Note: Descriptions are shown in the official language in which they were submitted.
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NLAMMALIAN CHEMOKINES
FIELD OF THE INVENTION
The present invention contemplates compositions
related to proteins which function in controlling
development, differentiation, trafficking, and physiology
of mammalian cells , a . g . , cells of a mammalian immune
system. In particular, it provides proteins which
regulate or evidence development, differentiation, and
function of various cell types, including hematopoietic
cells.
BACKGROUND OF THE INVENTION
The circulating component of the mammalian
circulatory system comprises various cell types,
including red and white blood cells of the erythroid and
myeloid cell lineages. See, e.g., Rapaport (1987)
Introduction to Hematoloav (2d ed.) Lippincott,
Philadelphia, PA; Jandl (1987) Blood: Textbookof
Hematoloav, Little, Brown and Co.) Boston, MA.; and Paul
(ed.) (1993) Fundamental Immunology (3d ed.) Raven Press,
N.Y.
For some time, it has been known that the mammalian
immune response is based on a series of complex cellular
interactions, called the "immune network." Recent
research has provided new insights into the inner
workings of this network. While it remains clear that
much of the response does, in fact, revol~.re around the
network-like interactions of lymphocytes, macrophages,
granulocytes, and other cells, immunologists now
generally hold the opinion that soluble proteins, known
as lymphokines, cytokines, or monokines, play a critical
role in controlling these cellular interactions. Thus,
there is considerable interest in the isolation,
characterization, and mechanisms of action of cell
modulatory factors, an understanding of which should lead
to significant advancements in the diagnosis and therapy
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of numerous medical abnormalities, e.g., immune system
and other disorders.
Lymphokines apparently mediate cellular activities
in a variety of ways. They have been shown to support
the proliferation, growth, and differentiation of the
pluripotential hematopoietic stem cells into vast numbers
of progenitors comprising diverse cellular lineages
making up a complex immune system. These interactions
between the cellular components are necessary for a
healthy immune response. These different cellular
lineages often respond in a different manner when
lymphokines are administered in conjunction with other
agents.
The chemokines are a large and diverse superfamily
of proteins. The superfamily is subdivided into two
classical branches, based upon whether the first two
cysteines in the chemokine motif are adjacent (termed the
"C-C" branch), or spaced by an intervening residue ("C-X-
C"). A more recently identified branch of chemokines
lacks two cysteines in the corresponding motif, and is
represented by the chemokines known as lymphotactins.
Another recently identified branch has three intervening
residues between the two cysteines, e.g., CX3C
chemokines. See, e.g., Schall and Bacon (1994) Current
Opinion in Immunoloav 6:865-873; and Bacon and Schall
(1996) Int. Arch. Allerav & Immunol. 109:97-I09.
Many factors have been identified which influence
the differentiation process of precursor cells, or
regulate the physiology or migration properties of
specific cell types. These observations indicate that
other factors exist whose functions in immune function
were heretofore unrecognized. These factors provide for
biological activities whose spectra of effects may be
distinct from known differentiation or activation
factors. The absence of knowledge about the structural,
biological, and physiological properties of the factors
which regulate cell physiology in vivo prevents the
modulation of the effects of such factors. Thus, medical
conditions where regulation of the development or
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physiology of relevant cells is required remains
unmanageable.
SUMMARY OF THE INVENTION
. The present invention reveals the existence of
previously unknown chemokine-motif containing molecules
which are hereby designated mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl~kine chemokine. Based on sequence
analysis of the chemokine protein sequences described
below, it is apparent that mpf4, mCTAP3, and Chrl9kine
belong to the CXC chemokine family, and m6Ckine and
h6Ckine belong to the CC chemokine family.
The present invention provides a composition of
matter selected from a composition comprising an antigen
binding site from an antibody which specifically binds to
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine;
an expression vector encoding mpf4, mCTAP3, m5Ckine,
h6Ckine, or Chrl9kine chemokine or fragment thereof; a
substantially pure protein which is specifically
recognized by the. respective antigen binding site; and a
substantially pure mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine or peptide thereof, or fusion protein
comprising a 30 amino acid fragment of mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine sequence. Also
provided are methods for making and using said reagents.
In the antigen binding site containing embodiments,
the antigen binding site may be: specifically
immunoreactive with a mature protein selected from the
group consisting of the polypeptides of SEQ ID NO: 2, 4,
6, 8, or 10; raised against a purified or recombinantly
produced mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine; in a monoclonal antibody, Fab, or F(ab)2; or
in a labeled antibody. In certain embodiments; the
antigen binding site is detected in a biological sample
by a method of: contacting a binding agent having an
affinity for the mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine protein with the biological sample;
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incubating the binding agent with the biological sample
to fo~n a binding agent:mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine protein complex; and detecting the
complex. In a preferred embodiment, the biological
sample is human or mouse, and the binding agent is an
antibody.
A kit embodiment is provided possessing a compound,
described above, with either instructional material for
the use of the compound; or a compartment into which the
compound is segregated.
A nucleic acid embodiment of the invention includes
an expression vector encoding a mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine protein, wherein the
protein specifically binds an antibody generated against
an immunogen selected from the mature polypeptide
portions of SEQ ID NO: 2, 4, 6, 8, or 10, more
particularly a natural protein. The vector may: encode a
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
polypeptide with complete sequence identity to a
naturally occurring mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine protein; encode a mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine protein
comprising sequence selected from the polypeptides of SEQ
ID NO: 2, 4, 6, 8, or 10, and particularly a natural
protein; or comprise sequence selected from the nucleic
acids of SEQ ID N0: 1, 3, 5, or 7. In other embodiments,
the vector is capable of selectively hybridizing to a
nucleic acid encoding a mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine protein, e.g., a mature protein
coding segment of SEQ ID NO: 1, 3, 5, or 7. In various
preferred embodiments, the isolated nucleic acid is
detected in a biological sample by a method of:
contacting a biological sample with a nucleic acid probe
capable of selectively hybridizing to the nucleic acid;
incubating the nucleic acid probe with the biological
sample to form a hybrid of the nucleic acid probe with
complementary nucleic acid sequences present in the
biological sample; and determining the extent of
hybridization of the nucleic acid probe to the
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complementary nucleic acid sequences. In such method,
preferably the nucleic acid probe is capable of
hybridizing to a nucleic acid encoding a protein
consisting of the polypeptides of SEQ ID NO: 2, 4, 6, 8,
or 10. Also provided are methods of making an expression
vector or making protein comprising construction or
expression of a vector encompassing said nucleic acids.
In various embodiments, the isolated mpf4, mCTAP3,
m6Ckine is from a mouse; the isolated h6Ckine is from a
human; consists of a polypeptide comprising sequence from
SEQ ID NO: 2, 4, 6, 8, or 10; recombinantly produced, or
a naturally occurring protein. Also provided are fusion
proteins comprising a sequence of SEQ ID NO: 2, 4, 6, 8,
or 10 and/or a sequence of another cytokine or chemokine.
The present invention also embraces a cell
transfected with the nucleic acid encoding a mpf4,
mCTAP3, m6Ckine, h6Ckine) or Chrl9kine chemokine, e.g.,
where the nucleic acid has SEQ ID NO: 1, 3, 5, or 7. The
cell may be either prokaryote or eukaryote.
The invention also provides a method of modulating
physiology or development of a cell by contacting the
cell with a mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine, or an antagonist, e.g., neutralizing antibody,
of the chemokine. In preferred embodiments, the
physiology is attraction of a cell which possesses a
natural receptor for the chemokine.
DETAILED DESCRIPTION
I. General
The present invention provides mouse and human DNA
sequences encoding mammalian proteins which exhibit
structural properties or motifs characteristic of a
~ cytokine or chemokine. For a review of the chemokine
family, see, e.g., Lodi, et al. (1994) Science 263:1762-
1767; Gronenborn and Clore (1991) Protein Enaineering
4:263-269; Miller and Kranger (1992) Proc. Nat'1 Acad.
Sci. USA 89:2950-2954; Matsushima and Oppenheim (1989)
Cytokine 1:2-13; Stoeckle and Baker (1990) New Biol.
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2:313-323; Oppenheim, et al. (1991) Ann. Rev. Immunol.
9:617-648; Schall (1991) ~:ytokine 3:165-183; and The
Cvtokine Handbook Academic Press, NY.
The novel cytokines described herein are designated
mpf4, mCTAP3) m6Ckine, h6Ckine, or Chrl9kine. The
descriptions below are directed, for exemplary purposes, to
mammalian embodiments, e.g., human and mouse, but are likewise
applicable to related embodiments from other, e.g., natural,
sources. These sources should include various vertebrates,
typically warm blooded animals, e.g., birds and mammals,
particularly domestic animals, and primates.
The nucleic acid and amino acid sequences of the novel
Mouse mpf4 chemokine are provided in SEQ ID NO: 1 and SEQ ID
N0: 2, respectively. The mpf4 coding sequence begins at base
141 and ends at base 458, the CXC motif is at amino acid
residues 5-7. Absence of an ELR motif immediately preceding
the CXC suggests likely anti-inflammatory properties. A
signal sequence is indicated, but based upon related genes,
variable forms may be produced by different cell types.
The nucleic acid and amino acid sequences of the novel
-- mouse CTAP3 (mCTAP3) are provided in SEQ ID NO: 3 and SEQ ID
NO: 4, respectively. The CXC motif corresponds to amino acid
residues numbered 10-22. A predicted signal sequence is
indicated, but may actually be of longer or shorter length
depending, in part, upon the cell expressing the protein. The
ELR motif immediately preceeding the CXC motif suggests
function in inflammatory mediator trafficking.
The nucleic acid and amino acid sequences of the novel
mouse 6Ckine (m6Ckine) chemokine are provided in SEQ ID N0: 5
and SEQ ID NO: 6, respectively. The predicted coding region
begins at about position 71 and ends at position 469. A
predicted signal sequence is indicated and the CC motif is at
residues numbered 7-8. m6Ckine has a total of 6 conserved
cysteine residues in the mature peptide, suggesting a new
subclass of CC chemokines exhibiting extended carboxy terminal
sequence.
The nucleic acid sequence and the corresponding
predicted amino acid sequence of the novel human 6Ckine
(h6Ckine) are provided in SEQ ID N0:7 and SEQ ID NO: 8,
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respectively. Predicted coding sequence runs from position 6
through position 407. The CC motif corresponds to residues
numbered 8-9; predicted signal sequence is indicated. h6Ckine
has 6 conserved cysteine residues in the mature peptide,
similar to m6Ckine, and exhibits carboxy terminal extension.
Table 1, below, depicts the alignment of porcine 6Ckine
. (p6Ckine) (SEQ ID NO: 9), mouse and human 6Ckine amino acid
sequences.
Table 1:
Alignment
of porcine
(SEQ ID
NO: 9),
mouse,
and human
6Ckine
amino acid sequences. "*" indicates identity) and ".' denotes
similarity.Only a portion of porcine 6Ckine is compared here.
Other
chemokines possess a structural domain which corresponds to
the sequence
from about G1u56 to Asp67. Immediately following this in the
6Ckines,
both mouse and human, is additional sequence unlike other chemokines.
Starting
after Lys69
of human,
are a series
of prolines,
which should
disrupt
helical
structure.
Thus the
additional
segment
which contains
two cysteines
(at positions
80 and
99) appear
peculiar
to these
molecules. These new structural features may define new biology
arid an
additional chemokine subclass.
p6Ckine MXQSLVLRILVLVLAFCIPHTQGSDGGAQDCCLKYSLRKIPTHVVRSYRK
m6Ckine MAQMMTLSLLSLVLALCIPWTQGSDGGGQDCCLKYSQKKIPYSIVRGYRK
h6Ckine MAQSLALSLLILVLAFGIPRTQGSDGGAQDCCLKYSQRKIPAKWRSYRK
* * , * ,* **** ** ******* ******** _*** .** ***
p6Ckine QEPSLGCPIPAILFSPRKXSQPELCAD-----------------------
m6Ckine QEPSLGCPIPAILFSPRKHSKPELCANPEEGWVQNLMRRLDQPPAPGKQS
h6Ckine QEPSLGCSIPAILFLPRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPA
******* ****** *** *, ****** * *** ** **
p6Ckine ____. _________-__________________
m6Ckine PGCRKNRGTSKSGKKGKGSKGCKRTEQTQPSRG-
h6Ckine QGCRKDRGASKTGKKGKGSKGCKRTERSQTPKGP
**** ** ** ************** * .*
Partial amino acid sequence of human Chrl9kine (CXC
chemokine; SEQ ID N0: 10). The CXC motif is underlined. The
absense of the ELR motif suggests anti-inflammatory
properties. Chrl9kine was found by search and careful
analysis of a public EST database. See, e.g.) gnl~dbest~14024
(H14024). This gene maps to human chromosome 19p12-13.1.
The chemokine proteins of this invention are defined
in part by their physicochemical and biological
properties. The biological properties of the chemokines
described herein, e.g., mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine, are defined, in part, by their amino acid
sequence, and mature size. They also should share at
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least some biological properties with other similar
chemokines. One of skill will readily recognize that
some sequence variations may be tolerated, e.g.,
conservative substitutions or positions remote from the
critical residues for receptor interaction or important
tertiary structure features, without altering
significantly the biological activity of the molecule.
Conversely, non-conservative substitutions may be adapted
to delete selected functions.
These chemokines are present in specific tissue
types, e.g., lymphoid tissues, and the interaction of the
protein with a receptor will be important for mediating
various aspects of cellular physiology or development.
The cellular types which express message encoding mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine suggest that
signals important in cell differentiation and development
are mediated by them. See, e.g., Gilbert (1991)
Developmental Biology (3d ed.) Sinauer Associates,
Sunderland, MA; Browder, et al. (1991) Developmental
Bioloav i3d ed.) Saunders, Philadelphia, PA.; Russo, et
al. (1992) Develobment: The Molecular Genetic Ap~~roach
Springer-Verlag, New York, N.Y.; and Wilkins (1993)
Genetic Analysis of Animal Development (2d ed.) Wiley-
Liss, New York, N.Y. Moreover, mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine expression or responsiveness should
serve as markers, e.g., to define certain cell
subpopulations.
The mpf4 and mCTAP3 chemokines were discovered
through searches and careful analysis of the GENBANK EST
(WashU-Merck EST project, St. Louis, MO) public database.
mpf4 exhibits approximately 71~ amino acid identity to
rat platelet factor 4 (pf4), which functions as a marker
for megakaryocyte differentiation. See, e.g., Doi, et
al. (1987) Mol. Cell. Biol. 7:898-904; and GenBank
Accession number M15254 (1987). Identity comparisons of
mCTAP3 sequence, see, e.g., Marra, et al. (1996) GenBank
Accession number W53807, revealed 65~ identity on the
nucleotide level to human neutrophil-stimulating peptide
2 (NAP-2), and 55~ identity on the amino acid level to
-9-
human connective tissue activating peptide 3 (hCTAP3).
mCTAP3 is likely to be the murine counterpart of the
human protein.
m6Ckine was also found by search and careful
analysis of the public EST database with sequences
exhibiting chemokine structure in the SWISS PROT
database. The nucleotide sequence of Table 3 and SEQ ID
NO 5 is found on three murine ESTs, see, e.g.,
gn1 dbest 17930 (W17930); gn1 ~ dbest ~ 67046 (W67046); and
gn1'dbest~08383 (W08383). h6Ckine was found in the
similar manner. The nucleotide sequence of Table 4 and
SEQ ID NO: 6 can be found, e.g., on gnl~dbest~366929;
gnl~dbest~302355; gnl~dbestl342967; gnl~dbest~356690; and
on a clone designated est703.
Chrl9kine was found by search and careful analysis
of a public EST database. See, e.g., gnl~dbest~14024
(H14024). This gene maps to human chromosome 29p12-13.1.
Northern blot analysis was performed for m6Ckine
using standard methods, see, e.g., Maniatis, et al.
(1982) Molecular ClQnina, A Laboratory Manual Cold Spring
Harbor Laboratory, Cold Spring Harbor Press, NY;
Sambrook, et al. (1989) Molecular Cloning: A Laboratory
Manual (2d ed.) Vols. 1-3, CSH Press, NY; Ausubel, et
al., Biologv Greene Publishing Associates, Brooklyn, NY;
and Ausubel, et al. (1987 and Supplements) Current
Protocols in Molecular Biology Wiley/Greene, NY.
Preliminary data indicates a transcript of approximately
900 by with high levels of expression in mouse lung and
spleen. Lower expression levels were found in mouse
heart, liver, skeletal muscle, kidney, testis, and RAG 1
thymus tissue. No message has been detected in human
fetal brain, lung, liver, or kidney tissues, nor has any
expression been detected in various T cell libraries.
However, analysis of human immune tissues yields
expression in lymph node, appendix, and at low level, in
spleen. This wide range of distribution in various
tissues may indicate a role in normal leukocyte
trafficking. Northern analysis of h6Ckine indicates
expression in human tonsil, lymph node, and fetal spleen
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~~o-
tissues, as well germinal center cells. A lower message
was found in fetal testis and small intestine.
Through standard Ca++ flux analysis, see, e.g.,
Coligan, et al (eds.)(1992 and periodic supplements)
Current Protocols_in Immunolocrv, Greene/Wiley, NY, it was
determined that the receptor for m6Ckine is CXCR3. This
receptor is shared by other known chemokines, e.g., IP-10
and Mig. See, e.g., Sgadari, et al. (1997) B o0
89:2635-2643; and Loetscher, et al. (1996) J. Exp. Med.
184:963-969. This is an interesting ligand-receptor pair
because as described above m6Ckine is a CC chemokine
while CXCR3 normally binds CXC chemokines.
IP-10 and Mig are known to have angiostatic
properties. See, e.g., Keane, et al. (1997) J. Immunol.
159:1437-l443; and Farber (1997) J. Leukoc. Biol. 61:246-
257. Because of this angiostatic effect, Mig and IP-10
prevent tumor related angiogenesis as well as regulate
the rate of wound healing and scar tissue formation.
Since 6Ckine shares a receptor with IP-10 and Mig, 6Ckine
may also play a role in preventing tumor formation, tumor
metastasis) and/or regulating wound healing.
Additionally, since 6Ckine is a T cell chemoattractant,
especially for activated T cells, it may serve to enhance
many T-cell mediated anti-tumor effects. This may occur
with 6Ckine alone, or in combination with IP-10, Mig,
lymphotactin, see, e.g., Kelner, et al. (1994) Science
266:1395-1399, and/or MIP3a, see, e.g., U.S.S.N.
08/675,814. And since h6Ckine binds to mouse CXCR3, it
is likely that the chemokine will also bind to human
CXCR3.
In addition, as for other ligands for the CXCR3
receptor, 6Ckine may mediate rapid lymphocyte adhesion.
This may be useful in enhancing anti-tumor responses.
Or, 6Ckine may be used to direct activated T cells to an
antigen, e.g., an infectious agent.
II. Definitions
The term "binding composition" refers to molecules
that bind with specificity and selectivity to a mpf4,
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mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine,
respectively, e.g., in an antibody-antigen interaction.
However, other compounds) e.g., receptor proteins) may
also specifically and/or selectively associate with mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokines to the
exclusion of other molecules. Typically, the association
will be in a natural physiologically relevant protein-
protein interaction, either covalent or non-covalent, and
may include members of a multiprotein complex, including
carrier compounds or dimerization partners. The molecule
may be a polymer, or chemical reagent. No implication as
to whether a mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine is either the ligancl or the receptor of a
ligand-receptor interaction is necessarily represented,
other than whether the interaction exhibits similar
specificity, e.g., specific affinity. A functional
analog may be a ligand with structural modifications, or
may be a wholly unrelated molecule, e.g., which has a
molecular shape which interacts with the appropriate
ligand binding determinants. The ligands may serve as
agonists or antagonists of the receptor, see, e.g.,
Goodman, et al . ( eds . ) ( 1990 ) Goo~m~zz & Gilmar~ s : The
Pharmacoloaical Bases of Therapeutics (8th ed.) Pergamon
Press, Tarrytown, N.Y.
The term "binding agent:mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine protein complex", as used
herein, refers to a complex of a binding agent and a
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
protein that is formed by specific binding of the binding
agent to the mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine protein. Specific binding of the binding agent
means that the binding agent has a specific binding site,
e.g., antigen binding site) that recognizes a site on the
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
protein. For example, antibodies raised to a mpf~;
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine protein
and recognizing an epitope on the mpf4, CTAP3, or 6Ckine
chemokine protein are capable of forming a binding
agent:mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
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chemokine protein complex by specific binding.
Typically, the formation of a binding agent:mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine protein complex
allows the measurement of mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine protein in a mixture of other
proteins and biologics. The term "antibody:mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine~chemokine protein complex"
refers to an embodiment in which the binding agent, e.g.,
is the antigen binding portion from an antibody. The
antibody may be monoclonal, polyclonal, or a binding
fragment of an antibody, e.g., an Fab or F(ab)2 fragment.
The antibody will preferably be a polyclonal antibody for
cross-reactivity testing purposes.
"Homologous" nucleic acid sequences, when compared,
exhibit significant similarity, or identity. The
standards for homology in nucleic acids are either
measures for homology generally used in the art by
sequence comparison andlor phylogenetic relationship, or
based upon hybridization conditions. Hybridization
conditions are described in greater detail below.
An "isolated" nucleic acid is a nucleic acid, e.g.,
an RNA, DNA, or a mixed polymer, which is substantially
separated from other biologic components which naturally
accompany a native sequence, e.g., proteins and flanking
genomic sequences from the originating species. The term
embraces a nucleic acid sequence which has been removed
from its naturally occurring environment, and includes
recombinant or cloned DNA isolates and chemically
synthesized analogs, or analogs biologically synthesized
by heterologous systems. A substantially pure molecule
includes isolated forms of the molecule. An isolated
nucleic acid will usually contain homogeneous nucleic
acid molecules, but will, in some embodiments, contain
nucleic acids with minor sequence heterogeneity. This
heterogeneity is typically found at the polymer ends or
portions not critical to a desired biological function or
activity.
As used herein, the term "mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine protein" shall encompass,
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when used in a protein context, a protein having amino
acid sequences, particularly from the chemokine motif
portions, shown in SEQ ID NO: 2, 4, 6, 8, or 10, or a
significant fragment unique to and/or characteristic of
such a protein, preferably a natural embodiment. The
invention also embraces a polypeptide which exhibits
similar structure to mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine, e.g., which interacts with mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine specific
binding components. These binding components, e.g.,
antibodies, typically bind to a mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine with high affinity, e.g.,
at least about 100 nM, usually better than about 30 nM,
preferably better than about 10 nM, and more preferably
at better than about 3 nM.
The term "polypeptide" or "protein" as used herein
includes a significant fragment or segment of chemokine
motif portion of a mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine, and encompasses a stretch of amino
acid residues of at least about 8 amino acids, generally
at least 10 amino acids, more generally at least 12 amino
acids, often at least--14 amino acids, more often at least
16 amino acids, typically at least 18 amino acids, more
typically at least 20 amino acids, usually at least 22
amino acids, more usually at least 24 amino acids,
preferably at least 26 amino acids, more preferably at
least 28 amino acids, and, in particularly preferred
embodiments, at least about 30 or more amino acids, e.g.,
35, 40, 45, 50, 60, 70, 80, etc. The segments may have
amino and carboxy termini, with appropriate lengths)
e.g., starting at residue 1, 2, 3, etc., and ending at
residue 134, 133, 132, etc. The invention encompasses
proteins comprising a plurality of said segments.
A "recombinant" nucleic acid is defined either by
its method of production or its structure. In reference
to its method of prvductivn, e.g., a product made by a
process, the process is use of recombinant nucleic acid
techniques, e.g., involving human intervention in the
nucleotide sequence, typically selection or production.
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Alternatively, it can be a nucleic acid made by
generating a sequence comprising fusion of two fragments
which are not naturally contiguous to each other, but is
meant to exclude products of nature, e.g., naturally
occurring mutants. Thus, for example, products made by
transforming cells with any non-naturally occurring
vector is encompassed, as are nucleic acids comprising
sequence derived using any synthetic oligonucleotide
process) Such is often done to replace a codon with a
redundant codon encoding the same or a conservative amino
acid, while typically introducing or removing a sequence
recognition site. Alternatively, it is performed to join
together nucleic acid segments of desired functions to
generate a single genetic entity comprising a desired
combination of functions not found in the commonly
available natural forms. Restriction enzyme recognition
sites are often the target of such artificial
manipulations, but other site specific targets, e.g.,
promoters, DNA replication sites, regulation sequences,
control sequences, or other useful features may be
incorporated by design. A similar concept is intended
for a recombinant, e.g., fusion, polypeptide.
Specifically included are synthetic nucleic acids which,
by genetic code redundancy, encode polypeptides similar
to fragments of these antigens, and fusions of sequences
from various different species variants.
"Solubility" is reflected by sedimentation measured
in Svedberg units, which are a measure of the
sedimentation velocity of a molecule under particular
conditions. The determination of the sedimentation
velocity was classically performed in an analytical
ultracentrifuge, but is typically now performed in a
standard ultracentrifuge. See, Freifelder (1982)
Phvsical Biochemistrv (2d ed.) W.H. Freeman & Co., San
Francisco, CA; and Cantor and Schimmel (1980) Biophysical
Chemistry parts 1-3, W.H. Freeman & Co., San Francisco,
CA. As a crude determination) a sample containing a
putatively soluble polypeptide is spun in a standard full
sized ultracentrifuge at about 50K rpm for about 10
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minutes, and soluble molecules will remain in the
supernatant. A soluble particle or polypeptide will
typically be less than about 30S, more typically less
than about 15S, usually less than about 10S, more usually
less than about 5S, and) in particular embodiments,
preferably less than about 4S, and more preferably less
than about 3S. Solubility of a polypeptide or fragment
depends upon the environment and the polypeptide. Many
parameters affect polypeptide solubility, including
temperature, electrolyte environment, size and molecular
characteristics of the polypeptide, and nature of the
solvent. Typically, the temperature at which the
polypeptide is used ranges from about 4~ C to about 65~
C. Usually the temperature at use is greater than about
25 18~ C and more usually greater than about 22~ C. For
diagnostic purposes, the temperature will usually be
about room temperature or warmer, but less than the
denaturation temperature of components in the assay. For
therapeutic purposes, the temperature will usually be
body temperature, typically about 37~ C for humans,
though under certain situations the temperature may be
raised or lowered in situ or in vitro.
The size and structure of the polypeptide should
generally be in a substantially stable state, and usually
not in a denatured state. The polypeptide may be
associated with other polypeptides in a quaternary
structure, e.g., to confer solubility, or associated with
lipids or detergents in a manner which approximates
natural lipid bilayer interactions.
The solvent will usually be a biologically
compatible buffer, of a type used for preservation of
biological activities, and will usually approximate a
physiological solvent. Usually the solvent will have a
. neutral pH, typically between about 5 and 10, and
preferably about 7.5. On some occasions, a detergent
. will be added, typically a mild non-denaturing one, e.g.,
CHS (cholesteryl hemisuccinate) or CHAPS (3-[3-
cholamidopropyl- dimethylammonio)-1-propane sulfonate),
or a low enough concentration as to avoid significant
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disruption of structural or physiological properties of
the protein.
"Substantially pure" in a protein context typically
means that the protein is isolated from other
contaminating proteins, nucleic acids, and other
biologicals derived from the original source organism.
Purity, or "isolation" may be assayed by standard
methods, and will ordinarily be at least about 50~ pure,
more ordinarily at least about 60~ pure, generally at
least about 70~ pure, more generally at least about 80~
pure, often at least about 85~ pure, more often at least
about 90~ pure, preferably at least about 95~ pure, more
preferably at least about 98~ pure, and in most preferred
embodiments, at least 99~ pure. Similar concepts apply,
e.g., to antibodies or nucleic acids.
"Substantial similarity" in the nucleic acid
sequence comparison context means either that the
segments, or their complementary strands, when compared,
are identical when optimally aligned, with appropriate
nucleotide insertions or deletions, in at least about 50~
of the nucleotides, generally at least 56~, more
generally at least 59~, ordinarily at least 62~, more
ordinarily at least 65~, often at least 68~, more often
at least 71~, typically at least 74~, more typically at
least 77~, usually at least 80~, more usually at least
about 85~, preferably at least about 90~, more preferably
at least about 95 to 98~ or more, and in particular
embodiments, as high at about 99~ or more of the
nucleotides. Alternatively, substantial similarity
exists when the segments will hybridize under selective
hybridization conditions) to a strand, or its complement,
typically using a sequence derived from SEQ ID NO: 1, 3,
5, or 7. Typically, selective hybridization will occur
when there is at least about 55~ similarity over a
stretch of at least about 30 nucleotides, preferably at
least about 65~ over a stretch of at least about 25
nucleotides, more preferably at least about 75~, and most
preferably at least about 90~ over about 20 nucleotides.
See Kanehisa (1984) Nuc. Acids Res. 12:203-213. The
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length of similarity comparison, as described, may be
over longer stretches, and in certain embodiments will be
over a stretch of at least about 17 nucleotides, usually
at least about 20 nucleotides, more usually at least
about 24 nucleotides, typically at least about 28
nucleotides, more typically at least about 40
nucleotides, preferably at least about 50 nucleotides,
and more preferably at least about 75 to 100 or more
nucleotides, e.g., 150, 200, etc.
"Stringent conditions", in referring to homology or
substantial similarity in the hybridization context, will
be stringent combined conditions of salt, temperature,
organic solvents, and other parameters, typically those
controlled in hybridization reactions. The combination
of parameters is more important than the measure of any
single parameter. See, e.g., Wetmur and Davidson (1968)
J. Mol. Biol. 31:349-370. A nucleic acid probe which
binds to a target nucleic acid under stringent conditions
is specific for said target nucleic acid. Such a probe
is typically more than 11 nucleotides in length, and is
sufficiently identical or complementary to a target
nucleic acid over the region specified by the sequence of
the probe to bind the target under stringent
hybridization conditions.
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokines from other mammalian species can be cloned and
isolated by cross-species hybridization of closely
related species. See, e.g., below. Similarity may be
relatively low between distantly related species, and
thus hybridization of relatively closely related species
is advisable. Alternatively, preparation of an antibody
preparation which exhibits less species specificity may
be useful in expression cloning approaches.
The phrase "specifically binds to an antibody" or
"specifically immunoreactive with", when referring to a
protein or peptide, refers to a binding reaction which is
determinative of the presence of the protein in the
presence of a heterogeneous population of groteins and
other biological components. Thus, under designated
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immunoassay conditions, the specified antibodies bind to
a particular protein and do not significantly bind other
proteins present in the sample. Specific binding to an
antibody under such conditions may require an antibody
that is selected for its specificity for a particular
protein. For example, antibodies raised to the mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine protein
immunogen with the amino acid sequence depicted in SEQ ID
NO: 2, 4, 6, 8, or 10 can be selected to obtain
antibodies specifically immunoreactive with mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine proteins and not
with other proteins. The antibodies may be species
specific, e.g., also recognizing polymorphic and splicing
or developmental variants.
III. Nucleic Acids
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine is exemplary of structurally and functionally
related proteins. These soluble chemokine proteins will
serve to transmit signals between different cell types.
The preferred embodiments, as disclosed, will be useful
in standard procedures to isolate genes from different
individuals or other species, e.g., warm blooded animals,
such as birds and mammals. Cross hybridization will
allow isolation of related genes encoding proteins from
individuals, strains, or species. A number of different
approaches are available to successfully isolate a
suitable nucleic acid clone based upon the information
provided herein. Southern blot hybridization studies can
qualitatively determine the presence of homologous genes
in human, monkey, rat, mouse, dog, cat, cow, and rabbit
genomes under specific hybridization conditions.
Complementary sequences will also be used as probes
or primers. Based upon identification of the likely
amino terminus, other peptides should be particularly
useful, e.g., coupled with anchored vector or poly-A
complementary PCR techniques or with complementary DNA of
other peptides.
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Techniques for nucleic acid manipulation of genes
encoding mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine proteins, such as subcloning nucleic acid
sequences encoding polypeptides into expression vectors,
labeling probes) DNA hybridization, and the like are
described generally in Sambrook, et al. (1989) Molecular
Clonincr: A L~boratorv Manual (2nd ed.) Vol. 1-3, Cold
Spring Harbor Laboratory, Cold Spring Harbor Press, NY,
which is incorporated herein by reference. This manual
is hereinafter referred to as "Sambrook, et al."
There are various methods of isolating DNA sequences
encoding mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine proteins. For example, DNA is isolated from a
genomic or cDNA library using labeled oligonucleotide
probes having sequences identical or complementary to the
sequences disclosed herein. Full-length probes may be
used, or oligonucleotide probes may be generated by
comparison of the sequences disclosed. Such probes can
be used directly in hybridization assays to isolate DNA
encoding mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine proteins, or probes can be designed for use in
amplification techniques such as PCR, for the isolation
of DNA encoding mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine proteins. Reverse translation
computer programs can also provide alternative nucleic
acid sequences which encode the same proteins.
To prepare a cDNA library, mRNA is isolated from
cells which expresses a mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine protein. cDNA is prepared from
the mRNA and ligated into a recombinant vector. The
vector is transfected into a recombinant host for
propagation, screening, and cloning. Methods for making
and screening cDNA libraries are well known. See Gubler
and Hoffman (1983) ne 25:263-269 and Sambrook, et al.
For a genomic library, the DNA can be extracted from
tissue and either mechanically sheared or enzymatically
digested to yield fragments of about 12-20 kb. The
fragments are then separated by gradient centrifugation
and cloned in bacteriophage lambda vectors. These
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vectors and phage are packaged in vitro, as described in
Sambrook, et al. Recombinant phage are analyzed by
plaque hybridization as described in Benton and Davis
(1977) Science 196:180-182. Colony hybridization is
carried out as generally described in e.g.) Grunstein, et
al. (1975) Proc. Natl. Acad. Sci. USA. 72:3961-3965.
DNA encoding a mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine protein can be identified in either
cDNA or genomic libraries by its ability to hybridize
with the nucleic acid probes described herein, e.g., in
colony or plaque hybridization assays. The corresponding
DNA regions are isolated by standard methods familiar to
those of skill in the art. See, e.g., Sambrook, et al.
Various methods of amplifying target sequences, such
as the polymerase chain reaction, can also be used to
prepare DNA encoding mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine proteins. Polymerase chain reaction
(PCR) technology is used to amplify such nucleic acid
sequences directly from mRNA, from cDNA, and from genomic
libraries or cDNA libraries. The isolated sequences
encoding mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine proteins may-also be used as templates for PCR
amplification.
Typically, in PCR techniques, oligonucleotide
primers complementary to two 5' regions in the DNA region
to be amplified are synthesized. The polymerase chain
reaction is then carried out using the two primers. See
Innis, et al. (eds.) (1990) _PCR Protocols: A Guide to
Methods and An~lications Academic Press, San Diego, CA.
Primers can be selected to amplify the entire regions
encoding a full-length mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine protein or to amplify smaller DNA
segments as desired. Once such regions are PCR-
amplified, they can be sequenced and oligonucleotide
probes can be prepared from sequence obtained using
standard techniques. These probes can then be used to
isolate DNA's encoding mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine proteins.
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Oligonucleotides for use as probes are usually
chemically synthesized according to the solid phase
phosphoramidite triester method first described by
Beaucage and Carruthers (1983) Tetrahedron Lett.
22(20):1859-1862, or using an automated synthesizer, as
described in Needham-VanDevanter, et al. (1984) Nucleic
Acids Res. 12:6l59-6168. Purification of
oligonucleotides is performed e.g., by native acrylamide
gel electrophoresis or by anion-exchange HPLC as
described in Pearson and Regnier (1983) J. Chrom.
255:137-149. The sequence of the synthetic
oligonucleotide can be verified using, e.g., the chemical
degradation method of Maxam, A.M. and Gilbert, W. in
Grossman, L. and Moldave (eds.) (1980) Methods in
Enz~moloay 65:499-560 Academic Press, New York.
An isolated nucleic acid encoding a mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine protein was
identified. The nucleotide sequence and corresponding
open reading frame are provided in SEQ ID NO: 1, 3, 5, or
7.
These mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokines exhibit limited similarity to portions of
chemokines. See, e.g., Matsushima and Oppenheirn (1989)
C~okine 1:2-13; Oppenheim, et al. (1991) Ann. Rev.
Immunol. 9:617-648; Schall (1991) Cytokine 3:165-183; and
Gronenborn and Clore (1991) Protein Enaineering 4:263-
269. Other features of comparison are apparent between
the mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine and chemokine families. See, e.g., Lodi, et
al. (1994) Science 263:1762-1766. In particular, (3-sheet
and oc-helix residues can be determined using, e.g.,
RASMOL program, see Sayle and Milner-White (1995) TzBS
20:374-376; or Gronenberg, et al. (1991) Protein
Enaineerinq 4:263-269; and other structural features are
defined in Lodi, et al. (1994) Science 263:1762-1767.
These secondary and tertiary features assist in defining
further the C, CC, CXC, and CX3C structural features,
along with spacing of appropriate cysteine residues. The
6Ckine embodiments provided herein exhibit peculiar
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carboxy termini for the chemokine class of molecules. In
particular, the sequence of h6Ckine extending beyond
residue Asp68 to Pro111 therein may be peculiar to, and
may represent a heretofore unrecognized subgroup of the
CC chemokines.
This invention provides isolated DNA or fragments to
encode a mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine protein. In addition, this invention provides
isolated or recombinant DNA which encodes a protein or
polypeptide which is capable of hybridizing under
appropriate conditions, e.g., high stringency, with the
DNA sequences described herein. Said biologically active
protein or polypeptide can be an intact ligand, or
fragment, and have an amino acid sequence as disclosed in
SEQ ID NO: 2, 4, 6, 8, or 10, particularly natural
embodiments. Preferred embodiments will be full length
natural sequences, from isolates, e.g., about 11,000 to
12,500 daltons in size when unglycosylated, or fragments
of at least about 6,000 daltons, more preferably at least
about 8,000 daltons. In glycosylated form, the protein
may exceed 12,500 daltons. The 6Ckine embodiments
exhibit a correspondingly larger size. Further, this
invention contemplates the use of isolated or recombinant
DNA, or fragments thereof, which encode proteins which
are homologous to a mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine protein or which were isolated using
cDNA encoding a mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine protein as a probe. The isolated DNA
can have the respective regulatory sequences in the 5'
and 3' flanks, e.g., promoters, enhancers, poly-A
addition signals, and others. Also embraced are methods
for making expression vectors with these sequences, or
for making, e.g., expressing and purifying, protein
products.
IV. Making mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokines
DNAs which encode a mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine or fragments thereof can be
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obtained by chemical synthesis, screening cDNA libraries,
or by screening genomic libraries prepared from a wide
variety of cell lines or tissue samples. Methods for
doing so, or making expression vectors are described
herein.
These DNAs can be expressed in a wide variety of
host cells for the synthesis of a full-length protein or
fragments which can in turn, e.g., be used to generate
polyclonal or monoclonal antibodies; for binding studies;
for construction and expression of modified molecules;
and for structure/function studies. Each mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine or its fragments
can be expressed in host cells that are transformed or
transfected with appropriate expression vectors. These
molecules can be substantially purified to be free of
protein or cellular contaminants, other than those
derived from the recombinant host, and therefore are
particularly useful in pharmaceutical compositions when
combined with a pharmaceutically acceptable carrier
and/or diluent. The antigen, e.g., mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine, or portions
thereof, may be expressed as fusions with other proteins
or possessing an epitope tag.
Expression vectors are typically self-replicating
DNA or RNA constructs containing the desired antigen gene
or its fragments, usually operably linked to appropriate
genetic control elements that are recognized in a
suitable host cell. The specific type of control
elements necessary to effect expression will depend upon
the eventual host cell used. Generally, the genetic
control elements can include a prokaryotic promoter
system or a eukaryotic promoter expression control
system, and typically include a transcriptional promoter,
an optional operator to control the onset of
transcription, transcription enhancers to elevate the
level of mRNA expression, a sequence that encodes a
suitable ribosome binding site, and sequences that
terminate transcription and translation. Expression
vectors also usually contain an origin of replication
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that allows the vector to replicate independently from
the host cell.
The vectors of this invention contain DNAs which
encode a mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine, or a fragment thereof, typically encoding,
e.g., a biologically active polypeptide, or protein. The
DNA can be under the control of a viral promoter and can
encode a selection marker. This invention further
contemplates use of such expression vectors which are
capable of expressing eukaryotic cDNA coding for a mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine protein
in a prokaryotic or eukaryotic host, where the vector is
compatible with the host and where the eukaryotic cDNA
coding for the protein is inserted into the vector such
that growth of the host containing the vector expresses
the cDNA in question. Usually, expression vectors are
designed for stable replication in their host cells or
for amplification to greatly increase the total number of
copies of the desirable gene per cell. It is not always
necessary to require that an expression vector replicate
in a host cell, e.g., it is possible to effect transient
expression of the protein or its fragments in various
hosts using vectors that do not contain a replication
origin that is recognized by the host cell. It is also
possible to use vectors that cause integration of a mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine gene or
its fragments into the host DNA by recombination, or to
integrate a promoter which controls expression of an
endogenous gene.
Vectors, as used herein, contemplate plasmids)
viruses, bacteriophage, integratable DNA fragments, and
other vehicles which enable the integration of DNA
fragments into the genome of the host. Expression
vectors are specialized vectors which contain genetic
control elements that effect expression of operably
linked genes. Plasmids are the most commonly used form
of vector, but many other forms of vectors which serve an
equivalent function are suitable for use herein. See,
e.g., Pouwels, et al. (1985 and Supplements) Clonincr
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Vec,~ors: A Laboratorv~,anu~l Elsevier, N.Y.; and
Rodriquez, et al. (eds.) (1988) Vectors: A Surv~v of - --
Molecu~ar Cloyi~na Vectors and Their Uses Buttersworth,
Boston, MA.
Suitable host cells include prokaryotes, lower
eukaryotes, and higher eukaryotes. Prokaryotes include
both gram negative and gram positive organisms, e.g., E.
coli and B. subtilis. Lower eukaryotes include yeasts,
e.g., S. cerevisiae and Pichia, and species of the genus
Dictyostelium. Higher eukaryotes include established
tissue culture cell lines from animal cells, both of
non-mammalian origin, e.g., insect cells, and birds, and
of mammalian origin, e.g., human, primates, and rodents.
Prokaryotic host-vector systems include a wide
variety of vectors for many different species. As used
herein, E. coli and its vectors will be used generically
- to include equivalent vectors used in other prokaryotes.
A representative vector for amplifying DNA is pBR322 or
its derivatives. Vectors that can be used to express
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokines
or mpf4,-mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
fragments include, but are not limited to, such vectors
as those containing the lac promoter (pUC-series); trp
promoter (pBR322-trp); Ipp promoter (the pIN-series);
lambda-pP or pR promoters (pOTS); or hybrid promoters
such as ptac (pDR540). See Brosius, et al. (1988)
"Expression Vectors Employing Lambda-, trp-, lac-, and
Ipp-derived Promoters", in Rodriguez and Denhardt (eds.)
Vectors: A Su~vev of Molecular Cloning Vectors and Their
Uses 10:205-235 Buttersworth, Boston, MA.
Lower eukaryotes, e.g., yeasts and Dictyostelium,
may be transformed with mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine sequence containing vectors. Far
purposes of this invention, the most common lower
eukaryotic host is the baker's yeast, Saccharomyces
cerevisiae. It will be used generically to represent
lower eukaryotes although a number of other strains and
species are also available. Yeast vectors typically
consist of a replication origin (unless of the
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integrating type), a selection gene, a promoter, DNA
encoding the desired protein or its fragments, and
sequences for translation termination, polyadenylation,
and transcription termination. Suitable expression
vectors for yeast include such constitutive promoters as
3-phosphoglycerate kinase and various other glycolytic
enzyme gene promoters or such inducible promoters as the
alcohol dehydrogenase 2 promoter or metallothionine
promoter. Suitable vectors include derivatives of the
following types: self-replicating low copy number (such
as the YRp-series), self-replicating high copy number
(such as the YEp-series); integrating types {such as the
YIp-series), or mini-chromosomes (such as the YCp-
series).
Higher eukaryotic tissue culture cells are typically
the preferred host cells for expression of the
functionally active mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine protein. In principle, many higher
eukaryotic tissue culture cell lines may be used, e.g.,
insect baculovirus expression systems, whether from an
invertebrate or vertebrate source. However, mammalian
cells are preferred to achieve proper processing, both
cotranslationally and posttranslationally.
Transformation or transfection and propagation of such
cells is routine. Useful cell lines include HeLa cells,
Chinese hamster ovary (CHO) cell lines, baby rat kidney
(BRK) cell lines, insect cell lines, bird cell lines, and
monkey (COS) cell lines. Expression vectors for such
cell lines usually include an origin of replication, a
promoter, a translation initiation site, RNA splice sites
(e. g.) if genomic DNA is used), a polyadenylation site,
and a transcription termination site. These vectors also
may contain a selection gene or amplification gene.
Suitable expression vectors may be plasmids, viruses, or
retroviruses carrying promoters derived, e.g., from such
sources as from adenovirus, SV40, parvoviruses, vaccinia
virus, or cytomegalovirus. Representative examples of
suitable expression vectors include pCDNAI; pCD, see
Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142;
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pMClneo Poly-A, see Thomas, et al. (1987) Cell 51:503-
512; and a baculovirus vector such as pAC 373 or pAC 610.
It is likely that mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokines need not be glycosylated to elicit
biological responses. However, it will occasionally be
desirable to express a mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine polypeptide in a system which
provides a specific or defined glycosylation pattern. In
this case, the usual pattern will be that provided
naturally by the expression system. However, the pattern
will be modifiable by exposing the polypeptide, e.g., in
unglycosylated form, to appropriate glycosylating
proteins introduced into a heterologous expression
system. For example, the mpf4, mCTAP3, m6Ckine) h6Ckine,
or Chrl9kine chemokine gene may be co-transformed with
one or more genes encoding mammalian or other
glycosylating enzymes. It is further understood that
over glycosylation may be detrimental to mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine biological
activity, and that one of skill may perform routine
testing to optimize the degree of glycosylation which
confers optimal biological activity.
A mpf4, mCTAP3, m6Ckine, h6Ckine) or Chrl9kine
chemokine, or a fragment thereof, may be engineered to be
phosphatidyl inositol (PI) linked to a cell membrane, but
can be removed from membranes by treatment with a
phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl
inositol phospholipase-C. This releases the antigen in a
biologically active form, and allows purification by
standard procedures of protein chemistry. See, e.g., Low
(1989) Biochem. Bio>'hYS. Acta 988:427-454; Tse, et a1.
(1985) Science 230:1003-1008; and Brunner, et al. (2991)
J. Cell Biol. 114:1275-1283.
Now that mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokines have been characterized, fragments
or derivatives thereof can be prepared by conventional
processes for synthesizing peptides. These include
processes such as are described in Stewart and Young
(1984) Solid Phase Pet~tide SSmthesis Pierce Chemical Co.,
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Rockford, IL; Bodanszky and Bodanszky (1984) The Practice
of PeBtide S,~mthesis Springer-Verlag, New York, NY; and
Bodanszky (1984) The Principles of Peptide Svnth sis
Springer-Verlag) New York, NY. For example, an azide
process, an acid chloride process, an acid anhydride
process, a mixed anhydride process, an active ester
process (for example, p-nitrophenyl ester, N-
hydroxysuccinimide ester, or cyanomethyl ester), a
carbodiimidazole process, an oxidative-reductive process,
~r a dicyclohexylcarbodiimide (DCCD)/additive process can
be used. Solid phase and solution phase syntheses are
both applicable to the foregoing processes. See also
chemical ligation, e.g., Dawson, et al. (1994) Science
266:776-779, a method of linking long synthetic peptides
by a peptide bond.
The prepared protein and fragments thereof can be
isolated and purified from the reaction mixture by means
of peptide separation, for example, by extraction,
precipitation, electrophoresis and various forms of
chromatography, and the like. The mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chr29kine chemokines of this invention can be
obtained in varying degrees of purity depending upon its
desired use. Purification can be accomplished by use of
known protein purification techniques or by the use of
the antibodies or binding partners herein described,
e.g., in immunoabsorbant affinity chromatography. This
immunoabsorbant affinity chromatography is carried out by
first linking the antibodies to a solid support and then
contacting the linked antibodies with solubilized lysates
of appropriate source cells, lysates of other cells
expressing the ligand, or lysates or supernatants of
cells producing the mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokines as a result of recombinant DNA
'techniques, see below.
Multiple cell lines may be screened for one which
expresses a mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine at a high level compared with other cells.
Natural mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
'-chemokines can be isolated from natural sources, or by
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expression from a transformed cell using an appropriate
expression vector. Purification of the expressed protein
is achieved by standard procedures, or may be combined
with engineered means for effective purification at high
efficiency from cell lysates or supernatants. Epitope or
other tags, e.g., FLAG or His6 segments, can be used for
such purification features.
V. Antibodies
Antibodies can be raised to various mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokines, including
individual, polymorphic, allelic, strain, or species
variants, and fragments thereof, both in their naturally
occurring (full-length) forms and in their recombinant
forms. Additionally, antibodies can be raised to mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokines in
either their active forms or in their inactive forms.
Anti-idiotypic antibodies may also be used. The
antibodies may exhibit various binding specificities for
species, individual or polymorphic variants
_. A. Antibody Production
A number of immunogens may be used to produce
antibodies specifically reactive with mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine proteins.
Recombinant protein is the preferred immunogen for the
production of monoclonal or polyclonal antibodies.
Naturally occurring protein may also be used either in
pure or impure form. Synthetic peptides, made using the
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
protein sequences described herein, may also used as an
immunogen for the production of antibodies to mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokines.
Recombinant protein can be expressed in eukaryotic or
prokaryotic cells as described herein, and purified as
described. Naturally folded or denatured material can be
used, as appropriate, for producing antibodies. Either
monoclonal or polyclonal antibodies may be generated for
subsequent use in immunoassays to measure the protein.
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Methods of producing polyclonal antibodies are known
to those of skill in the art. Typically, an immunogen,
preferably a purified protein, is mixed with an adjuvant
and animals are immunized with the mixture. The animal's
immune response to the immunogen preparation is monitored
by taking test bleeds and determining the titer of
reactivity to the mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine protein of interest. TnThen
appropriately high titers of antibody to the immunogen
are obtained, usually after repeated immunizations, blood
is collected from the animal and antisera are prepared.
Further fractionation of the antisera to enrich for
antibodies reactive to the protein can be done if
desired. See, e.g., Harlow and Lane; or Coligan.
Monoclonal antibodies may be obtained by various
techniques familiar to those skilled in the art.
Typically, spleen cells from an animal immunized with a
desired antigen are immortalized, commonly by fusion with
a myeloma cell (see, Kohler and Milstein (1976) Eur. J.
Immunol. 6:511-519, incorporated herein by reference).
Alternative methods of immortalization include
transforniation with Epstein Barr Virus, oncogenes, or
retroviruses, or other methods known in the art.
Colonies arising from single immortalized cells are
screened for production of antibodies of the desired
specificity and affinity for the antigen, and yield of
the monoclonal antibodies produced by such cells may be
enhanced by various techniques, including injection into
the peritoneal cavity of a vertebrate host.
Alternatively, one may isolate DNA sequences which encode
a monoclonal antibody or a binding fragment thereof by
screening a DNA library from human B cells according,
e.g., to the general protocol outlined by Huse, et aI.
(1989) Science 246:1275-1281.
Antibodies, including binding fragments and single
chain versions, against predetermined fragments of mpf4,
mCTAP3) m6Ckine) h6Ckine, or Chrl9kine chemokines can be
raised by immunization of animals with conjugates of the
fragments with carrier proteins as described above.
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Monoclonal antibodies are prepared from cells secreting
the desired antibody. These antibodies can be screened
for binding to normal or defective mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokines, or screened for
agonistic or antagonistic activity, e.g., mediated
through a receptor. These monoclonal antibodies will
usually bind with at least a KD of about 1 mM, more
usually at least about 300 E.LM, typically at least about
E.~M, more typically at least about 30 E1M, preferably at
10 least about 10 ~,M, and more preferably at least about 3
~tM or better.
In some instances, it is desirable to prepare
monoclonal antibodies from various mammalian hosts, such
as mice, rodents, primates, humans, etc. Description of
techniques for preparing such monoclonal antibodies may
be found in, e.g., Stites, et al. (eds.) Basic and
Clinical Immunoloav (4th ed.) Lange Medical Publications,
Los Altos, CA, and references cited therein; Harlow and
Lane (1988) Antibodi,gs: A Laboratory Manual CSH Press;
Goding (1986) Monoclonal Antibodies: Principles and
Practice (2d ed.) Academic Press, New York, NY; and
particularly in Kohle~ and Milstein (1975) pure
256:495-497, which discusses one method of generating
monoclonal antibodies. Summarized briefly, this method
involves injecting an animal with an immunogen. The
animal is then sacrificed and cells taken from its
spleen, which are then fused with myeloma cells. The
result is a hybrid cell or "hybridoma" that is capable of
reproducing in vitro. The population of hybridomas is
then screened to isolate individual clones, each of which
secrete a single antibody species to the immunogen. In
this manner, the individual antibody species obtained are
the products of immortalized and cloned single B cells
from the immune animal generated in response to a
specific site recognized on the immunogenic substance.
Other suitable techniques involve selection of
libraries of antibodies in phage or similar vectors.
See, e.g., Huse, et al. (1989) "Generation of a Large
Combinatorial Library of the Immunoglobulin Repertoire in
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Phage Lambda," Science 246:1275-1281; and Ward, et al.
(1989) Nature 341:544-546. The polypeptides and -
antibodies of the present invention may be used with or
without modification, including chimeric or humanized
antibodies. Frequently, the polypeptides and antibodies
will be labeled by joining) either covalently or non-
covalently, a substance which provides for a detectable
signal. A wide variety of labels and conjugation
techniques are known and are reported extensively in both
the scientific and patent literature. Suitable labels
include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent moieties, chemiluminescent
moieties, magnetic particles; and the like. Patents,
teaching the use of such labels include U.S. Patent Nos.
3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;
4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced. See, Cabilly, U.S.
Patent No. 4,816,567; and Queen, et al. (1989) Proc.
Nat'1 Acad. Sci. USA 86:10029-10033.
The antibodies of this invention are useful for
affinity chromatography in isolating mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine protein.
Columns can be prepared where the antibodies axe linked
to a solid support, e.g., particles, such as agarose,
SEPHADEX, or the like, where a cell lysate or supernatant
may be passed through the column, the column washed,
followed by increasing concentrations of a mild
denaturant, whereby purified mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine protein will be released.
Likewise, antibody binding to the chemokine may be
capable of neutralizing receptor binding, and may serve
as a receptor antagonist. They may also be useful as
Western blot detection reagents, or ELISA reagents.
The antibodies may also be used to screen expression
libraries for particular expression products. Usually
the antibodies used in such a procedure will be labeled
with a moiety allowing easy detection of presence of
antigen by antibody binding.
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Antibodies to mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokines may be used for the identification
of cell populations expressing mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokines. By assaying the
expression products of cells expressing mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokines it is possible
to diagnose disease, e.g., immune-compromised conditions.
Antibodies raised against each mpf4, mCTAP3,
mSCkine, h6Ckine, or Chrl9kine chemokine will also be
useful to raise anti-idiotypic antibodies. These will be
useful in detecting or diagnosing various immunological
conditions related to expression of the respective
antigens.
B . Immunoas says
A particular protein can be measured by a variety of
immunoassay methods. For a review of immunological and
immunoassay procedures in general, see Stites and Terr
(eds.) (1991) Basic and Clinical Immunoloav (7th ed.).
Moreover, the immunoassays of the present invention can
be performed in many configurations, which are reviewed
extensively in Maggio (ed.) (1980) ~nzvme Immunoassay CRC
Press, Boca Raton, Florida; Tijan (1985) "Practice and
Theory of Enzyme Immunoassays," Laboratory Technigues in
Biochemistry and Molecular Biolocrv, Elsevier Science
Publishers B.V., Amsterdam; and Harlow and Lane
Antibodies, A Laboratory Manual, supra, each of which is
incorporated herein by reference. See also Chan (ed.l
(1987) Immunoassay: A Practical Guide Academic Press,
Orlando, FL; Price and Newman (eds.) i1991) Principles
and Practice of ImmurLoassays Stockton Press, NY; and Ngo
(ed.) (1988) Non-isotopic Immunoassays Plenum Press, NY.
Immunoassays for measurement of mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine proteins can be
performed by a variety of methods known to those skilled
in the art. Tn brief, immunoassays to measure the
protein can be either competitive or noncompetitive
binding assays. In competitive binding assays, the
sample to be analyzed competes with a labeled analyte for
specific binding sites on a capture agent bound to a
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solid surface. Preferably the capture agent is an
antibody specifically reactive with mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine proteins
produced as described above. The concentration of
labeled analyte bound to the capture agent is inversely
proportional to the amount of free analyte present in the
sample.
In a competitive binding immunoassay, the mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine protein
present in the sample competes with labeled protein for
binding to a specific binding agent, for example, an
antibody specifically reactive with the mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine protein. The
binding agent may be bound to a solid surface to effect
separation of bound labeled protein from the unbound
labeled protein. Alternately, the competitive binding
assay may be conducted in liquid phase and a variety of
techniques known in the art may be used to separate the
bound labeled protein from the unbound labeled protein.
Following separation, the amount of bound labeled protein
is determined. The amount of protein present in the
sample is inversely proportional to the amount of labeled
protein binding.
Alternatively, a homogeneous immunoassay may be
performed in which a separation step is not needed. In
these immunoassays, the label on the protein is altered
by the binding of the protein to its specific binding
agent. This alteration in the labeled protein results in
a decrease or increase in the signal emitted by label, so
that measurement of the label at the end of the
immunoassay allows for detection or quantitation of the
protein.
mpf4, mCTAP3, m6Ckine) hSCkine, or Chrl9kine
chemokine proteins may also be determined by a variety of
noncompetitive immunoassay methods. For example, a two-
site, solid phase sandwich immunoassay may be used. In
this type of assay, a binding agent for the protein, for
example an antibody, is attached to a solid support. A
second protein binding agent, which may also be an
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antibody, and which binds the protein at a different
site, is labeled. After binding at both sites on the
protein has occurred, the unbound labeled binding agent
is removed and the amount of labeled binding agent bound
to the solid phase is measured. The amount of labeled
binding agent bound is directly proportional to the
amount of protein in the sample.
Western blot analysis can be used to determine the
presence of mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine proteins in a sample. Electrophoresis is
carried out, for example, on a tissue sample suspected of
containing the protein. Following electrophoresis to
separate the proteins, and transfer of the proteins to a
suitable solid support, e.g., a nitrocellulose filter,
the solid support is incubated with an antibody reactive
with the protein. This antibody may be labeled, or
alternatively may be detected by subsequent incubation
with a second labeled antibody that binds the primary
antibody.
The immunoassay formats described above employ
labeled assay components. The label may be coupled
directly or indirectly to the desired component of the
assay according to methods well known in the art. A wide
variety of labels and methods may be used.
Traditionally, a radioactive label incorporating 3H,
125D 355 14C or 32p was used. Non-radioactive labels
include ligands which bind to labeled antibodies,
fluorophores, chemiluminescent agents, enzymes, and
antibodies which can serve as specific binding pair
members for a labeled ligand. The choice of label
depends on sensitivity required, ease of conjugation with
the compound, stability requirements, and available
instrumentation. For a review of various labeling or
signal producing systems which may be used, see U.S.
Patent No. 4,391,904, which is incorporated herein by
reference.
Antibodies reactive with a particular protein can
also be measured by a variety of immunoassay methods.
For a review of immunological and immunoassay procedures
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applicable to the measurement of antibodies by
immunoassay techniques, see Stites and Terr (eds.) Basic
and Clinical Immunolow (7th ed.) supra; Maggio (ed.)
En~rme Immunoassay, supra; and Harlow and Lane
Antibodies. A Laboratory Manual, supra.
In brief, immunoassays to measure antisera reactive
with mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine proteins can be either competitive or
noncompetitive binding assays. In competitive binding
assays, the sample analyte competes with a labeled
analyte for specific binding sites on a capture agent
bound to a solid surface. Preferably the capture agent
is a purified recombinant mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine protein produced as described
above. Other sources of mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine proteins, including isolated or
partially purified naturally occurring protein, may also
be used. Noncompetitive assays include sandwich assays,
in which the sample analyte is bound between two analyte-
specific binding reagents. One of the binding agents is
-. used as a capture agent and is bound to a solid surface.
The second binding agent is labeled and is used to
measure or detect the resultant complex by visual or
instrument means. A number of combinations of capture
agent and labeled binding agent can be used. A variety
of different immunoassay formats, separation techniques,
and labels can be also be used similar to those described
above for the measurement of mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine proteins.
VI. Purified mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokines
Mouse nucleotide and amino acid sequences are
provided in SEQ ID NO: 1 to 6, and human nucleotide and
amino acid sequences for h6Ckine are provided in SEQ ID
NO: 7 and 8.
Purified protein or defined peptides are useful for
generating antibodies by standard methods, as described
above. Synthetic peptides or purified protein can be
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presented to an immune system to generate polyclonal and
monoclonal antibodies. See, e.g., Coligan (1991) current __
Protocols in Immuno_loav Wiley/Greene, NY; and Harlow and
Lane (1989) Antibodies: A Laboratory Manual Cold Spring
Harbor Press, NY, which are incorporated herein by
reference. Alternatively, a mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine receptor can be useful as
a specific binding reagent, and advantage can be taken of
its specificity of binding, for, e.g., purification of a
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
ligand.
The specific binding composition can be used for
screening an expression library made from a cell line
which expresses a mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine. Many methods for screening are
available, e.g., standard staining of surface expressed
ligand, or by panning. Screening of intracellular
expression can also be performed by various staining or
immunofluorescence procedures. The binding compositions
could be used to affinity purify or sort out cells
expressing the ligand.
The peptide segments, along with comparison to
homologous genes, can also be used to produce appropriate
oligonucleotides to screen a library. The genetic code
can be used to select appropriate oligonucleotides useful
as probes for screening. In combination with polymerase
chain reaction (PCR) techniques, synthetic
oligonucleotides will be useful in selecting desired
clones from a library, including natural allelic an
polymorphic variants.
The peptide sequences allow preparation of peptides
to generate antibodies to recognize such segments, and
allow preparation of oligonucleotides which encode such
sequences. The sequence also allows for synthetic
preparation, e.g., see Dawson, et al. (1994} Science
266:?76-779. Since mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokines may be secreted proteins, the gene
will normally possess an N-terminal signal sequence,
which is removed upon processing and secretion. However,
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the exact processing point may be vary in different cell
types, and forms of different lengths are often detected.
Prediction of the signal cleavage point can be performed,
e.g., using the methods of Nielsen, et al. (1997) Protein
Ena. 10:1-8. Analysis of the structural features in
comparison with the most closely related reported
sequences has revealed similarities with other cytokines,
particularly the class of proteins known as CC and CXC
chemokines. The longer carboxy terminus of the 6Ckine
embodiments suggest a distinct subfamily of CC
chemokines. This subfamily may exhibit additional
biological or structural features.
VII. Physical Variants
This invention also encompasses proteins or peptides
having substantial amino acid sequence similarity with an
amino acid sequence of a mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine. Natural variants include
individual, polymorphic, allelic, strain, or species
variants.
Amino acid sequence similarity, or sequence
identity, is determined by optimizing residue matches, if
necessary, by introducing gaps as required. This changes
when considering conservative substitutions as matches.
Conservative substitutions typically include
substitutions within the following groups: glycine,
alanine; valine, isoleucine, leucine; aspartic acid,
glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine.
Homologous amino acid sequences include natural
polymorphic, allelic, and interspecies variations in each
respective protein sequence. Typical homologous proteins
or peptides will have from 50-100 similarity (if gaps
can be introduced), to 75-100g similarity (if
conservative substitutions are included) with the amino
acid sequence of the mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine. Similarity measures will be at
least about 50~, generally at least 60~, more generally
at least 65~, usually at least 70~, more usually at least
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75~, preferably at least 80~, and more preferably at
least 80~) and in particularly preferred embodiments, at
least 85~ or more. See also Needleham, et al. (2970) J.
Mol. Biol. 48:443-453; Sankoff, et al. i1983) Time Wa,~ps,
Strir~g Edits, and Macromolecules: The Theory and Practice
of Sec~ence Comparison Chapter One, Addison-Wesley,
Reading, MA; and software packages from IntelliGenetics,
Mountain View, CA; and the University of Wisconsin
Genetics Computer Group, Madison, WI.
Nucleic acids encoding mammalian mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine proteins will
typically hybridize to the nucleic acid sequence of SEQ
ID NO: 1, 3, 5, or 7 under stringent conditions. For
example, nucleic acids encoding mpf4, mCTAP3, m6Ckine, or
h6Ckine chemokine proteins will normally hybridize to the
nucleic acid of SEQ ID NO: 1, 3, 5, or 7 under stringent
hybridization conditions. Generally, stringent
conditions are selected to be about 10~ C lower than the
thermal melting point (Tm) for the probe sequence at a
defined ionic strength and pH. The Tm is the temperature
(under defined ionic strength and pH) at which 50~ of the
target sequence hybridizes to a perfectly matched probe.
Typically, stringent conditions will be those 'in which
the salt concentration is about 0.2 molar at pH 7 and the
temperature is at least about 50~ C. Other factors may
significantly affect the stringency of hybridization,
including, among others, base composition and size of the
complementary strands, the presence of organic solvents
such as formamide, and the extent of base mismatching. A
preferred embodiment will include nucleic acids which
will bind to disclosed sequences in 50~ formamide and 200
mM NaCl at 42~ C .
An isolated mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine DNA can be readily modified by
nucleotide substitutions, nucleotide deletions,
nucleotide insertions, and short inversions of nucleotide
stretches. These modifications result in novel DNA
sequences which encode mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine antigens, their derivatives, or
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proteins having highly similar physiological,
immunogenic, or antigenic activity.
Modified sequences can be used to produce mutant
antigens or to enhance expression. Enhanced expression
may involve gene amplification, increased transcription,
increased translation, and other mechanisms. Such mutant
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
derivatives include predetermined or site-specific
mutations of the respective protein or its fragments.
"Mutant mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine" encompasses a polypeptide otherwise falling
within the homology definition of the human mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine as set forth
above, but having an amino acid sequence which differs
25 from that of a mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine as found in nature, whether by way of
deletion, substitution, or insertion. In particular,
"site specific mutant mpf4, mCTAP3,~m6Ckine, h6Ckine, or
Chrl9kine chemokine" generally includes proteins having
significant similarity with a protein having a sequence
of SEQ ID NO: 2, 4, 6, 8, or 20, e.g., natural
embodiments, and as sharing various biological
activities, e.g., antigenic or immunogenic, with those
sequences, and in preferred embodiments contain most or
a11 of the disclosed sequence. This applies also to
polymorphic variants from different individuals. Similar
concepts apply to different mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine proteins, particularly
those found in various warm blooded animals, e.g.,
mammals and birds. As stated before, it is emphasized
that descriptions are generally meant to encompass other
mpf4, mCTAP3, m6Ckine, hSCkine, or Chrl9kine chemokine
proteins, not limited to the mouse or human embodiments
specifically discussed.
Although site specific mutation sites are
predetermined, mutants need not be site specific. mpf4,
mCTAP3) m6Ckine, h6Ckine, or Chrl9kine chemokine
mutagenesis can be conducted by making amino acid
insertions or deletions. Substitutions, deletions,
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insertions, or any combinations may be generated to
arrive at a final construct. Tnsertions include amino-
or carboxyl- terminal fusions, e.g. epitope tags. Random
mutagenesis can be conducted at a target codon and the
expressed mutants can then be screened for the desired
activity. Methods for making substitution mutations at
predetermined sites in DNA having a known sequence are
well known in the art, e.g., by M13 primer mutagenesis or
polymerase chain reaction (PCR) techniques. See also,
Sambrook, et al. (1989) and Ausubel, et al. (1987 and
Supplements). The mutations in the DNA normally should
not place coding sequences out of reading frames and
preferably will not create complementary regions that
could hybridize to produce secondary mRNA structure such
as loops or hairpins.
The present invention also provides recombinant
proteins, e.g., heterologous fusion proteins using
segments from these proteins. A heterologous fusion
protein is a fusion of proteins or segments which are
naturally not normally fused in the same manner. Thus,
the fusion product of an immunoglobulin with a mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
polypeptide is a continuous protein molecule having
sequences fused in a typical peptide linkage, typically
made as a single translation product and exhibiting
properties derived from each source peptide. A similar
concept applies to heterologous nucleic acid sequences.
In addition, new constructs may be made from
combining similar functional domains from other proteins.
For example, protein-binding or other segments may be
"swapped" between different new fusion polypeptides or
fragments. See, e.g., Cunningham, et al. (1989) Science
243:1330-1336; and O'Dowd, et al. (2988) J. Biol. Chem.
263:15985-15992. Thus, new chimeric polypeptides
exhibiting new combinations of specificities will result
from the functional linkage of protein-binding
specificities and other functional domains.
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VIII. Binding Agent:mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine Protein Complexes
A mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine protein that specifically, or selectively,
binds to or that is specifically immunoreactive with an
antibody generated against a defined immunogen, such as
an immunogen consisting of the amino acid sequence of SEQ
ID NO: 2, 4, 6, 8, or 10, is typically determined in an
immunoassay. The immunoassay uses a polyclonal antiserum
which was raised to a protein of SEQ ID NO: 2, 4) 6, 8,
or 10. This antiserum is selected to have low
crossreactivity against other chemokines and any such
crossreactivity is removed by immunoabsorbtion prior to
use in the immunoassay.
In order to produce antisera for use in an
immunoassay, the protein of SEQ ID NO: 2, 4, 6, 8, or 10,
is isolated as described herein. For example,
recombinant protein may be produced in a mammalian cell
line. An inbred strain of mice such as balb/c is
immunized with the protein of SEQ ID NO: 2, 4, 6, 8, or
10, using a standard adjuvant, such as Freund's adjuvant,
and a standard mouse immunization protocol (see Harlow
and Lane, supra). Alternatively, a synthetic peptide,
preferably near full length, derived from the sequences
disclosed herein and conjugated to a carrier protein can
be used an immunogen. Polyclonal sera are collected and
titered against the immunogen protein in an immunoassay,
for example, a solid phase immunoassay with the immunogen
immobilized on a solid support. Polyclonal antisera with
a titer of 104 or greater are selected and tested for
their cross reactivity against C, CC, CX3C, and CXC
chemokines, using a competitive binding immunoassay such
as the one described in Harlow and Lane, supra, at pages
570-573. Preferably two chemokines are used in this
determination in conjunction with human mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine.
Immunoassays in the competitive binding format can
be used for the crossreactivity determinations. For
example, a protein of SEQ ID NO: 2, 4, 6, 8, or 10 can be
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immobilized to a solid support. Proteins added to the
assay compete with the binding of the antisera to the
immobilized antigen. The ability of the above proteins
to compete with the binding of the antisera to the
immobilized protein is compared to the protein of SEQ ID
NO: 2, 4, 6, 8, or 10. The percent crossreactivity for
the above proteins is calculated, using standard
calculations. Those antisera with less than 10~
crossreactivity with each of the proteins listed above
are selected and pooled. The cross-reacting antibodies
are then removed from the pooled antisera by
immunoabsorbtion with the above-listed proteins.
The immunoabsorbed and pooled antisera are then used
in a competitive binding immunoassay as described above
to compare a second protein to the immunogen protein
(e. g., the mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine motif of SEQ ID NO: 2, 4, 6, 8, or 10). In
order to make this comparison, the two proteins are each
assayed at a wide range of concentrations and the amount
of each protein required to inhibit 50~ of the binding of
the antisera to the immobilized protein is determined.
If the amount of the second protein required is less than
twice the amount of the protein, e.g., of SEQ ID NO: 2,
4, 6, 8, or 10 that is required, then the second protein
is said to specifically bind to an antibody generated to
the immunogen .
It is understood that each of mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine proteins are members of
respective families of homologous proteins that comprise
two or more genes. For a particular gene product, such
as the mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine protein, the term refers not only to the amino
acid sequences disclosed herein, but also to other
proteins that are polymorphic, allelic, or non-allelic
variants. It is also understood that the term "mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine" includes
nonnatural mutations introduced by deliberate mutation
using conventional recombinant technology such as single
site mutation, or by excising short sections of DNA
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encoding mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine proteins, or by substituting new amino acids,
or adding new amino acids. Such minor alterations should
substantially maintain the immunoidentity of the original
molecule and/or its biological activity. Thus, these
alterations include proteins that are specifically
immunoreactive with a designated naturally occurring
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
protein, for example, the mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine protein shown in SEQ ID NO: 2, 4,
6, 8, or 10. The biological properties of the altered
proteins can be determined by expressing the protein in
an appropriate cell line and measuring, e.g., a
chemotactic effect. Particular protein modifications
considered minor would include conservative substitution
of amino acids with similar chemical properties, as
- described above for the mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine as a whole. By aligning a protein
optimally with the protein of SEQ ID NO: 2, 4, 6, 8, or
10, and by using the conventional immunoassays described
herein to determine immunoidentity, or by using
lymphocyte chemotaxis assays, one can determine the
protein compositions of the invention.
IX. Functional Variants
The blocking of physiological response to mpf4,
mCTAP3, mSCkine, h6Ckine, or Chrl9kine chemokine may
result from the inhibition of binding of the protein to
its receptor) e.g., through competitive inhibition.
Thus, in vitro assays of the present invention will often
use isolated protein, membranes from cells expressing a
recombinant membrane associated mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine, soluble fragments
comprising receptor binding segments of these proteins,
or fragments attached to solid phase substrates. These
assays will also allow for the diagnostic determination
of the effects of either binding segment mutations and
modifications, or protein mutations and modifications,
e.g., protein analogs. This invention also contemplates
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the use of competitive drug screening assays, e.g.) where
neutralizing antibodies to antigen or receptor fragments
compete with a test compound far binding to the protein.
In this manner, the antibodies can be used to detect the
presence of a polypeptide which shares one or more
antigenic binding sites of the protein and can also be
used to occupy binding sites on the protein that might
otherwise interact with a receptor.
"Derivatives" of mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine antigens include amino acid sequence
mutants, glycosylation variants, and covalent or
aggregate conjugates with other chemical moieties.
Covalent derivatives can be prepared by linkage of
functionalities to groups which are found in mpf4,
mCTAP3, m6Ckine, h6Ckine, ar Chrl9kine chemokine amino
acid side chains or at the N- or C- termini, by means
which are well known in the art. These derivatives can
include, without limitation, aliphatic esters or amides
of the carboxyl terminus, or of residues containing
carboxyl side chains, 0-acyl derivatives of hydroxyl
group-containing residues, and N-acyl derivatives of the
amino terminal amino acid or amino-group containing
residues, e.g., lysine or arginine. Acyl groups are
selected from the group of alkyl-moieties including C3 to
C18 normal alkyl, thereby forming alkanoyl aroyl species.
Covalent attachment to carrier proteins may be important
when immunogenic moieties are haptens.
In particular, glycosylation alterations are
included, e.g., made by modifying the glycosylation
patterns of a polypeptide during its synthesis and
processing, or in further processing steps. Particularly
preferred means for accomplishing this are by exposing
the polypeptide to glycosylating enzymes derived from
cells which normally provide such processing, e.g.,
mammalian glycosylation enzymes. Deglycosylation enzymes
are also contemplated. Also embraced are versions of the
same primary amino acid sequence which have other minor
modifications, including phosphorylated amino acid
residues, e.g., phosphotyrosine, phosphoserine, or
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phosphothreonine, or other moieties, including ribosyl
groups or cross-linking reagents.
A major group of derivatives are covalent conjugates
of the mpf4) mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine or fragments thereof with other proteins or
polypeptides. These derivatives can be synthesized in
recombinant culture such as N- or C-terminal fusions or
by the use of agents known in the art for their
usefulness in cross-linking proteins through reactive
side groups. Preferred protein derivatization sites with
cross-linking agents are at free amino groups,
carbohydrate moieties, and cysteine residues.
Fusion polypeptides between mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine and other homologous or
heterologous proteins are also provided. Many growth
factors and cytokines are homodimeric entities) and a
repeat construct may have various advantages, including
lessened susceptibility to proteolytic degradation.
Moreover, many receptors require dimerization to
transduce a signal, and various dimeric proteins or
domain repeats can be desirable. Heterologous
polypeptides may be fusions between different surface
markers, resulting in, e.g., a hybrid protein exhibiting
receptor binding specificity. Likewise, heterologous
fusions may be constructed which would exhibit a
combination of properties or activities of the derivative
proteins. Typical examples are fusions of a reporter
polypeptide, e.g., luciferase, with a segment or domain
of a protein, e.g., a receptor-binding segment, so that
the presence or location of the fused protein may be
easily determined. See, e.g., Dull, et al., U.S. Patent
No. 4,859,609. Other gene fusion partners include
bacterial (3-galactosidase, trpE, Protein A, i3-lactamase,
alpha amylase, alcohol dehydrogenase, and yeast alpha
mating factor. See, e.g.) Godowski, et al. (l988)
Science 241:812-816.
Such polypeptides may also have amino acid residues
which have been chemically modified by phosphorylation,
sulfonation, biotinylation, or the addition or removal of
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other moieties, particularly those which have molecular
shapes similar to phosphate groups. In some embodiments,
the modifications will be useful labeling reagents, or
serve as purification targets, e.g., affinity ligands.
This invention also contemplates the use of
derivatives of mpf4, mCTAP3, m6Ckine, hSCkine, or
Chrl9kine chemokine other than variations in amino acid
sequence or glycosylation. Such derivatives may involve
covalent or aggregative association with chemical
moieties. These derivatives generally fall into the
three classes: (1) salts, (2) side chain and terminal
residue covalent modifications, and (3) adsorption
complexes) for example with cell membranes. Such
covalent or aggregative derivatives are useful as
immunogens, as reagents in immunoassays, or in
purification methods such as for affinity purification of
ligands or other binding ligands. For example, a mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine antigen
can be immobilized by covalent bonding to a solid support
such as cyanogen bromide-activated SEPHAROSE, by methods
_. which are well known in the art, or adsorbed onto
polyolefin surfaces, with or without glutaraldehyde
cross-linking, for use in the assay or purification of
anti-mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine antibodies or its receptor. The mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine can also be
labeled with a detectable group, e.g., radioiodinated by
the chloramine T procedure, covalently bound to rare
earth chelates, ar conjugated to another fluorescent
moiety for use in diagnostic assays. Purification of
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokines
may be effected by immobilized antibodies or receptor.
Isolated mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine genes will allow transformation of
cells lacking expression of corresponding mpf4, mCTAP3,
m6Ckine) h6Ckine, or Chrl9kine chemokine) e.g., either
species types or cells which lack corresponding proteins
and exhibit negative background activity. Expression of
transformed genes will allow isolation of antigenically
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pure cell lines, with defined or single specie variants.
This approach will allow for more sensitive detection and
discrimination of the physiological effects of mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine receptor
proteins. Subcellular fragments, e.g., cytoplasts or
membrane fragments, can be isolated and used.
X. Uses
The present invention provides reagents which will
find use in diagnostic applications as described
elsewhere herein, e.g., in the general description for
developmental abnormalities, or below in the description
of kits for diagnosis.
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine nucleotides, e.g., mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine DNA or RNA, may be used
as a component in a forensic assay. For instance, the
nucleotide sequences provided may be labeled using, e.g.,
32p or biotin and used to probe standard restriction
fragment polymorphism blots, providing a measurable
character to aid in distinguishing between individuals.
Such probes may be used in well-known forensic techniques
such as genetic fingerprinting. In addition, nucleotide
probes made from mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine sequences may be used in in situ
assays to detect chromosomal abnormalities. For
instance, rearrangements in the human chromosome encoding
a mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
gene may be detected via well-known in situ techniques,
using mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine probes in conjunction with--other known
chromosome markers.
Antibodies and other binding agents directed towards
mpf4, mCTAP3, m6Ckine, h6Ckine, or~Chrl9kine chemokine
proteins or nucleic acids may be used to purify the
corresponding mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine molecule. As described in the
Examples below, antibody purification of mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine components is
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both possible and practicable. Antibodies and other
binding agents may also be used in a diagnostic fashion
to determine whether mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine components are present in a tissue
sample or cell population using well-known techniques
described herein. The ability to attach a binding agent
to a mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine provides a means to diagnose disorders
associated with mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine misregulation. Antibodies and other
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
binding agents may also be useful as histological
markers. As described in the examples below, mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
expression is limited to specific tissue types. By
directing a probe, such as an antibody or nucleic acid to
a mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
it is possible to use the probe to distinguish tissue and
cell types in situ or in vitro.
This invention also provides reagents with
significant therapeutic value. The mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine (naturally
occurring or recombinant), fragments thereof, and
antibodies thereto, along with compounds identified as
having binding affinity to a mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine, are useful in the
treatment of conditions associated with abnormal
physiology or development, including abnormal
proliferation, e.g., cancerous conditions, or
degenerative conditions. Abnormal proliferation,
regeneration, degeneration, and atrophy may be modulated
by appropriate therapeutic treatment using the
compositions provided herein. For example, a disease or
disorder associated with abnormal expression or abnormal
signaling by a mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine is a target for an agonist or
antagonist of the protein. The proteins likely play a
role in regulation or development of neuronal or
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hematopoietic cells, e.g., lymphoid cells, which affect
immunological responses.
In particular, an antagonist for h6Ckine may have
potential for T cell mediated autoimmunity, e.g., joint
inflammation, organ/tissue inflammation. The fact that
h6Ckine is expressed in secondary lymphoid organs, e.g.,
lymph nodes, tonsils, indicates that h6Ckine may be a
preliminary molecule involved in the initial inflammatory
response.
Other abnormal developmental conditions are known in
cell types shown to possess mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine mRNA by northern blot
analysis. See Berkow (ed.) The Merck Manual of Diagnosis
and Therapy. Merck & Co., Rahway, NJ; and Thorn, et al.
Harrison's Principles of Internal Medicine, McGraw-Hill,
NY. Developmental or functional abnormalities, e.g., of
the neuronal or immune system, cause significant medical
abnormalities and conditions which may be susceptible to
prevention or treatment using compositions provided
herein.
_. Certain chemokines have also been implicated in
viral replication mechanisms. See, e.g., Cohen (1996)
Science 272:809-810; Feng, et al. (1996) Science 272:872-
877; and Cocchi, et al. (1995) Science 270:1811-1816.
The mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine may be useful in a similar context.
Alternatively, the stalk structure may be very important
in presentation of the ligand domain, and other
chemokines may be advantageously substituted for the
chemokine domain in this molecule.
Recombinant mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine or mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine antibodies can be purified and then
administered to a patient. These reagents can be
combined for therapeutic use with additional active or
inert ingredients, e.g., in conventional pharmaceutically
acceptable carriers or diluents, e.g., immunogenic
adjuvants, along with physiologically innocuous
stabilizers and excipients. These combinations can be
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sterile filtered and placed into dosage forms as by
lyophilization in dosage vials or storage in stabilized
aqueous preparations. This invention also contemplates
use of antibodies or binding fragments thereof, including
forms which are not complement binding.
Drug screening using antibodies or receptor or
fragments thereof can identify compounds having binding
affinity to mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine) including isolation of associated components.
Subsequent biological assays can then be utilized to
determine if the compound has intrinsic stimulating
activity and is therefore a blocker or antagonist in that
it blocks the activity of the protein. Likewise, a
compound having intrinsic stimulating activity can
activate the receptor and is thus an agonist in that it
simulates the activity of a mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine. This invention further
contemplates the therapeutic use of antibodies to mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine as
antagonists. This approach should be particularly useful
with other mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine species variants.
The quantities of reagents necessary for effective
therapy will depend upon many different factors,
including means of administration, target site,
physiological state of the patient, and other medicants
administered. Thus, treatment dosages should be titrated
to optimize safety and efficacy. Typically, dosages
used in vitro may provide useful guidance in the amounts
useful for in situ administration of these reagents.
Animal testing of effective doses for treatment of
particular disorders will provide further predictive
indication of human dosage. Various considerations are
described, e.g., in Gilman, et al. (eds.) (1990) Goodman
and Gilman's: The Pharmacological Bases of Therat~eutics
(8th ed.) Pergamon Press; and (1990) Remington's
Pharmaceutical Sciences (17th ed.) Mack Publishing Co.,
Easton, PA. Methods for administration are discussed
therein and below, e.g., for oral, intravenous,
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intraperitoneal, or intramuscular administration,
transdermal diffusion, and others. Pharmaceutically
acceptable carriers will include water, saline, buffers)
and other compounds described, e.g., in the Merck Index,
Merck & Co., Rahway, NJ. Dosage ranges would ordinarily
be expected to be in amounts lower than 1 mM
concentrations, typically less~than about 10 ~1.M
concentrations, usually less than about 100 nM,
preferably less than about 10 pM (picomolar), and most
preferably less than about 1 fM (femtomolar), with an
appropriate carrier. Slow release formulations, or a
slow release apparatus will often be utilized for
continuous administration.
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokines, fragments thereof, and antibodies to it or
its fragments, antagonists, and agonists, may be
administered directly to the host to be treated or,
depending on the size of the compounds, it may be
desirable to conjugate them to carrier proteins such as
ovalbumin or serum albumin prior to their administration.
Therapeutic formulations may be administered in any
conventional dosage formulation. While it is possible
for the active ingredient to be administered alone, it is
preferable to present it as a pharmaceutical formulation.
Formulations typically comprise at least one active
ingredient, as defined above, together with one or more
acceptable carriers thereof. Each carrier should be both
pharmaceutically and physiologically acceptable in the
sense of being compatible with the other ingredients and
not injurious to the patient. Formulations include those
suitable for oral, rectal, nasal, or parenteral
(including subcutaneous, intramuscular, intravenous and
intradermal) administration. The formulations may
conveniently be presented in unit dosage form and may be
prepared by any methods well known in the art of
pharmacy. See, e.g., Gilman, et al. (eds.) (1990)
Goodman and Gilman's~ The Pharmacological Bases of
Therapeutics (8th ed.) Pergamon Press; and (1990)
Reminaton's Pharmaceutical Sciences (17th ed.) Mack
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Publishing Co., Easton, PA; Avis, et al. (eds.) (1993)
Pharmaceutical DQsaae Forms: Parenteral Medications
Dekker, NY; Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosaae Forms: Tablets Dekker, NY; and
Lieberman, et al. (eds.) (1990) Pharmaceutical Dosaae
Forms: Dis,p~rse Systems Dekker, NY. The therapy of this
invention may be combined with'or used in association
with other therapeutic agents.
Both the naturally occurring and the recombinant
forms of the mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokines of this invention are particularly useful in
kits and assay methods which are capable of screening
compounds for binding activity to the proteins. Several
methods of automating assays have been developed in
recent years so as to permit screening of tens of
thousands of compounds in a short period. See, e.g.,
Fodor, et al. (1991) Science 251:?67-773, and other
descriptions of chemical diversity libraries, which
describe means for testing of binding affinity by a
plurality of compounds. The development of suitable
assays can be greatly facilitated by the availability of
large amounts of purified, soluble mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine as provided by this
invention.
For example, antagonists can normally be found once
the protein has been structurally defined. Testing of
potential protein analogs is now possible upon the
development of highly automated assay methods using a
purified receptor. In particular, new agonists and
antagonists will be discovered by using screening
techniques described herein. Of particular importance
are compounds found to have a combined binding affinity
for multiple mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine receptors, e.g., compounds which can serve as
antagonists for species variants of a mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine.
This invention is particularly useful for screening
compounds by using recombinant protein in a variety of
drug screening techniques. The advantages of using a
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recombinant protein in screening for specific ligands
include: (a) improved renewable source of the mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine from a
specific source; (b) potentially greater number of
ligands per cell giving better signal to noise ratio in
assays; and (c) species variant specificity
(theoretically giving greater biological and disease
specificity).
One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing a mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine receptor, e.g.,
CXCR3 for 6Ckine. Cells may be isolated which express a
receptor in isolation from any others. Such cells,
either in viable or fixed form, can be used for standard
ligand/receptor binding assays. See also, Parce, et al.
(1989) Science 246:243-247; and Owicki, et al. (1990)
Proc. Nat'1 Acad. Sci. USA 87:4007-4011, which describe
sensitive methods to detect cellular responses.
Competitive assays are particularly useful, where the
cells (source of mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine) are contacted and incubated with a
labeled receptor or antibody having known binding
affinity to the ligand) such as 125I-antibody, and a test
sample whose binding affinity to the binding composition
is being measured. The bound and free labeled binding
compositions are then separated to assess the degree of
ligand binding. The amount of test compound bound is
inversely proportional to the amount of labeled receptor
binding to the known source. Any one of numerous
techniques can be used to separate bound from free ligand
to assess the degree of ligand binding. This separation
step could typically involve a procedure such as adhesion
to filters followed by washing, adhesion to plastic
followed by washing, or centrifugation of the cell
membranes. Viable cells could also be used to screen for
the effects of drugs on mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine mediated functions, e.g., second
messenger levels, i.e., Ca++; cell proliferation;
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inositol phosphate pool changes; and others. Some
detection methods allow for elimination of a separation
step, e.g., a proximity sensitive detection system.
Calcium sensitive dyes will be useful for detecting Ca++
levels, with a fluorimeter or a fluorescence cell sorting
apparatus.
The 6Ckine-CXCR3 ligand receptor pair can also be
used as a positive control to identify other CXCR3
ligands. For example, CXCR3 can be attached to a solid
1.0 support, e.g., a chip, a bead in an affinity column, a
microtiter plate well. Unknown samples containing
potential ligands are incubated with the receptor.
6Ckine is treated similarly in a separate sample.
Binding affinities are measured as described above and
compared to 6Ckine. Similarly, cells expressing CXCR3,
which respond biologically to ligand binding, can be
assayed and responses compared to 6Ckine induced
activity.
Another method utilizes membranes from transformed
eukaryotic or prokaryotic host cells as the source of a
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine.
These cells are stably transformed with DNA vectors
directing the expression of a mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine, e.g., an engineered
membrane bound form. Essentially, the membranes would be
prepared from the cells and used in a receptor/ligand
binding assay such as the competitive assay set forth
above.
Still another approach is to use solubilized,
unpurified or solubilized, purified mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine from transformed
eukaryotic or prokaryotic host cells. This allows for a
"molecular" binding assay with the advantages of
increased specificity, the ability to automate, and high
drug test throughput.
Another technique for drug screening involves an
approach which provides high throughput screening for
compounds having suitable binding affinity to a mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine antibody
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i.6.1
and is described in detail in Geysen, European Patent
Application 84/03564, published on September 13, 1984.
First, large numbers of different small peptide test
compounds are synthesized on a solid substrate, e.g.,
plastic pins or some other appropriate surface, see
Fodor, et al., supra. Then a11 the pins are reacted with
solubilized, unpurified or solubilized, purified mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
antibody, and washed. The next step involves detecting
bound mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine antibody.
Rational drug design may also be based upon
structural studies of the molecular shapes of the mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine and
other effectors or analogs. See, e.g., Methods in
Enzvmolocw vols: 202 and 203. Effectors may be other
proteins which mediate other functions in response to
ligand binding, or other proteins which normally interact
with the receptor. One means for determining which sites
interact with specific other proteins is a physical
structure determination, e.g., x-ray crystallography or 2
dimensional NMR techniques. These will provide guidance
as to which amino acid residues form molecular contact
regions. For a detailed description of protein
structural determination, see, e.g., Blundell and Johnson
(1976? Protein Crvstallocrraphy Academic Press, NY.
A purified mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine can be coated directly onto plates
for use in the aforementioned drug screening techniques.
However, non-neutralizing antibodies to these ligands can
be used as capture antibodies to immobilize the
respective ligand on the solid phase.
XI. Kits
This invention also contemplates use of mpf4,
mCTAP3, m6Ckine, h6Ckine) or Chrl9kine chemokine
proteins, fragments thereof, peptides, and their fusion
products in a variety of diagnostic kits and methods for
detecting the presence of mpf4, mCTAP3, m6Ckine, h6Ckine,
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-~ y _
or Chrl9kine chemokine or a mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine receptor. Typically the
kit will have a compartment containing either a defined
mpf4) mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
peptide or gene segment or a reagent which recognizes ane
or the other, e.g., receptor fragments or antibodies.
A kit for determining the binding affinity of a test
compound to a mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine would typically comprise a test
compound; a labeled compound, e.g., a receptor or
antibody having known binding affinity for the mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine; a
source of mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine (naturally occurring or recombinant); and a
means for separating bound from free labeled compound,
such as a solid phase for immobilizing the mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine. Once compounds
are screened, those having suitable binding affinity to
the mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine can be evaluated in suitable biological assays,
as are well known in the art, to determine whether they
act as agonists or antagonists to the receptor. The
availability of recombinant mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chxl9kine chemokine polypeptides also provide
well defined standards for calibrating such assays.
A preferred kit for determining the concentration
of, for example, a mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine in a sample would typically comprise
a labeled compound, e.g., receptor or antibody, having
known binding affinity for the mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine, a source of mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine
(naturally occurring or recombinant), and a means for
separating the bound from free labeled compound, for
example, a solid phase for immobilizing the mpf4) mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine. Compartments
containing reagents, and instructions, will normally be
provided.
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-s g
Antibodies, including antigen binding fragments,
specific for the mpf4, mCTAP3, m6Ckine) h6Ckine, or __
Chrl9kine chemokine or ligand fragments are useful in
diagnostic applications to detect the presence of
elevated levels of mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine and/or its fragments. Such
diagnostic assays can employ lysates, live cells, fixed
cells, immunofluorescence, cell cultures, body fluids,
and further can involve the detection of antigens related
to the ligand in serum, or the like. Diagnostic assays
may be homogeneous (without a separation step between
free reagent and antigen-mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine complex) or heterogeneous (with a
separation step). Various commercial assays exist) such
as radioimmunoassay (RIA), enzyme-linked immunosorbent
assay (ELISA), enzyme immunoassay (EIA), enzyme-
multiplied immunoassay technique (EMIT), substrate-
labeled fluorescent immunoassay (SLFIA), and the like.
For example, unlabeled antibodies can be employed by
using a second antibody which is labeled and which
recognizes the antibody to a mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine or to a particular
fragment thereof. Similar assays have also been
extensively discussed in the literature. See, e.g.,
Harlow and Lane (1988) Antibodies: A Laboratory Manual,
CSH Press, NY; Chan (ed.) (1987) Immunoassay: A Practical
uide Academic Press, Orlando, FL; Price and Newman
(eds.) (1991) Principles and Practice of Immunoassay
Stockton Press, NY; and Ngo (ed.) (1988) Nonisotopic
Immunoassay Plenum Press, NY.
Anti-idiotypic antibodies may have similar use to
diagnose presence of antibodies against a mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine, as such may be
diagnostic of various abnormal states. For example,
overproduction of mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine may result in production of various
immunological or other medical reactions which may be
diagnostic of abnormal physiological states, e.g., in
cell growth, activation, or differentiation.
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Frequently, the reagents for diagnostic assays are
supplied in kits, so as to optimize the sensitivity of
the assay. For the subject invention, depending upon the
nature of the assay, the protocol, and the label, either
labeled or unlabeled antibody or receptor, or labeled
mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine is
provided. This is usually in conjunction with other
additives, such as buffers, stabilizers, materials
necessary for signal production such as substrates for
enzymes, and the like. Preferably, the kit will also
contain instructions for proper use and disposal of the
contents after use. Typically the kit has compartments
for each useful reagent. Desirably, the reagents are
provided as a dry lyophilized powder, where the reagents
may be reconstituted in an aqueous medium providing
appropriate concentrations of reagents for performing the
assay.
Many of the aforementioned constituents of the drug
screening and the diagnostic assays may be used without
modification, or may be modified in a variety of ways.
For example, labeling may be achieved by covalently or
non-covalently joining a moiety which directly or
indirectly provides a detectable signal. In any of these
assays, the protein, test compound, mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine, or antibodies
thereto can be labeled either directly or indirectly.
Possibilities for direct labeling include label groups:
radiolabels such as 125I, enzymes (U.S. Pat. No.
3,645,090) such as peroxidase and alkaline phosphatase,
and fluorescent labels (U. S. Pat. No. 3,940,475) capable
of monitoring the change in fluorescence intensity,
wavelength shift, or fluorescence polarization.
Possibilities for indirect labeling include biotinylation
of one constituent followed by binding to avidin coupled
to one of the above label groups.
There are also numerous methods of separating the
bound from the free ligand, or alternatively the bound
from the free test compound. The mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine can be immobilized on
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various matrices followed by washing. Suitable matrices
include plastic such as an ELISA plate, filters, and
beads. Methods of immobilizing the mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine to a matrix
include, without limitation, direct adhesion to plastic,
use of a capture antibody, chemical coupling, and biotin-
avidin. The last step in this approach involves the
precipitation of ligand/receptor or ligand/antibody
complex by any of several methods including those
utilizing, e.g., an organic solvent such as polyethylene
glycol or a salt such as ammonium sulfate. Other
suitable separation techniques include, without
limitation, the fluorescein antibody magnetizable
particle method described in Rattle, et al. (1984) lin.
Chem. 30:1457-1461) and the double antibody magnetic
particle separation as described in U.S. Pat. No.
4,659,678.
Methods for linking proteins or their fragments to
the various labels have been extensively reported in the
literature and do not require detailed discussion here.
Many of the techniques involve the use of activated
carboxyl groups either through the use of carbodiimide or
active esters to form peptide bonds, the formation of
thioethers by reaction of a mercapto group with an
activated halogen such as chloroacetyl, or an activated
olefin such as maleimide, for linkage, or the like.
Fusion proteins will also find use in these applications.
Another diagnostic aspect of this invention involves
use of oligonucleotide or polynucleotide sequences taken
from the sequence of a mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine. These sequences can be used as
probes for detecting levels of the mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine message in samples from
natural sources, or patients suspected of having an
abnormal condition, e.g., cancer or developmental
problem. The preparation of both RNA and DNA nucleotide
sequences, the labeling of the sequences, and the
preferred size of the sequences has received ample
description and discussion in the literature. Normally
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an oligonucleotide probe should have at least about 14
nucleotides, usually at least about 18 nucleotides, and
the polynucleotide probes may be up to several kilobases.
Various labels may be employed, most commonly
radionuclides, particularly 32P. However) other
techniques may also be employed, such as using biotin
modified nucleotides for introduction into a
polynucleotide. The biotin then serves as the site for
binding to avidin or antibodies, which may be labeled
with a wide variety of labels, such as radionuclides,
fluorophores, enzymes, or the like. Alternatively,
antibodies may be employed which can recognize specific
duplexes, including DNA duplexes, RNA duplexes, DNA-RNA
hybrid duplexes, or DNA-protein duplexes. The antibodies
in turn may be labeled and the assay carried out where
the duplex is bound to a surface, so that upon the
formation of duplex on the surface, the presence of
antibody bound to the duplex can be detected. The use of
probes to the novel anti-sense RNA may be carried out
using many conventional techniques such as nucleic acid
hybridization, plus and minus screening, recombinational
probing, hybrid released translation (HRT), and hybrid
arrested translation (HART). This also includes
amplification techniques such as polymerise chain
reaction (PCR).
Diagnostic kits which also test for the qualitative
or quantitative presence of these and other markers are
also contemplated. Diagnosis or prognosis may depend on
the combination of multiple indications used as markers.
Thus, kits may test for combinations of markers. See,
e.g., Viallet, et al. (1989) P~oaress in Growth Factor
Res. 1:89-97. Qualitative or quantitative expression of
each chemokine may be evaluated by standard methods at
the protein or mRNA levels.
XII. Receptor Isolation
Having isolated a binding partner of a specific
interaction, methods exist for isolating the counter-
partner. See, Gearing, et a1. (1989) EI~O J. 8:3667-
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3676. For example, means to label a mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine without
interfering with the binding to its receptor can be
determined. For example, an affinity label or epitope
tag can be fused to either the amino- or carboxyl-
terminus of the ligand. An expression library can be
screened for specific binding of the mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine, e.g., by cell
sorting, or other screening to detect subpopulations
which express such a binding component. See, e.g., Ho,
et al. (1993) Proc. Nat'1 Acad. Sci. USA 90:11267-11271.
Alternatively, a panning method may be used. See, e.g.,
Seed and Aruffo (1987) Proc. Nat'1 Acad. Sci. LISA
84:3365-3369. A two-hybrid selection system may also be
applied making appropriate constructs with the available
chemokine sequences. See, e.g., Fields and Song (1989)
Na a a 340:245-246. Standard Ca~'+ flux methods can also
be utilized. See, e.g., Coligan, et al. (eds.) (1992 and
periodic supplements) Current Protocols in Immunoloay
Greene/Wiley, New York, NY.
_. Protein cross-linking techniques with label can be
applied to isolate binding partners of a mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine. This would
allow identification of proteins which specifically
interact with a mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine, e.g., in a ligand-receptor like
manner. Typically, the chemokine family binds to
receptors of the seven transmembrane receptor family, and
the receptor for the mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine is likely to exhibit a similar
structure. Thus, it is likely that the receptor will be
found by expression in a system which is capable of
expressing such a membrane protein in a form capable of
exhibiting ligand binding capability.
The broad scope of this invention is best understood
with reference to the following examples, which are not
intended to limit the invention to specific embodiments.
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A ~~ ~,
ExAMPLEs
I. General Methods
Many of the standard methods below are described or
referenced, e.g., in Maniatis, et al. i1982) Molecular
C, lonincr, A Laboratoryr Manual Cold Spring Harbor
Laboratory, Cold Spring Harbor.Press, NY; Sambrook, et
' al. (1989) Molecular Clonina: A Laboratory anual (2d
ed.) Vols. 1-3, CSH Press, NY; Ausubel, et al., Bioloav
Greene Publishing Associates, Brooklyn, NY; or Ausubel,
et al. (1987 and Supplements) ~urxent Protocols a.r~
Molecular Bioloav Wiley/Greene, NY; Innis, et al. (eds.)
(1990) SCR ~rotoco~s: A Guide to Methods and A~nlica ions
Academic Press, NY. Methods for protein purification
include such methods as ammonium sulfate precipitation,
column chromatography, electrophoresis, centrifugation,
crystallization, and others. See, e.g., Ausubel, et al.
(1987 and periodic supplements); Deutscher (1990) "Guide
to Protein Purification," Methods in Enzy~olocrv vol. 182,
and other volumes in this series; and manufacturer's
literature on use of protein puxification products, e.g.,
Pharmacia, Piscataway, NJ, or Bio-Rad, Richmond, CA.
Combination with recombinant techniques allow fusion to
appropriate segments (epitope tags), e.g., to a FLAG
sequence or an equivalent which can be fused, e.g., via a
protease-removable sequence. See, e.g., Hochuli (1989)
~ emisc~e Industrie l2:69-70; Hochuli (1990)
"Purification of Recombinant Proteins with Metal Chelate
Absorbent" in Setlow (ed.) Senetic Enair~e=rina. Principle
and Methods 12:87-98, Plenum Press, NY; and Crowe, et al.
(1992) OIAexpress: The Hiah Level Exr~ression & Protein
Purification System QUIAGEN) Inc., Chatsworth, CA.
Standard immunological techniques are described,
e.g., in Coligan (1991) Current Protocols in Imm~,nology
Wiley/Greene, NY; and Methods in Enzvmoloav volumes. 70,
73, 74, 84, 92, 93, 108, 1l6, 121, 132, 150, 162, and
163. Assays for neural cell biological activities are
described) e.g., in Wouterlood (ed. 1995) Neuroscience
Protocols modules 10, Elsevier; Methods in Neurosciences
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6 ~-
Academic Press; and Neuromethods Humana Press, Totowa,
NJ. Methodology of developmental systems is described,
e.g., in Meisami (ed.) Handbook.of Human Growth and
Developmental Bioloav CRC Press; and Chrispeels (ed.)
Molecular Techniaues and Approaches in Developmental
Biolocrv Interscience .
FACS analyses are described in Melamed, et al.
(1990) Flow Cvtometrv and Sortina Wiley-Liss, Inc., New
York, NY; Shapiro (1988) Practical Flow Cvtometrv Liss,
New York, NY; and Robinson, et al. (1993) Handbook of
Flow Cytometrv Methods Wiley-Liss, New York, NY.
II. Isolation of mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine chemokine clone
A clone encoding the mpf4, mCTAP3, m6Ckine, h6Ckine,
or Chrl9kine chemokine is isolated from a natural source
by many different possible methods. Given the sequences
provided herein, PCR primers or hybridization probes are
selected and/or constructed to isolate either genomic DNA
segments or cDNA reverse transcripts. Appropriate cell
sources include listed tissues, e.g., brain libraries.
Tissue distribution below also suggests source tissues.
Genetic and polymorphic or allelic variants are isolated
by screening a population of individuals.
PCR based detection is performed by standard
methods, preferably using primers from opposite ends of
the coding sequence, but flanking segments might be
selected for specific purposes.
Alternatively, hybridization probes are selected.
Particular AT or GC contents of probes are selected
depending upon the expected homology and mismatching
expected. Appropriate stringency conditions are selected
to balance an appropriate positive signal to background
ratio. Successive washing steps are used to collect
clones of greater homology.
Further clones are isolated using an antibody based
selection procedure. Standard expression cloning methods
are applied including, e.g., FACS staining of membrane
associated expression product. The antibodies are used
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~s -
to identify clones producing a recognized protein.
Alternatively, antibodies are used to purify a mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine, with
protein sequencing and standard means to isolate a gene
encoding that protein.
Genomic sequence based methods will also allow for
identification of sequences naturally available, or
otherwise, which exhibit homology to the provided
sequences.
III. Isolation of a primate counterpart for mpf4, mCTAP3,
6Ckine, or Chrl9kine chemokine clone
Similar methods are used as above to isolate an
appropriate primate chemokine gene from another primate.
Similar source materials as indicated above are used to
isolate natural genes, including genetic, polymorphic,
allelic, or strain variants. Other species variants are
also isolated using similar methods. Alternatively, gene
databases may be searched for the appropriate motifs.
IV. Isolation of a rodent pf4, CTAP3, 6Ckine, or
Chrl9kine chemokine clone
An appropriate rodent source is selected as above,
e.g., rat, hamster, etc. Similar methods are utilized to
isolate a species variant, though the level of similarity
will typically be lower for rodent chemokine as compared
to a human to other primate sequence.
V. Expression; purification; characterization
With an appropriate clone from above, the coding
sequence is inserted into an appropriate expression
vector. This may be in a vector specifically selected
for a prokaryote, yeast) insect) or higher vertebrate,
e.g., mammalian expression system. Standard methods are
applied to produce the gene product, preferably as a
soluble secreted molecule, but will, in certain
instances, also be made as an intracellular protein.
Intracellular proteins typically require cell lysis to
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-6c'r
recover the protein, and insoluble inclusion bodies are a
common starting material for further purification.
With a clone encoding a mpf4, mCTAP3, m6Ckine,
h6Ckine, or Chrl9kine chemokine, recombinant production
means are used, although natural forms may be purified
from appropriate sources. The protein product is
purified by standard methods of protein purification, in
certain cases, e.g., coupled with immunoaffinity methods.
Immunoaffinity methods are used either as a purification
step, as described above, or as a detection assay to
determine the separation properties of the protein.
Preferably, the protein is secreted into the medium,
and the soluble product is purified from the medium in a
soluble form. Alternatively, as described above,
inclusion bodies from prokaryotic expression systems are
a useful source of material. Typically, the insoluble
protein is solubilized from the inclusion bodies and
refolded using standard methods. Purification methods
are developed as described above.
The product of the purification method described
above is characterized to determine many structural
features. Standard physical methods are applied, e.g.,
amino acid analysis and protein sequencing. The
resulting protein is subjected to CD spectroscopy and
other spectroscopic methods, e.g., NNgt, ESR, mass
spectroscopy, etc. The product is characterized to
determine its molecular form and size, e.g., using. gel
chromatography and similar techniques. Understanding of
the chromatographic properties will lead to more gentle
or efficient purification methods.
Prediction of glycosylation sites may be made, e.g.,
as reported in Hansen, et al. (1995) Biochem. J. 308:801-
813.
VI. Preparation of antibodies against mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine
With DNA for expression, or protein produced, e.g.,
as above, animals are immunized to produce antibodies.
Polyclonal antiserum is raised, in some cases, using non-
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-~7-
purified antigen, though the resulting serum will exhibit
higher background levels. Preferably, the antigen is
purified using standard protein purification techniques,
including, e.g., affinity chxomatography using polyclonal
serum indicated above. Presence of specific antibodies
is detected using defined synthetic peptide fragments.
Polyclonal serum is raised against a purified
antigen, purified as indicated above, or using, e.g., a
plurality of, synthetic peptides. A series of
overlapping synthetic peptides which encompass a11 of the
full length sequence, if presented to an animal, will
produce serum recognizing most linear epitopes on the
protein. Such an antiserum is used to affinity purify
protein, which is, in turn, used to introduce intact full
length protein into another animal to produce another
antiserum preparation.
- Similar techniques are used to generate induce
monoclonal antibodies to either unpurified antigen, or,
preferably, purified antigen. The antiserum or
antibodies may recognize native protein, or may recognize
denatured antigen,
VII. Cellular and tissue distribution
Distribution of the protein or gene products are
determined, e.g., using immunohistochemistry with an
antibody reagent, as produced above, or by screening for
nucleic acids encoding the chemokine. Hybridization or
PCR methods are used to detect DNA, cDNA, or message
content. Histochemistry allows determination of the
specific cell types within a tissue which express higher
or lower levels of message or DNA. Antibody techniques
are useful to quantitate protein in a biological sample,
including a liquid or tissue sample. Immunoassays are
developed to quantitate protein. Also, FACS analysis may
be used to evaluate expression in a cell population.
VIII. Microchemotaxis assays
The pro-migratory activities of mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine are assessed in
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microchemotaxis assays. See, e.g., Bacon, et al. (1988)
Br. J. Pharmacol. 95:966-974.
Chemokines may also be assayed for activity in
hemopoietic assays as described, e.g., by H. Broxmeyer.
They may be assayed for angiogenic activities as
described, e.g., by R. Streiter.
IX. Biological activities, direct and indirect
A robust and sensitive assay is selected as
described above, e.g., on immune cells, neuronal cells)
or stem cells. Chemokine is added to the assay in
increasing doses to see if a dose response is detected.
For example, in a proliferation assay, cells are plated
out in plates. Appropriate culture medium is provided,
and chemokine is added to the cells in varying amounts.
Growth is monitored over a period of time which will
detect either a direct effect on the cells, or an
indirect effect of the chemokine.
Alternatively, an activation assay or attraction
assay is used. An appropriate cell type is selected,
e.g., hematopoietic cells, myeloid (macrophages,
neutrophils, polymorphonuclear cells, etc.) or lymphoid
(T cell, B cell, or NK cells), neural cells (neurons,
neuroglia, oligodendrocytes, astrocytes, etc.), or stem
cells, e.g., progenitor cells which differentiate to
other cell types, e.g., gut crypt cells and
undifferentiated cell types.
Other assays will be those which have been
demonstrated with other chemokines. See, e.g., Schall
and Bacon (1994) Current Opinion in Immunoloav 6:865-873;
and Bacon and Schall (1996) Int. Arch. Allerav & Immunol.
109:97-109.
IX. Structure activity relationship
Information on the criticality of particular
residues is determined using standard procedures and
analysis. Standard mutagenesis analysis is performed,
e.g., by generating many different variants at determined
positions) e.g., at the positions identified above, and
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_6g_
evaluating biological activities of the variants. This
may be performed to the extent of determining positions
which modify activity, or to focus on specific positions
to determine the residues which can be substituted to
either retain, block, or modulate biological activity.
Alternatively, analysis of natural variants can
indicate what positions tolerate natural mutations. This
may result from populational analysis of variation among
individuals, or across strains or species. Samples from
selected individuals are analysed, e.g., by PCR analysis
and sequencing. This allows evaluation of population
polymorphisms.
X. Screening for agoniststantagonists
Agonists or antagonists are screened for ability to
induce or block biological activity. The candidate
compounds, e.g., sequence variants of natural mpf4,
mCTAP3, m6Ckine, h6Ckine, or Chrl9kine chemokine, are
assayed for their biological activities. Alternatively,
compounds are screened, alone or in combinations, to
determine effects on biological activity.
XI. Isolation of a Receptor for mpf4, mCTAP3, m6Ckine,
hSCkine, or Chrl9kine chemokine
A mpf4, mCTAP3, m6Ckine, h6Ckine, or Chrl9kine
chemokine can be used as a specific binding reagent to
identify its binding partner, by taking advantage of its
specificity of binding, much like an antibody would be
used. A binding reagent is either labeled as described
above, e.g., fluorescence or otherwise, or immobilized to
a substrate for panning methods. The typical chemokine
receptor is a seven transmembrane receptor.
The binding composition, e.g., chemokine, is used to
screen an expression library made from a cell line which
expresses a binding partner, i.e. receptor. Standard
staining techniques are used to detect or sort
intracellular or surface expressed receptor, or surface
expressing transformed cells are screened by panning.
Screening of intracellular expression is performed by
CA 02267092 1999-03-30
WO 98I14581 PCT/US97/I7122
various staining or immunofluorescence procedures. See
also McMahan, et al. (1991) EMBO J. 10:2821-2832. _ _
Using standard Ca'~+ flux protocols, see, e.g.,
Coligan, et al. (eds.)(1992 and periodic supplements)
Current Protocols in Immunol. Greene/Wiley, New York, NY,
a receptor for m6Ckine was found to be a CXC chemokine
receptor, designated CXCR3. This receptor is shared with
IP-10 and Mig, chemokines which possess potent
angiostatic and antitumor properties. See, e.g., Keane,
et al. (1997) J. Immunol. 159:1437-1443; and Farber
(1997) J. Leukoc. Biol. 61:246-257.
For example, on day 0, precoat 2-chamber permanox
slides with 1 ml per chamber ~of fibronectin, 10 ng/ml in
PBS) for 30 min at room temperature. Rinse once with
PBS. Then plate COS cells at 2-3 x 105 cells per chamber
in 1.5 ml of growth media. Incubate overnight at 37' C.
On day 1 for each sample, prepare 0.5 ml of a
solution of 66 ~l,g/ml DEAF-dextran, 66 ~lM chloroquine, and
4 ~.g DNA in serum free DME. For each set, a positive
control is prepared, e.g., of human mpf4, mCTAP3,
m6Ckine, h6Ckine, or Chrl9kine chemokine cDNA at 1 and
1/200 dilution, and a negative mock. Rinse cells with
serum free DME. Add the DNA solution and incubate 5 hr
at 37' C. Remove the medium and add 0.5 ml 10~ DMSO in
DME for 2.5 min. Remove and wash once with DME. Add 1.5
ml growth medium and incubate overnight.
On day 2, change the medium. On days 3 or 4, the
cells are fixed and stained. Rinse the cells twice with
Hank's Buffered Saline Solution (HBSS) and fix in 4~
paraformaldehyde (PFA)/glucose for 5 min. Wash 3X with
HBSS. The slides may be stored at -80' C after a11
liquid is removed. For each chamber, 0.5 ml incubations
are performed as follows. Add HBSS/saponin (0.1~) with
32 ).~.1/ml of 1 M NaN3 for 20 min. Cells are then washed
with HBSS/saponin 1X. Add chemokine or
chemokine/antibody complex to cells and incubate for 30
min. Wash cells twice with HBSS/saponin. If
appropriate, add first antibody for 30 min. Add second
antibody, e.g., Vector anti-mouse antibody) at 1/200
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dilution, and incubate for 30 min. Prepare ELISA
solution, e.g., Vector Elite ABC horseradish peroxidase
solution, and preincubate for 30 min. Use, e.g., 1 drop
of solution A (avidin) and 1 drop solution B (biotin) per
2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin.
Add ABC HRP solution and incubate for 30 min. Wash cells
twice with HBSS, second wash fox 2 min, which closes
cells. Then add Vector diaminobenzoic acid (DAB) for 5
to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2
drops of H202 per 5 ml of glass distilled water.
Carefully remove chamber and rinse slide in water. Air
dry for a few minutes, then add 1 drop of Crystal Mount
and a cover slip. Bake for 5 min at 85-90' C.
Evaluate positive staining of pools and
progressively subclone to isolation of single genes
responsible for the binding.
Alternatively, chemokine reagents are used to
affinity purify or sort out cells expressing a receptor.
See, e.g., Sambrook, et al. or Ausubel, et al.
Another strategy is to screen for a membrane bound
receptor by panning. The receptor cDNA is constructed as
described above. The ligand can be immobilized and used
to immobilize expressing cells. Immobilization may be
achieved by use of appropriate antibodies which
recognize, e.g., a FLAG sequence of a chemokine fusion
construct, or by use of antibodies raised against the
first antibodies. Recursive cycles of selection and
amplification lead to enrichment of appropriate clones
and eventual isolation of receptor expressing clones.
Phage expression libraries can be screened by
chemokine. Appropriate label techniques, e.g., anti-FLAG
antibodies, will allow specific labeling of appropriate
clones.
XII. Immunohistochemical localization
The antibody described above is used to identify
expression of mpf4, mCTAP3, m6Ckine, h6Ckine, or
Chrl9kine in various tissues. Methods for
immunohistochemical staining are described, e.g., in
CA 02267092 1999-03-30
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_ 7z _
Sheehan, et al. (eds.) (1987) Theory and Practice of
Histotechnoloav, Battelle Press, Columbus, OH.
All references cited herein are incorporated herein
by reference to the same extent as if each individual
publication or patent application was specifically and
individually indicated to be incorporated by reference in
its entirety for a11 purposes.
Many modifications and variations of this invention
can be made without departing from its spirit and scope,
as will be apparent to those skilled in the art. The
specific embodiments described herein are offered by way
of example only, and the invention is to be limited only
by the terms of the appended claims, along with the full
scope of equivalents to which such claims are entitled.
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- 73 -
SEQUENCE SUBMISSION
SEQ ID NO: 1 is mpf4 mouse nucleic acid sequence.
SEQ ID NO: 2 is mpf4 mouse amino acid sequence.
SEQ ID NO: 3 is mCTAP3 mouse nucleic acid sequence.
SEQ ID NO: 4 is mCTAP3 mouse amino acid sequence.
SEQ ID NO: 5 is m6Ckine mouse nucleic acid sequence.
SEQ ID NO: 6 is m6Ckine mouse amino acid sequence.
SEQ ID NO: 7 is h6Ckine human nucleic acid sequence.
SEQ ID NO: 8 is h6Ckine humanlamino acid sequence.
SEQ ID NO: 9 is 6Ckine porcine amino acid sequence.
SEQ ID NO: 10 is Chrl9kine human amino acid sequence.
20
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Schering-Corp.
(ii) TITLE OF INVENTION: MAMMALIAN CHEMOKINES
(iii) NUMBER OF SEQUENCES: 10
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Schering-Plough Corporation
(B) STREET: 2000 Galloping Hill Road
(C) CITY: Kenilworth
(D) STATE: New Jersey
(E) COUNTRY: USA
(F) ZIP: 07033-0530
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: Apple Macintosh
(C) OPERATING SYSTEM: Macintosh 7.5.3
(D) SOFTWARE: Microsoft Word 6.1
(vi) CURRENT APPLICATION DATA:
4O (A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60l027,242
(B) FILING DATE: 02-OCT-1996
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/028,042
(B) FILING DATE: 09-OCT-1996
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/05B,007
(B) FILING DATE: 28-AUG-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Thampoe, Immac J.
(B) REGISTRATION NUMBER: 36,322
(C) REFERENCE/DOCKET NUMBER: DX0645
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (908)298-5061
(B) TELEFAX: (908)298-5388
CA 02267092 1999-03-30
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_7~_
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 540 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 141..455
(ix) FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION: 258..455
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CCTGGGTTTC CGGACTGGGC AGGCAGTGAA GATAAAACGT GCTTGGGAAG TCCCAGGAGC
60
TGCTGGCCTG CACTTAAGAG CCCTAGACCC ATTTCCTCAA GGTAGAACTT TATCTTTGGG
120
3O TCCAGTGGCA CCCTCTTGAC ATG AGC GTC GCT GCG GTG TTT CGA GGC CTC
l70
Met Ser Val Ala Ala Val Phe Arg Gly Leu
-39 -35 -30
CGG CCA AGT CCT GAG CTG CTG CTT CTG GGC CTG TTG TTT CTG CCA GCG
218
Arg ProSer ProGluLeu LeuLeuLeuGly LeuLeuPhe LeuProAla
-25 -20 -15
GTG GTTGCT GTCACCAGC GCTGGTCCCGAA GAAAGCGAT GGAGATCTT
266
Val ValAla ValThrSer AlaGlyProGlu GluSerAsp GlyAspLeu
-10 -5 1
AGC TGTGTG TGTGTGAAG ACCATCTCCTCT GGGATCCAT CTTAAGCAC
314
Ser CysVal CysValLys ThrIleSerSer GlyIleHis LeuLysHis
5 10 15
ATC ACCAGC CTGGAGGTG ATCAAGGCAGGA CGC-CACTGT GCGGTTCCC
362
Ile ThrSer LeuGluVal IleLysAlaGly ArgHisCys AlaValPro
20 25 30 35
CAG CTCATA GCCACCCTG AAGAATGGGAGG AAAATTTGC CTGGACCGG
410
Gln LeuIle AlaThrLeu LysAsnGlyArg LysIleCys LeuAspArg
40 45 50
6O CAA GCACCC CTATATAAG AAAGTAATCAAG AAAATCCTG GAGAGT
455
Gln AlaPro LeuTyrLys LysValIleLys LysIleLeu GluSer
55 60 65
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- 7S-
TAGGTATCAG CTGCCTAAAT GTCAATTGTG TTACAAGACT CCTGGAATCT TGTCTACTTT
515
TAATGTAACT GCAATCTTCC GATGT
54o
(2) INFORMATION FORSEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:
105
amino
acids
' (B) TYPE:
amino
acid
(D) TOPOLOGY:
linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ser Ala AlaVal Phe Arg Gly Leu Arg Pro Ser Pro
Val Glu Leu
-39 -35-30 -25
Leu Leu Gly LeuLeu Phe Leu Pro Ala Val Val Ala Val
Leu Thr Ser
-20 -15 -10
Ala G1y Pro Glu Glu Ser Asp Gly Asp Leu Ser Cys Val Cys Val Lys
-5 1 5
Thr Ile Ser Ser Gly Ile His Leu Lys His Ile Thr Ser Leu Glu Val
10 15 20 25
Ile Lys Ala Gly Arg His Cys Ala Val Pro Gln Leu Ile Ala Thr Leu
30 35 40
Lys Asn Gly Arg Lys Ile Cys Leu Asp Arg Gln Ala Pro Leu Tyr Lys
45 50 55
Lys Val Ile Lys Lys Ile Leu Glu Ser
60 65
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 442 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 74..412
(ix) FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION: 200..412
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
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98I14581
GTGTTCTGGG CAGCTCAG CC TCACGTTGTT TGGC GTTCTAGCTC
CCCTCC TCTGGGGACA
60
ACTGCTCTTC GGC TTC AGA CTC CCT TCCTGC
ATT AGA ACA ACC
ATG TCG
109
Met Gly Phe Arg Leu Pro SerSerCys
Arg Thr Thr
-42 -40 -35
1O AGG GCC TGCCCA CTTCAT AAC CTC CAG_ATCTTG CTGCTGGGC CTG
CTG
157
Arg Ala CysPro LeuHis Asn Leu Gln Leu LeuLeuGly Leu
Ile Leu
-30 -25 -20 -15
Z5 ATC CTT GTTGCG CTGGCT CCC CTT ACA GGA TCTGATGGC ATG
GCT AAA
205
Ile Leu ValAla LeuAla Pro Leu Thr Gly SerAspGly Met
Ala Lys
-10-5 1
20 GAC CCA TATATC GAACTG CGC TGC AGA ACG ACCATCTCT GGA
TGT AAT
253
Asp Pro TyrIle GluLeu Arg Cys Arg Thr ThrIleSer Gly
Cys Asn
5 20 15
25 ATC CCA TTCAAT TCTATC TCC CTT GTG GTG AGGCCAGGA GTT
AAT TAC
301
Ile Pro PheAsn SerIle Ser Leu Val Val ArgProGly Val
Asn Tyr
20 25 30
30 CAC TGT GCTGAT GTGGAA GTG ATA GCC CTG AATGGACAA AAA
ACA AAG
349
His Cys AlaAsp ValGlu Val Ile Ala Leu AsnGlyGln Lys
Thr Lys
35 40 45 50
3 ACG TGC CTGGAC CCAAAT GCC CCT GGC AAG ATCGTCATG AAA
5 GTC AGA
397 ~.
Thr Cys LeuAsp ProAsn Ala Pro Gly Lys IleValMet Lys
Val Arg
5560 65
4O ATC TTG GAAGGT TACTGACCAGCTG CTTCATCTGT
GCCAAACCAT
442
Ile Leu GluGly Tyr
70
45
(2) INFORMATION FORSEQ ID N0:4:
(i) CHARACTERISTICS:
SEQUENCE
(A} LENGTH:
113
amino
acids
50 (B) TYPE:
amino
acid
(D) TOPOLOGY:
linear
(ii) TYPE: protein
MOLECULE
55 (xi) DESCRIPTION: SEQ N0:4:
SEQUENCE ID
Met Gly PheArg LeuArg Pro Thr Ser Cys ArgAlaCys Pro
Ser Thr
-42 -40 -35 -30
60 Leu His AsnLeu GlnIle Leu Leu Leu Gly IleLeuVal Ala
Leu Leu
-25 -20 -15
Leu Ala ProLeu ThrAla Gly Lys Ser Gly AspProTyr Ile
Asp Met
-10 -5 1 5
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- 77~
G1u Leu Arg Cys Arg Cys Thr Asn Thr Ile Ser Gly Ile Pro Phe Asn
15 20
5 Ser Ile Ser Leu Val Asn Val Tyr Arg Pro Gly Val His Cys Ala Asp
25 30 35
Val Glu Val Ile Ala Thr Leu Lys Asn Gly Gln Lys Thr Cys Leu Asp
40 45 50
10 _
Pro Asn Ala Pro Gly Val Lys Arg Ile Val Met Lys Ile Leu G1u Gly
55 60 65 70
Tyr
(2) INFORMATION
FOR
SEQ
ID
N0:5:
(i)SEQUENCE CHARACTERISTICS:
(A} LENGTH: 893 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: cDNA
(ix)FEATURE:
(A) NAME/KEY: CDS
3 (B) LOCATION: 71..469
0
(ix)FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION: 140..469
(xi)SEQUENCE DESCRIPTION: SEQ
ID N0:5:
ATTCGGATCC TCTGGTCTCA TCCTCAACTC
ATCCTTGCGG
CTGTCCATCT
CACCTACAGC
60
AACCACAATC CTC CTT AGC CTG
ATG GTC
GCT
CAG
ATG
ATG
ACT
CTG
AGC
109
Met Ala Gln Met Met Thr Leu Leu Leu Ser Leu
Ser Val
-23 -20 -15
CTG GCTCTC TGC ATC CCC TGG ACC CAA GAT GGA GGG GGT
GGC AGT CAG
157
Leu AlaLeu Cys Ile Pro Trp Thr Gln Asp Gly Gly Gly
Gly Ser Gln
-10 -5 1 5
GAC TGCTGC CTT AAG TAC AGC CAG AAG CCC TAC AGT ATT
AAA ATT GTC
205
Asp CysCys Leu Lys Tyr Ser Gln Lys Pro Tyr Ser Ile
Lys Ile Val
10 15 20
CGA GGCTAT AGG AAG CAA GAA CCA AGT TGT CCC ATC CCG
TTA GGC GCA
253
Arg GlyTyr Arg Lys Gln G1u Pro Sex Cys Pro Ile Pro
Leu Gly Ala
50 25 30 35
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ATC CTG TTCTCACCC CGGAAGCAC TCT CCT GAGCTATGT GCAAAC
AAG
301
Ile Leu PheSerPro ArgLysHis Ser Pro GluLeuCys AlaAsn
Lys
40 45 50
CCT GAG GAAGGCTGG GTGCAGAAC CTG CGC CGCCTGGAC CAGCCT
ATG
349
Pro Glu GluGlyTrp ValGlnAsn Leu Arg ArgLeuAsp GlnPro
Met
55 60 65 70
CCA GCC CCAGGGAAA CAAAGCCCC GGC AGG AAGAACCGG GGAACC
TGC
397
Pro Ala ProGlyLys GlnSerPro Gly Arg LysAsnArg GlyThr
Cys
75 80 85
TCT AAG TCTGGAAAG AAAGGAAAG GGC AAG GGCTGCAAG AGAACT
TCC
445
Ser Lys SerGlyLys LysGlyLys Gly Lys GlyCysLys ArgThr
Ser
90 95 100
GAA CAG ACACAGCCC TCAAGAGGA TAGCCCAGTA
GCCCGCCTGG
AGCCCAGGAG
499
Glu Gln ThrGlnPro SerArgGly
205 110
ATCCCCCACG AACTTCAAGC TGGGTGGTTC ACGGTCCAAC TCACAGGCAA AGAGGGAGCT
559
3 O AGAAAACAGA CTCAGGAGCC CAAAGCAGCC ACCTCATGCT GGCCTCCGTC CACACCCTTG
619
CCCTGCTTCA ACCATTACAT TTGCACGGCC ATCCCTTTTT TACCTGGCGG AGCTGCCTTC
679
CCCTGGGGTA GACCTAGAGA GTCAGAAGAA AGAGTGTTTC CCAGGGAATG AGGAAGGAGA
739
CAGCAGGACT GTCCCCTCTA GGAGGTCACT CAGGTCCCAA GACCTGAACC TGTTTTCCAT
799
GGCGCCCTTC CCTTGTCCTT GCACCTATGA TTTATACCTA ACTGAATAAA AAGGTGATCC
859
AGCCTCAAAA F~~~1AAAAAP.A AAAAAAAAAA AAAA
893
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 133 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Ala Gln Met Met Thr Leu Ser Leu Leu Ser Leu Val Leu Ala Leu
-23 -20 -15 -10
Cys Ile Pro Trp Thr Gln Gly Ser Asp Gly Gly Gly Gln Asp Cys Cys
-5 1 5
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- ?~1 '-
Leu Lys Tyr Ser Gln Lys Lys Ile Pro Tyr Ser Ile Val Arg Gly Tyr
15 20 25
5 Arg Lys Gln Glu Pro Ser Leu Gly Cys Pro Ile Pro Ala Ile Leu Phe
30 35 40
10 Ser Pro Arg Lys His Ser Lys Pro Glu_Leu Cys Ala Asn Pro Glu Glu
45 50 55
Gly Trp Val Gln Asn Leu Met Arg Arg Leu Asp Gln Pro Pro Ala Pro
60 65 70
Gly Lys Gln Ser Pro Gly Cys Arg Lys Asn Arg Gly Thr Ser Lys Ser
75 80 85
Gly Lys Lys Gly Lys Gly Ser Lys Gly Cys Lys Arg Thr Glu Gln Thr
90 95 100 105
Gln Pro Sex Arg Gly
110
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 814 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 6..407
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 75..407
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
CAGAC ATG GCT CAG TCA CTG GCT CTG AGC CTC CTT ATC CTG GTT CTG
47
Met Ala Gln Ser Leu Ala Leu Ser Leu Leu Ile Leu Val Leu
-23 -20 -15 -10
GCC TTT GGA ATC CCC AGG ACC CAA GGC AGT GAT GGA GGG GCT CAG GAC
55 Ala Phe Gly Ile Pro Arg Thr Gln Gly Ser Asp Gly Gly Ala Gln Asp
-5 1 5
TGT TGC CTC AAG TAC AGC CAA AGG AAG ATT CCC GCC AAG GTT GTC CGC
143
60 Cys Cys Leu Lys Tyr Ser Gln Arg Lys Ile Pro Ala Lys Val Val Arg
10 15 20
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AGC TAC CGGAAGCAG GAACCAAGC TTAGGCTGCTCC ATCCCAGCT ATC
191
Ser Tyr ArgLysGln GluProSer LeuGlyCysSer IleProAla Ile
25 30 35
CTG TTC TTGCCCCGC AAGCGCTCT CAGGCAGAGCTA TGTGCAGAC CCA
239
Leu Phe LeuProArg LysArgSer GlnAlaGluLeu CysAlaAsp Pro
40 45 50 55
AAG GAG CTCTGGGTG CAGCAGCTG ATGCAGCATCTG GACAAGACA CCA
287
Lys Glu LeuTrpVal GlnGlnLeu MetGlnHisLeu AspLysThr Pro
60 65 70
TCC CCA CAGAAACCA GCCCAGGGC TGCAGGAAGGAC AGGGGGGCC TCC
335
Ser Pro GlnLysPro AlaGlnGly CysArgLysAsp ArgGlyAla Ser
75 80 85
AAG ACT GGCAAGAAA GGAAAGGGC TCCAAAGGCTGC AAGAGGACT GAG
383
Lys Thr GlyLysLys GlyLysGly SerLysGlyCys LysArgThr Glu
90 95 100
CGG TCA CAGACCCCT AAAGGGCCA TAGCCCAGTG
AGCAGCCTGG
AGCCCTGGAG
437
Arg Ser GlnThrPro LysGlyPro
105 110
ACCCCACCAG TGAACCCAAG ATGCAAGAAG
CTTCACCAGC GAGGCTATGC
GCTTGAAGCC
497
TCAGGGGCCC GGCCTTGCCA CACTCTTTCT
TGGAGCAGCC CCTGCTTTAA
ACCCCATGCT
557
CCACCCCATC GCATGGCTGA GCTGCCCACA
TGCATTCCCA GCAGGCCAGG
GCTCTACCCT
617
TCCAGAGAGA CCGAGGAGGG AGAGTCTCCC AGGGAGCATG AGAGGAGGCA GCAGGACTGT
677
CCCCTTGAAG GAGAATCATC AGGACCCTGG ACCTGATACG GCTCCCCAGT ACACCCCACC
737
TCTTCCTTGT AAATATGATT TATACCTAAC TGAATAAAAA GCTGTTCTGT CTTCCCACCC
797
5 0 AAAAA.AAAAA AAAAAAA
814
60
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 134 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
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Met Ala Gln Ser Leu Ala Leu Ser Leu Leu Ile Leu Val Leu Ala Phe
-23 -20 -15 -10
Gly Ile Pro Arg Thr Gln Gly Ser Asp Gly Gly Ala Gln Asp Cys Cys
-5 1 5
Leu Lys Tyr Ser Gln Arg Lys Ile Pro Ala Lys Val Val Arg Ser Tyr
15 20 25
10 Arg Lys Gln Glu Pro Ser Leu Gly Cys_Ser Ile Pro Ala Ile Leu Phe
30 35 40
Leu Pro Arg Lys Arg Ser Gln Ala Glu Leu Cys Ala Asp Pro Lys Glu
45 50 55
Leu Trp Val Gln Gln Leu Met Gln His Leu Asp Lys Thr Pro Ser Pro
60 65 70
Gln Lys Pro Ala Gln Gly Cys Arg Lys Asp Arg Gly Ala Ser Lys Thr
75 80 85
Gly Lys Lys Gly Lys Gly Sex Lys Gly Cys Lys Arg Thr Glu Arg Ser
90 95 100 105
Gln Thr Pro Lys Gly Pro
110
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 77 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 24..77
(D) OTHER INFORMATION: /note= "mature peptide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Met Xaa Gln Ser Leu Val Leu Arg Ile Leu VaI Leu Val Leu Ala Phe
1 5 10 15
Cys Ile Pro His Thr Gln GIy Ser Asp Gly Gly Ala Gln Asp Cys Cys
20 25 30
Leu Lys Tyr Ser Leu Arg Lys Ile Pro Thr His Val Val Arg Ser Tyr
35 40 45
Arg Lys Gln Glu Pro Ser Leu Gly Cys Pro Ile Pro Ala Ile Leu Phe
50 55 60
Ser Pro Arg Lys Xaa Ser Gln Pro Glu Leu Cys Ala Asp
70 75
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o 'b Z -
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
{C) STRANDEDNESS: not relevant
{D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Arg Phe Glu Gly Tyr Arg Pro Asn Leu Ala Glu Ser Cys Pro Cys Arg
1 5 10 15
2 ~ Gln Tyr Arg Ala Tyr Val Leu Thr His Ser Gly Glu Leu Tyr Glu Arg
25 30
Pro Glu Arg Ser Asp Arg Gln Ile Cys Val
35 40
