
Drug Delivery, V.H.L. Lee (Ed.), Marvel Dekker, Inc., New York, pp. 785-806
{1991)]. For
example, MDC is incorporated into biodegradable microspheres, such as
poly(laetic-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
transderma! delivery made available by Atza Corporation, Palo Alto, Calif.
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
andlor to promote the activities of endogenously produced or exogenously
administered MDC,
have therap~tic utility. Such modulators typically include small molecules,
peptides, chemokine
2 0 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
2 5 methods of the invention also are considered aspects of the invention.
In one embodiment, the imrention 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 delectably-labeled MDC; (c) providing a test composition comprising
detectably-
3 0 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
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to MDC receptors; (e) contacting the second receptor composition with the test
composition
under conditions wherein MDC is capable of binding to 1V>DC receptors; (f)
washing the first and
second receptor compositions to remove detestably-labeled MDC that is unbound
to MDC
receptors; (g) measuring detestably-labeled IViDC 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 detestably-labeled MDC
between the first
second receptor compositions.
As reported herein, the chemokine receptor CCR4 has been demonstrated to be
a high affinity receptor for NiDC. 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 fiaction of cells that express CCR4. Cells that naturally express
CCR4 and cells that
have been transformed or transfected to express CCR4 recombinantly are
contemplated. In an
alternative embodiment, cells (e.g., eosinophils) that express an lVmC
receptor other than CCR4
are used to provide the composition comprising IvIDC receptors.
In a related aspect, the invention provides a method for identifying a
modulator
of binding between NII')C and CCR4, comprising the steps of (a) contacting
IV~C and CCR4
2 0 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 NB'~C 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
2 5 buffered aqueous suspension.
In one embodiment, the method is performed with labeled MDC. In step (b),
binding between MDC and CCR4 is detected by detecting labeled 11~C bound to
CCR4. In a
preferred embodiment, the comacting step comprises contacting a suspension of
cell membranes
comprising CCR4 with a solution containing MDC. In a highly preferred
embodiment, the
3 0 method fiuther comprises the steps of recovering the cell membranes from
the suspension after
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the contacting step (e.g., via filtration of the suspension); and washing the
cell membranes prior
to the detecting step to remove unbound 1VIDC.
In an alternative embodiment, the method is performed with a host cell
expressing
CCR4 on its stuface. In step (b), binding between MDC and CCR4 is detected by
measuring the
conversion of GTP to GDP in the host ce-71.
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 cAIVIP levels in the host cell.
It will be appreciated that assays for modulators such as those described
above are
often performed by itnmobilizmg (e.g., on a solid support) one of the binding
partners (e.g.,1V)DC
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.
In yet another embodiment, the invention provides a method for identifying a
chemical compound having IVjDC modulating activity, comprising the steps of
(a) providing first
and second receptor compositions comprising MDC receptors; (b) contacting the
first receptor
compo~tion with a control composition comprising detectably-labeled Iv>DC; (c)
contacting the
second receptor composition with a test composition comprising delectably-
labeled IvmC and
further comprising the chemical compound; (d) washing the first and second
receptor
2 5 compositions to remove delectably-labeled IVmC that is unbound to IvmC
receptors; (e)
measuring delectably-labeled 11~C in the first and second receptor
compositions after the
washing; and (f) identifsring a chemical compound having MDC modulating
activity, wherein
N117C modulating activity is correlated with a difference in delectably-
labeled NIDC between the
first and the second receptor compositions.
3 0 In yet another embodiment, N>DC binding to its receptor is measured by
measurement of the activation of a reporter gene that has been coupled to the
receptor using
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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).
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.
As taught herein in detail, 11~C stimulates eosinophil chemotaxis through a
pathway that apparently does not involve the chanokine receptor CCR4. This
discovery provides
for the design of assays to identify modulators of MDC activity that have
specificity for CCR4-
mediated activities without affecting lvmC-induced stimulation of eosinophils,
and vice versa.
~ For example, in one anbodiment, the invention provides a method for
identifying
a modulator of binding between lVmC and eosinophils, comprising the steps of
(a) contacting
MDC and a composition comprising an MDC receptor that is expressed on
eosinophil cell
membranes in the presence and in the absence of a putative modulator compound;
(b) detecting
binding between MDC and the composition; and (c) identifying a putative
modulator compound
in view of decreased or increased binding between MDC and the composition in
the presence of
the putative modulator, as compared to binding in the absence of the putative
modulator.
To identify modulators with eosinophil-specificity, the method, in a preferred
embodiment, fiuther comprising the steps of (d) contacting lVmC and a
composition comprising
CCR4 in the presence and in the absence of the putative modulator compound;
(e) detecting
2 0 binding between IvmC and CCR4; (f) 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;
and (g) selecting a
modulator identified in step (c) as causing increased or decreased binding and
identified in step
(f) as failing to cause increased or decreased binding. To identify modulators
with specificity
2 5 towards CCR4, in step (g) one selects a modulator identified in step (f)
as causing increased or
decreased binding and identified in step (c) as failing to cause increased or
decreased binding.
MDC's involvement in various aspects of immune responses is described in
detail
below. Based on the involvement of Nll~C in immune response, the
administration of IV1DC
antagonists is indicated, for example, in the treatment anaphylaxis [Brown,
A.F., .l. Accic~ Emerg.
30 Med, 12(2):89-100 (1995)], shock [Brown (1995) supra], ischemia,
reperfixsion injury and
cerrxral ischemia [Lindsberg et al., Ann. Neurol., 30(2):117-129 (1991)],
atherogenesis [Handley
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et al., Drug Dev Res., 7:361-375 (1986)], Crohn's disease [Denizot et al.,
Digestive Diseases
a»d Sciences, 37(3):432-437 (1992)], ischemic bowel necrosis/necrotizing
enterocolitis [Denizot
et al. (1992), suprcx 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., StroJre, 21:1452-1457 (1990) and
Lindsberg et al. (1991),
supra], systemic lupus erythematosus {Matsuzaki et al., Clinica Chimica Acts,
210:139-144
(1992)], acute pancreatitis [Kald et al., Pancreas, 8(4):440-442 (I993)],
septicemia (Kald et al.
(1993), supra), acute post-streptococcal glomerulonephritis [Mezzano et al.,
J. Am. Soc.
Nephro~, 4:235-242 (1993)], pulmonary edema resulting from IL-2 therapy
[Rabinovichi et al.,
J. Clin. Irrvest., 89:1669-1673 (1992)], ischemic renal failure [Grino et aL,
Annals of Internal
Medicine, 121(5):345-347 (1994)]; pre-term labor [Hoffrnan et al., Am. J.
Obstet. Gynecol.,
162(2):525-528 (1990) and Maki et al., Proc. Natl. Acad Sci. USA, 85:728-?32
(1988)], adult
respiratory distress syndrome [Rabinovichi et al., J. Appl. Phsiol.,
74(4):1791-1802 (1993);
Matsumoto etal., Clip. Exp. Pharmocol. Physiol., 19:509-515 (1992); and
Rodriguez Roisin et
arl, J. Clir~ Irrv~es~, 93:188-194 (1994)]. "Treatment" as used herein
includes both prophylactic
and therapeutic treatment.
MDC acts as a chemoattractant for TH2 differerniated memory cells, which
produce the cytokines ILr4, IL-5, IL-10 and others. It is expected that, in
some instances, MDC
leads to an immune state in which THl cytokine driven responses are reduced.
In such instances,
2 0 antagonism of MDC would lead to a state in which THl cytokine driven
responses are enhanced.
Modulation of the THl-TH2 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 assoaated with undesirable or excessive TH2 responses and/or less-
than-desirable THl
responses are contemplated as additional aspects of the invemion.
Administration of sui~cient
MDC antagonists to substa~ially reduce ex~dogenous IL-10, a THl immune
suppressing cytokine,
would lead to enhanced cytotoxic T-lymphocyte mediated immunity and immune
surveillance [see
Muller et al., J. Infect. Dis , 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
CA 02302806 2000-03-08
wo ~nst~s Prrms9snoz~o
-22-
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 [Mosey et al., APMIS, 105(11):838-42 (1997)], to delay HIV-1
induced
immunodefic~ency [Bergen et al., Rer. Yiro~, 147(2-3) :103-108 ( 1996); Barker
et al., Proc. Natl.
Acad Sci. USA, 92(24):11135-9 (1995); Jason et al., J. Acquir. Immune. Defic.
Syndrome
Retrovirol., 10(4): 471-6 (1995); Maggi et al., J. Biol. Regal. Homeost.
Agents, 9(3): 78-81
(1995)], chronic interstitial lung disease [Kunkel et al., Sarcoidosis Yasc.
Dose Lung Dis , 13:
120-128 (1996)], in neurological disorders associated with a TH2 response
[Windhagen et al.,
Chem. Immunol., 63: 171-86 (1996); Bai et al., Clin. Immureol. Immunopathol.,
83(2): 117-26
(1997)], colorectal cancer [Pellegrini et al., Cancer Immunol. Immunother.,
42(1): 1-8 (1996)],
viral infection, for example various speaes of herpes and hepatitis [Spruance
et al., Aretiviral Res ,
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 etat., J. Infect. Dis, 176(3):
728-39 (1995);
Cenci etal., J. IrfectDi~, 171(5): 1279-88 (1995)j, chronic pneumonia
[Johansen et al., Behring
Inst. Mitt., 98: 269-73 (199'1)], solid tumor cancer [Khan eta~, CytoldnesMol.
They., 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 pathog~esis (Deptia et a1, Arch. Dermatol Res , 289(12):
667-70 (1997)],
glomerulonephritis [Kitching et al., Kidney Inf., 53(1): 112-8 (1998); Huang
et al., J. Am Soc.
Nepro~, 8(7): 1101-8 (1997); Tipping et al., Eur. J. Immunol., 27(2): 515-21
(1997)], pulmonary
respiratory syncytial virus infection [Russell et al., Eur. J. Immunol.,
27(12): 3341-9 (1997)],
complications of trauma associated with surgical stress [Decker et a~,
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)], ameoboc3rte
infection, for example
Plasmodium falciparum [Elghazali et al., Clin. Exp. Immunol., 109(1): 84-9
(1997)] and
schistosomamansoni[Wolowczuketa~, Immunol., 91(1): 35-44 (1997)], and B-cell
lymphoma,
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especially mucosa-assoaated lymphoid tissue type [Greiner et al., Am .I.
Pathol., 150(5): 1583-93
(1997)]. "Treatment" as used herein includes both prophylactic and therapeutic
treatment.
In fad, the expression pattern of MDC (or TARC) and its receptor CCR4 provide
a unique indication for IVmC in viva in inducing a cellular complex (e.g.,
dendritic and/or
macrophage cells; TH2 antigen-specific memory cells, and antigen-specific B
cells} geared to
producing a strong humoral immune r~eesponse. The induced complex is
contemplated to produce
antigen-specific antibodies and TH2-specific cytokines (ILr2, IL,-4, IL,-5,
and/or IL-10) and
additional chemokines, including additional lvmC, with local concentrations of
the chemokines
and cytokines that potentiate the activity of the complex possibly being quite
high. The cellular
complex is specifically contemplated to be involved in the establishment of a
humoral response
to "recall antigens," since another chemokine/receptor pair (M>P3aJCCR6)
appears to be specific
for "naive" responses to new antigens. Thus, administration of MDC or IVmC
agonists for the
purpose of inducing or augmenting a response to "recall antigens" is
specifically contemplated as
an aspect of the intention. Similarly, administration of MDC antagonists is
indicated when
suppression of such an immune response is desired. Administration of Ivfi3C
antagonists to treat
conditions and disorders mediated (directly or indirectly) by TH2 cell
migration, including but not
limited to autoimmune conditions, lupus erythematosus, multiple sclerosis,
scleroderma, asthma,
and atopic allergy, is specifically contemplated.
With respect to any of the conditions, disorders, and disease states
identified in the
2 0 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 1V87C antagonist compound. By
"therapeutically
effective amount" is meant a dose and dosing schedule that is sufficient to
cure the disease state,
2 5 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 states 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 lVmC
antagonist.
Therapeutically effective amounts are readily determined by dose-response
studies that are
3 0 conventionally performed in the art.
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In one highly preferred embodiment, the invention includes a method of
inhibiting
proliferation of a mammalian immunodeficiency virus comprising the step of
contacting
marrnnalian 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
s 0 an infecxed 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.)
The term "MDC-IV antagonist compound" refers to compounds thax antagonize
the apparent Immunodeficiency Virus-proliferative effects of MDC in infected
cells. Thus, the
term "MDGIV 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 1VIDC neutralizing antibodies or the inhibition
reported herein for
certain 1VIDC analogs (e.g., analogs having amino terminal additions or
truncations). For example,
2 0 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-1VIDC
antibody and that are
capable of binding to 11~C are preferred 1VIDGIV antagonist compounds.
As described elsewhere herein in greater detail, amino-terminal truncations of
mat<we human 1~C(1-69) possess antiproliferative activity against HIV-1. Thus,
another set of
preferred lVmC-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 su~cient 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
3 0 lViDC(1-69). Amino tenrucsal deletion analogs that have been further
modified, e.g., by including
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an oligopeptide tag to facilitate purification, or by including an initiator
methionine for bacterial
expression, are also contemplated.
Amino-terminal additions to mature MDC also result in analogs possessing
antiproliferative activity against HIV-1. Thus, another set of preferred 1VIDC-
IV antagonist
compounds are polypeptides that comprise a mature MDC sequence (e.g., amino
acids 1-69 of
SEQ ID N0: 1), and that further comprise a chemical addition to the amino
terminus of the
mottos NL<~C sequence to rider said polypeptide antagonistic to IvmC.
Additions of additional
amino acids and other chemical moieties are contemplated.
It will fiirther be appreciated that substitution of amino acids in a mature
MDC
sequence (especially substitutions in the amino terminus of mature 1VIDC) may
be expected, in
some instances, to result in analogs possessing antiproliferative activity
against HIV-1. Such
analogs also are intended IVLDC-IV antagonist compounds, and are identifiable
using HIV
proliferation assays described herein.
It is postulated that MDC's HIV-prolife~rative 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 fi~agment 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
2 0 contemplated are anti-CCR4 antibodies, which are expected to block MDC-
CCR4 interactions,
thereby inhibiting IVmC-induced HIV proliferation.
As descn'bed herein in detail, the chemokine TARC possesses sequence
similarity
to MDC, possesses various overlapping biological activities, and, like IvIDC,
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.
3 0 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
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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 1D 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.
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.
The term "IVmGPA antagonist compound" refers to compounds that antagonize
2 0 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 1VIDC to a mammalian subject (e.g.,
to a mouse or rat).
Those compounds described above as MDC-IV antagonist compounds are
specifically
contemplated as lVmC PA antagonist compounds as well. For example, anti-MDC
antibodies are
2 5 highly preferred lVmGPA antagonist compounds. For treatment of humans,
humanized
antibodies are highly pref~Ted. Similarly, polypeptides that comprise an
antigen-binding fragment
of an anti-NmC antibody and that are capable of binding to Nn7C are preferred
IvmC-PA
antagonist compounds. All 1VlDC analogs that inlu'b'rt the platelet
aggregating effects of lVmC also
are preferred. Analogs having additions, deletions, and/or substitutions in
the amino terminus are
3 0 specifically contemplated.
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The structural and functional similarities between M'DC and TARC reported
herein
indicate that compounds that intu'bit TARC-CCR4 interactions will be useful
for inhibiting platelet
aggegation. Compounds that inhibit TARC-induced platelet aggregation are
collectively referred
to as "TARC PA antagonist compounds." Such compounds include anti-CCR4
antibodies, anti-
s 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.
As described herein in detail, the expression patterns of 1V1DC 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 earner, 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. As
explained above, MDC is contemplated to preferentially augment an immune
response to "recall
antigens." Accordingly, in a preferred embodiment, IvIDC is included in a
booster vaccine
composition.
2 0 For any of the therapeutic indications and methods descn'bed above,
another aspect
of the invention relates to the use of indicated compounds (e.g., MDC, MDC
fragments or
analogs,1VR7C agonists, or MDC antagonists) for preparation of a medicament
for the therapeutic
indication. For example, the invention includes the use of an MDC antagonist
for preparation of
a medicament for suppressing a humoral response to recall arnigens.
2 5 The foregoing aspects and numerous additional aspects will be apparent
from the
drawing and detailed description which follow.
BRIEF DESC'RIp'fION OF THE DRAWING
FIGURE I is a comparison of the amino acid sequence of human M'DC (SEQ ID
3 0 NO: 2) with the amino acid sequences of other, previously characterized
human C-C chemokines:
MCP-3 [Van Damme et al., .I. Exp. Med, 176:59 (1992)] (SEQ ID N0: I8); MCP-1
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[Matsushima et al., J. Exp. 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);
RAN'TES [Schall et al., J. Immunol., 141:1018 (1988)] (SEQ ID NO: 21); MIP-lei
[Brown et
al., J. Immunol., 142:679 (1989)] (SEQ ID NO: 22); MIP-la [Nakao et al., Mol.
Cell Biol.,
10:3646 (1990)] (SEQ ff~ NO: 23); ana 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.
Figure 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
THIS-1. The op~ diamond shows the response to the positive control, zymosan
activated serum
(ZAS).
Figure 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.
Figure 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
2 0 show the response to MDC of monocytes derived from the cell line THP-1.
Figure 5 is a graph depicting the chemotactic effect (measured in fluorescence
units) of increasing concentrations of MDC on guinea pig peritoneal macrophage
migration.
Closed arcles show the response of macxophages to MDC. An open triangle shows
the response
to the positive control, zymosan activated serum (ZAS).
2 5 Figure 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 chemotactio-inhibitory effects of MDC
where chemotaxis
has been induced by MCP-1. Open circles depict the chemotactio-inhibitory
effects of MDC in
a control experiment wherein only the basal medium (RPMI with 0.2% BSA (RBSA),
no MCP-1)
3 0 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.
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Figure 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 lOF). Open circles depict the response with chemically synthesized
MDC (Example
11).
Figure 8 schematically depicts the construction of mammalian expression vector
pDC 1.
Figure 9 depicts the nucleotide and deduced amino acid sequence (SEQ m NOs:
39 and 40) of a S. cerevisiae alpha factor pre-pro/human MDC cDNA chimeric
construct used
to express human MDC in yeast.
Figure 10 depicts the structure of plasmid pYGL/preproMDC, used to express
human MDC in yeast.
Figure 11 depicts the inlu'bitory effects of the anti-MDC a~'bodies 252Y and
2522
on the binding of the fusion protein MDC-SEAP to the RFC receptor designated
CCR4. Binding
(depicted as percent of maximal binding) is plotted as a function of increased
concentrations of
antibody
Figure 12 depicts the inhibitory effects of the anti-MDC antibodies 252Y and
2522
on the NIDC-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
2 0 concentrations of antibody.
The preset 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 chemolcine"). 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 Z
presents a composite MDC cDNA nucleotide sequence and presents a
characterization of the
3 0 deduced amino acid sequence of the chemohine (1VIDC) encoded thereby. In
Example 3,
experiments are described which reveal the level of MDC gene expression in
various human
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tissues. The geatest 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.
Since MDC gene expression was detected in thymus and spleen in Example 3, in
situ hybridization studies were conducted to locaiize further the MDC gene
expression in these
tissues. Moreover, in situ 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.
Example 6 descxibes 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.
Example 8 provides experimental protocols for purification of recombinant MDC
produced, e.g., as descn'bed in Example 7. Examples 9 and 10 provide protocols
for the
recombinant production ofMDC 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.
2 0 Example 11 describes production of MDC and MDC polypeptide analogs by
peptide synthesis.
Certain preferred analogs are specifically described in Example 11.
Examples 12-17 provide protocols for the determination of MDC biological
activities. For instance, Example 12 provides an assay of IvlI3C effects upon
basophils, mast cells,
and eosinophils. NmC-induced chemotaxis of eosinophils is specifically
demonstrated. Example
2 5 13 describes assays of chemoattractant and cell-activation properties of
MDC on
monocyteslmacrophages, n~trophils, and ganulocytes. MDC induced macrophage
ehemotaxis,
but inhibited monocyte chemotaxis.
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
3 0 and 16 provide protocols for assaying MDC activity via intraperitoneal and
subcutaneous
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injection, respectively. Example 17 provides protocols for determining the
myelosuppressive
activity of MDC.
Example 18 provides protocols for generating antibodies that are specifically
immunoreactive with NmC, including polyclonal, monoclonal, and humanized
antibodies. Uses
of the antibodies also are described.
Example 19 provides a calcium flux assay for determining the ability of MDC to
induce cellular activation.
Example 20 provides assays and experimental results relating to the HIV
proliferative and anti-proliferative effects of human mature MDC and NmC
antagonists.
Example 21 demonstrates the anti-proliferative effects of lVmC 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 viva assay for determining the anti-
proliferative effects of
lVmC on fibroblasts.
Example 24 describes the chromosomal localization of the human MDC gene.
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.
Example 28 describes the isolation of cDNAs encoding rat, mouse, and macaque
MDC, and characterizes the MDC proteins encoded thereby. Example 29 further
characterizes
2 0 selected MDC analogs.
Example 30 describes experiments that demonstrate that anti-IVmC monoclonal
antibodies are effective for neutralizing biological activities of MDC that
were elucidated in other
examples.
Example 31 describes experiments that demonstrate that 11~C induces chemotaxis
of TH2 helper cells, a discovery with therapeutic implications as discussed in
Example 31 and
elsewhere herein.
Example 32 describes platelet-aggregating activities of MDC, and describes the
use of MDC and MDC antagonists to modulate platelet aggregation.
Example 33 provides exemplary assays to demonstrate the therapeutic efficacy
of
3 0 an MDC antagonist to modulate immune responses in a mammalian host.
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A partial cDNA for a new C-C chemokine, designated pMP390, was isolated from
a ma~xophage cDNA library as described in U.S. Patent Application Serial No.
08/939,107, filed
September 26, 1997, and in related international publication number WO
96140923, both of which
are incorporated herein by reference. Sequence comparisons were performed on
December 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~/0
identity with previously-identified chemokine genes, including the human MCP-3
gene and rat
MIP-lei gene.
The 2.85 kb cDNA insert of pMP390 was subcloned into the vector pBluescript
SK (Stratagene, La Jolla CA) to facilitate complete sequencing. The complete
sequence of this
pMP390 cDNA corresponds to nucleotides 73 to 2923 of SEQ B? 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.
Isolation of additional cDNA clones having
2 0 the c;omRlete MDC codin$~~e_~auence
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.S.N. 08/939,107 and WO 96/40923, incorporated herein by reference.
Ofthe 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
3 0 pMP390-12 cDNA sequences. This 2923 base pair composite cDNA sequence, and
the deduced
amino aad sequence of the chemokine MDC, are set forth in SEQ TL7 NOs: 1 and
2, respectively.
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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 (Figure 1). As aligned in Figure 1, MDC shares 29% amino acid
identity with MCP-I
and MIP-la, 28% identity with MCP-2, 32% identity with I-309, 33% identity
with MCP-3 and
MIP-lei, 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.
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 Res ,
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 ofMDC beginning with the glycine residue at position 1
of SEQ m NO:
2. This prediction was confirmed by direct sequencing of MDC protein produced
recombinantly
I5 in mammalian cells, as described below in Example 10. The MDC composite
cDNA sequence
shown in SEQ ID NO: I extends nineteen nucleotides upstream of the predicted
initiating
methionine codon, and 2.6 kb downstream of the termination codon.
T~eternination ofMDC CTe_n_e Expression in Human Tissues
Northern blot analysis were conducted to determine the tissues in which the
MDC
gene is expressed.
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,
CA) 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°C with 2 intensifying
screens.
The greatest MDC gene expression was observed in the thymus, with much weaker
3 0 expression detectable in spleen and lung tissues. Expression of MDC in
tissue from the small
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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.
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
far the
chemokine TARC. See Imai et al., J. Biol.'Chem., 272: 15036-15042 (I997). 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'" 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. F.xp. 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.
These expression pattern data suggest therapeutic utilities of MDC (or MDC
2 0 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.
Conversely, the expression pattern data also indicates a therapeutic utility
for
2 5 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.
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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.
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. Bio~
Chem., 265: 9682-9687 (1990)J.
A Northern blot of RNA (10 pg 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.
To confirm the expression of MDC in differentiated human macrophages, culture
2 0 supernatants were analyzed by western blotting with anti-MDC monoclonal
antibodies produced
as described below in Example I8. 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, Nhnnesota).
The medium from the differentiated macrophage cell cultures was removed and
2 5 replaced with similar medium or with medium containing low density
lipoprotein (LDL, Sigma),
oxidized LDL (oxidized by incubation in SpM CuS04~SHZO 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 HCI, and passed over a Heparin-Sepharose CL-6B column (Phannacia,
Piscataway,
3 0 Nn. The column was washed with 0.2 M NaCI in 20 mM Tris, pH 8, and eluted
with 0.6 M NaCI
in 20 mM Tris, pH 8. The eluted material was fractionated on an 18% acrylamide
SDS-PAGE
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gel (NOVE3~ and electroblotted to PVDF membrane (Millipore, Bedford MA). The
filter was
blocked, washed, and reacted with monoclonal antibodies against MDC using
standard techniques
(Sacnbrook et al.). In each of the culture media analyzed, MDC protein was
detected at a
concentration of approximately 0.5 pg/ml, thus confirming expression of MDC in
differentiated
human macrophages.
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.
In addition, samples of the T84 cell line were treated for 1 day with TNFa (5
ng/ml, PeproTech, Rocky Hill, New Jersey), TGF-~i (1 ng/ml, R&D Systems), or
interferon-'y
(200 U/ml, PeproTech), 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 Ch~nical 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.
2 0 C.
Further examination of MDC gene expression in macrophages was conducted by
treating the human cel! line HI.60 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., Blooa;
S8: 836-843 (1981)]. RNA was isolated from untreated cells and from cells
treated for one or
three days with DMSO or PMA, electrophoresed (lOpg/Iane), and blotted. The
Northern blot
of the RNA was probed with the radiolabeled pMP390 5' fragment described in
Example 3.
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
3 0 days of culture (see above). No expression was seen after one day of
treatment or in untreated
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cells. Fucth~, no detectable expression of MDC was induced by treatment with
DMSO for one
or three days.
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.S.N. 08/939,107 and WO 96/40923,
both of which are
incorporated by reference.
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 din 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.
Colon samples from patients with Crohn's disease exhibited hybridization in
cells
of the epithelium, lamina propria, Payees patches, and smooth muscle. In
contrast, normal human
2 0 colon showed no hybridization above background. The observed pattern of
1V1DC expression in
the colons of Crohn's disease patients closely correlates with the expression
of a maerophage-
specific gene, Platelet Activating Factor Acetylhydrolase (PAF-AITj [Tjoelker
et al., supra]. This
result, together with the data presented in Example 4, suggest that
macrophages express MDC
cDNA in viva during pathogenic inflammation. Moreover, the identification of
MDC in Crohn's
2 5 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.
3 0 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-3X
(Pharmacia,
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Piscataway, NJ). 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.
An MDC cDNA fragment was amplified by PCR using the primers 390-2R (SEQ
ID NO: 8) and 390-FX2 (SEQ lI? NO: 1 I~. Primer 390-FX2 contains a BamH I
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 fission
protein, releasing the
mature chemolcine from the GST fusion.
The PCR product was purified by agarose gel electrophoresis, digested with
BamH
I endonuclease, and cloned into the BamH I site of pGEX-3X. This pGEX-3X/MDC
construct
was transformed into E. coli XL-1 Blue cells (Stratagerie, La Jolla CA), and
individual
transformants were isolated and grown. Plasmid DNA fibm individual
transformants was purified
and partially sequenced using an automated sequencer and primer GEXS (SEQ ID
NO: 12),
which hybridizes to the pGEX-3X vector near the BamHI cloning site. The
sequence obtained
with this primer confirmed the presence of the desired N1DC insert in the
proper orientation.
Induction of the GST-MDC fusion protein was achieved by growing the
transformed XL-1 Blue culture at 37°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 ~i-D-Thiogalactopyranoside (Sigma
Chemical Co., St.
Louis MO).
The fusion protein, produced as an insoluble inclusion body in the bacteria,
was
2 5 purified as follows. Cells were harvested by centrifugation; washed in
0.15 M NaCI, 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 X 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 X g. The pellet was resuspended in standard phosphate
buffered saline
solution (PBS) free of Mg~ and Ca~. The fusion protein, which remained
insoluble, was
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approximately 80-90'/n 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/1 of E. coli culture.
The fusion protein was subjected to thrombin digestion to cleave the GST from
the mature MDC protein. The digestion~eaction (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.
The identity of the 7 kD SDS PAGE fragtnent was conf rmed 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, CA). Subjecting the sample to automated sequencing
(Applied
Biosystems Model 473A, Foster City, CA) 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).
2 0 MDC peptides and analogs can be expressed using a variety of bacterial
expression
systems including E. coli, Bacillus subtilis, streptomyces lividans, and many
others. [For a
general review see "Gene Expression Technology" in Vol. 185: pp. 1-
283, Ed. D.V. Goeddel, Academic Press, San Diego, CA (1990).] In general, an
expression
cassette comprised of a transcription el~nent (a promoter), a translation
element, a coding region
to be expressed (for example MDC), and a transcription ternnination 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.,
peXB, ompA, esl2) to
3 0 direct secretion of the gene product out of the cytoplasm into either the
periplasmic space or
media, or to other leader sequ~ces (e.g., ubiquitin) to enhance the folding or
otherwise stabilize
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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.
A. Conctn~ction and testing o~Bacterial_ MI~C Expression Vector P2-390
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)].
More pardcx~larly, an MDC cDNA was amplified by PCR using approximately 0.1
pg of pMP390-12 as template and synthetic oligonucleotide primers 390-2R (SEQ
ID N0:8) and
390-Pel (SEQ D7 NO: 13). Primer 390-Pel contains an Nco I restriction site,
followed by two
cytosine residues, followed by bases 92 to 1 I S of SEQ ID NO: 1.
The expected PCR product of 232 by was purified by agarose gel
electrophoresis,
digested with Nco I and BamH I, and cloned along with a portion of the
arabinose operon and
peIB leader sequence (Better et al., supra) into the vector pUCl9 (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 Aral (SEQ ID N0:28) and Ara2 (SEQ ID
NO:29),
which annul to the vector adjacent to the cloning site. The plasmid P2-390 was
transformed into
the E. coli strain MC1061 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,
CA) and MDC production was induced by the addition of 50% arabinose to a final
concentration
of O.I%. 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
2 5 pg/g of cell paste and was secreted into the culture medium to a level of
approximately 1 pg/ml.
B. Protocol for bacterial expression of MDC using pl a id 2-390
The plasmid P2-390 was transformed into E. coli strain SB7219 (Sheppard and
Englesbexg, J. Molec. Biol., 25:443-454 (1967) and Wilcox et al., J. Biol.
Chem., 249:2946-2952
3 0 (I974)). 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.
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coliKl2 F' del(cealb-lac)3 del(ara735) rpsL150(strR) ~,'. 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:
Basal Medium
Component Quantity per L
Na3citrate lg
5.4% FeCl3 6Hz0 2 ml
glucose 2 g
NaHzPO,; H20 3 g
KzHP04 6 g
(NH,~SO~ 5 g
20% yeast extract solution5 ml
1 M CaCIZ 0.5 ml
1 M MgCl2 2.0 ml
trace elements 4 ml
trace vitamins 2 ml
1% thiamine 1 ml
tetracycline 5 mg
2 0 pH is set to 7.0
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Trace Elements Solution
Component Quantity per L
Boric Acid 5.0 g
Copper Sulfate SHiO 2.0 g
Potassium Iodide 1.0 g
Manganese sulfate 10 g
Molybdic acid o.s g
ZnCl2 {Anhydrous) 5.2 g
Cobalt chloride 0.5 g
Tr$ce Vitamin Solution
Component Quantity per L
Sodium Hydroxide, 50% 1.3 ml
Riboflavin 0.42 g
Folic Acid 0.04 g
D-Pantothenic Acid (hemicalcium5.4 g
salt)
Ncotiinic Acid (niacin) 6.1 g
Pyridoxine HCl 1.4 g
Biotin 0.06 g
The shake flask culture is grown at 3TC and 220 RPM to an optical density
corresponding to
mid-exponential growth (approximately ODD ~ 0.7). The inocuium is added to the
fermentor
containing 1.5 L of basal media and grown at 30'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
2 5 of feed is achieved.
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Feed Medium
Component Quantity per L
Na3citrate Sg
5.4%FeCl3 . 6H20 10 ml
glycerol 500 g
~4)2SO4 5 g
1 M CaCl2 4 ml
1 M MgCiz 100 ml
1 M MnClz 0.4 ml
trace elements 10 ml
When the wet cell mass is approximately 100gIL, 20 ml of 50% arabinose
solution is added to
induce expression of MDC. The temperature is raised to 37°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°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.
C. Direct Exrrcession ~f MDC in E. coli
2 0 In a similar way, MDC that is directly expressed (i. e., without a fused
in-fr ame
leader sequence) is engineered into the same vector. The plasmid pBARS/MDC/RC
is a plasmid
id~tical to P2-390 except for the elimination of the pelB leader sequence. In
addition, the first
fourteen perc~t 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
2 5 nucleotide (while preserving the encoded amino acid). Additionally, a
translation initiation codon
was added. Thus, the coding sequence in pBARS/MDC/RC begins:
5' ATG GGA CCA TAT GGA GCA AAT ATG GAA GAT AGT ...... (SEQ ID NO: 44 )
E. coli strain SB7219 harboring this plasmid is gown in a fermentor
essentially as described
above and the MDC that is produced is similarly recovered.
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_ 4,4, _
D. P2-390 Slariant e~ , ression vector
In addition, a derivative of P2-390 pBARS/PeIB/MDCIRC 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.
Purification of Recombinant MDC from Bacteria and Culture Medium
The following are experim~tal protocols for purification of the recombinant
MDC
l0 produced as described in Example 7.
A. Recovery and Purification of secreted recombinant MDC.
The secxeted recombinant MDC protein is purified from the bacterial culture
media
by, e.g., adapting methods previously described for the purification of
recombinantly produced
RAN'TES chemokine [Kuna et al., J. Immunol., 149:636-642 (1992)], MGSA
chemokine [Horuk
et a~, J. Bio~ Chem. 268: 541-46 (1993)], and IP-10 chemokine (expressed in
insect cells) [Sarris
et al., J. Exp. Med, 178:1127-1132 (1993)].
B. Recover;t and Re-folding of~~,~ Bound in Inclusion Bodies
2 0 Methods for recovery of inclusion bodies from E. coli paste has been weU
d[see Lin et a~, Biotechniques, Il (6): 748-52 (1991); Myers et al., Prot.
Express. Purl, f.,
2: 136-143 (1991); Krueger et al., BioPharm., pp. 40-45 (March, 1989); Marston
et a~,
"Solubilization ofProtein Aggregates," Methods in Enzymology, MP 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% wlv) in
buffer [for example, containing 50 mM Tris HCI, pH 8.0, 1 mM EDTA, 50 mM NaCI,
0.2 mglml
lysozymey 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°C. The soluble components
of the cell are
separated from MDC and the other cellular-derived insoluble components by
applying a
3 0 centrifugal force of approximately 12,000 X g for a period of about 5-10
minutes. The insoluble
pelleted material is then re-suspended and re-centrifixged using dilute
solutions of detergent [for
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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)].
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 O.1M TrisHCl, pH 8.6, 20% (3-mercaptoethanol, for 1 hour at
37°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.
The pH of the solution containing the fully reduced MDC is gradually lowered
to
4.0 with 10% HCI. 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
TiisHCl, 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
2 0 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].
2 5 C. Purification of refolded MDC
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
NaCI, 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
3 0 solution is filtered to remove insoluble materials and applied to the
column to a capacity of 0.5
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Feed
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mg MDC/ml of resin. Loading buffer is then used until the OD2,o returns to
baseline. MDC is
eluted using a higher salt buffer (0.6 M NaCI, 20 mM Tris, pH 7.5).
The SP-Sephadex elution peak is then chromatographed on an WP I~-Propyl (C3)
hydrophobic interaction column (JT Baker #7585-02). The column is equilibrated
with 2.4 M
NaCI, 20 mM Tris, pH 7.5. The 0.6 MlVaCI containing S-P eluate is then
adjusted with the
appropriate amount of 5 M NaCI to bring the salt concentration of the eluate
to 2.4M NaCI. The
adjusted eluate is loaded onto the propyl column at 2 mg of MDC/ml and washed
with 2.4 M
NaCI, 20 mM Tris, pH 7.5, until the OD28o returns to baseline. The column is
then washed with
two column volumes of 2.0 M NaCI, 20 mM NaCI. The purified MDC is eluted from
the column
with 0.8 MNaCI, 20 mM Tris, pH 7.5. Purified MDC is then filter sterilized and
stored at -70°C.
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))
Kluy~eromyces lactis (Fleer et al., BiolTeclmology, 9: 968-975 (1991)), Pichia
pastoris, (Cregg
et al., BiolTechnology, ll: 905-910 (1993)), Schizosaccharomyces pombe (Broker
et al., FEES
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.
2 5 The mRNA typically encodes a coding region of interest preceded by an in
fi-ame leader sequence,
e.g., S. cerevisiae pre-pro alpha factor (Brake et al., Proc. Nat. Acac~ 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.
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 390-1F (SEQ m NO: 7) and 390-2R (SEQ B7 NO: 8). A
DNA
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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 255-235 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. A's taught by Rose and Broach, Meth. Enz , 185:
234-279, D.
Goeddel, ed., Academic Press, Inc., San Diego, CA (1990), the vector fiuther
includes an ADH2
transcription terminator downstream of the cloning site, the yeast "2-nucron"
replication origin,
the yeast leu-2d gene, the yeast REP 1 and REP2 genes, the ~ coli beta-
lactamase gene, and an
E coli origin of replication. The beta-lactamase and l~-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 REP 1 and REP2 genes encode
proteins involved
in regulation of the plasmid copy number.
The DNA construct described in the preceding paragraph is transformed into
yeast
cells using a known method, e.g., lithium acetate treatment [Steams et al.,
Meth. Enz , supra, pp.
280-297]. The ADH2 promoter is induced upon exhaustion of glucose in the
growth media [Price
et a~, Gene, 55:287 (1987)]. The pre-pro-alpha s~uence 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. Nato Acad Sci. USA, 81:5330-5334
(1984)].
Alternatively, IvmC is recombinantly expressed in yeast using a commercially
available expression system, e.g., the Pichia Expression System (Invitrogen,
San Diego, CA),
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 (AOXl)
2 5 promoter upon induction by methanol.
The secreted IVmC is purified from the yeast growth medium by, e.g., the
methods
used to purify IVmC from bacterial and mammalian cell supernatants (see
Examples 8 and 10).
N~7C was expressed in yeast as follows. Using standard molecular biological
methods (Sambrook et al., Molecular Cloning: a Laboratory Manual, Second
Edition, Cold
3 0 Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)) such as
those described above,
the S. cerevisiae alpha factor pre-pro sequence (codons 1-85 in Figure 9) was
fused to the
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presumptive mature form of MDC (SEQ m 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 Figure 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 deriv~l from the plasmid pKDl, a 21t like
plasmid from in K.
drosophilarium (Chen et al., Nucleic Acids Research, 14: 447-81 (1986)). These
vectors are
autonomously replicated and mauitained 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 6418 (200 mglL) and the
giycolytic
enzyme phosphoglucokinase (PGK). E~cient 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.
Plasmid pYGLIpreproMDC was transformed into the pgko deficient host strain
FBOS (Delta Biotechnology Limited) by selecting for 6418 resistance in
YEPPglycerol/ethanol
medium (0.5% yeast extract, 1% peptone, 1 M KPO,, pH 7.0, containing 3%
glycerol and 2%
ethanol). Following clonal isolation, the transformed seed was grown in shake
flask production
medium YEPPgaI (0.5% yeast extract, 1% peptone, 1 M KP04, pH 7.0, containing
2% galactose
as sole carbon source). SDS-PAGE analysis of the culture medium indicated that
a protein
2 0 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.
Western analyses using an anti MDC monoclonal antibody did not reveal the
2 5 presence of MDC-related degrad~ion 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.
The MDC production seed was used to inoculate a fermentor maintained at
26°C
3 0 contairring a batch medium. The composition of the batch medium ( 1200 ml)
was as follows: 7.5
g Yeast extract; 0.6 g MgSO,; 6.0 g NH4S04; 9.6 g KHiF'O,; 26.4 g KzHP04; 11
mg CaCl2; 5.0
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ml 1000X vitamins [Bitter et al., J.Mec~ Yirol., 25(2):123-140 (1988)]; 2.5 ml
1000X trace
elements [Bitter et al. (1988)]; and 1.2 g 30% galactose.
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; MgS04, 4 g; NH4SO4, 40 g; ICHzP04, 60 g; KZHPO4,
165 g; 1000X
trace elements, i 5 ml; 1 OOOX vitamins, 30 ml; 4% CaClz solution, 20 ml.
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.
Yeast-produced MDC was fiuther 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.
Recombinant Production of MDC in Mammalian Cells
MDC was recombinantly producxd in mammalian cells according to the following
procedures.
A. Synthesis of Expression Vector 390HXE
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
394RcX (SEQ
D7 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.
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The expected 423 by PCR product was purified by agarose gel electrophoresis
and
cloned into Hind 1TIlXba I-digested pRc/CMV ((InVitrogen, San Diego CA) a
vector which
allows for direct expression in mammalian cells). The resulting plasmid,
designated 390I-1~,
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 D7 NO: 16) and JHSP6 (SEQ 1D
NO: 3).
Primer DC03 anneals to the pRcICMV vector sequence adjacent to the cloning
site.
B. g3mthesis of~F pression Vector 390 mX
Another MDC cDNA construct was generated by PCR, using pMP390-12 as
template and the primers 390RcH (SEQ 1D NO: 14) and 390mycRX (SEQ 1D 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 B7 NO: 1. This reaction amplified the
expected 354
by fragment containing bases 1 to 298 of SEQ 1D NO: 1 fused to a "myc" epitope
at the lVmC
carboxy ten:ninus. This epitope can be used to facilitate immunoprecipitation,
affinity purification,
and detection ofthe N~7C-myc fusion protein by Western blotting. The fragment
was cloned into
pRc/CMV to generate the plasmid 390IdrnX. The sequence of the insert was
confirmed by
automated sequencing using the primer DC03 (SEQ ID NO: 16).
2 0 C. Expression ofMDC in 293T and N 0 Ce lc
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 NSO
(ECAGC 85110503).
Transient transfection of 293T cells was carried out by the calcium phosphate
precipitation protocol of Chen and Okayama, BioTechniques, 6:632-638 (1988)
andMol. Cel.
Bio~, 87:2745-2752 (1987). Cells and supernatants were harvested four days
after transfection.
A Northern blot was prepared from 4 p,g of total RNA from each cell lysate and
probed with a
radiolabeled lViDC fi~ag~ment prepared by PCR The template for the labeling
reaction was a PCR
3 0 fi~agnent previously g~erated by amplifying pMP390 with the primers 390-1F
(SEQ 117 NO: 17)
and 390-4R (SEQ TD NO: 9). Approximately 30 ng of this fragment was employed
in a PCR
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reaction containing the following: 1.5 mM MgCl2, 50 mM KC1, 10 mM Tris, pH
8.4, 0.2 mM
dATP, 0.2 mM dTTP, 0.2 mM dGTP, 1 pM dCTP, 50 pCi a32P-dCTP (DuPont/New
England
Nuclear, Boston MA), 2.5 U Taq polymerise, and 10 pg/ml each of primers 390-1F
and 390-2R.
The reaction was denatured by heating for 4 minutes at 94°C, followed
by 15 cycles of
amplification (denaturation for 15 seconds at 94°C, annealing for 15
seconds at 60°C, and
extension for 3 0 seconds at 72°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°C
for 16 hours with 5 x 10' counts per minute {cpm) of the probe, in 40-50 ml of
a solution
containing 50% formamide, SX Denhardt's solution, SX SSC (1X SSC is 0.15 M
NaCI, 15 mM
sodium citrate), SO mM sodium phosphate, pH 6.5, and 0.1 mg/ml sheared salmon
sperm DNA
(Sigma, St. Louis MO).
Filters were subsequently washed in 0.5 X SSC and 0.2% SDS at 42°C
for 30
minutes. Autoradiography was carried out at -80°C with one
intensif5ring screen for sixteen hours.
The MDC DNA constructs were vezy highly expressed in the transfected cells and
not detectable
in the non-transfected cells.
For stable transfections, NSO cells were grown to 80% confluency in D-MEM
(Gibco), collected by centrifugation, and washed with PBS. Twenty pg of
plasmid DNA was
iinearized 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 CA). The cells were
eleetroporated with
2 0 two pulses of 3 microfarad at 1.5 kilovolts. Cells were diluted into 20 ml
D-MEM, incubated at
37°C in 5% COZ for 24 hours, and selected by plating into 96-well
plates at various dilutions in
D-MEM containing 800 pg/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.
2 5 MDC is purified from mammalian culture supernatants by, e.g., adapting
methods
described for the purification of recombinant TCA3 chemokine [Wilson et al.,
J. Immu»ol.,
145:2745-2750 (1990], or as described below in subpart F.
D. F~xpression of MDC in C O Cellc
3 0 PCR was used to amplify bases 1 to 403 of the MDC cDNA clone (SEQ >D NO:
1) using primers 390RcH and 390RcX (SEQ. ll7 NOs: 14 and 15), as described
above in subpart
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A. The fragment was cloned into the I~ndIII and XbaI sites of the expression
vector pDCI, a
pUC 19 derivative that contains the cytomegalovirus (CMV) promoter to drive
expression of the
insert. More specifically, vector pDC 1, depicted in Figure 8, was derived
from pRc/CMV and
pSV2-dhfr {ATCC vector #37146). Vector pDCI is similar to the mammalian
expression vector
pRdCMV (Invitrogen, San Diego) except that pDCl carries the mouse
dihydrofolate reductase
(dhfr) gene as a selectable marker, in place of the neomycin
phosphotransferase gene.
Transcription of the target gene in pDC 1 is under the control of the strong
CMV promoter. See
St~berg eta~, J. Virology, 49:190-199 (1984). Additionally, a polyadenylation
sequence from
the bovine growth hormone gene [Goodwin and Rottman, J. Biol. Chem., 267:16330-
16334
(1992)] is provided on the 3' side of the target gene. The dhfr expression
cassette [Subramani et
a~, Mol. CeIX Biol. l: 854-864 (1981)] allows selection for pDCl in cells
lacking a functional dhfr
gene.
XI~l Blue bacteria (Stratagene) were transformed with the.pDCl/MDC plasmid
using standard techniques of CaCl2 incubation and heat shock (Sambrook et al.
). Transformants
were grown in LB medium containing 100 lrg/ml carbenicillin. Plasmid DNA from
individual
transformed clones was isolated using the Promega Wizard Maxiprep system
(Madison, WI) and
its sequence was confirmed by automated sequencing using the primers 390-IF
and 390-2R (SEQ
B7 NOs: 7 & 8). The plasmid was linearized by restriction digestion with Pvu I
endonuclease
(Boehringer Mannheim), which cuts once within the vector sequence.
2 0 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 ug of linearized plasmid, and transferred to a 0.4 cm cuvette. The
suspension was
electroporated with a Biorad Gene Pulser {Richmond, CA) at 290 volts, 960
pFarad.
2 5 Transfectants were selected by growth in a medium (Cat. No. 12000, Gibco,
Gaithersburg, MD)
containing 10~~o dialyzed fetal bovine serum (FBS) (Hyclone, Logan, UT) 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
3 0 methotrexate.
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E. ' Purification of MDC for pro in q, ~en,~cing
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 PS
medium containing 0.2% to 1.0% FBS. PS medium consists of the components
listed in Table
2, below (purchased as a premixed powder form Hycione, Logan UT), supplemented
with the
following additional components: (1) 3 g/1 sodium bicarbonate (Sigma, St.
Louis, MO); (2) 2 ~tg/l
sodium selenite (Sigma); (3) 1% soy bean hydrolysate (Quest International,
Naarden, The
Netherlands); (4) lx ferrous sulfate/EDTA solution (Sigma); (5) 1.45 ml/1 EX-
CYTE VLE
solution (Bayer, Kankakee, IL); (6) 10 pg/ml recombinant insulin (Nucellin,
EIi Lily,
Indianapolis, IN); (7) 0.1% pluronic F-68 (Sigma); (8) 30 gg/ml glycine
(Sigma); (9) 50 pM
ethanolamine (Sigma); and (10) 1 mM sodium pyruvate (Sigma}.
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TABLE 2
Com onent Powder #5 m/L
Sodium Chloride 4.0
Potassium Chloride 0.4
Sodium Phos hate Dibasic, 0.07102
Anhydrous
Sodium Phos hate Monobasic 0.0625
0
Ma esium Sulfate, Anh drous 0.1
U
Cupric sulfate 5 Hz0 0.00000125
o Ferrous Sulfate 7 0 0.000417
z
Zinc Sulfate 7 H20 0.0004315
Ferric Mtrate 9 H 0 0.00005
Calcium Chloride, Anhydrous 0.11661
Masznesium Chloride. Anhydrous0 I
I
L-Alanine 0
L-Ar ' 'ne HCl 0.15
L-As ara ' a 0 0.075
L-As antic Acid 0.04
L-C teine HCl 0 0.03
5
d
O L-C stine 2 HCl 0.12
L-Glutamic Acid 0.02
L-Glutamine 0.5
846
Gl cine 0.02
L-Histidine HCl 0 0.04
L-Isoleucine 0.15
L-Leucine 0.15
L-L sine HCl 0.1
L-Methionine 0.05
L-Proline I 0.05
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L-Phenylainine 0.05
L-Serine 0.075
L-Threonine 0.075
L-T to han 0.02
L- osine 2 Na 2 0 0.075
L-Valine 0.12$
Biotin 0.001
D-Calcium Pantothenate 0.0025
Choline Chloride 0.015
Folic Acid 0.005
i-Inositol 0.175
Ncotinamide 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
H oxanthine Na 0.005
Th 'dine 0.005
Putrescine 2HC1 0.000081
Sodium vate 0.11004
Linoleic Acid 0.0001
DL-A1 ha Li oic Acid 0.0002
Phenol Red Na Salt 0.0086022
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 (NOVEL, and blotted to a PVDF membrane (Mllipore,
Bedford, MA).
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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 Itg/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 ofPS
medium were added to the culture. Proteins were precipitated from the culture
supernatant by
addition of polyethylene glycol (MW 8000, Union Carbide, Danbury, CT) to 20
(weight/volume), fractionated on an 18% Tris glycine gel, and electroblotted
to a PVDF
membrane (lVfillipore, Bedford, MA) 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.
Using an automated sequencer (Applied Biosystems, Model 473A, Foster City,
CA), 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 m NO. 2,
corresponding to the N-
terminus of the predicted mature form of MDC.
F. p~cation of MDC for biologica~i~s_
2 0 For gowth of larger cultures, MDC-expressing CHO cells were grown to 80%
confluence on tissue culture plates in a' medium. The cells were removed from
the plates by
treatment with trypsin and EDTA and resuspended at a density of 3 x 105
cellslml in PS medium
plus 1% FBS in a spinner flask at 37 °C. Additional P5/1% FBS medium
was added as needed
to keep the cell density in the range of 1 x 106 to 3 x 106.
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 (Phannacia, Piscataway, Nn. After washing with 0.2 M NaCI in
potassium
phosphate buffer, pH 7, the column was eluted with a linear gradient of 0.2 to
0.7 M NaCI.
Fractions were analyzed by SDS-PAGE and Coomassie stained to determine which
of them
3 0 contained MDC. MDC eluted from the column at approximately 0.6 M NaCI.
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The fractions cornaining MDC were pooled and concentrated by ultrafiltration
in
stirred-cell chamber (Amicon, Beverly, MA) 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, Nn. 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.
Production of MDC a_n_d MDC nalogc by Pelt it de S, h is
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 II,-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.
For example, MDC and MDC analogs were chemically synthesized using
optimized stepwise solid-phase methods [Schnolzer et al., Int. J. Pept.
Protein Res , 40:180
( 1992)] based on t-butyloxycarbonyl (Boc) chemistries of Merrifield [J. Am.
Chem. Soc.,
85:2149-2154 (1963)] on an Applied Biosystems 430A Peptide Synthesizer (Foster
City, CA).
The proteins were purified by reverse-phase HPLC and characterized by standard
methods,
including electrospray mass spectrometry and nuclear magnetic resonance.
The chemically synthesized MDC corresponded to the mature form of recombinant
MDC, consisting of residues 1 to 69 of SEQ ll~ 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, NOVEL. 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
3 0 immunoprecipitation assays with the anti-MDC monoclonal antibodies. These
studies indicate
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that the denatured and the non-denatured structures of chemically synthesized
IVmC are similar
to those of recombinant MDC.
The following MDC analogs also have been chemically synthesized:
1. "MDC (n+1)" (SEQ m NO: 30) consists of Ixucine followed by residues 1 to 69
of SEQ
m NO. 2. This analog has alternatively been referred to herein as "IVmC(0-
69)."
2. "MDC (9-69)" consists of residues 9 to 69 of SEQ m NO. 2.
3. "MDC-yl" (SEQ m 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 "NlDC-wvas" consists of residues 1 to 69 of SEQ m NO. 2,
with
the following substitution: Residues 59-60 (Trp-Val) were replaced with the
sequence
Ala-Ser.
4. "1VB7C-eyfy" (SEQ m NO: 32) consists of residues 1 to 69 of SEQ ID NO. 2,
with the
following substitution: Residues 28-31 (T~s-Phe-Tyr-Trp) were replaced with
the
sequence Glu-Tyr-Phe-Tyr, derived from the amino acid sequence of the
chemokine
RArTTES (residues 26-29 of SEQ m NO: 21).
The analogs "MDC (n+1)", "MDC (9-69)", and "lVmC-yl" are expected to be
antagonists
of MDC activity, inhibiting MDC activity by competitively binding to the same
receptor that
recognizes IVlI7C. Alternatively, they may effect inhibition by forming
inactive heterodimers with
the native IvmC. Possible activities of the analog "IVmC-eyfy" include
inhibition of MDC as
2 0 described for the previous analogs. Alternatively, "MDC-eyfy" may confer
some of the activities
typical of the chemolcine RAN'fES, such as chemotaxis of T lymphocytes,
monocytes, or
eosinophils.
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
2 5 and/or lysine amino acids at positions 24 and 27, respectively, of SEQ m
NO: 2; (2) substitution
of a charged or polar amino acid (e.g., serine, lysine, arginine, histidine,
aspastatc, glutamate,
asparagine, glutamine or cysteine) for the tyrosine amino acid at position 30
of SEQ m NO: 2,
the tryptophan amino acid at position 59 of SEQ m NO: 2, and/or the valine
amino acid at
position 60 of SEQ m NO: 2; and (3) substitution of a basic or small,~non-
charged amino acid
3 0 (e.g., lysine, arginine, histidine, glycine, alanine) for the glutamic
acid amino acid at position 50
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of SEQ m NO: 2. Specific analogs having these amino acid alterations are
encompassed by the
following formula (SEQ m NO: 25):
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 err Gly Ala Aan Met Glu
-5 1 5
Asp Ser Val Cars Cars Arg Aep T~rr Val Arg err Arg Leu Pro Leu Xaa
10 15 20
Val Val Xaa His Phe Xaa Trp Thr Ser Asp Ser G'ye Pro Arg Pro Gly
25 30 35 40
Val Val Leu Leu Thr Phe Arg Asp Lys Xaa Ile Gars Ala Asp Pro Arg
45 50 55
Val Pro Xaa Xaa Lys Met Ile Leu Rsn Lys Leu Ser Gln
60 65
wherein the amino acid at position 24 is selected from the group consisting of
arginine, glycine,
alanine, valine, leucine, isol~cine, 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,
2 5 leucine, isoleucine, proline, serine, threonine, phenylalanine, tyrosine,
tryptophan, aspartate,
glutamate, asparagine, glutamine, cysteine, and methionine; wherein the amino
acid at position
is independently selected from the group consisting of tyrosine, serine,
lysine, arginine,
histidine, aspartate, glutamate, asparagine, giutamine, and cysteine; wherein
the amino acid at
position 50 is independently selected from the group consisting of glutamic
acid, lysine, arginine,
3 0 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
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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.
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~Pro2 polypeptides." As described below in
Example 20,
MDC (3-69) derived from an H1V-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. F.xper. Med,
186:1865 (1997)] possesses a specificity for the sequence NHZ-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 NH2-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 lldDC~Pro2
polypeptides, rendering
such mutants more stable than MDC(1-69) in vivo. MDCAPro2 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~Pro2 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
2 0 contemplated. Introducing the MDCOPro2 mutation into any of the analogs
described above is
also specifically contemplated.
After synthesis, synthetic MDC or MDC analogs may be reduced and refolded
substanbaily 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, NY (1992): van Kimmenade et al., Eur. J.
Biochem.,
173: 109-114 (1988); and PCT publication no. WO 89/01046.
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
3 0 techniques, e.g., site-directed mutagenesis and the polymerase chain
reaction. See generally
Sambrook et al., supra, Chapter 15. The modified polynucleotides are expressed
recvmbinantly,
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and the recombinant MDC polypeptide analogs are purified, as described in the
preceding
examples.
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, bas~phils, eosinophds, neutrophils, mast
cells, and natural
killer cells), on endothelial cells, epithelial cells, fibroblasts, or others
are assayed by art-
recognized t~hniques 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.
~lnaL L
Assay of MDC Effects upon
~soyhils, Mast Cells~and Eosinoyhils
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-
1/213 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
2 0 allergic reactions, in which secretion of proin$amznatory mediators plays
a significant role [Weber
et al., supra].
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
2 5 chemotactic factors of eosinophils, was as follows: MDC # eotaxin <
RANZ'ES < MCP-4 s
eotaxin-2. Eotaxin-2 and MCP-4 were especially potent, whereas RArTTES effects
were
intermediate, about one log less potent than MCP-4 or eotaxin 2. MDC induced
eosinophil
migration and shape change even though it did not elicit measurable cytosolic
calcium elevations
in the eosinophils during these responses. In contrast, the MDC analog MDC(9-
69) displayed no
3 0 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.
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In reported studies with human eosinophils, CCR3 was identified as a critical
receptor for a variety of CC chemokines that exert effects on eosinophils,
including eotaxin,
RA1VTES, MCP-4 and eotaxin-2. See, e.g., Garcia Zepeda, et al., J. Immunol.
157:5613 (1996);
Forssman et al.; J. Exp. Med, 185:2171 (1997); Stellato et al., J. Clin.
Invest , 99:926 (1997);
and White et al., J. Leukoc. Biol. 62:667 (1997). Also, as reported elsewhere
herein, the
chemokine MDC binds and signals through the chemokine receptor CCR4. However,
it was
determined that the eosinophil-chemotactic activity of MDC appears to operate
in a manner
independent ofthe chemokine receptors CCR3 and CCR4. 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/mI 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). In
addition, a CCR3 blocking monoclonal antibody was found to inhibit eotaxin-
and eotaxin-2-
induced chemotaxis of eosinophils, but not chemotaxis induced by MDC.
Two lines of evidence suggest that NIDC-induced chemotaxis of eosinophils
operates independently of CCR4. F'ust, ~sinophil cDNA (generated from
eosinophil RNA using
oligo-dT or random primers) was screened via PCR. CCR4 could not be detected
in either the
oligo-dT or random primed cDNA, even though the same PCR primers amplif ed
CCR4 fram
2 0 genomic DNA, and even though CCR3 mRNA was readily amplifyable. Thus, it
appears that
eosinophils do not express CCR4. S~;ond, chemotaxis experiments with TARC, a
chemokine
known to signal through CCR4, have failed (at concentrations up to 100 nlVl)
to induce
chemotaxis of eosinophils.
The fact that IvlDC apparently exerts its effects on eosinophils in a CCR4-
2 5 independent manna indicates that, when selecting 1VIDC modulators to treat
allergic reactions in
which eosinophils play a role, modulators that will have fewer side-effects
are those that modulate
MDC-induced chemotaxis of eosinophils without modulating MDC's signaling
through CCR4.
Assays are provided herein to select such modulators.
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Assay of Chemoattractant and Cell-Activation
Properties of MDC upon Human
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, ch~notaxis, induction of reactive nitrogen intermediates,
respiratory burst (superoxide
1o 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,
76:301-3I4 (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.
In one art-recognized chemotaxis assay, a modified Boyden chamber assay,
leukocytes to be tested are fluorescemly labeled with calcein by incubating
for 20 minutes at room
temperature. The labeled cells are washed twice with serum-fi-ee RPMI,
resuspended in RPMI
containing 2 mg/mI of BSA, and then added quantitatively to the upper wells of
the chambers,
which are separated finm the lower wells by a polycarbonate filter (Neuroprobe
Inc. Cabin John,
IVm). IBC 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 °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
2 5 with a rubber policeman. Cells that have migrated through the filter are
quantitated by reading
fluorescence per well in a fluorescent plate reader (Cytoffuor, Nflllipore
Inc., Boston, MA).
A series of experiments were performed using art-recognized procedures to
determine the chemotactic properties ofMDC. Initially, the response of human
mononuclear cells
to MDC was determined. The effect of 1~C on the chemotactic response of
polymorphonuclear
3 0 leukocytes (granulocytes) also was examined.
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
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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 dece~easing as these cells differentiate.
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 e~cpression pattern correlates to the
biological response
to MDC, the effects of MDC on the migration of monocytes and macrophages were
compared.
1o
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 TI3P-1
cells were employed, and in others monocytic THP-1 cells were differentiated
to macrophages by
2 0 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
2 5 by centrifugation and then utilized in chemotaxis assays as described
below.
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, OR). Polycarbonate filters
used in this assay
3 0 were PVP-free (Neuroprobe Inc.); filter pore sizes used for different cell
types were: 5 pm for
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monocytes and THP-1 cells, 3 pm for polymorphonuclear leukocytes, and 8 p.m
for guinea pig
macrophages.
Fifty thousand calceirt labellal 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 °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. lvfigration of cells was quantitated on a fluorescent
plate reader (Cytofluor,
Millipore Inc. Bedford, MA) and the number of cells migrated expressed as
fluorescent units.
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- I 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 ofMDC. Preparation of cells and quantitation of
assays was performed
exactly as described above for the chemotaxis assays. The number of cells
migrated was
2 0 expressed as fluorescent units.
As indicated in Figure 2, IvIDC did not induce THP-I-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.
2 5 As shown in Figure 3, MDC, at concentrations of .001 to 100 ng/ml had no
net
effect on gcanulocyte migration. In respect to this lack of effect on
granulocyte chemotaxis, MDC
is similar to other previously described C-C chemokines.
The response of both macrophage and monocyte THP-1 cells to MDC is shown
in Figure 4. Macrophages (closed circles) migrated to MDC in a dose dependent
manner, with
3 0 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
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-
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.
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 {Figure 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}.
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. Immu»oZ, 150:5025-5032 (1993)], MDC induces macrophage but not monocyte
chemotaxis.
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)].
In certain conditions such as arthritis, the accumulation of macrophages is
understood to be detrimental and destructive. The ability of MDC to promote
macrophage
2 0 chemotaxis indicates a therapeutic utility for MDC inhibitors of the
invention, to prevent, reduce,
or eliminate macrophage accumulation in tissues.
The results of the chemotaxis assays with human mononuclear cells, presented
in
Figure Z, suggested that MDC might inhibit cell migration. In the absence of
MDC, monocytic
THP-I cells migrate to MCP-1, as shown in Figure 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
3 0 of MDC.
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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.
Tumor growth-inhibition properties of MDC are assayed, e.g., by modifying the
protocol described by Laving et al., J. Immunol., 153: 4625-463 5 ( 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
generaxed
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
2 0 identified by histologic means. In addition, recombinant MDC (20 ng) is
mixed with non-
transfec~ed J558 cells and injected (20 ng/day) into tumors derived from such
cells, to assay the
effect of MDC administered exogenously to tumor cells.
2 5 Intraneritoneal Ini ec,~tion Assav
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., .I. Immunol., 153:4616-4624
(1994). Following
injection, leukocytes are isolated from peripheral blood and from the
peritoneal cavity and
3 0 identified by staining with the Diff Quick kit (Baxter, McGraw, IL). The
profile of leukocytes is
measured at various times to assess the kinetics of appearance of different
cell types. In separate
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experiments, neuaalizing 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.
In vivo Activil~r - Subcutpnenna Tn; .rrin~
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 injintradennally 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 NJDC.
The nature of the cellular inf prate is identified by staining with Baxter's
Diff Quick kit.
The myelosuppressive activity of MDC is assayed by injection of MDC into mice
or another mammsi (e.g. rabbits, guinea pigs), e.g., as described by Maze et
al., J. Immurrol.,
149:1004-1009 (1992) for the measurement of the myelosuppressive action of MIP-
1 a. 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
2 0 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.,
Bloom 79:2221 (1992).
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 (a,-8) and platelet factor 4 (PF-4). See Daly ei 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 10% fetal bovine serum (HyClone, Logan, tTT)
with 100
3 0 units/ml recombinant human GM-CSF (R&D Systems, Minneapolis, MIA plus 50
ng/ml
recombinant human Steel factor (Immunex Corp., Seattle, WA) in the absence
(control) and
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presence of MDC for assessment of granulocyte-macrophage precursors. For
assessment of
granulocyte ersrthroid 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 ofrecombinant human erythropoietin (1-~ units/ml) in combinationwith
50 nglml Steel
factor. Plates are scored for colonies aftei incubation at 37 °C in
lowered (5%) 02 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-G11~, CFU-GEMM, and BFU-E.
Eaam In a 18
A Monoclonal antibodies
Recombinant MDC, produced by cleavage of a GST-lVmC 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 synthe~zed
peptide corresponding
to the N termunus of the mature form of Iv>DC (residues 1 to 12 of SEQ ID NO.
2). The peptide
was synthesized on an Applied Biosystem Model 473A Peptide Synthesizer (Foster
City, CA),
and conjugated to Keyhole Lympet Hemocyanine (Pierce), according to the
manufacturer's
recommendations. For the initial injection to produce "Fusion 191" hybridomas,
approximately
2 0 10 ltg 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 1V1DC
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 1V1DC protein.
For a prefusion
boost, the mouse was injected with I1~C in PBS, and four days later the mouse
was sacrificed
and its spleen removed.
For the production of "Fusion 252" hybridomas, a mouse was immunized with the
11~C(0-69) chemically syrnhesized peptide (See Example 11). On Day 0, the
mouse was pre-bl~i
and injected subcutaneously at two sites with 10 ug of IvmC(0-69) in 200 ul
complete Freund's
3 0 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 IvIDC(0-69) in
100 ul of
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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 u( PBS. On day 131, the mouse was sacrificed and the spleen was removed
for a fusion.
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 lVmC(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 pg injections of
MDC(0-69). On
day 389 the spleen was removed for a fusion.
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 p.g/ml streptomycin
(RPMI) (Gibco,
Canada). The cell ions were filtered through a sterile 70-mesh Nitex cell
strainer (Becton
Dickinson, Parsippany, New Jersey), 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~/o 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
2 0 pellet was washed twice as described above.
Spleen cells (2 x 10~ were combined with 4 x 10' NS-I cells and centrifuged,
and
the supernatant was aspirated. The cell pellet was dislodged by tapping the
tube, and 2 ml of 37°C
PEG 1500 (50~/o in 75mM Hepes, pH 8.0) (Boehringer MaMheim) was added with
stirring over
the course of 1 minute, followed by the addition of 14 ml of serum-free RPMI
over 7 minutes.
2 5 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 iiM sodium hypoxanthine, 0.4 pM aminopterin, 16 ~.M thymidine (HAT)
(Gibco), 25
units/mI II,-6 (Boehringer Mannheim) and 1.5 x 106 thymocytes/ml and plated
into 10 Corning
flat-bottom 96-well tissue culture plates (Corning, Corning New York).
3 0 On days 2, 4, and 6, after the fusion, 100 pl of medium was removed from
the
wells of the fusion plates and replaced with fresh medium. On day 8, Fusion
191 was screened
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by ELISA, testing for the presence of mouse IgG binding to lVmC as follows.
Immulon 4 plates
(Dynatech, Cambridge, MA) were coated for 2 hours at 37°C with 100
ng/well of MDC diluted
in 25mM Tris, pH 7.5. The coating solution was aspirated and 200 ul/well of
blocking solution
[0.5% fish skin gelatin (Sigma) diluted in CMF-PBS] was added and incubated
for 30 min. at
37°C. The blocking solution was aspirated and 50 ~1 culture supernatant
was added. After
incubation at 37°C for 30 minutes, and washing three times with PBS
containing 0.05% Tween
20 (PBST), 50 ul of horseradish peroxidase conjugated goat anti-mouse IgG(fc)
(Jackson
ImmunoResearch, West Grove, Pennsylvania) diluted 1:7000 in PBST was added.
Plates were
incubated as above, washed four times with PBST, and 100 uL substrate,
consisting of 1 mg/ml
o-phenylene diamine (Sigma) and 0.1 ~1/ml 30% H2O2 m 100 mM Citrate, pH 4.5,
was added.
The color reaction was stopped after 5 minutes with the addition of 50 pl of
15% HZS04. A,4~
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.
Selected fusion wells wexe Boned 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, III.
Anti-MDC antibodies were characterized further by western blotting against
recombinant MDC produced as desc~ed above in E. coli or mammalian CHO cells.
To prepare
2 o the blot, approximately 3 ltl of sedimented cells (transformed E. coli
producing MDC; transfected
CHO cells producing MDC; untransformed ~ toll (control); and untransfected CHO
cells
(control)) were dissolved in standard sample preparation buffer containing SDS
(sodium dodecyl
sulfate) and DTT (dithiolthreitol) (Sambrook et al.). After boiling, the
lysates were fractionated
via denaturing SDS-PAGE (18% acrylamide, Tris Glycine gel, NOVEL and
electroblotted to
2 5 PVDF membranes (ll~~llipore, Bedford, MA). MDC monoclonal antibodies were
diluted to 0.7
pg/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, arid thymus.
One anti-MDC monoclonal antibody, designated monoclonal antibody l9iD,
3 0 reacted strongly with recombinant MDC produced by both bacteria and
mammalian cells.
Further, this ant~ody displayed very little background reactivity in
preliminary screening against
CA 02302806 2000-03-08
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-72-
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.).
Some background reactivity was observed in subsequent western analyses using
the anti-MDC monoclonal antibody 191D. further anti-MDC monoclonal antibodies
designated
252Y and 2522 (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 2522
have been
designated "hybridoma 252Y" and "hybridoma 2522," respectively.
Monoclonal antibody 272D, at 1 ~.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.
The hybridoma cell line which produces monoclonal antibody 191D (designated
hybridoma 19ID) has been deposited with the American Type Culture Collection
(ATCC), 10801
University Blvd., Manassas, V'~rginia 20110-2209 (USA) pursuant to the
provisions of the
Budapest Treaty (ATCC Deposit date: June 04, 1996; ATCC Accession No. HB-
12122). The
hybridoma cell lines that produce monoclonal antibodies 252Y and 2522
(designated "hybridoma
2 0 252Y" and "hybridoma 252Z") were also deposited with the ATCC pursuant to
the provisions
of the Budapest Treaty {ATCC Deposit date: November 19, 1997; ATCC Accession
Nos. HB-
12433 and HB-12434, rvely). The hybridoma cell line that produces monoclonal
antibody
272D was deposited with the ATCC pursuant to the provisions of the Budapest
Treaty on March
27, 1998 (ATCC Accession No. HB-12498). Availability of the deposited
materials is not to be
2 5 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.
B. ~3iclonal_ antibodies.
Polyclonal antibodies against MDC were raised in rabbits following standard
3 0 protocols (Sambrook et al.). Recombinant MDC produced as a GST fusion
protein as descn'bed
above was diluted in PBS, emulsified with Freund's Complete Adjuvant, and
injected
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72/1
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PC"T Rule 136is)
A. The indications made below relate
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to in the description
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sheet
Name of depositary institution
American Type Culture Collection
(ATCC)
Address of depositary institution
including postal code and country)
10801 University Blvd.
Manassas, VA 20110-2209
Date of deposit Accession Number
04 June 1998; 19 November 1997; ATCC HB-12122, -12433, -12434, -12498
27 March 1998
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CA 02302806 2000-03-08
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72/2
INDICATIONS RELATING TO A DEPOSTTED MICROORGANISM
(PCT Rule 13 bis)
A. The indications made below relate
to the microorganism referred to
in the description
on page 72 , line 15-24
B. IDENTIFICATION OF DEPOSiiT Further
deposits are identified ~ an additional
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Name of depository institution
American Type Culture Collection
(ATCC)
Address of depository institution
(including postal code and country)
10801 University Blvd.
Mantissas, VA 20110-2209
Date of deposit Accession Number
04 June 1996; 19 November 1997; ATCC Hl3-12122, -12433, -12434,
27 March 1998 - 12498
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this Patent Request
are only to be provided before:
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CA 02302806 2000-03-08
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- 73 -
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.
In a similar set of experiments, polyclonal antisera was generated in a rabbit
against
a 12-mer peptide corresponding to the amino-terniinus 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 hut the MDC(9-69) peptide.
C. MDC Detection . eAv
Monoclonal a~'bodies 252Y and 2522 were employed in an MDC detection assay
as follows: Aliquots of the antibodies 252Y and 2522 were biotinylated using
NHS-LC-Biotin
(Pierce) according to menu's instructions. Immulon 4 ELISA plates were coated
with one
monoclonal ant~'body (252Y or 2522, unbiotinylated) overnight at 4°C.
The next day, the plates
were blocked with 0.5% fish skin for 30 minutes at 37°C. Known
quantities of MDC were loaded
onto the plate for 30 minutes at 37°C. The plates were washed and
coated with the other
monoclonal antibody (biotinylated) for 30 minutes at 37°C. The plates
were washed and loaded
2 0 with streptavidin-HRP for 30 minutes at 37°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 2522, MDC is detectable in the conceon range of low monograms to high
picograms per
milliliter.
In a related set of experiments, an ELISA format was employed to examine the
relative affinity of antibodies 191D, 252Y, and 2522 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 Ago 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
3 0 described above, each antibody was added at a constant concentration of
250 ng/ml, and A~
measurements were taken to quantify antibody bound to the plates. The
absorbance values for
CA 02302806 2000-03-08
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antibodies 252Y and 2522 were more than five-fold higher than those of
antibody 191D (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 Ago measurements were at background levels for all
three
antibodies. -
D. ~a_rarteri~tion of epi open reco, , ize~j bX antibodies 252Y a_nd 2527
The ability of monoclonal antibodies 252Y and 2522 to recognize synthetic MDC
(mature form, residues 1-69) and MDC variants (MDC(0-69); MDC(9-69); MDC-eyfy;
and
MDGwvas (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 2522 at a concentration of I pg/ml.
Immunoreactivity was visualized by incubating the probed blot with horseradish
peroxidase-
conjugated goat anti-mouse immunoglobulin G {Transduction Laboratories
#M15345) at a
concentration of 0.2 ug/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 salineJO.1% Tween 20 solution and detection
of antibody binding
was measured by autoradiography (Kodak Hyperfihn) using electro-
chemiluminescence (NEN
Renaissance ECL # NEL 102). Both monoclonal antibodies were observed to
recognize wildtype
2 0 1VIDC and the analogs MDC(0-69), MDC(9-69), and MDGeyfy. However, antibody
252Y and
antibody 2522 both failed to recognize I~C-wvas, suggesting that the
epitope(s) recognized by
these antibodies includes) the wv motif near the carboxyl-terminus of lVmC.
This motif tends
to be highly conserved in atl CC chemokines (see Fig. 1).
To further characterize the epitope(s) recognized by antibodies 252Y and 2522,
2 5 an Immulon 4 plate was coaxed with lVft7C at 1.0 pg/ml. After blocking the
plate with fish skin
as described above in part C, unlabeled antibody 252Y, 2522, or an isotype-
matched control was
added at 5 lcg/ml and incubated for 30 minutes at 37°C. Without
washing, either biotinlyated
antibody 252Y or 2522 was added at a concentration of 0.25 ~glml, and the
plate was incubated
an additional 30 minutes at 37°C. Thereafter, the plate was washed and
developed with
3 0 streptavidin-HRP. The results showed that either 252Y or 2522 was capable
of reducing the
signal of either biotinlyated antibody ten-fold, as compared with the signal
of either biotynilated
CA 02302806 2000-03-08
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-75-
antibody blocked with the control antibody. These results further indicate
that antibodies 252Y
and 2522 recognize similar or overlapping epitopes.
In contrast, unpurified supernatant from hydriboma 272D was tested in a
similar
experiment for its ability to compete with biotynilated 252Y or biotynilated
2522, 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 2522.
E. A_n_tibodies 252Y nd 252Z mpWi for i m m~~,; a ine 1~~C
The following experiments were conducted which demonstrate a utility for
antibodies 252Y and 2522 for immuprecipitation of MDC. Antibodies 252Y, 2522,
and an
irrelevant isotype-matched control were added separately at a concentration of
10 pg/ml to an
extraction buffer (1% triton X-100, 10 mM Tris base, 5 mM EDTA, 10 mM NaCI, 30
mM Na
pyrophospate, 50 mM NaF, 100 pM 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 pl of
protein G sepharose (Pharmacia Biotech # I7-0618-O1) were added to each sample
and incubated
on a rotation apparatus at 4°C for 30 minutes. The samples were then
centrifuged to collect the
protein G sepharoseJ'immune complexes, washed three times (1 ml each) in
extraction buffer,
boiled/solubilized in 2X SDS-PAGE buffer, electrophoresed on an 18% SDS-PAGE
gel, and
western blotted to PVDF membrane (Novex # LC2002). Nonspecific binding sites
on the PVDF
2 0 membrane were blocked with TBS Tw20/1% BSA for 30 minutes at room
temperature. The blot
was then probed with 1 pg/ml of antibody 252Y in TBS Tw20/1% BSA for 1 hour,
washed three
times with TBS Tvr~O, probed with horseradish peroxidase-conjugated goat anti-
mouse 1gG 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
2 5 252Y and 2522 lanes but not in the negative isotype-matched control lane.
Additionally, MDC
was immunprecipitated from cell culture supernatants containing RPMI (Rosell
Park Memorial
Institute - Gibco) medium with 10~/o fetal bovine serum spiked with 25 ng/ml
MDC using the
same conditions stated above.
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-76-
F.
The activities of MDC as reported herein suggest numerous the~rap~tic
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).
The principles of humanizaxion 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.
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 ingest. The V region gene fiagments 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).
To achieve an even greater level of humanization, only those portions of the
2 0 variable region gene fragm~ts 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.,
BiolT'echnology, 9:266-71 (1991). If necessary, the ~i-sheet framework of the
human antibody
2 5 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).)
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
3 0 site-directed mutagenesis, while retaining all of the interior and
contacting residues of the non-
human antibody. See Padlan, Molecular Immunol., 28(4/Sj:489-98 (1991).
CA 02302806 2000-03-08
WO 99/15666 PCT/US98/20270
The foregoing approaches are employed using MDC-neutralizing anti-MDC
monoclonal a~'bodies and the hybridomas that produce them, such as antibodies
252Y and 2522,
to generate humanized MDC-neutralizing antibodies useful as therapeutics to
treat or palliate
conditions wherein MDC expression is detrimental.
G. Human MDC-Neutrali~ng t~n_tibodies from ~hage di~~lav
Human lVmC-neutralizing antibodies are generated by phage display techniques
such as those described in Aujame et a~, HumanAretibariies, 8(4):155-168
(1997); Hoogenboom,
TTBTECH, 15:62-70 (199'n; and Ra,der et al., Curr. Opin. Biotechtio~, 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 pIII. 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.
H. Humarn_ MDC-neutrat_i_~ng a_n_tibodie from ~r~enic min
Human 1V)DC-neutralizing antibodies are generated in transgenic mice
essentially
as described in Bruggemann and N~berger, Immunol. Today, 17(8):391-97 (1996)
and
Bruggemann and Taussig, Cun: 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 IvmC 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-IVmC human
antibodies (e.g., as
described above).
I. FI,ISA for detecting and monito 'ng sen3m conc"-ntration of h"~C
The measurement of endogenous levels of MDC is useful to monitor the immune
3 0 state of a patient, especially a patient who is immunocompromized, in a
hyperimmune state, or
undergoing treatment with Nll~C neutralizing antibodies or other 1V>DC
antagonists.
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_78_
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 2522 were employed in an MDC detection
assay as
described below. -
Aliquots of the antibodies 252Y and 2522 were biotinyIated using NHS-LC-Biotin
(Pierce) according to manufacturer's instructions. Immulon 4 ELISA plates were
coated with
antibody 272D overnight at 4°C. The next day, the plates were blocked
with 0.5% fish skin for
30 minutes at 37°C. Known quantities of MDC(1-69) were loaded onto the
plate for 30 minutes
at 37°C. The plates were washed and coated with either 252Y or 2522
(biotinylated) for 30
minutes at 37°C. The plates were washed and loaded with streptavidin-
HRP for 30 minutes at
37°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.
Changes in intracellular calcium concentrations, indicative of cellular
activation by
2 0 chemokines, were monitored in several cell lines by an art-recognized
calcium flux assay. Cells
were incubated in 1 ml complete media containing I uM Fura-2/AM (Molecular
Probes, Eugene,
OR) for 30 minutes at room temperature, washed once, and resuspended in D-PBS
at 106
cells/ml.
Two ml of suspended cells were placed in a continuously stirred cuvette at
37°C
2 5 in a fluorimeter (AMiNCO Bowman Series 2, Rochester, NY). 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 ofthe emissions from the 340 nm relative to the 380 mn excitation
wavelengths corresponds
to the level of intracellular calcium.
3 0 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
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-79-
[Report et al., Gene, 163:295-299 (1995)]; HEK 293 cells stably transfected
with the chemokine
receptor gene CCRS [Samson et al., Biochemisby, 35:3362-3367 (1996); see also
co-owned, co-
pending U.S. Patent Application Serial No. 08/575,967, filed December 20,
1995, incorporated
herein by reference, disclosing chemokine receptor materials and methods,
including CCRS
(identified therein as "88C")], the human manocytic cell line THP-1, the human
lung epithelial cell
line A 549; and the human fibroblast cell line ZIVVIR 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 Ifill, Nn at a final concentration of 2S ng/ml. No additional stimuli
were tested on the A
549 or IZVIR 90 cell lines.
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 (I99S);
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+ T cell line is
acutely infected with an
2 0 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 S 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, FI,).
One technical report teaches that MDC possesses an HIV antiproliferative
activity.
2 5 See Pat et a~, Science, 278: 695-698 (1997). The agert used in the study
consisted of purified
polypeptides that had been secreted fi~om an immortalized cell line derived
from CD8+ T cells from
an HIV 1-infected individual. Pal et a~ reported that the purified "native
MDC" from this cell line
possessed an NH2-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 11? NO: 1
also was detected.
3 0 The authors did not detect a peptide beginning with the glycine at
position I of SEQ ID NO: 1
in their "native MDC" composition. According to Pal et al., a reversed-phase
HPLC fraction
CA 02302806 2000-03-08
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containing the "native MDC" suppressed the acute infection of CD8+ cell-
depleted PBMCs by
HIV-1~ 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. IJse ofMDC an~agoniats to in_h~bit HIV~~roliferation
An acute HIV 1H,, infectivity assay reported in Pal et al. was repeated (100
TCID~
units/well) using the macrophage cell line PM-1 (1 x 105 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 I0). 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 pg/ml, MDC(0-69) conferred a 58%
and 67%
reduction in the production of infectious particles (TCID~ 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 matiue MDC (1-69)
to stimulate HIV-
2 0 1 production.
The effects of mature MDC and of MDC-neutralizing antibodies were analyzed
in Pal et al.'s acute HIV-1~ (0.01 MOI/well) infectivity assay using
peripheral blood
mononuclear cells (PBMC, 1 x 106 cells/well) depleted of CD8'" cells. The
mature MDC (1-69)
failed to inhibit p24 production, as compared to a control marine IgGl
antibody. However, the
marine monoclonal anti-MDC neutralizing antibodies 252Y (IgGl) and 2522 each
inhibited p24
production when tested separately at a concentration of 2 pg/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 HTV 1 functions, and that IvB?C
antagonists inhibit
this elect.
3 0 To confirm the apparent role of MDC as an HIV-1 agonist, an infectivity
assay
(such a~s that described in Pal et al.) is repeated using MDC neutralizing
antibody and titrating
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exogenous mature 1~'?C(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 11~C,
and will be
overwhelmed with the addition of a molar excess of MDC.
Collectively, these results provide a therapeutic indication for lVmC
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-11~C amibodies (especially neutralizing antibodies, and
preferably humanized
antibodies) are highly preferred 1VIDC antagonists. Similarly, polypeptides
that are capable of
binding to MDC that comprise an antigen-binding fragment of an anti-lVmC
antibody are
contemplated. Effective MDC analogs also are contemplated as MDC antagonists.
For example,
N-terminal deletion analogs of IvmC are contemplated, especially deletion
analogs having an
amino acad sequence consisting of a portion of the amino acid sequence set
forth in SEQ ID NO:
2 that is ~cient to bind to the chemokine receptor CCR4, the portion having an
amino-ternunus
between residues 3 and 12 of SEQ ID NO: Z. Likewise, analogs comprising a
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 IvmC(1-69) to form the
analog, or to
2 0 the amino terminus of an 11~C 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).
Additional classes of MDC antagonists useful in anti-HIV therapeutic methods
include antagonists derived from CCR4 or from other 1VIDC receptors. For
example, a
solubilized, NIDGbinding 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 th~ap~tics. 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. Fxp.
MeaC, 185:1681
3 0 1691 (1997).
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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 irr viva, e.g., by modifying the amino terminus of MDC. For example,
administration of a
therapeutically effective amount of the dipeptidyl aminopeptidase CD26 is
contemplated.
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 su~aent quantity and with sufficient frequency so as
to maintain serum
concentrations ofMDC 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.
Moreover, determination of therapeutically effecEive MDC antagonists, doses,
and
dosing schedules is faalitated by dose-response studies in art-recognized in
viva models for HIV
2 0 infection and proliferation, such as studies in appropriate mice
jPettoello-Mantovani et al., J.
Irfec~ Diseases, 177:337 (1998); J.M. McCune et al., "The Hematophtology ofHIV-
1 Disease:
Experimental Analysis in viva," in Huma~ee Hematopoiesis in SLID Mice, M.
Roncarolo et al.
(eds.), Landes Publishing Co., New York, NY, 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
Immurtol., Yol. Yll, Melchers et al. (eds.), Springer-Verlag Berlin
Heidelberg, pp. 1046-1049
( 1989)] or primate models.
B. Use of TA_RC ~n~gonQts to inhibit HIV~rofferation
The foregoing experiments also suggest further analysis wherein an HIV-1
3 0 infectiv'rty assay is repeated using neutralizing antibodies directed
against other beta chemokines.
For those (i-chemokines lacking an activity towards TH2 cells (analogous to
MDC's activity
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toward such cells), it is expected that chemokine-specific n~trali~ng
antibodies will behave much
like the marine control IgGl antibody above. However, for those ~3 chemokines
that possess an
activity toward TH2 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 infeetivity and/or proliferation
of immunodeficiency
viruses is specifically contemplated as an aspect of the invention.
The nucleotide and deduced amino acid sequences of TARC have been reported
in the literature and are set forth herein in SEQ m NOs: 42 and 43. See Imai
et al., J. Biol.
Chem. 271: 21514-21521 (1996); GENBANK ACCESSION NO. D43767. TARC golypeptides
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. Cltem., 272: 15036-15042 (1997); and Imai et al.,
J. Bio~ Chem., 271:
21514-21521 (1996).] The HIV-proliferativeJanti-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.
Based on the theory that the HIV antiproliferative e$ycacy of 1VIDC
antagonists
is mediated by blocking the signaling of MDC through CCR4 in target cells that
express CCR4,
2 0 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.
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 heating and response to injury but can be deleterious as well, as in
the case of chronic
inflammatory diseases, such as pulmonary fibrosis [Phan, in: ImmunoloQV of
nflamma inn
Elsevier (1983), pp. 121-162].
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In vitro ceU 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 x 103 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 finm cells and replaced
with MDC
diluted in DMEM comaining 0.4% FBS. Day 5: one microCurie of 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 ~i-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% FB S as the positive control.
As shown in Figure 7, MDC treatment decreased the proliferation of fibroblasts
in a dose dep~dent 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.
The effects of MDC upon the proliferation of epithelial cells, T cells,
fibroblasts,
endothelial cells, macrophages, antl tumor cells are assayed by methods known
in the art, such as
those described in Denholm et al., Amer. J. Paihol., 134:355-363 (1989), and
"In Vitro Assays
2 5 of Lymphocyte Functions," in: Current Protocols Immunology, Sections 3-4,
Whey 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:
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).
3 0 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
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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 pg/ml PHA with or without 50, 125, 250 or 500
ng/ml of
MDC. One microCurie of 3H-thymidine is added during the last 18 hours of
incubation. Cells are
harvested and proliferations expressed as described for fibroblasts in Example
21.
The effects of MDC on macrophage proliferation are determined using elicited
guinea pig peritoneat 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.
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.
2 0 In Pivo FibrQblast Proliferation Aesav
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
2 5 proliferation and collagen synthesis during all stages of this disease.
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 ofMDC in saline; 3) bleomycin-treated, given an
intratracheal injection of 1.5
mg/kg bleomycin (Calbiochem, Palo Alto, CA); and 4) bleomycin-treated rats
which also are
3 0 given a daily intraperitoneal injection of 500 ng of MDC.
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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.
~aam In a 24
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%
fonmamide, 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-
phemrlindole. An
initial hybridization experiment localized the gene to the q terminus of a
group E chromosome.
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 biting specific labeling. The lVmC
probe hybridized
to a region immediately adjacent to the heterochromaticJeuchromatic boundary,
corresponding
2 0 to band 16q13. The gene encoding TARC also is localized in this region.
See Nomiyama et al.,
Genomics, 40: 211-213 (1997).
These chromosomal mapping data indicate a utility of lVmC-encoding
polynucleotides as a chromosomal marker. Contiguous fragments of SEQ m NO: 1
of at least
15 nucleotides, and more preferably at least 20, 25, 50, 75, 100, 150, 200,
500, or more
2 5 nucleotides, and the complements of such fragments, are specifically
contemplated as probes of
the invention. Moreover, probes having partial degeneracy from SEQ m 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 B7 NO: 1 are preferred as probes of
the invention.
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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 ('I'l~nus and Activation-Regulated Chemokine, Genbank Accession No.
043767). 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.
A. prgpsytion of CCR4-transfected cells
The murine pre-B cell line L1.2 [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
descn'bed previously by elecrroporation with 10 pg linearized plasmid at 260
V, 960 microfarads
using a Gene Pulser (BioRad). See Imai et a1, 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
2 0 assays. For example, 293 cell lines have been transfected with CCR4 cDNA
and employed
effectively in calcium Flux assays.
B. Preparation of Recombinant Chemokines
The mature sequences of both 1V11?C and TARC were chemically synthesized by
Gryphon Sciences (South San Francisco CA) using t-butyl-oxycarbonyl
chemistries on a peptide
synthesizer (430A; Applied Biosyste<ns). Lyophilized protein was dissolved at
10 mgrml in 4 mM
HCl and immediately diluted to 0.1 mglml in phosphate-buffered saline plus
0.1% bovine serum
albumin (BSA) for storage at -80° C.
Recombinant 11~C also was expressed as a fusion protein with the secreted form
3 0 of placental alkaline phosphatase (SEAP) in the expression vector pcDNA3
(Clontech, Palo Alto
CA). A similar TARO-SEAP fusion protein is described in Imai et al. (1997).
Briefly, the coding
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region ofMDC, 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)6 tag. The MDC-SEAP expression plasmid was transfected into COS cells by
the DEAF
Dextran method. See Sambrook et al., Molecular Ctonireg: A LaboratoryMamral,
Cold Spring
Harbor Laboratory, Cold Spring Harbor; NY (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 1 %. After 3-4
days, the culture
supernatants were collected, centrifuged, filtered through a 0.45 micron
membrane, and stored
at 4 ° 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.
C. CCR4 Binding
The 1V1DC-SEAP was used as a probe to examine MDC binding to CCR4-
transfected L 1.2 cells. For displacement and saturation experiments,
transfected L 1.2 cells
(approx. 3 X I03) were incubated for one hour ax 16'C in the presence of 0.5
nM MDC-SEAP
in the presence or absence of various concentrations of unlabeled chemokines
in 200 ~tI binding
buffer (RPMI 1640 media containing 25 mM HEPES, pH 7.4, 1% BSA, and 0.02%
sodium
2 0 azide). Following incubation, the cells were washed four times in binding
buffer and lysed in 50
pl of 10 mM Tris-HCI, pH 8.0, and 1% Triton X-100. Samples were heated at bS~C
for 15
minutes to inactivate cellular phosphatases, centrifuged, and stored at -20
° C until assayed.
Alkaline phosphatase activity in 10 ~1 of sample was determined by a
chemiluminescence assay using the Great Escape Detection kit (Clontech, Palo
Alto, CA)
2 5 according to the manufacturer's instructions. The saturation binding curve
was fitted (Table
G~uveT~ using the Ill equation y = a(~)l(~ + b'), 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
Iigand at which 50% of receptor sites are occupied (KD), and c is the I~11
coeffcient. Binding
competition curves were fitted (TabIeCurveTM) using a three-parameter logistic
model described
3 0 by the equation y = al(I + (xlb)'J, where y is the amount of labelled
ligand bound, a is the
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maximum amount of labelled ligand bound; x is the concentration of the
competitive chemokine,
b is the ICso, and c is a parameter that determines the slope of the curve at
the ICso.
These binding assays demonstrated that MDC-SEAP bound to CCR4-expressing
cells. This binding was to a single high affinity site with a ICd of 0.18 nM,
as demonstrated by
Scatchard analysis. Binding of MDGSEAP was competitively inhibited with
increasing
concons of unlabeled MDC or TARC. The ICso for 1V1DC was 0.65 nM, while the
ICS 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.
To examine the specificity of MDC binding to CCR4, six additional chemokines
(MCP-1, MCP-3, MCP-4, RANTES, MIP-la, and MIP-lp) 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.
D. Calcium mobiI'~tion ascav
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 eacamined calcium mobilization in L1.2 cells recombinantly expressing CCRI,
CCR2B, CCR3,
2 0 CCR4, CCRS, CCR6, or CCR7.
Transfected L1.2 cells were suspended at a concentration of 3 x 106 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 pM fore-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 x 106
cells/mI. To measure intracellular calcium, 2 ml of cells were placed in a
quartz cuvette in a
Perkin-Elmer LS SOB spectrofluorimeter. Fluorescence was monitored at 340 nm
(excitation
wavelength 1), 380 nm (excitation wavelength 2), and 510 nm (emission
wavelength) every 200
ms.
In these experiments, MDC did not cause calcium flux in Ll .2 cells
transfected
3 0 with CCRI, CCR2B, CCR3, CCRS, 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
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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 1~C 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 lvIDC. 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 N117C is a
ligand for CCR4.
E. Chemotaxis assay
. We next examined the ability of lViDC and TARC to induce migration of CCR4-
transfected L1.2 cells. Approximately 106 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 pm
pore size, Costar). Untransfected L1.2 cells were used as a control. Test
chemolcines 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°C,
cells in the lower chamber were collected and counted by FACS.
Both lViDC and TARC induced migration of CCR4-transfected L1.2 cells. Both
chemolcines 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
N117C inducing migration of greater than 7% of input cells versus less than 3%
migration for
2 0 TARC. Untransfected L1.2 cells failed to migrate when treated with Iv)DC.
These chemotaxis
results fizrther confirm that both IvmC and TARC are functional Iigands for
CCR4.
F. Conclusion
Collectively, the foregoing experiments provide compelling evidence that MDC
2 5 acts as a high affinity ligand for the chemoldne receptor CCIt4.
As described below in Example 32, CCR4 has been found to be abundantly and
nearly exclusively expressed on antigen-specific TH2 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 1VZDC receptor on HIV-susceptible T
cells indicates a
3 0 putative mechanism/pathway through which 1VIDC(1-69) exerts its agonistic
activity relating to
enhanced HIV-1 infectivity and or viral production in infected cells (see
Example 20), and
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likewise indicates a target for therapeutic intervention. Without intending to
be limited to a
particular theory, MDC-mediated activation of TH2 cells, through the CCR4
receptor, is
postulated to enhance infectivity and/or production of HIV-1 vines, 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 (199'x; and Roederer et al., J. Clin. Invest., 99(7):
1555-1564 (1997).
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.
MDC is labelled with a detectable label such as "~I, 3H, '4C, 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'~I-labelled MDC, for example,
and incubated
2 0 under suitable conditions (e.g., ten minutes at 37°C). The
membranes, with any bound'uI-MDC,
are then collected on a filter by vacuum filtration and washed to remove
unbound'~I-MDC. The
radioactivity associated with the bound MDC is then quantitated by subjecting
the filters to liquid
scintillation spectrophotometry.
The specificity of MDC binding may be confirmed by repeating the foregoing
assay
2 5 in the presence of incxeasing 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.
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
3 0 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
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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.
-
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.
A. Direct Assav
In one embodiment, the invention comprehends a direct assay for modulation
(potentiation or inhibition) of MDC-receptor binding. In one direct assay,
membrane preparations
preseribng the chemokine receptor CCR4 in a functional conformation are
exposed to either MDC
alone or MDC in combination with potential modulators.
For suitable meanbrane 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
2 0 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-HCI, pH 7.4, I mM EDTA, 6 mM MgC h 10 pM PMSF, 1 pg/ml
leupeptin).
The homogenate is centrifuged at 800 x g for 10 minutes. The resulting pellet
is homogenized
again in TEM and re-pelleted. The combined supernatants are then centrifuged
at 100,000 x g
2 5 for one hour. The pellets containing the membrane preparations are
resuspended in TEM at 1.5
mg/ml.
Membrane preparations are exposed to labelled MDC (e.g., MDC labelled with
h~ 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)
3 0 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 pg of the
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membrane preparation, 0.06 nM of radio-labelled MDC, and 0.01 to 100 ~M of one
or more test
compounds, in a reaction buffer comprising 50 mM HEPES, pH 7.4, 1 mM CaCl2, 5
mM MgCl2,
and 0.1% BSA. The reactions are then incubated under suitable conditions
(e.g., for I-120
minutes, or more preferably 10-60 minutes, at a temperature from about room
temperature to
about 37°C).
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 ~nbodiment wherein the label is a
radioisotope, then bound MDC
preferably is quantified by subjecting the filters to liquid scintillation
spectrophotornetry. In an
embodiment wherein an MDC-alkaline phosphatase fixsion protein is employed,
alkaline
phosphatase activity is measured using, for example, the "Great Escape"
detection kit (Clontech,
Palo Alto, California) 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
2 0 experimental reaction is scored as containing one or more putative
antagonists (inhibitors) of
MDC receptor binding.
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
2 5 compounds, to determine whether the modulator compound operates by binding
with the MDC
receptor.
B. Tndirect GT~P aceav
In another embodiment, the invention comprehends indirect assays for
identifying
3 0 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,
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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
3zp; from ['y 3zP]-GTP.
For example, approximatelg 5 x 10' 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 5 mCi/ml [~PJ-
sodium
phosphate for 2 hours to uniformly label nucleotide pools. The cells are
subsequently washed in
a low-phosphate isotonic buffer.
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., 10
minutes, 37°C); cells
are pelleted and lysed, and nucleotide compounds are fractionated using, e.g.,
thin layer
chromatography (TLC) developed with 1 M LiCI. 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.
In this assay, the extent of MDC irneraction with its receptor is proportional
to the
levels of~P-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,
2 0 producing 3zP-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 ~P-
labelled GDP,
indicative of decreased rector activity, identifies the pot~tial modulator as
an inhibitor of MDC
receptor binding or an inhibitor of MDC-induced CCR4 signal transduction.
C. ~8,~
The ales of G protein effector molecules (e.g., adenylyl cycdase,
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
3 0 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
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activates adenylyl cyclase, can be detected by monitoring cAMP levels in a
host cell that
recombinantly expresses CCR4.
Host cells that recombinarnly express CCR4 are prefeixed 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 descn'bed 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, IL) provides reagents for a suitable
competitive
immunoassay for cAMP.
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.
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 1V>DC and CCR4,
and especially those
variations which utilize MDC and cells that recombinantly express CCR4, are
intended as aspects
of the invention.
While the use of human MDC and CCR4 comprises a highly preferred
2 0 embodiment, it will be apparent that the source organism for 1V>DC 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. Biophy~ Res Comm., 218(1): 337-343, and GenBank
Accession No.
2 5 X90862. Moreover, the methods used herein to obtain rat and mouse MDC are
employable to
obtain MDC or CCR4 from other organisms.
Moreover, evidence exists thlK there is at least one additional receptor that
recognizes MDC. For example, MDC stimulates migration of dendritic cells and
IIr2 activated
natural killer cells. Godiska et al., J. Fxp. Med, 185: 1595-1604 (1997),
incorporated herein by
3 0 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).
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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.
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.
A Isc~1_ation of cl~NA Encoding Rat and Mouse MDC Pnp~ein_s
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.
To clone a rat MDC cDNA, a labelled probe was prepared using standard random
2 0 primer extension techniques. A fragment of the human MDC cDNA was
generated by PCR,
which fi~nent includes the MDC coding region plus approximately 300 bases of
3' untranslated
sequence. This fi-agment was labelled with ~P-deoxyribonucleotides using the
Random Primed
DNALabeling kit (Boehringer Mannhein, Indianapolis, IN). The labelled MDC
probe was used
to screen approximately 106 bacteriophage lambda clones from a commercially-
available rat
2 5 thymus cDNA fbrary (Stratagene, La. Jolla, California, 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 m NO: 37) that included an MDC open
reading
fi~ame (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)
3 0 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
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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.
To clone a mouse MDC cDNA, approximately 106 bacteriophage lambda clones
of a commercially-available mouse thymes cDNA library (Stratagene, Cat. No.
935303) were
scxeened 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); 32P-labelled
deoxyribonucleotides; and
the Klenow fragment of E. coli DNA polymerase. Twelve positive clones were
isolated.
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 of3' uMranslated sequence. The cDNA and deduced amino acid sequences for
this murine
MDC clone are set forth in SEQ m NOs: 35 and 36, respectively. The mouse MDC
has a
I5 putative 24 amino acid signal sequence followed by a 68 amino acid A~II3C
sequence.
Comparisons of the human, rat, and mouse MDC protein and DNA (coding
region) sequences reveal the following levels of similarity:
Human vs. rat protein: 65% identity;
Human vs. rat DNA: 74% identity;
2 0 Human vs. mouse protein: 64% identity;
Human vs. mouse DNA: 72% identity;
Rat vs. mouse protein: 88% identity;
Rat vs. mouse DNA: 92% identity.
The four cysteines characteristic of C-C chemokines are conserved in all three
MDC proteins.
2 5 It is contemplated that the encoded rat and mouse lVmC polypeptides
corresponding to SEQ ll? NOs: 38 and 36 are processed into mature mouse MDC
proteins, in a
manner analogous to the processing of the human lVlDC precursor, by cleavage
of a signal
peptide. The signal peptides for both human and murine 1VIDC are 24 amino
acids. The exact
length of the rat MDC signal peptide will be readily apparent upon isolation
of a full length rat
3 0 MDC cDNA. It will be appreciated that these proteins can be synthesized
recombinantly or
synthetically and assayed for NIDC biological activities as described herein
for human 1VIDC.
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Likewise, it will be appreciated that any analogs descn'bed herein for human
MDC can be similarly
prepared for these other mammalian MDC proteins.
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
polynucieotides, in turn,
can be expressed (using procedures similar to those described in preceding
examples) to produce
recombinant polypeptides corresponding to other vertebnite 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.
B. SSmthesis of mLrine MDC and demon~ration of biological activit~
The inteniction between marine MDC and human CCR4 was demonstrated using
synthetic marine MDC in a chemotaxis assay. Marine L1.2 cells transfected with
human CCR4
(Example 25) were tested to determine if such cells would migrate towards
synthetic full-length
mature marine MDC (SEQ ID NO: 36, residues 1 to b8) (Gryphon Sciences and Ian
Clark-
Lewis), and/or toward a synthetic marine MDC analog designated "Lea-MDC" which
consists
of a l~cine residue attached to the amino terminus of mature marine MDC.
(Marine Leu-MDC
is thus analogous to 'W.DC(n+1)" described in Example 11. Costar Transwells
with 3 pm filters
were used for the assay.
Varying amounts of the synthetic I193C polypeptides (ten-fold dilutions from
10000 to 1 ng/ml final concentrations) were added to 600 pl RPMI/0.5% BSA
(endotoxin-free)
in the lower wells and 106 cells in 100 ~.l RPMI/0.5% BSA (endotoxin-free)
were added to the
uppex chambers. After incubating the transwells at 37°C for 4 hours,
the upper chambers were
transferred to 500 p.l ice-cold PBS/0.5 mM EDTA to release any migrated cells
still clinging to
the underside of the filter. Cells which had migrated to the lower chambers
were harvested by
combining the 600 lil medium from the lower chamber with the 500 pl PBS/EDTA
for each well.
Cells were centrifuged, resuspended in 200 pl of 1% formaldehyde, and then
counted for 30
3 0 seconds on the FACSCAN (Becton-Dickinson).
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The number of Ll.2/huCCR4 cells that were observed to have migrated toward
full-length mature marine MDC showed a characteristic dose-response curve,
with chemotaxis
observed at 1 ng/ml MDC and with peak chemotaxis occurring at 100 ng/mI marine
MDC. The
same number of cells migrated towards the 100 ng/ml full-lenght mature marine
MDC from
Gryphon Sciences and Ian Clark-Lewis,-indicating that the two preparations had
equivalent
activity. The responses of Ll.Z/huCCR4 cells to marine Leu-MDC were
approximately 20%
lower than to full-length MDC.
C. brine MDC comyetes with human MDC for binding to uma_n CCR4.
In duplicate, 5 x lOs L1.2/huCCR4 cells were incubated with 0.1 nM'ZSI-labeled
human mature MDC, alone or with unlabeled human mature MDC ( 10 nM or 100 nMJ,
marine
mature MDC (100 nM), or the the chemokine LARC (100 nM, control), for one hour
in 200 pl
binding buffer (50 mM HEPES, pH 7.5, 1 mM CaCl2, 5 mM MgCh, 0.5% BSA, and
0.05%
azide). Cells were spun down at slow speed and washed twice with binding
buffer plus 0.5 M
NaCI. Fifty microliters of scintillant fluid was added and samples were
counted with a beta
counter. Unlabeled human and marine MDC both substantially reduced the amount
of labeled
MDC that bound to the CCR4-expressing cells (approx. 4000 cpm versus less than
500 cpm),
with 100 nM marine MDC displaying a level of competition imermediate to that
of 10 nM and
100 nM human MDC. The contol chemokine LARC (which specifically binds CCR6)
diplayed
substantially no competitive binding ability (approx. 3800 cpm).
The foregosng assay results demonstrate that a nonhuman form of MDC (marine
MDC) is capable of binding and stimulating cells expressing a human MDC
receptor. This data
demonstrates an indication for vertebrate MDC, MDC fi~agtnents and analogs,
and MDC
modulators for human treatments and treatment formulations, as described
elsewhere herein for
human MDC, MDC fragments and analogs, and MDC modulators.
D. M~que MDC cDNA and po~y~gptide sequences.
Polymerise chain reaction (PCR), using oligonucleotides died from the human
MDC cDNA as primers, was performed in order to amplify and isolate a cDNA
encoding
3 0 macaque MDC from a macaque thymus cDNA library. The macaque MDC amino acid
sequence
with sec~ory signal sequence is 93 amino acids and shares about 94% amino acid
identity with
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human MDC. Referring to SEQ m NO: 2, the macaque MDC amino acid sequence is
identical
to that ofthe human sequence, with the following variations: valine at
position -18; phenylalanine
at position -17; glycine at position -15; isoleucine at position -12;
methionine at position 21; and
serine at position 46. The macaque cDNA and deduced amino acid sequences are
set forth in
SEQ m NOs: 45 and 46.
E. Use of multiple vertebrate MDC syuences to desjgn h~G a_nal_oqc_
The amino acid sequences for human, macaque, mouse, rat and/or other animals
can be aligned using any alignment algorithm known in the art. Such an
alignment will identify
positions and regions within the MDC sequences that are highly conserved
(e.g., that are identical
in different species), moderately conserved (e.g., identical in some species
with substitutions in
other species of amino acids of similar character (e.g., acidic, basic,
aliphatic, aromatic)), or
variable (e.g., different in most or all species, including substitutions of
amino acids of different
character). Such an alignment provides significant guidance for the design of
1V1DC analogs that
will act as MDC mimetics as well as analogs that may act as 11~C inhibitors.
Substitution or
deletion of variable residues is more likely to result in analogs that retain
MDC biological
activities, whereas highly conserved residues are targets for alteration or
deletion to design
analogs having different activities or having MDC inhibitory activity.
~~~pile 29
Using procedures essentially as described in Example 25, selected IVmC analogs
descn'bed in Example 11 were for the ability to bind CCR4 and/or induce
calcium (Ca'"")
flux and chemotaxis in Ll .2 cells transfected with CCR4.
The analog lVmC{n+1) bound CCR4 with similar affnity to lva3C, but induced
calcium 8ux and chemotaxis in L1.2/CCR4 cells with a slightly Lower potency
than MDC. For
example, in chemotaxis, the peak activity for lVmC(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 lVmC.
MDC(9-69) bound CCR4 with reduced amity relative to that of MDC (0-69).
lVmC(9-69) did not induce calcium flux in L1.2/CCR4 cells, and it was much
less potent in
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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.
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.
The analog "MDC-wvas"-hound CCR4 with 500-fold less amity than MDC,
induced only a very small calcium flux, and did not induce any chemotaxis. The
analog "MDC-
eyfy" acted similar to lVmC in CCR4-binding, chemotaxis, and calcium flux
assays.
Monoclonal Antibodies 252Y & 2522 Inhibit
CCR4-Mediated Cellular Resnonsrs to MDC
Using procedures similar to those described in Example 25, the monoclonal
antibodies 252Y and 2522 described in Example 18 were screened for the ability
to modulate
MDC-CCR4 binding and modulate the CCR4-mediated biological activities of MDC.
A. Antibodies 252Y and 2522 inhibit MDC binding to CCR4
The fusion protein MDC-SEAP (Example 25) was employed to evaluate the ability
of the antt'bodies 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
2 0 (0.01-10 pg/ml, shown in Fig. I 1) of antibody 252Y, antibody 2522, or an
isotype control (final
reaction volume 100 pl). Thereafter, the mixtures were added to CCR4-
expressing L1.2 cells
(100 ul, 4000 cells per ul), and incubated at 4 ° 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
2 5 (defined as the amount of binding that could not be competed by a 200-fold
molar excess of native
MDC) was detennined and subtracted from experimental measurements. Figure 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
Figure 11, both
3 0 antt'body 252Y and arnx'body 2522 (but not the isotype control) inhibited
MDC-SEAP binding to
CCR4-infected cells in a dose-dependem manner. Fifty percent inhibition of
binding was observed
for both antibodies at an a~'body concentration of about 2 pglml.
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B. Antibodies 252Y and 2522 irLh_ibit MDC-indLC.~d chemotaxis
To confnm that antibodies 252Y and 2522 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.
For the calaum flux assay, the transfected L1.2 cells were labelled with Fura-
2/AM
(see Example 19) and monitored for Ca~-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''~* levels. This Ca''+ flux response was
completely inhibited when either
antibody 252Y or antibody 2522 were added to the cells at a concentration of
10 lig/ml one
minute before contacting the cells with the MDC solution. An isotype-matched
control antibody
had no effect on the MDC-induced Ca** flux. Thus, both antibodies blocked the
calcium flux
response to MDC in CCR4-transfected L1.2 cells.
For the chemotaxis assay, CCR4 transferred L1.2 cells (approx. 10 million
cellslml
in a volume of 0.1 ml) were preincubated with antibody 252Y, antibody 2522, or
an isotype-
matched control in RPMI-1640 media (Cribco) at various concentrations ranging
from 0.5 to 50
lig/ml for 30 minutes at room temperature. Thereafter, the cells were exposed
to 100 nglml
11~C (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 Figure 12, MDC-induced chemotaxis of these
cells was totally
2 0 inhibited by either antibody 252Y or antibody 2522 at concentrations of 2 -
5 ~,g/ml, but not by
the isotype-matched control. The ICS antibody concentration (required to
inhibit 50% migration)
was 1 lig/ml. The same antibodies did not inhibit chemotaxis ofthe CCR4/L1.2
cells toward the
C-C chemokine TARC, indicating that the inhibitory effect was specific for
MDC.
In a similar set of experiments, antibody 272D was sfor its ability to inhibit
MDC stimulated chemotaxis. Ten ~g/ml of antibody 272D was required to inhibit
chemotaxis
toward recombinant MDC (30 ng/ml) by greater than 90%. Only 2 pg/ml of
antibody 2522 was
required to achieve a similar level of inhibition, indicating that antibody
2522 is a more potent
inhibitor of 1V1DC induced chemotaxis.
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A transendothelial migation assay was performed essentially as described in
the
art [Ponath, et al., J. Clin. Invest., 97: 604-612 (1996); Ponath et al., J.
Exp. Mec~, 183: 2437-
2448 (1996); and Imai, et al., Cell, 91: 521-530 (1997)] to determine the
presence and the
phenotype of T cells that migate toward the chemokines TARC and MDC. Briefly,
about 2 x
10' cells of the endothelial cell line ECV304 (ATCC CRL-1998 or European Cell
Culture
Colleckion, Portions Down, LJK) were added to Transwell inserts (Coaster) with
a 5 pm pore size
and cultured at 37°C for 48-96 hours in M199 medium (GIBCOBRL)
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:M199, 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 pl. Endothelial cell-
coated inserts were
placed in each well and 106 peripheral blood mononuclear cells (PBMC) or T
cell lines in 100 pl
were added to the upper chambers. The cells were allowed to migrate through
the endothelial
cells into the lower chambers at 37°C for 4 hours (PBMC) or 90 minutes
(T cell lines). The
migated 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.
In the tru~sendothelial cell migration assay, both TARC and MDC induced dose-
dependent vigorous migration of CD14' lymphocytes but not of CD14+ monocytes,
with MDC
2 0 consistently inducing cell migration about 2 times more e~ciently than
TARC. IVfigration activity
was detected with chemolane conons as low as 1 nM. Significant migration
occurred with
10 nM TARC and 10 nM MDC. Analysis of the migating lymphocytes revealed that
10 nM of
either TARC or MDC attracted predominantly CD4'' T cells. Neither TARC nor MDC
induced
migarion of CD19+ B calls or CD16+ NK cells. Furthermore; TARC and NmC
attracted almost
2 5 exclusively CD45RA /CD45R0+ effectorlmemory T cells. This observation was
consistent with
the observation that a murine {IgG) monoclonal antibody to CCR4 stained highly
selectively a
fraction 020~/0) of CD45R0+CD4+ memory helper T cells.
Effector/memory helper T cells represent a population of cells that have
encountered cognate antigens irm vivo and have differentiated into THl or TH2
cells. Since CCR4
3 0 is expressed on about 20% of effector/memory helper T cells, additional
experiments were
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conducted to determine whether CCR4 is selectively expressed on certain
subsets of helper T
cells.
First, CD4+CD45R0+ T cells (obtained from PBMC by negative selection with
Dynabeads (Dynal) after incubation with anti-CD 16, anti-CD 14, anti-CD20,
anti-CDB, and anti-
CD45RA antibodies) were fractionated into CCR4+ and CCR4' 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 ° 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
l0 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 cytolcine production. Since helper T cells are classified into THI and TH2
subsets based on their
profiles of cytoIcine 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.
Analysis of the culture medium revealed that the CCR4+ T cells produced
significantly larger amounts of IL-4 and IL-5 than the cultured CCR4' T cells
(>12 ng/ml for
CCR4+ T cells versus < 2.5 ng/ml for CCR4' T cells for each cytokine).
Conversely, CCR4' T
cells produced IFN-Y at levels much higher than CCR4+ T cells (> 300 ng/ml vs.
< 25 ng/ml).
2 0 These cytolane acpression patterns indicate that the CCR4'" population of
cells contained almost
exclusively TH2 cells, whereas CCR4' cells were enriched for THl cells.
To support the conclusion that CCR4+ T cells are predominantly TH2 cells, the
CD4+CD45R0+ T cells that had been attracted by TARC or NmC in the
transendothelial
migration assay were expanded by wlturing in PHA and ILr2 and then examined
for their pattern
2 5 of cytoldne production as described above. Compared to total CD4*CD45R0+ 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-y.
To further confirm the observed selective expression of CCR4 on TH2 cells,
experiments were performed to polarize CD4+CD45RA+ naive T cells in vitro, and
the artificially
3 0 polarized cell populations were acamined for CCR4 expression. The naive T
cells (obtained from
PBMC by negative selection with Dynabeads after incubation with anti-CD 16,
anti-CD 14, anti-
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CD20, anti-CDB, and anti-CD45R0 antibodies) were polarized into THl 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 TH2 cells by culturing with PHA ( 1:100), 10 ng/ml IL-4,
and 2 ~glml anti-
IG 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.
Analysis of the cultured cells with an anti-CCR4 monoclonal antibody revealed
that 60% of cells polarized into TH2 cells expressed CCR4, compared to only 4%
of cells
polarized into THl cells. Northern blot analysis of the RNA isolated from
these cell populations
also demonstrated that TH2 cells expressed CCR4 mRNA at levels much higher
than TH1 cells.
As controls, CCR7 mRNA was expressed in both types of cells whereas CCR3 mRNA
was not
detected in either type of cell.
In the endothelial transmigration assay, the artificially polarized TH2 cells,
but not
those polarized into THl, 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
chemoldne secondary lymphoid-tissue chemolcine that is a potent
chemoatrcactant for
lymphocytes and mapped to chromosome 9p13," J. Biol. Chem., 272: 19518-19524
(1997).)
Neither population of cells migrated toward eotaxin, a ligand for CCR3.
Collectively, the foregoing experiments demonstrate that a significant
population
of TH2 cells express the chemolcine receptor CCR4, and that the chemolcines
TARC and MDC
2 0 represent selective chemoattractants of TH2 cells, an effect that
presumably is mediated at least
in part through CCR4. Tissues of allergic inflammation are infiltrated by TH2
cells, as well as by
eosinophils, another cell type selectively attracted by MDC {see Example 12).
Furthermore, T
calls migrating into tissues after antigen challenge have been reported to be
involved in localized
production of the TH2 cytolW es, IL.-4 and TL-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.,
Armu. Reu Immunol., 9: 271-296 (1991).) Enhanced TARC and MDC production from
antigen
presenting cells in TH2 responses would be expected to lead to further
recruitment of TH2 cells
3 0 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
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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 TH2 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 TH2 T cells, is
specifically contemplated.
, Use of MDC aid MDG antagonists to modLlate,platelet aaggL~ga ion
The following experimental data indicates that 11~C promotes platelet
aggregation, and suggests a therapeutic indication for MDC and lvmC
antagonists to modulate
platelet aggregation.
Female Lewis rats, six to eight weeks old, were administered 0.5 pg of
synthetic
mature human IvmC(1-69) intravenously in a saline solution, via the tail vein.
At various time
points, the animals (4) were anesthetized with 100 lcl ACE cocktail (Ketamine,
ACE promazine
and Rompon) and blood samples were collected into Nficrocontainers containing
EDTA
(Beckton Dickinson). Samples (300-400 pl) were stored overnight at 4-8
°C. A CBC with
Differential analysis was conducted to identify changes in cell number in the
rats compared to
2 0 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 oflvmC administration and dissipated with time after the bolus. A
similar phenomenon
was observed in mice using an analogous protocol.
Receptor analyses have indicated that platelets express detectable levels of
the
2 5 1~C receptor CCR4. These experiments suggest a receptor through which 11~C
may exert its
platelet-aggregating effects.
The foregoing observations suggest that mature MDC stimulates platelet
aggregation, and suggests that Iv~C antagonists are useful for inhibiting
coagulation. Such use
is indicated, e.g., in myocardial infarction patients to prevent further
inappropriate blood clotting,
3 0 and in patients for the therapeutic or prophylactic treatment of stroke.
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The concentrations at which MDC induces platelet aggregation and at which
lVmC antagonists prevent platelet aggregation are determined in vitro using
purified platelets and
serial dilutions of MDC and NmC 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 a~f.Jpn. J. Phmmaco~, 75(3):295-298
(1997). Putative
MDC antagonists for screening in such assays include all of the putative IvR7C
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.
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.
The following procedures are performed to demonstrate that MDC antagonists,
such as MDC neutralizing antibodies, are capable of modulating an immune
respone in a
mammalian host.
A. Antigen-induced asthma model
Laboratory animals (e.g., BaIb/C mice) are challenged with ovalbumin using the
following regimen: Day 0: 100 pg ovalbumin (Sigma), 4.5 mg alum (Lnject~,
Pierce),
administered by 200 ~1 intraperitoneal injection; Day 14: 100 pg ovaibumin,
4.5 mg alum
administered by 200 gl intraperitoneal injection, plus 100 p.g ovalbumin in 50
ul saline,
administered infra-nasally, days 25, 26, and 27: 50 pg ovalbumin in 50 gl
saline, administered
2 5 infra-nasally. As a contol, saline is administered to animals in lieu of
ovalbumin. To test the
effect of a putative ABC modulator (such as an MDC-neutralizing antibody) on
the animal's
allergio-type response to the ovalbumen, the modulator (or a control, e.g.,
saline) is administered
to test animals intraperitoneally on days 25, 26, and 27, one hour prior to
challenge with
ovalbumin. Exemplary dosing of an anti-MDC antibody is 0.1 to 5 mg/kg body
weight.
3 0 On day 28, the mice are sacrificed, blood is collected, and
bronchioalveolar lavage
is performed. Cells from the lavage fluid are collected and counted, and a
white blood cell
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differential is performed. Reduction in eosinophils and/or neutrophils in the
lavage fluid of
treated animal versus contol animals is indicative of the therapeutic efl~cacy
of the MDC
antagonist treatment. Reduction in anti-ovalbumen antibodies (especially IgE
antibodies) in the
blood {assayed by ELISA, for example) is further indicative of the therapeutic
efficacy of the
MDC antagonist.
B. Modulation of a Ta ~ r~.cnonse
To demonstrate the ability of an MDC antagonist to suppress an immune
response, laboratory animals are immunized subcutaneously or intraperitoneally
with a suitable
antigen, such as ovalbumin or tetanus toxoid, or with a saline control.
Aluminum hydroxide
(alum), which preferentially promotes a TH2 response, or Freund's complete
adjuvant, which
tends to drive a THl response, are used as adjuvants in some of the animals.
Animals are
immunized on day 0 (e.g., with 100 pg ovalbumin + 4.5 mg alum), followed by
booster
immunizations at, e.g., days 14 and 28. The antibody titer against the
selected antigen is
permitted to drop to normal levels in the animals, e.g., for 1-2 months,
monitored via ELISA.
After arna'body levels have dropped to normal, the animals are re-challenged
with
the selected antigen. An MDC antagonist, such as a an MDC-neutralizing
antibody, is
administered contemporaneously with the antigen, two, six, and/or twenty-four
hours later. One
week later, blood from the animals is drawn, white blood cells are analyzed,
and antibodies to
2 0 the antigen are titered and isotyped. Reduced levels of IgGI antibody, IgE
antibody, and TH2
cells in the treated animals versus the control animals is indicative of a
therapeutically effective
MDC antagonist, where immunosuppression is desired. A more pronounced
thereapeutic effect
in the alum-administered animals than the animals injected with Freund's
aduvant is expected.
C. Murine lupus model
The therapeutic e~cacy of an MDC antagonist for the treatment of Iupus
erythematosus is demonstrated in animal models, such as NZB/NZW Fl mice, that
are known
in the art and have been described in the literature. See, e.g., Wofsy, D. et
al., J. Immu»ol.,
138(10): 3247-3253 (May, 1987); and Daikh et al., J. Immunol., 159(7): 3104-
3108 (Oct.,
3 0 1997).
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D. Use of a_n- MDC antrgorist to treat huma_n_ lupLS ervt-h_emAtosus
An MDC antagonist such as a humanized or human anti-MDC antibody or anti-
CCR4 antibody is employed in a standard dose-escalation study to demonstrate
efficacy in the
treatment of lupus erythematosus in affected human individuals. Exemplary
dosing regimens for
an antibody range from 0.01 to 50 mg/kg body weight, and preferably 0.1 to 5
mg/kg,
administered weekly, or bi-weekly, or monthly. Treatment e~cacy is detenmined
by monitoring
standard indices. See, e.g., Bombardier et al., "Derivation of the SLEDAI: a
disease activity
index for lupus patients," Arthritis Rheum, 35: 630-640 (1992); Liang et al.,
"Measurement of
systemic lupus erythematosus activity in clinical research," ArthritisRheum.,
31: 817-825 (1988).
Optimal dosing is determined by standard dose-response studies after eff cacy
is demonstrated.
E. Use of an MDC a_ntagoni_st to treat huma_n_ multiple sclerosis
An MDC antagonist such as a humanized or human anti-MDC antibody or anti-
CCR4 antibody is employed in a standard dose-escalation study to demonstrate
efficacy in the
treatment of multiple sclerosis in affected human individuals. Exemplary
dosing regimens for an
antibody-based therapeutic are as set forth in Seciton D, above. Treatment
efficacy is determined
by monitoring standard MS indices. See, e.g., Kurtzke, J.F., "Rating
neurologic impairment in
multiple sclerosis: An expanded disability status scale (EDSS)," Neurology,
33: 1444 (1983).
The biological functions of MDC, elucidated as described above, suggest
several
clinical applications.
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
3 0 directly demonstrated in experiments involving 13,,-8, a C-X-C chemokine
that attracts and
activates neutrophils. Antibodies directed against IL-8 have a profound
ability to inhibit
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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.
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 NIDC or MI7C agonists may be beneficial in
promoting recovery
from such diseases.
In addition, the myelosuppressive effect demonstrated for the C-C chemokine
MIP-la (Maze et al., supra) suggests that MDC may have a similar activity.
Such activity,
provided by 1VIDC or 11~C 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.
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 Laving 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.
2 0 While the present invention has been described in terms of specific
embodiments,
it is understood that variations and modifications will occur to those skilled
in the art.
Accordingly, only such limitations as appear in the appended claims should be
placed on the
invention.
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-1-
SEQUENCE LISTING
<110> ICOS Corporation, et al.
<120> MACROPHAGE DERIVED CHEMOKINE (MDC), MDC ANALOGS, MDC
INHIBITOR SUBSTANCES, AND USES THEREOF
<130> 27866/34810PCT
<140>
<141>
<150> 09/067,447
<151> 1998-04-28
<150> 08/939,107
<151> 1997-09-26
<150> 08/660,542
<151> 1996-06-07
<150> 08/558,658
<151> 1995-11-16
<150> 08/479,620
<151> 1995-06-07
<160> 46
<170> PatentIn Ver. 2.0
<210> 1
<211> 2923
<212> DNA
<213> Homo sapiens - human MDC cDNA
<220>
<221> CDS
<222> (20)..(298)
<220>
<221> mat~eptide
<222> (92)..(298)
<400> 1
gagacataca ggacagagc atg get cgc cta cag act gca ctc ctg gtt gtc 52
Met~Ala Arg Leu Gln Thr Ala Leu Leu Val Val
-20 -15
ctc gtc ctc ctt get gtg gcg ctt caa gca act gag gca ggc ccc tac 100
Leu Val Leu Leu Ala Val Ala Leu Gln Ala Thr Glu Ala Gly Pro Tyr
-10 -5 -1 1
SUBSTfTUTE SHEET (RULE 26)
CA 02302806 2000-03-08
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-2-
ggcgcc aacatggaa gacagc gtctgctgc cgtgat tacgtccgt tac 148
GlyAla AsnMetGlu AspSer ValCysCys ArgAsp TyrValArg Tyr
5 10 15
cgtctg cccctgcgc gtggtg aaacacttc tactgg acctcagac tcc 196
ArgLeu ProLeuArg ValVal LysHisPhe TyrTrp Thr'SerAsp Ser
20 25 30 35
tgcccg aggcctggc gtggtg ttgctaacc ttcagg gataaggag atc 244
CysPro ArgProGly ValVal LeuLeuThr PheArg AspLysGlu Ile
40 45 50
tgtgcc gatcccaga gtgccc tgggtgaag atgatt ctcaataag ctg 292
CysAla AspProArg ValPro TrpValLys MetIle LeuAsnLys Leu
55 60 65
agccaa tgaagagcct ggaaggctca 348
actctgatga
ccgtggcctt
ggctcctcca
SerGln
ggagccctac ctccctgcca ttatagctgc tccccgccag aagcctgtgc_caactctctg 408
cattccctga tctccatccc tgtggctgtc acccttggtc acctccgtgc tgtcactgcc 468
atctcccccc tgacccctct aacccatcct ctgcctccct ccctgcagtc agagggtcct 528
gttcccatca gcgattcccc tgcttaaacc cttccatgac tccccactgc cctaagctga 588
ggtcagtctc ccaagcctgg catgtggccc tctggatctg ggttccatct ctgtctccag 648
cctgcccact tcccttcatg aatgttgggt tctagctccc tgttctccaa acccatacta 708
cacatcccac ttctgggtct ttgcctggga tgttgctgac actcagaaag tcccaccacc 768
tgcacatgtg tagccccacc agccctccaa ggcattgctc gcccaagcag ctggtaattc 828
catttcatgt attagatgtc ccctggccct ctgtcccctc ttaataaccc tagtcacagt 888
ctccgcagat tcttgggatt tgggggtttt ctcccccacc tctccactag ttggaccaag 948
gtttctagct aagttactct agtctccaag cctctagcat agagcactgc agacaggccc 1008
tggctcagaa tcagagccca gaaagtggct gcagacaaaa tcaataaaac taatgtccct 1068
cccctctccc tgccaaaagg cagttacata tcaatacaga gactcaaggt cactagaaat 1128
gggccagctg ggtcaatgtg aagccccaaa tttgcccaga ttcacctttc ttcccccact 1188'
cccttttttt tttttttttt tttgagatgg agtttcgctc ttgtcaccca cgctggagtg 1248
caatggtgtg gtcttggctt attgaagcct ctgcctcctg ggttcaagtg attctcttgc 1308
ctcagcctcc tgagtagctg ggattacagg ttcctgctac cacgcccagc taatttttgt 1368
atttttagta gagacgaggc ttcaccatgt tggccaggct ggtctcgaac tcctgtcctc 1428
SUBSTITUTE SHEET (RULE 26)
CA 02302806 2000-03-08
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-3-
aggtaatccg cccacctcag cctcccaaag tgctgggatt acaggcgtga gccacagtgc 1488
ctggcctctt ccctctcccc actgcccccc ccaacttttt tttttttttt atggcagggt 1548
ctcactctgt cgcccaggct ggagtgcagt ggcgtgatct cggctcacta caacctcgac 1608
ctcctgggtt caagtgattc tcccacccca gcctcccaag tagctgggat tacaggtgtg 1668
tgccactacg gctggctaat ttttgtattt ttagtagaga caggtttcac catattggcc 1728
aggctggtct tgaactcctg acctcaagtg atccaccttc cttgtgctcc caaagtgctg 1788
agattacagg cgtgagctat cacacccagc ctcccccttt ttttcctaat aggagactcc 1848
tgtacctttc ttcgttttac ctatgtgtcg tgtctgctta catttccttc tcccctcagg 1908
ctttttttgg gtggtcctcc aacctccaat acccaggcct ggcctcttca gagtaccccc 1968
cattccactt tccctgcctc cttccttaaa tagctgacaa tcaaattcat gctatggtgt 2028
gaaagactac ctttgacttg gtattataag ctggagttat atatgtattt gaaaacagag 2088
taaatactta agaggccaaa tagatgaatg gaagaatttt aggaactgtg agagggggac 2148
aaggtgaagc tttcctggcc ctgggaggaa gctggctgtg gtagcgtagc gctctctctc 2208
tctgtctgtg gcaggagcca aagagtaggg tgtaattgag tgaaggaatc ctgggtagag 2268
accattctca ggtggttggg ccaggctaaa gactgggagt tgggtctatc tatgcctttc 2328
tggctgattt ttgtagagac ggggttttgc catgttaccc aggctggtct caaactcctg 2388
ggctcaagcg atcctcctgg ctcagcctcc caaagtgctg ggattacagg cgtgaatcac 2448
tgcgcctggc ttcctcttcc tcttgagaaa tattcttttc atacagcaag tatgggacag 2508
cagtgtccca ggtaaaggac ataaatgtta caagtgtctg gtcctttctg agggaggctg 2568
gtgccgctct gcagggtatt tgaacctgtg gaattggagg aggccatttc actccctgaa 2628
cccagcctga caaatcacag tgagaatgtt caccttatag gcttgctgtg gggctcaggt 2688
tgaaagtgtg gggagtgaca ctgcctaggc atccagctca gtgtcatcca gggcctgtgt 2748
ccctcccgaa cccagggtca acctgcctgc cacaggcact agaaggacga atctgcctac 2808
tgcccatgaa cggggccctc aagcgtcctg ggatctcctt ctccctcctg tcctgtcctt 2868
gcccctcagg actgctggaa aataaatcct ttaaaatagt aaaaaaaaaa aaaaa 2923
<210> 2
<211> 93
<212> PRT
<213> Homo sapa.ens - human NmC
SUBSTITUTE SHEET (RULE 26)
II
CA 02302806 2000-03-08
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<400> 2
Met Ala Arg Leu Gln Thr Ala Leu Leu Val Val Leu Val Leu Leu Ala
-20 -15 -10
Val Ala Leu Gln Ala Thr Glu Ala Gly Pro Tyr Gly Ala Asn Met Glu
-5 -1 1 5
Asp Ser Val Cys Cys Arg Asp Tyr Val Arg Tyr Arg Leu Pro Leu Arg
15 20
Val Val Lys His Phe Tyr Trp Thr Ser Asp Ser Cys Pro Arg Pro Gly
25 30 35 40
Val Val Leu Leu Thr Phe Arg Asp Lys Glu Ile Cys Ala Asp Pro Arg
45 50 55
Val Pro,Trp Val Lys Met Ile Leu Asn Lys Leu Ser Gln
60 65
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer JHSP6
<400> 3
gacactatag aatagggc lg
<210> 4
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer M13
<400> 4
gtaaaacgac ggccagt
17
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer T3.1
<400> 5
aattaaccct cactaaaggg 20
SUBSTITUTE SHEET (RULE 26)
CA 02302806 2000-03-08 n
WO 99/15666 PCT/US98/20270
-5-
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer T7.1
<400> 6
gtaatacgac tcactatagg gc 22
<210> 7
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 390-1F
<400> 7
tctatctaga ggcccctacg gcgccaacat ggaag 35
<210> 8
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 390-2R
<400> 8
caccggatcc tcattggctc agcttattga gaa 33
<210> 9
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 390-4R
<400> 9
aatggatcca cagcacggag gtgaccaag 29
<210> 10
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 390-3R
SUBSTITUTE SHEET (RULE 26)
i
CA 02302806 2000-03-08
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-6-
<400> to
agtcaagctt agggcactct gggatcggca c 31
<210> 11
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 390-FX2
<400> 11
tatcggatcc tggttccgcg tggcccctac ggcgccaaca tggaa 45
<210> 12
c211> 22
<212> DNA
c213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer GEX5
<400> 12
gaaatccagc aagtatatag ca 22
<210> 13
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 390-Pel
<400> 13
attgccatgg ccggccccta cggcgccaac atggaa 36
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 390RcFI
<400> 14
gaccaagctt gagacataca ggacagagca 30
<210> 15
<211> 29
<212> DNA
SUBSTITUTE SHEET (RULE 26)
~~
__ "",.~"~,
CA 02302806 2000-03-08
WO 99115666 PCT/US98/Z0270
_'7_
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 390RcX
<400> 15
tggatctaga agttggcaca ggcttctgg 29
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer DC03
<400> 16
cgaaattaat acgactcact 20
<210> 17
<211> 67
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
390mycRX
<400> 17
tggatctaga tcaattcaag tcctcctcgc tgatcagctt ctgctcttgg ctcagcttat 60
tgagaat 67
<210> 18
<211> 99
<212> PRT
<213> Homo sapiens - Hu MCP-3
<220>
<400> 18
Met Lys Ala Ser Ala Ala Leu Leu Cys Leu Leu Leu Thr Ala Ala Ala
-20 -15 -10
Phe Ser Pro Gln Gly Leu Ala Gln Pro Val Gly Ile Asn Thr Ser Thr
-5 1 5
Thr Cys Cys Tyr Arg Phe Ile Asn Lys Lys Ile Pro Lys Gln Arg Leu
15 20 25
Glu Ser Tyr Arg Arg Thr Thr Ser Ser His Cys Pro Arg Glu Ala Val
30 35 40
SUBSTITUTE SH~B'~ (RUtE2S~
CA 02302806 2000-03-08
WO 99/I5666 PCT/US98/20270
_g_
Ile Phe Lys Thr Lys Leu Asp Lys Glu Ile Cys Ala Asp Pro Thr Gln
45 50 55
Lys Trp Val Gln Asp Phe Met Lys His Leu Asp Lys Lys Thr Gln Thr
60 65 70
Pro Lys Leu
<210> 19
<211> 99
<212> PRT
<213> Homo spapiens - Hu MCP-1
<400> 19
Met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu Leu Ile Ala Ala Thr
-20 -15 -10
Phe Ile Pro Gln Gly Leu Ala Gln Pro Asp Ala Ile Asn Ala Pro Val
-5 1 5
Thr Cys Cys Tyr Asn Phe Thr Asn Arg Lys Ile Ser Val Gln Arg Leu
10 15 20 25
Ala Ser Tyr Arg Arg Ile Thr Ser Ser Lys Cys Pro Lys Glu Ala Val
30 35 40
Ile Phe Lys Thr Ile Val Ala Lys Glu Ile Cys Ala Asp Pro Lys Gln
45 50 55 80
Lys Trp Val Gln Asp Ser Met Asp His Leu Asp Lys Gln Thr Gln Thr
60 65 70
Pro Lys Thr
<210> 20
<211> 76
<212> PRT
<213> Homo sapiens - Hu MCP-2
<220>
<400> 20
Gln Pro Asp Ser Val Ser Ile Pro Ile Thr Cys Cys Phe Asn Val Ile
1 5 10 15
Asn Arg Lys Ile Pro Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr
20 25 30
Asn Ile Gln Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Lys Arg Gly
35 40 45
Lys Glu Val Cys Ala Asp Pro Lya Glu Arg Trp Val Arg Asp Ser Met
50 55 60
SUBSTITUTE SHEET (RULE 26)
CA 02302806 2000-03-08
WO 99/15666 PCT/US98/20270
-9-
Lys His Leu Asp Gln Ile Phe Gln Asn Leu Lys Pro
65 70 75
<210> 21
<211> 91
<212> PRT
<213> Homo sapiens - RANTES
<220>
<400> 21
Met Lys Val Ser Ala Ala Ala Leu Ala Val Ile Leu Ile Ala Thr Ala
-20 -15 -10
Leu Cys Ala Pro Ala Ser Ala Ser Pro Tyr Ser Ser Asp Thr Thr Pro
-5 1 5
Cys Cys Phe Ala Tyr Ile Ala Arg Pro Leu Pro Arg Ala His Ile Lya
15 20 25
Glu Tyr Phe Tyr Thr Ser Gly Lys Cys Ser Asn Pro Ala Val Val Phe
30 35 40
Val Thr Arg Lys Asn Arg Gln Val Cys Ala Asn Pro Glu Lys Lys Trp
45 50 55
Val Arg Glu Tyr Ile Asn Ser Leu Glu Met Ser
60 65
<210> 22
<211> 9I
<212> PRT
<213> Homo sapiens - MIP-1 beta
<220>
<400> 22
Met Lys Leu Cys Val Thr Val Leu Ser Leu Leu Met Leu Val Ala Ala
-20 -15 -10
Phe Cys Ser Pro Ala Leu Ser Ala Pro Met Gly Ser Asp Pro Pro Thr
-15 1 5
Ala Cys Cys Phe Ser Tyr Thr Arg Glu Ala Ser Ser Asn Phe Val Val
10 15 20
Asp Tyr Tyr Glu Thr Ser Ser Leu Cys Ser Gln Pro Ala Val Val Phe
30 35 40
Gln Thr Lys Arg Ser Lys Gln Val Cys Ala Asp Pro Ser Glu Ser Trp
45 50 55
Val Gln Glu Tyr Val Tyr Asp Leu Glu Leu Asn
60 65
SUBSTITUTE SI-~~ET RULE ~~)
CA 02302806 2000-03-08
WO 99/15666 PCT/IJS98I20270
-10-
<210> 23
<211> 92
<212> PRT
<213> Homo sapiens - MIP-1 alpha
<220>
<400> 23
Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala
-20 -15 -10
Leu Cys Asn Gln Phe Ser Ala Ser Leu Ala Ala Asp Thr Pro Thr Ala
-5 1 5 10
Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile Ala
15 20 25
Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Gly Val Ile Phe
30 35 40
Leu Thr Lys Arg Ser Arg Gln Val Cys Ala Asp Pro Ser Glu Glu Trp
45 50 55
Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala
60 65 70
<210> 24
<211> 96
<212> PRT
<213> Homo sapiens - I-309
<220>
<400> 24
Met Gln Ile Ile Thr Thr Ala Leu Val Cys Leu Leu Leu Ala Gly Met
-20 -15 -10
Trp Pro Glu Asp Val Asp Ser Lys Ser Met Gln Val Pro Phe Ser Arg
-5 1 5
Cys Cys Phe Ser Phe Ala Glu Gln Glu Ile Pro Leu Arg Ala Ile Leu
15 20 25
Cys Tyr Arg Asn Thr Ser Ser Ile Cys Ser Asn Glu Gly Leu Ile Phe
30 3S 40
Lys Leu Lys Arg Gly Lys Glu Ala Cys Ala Leu Asp Thr Val Gly Trp
45 50 55
Val Gln Arg His Arg Lys Met Leu Arg His Cys Pro Ser Lys Arg Lys
60 65 70
SUBSTITUTE SHEET (RUtE 26)
CA 02302806 2000-03-08
WO 99/15666 PCT/US98/20270
-11-
<210> 25
<211> 93
<212> PRT
<213> Artificial Sequence - Human MDC analog
<220>
<223> The amino acid at position 24 is selected from the
group consisting of arg, gly.~ ala, val, leu, ile,
pro, ser, thr, phe, tyr, trp, aspartate,
glutamate, asn, gln, cys, and met
<220>
<223> The amino acid at position 27 is independently
selected from the group consisting of lys, gly,
ala, val, leu, ile, pro, ser, thr, phe, tyr, trp,
aspartate, glutamate, asn, gln, cys, and met
<220>
<223> The amino acid at position 30 is independently
selected from the group consisting of tyr, ser,
lys, arg, his, aspartate, glutamate, asn, gln, and
cys
<220>
<223> The amino acid at position 50 is independently
selected from the group consisting of glu, lys,
arg, his, gly, and ala
<220>
<223> The amino acid at position 59 is independently
selected from the group consisting of trp, ser,
lys, arg, his, aspartate, glutamate, asn, gln, and
cys
<220>
<223> The amino acid at position 60 is independently
selected from the group consisting of val, ser,
lys, arg, his, aspartate, glutamate, asn, gln, and
cys
<400> 25
Met Ala Arg Leu Gln Thr Ala Leu Leu Val Val Leu Val Leu Leu Ala
-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
15 20
val Val Xaa His Phe Xaa Trp Thr Ser Asp Ser Cys Pro Arg Pro Gly
25 30 35 40
SUBSTITUTE SHEET (RULE 26)
ni
CA 02302806 2000-03-08
WO 99/15666 PCT/US98/20270
-12-
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
<210> 26
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 390-7F
<400> 26
tattggatcc gttctagctc cctgttctcc 30
<210> 27
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 390-8R
<400> 27
ccaagaattc ctgcagccac tttctgggct c 31
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer ARA1
<400> 28
gcgactctct actgtttctc 2p
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer ARA2
<400> 29
cacaggaaac agctatgacc 20
SUBSTITUTE SHEET (RULE 26)
CA 02302806 2000-03-08
WO 99/15666 PCT/US98/20270
-13-
<210> 30
<211> 70
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Human MDC analog
<400> 30
Leu Gly Pro Tyr Gly Ala Asn Met Glli Asp Ser Val Cys Cys Arg Asp
1 5 10 15
Tyr Val Arg Tyr Arg Leu Pro Leu Arg Val Val Lys His Phe Tyr Trp
20 25 30
Thr Ser Asp See Cys Pro Arg Pro Gly Val Val Leu Leu Thr Phe Arg
35 40 45
Asp Lys Glu Ile Cys Ala Asp Pro Arg Val Pro Trp Val Lys Met Ile
50 55 60
Leu Asn Lys Leu Ser Gln
65 70
<210> 31
<211> 69
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Human MDC analog
<400> 31
Gly Pro Tyr Gly Ala Asn Met Glu Asp Ser Val Cys Cys Arg Asp Tyr
1 5 10 15
Val Arg Tyr Arg Leu Pro Leu Arg Val Val Lys His Phe Tyr Trp Thr
20 25 30
Ser Asp Ser Cys Pro Arg Pro Gly Val Val Leu Leu Thr Phe Arg Asp
35 40 45
Lys Glu Ile Cys Ala Asp Pro Arg Val Pro Tyr Leu Lys Met Ile Leu
50 55 60
Asn Lys Leu Ser Gln
<210> 32
<211> 69
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Human MDC analog
<400> 32
SUBSTI~'UTE SIi~EET (RULE 26)
CA 02302806 2000-03-08
WO 99/15666 PCTIUS98/20270
-14-
Gly Pro Tyr Gly Ala Asn Met Glu Asp Ser Val Cys Cys Arg Asp Tyr
1 ' 5 10 15
Val Arg Tyr Arg Leu Pro Leu Arg Val Val Lys Glu Tyr Phe Tyr Thr
20 25 30
Ser Asp Ser Cys Pro Arg Pro Gly Val Val Leu Leu Thr Phe Arg Asp
35 40 45
Lys Glu Ile Cys Ala Asp Pro Arg Val Pro Trp Val Lys Met Ile Leu
50 55 60
Asn Lys Leu Ser Gln
<210> 33
<211> 1677
<212> DNA
<213> Homo sapiens - human CCR4 cDNA
<220>
<221> CDS
<222> (183)..(1262)
<400> 33
cgggggtttt gatcttcttc cccttctttt cttccccttc ttctttcctt cctccctccc 60
tctctcattt cccttctcct tctccctcag tctccacatt caacattgac aagtccattc 120
agaaaagcaa gctgcttctg gttgggccca gacctgcctt gaggagcctg tagagttaaa 180
as atg aac ccc acg gat ata gca gat acc acc ctc gat gaa agc ata 227
Met Asn Pro Thr Asp Ile Ala Asp Thr Thr Leu Asp Glu Ser Ile
1 5 10 15
tac agc aat tac tat ctg tat gaa agt atc ccc aag cct tgc acc aaa 275
Tyr Ser Asn Tyr Tyr Leu Tyr Glu Ser Ile Pro Lys Pro Cys Thr Lys
20 25 30,
gaa ggc atc aag gca ttt ggg gag ctc ttc ctg ccc cca ctg tat tcc 323
Glu Gly Ile Lys Ala Phe Gly Glu Leu Phe Leu Pro Pro Leu Tyr Ser
35 40 45
ttg gtt ttt gta ttt ggt ctg ctt gga aat tct gtg gtg gtt ctg gtc 371
Leu Val Phe Val Phe Gly Leu Leu Gly Asn Ser Val Val Val Leu Val
50 55 60
ctg ttc aaa tac aag cgg ctc agg tcc atg act gat gtg tac ctg ctc 419
Leu Phe Lys Tyr Lys Arg Leu Arg Ser Met Thr Asp Val Tyr Leu Leu
65 70 75
aac ctt gcc atc tcg gat ctg ctc ttc gtg ttt tcc ctc cat ttt tgg 467
Asn Leu Ala Ile Ser Asp Leu Leu Phe Val Phe Ser Leu Pro Phe Trp
80 85 90 95
SUBST'1TUTE SHEET (RULE 26)
CA 02302806 2000-03-08
WO 99/15666 PCTNS98/ZOZ70
-15-
ggc tac tat gca gca gac cag tgg gtt ttt ggg cta ggt ctg tgc aag 515
Gly Tyr Tyr Ala Ala Asp Gln Trp Val Phe Gly Leu Gly Leu Cys Lys
100 105 110
atg att tcc tgg atg tac ttg gtg ggc ttt tac agt ggc ata ttc ttt 563
Met Ile Ser Trp Met Tyr Leu Val Gly Phe Tyr Ser Gly Ile Phe Phe
115 120 125
gtc atg ctc atg agc att gat aga tac ctg gcg ata gtg cac gcg gtg 611
Val Met Leu Met Ser Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val
130 135 140
ttt tcc ttg agg gca agg acc ttg act tat ggg gtc atc acc agt ttg 659
Phe Ser Leu Arg Ala Arg Thr Leu Thr Tyr Gly Val Ile Thr Ser Leu
145 150 155
get aca tgg tca gtg get gtg ttc gcc tcc ctt cct ggc ttt ctg ttc 707
Ala Thr Trp Ser Val Ala Val Phe Ala Ser Leu Pro Gly Phe Leu Phe
160 165 170 175
agc act tgt tat act gag cgc aac cat acc tac tgc aaa acc aag tac 755
Ser Thr Cys Tyr Thr Glu Arg Asn His Thr Tyr Cys Lya Thr Lys Tyr
180 185 190
tct ctc aac tcc acg acg tgg aag gtt ctc agc tcc ctg gaa atc aac 803
Ser Leu Asn Ser Thr Thr Trp Lys Val Leu Ser Ser Leu Glu Ile Asn
195 200 205
att ctc gga ttg gtg atc ccc tta ggg atc atg ctg ttt tgc tac tcc 851
Ile Leu Gly Leu Val Ile Pro Leu Gly Ile Met Leu Phe Cys Tyr Ser
210 215 220
atg atc atc agg acc ttg cag cat tgt aaa aat gag aag aag aac aag 899
Met Ile Ile Arg Thr Leu Gln His Cys Lys Asn Glu Lys Lys Asn Lys
225 230 235
gcg gtg aag atg atc ttt gcc gtg gtg gtc ctc ttc ctt ggg ttc tgg 947
Ala VaI Lys Met Ile Phe Ala Val Val Val Leu Phe Leu Gly Phe Trp
240 245 250 255
aca cct tac aac ata gtg ctc ttc cta gag acc ctg gtg gag cta gaa 995
Thr Pro Tyr Asn Ile Val Leu Phe Leu Glu Thr Leu Val Glu Leu Glu
260 265 270
gtc ctt cag gac tgc acc ttt gaa aga tac ttg gac tat gcc atc cag 1043
Val Leu Gln Asp Cys Thr Phe Glu Arg Tyr Leu Asp Tyr Ala Ile Gln
275 280 285
gcc aca gaa act ctg get ttt gtt cac tgc tgc ctt aat ccc atc atc 1091
Ala Thr Glu Thr Leu Ala Phe Val His Cys Cys Leu Asn Pro Ile Ile
290 295 300
tac ttt ttt ctg ggg gag aaa ttt cgc aag tac atc cta cag ctc ttc 1139
Tyr Phe Phe Leu Gly Glu Lys Phe Arg Lys Tyr Ile Leu Gln Leu Phe
305 310 315
SUBSTITUTE SHEET (RULE 26)
CA 02302806 2000-03-08
WO 99/1566b PCT/US98/20270
-16-
aaa acc tgc agg ggc ctt ttt gtg ctc tgc caa tac tgt ggg ctc ctc 1187
Lys Thr Cys Arg Gly Leu Phe Val Leu Cys Gln Tyr Cys Gly Leu Leu
320 325 330 335
caa att tac tct get gac acc ccc agc tca tct tac acg cag tcc acc 1235
Gln Ile Tyr Ser Ala Asp Thr Pro Ser Ser Ser Tyr Thr Gln Ser Thr
340 345 350
atg gat cat gat ctt cat gat get ctg taggaaaaat gaaatggtga 1282
Met Asp His Asp Leu His Asp Ala Leu
355 360
aatgcagagt caatgaactt ttccacattc agagcttact ttaaaattgg tatttttagg 1342
taagagatcc ctgagccagt gtcaggagga aggcttacac ccacagtgga aagacagctt 1402
ctcatcctgc aggcagcttt ttctctccca ctagacaagt ccagcctggc aagggttcac 1462
ctgggctgag gcatccttcc tcacaccagg cttgcctgca ggcatgagtc agtctgatga 1522
gaactctgag cagtgcttga atgaagttgt aggtaatatt gcaaggcaaa gactattccc 1582
ttctaacctg aactgatggg tttctccaga gggaattgca gagtactggc tgatggagta 1642
aatcgctacc ttttgctgtg gcaaatgggc ccccg 1677
<210> 34
<211> 360
<212> PRT
<213> Homo sapiens - human CCR4
<400> 34
Met Asn Pro Thr Asp Ile Ala Asp Thr Thr Leu Asp Glu Ser Ile Tyr
1 5 10 15
Ser Asn Tyr Tyr Leu Tyr Glu Ser Ile Pro Lys Pro Cys Thr Lys Glu
20 25 30
Gly Ile Lys Ala Phe Gly Glu Leu Phe Leu Pro Pro Leu Tyr Ser Leu
35 40 45
Val Phe Val Phe Gly Leu Leu Gly Asn Ser Val Val Val Leu Val Leu
50 55 60
Phe Lys Tyr Lys Arg Leu Arg Ser Met Thr Asp Val Tyr Leu Leu Asn
65 70 75 80
Leu Ala Ile Ser Asp Leu Leu Phe Val Phe Ser Leu Pro Phe Trp Gly
85 90 95
SUBSTITUTE SHEET (RULE 26)
CA 02302806 2000-03-08
WO 99/15666 PCT/US98/20270
-17-
Tyr Tyr Ala Ala Asp Gln Trp Val Phe Gly Leu Gly Leu Cys Lys Met
100 105 110
Ile Ser Trp Met Tyr Leu Val Gly Phe Tyr Ser Gly Ile Phe Phe Val
115 120 125
Met Leu Met Ser Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val Phe
130 135 140
Ser Leu Arg Ala Arg Thr Leu Thr Tyr Gly Val Ile Thr Ser Leu Ala
145 150 155 160
Thr Trp Ser Val Ala Val Phe Ala Ser Leu Pro Gly Phe Leu Phe Ser
165 170 175
Thr Cys Tyr Thr Glu Arg Asn His Thr Tyr Cys Lys Thr Lys Tyr Ser
180 185 190
Leu Asn Ser Thr Thr Trp Lys Val Leu Ser Ser Leu Glu Ile Asn Ile
195 200 205
Leu Gly Leu Val Ile Pro Leu Gly Ile Met Leu Phe Cys Tyr Ser Met
210 215 220
Ile Ile Arg Thr Leu Gln His Cys Lys Asn Glu Lys Lys Asn Lys Ala
225 230 235 240
Val Lys Met Ile Phe Ala Val Val Val Leu Phe Leu Gly Phe Trp Thr
245 250 255
Pro Tyr Asn Ile Val Leu Phe Leu Glu Thr Leu Val Glu Leu Glu Val
260 265 270
Leu Gln Asp Cys Thr Phe Glu Arg Tyr Leu Asp Tyr Ala Ile Gln Ala
275 280 285
Thr Glu Thr Leu Ala Phe Val His Cys Cys Leu Asn Pro Ile Ile Tyr
290 295 300
Phe Phe Leu Gly Glu Lys Phe Arg Lys Tyr Ile Leu Gln Leu Phe Lys
305 310 315 320
Thr Cys Arg Gly Leu Phe Val Leu Cys Gln Tyr Cys Gly Leu Leu Gln
325 330 335
Ile Tyr Ser Ala Asp Thr Pro Ser Ser Ser Tyr Thr Gln Ser Thr Met
340 345 350
Asp His Asp Leu His Asp Ala Leu
355 360
<210> 35
<211> 1784
SUBSTITUTE SHEET (RULE 2fi)
CA 02302806 2000-03-08
WO 99/15666 PCT/US98/20270
-18-
<212> DNA
<213> murine MDC cDNA
<220>
<221>
CDS
<222> (276)
(1)
. .
<220>
<221>
mat~eptide
<222>
(73)..(276)
<400>
35
atg aatctg cgtgtccca ctcctggtg getctcgtc cttctt get 48
tct
Met AsnLeu ArgValPro LeuLeuVal AlaLeuVal LeuLeu Ala
Ser
-20 -15 -10
gtg attcag acctctgat gcaggtccc tatggtgcc aatgtg gaa 96
gca
Val IleGln ThrSerAsp AlaGlyPro TyrGlyAla AsnVal Glu
Ala
-5 -1 1 5
gac atctgc tgccaggac tacatccgt caccctctg ccatca cgt 144
agt
Asp IleCys CysGlnAsp TyrIleArg HisProLeu ProSer Arg
Ser
15 20
tta aaggag ttcttctgg acctcaaaa tcctgccgc aagcct ggc 192
gtg
Leu LysGlu PhePheTrp ThrSerLys SerCysArg LysPro Gly
Val
25 30 35 40
gtt ttgata accgtcaag aaccgagat atctgtgcc gatccc agg 240
gtt
Val LeuIle ThrValLys AsnArgAsp IleCyaAla AspPro Arg
Val
45 50 55
cag tgggtg aagaagcta ctccataaa ctgtcctagggaggag 286
gtc
Gln TrpVal LysLysLeu LeuHisLys LeuSer
Val
60 65
gacctgatga ccatgggtct ggtgtggtcc agggaggctc agcaagccct attcttctgc 346
cattccagca agagccttgc caacgacgcc acctttactc acctccatcc cctgggctgt 406
cactctgtca ggctctggtc cctctacctc ccctctatcc cttccagctt ateccccttc 466
aatgtggcag ctgggaaaca cattcaggcc agccttaccc aatgcctact ccccactgct 526
ttagatgaga ccagcgtcct tgttttgatg ccctgatcct atgatgcctt ccccatcccc 586
agccttggcc cccttctctt cttgcatgta gggaaggccc ataggtttca aatatgtgct 646
acctacttcc ctttctgggg ggttctaata cccagcatgt ttttcctgct gcaggcacct 706
atccagtgcc acacacctcc caagtttcta tcagtcccag tgggcatcca ccaagcccca 766
aacttcagac ttccttggcc tccacctact ctcagtagaa ttctgggagt ttcaggctgg 826
tccaccaggc cccccagggt taggccaagg tccccaccag agctcctcct gtttcttggt 886
SUBSTITUTE SH~~T (RULE 28)
CA 02302806 2000-03-08
WO 991156b6 PCT/US98/Z0270
-19-
ctgcagcacg gggcagggag caaggagcag gctcagaatc agatttctta aaggagctgc 946
agactccatc agtaaaagga atctttctcc catccctgaa tataaggcag ttttctgtca 1006
acacagagac tcaggttgtt agaaatggcc acatagatca actgtgaaac cctaaattta 1066
ccaagaatca acttccaccc ctcttcaacc acatgctagg gtcttttact ttctctgccc 1126
cacacctttg actccttgcc tgtgtagctg atagtcgaag ttatgctatg gtgtcagtga 1186
ctgccacagt ttgtttggta ttataagcta tagttatatt tatataggaa agaggataaa 1246
tatatgtggg ccaaatagac gaactggaga gttttaggat ctgggggcag gaagggccat 1306
acaaagtgat acctcagaaa atagatggtt gtgggagctg ctgccagtgg cagagttaac 1366
ttaaagaact taattgaaat tattcttgag tggctgaggc caagacaaga atatagaacc 1426
cattcttgct tccctggaga caacagtggt cccaggggaa ggaataaacc ttcttgctcc 1486
tctggaggga gcatggcctg rcttagccga gtgactggac tgtgtgagat tgggggcatc 1546
gcttttccty tctgagcctc agctgacagc atatgggacc acaaagggct tgatccaaac 1606
cacagggatt gacagtgcca gccacagctg tgtccagggc tcgtgttctg ccagaaggag 1666
cacctggacg accagggcca ccactagtgc tactttgctc actgcccatg catgtcctga 1726
aggtccctcc ccctcctctc ctacttctgg gaaaataaat gctcgccaat aatacctg 1784
<210> 36
<211> 92
<212> PRT
<213> murine
MDC
<400> 36
Met Ser Asn ArgVal ProLeu Leu Ala Leu Val Leu
Leu Val Leu Ala
-20 -I5 -10
Val Ala Ile ThrSer AspAla Gly Tyr Gly Ala Val
Gln Pro Asn Glu
-5 -1 1 5
Asp Ser Ile CysGln AspTyr Ile His Pro Leu Ser
Cys Arg Pro Arg
15 20
Leu Val Lys PhePhe TrpThr Ser Ser Cys Arg Pro
Glu Lys Lys Gly
25 30 35 40
Val Val Leu ThrVal LysAsn Arg Ile Cys Ala Pro
Ile Asp Asp Arg
45 50 55
Gln Val Trp LysLys LeuLeu His Leu Ser
Val Lys
60 65
SUBSTITUTE SHEET (RULE 26)
CA 02302806 2000-03-08
WO 99/15666 PCT/US98/20270
-20-
<210> 37
<211> 958
<212> DNA
<213> rat MDC cDNA
<220>
<221> CDS
<222> (1) .. (243)
<220>
<221> mat~eptide
<222> (40)..(243)
<400> 37
ctc gtc ctt ctt get gtg gca ctt cag acc tcc gat gca ggt ccc tat 48
Leu Val Leu Leu Ala Val Ala Leu Gln Thr Ser Asp Ala Gly Pro Tyr
-10 -5 -1 1
ggt gcc aat gtg gaa gac agt atc tgc tgc cag gac tac atc cgt cac 96
Gly Ala Asn Val Glu Asp Ser Ile Cys Cys Gln Aap Tyr Ile Arg His
10 15
cct ctg cca cca cgt ttc gtg aag gag ttc tac tgg acc tca aag tcc 144
Pro Leu Pro Pro Arg Phe Val Lys Glu Phe Tyr Trp Thr Ser Lys Ser
20 25 30 35
tgc cgc aag cct ggc gtc gtt ttg ata acc atc aag aac cga gat atc 192
Cys Arg Lys Pro Gly Val Val Leu Ile Thr Ile Lys Asn Arg Asp Ile
40 45 50
tgt get gac ccc ang atg etc tgg gtg aag aag ata ctc cac aag ttg 240
Cys Ala Asp Pro Xaa Met Leu Trp Val Lys Lys Ile Leu His Lys Leu
55 60 65
gcc tagggagaag ggcctgatga ccacgggtct ggtgtctcca caaggctcag 293
Ala
caaaccctat ccttctgcca tccagcaaga gccttgccaa caactccacc tttgctcacc 353
tccatcccct gggttgtcac tctgtgaagc ctcgggtccc tgtacttcct gtccgtcccc 413
tccagctcat tctcttccaa cgtggcagcc gggaagcact tctggctagc cttacccaat 473
actactcccc actgctttaa atgagaccag ggtccttgtt ttggtgcctt tggatcctat 533
gatgccttcc cagtctccag ccttggcccc cttctcttct tacatgtagg gaacaccaat 593
atctttcaag tatgtgctac ccaattcctc ttcctcggag gctgctggga cccggaatat 653
tatcccctgc tgcaggcctc tccaagcacc actcacctcc caggctttcc atccgtccca 713
gtcccaagcc ccatgcttca gaacttccct tggccccccc ctacactcca caaattctgg 773
ggaagtctca cnaactgggt cccctcaggc ccccacggga aggaaggtcc cccnccaaca 833
SUBSTITUTE SHEET (RULE 26)
CA 02302806 2000-03-08
WO 99/15666 PCT/US98/20270
-21-
acntcctcct gttttccccg gtctcccncc nccgggantt gggcncccna atccccaatt 893
tctgaanang aacngcecat tcntcccntt aaaattaacc tttccccccc tccctgangt 953
tagga 958
<210> 38
<211> 81
<212> PRT
<213> rat
<400> 38
Leu Val Leu Leu Ala Val Ala Leu Gln Thr Ser Asp Ala Gly Pro Tyr
-10 -5 -1 1
Gly Ala Asn val Glu Asp Ser Ile Cys Cys Gln Asp Tyr Ile Arg His
10 15
Pro Leu Pro Pro Arg Phe Val Lys Glu Phe Tyr Trp Thr Ser Lys Ser
20 25 30 35
Cys Arg Lys Pro Gly Val Val Leu Ile Thr Ile Lys Asn Arg Asp Ile
40 45 50
Cys Ala Asp Pro Xaa Met Leu Trp Val Lys Lys Ile Leu His Lys Leu
55 60 65
Ala
<210> 39
<211> 506
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: S. cerevisiae alpha factor pre-
pro/human MDC cDNA chimeric construct
<221> CDS
<222> (15)..(476)
<220>
<221> mat_peptide
<222> (270)..(476)
<400> 39
atctcgagct cacg atg aga ttt cct tca att ttt act gca gtt tta ttc 50
Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe
-85 -80 -75
gca gca tcc tcc gca tta get get cca gtc aac act aca aca gaa gat 98
Ala Ala Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp
-70 -65 -60
SUBSTITUTE SHEET (RULE 26)
CA 02302806 2000-03-08
WO 99/15666 PCT/US98/20Z70
-22-
gaa acg gca caa att ccg get gaa get gtc atc ggt tac tta gat tta 146
Glu Thr Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu
-55 -50 -45
gaa ggg gat ttc gat gtt get gtt ttg cca ttt tcc aac agc aca aat 194
Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn
-40 -35 -30
aac ggg tta ttg ttt ata aat act act att gcc agc att get get aaa 242
Asn Gly Leu Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys
-25 -20 -15 -10
gaa gaa ggg gta cct ttg gat aaa aga ggc ccc tac ggc gcc aac atg 290
Glu Glu Gly VaI Pro Leu Asp Lys Arg Gly Pro Tyr Gly Ala Asn Met
-5 -1 1 5
gaa gac agc gtc tgc tgc cgt gat tac gtc cgt tac cgt ctg ccc ctg 338
Glu Asp Ser Val Cys Cys Arg Asp_Tyr Val Arg Tyr Arg Leu Pro Leu
15 20
cgc gtg gtg aaa cac ttc tac tgg acc tca gac tcc tgc ccg agg cct 386
Arg Val Val Lys His Phe Tyr Trp Thr Ser Asp Ser Cys Pro Arg Pro
25 30 35
ggc gtg gtg ttg cta acc ttc agg gat aag gag atc tgt gcc gat ccc 434
Gly Val Val Leu Leu Thr Phe Arg Asp Lys Glu Ile Cys Ala Asp Pro
40 45 50 55
aga gtg ccc tgg gtg aag atg att ctc aat aag ctg agc caa 476
Arg Val Pro Trp Val Lys Met Ile Leu Asn Lys Leu Ser Gln
60 65
tgaaggcctt ctagagcggc cgcatcgata 506
<210> 40
<211> 154
<212> PRT
<213> CDNA
<400> 40
Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
-85 -80 -75 -70
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
-65 -60 -55
Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe
-50 -45 -40
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
-35 -30 -25
Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val
-20 -15 -10
SUBSTITUTE SHEET (RULE 26)
CA 02302806 2000-03-08
WO 99/15666 . PCT/US98/20270
-23-
Pro Leu Asp Lys Arg Gly Pro Tyr Gly Ala Asn Met Glu Asp Ser VaI
-5 -1 1 5 10
Cys Cys Arg Asp Tyr Val Arg Tyr Arg Leu Pro Leu Arg Val Val Lys
15 20 25
His Phe Tyr Trp Thr Ser Asp Ser Cys Pro Arg Pro Gly Val Val Leu-
30 35 - 40
Leu Thr Phe Arg Asp Lys Glu Ile Cys Ala Asp Pro Arg Val Pro Trp
45 50 55
Val Lys Met Ile Leu Asn Lys Leu Ser Gln
60 65
<210> 41
<211> 93
<212> PRT
<213> Artificial Human MDC analog
<220>
<223> The amino acid at position 2 is not proline
<220>
<400> 41
Met Ala Arg Leu Gln Thr Ala Leu Leu Val Val Leu Val Leu Leu Ala
-20 -15 -10
Val Ala Leu Gln Ala Thr Glu Ala Gly Xaa Tyr Gly Ala Asn Met Glu
-5 1 5
Asp Ser Val Cys Cys Arg Asp Tyr Val Arg Tyr Arg Leu Pro Leu Arg
15 20
Val Val Lys His Phe Tyr Trp Thr Ser Asp Ser Cys Pro Arg Pro Gly
25 30 35 40
Val Val Leu Leu Thr Phe Arg Asp Lys Glu Ile Cys Ala Asp Pro Arg
45 50 55
Val Pro Trp Val Lys Met Ile Leu Asn Lys Leu Ser Gln
60 65
<210> 42
<211> 538
<212> DNA
<213> Homo sapiems
<220>
<221> CDS
<222> {53)..(334)
<220>
SUBS1lTUTE SHfET (RULE 26)
CA 02302806 2000-03-08
WO 99/15666 . PCT/US98f20270
-24-
<221> mat_peptide
<222> (122)..(334)
<400> 42
ccctgagcag agggacctgc acacagagac tccctcctgg gctcctggca cc atg gcc 58
Met Ala
cca ctg aag atg ctg gcc ctg gtc arc ctc ctc ctg ggg get tct ctg 106
Pro Leu Lys Met Leu Ala Leu Val Thr Leu Leu Leu Gly Ala Ser Leu
-20 -15 -10
cag cac atc cac gca get cga ggg acc aat gtg ggc cgg gag tgc tgc 154
Gln His Ile His Ala Ala Arg Gly Thr Asn Val Gly Arg Glu Cys Cys
-5 -1 1 5 10
ctg gag tac ttc aag gga gcc att ccc ctt aga aag ctg aag acg tgg 202
Leu Glu Tyr Phe Lys Gly Ala Ile Pro Leu Arg Lys Leu Lys Thr Trp
15 20 25
tac cag aca tct gag gac tgc tcc agg gat gcc atc gtt ttt gta act 250
Tyr Gln Thr Ser Glu Asp Cys Ser Arg Asp Ala Ile Val Phe Val Thr
30 35 40
gtg cag ggc agg gcc atc tgt tcg gac ccc aac aac aag aga gtg aag 298
Val Gln Gly Arg Ala Ile Cys Ser Asp Pro Asn Asn Lys Arg Val Lys
45 50 55
aat gca gtt aaa tac ctg caa agc ctt gag agg tct tgaagcctcc 344
Asn Ala Val Lys Tyr Leu Gln Ser Leu Glu Arg Ser
60 65 70
tcaccccaga ctcctgactg tctcccggga ctacctggga cctccaccgt tggtgttcac 404
cgcccccacc ctgagcgcct gggtccaggg gaggccttcc agggacgaag aagagccaca 464
gtgagggaga tcccatcccc ttgtctgaac tggagccatg ggcacaaagg gcccagatta 524
aagtctttat cctc 538
<210> 43
<211> 94
<212> PRT
<213> Homo sapiens
<400> 43
Met Ala Pro Leu Lys Met Leu Ala Leu Val Thr Leu Leu Leu Gly Ala
-20 -15 -10
Ser Leu Gln His Ile His Ala Ala Arg Gly Thr Asn Val Gly Arg Glu
-5 -1 1 5
Cys Cys Leu Glu Tyr Phe Lys Gly Ala Ile Pro Leu Arg Lys Leu Lys
15 20 25
SUBSTITUTE, SHEET (RULE 26)
CA 02302806 2000-03-08
WO 99/15666 . PCT/US98/20270
-25-
Thr Trp Tyr Gln Thr Ser Glu Asp Cys Ser Arg Asp Ala Ile Val Phe
30 35 40
Val Thr Val Gln Gly Arg Ala Ile Cys Ser Asp Pro Asn Asn Lys Arg
45 50 55
Val Lys Asn Ala Val Lys Tyr Leu Gln Ser Leu Glu Arg Ser
60 65 , 70
<210> 44
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer
<400> 44
atgggaccat atggagcaaa tatggaagat agt 33
<210> 45
<211> 335
<212> DNA
<213> Macaque MDC
<220>
<221> CDS
<222> (19)..(297)
<400> 45
agacatacag gacagagc atg get cgc cta cag act gtg ttc ctg ggt gtc 51
Met Ala Arg Leu Gln Thr Val Phe Leu Gly Val
-20 -15
ctc atc ctc ctt get gtg gcg ctt caa gca act gag gca ggc ccc tat 99
Leu Ile Leu Leu Ala Val Ala Leu Gln Ala Thr Glu Ala Gly Pro Tyr
-10 -5 -1 1
ggc gcc aac atg gaa gac agc gtc tgc tgc cgt gat tac gtc cgt tac 147
Gly Ala Asn Met Glu Asp Ser Val Cys Cys Arg Asp Tyr Val Arg Tyr
10 15
cgt atg ccc ctg cgt gtg gtg aaa cac ttc tac tgg acc tca gac tcc 195
Arg Met Pro Leu Arg Val Val Lys His Phe Tyr Trp Thr Ser Asp Ser
20 25 30 35
tgc ccg agg cct ggc gtg gtg ttg cta acc tcc agg gat aag gag atc 243
Cys Pro Arg Pro Gly Val Val Leu Leu Thr Ser Arg Asp Lys Glu Ile
40 45 50
tgt gcc gat ccc aga gtg ccc tgg gtg aag atg att ctc aat aag ctg 291
Cys Ala Asp Pro Arg Val Pro Trp Val Lys Met Ile Leu Asn Lys Leu
55 60 65
SUBSTITUTE SHEET (RULE 26~
CA 02302806 2000-03-08
WO 99/15666 PCT/US98/20270
-26-
agc caa tgaagagcct actatgatga ccgtggccta agcaagcc 335
Ser Gln
<210> 46
<211> 93
<212> PRT
<213> Macaque MDC
<400> 46
Met Ala Arg Leu Gln Thr Val Phe Leu Gly Val Leu Ile Leu Leu Ala
-24 -20 -15 -10
Val Ala Leu Gln Ala Thr Glu Ala Gly Pro Tyr Gly Ala Aan Met Glu
-5 -1 1 5
Asp Ser Val Cys Cys Arg Asp Tyr Val Arg Tyr Arg Met Pro Leu Arg
15 20
Val Val Lys His Phe Tyr Trp Thr Ser Aap Ser Cys Pro Arg Pro Gly
25 30 35 40
Val Val Leu Leu Thr Ser Arg Asp Lys Glu Ile Cys Ala Asp Pro Arg
45 50 55
Val Pro Trp Val Lys Met Ile Leu Asn Lys Leu Ser Gln
60 65
SUBSTITUTE SHEET (RULE 26)
