Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Th2 CELL DEPLETION; COMPOSITIONS; METHODS
The present filing claims priority from provisional
U.S. patent application USSN 60/065,392, filed November 13,
1997.
Field of the Invention
The invention relates generally to methods of
purifying or depleting Th2 cells and compositions used for
such, and more particularly, to methods of treating diseases
or conditions associated with elevated or depleted
populations of Th2 cells.
BACKGROUND
The immune system consists of a wide range of distinct
cell types, each with important roles to play. See Paul
(ed. 1993) Fundamental Immunology, 3d ed, Raven Press, New
York. The lymphocytes occupy central stage because they are
the cells that determine the specificity of immunity, and it
is their response that orchestrates the effector limbs of
the immune system. Two broad classes of lymphocytes are
recognized: the B lymphocytes, which are precursors of
antibody secreting cells, and the T (thymus-dependent)
lymphocytes. T lymphocytes express important regulatory
functions, such as the ability to help or inhibit the
development of specific types of immune response, including
antibody production and increased microbicidal activity of
macrophages. Other T lymphocytes are involved in direct
effector functions, such as the lysis of virus infected-
cells or certain neoplastic cells.
T cells may be subdivided into two distinct classes
based on the cell-surface receptors they express. T cells,
particularly the CD4+ T cells, are the major regulatory
cells of the immune system. These CD4+ cells tend to
differentiate into the Th2 or Th1 subsets. The Th2 cells
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are effective in helping B cells develop into antibody
producing cells, while the Thl cells effectively induce
cellular immune responses, e.g., cell mediated immunity,
delayed type hypersensitivity, macrophage activation, and
production of opsonizing antibodies. The Thl cell subsets
produce IL-2, IFN-y, TNF-oc, and lymphotoxin, but little or
no IL-4, IL-5, or IL-10. In contrast, Th2 cell subsets
produce IL-4, IL-5, IL-6, IL-10, and IL-13, but little or no
IL-2 or IFN-y. The different cytokine patterns lead to
markedly different immune responses.
Another subset of the T cells, the Trl, have also been
described. See, e.g., Groux, et al. (1997) Nature 389:737-
742 (1997); Groux, et al. (1997) J. Immunol. 158;5627-5631;
Groux, et al. (1996) J. Exp. Med. 184;19-29.
While the different types of cells can be
distinguished, practical positive selection markers have not
yet been identified. The availability of methods and/or
reagents useful to specifically identify, select, or
eliminate one or the other subset will find use both
diagnostically and therapeutically. The present invention
provides these.
SUMMARY OF THE INVENTION
The present invention is based, in part, upon the
surprising discovery that the CCR8 chemokine receptor is
specifically expressed on the Th2 subsets of T cells,
virtually to the exclusion of other cell types. Thus,
reagents which specifically bind to the chemokine receptor,
e.g., ligand or antibodies, will affect only these cell
subsets.
In particular, the agonist signaling of Th2 cells can
be achieved using CCR8 agonists. Conversely, antagonists of
CCR8 should block Th2 signaling. So the reagents will be
useful in specifically activating or blocking the Th2
subset. This provides means to specifically attract or
block Th2 trafficking, apoptosis, etc., and to redirect a
Th2 to Thl response, or vice versa.
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Thus, the invention provides a substantially pure
CCRB+ population of cells which consists essentially of
activated Th2 cells. In various embodiments, the population
will produce low amounts of the cytokines IL-2 and IFN-y,
and produce high amounts of IL-4 and IL-10; will bind anti-
CCR8 antibody or antiserum; or will be prepared by
Fluorescent Activated Cell Sorting with a labeled CCR8
selective: ligand; antibody; or binding compound comprising
the antigen binding portion from an antibody which
selectively binds CCR8. Preferably, the ligand is I-309 or
TCA3; or the antibody binds to human CCR8.
In other embodiment, the invention provides a method
of selectively labeling activated Th2 cells comprising
contacting a population containing those cells to a reagent
which specifically binds to a CCR8. In certain embodiments,
the population comprises Thl cells; or the method further
involves selectively depleting the activated Th2 cells from
the population, e.g., by killing of those cells expressing
CCR8; or by FACS sorting of those activated Th2 cells. The
depletion may use a toxic conjugate with: a ligand specific
for CCR8 binding; a binding compound comprising an antigen
binding portion from an antibody which selectively binds to
a mammalian CCR8; or a binding composition comprising
antigen binding portions from antiserum which selectively
binds to a mammalian CCR8. Preferably, the mammalian CCR8
is human CCR8 and the antibody is a monoclonal antibody.
Cells made by the method are also embraced.
Another embodiment includes a method of modulating a
physiological signal specifically to activated Th2 cells,
comprising contacting the activated Th2 cells with a CCR8
agonist or antagonist. For example, the modulating may be
blocking, e.g., by contacting with a CCR8 antagonist, e.g.,
an antibody. The antagonist antibody may bind to either a
CCR8 ligand or to CCR8. Alternatively, the modulating may
be inducing with an agonist, e.g., by contacting with a CCR8
signaling ligand, e.g., I-309 or TCA3. The modulating may
be directing a response between a Thl and Th2 response,
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where the contacting is with a CCRB antagonist or CCR8
signaling agonist. In preferred embodiments, the
physiological signal is a proliferation, apoptotic, or
differentiation signal. The contacting may be in
combination with another chemokine or cytokine agonist or
antagonist, including IL-12, an IL-12 antagonist, IL-ly, or
an IL-l~y antagonist .
DETAILED DESCRIPTION OF THE INVENTION
I. General
The invention is based, in part, on the surprising
discovery that a chemokine receptor, CCR8, is expressed
predominantly on Th2 cells. Its expression level on other
cells is very low or even undetectable, especially on other
T cell types.
The chemokines are a sub-family of chemoattractant
cytokines that were classically characterized by their
ability to mediate leukocyte trafficking by binding to
specific G-protein linked seven transmembrane spanning
receptors, or GPCRs. Chemokines are divided into four
groups based on the primary sequence of the first two
cysteines: the CXC, CC, C, and the newly discovered, CX3C
families. The CXC and C families are effective
predominantly on neutrophils and lymphocytes, respectively.
The CC chemokines are preferentially effective on
macrophages, lymphocytes, and eosinophils.
Only about half of the chemokines have been paired to
respective receptors. Some seem to bind to more than one
receptor. The matching of orphan receptors with the many
chemokines is an ongoing process. The matching of the
ligands with receptors often provide useful insight into the
physiological functions of the individual chemokines, often
because the distribution of the receptors is quite limited.
The human CCR8 receptor, which was an orphan GPCR
known under the names Terl (Napolitano, et al. (1996) ,7.
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Ircttnunol. 157:2759-2763), ChemRl (Samson, et al. (1996!
~enomics 36:522-526), or CKR-L1 (Zabellos, et al. (1996)
Biochem. Biobhvs. Res. Cc~amun. 227:846-853), has been known
for some time. See GenBank accession numbers AF005210
(partial) and U45983. The ligand for the CCR8 chemokine
receptor has been identified, in humans, the I-309 protein
(Boos, et al. (1997) J. Biol. Chem. 272:17251-17254; and
Tiffany, et al. (1997) J. Exn. Med. 186:165-170; GenBank
Accession Numbers M57502 and M57506), and in mouse, the TCA-
3 (GenBank Accession Numbers M17957 and X52401). However,
the distribution of the CCR8 had been characterized as
lymphoid specific, and particularly to the thymus.
Napolitano, et al. (1996) J. Immunol. 157:2759-2763. The
CCRS distribution in the peripheral lymphocytes has not yet
been well characterized until the present studies leading to
this invention.
II. Specific Binding Compositions
A. Antibodies
The present invention provides for the use of an
antibody or binding composition which specifically binds to
a CCR8, preferably mammalian, e.g., primate, human, cat,
dog, rat, or mouse. Antibodies can be raised to various
CCR8 proteins, including individual, polymorphic, allelic,
strain, or species variants, and fragments thereof, both in
their naturally occurring (full-length) forms or in their
recombinant forms. Additionally, antibodies can be raised
to CCR8 proteins in both their native (or active) forms or
in their inactive, e.g., denatured, forms. Anti-idiotypic
antibodies may also be used.
A number of immunogens may be selected to produce
antibodies specifically reactive, or selective for binding,
with CCR8 proteins. Recombinant protein is a preferred
immunogen for the production of monoclonal or polyclonal
antibodies. Naturally occurring protein, from appropriate
sources, e.g., primate, rodent, etc., may also be used
either in pure or impure form. Synthetic peptides, made
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using the CCR8 protein sequences described herein, may also
be used as an immunogen for the production of antibodies to
CCR8 proteins. Recombinant protein can be expressed and
purified in eukaryotic or prokaryotic cells as described,
e.g., in Coligan, et al. (eds.) (1995 and periodic
supplements) Current Protocols in Protein Science John Wiley
& Sons, New York, NY; and Ausubel, et al (eds.) (1987 and
periodic supplements) Current Protocols in Molecular
B_ioloav, Greene/Wiley, New York, NY. Naturally folded or
denatured material can be used, as appropriate, for
producing antibodies. Either monoclonal or polyclonal
antibodies may be generated, e.g., for subsequent use in
immunoassays to measure the protein, or for
immunopurification methods.
Methods of producing polyclonal antibodies are well
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 CCR8 protein or peptide of interest. For
example, when 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 performed, if desired. See, e.g., Harlow and Lane
Antibodies. A Laboratory Manual; or Coligan (ed.) Current
Protocols in Immunoloav. Immunization can also be performed
through other methods, e.g., DNA vector immunization. See,
e.g., Wang, et al. (1997) Viroloav 228:278-284.
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.
Alternative methods of immortalization include
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transformation with Epstein Barr Virus, oncogenes, or
retroviruses, or other methods known in the art. See,
e.g., Doyle, et al. (eds. 1994 and periodic supplements)
Cell and Tissue Culture: Laboratory Procedures, John Wiley
and Sons, New York, NY. 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 al. (1989)
Science 246:1275-1281.
Antibodies or binding compositions, including binding
fragments and single chain versions, against predetermined
fragments of CCR8 proteins can be raised by immunization of
animals with conjugates of the fragments with carrier
proteins as described above. Monoclonal antibodies are
prepared from cells secreting the desired antibody. These
antibodies can be screened for binding to normal or
defective CCR8 protein, or screened for Th2 cell depleting
ability. These monoclonal antibodies will usually bind with
at least a KD of about 1 mM, more usually at least about 300
E1I~, typically at least about 10 ~.M, more typically at least
about 30 E1M, preferably at least about 10 ~1M, and more
preferably at least about 3 N.M or better.
In some instances, it is desirable to prepare
monoclonal antibodies (mAbs) 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
~mmunoloary (4th ed.) Lange Medical Publications, Los Altos,
CA, and references cited therein; Harlow and Lane (1988)
Antibodies: A Laboratorv Manual CSH Press; Goding (1986)
monoclonal Antibodies Principles and Practice (2d ed.)
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Academic Press, New York, NY; and particularly in Kohler and
Milstein (1975) Nature 256:495-497, which discusses one
method of generating monoclonal antibodies. Summarized
briefly, this method involves injecting an animal with an
immunogen. The animal is then sacrificed and cells taken
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
Phage Lambda," ,$c_ience 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; or made in transgenic mice, see Mendez, et
al. (1997) Nature Genetics 15:146-156.
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Antibody binding compounds, including binding
fragments, of this invention can have significant diagnostic
or therapeutic value. They can be useful as non-
neutralizing binding compounds and can be coupled to toxins
or radionuclides so that when the binding compound binds to
the antigen, a cell expressing it, e.g., on its surface, is
killed. Further, these binding compounds can be conjugated
to drugs or other therapeutic agents, either directly or
indirectly by means of a linker, and may effect drug
targeting.
B. Other Molecules
Antibodies are merely one form of specific binding
compositions. Other binding compositions, which will often
have similar uses, include molecules that bind with
specificity to CCR8 receptor, e.g., in a binding partner-
binding partner fashion, an antibody-antigen interaction, or
in a natural physiologically relevant protein-protein
interaction, either covalent or non-covalent, e.g., proteins
which specifically associate with CCR8 receptor protein.
The molecule may be a polymer, or chemical reagent. A
functional analog may be a protein with structural
modifications, or may be a structurally unrelated molecule,
e.g., which has a molecular shape which interacts wit. the
appropriate binding determinants.
Drug screening using antibodies or CCR8 or fragments
thereof can be performed to identify compounds having
binding affinity to CCR8, or can block the natural
interaction with ligand. Subsequent biological assays can
then be utilized to determine if the compound has int=insic
blocking activity and is therefore an antagonist. Likewise,
a compound having intrinsic stimulating activity can signal
to the cells via the CCR8 and is thus an agonist in that it
simulates the activity of a ligand.
As indicated above, natural ligands for the CCR8
chemokine receptor have been identified. In humans, the I-
309 protein (Boos, et al. (1997) J. Biol. Chem. 272:17251-
17254; and Tiffany, et al. (1997) J. Ex~. Med. 186:165-170;
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GenBank M57502 and M57505), the TARO (see Imai, et al.
(1997) sT. Biol. Chem. 272:15036-15042; GenBank D43767), and
in mouse, the TCA-3 (GenBank M17957 and X52401). Mutein
antagonists may be developed which maintain receptor binding
but lack signaling.
Structural studies of the ligands will lead to design
of new variants, particularly analogs exhibiting agonist or
antagonist properties on the receptor. This can be combined
with previously described screening methods to isolate
muteins exhibiting desired spectra of activities.
As receptor specific binding molecules are provided,
also included are small molecules identified by screening
procedures. Various ligands for the receptor have been
identified. In particular, it is well known in the art how
to screen for small molecules which interfere, e.g., with
ligand binding to the receptor, often by specific binding to
the receptor and blocking of binding by natural ligand.
See, e.g., meetings on High Throughput Screening,
International Business Communications, Southborough, MA
01772-1749. Such molecules may compete with natural
ligands, and selectively bind to the CCR8. Such specific
binding compounds may be labeled or conjugated to toxic
reagents, which are targeted to CCR8+ cells.
These specific binding reagents may similarly be used
to target Th2 cells.
I I I . Immunoas s ays
Immunoassays are valuable in diagnosing a disease or
disorder associated with Th2 imbalance or pathology.
Qualitative or quantitative measurement of a particular
protein can be performed 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 Immunology (7th ed.). Moreover, the
immunoassays of the present invention can be performed in
many configurations, which are reviewed extensively in
Maggio (ed. 1980) Enzyme Immunoassay CRC Press, Boca Raton,
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Florida; Tijan (1985) "Practice and Theory of Enzyme
Immunoassays," Laboratory Technicrues in Bioche_m;~trv and
~rn~Pr-"gar Biolocrv, Elsevier Science Publishers B.V.,
Amsterdam; and Harlow and Lane Antibodies. A Laboratory
Manual, supra. See also Chan (ed. 1987) Immunoassay: A
gs actical Guide Academic Press, Orlando, FL; Price and
Newman (eds. 1991) Principles and Practice of Immunoassays
Stockton Press, NY; and Ngo (ed. 1988) Non-isotopic
Immunoassays Plenum Press, NY.
Immunoassays for measurement of CCR8 proteins or
peptides can be performed by a variety of methods known to
those skilled in the art. In 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 solid surface.
Preferably the capture agent is an antibody specifically
reactive with CCR8 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 CCR8 protein
present in the sample competes with labeled protein for
binding to a specific binding agent, for example, an
antibody specifically reactive with the CCR8 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
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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.
CCRB 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 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 CCR8 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 may 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, 125I~ 35g 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
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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.
Antibodies reactive with a particular protein can also
be measured by a variety of immunoassay methods. Thus
modifications of the above procedures may be used to
determine the amounts or affinities of various CCR8
antibodies or antibody preparation. For a review of
immunological and immunoassay procedures applicable to the
measurement of antibodies by immunoassay techniques, see
Stites and Terr (eds.) Basic and Clinical Immunology (7th
ed.) supra; Maggio (ed.) Enzyme Immunoassay, supra; and
Harlow and Lane Antibodies A Laboratory Manual, supra.
Screens to evaluate the binding and activity of mAbs
and binding compositions encompass a variety of methods.
Binding can be assayed by detectably labeling the antibody
or binding composition as described above. Cells expressing
a CCR8 receptor are incubated with this antibody or binding
composition, and binding is assayed by Fluorescence
Activated Cell Sorting (FACS) analysis.
To evaluate Th2 depletion ability, experimental
animals, e.g., mice, are preferably induced to produce those
cell types, e.g., by infection with a parasite. Th2 cell
counts are made prior to and at various time points after
administration of a bolus of the candidate depleting mAb or
binding composition. Levels are analyzed in various
samples, e.g., blood, serum, nasal or pulmonary lavages, or
tissue biopsy staining. A successful depleting mAb or
binding composition will significantly lower the level of
circulating Th2 cells. Thus, a substantially pure or
depleted population will be at least about 85~ pure, more
preferably at least about 90~ pure, and even more preferably
at least about 95, 97, or 99~ pure.
Evaluation of antibodies can be performed in other
animals, e.g., humans using various methods. For example,
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blood samples are withdrawn from patients suffering from a
Th2 related disease or disorder before a.nd after treatment
with a candidate mAb. The antibodies can be used in a
diagnostic context to evaluate the extent of Thl or Th2
polarization, e.g., by FAGS, tissue staining, in vitro
culture.
IV. Uses
The present invention is useful in the treatment of
medical conditions or diseases associated with a Thl or Th2
cell imbalance. See, e.g., Frank, et al. (eds.1995)
Samter's Immunoloaic Diseases, 5th Ed., vols. I-II, Little,
Brown and Co., Boston, MA; Coffman, et al (1989) Science
245:308-310; and Frick, et al. (1988) ~. Allerov Clin.
Immunol. 82:199-225; each of which is incorporated herein by
reference. The binding specificities of the compositions
described herein can be administered alone or in combination
with another modulator of Th balance, including, e.g., IFN-
y, IL-2, IL-4, antagonists, or other compounds used for the
treatment of symptoms, e.g., steroids such as
glucocorticoids.
In particular, the selectivity of the CCR8 receptor on
these cell types of the CD4+ lineage suggests means to block
the functions of the respective cell types. The expression
seems to be on the CD4+CD8+ subset (double positive or DP).
The expression in this subset may correspond to cells that
have already undergone positive selections and are destined,
e.g., committed, to become CD4+ thymocytes. As I-309 has
been reported to prevent apoptosis in a thymoma (Van Snick,
et al. (1996) J. Immunol. 157:2570-2576), the CCR8 may be
involved in positive selection in the double positive subset
in T cell development. Ligands which agonize or antagonize
this may have importance in the regulation of apoptosis in T
cell development, as well as Th2 or Th1 dominant response
shifting. CCR8 agonists may be used to activate, initiate,
or strengthen Th2 mediated responses. Alternatively,
antagonists may block the recruitment or attraction of CCRB+
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cells to the sites of ligand production, e.g., to the lung
or other sites of allergic or asthmatic effects, or to block
activation or maintenance of Th2 signaling through the CCRB
receptor.
In a like manner, CD8+ cytotoxic T cells may also have
a similar developmental pathway. See 0'Garra and Murphy
(1994) Curr. Opinion in Innmunol. 6:458-466; Palliard, et al.
(1988) ~. Immunol. 141:849-855; Erard, et al. (1993) science
260:1802-1805. Whether those cells may signal though the
same ligand and receptor pathway remains to be detei~nined.
For example, the iigands for the CCR8 would thus be
expected to signal specifically to the cell types expressing
the receptor. Thus, it will be possible to block signaling
to the Th2 subsets by reagents which block receptor
signaling, e.g., antibodies to receptor, antibodies to
ligand, and small drug antagonists. The knowledge of the
CCR8 mediation of Th2 trafficking provides means to block
the attraction by the CCRB ligands.
Particular medical conditions which are Thl or Th2
mediated, and are subject to treatment with this invention
include, e.g., asthma, allergies, allergic bronchopulmonary
aspergillosis, arthritis, inflammatory diseases, IBD, atopic
dermatitis, viral infections, various helminthic and
parasitic infections, and related conditions. See, e.g.,
Frank, et al. (eds.1995) ~amter's Immunoloaic Diseases, 5th
Ed., vols. I-II, Little, Brown and Co., Boston, MA; Coffman,
et al (1989) Science 245:308-310; and Frick, et al. (1988)
J. Allerw Clin. Immunol. 82:199-225.
Conversely, it is an unexpected prediction that it
will be possible to use known ligands to selectively affect
specific functions mediated by Th2 cells. Thus, positive
effects, e.g., blocking of apoptotic signals to these cell
types become feasible. One known ligand to the CCRB, I309,
is expressed in the thymus and there exists some evidence
that the ligand can signal an anti-apoptotic signal. Thus,
antagonists of the ligand may block the protection,
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resulting in cell death. Conversely, blocking of attractive
signals to CCRB+ cells may be effected.
Standard immunological techniques are described, e.g.,
in Hertzenberg, et al. (eds. 1996) Weir's Handbook of
~er;mPntal Immunolow vols 1-4, Blackwell Science; Coligan
(1991) Current Protocols in Immunolo_c~ Wiley/Greene, NY; and
ho s in Enzvmoloc~v volumes. 70, 73, 74, 84, 92, 93, 108,
116, 121, 132, 150, 162, and 163. These will allow use of
the reagents for purifying cell subpopulations, etc.
To prepare pharmaceutical or sterile compositions
including the CCR8 specific binding composition, the
antibody or binding composition is admixed with a
pharmaceutically acceptable carrier or excipient which is
preferably inert. Preparation of such pharmaceutical
compositions is known in the art, see, e.g., Reminaton's
pharmaceutical Sciences and U.S. Pharmaconeia: National
Forznularv, Mack Publishing Company, Easton, PA (1984).
Antibodies or binding compositions are normally
administered parenterally, preferably intravenously. Since
such protein or peptide antagonists may be immunogenic they
are preferably administered slowly, either by a conventional
IV administration set or from a subcutaneous depot, e.g. as
taught by Tomasi et al, U.S. patent 4,732,863.
When administered parenterally the antibodies or
fragments will be formulated in a unit dosage injectable
form (solution, suspension, emulsion) in association with a
pharmaceutically acceptable parenteral vehicle. Such
vehicles are inherently nontoxic and nontherapeutic. The
antagonist may be administered in aqueous vehicles such as
water, saline or buffered vehicles with or without various
additives and/or diluting agents. Alternatively, a
suspension, such as a zinc suspension, can be prepared to
include the peptide. Such a suspension can be useful for
subcutaneous (SQ) or intramuscular (IM) injection. The
proportion of antagonist and additive can be varied over a
broad range so long as both are present in effective
amounts. The antibody is preferably formulated in purified
16
CA 02309761 2000-OS-11
WO-99/25734 PCT/US98/23244
form substantially free of aggregates, other proteins,
endotoxins, and the like, at concentrations of about 5 to 30
mg/ml, preferably 10 to 20 mg/ml. Preferably, the endotoxin
levels are less than 2.5 EII/ml. See, e.g., Avis, et al.
(eds.)(1993) Pharmaceutical Dosaae Forms: Parenteral
Medications 2d ed., Dekker, NY; Lieberman, et al.
(eds.)(1990) Pharmaceutical Dosaae Forms: Tablets 2d ed.,
Dekker, NY; Lieberman, et al. (eds.)(1990) Pharmaceutical
Dosaae Forms: Disperse Systems Dekker, NY; Fodor, et al.
(1991) ~c'ei nce 251:767-773, Coligan (ed.) Current Protocols
in Immunoloav; Hood, et al. Immunoloav Benjamin/Cummings;
Paul (ed.) Fundamental Immunology; Academic Press; Parce, et
al. (1989) tScience 246:243-247; Owicki, et al. (1990) oc.
Na~'1 Acad. Sci. USA 87:4007-4011; and Blundell and Johnson
(1976) Protein Crvstalloaranhv, Academic Press, New York.
Selecting an administration regimen for an antagonist
depends on several factors, including the serum or tissue
turnover rate of the antagonist, the level of Th2 depletion,
the immunogenicity of the antagonist, the accessibility of
the target Th2 cells (e.g., if non-serum Th2 cells are to be
blocked). Preferably, an administration regimen maximizes
the amount of antagonist delivered to the patient consistent
with an acceptable level of side effects. Accordingly, the
amount of antagonist delivered depends in part on the
particular antagonist and the severity of the condition
being treated. Guidance in selecting appropriate doses is
found in the literature on therapeutic uses of antibodies,
e.g. Bach et al., chapter 22, in Ferrone et al., (eds.)
(1985), Handbook of Monoclonal Antibodies Noges
Publications, Park Ridge, NJ; and Russell, pgs. 303-357, and
Smith et al., pgs. 365-389, in Haber, et al. (eds.) (1977)
Antibodies in Human Diaanosis and Therapy, Raven Press, New
York, NY.
Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known in the
art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than
17
CA 02309761 2000-OS-11
WO'99/25734 PCTNS98/23240
the optimum dose and it is increased by small increments
thereafter until the desired or optimum effect is achieved
relative to any negative side effects. Circulating
activated Th2 levels would be important indicators of when
an effective dose is reached. Preferably, a CCR8 antibody
or binding composition thereof that will be used is derived
from the same species as the animal targeted for treatment,
thereby minimizing a humoral response to .the reagent.
The total weekly dose ranges for antibodies or
fragments thereof, which specifically bind to CCR8, range
generally from about 1 ng, more generally from about 10 ng,
typically from about 100 ng; more typically from about 1 ~tg,
more typically from about 10 ~,g, preferably from about 100
~.g, and more preferably from about 1 mg per kilogram body
weight. Although higher amounts may be more efficacious,
the lower doses typically will have fewer adverse effects.
Generally the range will be less than 100 mg, preferably
less than about 50 mg, and more preferably less than about
mg per kilogram body weight.
20 The weekly dose ranges for antagonists, e.g.,
antibody, binding fragments, range from about 10 ~,g,
preferably at least about 50 fig, and more preferably at
least about 100 ~1g per kilogram of body weight. Generally,
the range will be less than about 1000 fig, preferably less
25 than about 500 fig, and more preferably less than about 100
~g per kilogram of body weight. Dosages are on a schedule
which effects the desired treatment and can be periodic over
shorter or longer term. In general, ranges will be from at
least about 10 ~g to about 50 mg, preferably about 100 ~.g to
about 10 mg per kilogram body weight.
Other antagonists of the ligands, e.g., muteins, are
also contemplated. Hourly dose ranges for muteins range
from at least about 10 ~lg, generally at least about 50 fig,
typically at least about 100 ~tg, and preferably at least 500
~g per hour. Generally the dosage will be less than about
100 mg, typically less than about 30 mg, preferably less
than about 10 mg, and more preferably less than about 6 mg
18
CA 02309761 2000-OS-11
WU'99125734 ~ PCT/fJS98/23240
per hour. General ranges will be from at least about 1 ~1g
to about 1000 ~tg, preferably about 10 ~g to about 500 (1g per
hour.
The present invention also provides for administration
of CCR8 antibodies or binding compositions in combination
with known therapies, e.g., steroids, particularly
glucocorticoids, which alleviate the symptoms associated
with excessive Thl or Th2 responses. Daily dosages for
glucocorticoids will range from at least about 1 mg,
generally at least about 2 mg, and preferably at least about
5 mg per day. Generally, the dosage will be less than about
100 mg, typically less than about 50 mg, preferably less
than about 20 mg, and more preferably at least about 10 mg
per day. In general, the ranges will be from at least about
1 mg to about 100 mg, preferably from about 2 mg to 50 mg
per day.
The phrase "effective amount" means an amount
sufficient to ameliorate a symptom or sign of the Th2 or Th2
condition. Typical mammalian hosts will include mice, rats,
cats, dogs, and primates, including humans. An effective
amount for a particular patient may vary depending on
factors such as the condition being treated, the overall
health of the patient, the method, route, and dose of
administration and the severity of side affects. When in
combination, an effective amount is in ratio to a
combination of components and the effect is not limited to
individual components alone.
An effective amount of antagonist will decrease the
symptoms typically by at least about 10~; usually by at
least about 20~; preferably at least about 30~; or more
preferably at least about 50~. The present invention
provides reagents which will find use in therapeutic
applications as described elsewhere herein, e.g., in the
general description for treating disorders associated with
Th1/Th2 imbalances. See, e.g., Berkow (ed.) The Merck
Manual of Diaanosis and Therabv, Merck & Co., Rahway, N.J.;
Thorn, et al. Harrison's Princ~~les of Internal Medicine,
19
CA 02309761 2000-OS-11
- WO'99/25734 ~ PCT/US98/23240
McGraw-Hill, NY; Gilman, et al. (eds.) (1990) Goodmar_ and
Gilman' s : The Pharmacol oai cal Bases of Theraneu~,j,~, 8th
Ed., Pergamon Press; Remincrton's PharmacPm ti~a1 Sciences,
17th ed. (1990), Mack Publishing Co., Easton, Penn; Langer
(1990) Science 249:1527-1533; and Merck Index, Merck & Co.,
Rahway, New Jersey.
Antibodies to CCR8 proteins may be used for the
identification or sorting of cell populations expressing
CCR8 protein, e.g., activated T helper cells. Methods to
sort such populations are well known in the art, see, e.g.,
Melamed, et al. (1990) Flow Cvtometrv and Sorting Wiley-
Liss, Inc., New York, NY; Shapiro (1988) Practical Flow
ytometrv Liss, New York, NY; and Robinson, et al. (1993)
Handbook of Flow Cytometrv Methods Wiley-Liss, New York, NY.
Populations of cells expressing the CCR8 receptor can also
be purified using magnetic beads as described, e.g., in
Bieva, et al. (1989) gyp. Hematol. 17:914-920; Hernebtub, et
al. (1990) Bioconi. Chem. 1:411-418; Vaccaro (1990) Win.
Biotechnol. Lab. 3:30.
Moreover, antisense nucleic acids may be used. For
example, antisense against the ligands may function in a
manner like ligand antagonists, and antisense against the
CCR8 receptor may function like receptor antagonists. Thus,
it may be possible to block the signaling through the
pathway with antisense nucleic acids. Conversely, nucleic
acids for the receptor may serve as agonists, increasing the
numbers of receptor on the cell, thereby increasing cell
sensitivity to ligand, and perhaps blocking the normal
apoptotic signal described.
Using the assay methods described above, the
antibodies or binding compositions are useful in diagnosing
diseases states which result in Thl or Th2 imbalances.
Labeled antibodies can also be utilized in analyzing Th2
infiltration in tissues. Antibodies raised against each
CCR8 protein will also be useful to raise anti-idiotypic
antibodies. These will be useful in detecting or diagnosing
various immunological conditions related to expression of
CA 02309761 2000-OS-11
W0~99/25734 ~ PCT/US98/23240
the respective antigens. Combinations of these signals may
be also pursued.
The broad scope of this invention is best understood
with reference to the following examples, which are not
intended to limit the inventions to the specific
embodiments.
EXAMPLES
I. General Methods
Some of the standard methods are described or
referenced, e.g., in Maniatis, et al. (1982) Molecular
Cloning, A Laboratorv Manual, Cold Spring Harbor Laboratory,
Cold Spring Harbor Press; Sambrook, et al. (1989) Molecular
Cloning: A Laboratorv Manual, (2d ed.), vols. 1-3, CSH
Press, NY; Ausubel, et al., Biolocrv, Greene Publishing
Associates, Brooklyn, NY; or Ausubel, et al. (1987 and
Supplements) Current Protocols in Molecular Biology,
Greene/Wiley, New York; Innis, et al. (eds.)(1990)
Protocols: A Guide to Methods and Ap~oli~atinn~ Academic
Press, N.Y. 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" in Methods in En~ymology, vol. 182,
and other volumes in this series; manufacturer's literature
on use of protein purification products, e.g., Pharmacia,
Piscataway, N.J., or Bio-Rad, Richmond, CA; and Coligan, et
al. (eds.) (1995 and periodic supplements) Current Protocols
in Protein Science, John Wiley & Sons, New York, NY.
Combination with recombinant techniques allow fusion to
appropriate segments, e.g., to a FLAG sequence or an
equivalent which can be fused via a protease-removable
sequence. See, e.g., Hochuli (1989) Chemische Industrie
12:69-70; Hochuli (1990) "Purification of Recombinant
Proteins with Metal Chelate Absorbent" in Setlow (ed.)
21
CA 02309761 2000-OS-11
WO-99/25734 . PCT/US98/Z3240
Genetic Ena~neerir~a Principle and Methods 12:87-98, Plenum
Press, N.Y.; and Crowe, et al. (1992) OIAexnress: The High
Level Expression & Protein Purification SvstPm QIAGEN, Inc.,
Chatsworth, CA.
Standard immunological techniques are described, e.g.,
in Hertzenberg, et al. (eds. 1996) Weir's Handbook of
~erimental Immunoloav vols 1-4, Blackwell Science; Coligan
(1991) Current Protocols in Immu_n~~~~ Wiley/Greene, NY; and
Methods in Enzvmoloav volumes. 70, 73, 74, 84, 92, 93, 108,
116, 121, 132, 150, 162, and 163. Methodology of cell
biology techniques are described, e.g., in Celis (ed. 1998)
Cell Biolo~r: A Laboratory Handbook Academic Press, San
Diego; and Doyle, et al. (eds. 1994 and periodic
supplements) rPl~ and Tissue Culture Laboratory
Procedures, John Wiley and Sons, New York, NY. Techniques
in developmental systems are described, e.g., in Meisami
(ed.) Handbook of Human Growth and Developmental Biology CRC
Press; and Chrispeels (ed.) Molecular Techniaues and
An~roaches in Developmental Biology Interscience.
FRCS analyses are described in Melamed, et al. (1990)
Flow Cytometrv and Sortincr Wiley-Liss, Inc., New York, NY;
Shapiro (1988) Practical Flow Cytometrv Liss, New York, NY;
and Robinson, et al. (1993) Handbook of Flow Cvtometrv
Methods Wiley-Liss, New York, NY.
II. Isolation of mammalian CCR8 encoding sequences
The human CCR8 sequence is readily available. See,
e.g., Roos, et al. (1997) J. Biol. Chem. 272:17251-17254;
and Tiffany, et al. (1997) J. Exp. Med; 186:165-170; GenBank
Accession Numbers AF005210 and U45983. See also WO
96/39434.
To isolate the mouse sequence, a 1057 by fragment
containing the entire ORF of the human CCRB gene was used as
probe to screen the murine 129/SV genomic library in the
1/fix vector (Stratagene, La Jolla, CA). 350,000 phage
clones were plated. Hybridization with the labeled human
CCRB cDNA was carried out at 37° C in 50~ formamide, 5X
22
CA 02309761 2000-OS-11
Wa99/25734 ~ PCT/US98/23240
SSPE, lOX Denhardt's, 0.1~ SDS, and 100 mg/ml salmon sperm
DNA for 16 hrs. Filters were washed three times at 50° C in
2X SSC, 0.1~ SDS, 20 min. each. Positive genomic phage
clones were isolated according to the plaque purification
procedure. DNA was extracted, digested by restriction
endonucleases, and Southern blot analysis was performed.
Hybridizing restriction fragments were subcloned in the
pGemll plasmid (Promega) and sequenced using Sequenase 2.0
(USB). DNA sequence analysis was perfornzed using the
DNAsis/Prosis software (Hitachi).
III. Production of cell lines expressing CCR8
Mammalian cells, e.g., NIH3T3, are transfected by
electroporation or lipofectamine (Gibco BRL, Gair_hersburg,
MD) and selected in neomycin supplemented media for two
weeks. Resistant colonies are sorted by FACS into 96 well
plates and allowed to proliferate. RNA is isolated from
individual clones using RNAzol (Friendswood, TX) and
analyzed using RT PCR, see, e.g., Ausubel, et al. (1987 and
Supplements) Current Protocols in Mole liar Bioloav,
Greene/Wiley, New York; Innis, et al. (eds.)(1990) _P~R_
Protocols: A Guide to Methods and Applications Academic
Press, N.Y., following treatment with DNase.
Positive clones are subject to further analysis in a
Ca++ flux assay as described, e.g., in Kelner, et al. (1994)
Science 266:1395-1399, using I309 or TCA3 as ligand for
CCR8. The clone exhibiting the highest Ca++ flux is
expanded for use in generating mAbs.
Other methods of evaluation of expression can also be
utilized, e.g., staining and FACS analysis, tissue staining,
northern analysis, etc.
Similarly, the ligands can be recombinantly produced,
purchased, or fragments synthetically produced. The ligands
will be useful in generating further mutein antagonists or
antibodies blocking their effector functions.
IV. Antibody Production
23
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WO'99/25734 PGT/US98/23240
Appropriate mammals are immunized with appropriate
amounts of CCR8 transfected cells, e.g., intraperitoneally
every 2 weeks for 8 weeks. Typically, rodents are used,
though other species should accommodate production of
selective and specific antibodies. The final immunization
is given intravenously (IV) through the tail vein.
Generic polyclonal antibodies may be collected.
Alternatively, monoclonal antibodies can_be produced. For
example, four days after the IV injection, the spleen is
removed and fused to SP2/0 and NS1 cells. HAT resistant
hybridomas are selected, e.g., using a protocol designed by
Stem Cell Technologies (Vancouver, BC). After 10 days of
HAT selection, resistant foci are transferred to 96 well
plates and expanded for 3 days. Antibody containing
supernatants are analyzed, e.g., by FRCS for binding to
NIH3T3/CCR8 transfectants. Many different CCR8 mAbs are
typically produced. Those antibodies may be isolated and
modified, e.g., by labeling or other means as is standard in
the art. See, e.g., Harlow and Lane (1988) Antibodies: A
7~aboratorv Manual CSH Press; Goding (1986) Monoclonal
Ar~tibod?es: Principles and Practice (2d ed.) Academic
Press, New York, NY. Methods to conjugate magnetic
reagents, toxic entities, labels, attach the antibodies to
solid substrates, to sterile filter, etc., are known in the
art.
Additionally, some of the mAbs are used to sort CCR8
positive cells in spleens from mice or primates. Various
treatments of the mice are evaluated to determine the
relative proportions of Thl and Th2 cells.
V. Specific expression on Th2 cells
The mAbs may be isotyped, e.g., as directed in an
ELISA based kit from Zymed, Inc. (So. San Francisco, CA)
and/or in an Ouchterlony based kit from ICN (Aurora, OH).
Specific isotypes are selected for the appropriate studies,
e.g., for labeling studies or for the complement killing
effector function.
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CA 02309761 2000-OS-11
WO-99/25734 PCT/US98/23240
Alternatively, the distribution of CCR8, either the
human or mouse counterpart, may also be determined by
nucleic acid expression analysis. This will indicate the
locations of message expression, which typically reflects
protein expression levels.
Fluorescent labeling reagents are prepared according
to standard methods, e.g., labeled with a fluorescent probe.
See, e.g., Melamed, et al. (1990) Flow ytometrv and Sorting
Wiley-Liss, Inc., New York, NY; Shapiro (1988) Practical
Flow Cvtometrv Liss, New York, NY; and Robinson, et-al.
(1993) Handbook of Flow ~rtometry Methods Wiley-Liss, New
York, NY.
Analysis of human samples can be evaluated in a
similar manner. A biological sample, e.g., blood, tissue
biopsy sample, lung or nasal lavage, skin punch, is obtained
from an individual suffering from a disease or disorder
associated with a Th2 mediated disorder. Th2 cell subtype
diagnosis is performed, e.g., by FACS analysis, or similar
means. A mAb or binding composition, e.g., ligand, which
specifically binds to the human CCR8 receptor, is used to
label CCRB+ cells.
Mouse counterpart methods may be developed. Highly
Thl or Th2 polarized cell populations may be made as
described, e.g. , by Openshaw, et al. (1995) J-Ex». Med.
182:1357-1367. Specificity of labeling with the CCRB
reagent can be established.
In human the only significant expression observed was
in thymus. There is a slight signal in human spleen, and
barely anything in lymph nodes. Mouse data is extrapolated
to suggest that there is only significant levels of
expression in some T cells; and that the T cell subsets that
have been identified as having very high expression of CCR8
in mouse are activated polarized Th2 cells, with some signal
in activated NK1.1+ T cells. The latter subset has not been
characterized in humans, but we know it is extremely rare in
mice, probably <1~ of T cells in humans. Thus, the main
CA 02309761 2000-OS-11
WO 99/25734 PCT/US98/Z3240
significant expression of CCR8 in human T cells, as in
mouse, would be Th2-polarized cells.
In addition, the CCR8 goes up very quickly following
beginning of Th polarization. So it probably participates
in the differentiation and/or commitment of Th2 precurser
cells as well as in their recruitment to sites of Th2
accumulation. Also, Th2 cells produce TCA3, the ligand of
CCR8 in mouse, suggesting that there is an autologous loop
of recruitment of Th2 T cells. Thus, an activated,
polarized Th2 T cell will probably be able to recruit other
similar cells through its production of TCA-3 (mouse) or 1-
309 (equivalent ligand in humans).
This polarization process may involve many other
factors, and may be assisted or prevented by various other
cytokine or chemokine factors. The cytokines involved are
well recognized and described above, while the chemokine
components include IP-10, MIP-la, MIP-lei, JE, RANTES, and
eotaxin. See Pearlman, et al. (1997) J. Immunol. 158:827-
833.
The highly specific labeling of Th2 cells will be
useful in various contexts. It provides a means for
positive selection of CCR8 bearing cells, being of the T
cell Th2 subtype. The specificity of expression on Th2
cells also allows for the targeting of CCR8 specific
reagents. Thus, antibodies which bind to CCR8 will bring
them specifically to the Th2 subset. Thus, toxic conjugates
may be used to selectively or preferentially kill the Th2
subset. Alternatively to antibodies, a CCR8 specific
ligand, e.g., the I-309 or TCA3 ligands may be conjugated to
a toxic compound.
Blocking of the ligand-CCR8 interaction or signaling
will likely have specific effects on Th2 cells, e.g.,
chemoattraction, development, or physiology of the subtype.
Thus, agonists or antagonists of the I-309 or TCA3 ligands
should have specific effects on Th2 cells.
VI. Selecting Th2 cells
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CA 02309761 2000-OS-11
WO 99125734 PCT/US98rZ3240
CCRB+ cells may be isolated. Standard methods exist to
isolate T cells, and other cells expressing specific surface
markers. FRCS and magnetic bead methods will be applicable.
See, e.g., Hertzenberg, et al. (eds. 1996) Weir's Handbook of
~Qerimental Immunoloav vols 1-4, Blackwell Science; and
Coligan (1991) Current Pr~toco~s in Immunoloav Wiley/Greene,
NY.
The isolated Th2 cells may be proliferated ex vivo, where
appropriate, e.g., for reintroduction back to the same or
another patient. The Th2 cells may be transfected with a
desired gene or vector.
The CCR8+ specificity may be taken advantage of, e.g., to
specifically deplete the Th2 cells. Conjugation of a CCR8
specific binding agent to a toxic moiety may allow for
selective destruction of Th2 cells, where appropriate.
Antagonists may block chemoattraction of Th2 cells to the
sites of ligands for the receptor.
VII. CCR8 Antagonists
Various antagonists of the CCR8 are available. For
example, antibodies against the receptor itself may block the
binding of ligand, thereby serving as a direct receptor
antagonist. Other antagonists may.function by blocking the
binding of ligand to receptor, e.g., by binding to the ligand
in a way to preclude the possibility of binding to the
receptor. Other antagonists, e.g., mutein antagonists, may
bind to the receptor without signaling, thereby blocking a
true agonist from binding. Many of these may serve to block
the signal transmitted to the CCRB bearing cells, specifically
Th2 cells. Thus, means are provided to block physiological
signals transduced through CCR8, which is selectively
expressed on Th2 cells.
In addition, the CCRB specific reagents are useful in
targeting CCRB+ cells, which are also TH2 cells. Thus, toxic
conjugates will selectively deplete CCRB+ cells from a
population. This may be useful in treating parasitic
27
CA 02309761 2000-OS-11
wowns~3a ~ rcrnJS9sn3~so
infections, which typically involve a cellular response (Thl
type). The deletion of Th2 cells removes the accompanying
suppression of Thl effector functions.
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 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 analyzed, e.g., by PCR analysis and
sequencing. This allows evaluation of population
polymorphisms.
All citations 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.
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 by the terms of the
appended claims, along with the full scope of equivalents to
which such claims are entitled; and the invention is not to
be limited by the specific embodiments that have been
presented herein by way of example.
28
