Note: Descriptions are shown in the official language in which they were submitted.
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Title: Use of OX-2 Inhibitors For The Treatment of Cancer
FIELD OF THE INVENTION
The present invention relates to methods and compositions for
modulating tumor growth. In particular, the invention includes the use of
inhibitors to the protein OX-2 to treat cancer.
BACKGROUND OF THE INVENTION
Interest in the field of tumor immunology has increased in the
last several years with the growing evidence that it is possible to identify,
and
immunize animals for protection with, discrete tumor antigens (15-17). A
number of groups have reported on the use of DC coated with tumor antigen,
or expressing tumor antigen (following transfection with cDNAs encoding the
antigen), as immunogens to induce tumor rejection (18-22). An alternative
approach has used tumor cells themselves, transfected with gene(s) such as
those controlling cytokines or expression of costimulatory molecules
(CD80/CD86) on the cell surface, as an immunogen (11, 23-26). In an
interesting model using as recipients animals receiving allogeneic bone
marrow transplantation (BMT), which mimicks a therapy used for treatment of
leukemia in man, Blazar et al reported that it was possible to promote a Graft-
versus-Leukemia (GVL) effect, without unwanted Graft-versus-Host Disease
(GVHD), by pre-immunizing tumor transplanted mice with tumor cells
transfected to express CD80, but not CD86 (27).
There is a need in the art to develop new immunotherapeutic
approaches to treating cancer.
SUMMARY OF THE INVENTION
The present invention provides a method of inhibiting,
preventing or reducing the growth of a tumor cell comprising administering an
effective amount of an agent that inhibits OX-2 to a cell or animal in need
thereof.
The inventors also includes a use of an agent that inhibits OX-2
to prepare a medicament to inhibit, reduce or prevent the growth of a tumor
cell.
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The invention further includes pharmaceutical compositions
containing an OX-2 inhibitor for use in inducing or augmenting an immune
response to a tumor to inhibit tumor growth.
Other features and advantages of the present invention will
become apparent from the following detailed description. It should be
understood, however, that the detailed description and the specific examples
while indicating preferred embodiments of the invention are given by way of
illustration only, since various changes and modifications within the spirit
and
scope of the invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in
which:
Figure 1 is a graph showing Inhibition of EL4 or C1498 tumor
growth in C3H bone marrow reconstituted C57BL/6 mice. Groups of 6 BL/6
mice received 20x106 T-depleted BL/6 or C3H bone marrow cells 24hrs
following cyclophosphamide treatment. 5x106 EL4 or 5x105 C1498 tumor cells
were injected 28 days later into these mice, and control BL/6 or C3H mice.
>85% of PBL from C3H reconstituted BL/6 were stained by FITC anti-H2Kk
mAb at this time.
Figure 2 is a graph showing EL4 tumor growth in BL/6 mice
immunized twice, at 14 day intervals, with 5x106 EL4 cells transfected to
express CD80 or CD86. 5x106 EL4 cells were injected as tumor challenge 10
days after the last immunization .
Figure 3 is a graph showing suppression of growth inhibition in
C57BL/6 BMT recipients of EL4 or C1498 tumor cells (see Figure 19)
following 4 weekly infusions of 100 g/mouse anti-CD4 or anti-CD8 mAb,
beginning on the day of BMT (tumor cells were injected at 28 days post BMT).
Data are shown for 6 mice/group.
Figure 4 is a graph showing inhibition of immunity to EL4 or
C1498 tumor challenge following infusion of CD200Fc in C57BL/6 mice
reconstituted with C3H bone marrow -see Figure 19 and text for more details.
Cyclophosphamide treated BL/6 mice received bone marrow rescue with T-
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depleted C3H or BL/6 cells. 28 days later all mice, and groups of control
normal C3H mice, received ip injection with 5x106 EL4 or 5x105 C1498 tumor
cells. Bone marrow reconstituted mice received further iv infusion of normal
mouse IgG or CD200Fc (10 g/mouse/injection) 5 times at 2 day intervals
beginning on the day of tumor injection.
Figure 5 is a graph showing inhibition of immunity to EL4 tumor
cells in EL4-CD80 immunized BL/6 mice using CD200Fc-see Figure 2 and
text for details. Mice received iv infusion of control IgG or CD200Fc as
described in Figure 4.
Figure 6 is a graph showing improved tumor immunity in EL4-
CD86 or C1498-CD86 immunized C57BL/6 mice following infusion of anti-
CD200 mAb. See legend to Figure 2 and text for more details. Where shown,
groups of mice received iv. infusion of anti-CD200, 100mg/mouse, on 3
occasions at 3 day intervals beginning on the day of tumor injection.
Figure 7 is a graph showing Iog10 relative concentrations of
CD200 mRNAs compared with standardized control mRNA-see Materials and
Methods for technique used for quantitation. All samples were first normalized
for equivalent concentrations of GAPDH mRNA. Values shown represent
arithmetic means SD for 3 individual samples for each time point. Mice were
preimmunized with CD80/CD86-transfected tumor cells as described in the
text. *, p<0.02 compared with untreated control, or mice immunized with EL4
or EL4-transfected with CD80.
Figure 8 is a graph showing increased inhibition of tumor
immunity using infusion of CD200Fc with CD200r+ cells in C57BL/6 recipients
of C3H bone marrow -see text and Materials and Methods for more details. In
this experiment some mice received not only CD200Fc with EL4 or C1498
tumor, but in addition a lymphocyte-depleted, LPS-stimulated, macrophage
population stained (>65%) with anti-CD200r mAb (2F9).
Figure 9 is a graph showing combinations of CD200Fc and anti-
CD4 or anti-CD8 mAb produce increased suppression of tumor growth
inhibition in C57BL/6 recipients of C3H BMT. Groups of 6 mice received
weekly iv infusions of 100 g anti-T cell mab or 5 iv infusions of 10 g/mouse
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CD200Fc, alone or in combination, beginning on the day of tumor injection
(28 days post BMT).
Figure 10 is a graph showing effect of combined CD200Fc and
anti-CD4 or anti-CD8 mAb on suppression of EL4 tumor growth inhibition in
C57BL/6 recipients preimmunized with EL4-CD80 transfected cells (see
Figure 20). Data are shown for groups of 6 mice/group. Weekly iv infusions of
100 g anti-T cell mab or 5 iv infusions of 10 g/mouse CD200Fc, alone or in
combination, were begun on the day of tumor injection (10 days after the final
immunization with EL4-CD80 cells).
Figures 11A and B are bar graphs showing the median number
of lung nodules in mice receiving allogeneic blood by tail vein.
Figures 12A and B are bar graphs showing the number of lung
nodules in the presence of anti-OX2 in mice receiving allogeneic blood by tail
vein.
Figures 13A and B are bar graphs showing the number of lung
nodules in the presence of anti-OX-2, DEC205 or anti-CD11c in mice
receiving allogeneic blood by tail vein.
Figure 14 shows the cDNA sequence of rat (SEQ.ID.NO.:1),
mouse (SEQ.ID.NO.:3) and human MRC OX-2 (SEQ.ID.NO.:5).
Figure 15 shows the deduced protein sequence of rat
(SEQ.ID.NO.:2), mouse (SEQ.ID.NO.:4) and human MRC OX-2
(SEQ.ID.NO.:6) protein.
DETAILED DESCRIPTION OF THE INVENTION
(a) Preventing Tumor Growth
The present inventors have previously disclosed that OX-2 (or
CD200) plays a role in the development of immune suppression or tolerance
and may be useful in developing therapies for the prevention and treatment of
transplant rejection, fetal loss, autoimmune disease or allergies (WO
99/24565).
The inventors have now shown that OX-2 promotes tumor cell
growth and inhibiting OX-2 inhibits tumor cell growth.
Accordingly, in one aspect, the present invention provides a
method of inhibiting tumor cell growth comprising administering an effective
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amount of an agent that inhibits OX-2 to an animal in need thereof. The
invention also provides a use of an effective amount of an agent that inhibits
OX-2 to inhibit tumor growth or to prepare a medicament to inhibit tumor cell
growth.
The term "effective amount" as used herein means an amount
effective, at dosages and for periods of time necessary to achieve the desired
result (e.g. to inhibit tumor growth).
The term "animal" includes all members of the animal kingdom
and is preferably a mammal, more preferably a human.
The term "OX-2 protein" includes OX-2 or CD200 from any
species or source and includes a full length OX-2 protein as well as fragments
or portions of the protein. The term "OX-2" is also generally referred to as
"CD200" due to a change in nomenclature. Both "OX-2" and "CD200" may be
used interchangeably in the application.
The agent that inhibits OX-2 can be any agent that decreases
the expression or activity of an OX-2 protein such that the immune
suppression caused by OX-2 is reduced, inhibited and/or prevented. Such
agents can be selected from agents that inhibit OX-2 activity (such as
antibodies, OX-2 ligands, small molecules), agents that inhibit OX-2
expression (such as antisense molecules) or agents that inhibit the
interaction
of OX-2 with its receptor (such as soluble OX-2 receptor and antibodies that
bind the OX-2 receptor).
One of skill in the art can readily determine whether or not a
particular agent is effective in inhibiting OX-2. For example, the agent can
be
tested in in vitro assays to determine if the function or activity of OX-2 is
inhibited. The agent can also be tested for its ability to induce an immune
response using in vitro immune assays including, but not limited to, enhancing
a cytotoxic T cell response; inducing interleukin-2 (IL-2) production;
inducing
IFNy production; inducing a Thl cytokine profile; inhibiting IL-4 production;
inhibiting TGFI3 production; inhibiting IL-10 production; inhibiting a Th2
cytokine profile and any other assay that would be known to one of skill in
the
art to be useful in detecting immune activation.
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One of skill in the art can determine whether a particular agent
is useful in inhibiting tumor cell growth. As mentioned above, one can test
the
agent for its ability to induce an immune response using known in vitro
assays. In addition, the agent can be tested in an animal model, for example
as described in Examples 1 and 2, wherein the agent is administered to an
animal with cancer.
The term "inhibiting or reducing tumor cell growth" means that
the agent that inhibits OX-2 causes an inhibition or reduction in the growth
or
metastasis of a tumor as compared to the growth observed in the absence of
the agent. The agent may also be used prophylactically to prevent the growth
of tumor cells.
The tumor cell can be any type of cancer including, but not
limited to, hematopoietic cell cancers (including leukemias and lymphomas),
colon cancer, lung cancer, kidney cancer, pancreas cancer, endometrial
cancer, thyroid cancer, oral cancer, laryngeal cancer, hepatocellular cancer,
bile duct cancer, squamous cell carcinoma, prostate cancer, breast cancer,
cervical cancer, colorectal cancer, melanomas and any other tumors which
are antigenic or weakly antigenic. This could include, for example, EBV-
induced neoplasms, and neoplasms occurring in immunosuppressed pateints,
e.g. transplant patients, AIDS patients, etc.
(i) Antibodies
In a preferred embodiment, the agent that inhibits OX-2 is an
OX-2 specific antibody. The present inventors have prepared antibodies to
OX-2 which are described in WO 99/24565. Antibodies to OX-2 may also be
obtained commercially or prepared using techniques known in the art such as
those described by Kohler and Milstein, Nature 256, 495 (1975) and in U.S.
Patent Nos. RE 32,011; 4,902,614; 4,543,439; and 4,411,993. (See also
Monoclonal Antibodies, Hybridomas: A New Dimension in Biological
Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and
Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring
Harbor Laboratory Press, 1988).
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Conventional methods can be used to prepare the antibodies.
For example, by using the OX-2 protein, polyclonal antisera or monoclonal
antibodies can be made using standard methods. A mammal, (e.g., a mouse,
hamster, or rabbit) can be immunized with an immunogenic form of the OX 2
receptor which elicits an antibody response in the mammal. Techniques for
conferring immunogenicity on a peptide include conjugation to carriers or
other techniques well known in the art. For example, the peptide can be
administered in the presence of adjuvant. The progress of immunization can
be monitored by detection of antibody titers in plasma or serum. Standard
ELISA or other immunoassay procedures can be used with the immunogen
as antigen to assess the levels of antibodies. Following immunization,
antisera can be obtained and, if desired, polyclonal antibodies isolated from
the sera.
To produce monoclonal antibodies, antibody producing cells
(lymphocytes) can be harvested from an immunized animal and fused with
myeloma cells by standard somatic cell fusion procedures thus immortalizing
these cells and yielding hybridoma cells. Such techniques are well known in
the art, (e.g., the hybridoma technique originally developed by Kohler and
Milstein (Nature 256, 495-497 (1975)) as well as other techniques such as the
human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72
(1983)); the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy (1985) Allen
R. Bliss, Inc., pages 77-96); and screening of combinatorial antibody
libraries
(Huse et al., Science 246, 1275 (1989)). Hybridoma cells can be screened
immunochemically for production of antibodies specifically reactive with the
OX-2 receptor and the monoclonal antibodies can be isolated. Therefore, the
invention also contemplates hybridoma cells secreting monoclonal antibodies
with specificity for OX-2.
The term "antibody" as used herein is intended to include
fragments thereof which also specifically react with OX-2 or a peptide
thereof.
Antibodies can be fragmented using conventional techniques and the
fragments screened for utility in the same manner as described above. For
example, F(ab)2 fragments can be generated by treating antibody with
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pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide
bridges to produce Fab' fragments.
Chimeric antibody derivatives, i.e., antibody molecules that
combine a non-human animal variable region and a human constant region
are also contemplated within the scope of the invention. Chimeric antibody
molecules can include, for example, the antigen binding domain from an
antibody of a mouse, rat, or other species, with human constant regions.
Conventional methods may be used to make chimeric antibodies containing
the immunoglobulin variable region which recognizes an OX-2 receptor (See,
for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81,6851 (1985);
Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S. Patent No.
4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European
Patent Publication EP171496; European Patent Publication 0173494, United
Kingdom patent GB 2177096B).
Monoclonal or chimeric antibodies specifically reactive with the
OX-2 as described herein can be further humanized by producing human
constant region chimeras, in which parts of the variable regions, particularly
the conserved framework regions of the antigen-binding domain, are of
human origin and only the hypervariable regions are of non human origin.
Such immunoglobulin molecules may be made by techniques known in the art
(e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983);
Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth.
Enzymol., 92, 3-16 (1982); and PCT Publication WO 92/06193 or EP
0239400). Humanized antibodies can also be commercially produced
(Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.)
Specific antibodies, or antibody fragments reactive against OX-2
may also be generated by screening expression libraries encoding
immunoglobulin genes, or portions thereof, expressed in bacteria with
peptides produced from nucleic acid molecules of the present invention. For
example, complete Fab fragments, VH regions and FV regions can be
expressed in bacteria using phage expression libraries (See for example
Ward et al., Nature 341, 544-546: (1989); Huse et at., Science 246, 1275-
1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990)).
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Accordingly, the present invention provides a method of
inhibiting, preventing or reducing tumor cell growth comprising administering
an effective amount of an antibody that inhibits OX-2 to an animal in need
thereof.
(ii) Antisense Oligonucleotides
In another embodiment, the OX-2 inhibitor is an antisense
oligonucleotide that inhibits the expression of OX-2. Antisense
oligonucleotides that are complimentary to a nucleic acid sequence from an
OX-2 gene can be used in the methods of the present invention to inhibit OX-
2. The present inventors have prepared antisense oligonucleotides to OX-2
which are described in WO 99/24565.
Accordingly, the present invention provides a method of
inhibiting, preventing or reducing tumor cell growth comprising administering
an effective amount of an antisense oligonucleotide that is complimentary to a
nucleic acid sequence from a OX-2 gene to an animal in need thereof.
The term "antisense oligonucleotide" as used herein means a
nucleotide sequence that is complimentary to its target, the sense strand of
messenger RNA that is translated into protein at the ribosomal level.
In one embodiment of the invention, the present invention
provides an antisense oligonucleotide that is complimentary to a nucleic acid
molecule having a sequence as shown in Figure 14 (SEQ.ID.NO.:1, 3 or 5),
wherein T can also be U, or a fragment thereof.
The term "oligonucleotide" refers to an oligomer or polymer of
nucleotide or nucleoside monomers consisting of naturally occurring bases,
sugars, and intersugar (backbone) linkages. The term also includes modified
or substituted oligomers comprising non-naturally occurring monomers or
portions thereof, which function similarly. Such modified or substituted
oligonucleotides may be preferred over naturally occurring forms because of
properties such as enhanced cellular uptake, or increased stability in the
presence of nucleases. The term also includes chimeric oligonucleotides
which contain two or more chemically distinct regions. For example, chimeric
oligonucleotides may contain at least one region of modified nucleotides that
confer beneficial properties (e.g. increased nuclease resistance, increased
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uptake into cells), or two or more oligonucleotides of the invention may be
joined to form a chimeric oligonucleotide.
The antisense oligonucleotides of the present invention may be
ribonucleic or deoxyribonucleic acids and may contain naturally occurring
bases including adenine, guanine, cytosine, thymidine and uracil. The
oligonucleotides may also contain modified bases such as xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines,
5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza
thymine,
pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine,
8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines,
8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines,
8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza
uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl
uracil
and 5-trifluoro cytosine.
Other antisense oligonucleotides of the invention may contain
modified phosphorous, oxygen heteroatoms in the phosphate backbone,
short chain alkyl or cycloalkyl intersugar linkages or short chain
heteroatomic
or heterocyclic intersugar linkages. For example, the antisense
oligonucleotides may contain phosphorothioates, phosphotriesters, methyl
phosphonates, and phosphorodithioates. In an embodiment of the invention
there are phosphorothioate bonds links between the four to six 3'-terminus
bases. In another embodiment phosphorothioate bonds link all the
nucleotides.
The antisense oligonucleotides of the invention may also
comprise nucleotide analogs that may be better suited as therapeutic or
experimental reagents. An example of an oligonucleotide analogue is a
peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate
backbone in the DNA (or RNA), is replaced with a polyamide backbone which
is similar to that found in peptides (P.E. Nielsen, et al Science 1991, 254,
1497). PNA analogues have been shown to be resistant to degradation by
enzymes and to have extended lives in vivo and in vitro. PNAs also bind
stronger to a complimentary DNA sequence due to the lack of charge
repulsion between the PNA strand and the DNA strand. Other
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oligonucleotides may contain nucleotides containing polymer backbones,
cyclic backbones, or acyclic backbones. For example, the nucleotides may
have morpholino backbone structures (U.S. Pat. No. 5,034,506).
Oligonucleotides may also contain groups such as reporter groups, a group
for improving the pharmacokinetic properties of an oligonucleotide, or a group
for improving the pharmacodynamic properties of an antisense
oligonucleotide. Antisense oligonucleotides may also have sugar mimetics.
The antisense nucleic acid molecules may be constructed using
chemical synthesis and enzymatic ligation reactions using procedures known
in the art. The antisense nucleic acid molecules of the invention or a
fragment thereof, may be chemically synthesized using naturally occurring
nucleotides or variously modified nucleotides designed to increase the
biological stability of the molecules or to increase the physical stability of
the
duplex formed with mRNA or the native gene e.g. phosphorothioate
derivatives and acridine substituted nucleotides. The antisense sequences
may be produced biologically using an expression vector introduced into cells
in the form of a recombinant plasmid, phagemid or attenuated virus in which
antisense sequences are produced under the control of a high efficiency
regulatory region, the activity of which may be determined by the cell type
into
which the vector is introduced.
(iii) Other OX-2 Inhibitors
In addition to antibodies and antisense molecules, other agents
that inhibit OX-2 may also be used in the present invention.
Accordingly, the present invention also includes the isolation of
other ligands or molecules that can bind to OX-2 or the OX-2 receptor.
Biological samples and commercially available libraries may be tested for
proteins that bind to OX-2 or the OX-2 receptor. In addition, antibodies
prepared to the OX-2 or the OX-2 receptor may be used to isolate other
peptides with OX-2 or OX-2 receptor binding affinity. For example, labelled
antibodies may be used to probe phage displays libraries or biological
samples.
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Conditions which permit the formation of protein complexes may
be selected having regard to factors such as the nature and amounts of the
substance and the protein.
The substance-protein complex, free substance or non-
complexed proteins may be isolated by conventional isolation techniques, for
example, salting out, chromatography, electrophoresis, gel filtration,
fractionation, absorption, polyacrylamide gel electrophoresis, agglutination,
or
combinations thereof. To facilitate the assay of the components, the
antibodies, proteins, or substances may be labelled with a detectable
substance.
Once potential binding partners have been isolated, screening
methods may be designed in order to determine if the molecules that bind to
the OX-2 peptide or OX-2 receptor and are useful in the methods of the
present invention.
Therefore, the invention also provides methods for identifying
substances which are capable of binding to the OX-2. In particular, the
methods may be used to identify substances which are capable of binding to
and which suppress the effects of OX-2. Accordingly the invention provides a
method of identifying substances which bind with OX-2, comprising the steps
of:
(a) reacting OX-2 and a substance, under conditions which
allow for formation of a complex, and
(b) assaying for complexes, for free substance, and for non
complexed OX-2.
Substances which can bind with the OX-2 of the invention may
be identified by reacting OX-2 with a substance which potentially binds to the
OX-2, and assaying for complexes, for free substance, or for non-complexed
OX-2. Any assay system or testing method that detects protein-protein
interactions may be used including co-immunoprecipitation, crosslinking and
co-purification through gradients or chromatographic columns may be used.
Additionally, x-ray crystallographic studies may be used as a means of
evaluating interactions with substances and molecules. For example, purified
recombinant molecules in a complex of the invention when crystallized in a
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suitable form are amenable to detection of intra-molecular interactions by x-
ray crystallography. Spectroscopy may also be used to detect interactions
and in particular, Q-TOF instrumentation may be used. Biological samples
and commercially available libraries may be tested for OX-2-binding peptides.
In addition, antibodies prepared to the peptides of the invention may be used
to isolate other peptides with OX-2 binding affinity. For example, labelled
antibodies may be used to probe phage display libraries or biological
samples. In this respect peptides of the invention may be developed using a
biological expression system. The use of these systems allows the
production of large libraries of random peptide sequences and the screening
of these libraries for peptide sequences that bind to particular proteins.
Libraries may be produced by cloning synthetic DNA that encodes random
peptide sequences into appropriate expression vectors. (see Christian et al.
1992, J. Mol. Biol. 227:711; Devlin et al., 1990 Science 249:404; Cwirla et
al.
1990, Proc. Natl. Acad, Sci. USA, 87:6378). Libraries may also be
constructed by concurrent synthesis of overlapping peptides (see U.S. Pat.
No. 4,708,871).
It will be understood that the agonist and antagonist that can be
assayed using the methods of the invention may act on one or more of the
binding sites on the protein or substance including agonist binding sites,
competitive antagonist binding sites, non-competitive antagonist binding sites
or allosteric sites.
The invention also makes it possible to screen for antagonists
that inhibit the effects of an agonist of the interaction of OX-2 with a
substance which is capable of binding to OX-2. Thus, the invention may be
used to assay for a substance that competes for the same binding site of OX-
2. As such it will also be appreciated that intracellular substances which are
capable of binding to OX-2 may be identified using the methods described
herein.
The reagents suitable for applying the methods of the invention
to evaluate substances and compounds that affect or modulate a OX-2 may
be packaged into convenient kits providing the necessary materials packaged
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into suitable containers. The kits may also include suitable supports useful
in
performing the methods of the invention.
(b) Inducing Tumor Cell Growth
In another aspect, the present invention provides a method of
inducing tumor cell growth or metastasis comprising administering an
effective amount of an OX-2 protein or fragment thereof or a nucleic acid
sequence encoding an OX-2 protein or fragment thereof to an animal in need
of such treatment. The invention includes a use of an effective amount of an
OX-2 protein or fragment thereof or a nucleic acid sequence encoding an OX-
2 protein or fragment thereof to induce tumor cell growth or metastasis. The
method of inducing tumor growth can be used to study tumor cell growth or
metastasis. The method can also be used to develop an animal model to
study or test cancer therapies or chemotherapeutic agents. The method is
generally conducted on non-human animals.
The OX-2 protein or nucleic acid encoding the OX-2 protein for
use in the method can be obtained from any species or source. The nucleic
acid sequence and amino acid sequence of an OX-2 protein from human,
mouse and rat are shown in Figures 14 and 15 and in SEQ.ID.NOs.1-6.
Preferred fragments or portions of the OX-2 or CD200 protein are those that
are sufficient to suppress an immune response. Determining whether a
particular OX-2 or CD200 protein can suppress an immune response and
result in increased tumor growth can be assessed using known in vitro
immune assays including, but not limited to, inhibiting a mixed leucocyte
reaction; inhibiting a cytotoxic T cell response; inhibiting interleukin-2
production; inhibiting IFNy production; inhibiting a Thl cytokine profile;
inducing IL-4 production; inducing TGFfi production; inducing IL-10
production; inducing a Th2 cytokine profile; and any other assay that would
be known to one of skill in the art to be useful in detecting immune
suppression.
The term "administering an OX-2 protein" includes both the
administration of the OX-2 protein as well as the administration of a nucleic
acid sequence encoding an OX-2 protein. In the latter case, the OX-2 protein
is produced in vivo in the animal.
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In a preferred embodiment, the OX-2 protein is prepared and
administered as a soluble fusion protein. The fusion protein may contain the
extracellular domain of OX-2 linked to an immunoglobulin (Ig) Fc Region. The
OX-2 fusion may be prepared using techniques known in the art. Generally, a
DNA sequence encoding the extracellular domain of OX-2 is linked to a DNA
sequence encoding the Fc of the Ig and expressed in an appropriate
expression system where the OX-2 - FcIg fusion protein is produced.
The OX-2 or protein may be obtained from known sources or
prepared using recombinant DNA techniques. The protein may have any of
the known published sequences for OX-2 or CD200. The sequences can be
obtained from GenBank. The human sequence has accession no. M17226
X0523; the rat sequence has accession no. X01785; and the mouse
sequence has accession no. AF029214. The nucleic acid and protein
sequences of OX-2 (CD200) from human, mouse and rat are also shown in
SEQ.ID.Nos.: 1, 3 and 5 (nucleic acid) and SEQ.ID.Nos.:2, 4 and 6 (protein).
The OX-2 protein may also be modified to contain amino acid
substitutions, insertions and/or deletions that do not alter the
immunosuppressive properties of the protein. Conserved amino acid
substitutions involve replacing one or more amino acids of the OX-2 amino
acid sequence with amino acids of similar charge, size, and/or hydrophobicity
characteristics. When only conserved substitutions are made the resulting
analog should be functionally equivalent to the OX-2 protein. Non-conserved
substitutions involve replacing one or more amino acids of the OX-2 amino
acid sequence with one or more amino acids which possess dissimilar
charge, size, and/or hydrophobicity characteristics.
The OX-2 protein may be modified to make it more
therapeutically effective or suitable. For example, the OX-2 protein may be
cyclized as cyclization allows a peptide to assume a more favourable
conformation. Cyclization of the OX-2 peptides may be achieved using
techniques known in the art. In particular, disulphide bonds may be formed
between two appropriately spaced components having free sulfhydryl groups.
The bonds may be formed between side chains of amino acids, non-amino
acid components or a combination of the two. In addition, the OX-2 protein or
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peptides of the present invention may be converted into pharmaceutical salts
by reacting with inorganic acids including hydrochloric acid, sulphuric acid,
hydrobromic acid, phosphoric acid, etc., or organic acids including formic
acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid,
oxalic
acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid,
salicylic
acid, benzenesulphonic acid, and tolunesulphonic acids.
Administration of an "effective amount" of the OX-2 protein and
nucleic acid of the present invention is defined as an amount effective, at
dosages and for periods of time necessary to achieve the desired result. The
effective amount of the OX-2 protein or nucleic acid of the invention may vary
according to factors such as the disease state, age, sex, and weight of the
animal. Dosage regima may be adjusted to provide the optimum therapeutic
response. For example, several divided doses may be administered daily or
the dose may be proportionally reduced as indicated by the exigencies of the
therapeutic situation.
(c) Compositions
The invention also includes pharmaceutical compositions
containing OX-2 proteins or nucleic acids for use in inducing tumor cell
growth
as well as pharmaceutical compositions containing an OX-2 inhibitor for use
in preventing tumor cell growth.
Such pharmaceutical compositions can be for intralesional,
intravenous, topical, rectal, parenteral, local, inhalant or subcutaneous,
intradermal, intramuscular, intrathecal, transperitoneal, oral, and
intracerebral
use. The composition can be in liquid, solid or semisolid form, for example
pills, tablets, creams, gelatin capsules, capsules, suppositories, soft
gelatin
capsules, gels, membranes, tubelets, solutions or suspensions.
The pharmaceutical compositions of the invention can be
intended for administration to humans or animals. Dosages to be
administered depend on individual needs, on the desired effect and on the
chosen route of administration.
The pharmaceutical compositions can be prepared by per se
known methods for the preparation of pharmaceutically acceptable
compositions which can be administered to patients, and such that an
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effective quantity of the active substance is combined in a mixture with a
pharmaceutically acceptable vehicle. Suitable vehicles are described, for
example, in Remington's Pharmaceutical Sciences (Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA
1985).
On this basis, the pharmaceutical compositions include, albeit
not exclusively, the active compound or substance in association with one or
more pharmaceutically acceptable vehicles or diluents, and contained in
buffered solutions with a suitable pH and iso-osmotic with the physiological
fluids. The pharmaceutical compositions may additionally contain other
agents such as immunosuppressive drugs or antibodies to enhance immune
tolerance or immunostimulatory agents to enhance the immune response.
In one aspect, the pharmaceutical composition for use in
inhibiting tumor cell growth comprises an effective amount of a OX-2 inhibitor
in admixture with a pharmaceutically acceptable diluent or carrier. Such
compositions may be administered as a vaccine either alone or in
combination with other active agents.
In one embodiment, the pharmaceutical composition for use in
inhibiting tumor cell growth comprises an effective amount of an antibody to
OX-2 in admixture with a pharmaceutically acceptable diluent or carrier. The
antibodies may be delivered intravenously.
In another embodiment, the pharmaceutical composition for use
in inhibiting tumor cell growth comprises an effective amount of an antisense
oligonucleotide nucleic acid complimentary to a nucleic acid sequence from a
OX-2 gene in admixture with a pharmaceutically acceptable diluent or carrier.
The oligonucleotide molecules may be administered as described below for
the compositions containing OX-2 nucleic acid sequences.
When used in inhibiting tumor cell growth or in treating cancer
the composition can additionally contain other agents such as other immune
stimulants (including cytokines and adjuvants) as well as chemotherapeutic
agents.
In another embodiment, the pharmaceutical composition for use
in inducing tumor cell growth comprises an effective amount of a OX-2 protein
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in admixture with a pharmaceutically acceptable diluent or carrier. - The OX-2
protein is preferably prepared as an immunoadhesion molecule in soluble
form which can be administered to the patient.
In another embodiment, the pharmaceutical composition for use
in inducing tumor cell growth comprises an effective amount of a nucleic acid
molecule encoding a OX-2 protein in admixture with a pharmaceutically
acceptable diluent or carrier.
The nucleic acid molecules of the invention encoding a OX-2
protein may be used in gene therapy to induce inducing tumor cell growth. In
addition antisense oligonucleotides to OX-2 may be used in gene therapy to
prevent or inhibit tumor cell growth. Recombinant molecules comprising a
nucleic acid sequence encoding a OX-2 protein, or fragment thereof, may be
directly introduced into cells or tissues in vivo using delivery vehicles such
as
retroviral vectors, adenoviral vectors and DNA virus vectors. They may also
be introduced into cells in vivo using physical techniques such as
microinjection and electroporation or chemical methods such as
coprecipitation and incorporation of DNA into liposomes. Recombinant
molecules may also be delivered in the form of an aerosol or by lavage. The
nucleic acid molecules of the invention may also be applied extracellularly
such as by direct injection into cells. The nucleic acid molecules encoding
OX-2 are preferably prepared as a fusion with a nucleic acid molecule
encoding an immunoglobulin (Ig) Fc region. As such, the OX-2 protein will be
expressed in vivo as a soluble fusion protein.
The following non-limiting examples are illustrative of the
present invention:
EXAMPLES
EXAMPLE 1
Evidence of a role for CD200 in regulation of immune rejection of
leukemic tumor cells in C56BL16 mice
MATERIALS and METHODS
Mice: Male C3H/HeJ, BALB/c and C57BL/6 mice were purchased from the
Jackson laboratories, Bar Harbour, Maine. Mice were housed 5/cage and
allowed food and water ad libitum. All mice were used at 8-12 weeks of age.
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Monoclonal antibodies: The following monoclonal antibodies (mAbs) were
obtained from Pharmingen (San Diego, CA, USA) unless stated otherwise:
anti-IL-2 (S4B6, ATCC; biotinylated JES6-5H4); anti-IL-4 (11 B11, ATCC;
biotinylated BVD6-24G2); anti-IFNy (R4-6A2, ATCC; biotinylated XMG1.2);
anti-IL-10 (JES5-2A5; biotinylated, SXC-1); anti-IL-6 (MP5-20F3; biotinylated
MP5-32C11); anti-TNFa (G281-2626; biotinylated MP6-XT3); FITC anti-
CD80, FITC anti-CD86 and FITC anti-CD40 were obtained from Cedarlane
Labs, Hornby, Ontario. The hybridoma producing DEC205 (anti-mouse
dendritic cells) was a kind gift from Dr.R.Steinman, and was directly labeled
with FITC. FITC anti-H2Kb, FITC anti-H2Kk, and anti-thyl.2 monoclonal
antibodies (mAbs) were obtained from Cedarlane Labs, Hornby, Ontario.
Unconjugated and PE-conjugated rat anti-mouse CD200 was obtained from
BioSpark Inc., Mississauga, Ontario, Canada (28). CD200Fc was prepared in
a Baculovirus expression system, using a cDNA encoding a murine IgG2aFc
region (a kind gift from Dr. T Strom, Harvard, USA) which carried mutations to
delete complement binding and FcR sites, as we described elsewhere (5).
Rat monoclonal antibody to CD200r was prepared from rats immunized with
CHO cells transfected to express a cDNA encoding CD200r (29). Anti-CD4
(GK1.5, rat IgG2b) and anti-CD8 (2.43, rat IgG2b) were both obtained from
ATCC, and used for in vivo depleteion by iv infusion of 100 g Ig/mouse
weekly. A control IgG2b antibody (R35.38), as well as strepavidin horse
radish peroxidase and recombinant mouse GM-CSF, was purchased from
Pharmingen (San Diego, CA).
Preparation of cells: Single cell spleen suspensions were prepared aseptically
and after centrifugation cells were resuspended in a-Minimal Essential
Medium supplemented with 2-mercaptoethanol and 10% fetal calf serum
((xF10). CD200r+ LPS splenic Mph, stained (>20%) with FITC-CD200Fc, were
obtained by velocity sedimentation of cells cultured for 48 hrs with 1 g/mI
LPS (13). Bone marrow cells were flushed from the femurs of donor mice,
washed and resuspended in aF10. Cells were depleted of mature T
lymphocytes using anti-thyl.2 and rabbit complement.
C1498 (a spontaneous myeloid tumor) and EL4 (a radiation
induced thymoma tumor) cells were obtained from The American Type
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Culture Collection (ATCC, Rockville, MD). Cells used for transplantation into
mice were passaged weekly (5x106 cells/mouse) intraperitoneally in stock 8-
week old C57BL/6 recipients. For experimental tumor challenge either 5x106
EL4 tumor cells, or 5x105 C1498 cells, were given intraperitoneally to groups
of 6 mice (see results)-animals were sacrificed when they became moribund.
EL4 cells stably transfected to express CD80 or CD86 were obtained from
Dr.J.Allison, Cancer Research Labs, UC Berkeley, CA, while C1498
transfected with CD80/CD86 (cloned into pBK vectors) were produced in the
author's laboratory. Tumor cells (parent and transfected) were stored at
-80 C and thawed and cultured prior to use. Cells used for immunization,
including the tumor cells transfected with CD80/CD86, were maintained in
culture in aMEM medium supplemented with 10% FCS. Untransfected and
transfected cells of each tumor line were used for immunization within 2
passages in culture. Over this time in culture transfected cells repeatedly
showed stable expression (by FACS) of CD80/CD86 (>80% positive for each
tumor assayed over a 6 month period with multiple vials thawed and cultured).
Non-transfected tumor cells did not stain with these mAbs (<2%).
CD200r+ cells were obtained from lymphocyte-depleted murine
spleen cells. Cells were treated with rabbit anti-mouse lymphocyte serum and
complement (both obtained from Cedarlane Labs. Hornby, Ontario), cultured
with LPS (10 g/ml) for 24 hours, and separated into populations of different
size by velocity sedimentation (13). Small CD200r+ cells stained >65% by
FACS with anti-CD200r antibody (29).
Bone marrow transplantation (BMT): C57BL/6 mice received 300mg/Kg
cyclophosphamide iv 24 hrs before intravenous infusion of 20x1 06 T-depleted
C3H or C57BL/6 bone marrow cells. Immediately prior to use for tumor
transplantation (28 days following bone marrow engrafting), a sample of PBL
(50 I/mouse) was obtained from the tail vein of individual mice and analysed
by FACS with FITC-anti-H2Kk or FITC-anti-H2Kb mAb. Cells from normal
C57BL/6 or C57BL/6 reconsituted C57BL/6 mice were 100% H2Kb positive,
as expected. In similar fashion, PBL from C3H mice were 100% H2Kk
positive. H2Kk positive cells in the C31-1-reconstituted C57BL/6 mice by FACS
comprised 85% 8.5% of the total cell population (mean over - 100 mice used
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in the studies described below). Mice in all groups were gaining weight and
healthy.
Cytotoxicity and cytokine assays:
In allogeneic mixed leukocyte cultures (MLC) used to assess
cytokine production or CTL, responder spleen cells were stimulated with
equal numbers of mitomycin-C treated (45 min at 37 C) spleen stimulator
cells in triplicate in a1=10. Supernatants were pooled at 40hr from replicate
wells and assayed in triplicate in ELISA assays for lymphokine production as
follows, using capture and biotinylated detection mAbs as described above.
Varying volumes of supernatant were bound in triplicate at 4 C to plates pre-
coated with 100ng/ml mAb, washed x3, and biotinylated detection antibody
added. After washing, plates were incubated with strepavidin-horse radish
peroxidase (Cedarlane Labs), developed with appropriate substrate and
OD405 determined using an ELISA plate reader. Recombinant cytokines for
standardization were obtained from Pharmingen (U.S.A.). All assays showed
sensitivity in the range 40 to 4000 pg/ml. CTL assays were performed at 5
days using cells harvested from the same cultures (as used for cytokine
assays). Various effector:target ratios were used in 4hr 51Cr release tests
with
72 hr ConA activated spleen cell blasts of stimulator genotype.
Quantitation of CD200 mRNA by PCR:
RNA extraction from spleen tissue of tumor injected mice was
performed using TrizolTM reagent. The OD280/260 of each sample was
measured and reverse transcription performed using oligo (dT) primers (27-
7858: Pharmacia, USA). cDNA was diluted to a total volume of 100 I with
water and frozen at -70 C until use in PCR reactions with primers for mouse
CD200 and GAPDH (3). Different amounts of standard cDNA from 24hr
cultures of LPS stimulated peritoneal macrophages (known to express CD200
and GAPDH) were amplified in six serial 1:10 dilutions for 30 cycles by PCR,
in the presence of a tracer amount of 32P. Samples were analysed in 12.5%
polyacrylamide gels, the amplicons cut from the gel, and radioactivity
measured in a 13-counter. A standard curve was drawn for each set of primer
pairs (amplicons). cDNAs from the various experimental groups were assayed
in similar reactions using 0.1 l cDNA, and all groups were normalized to
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equivalent amounts of GAPDH. CD200 cDNA levels in the different
experimental groups were then expressed relative to the cDNA standard
(giving a detectable 32P signal over five loglo dilutions). Thus a value of 5
(serial dilutions) indicates a test sample with approximately the same cDNA
content as the standard, while a value of 0 indicates a test sample giving no
detectable signal in an undiluted form (<1/105 the cDNA concentration of the
standard).
RESULTS
Growth of EL4 or C1498 tumor cells in C57BU6 mice, and in allogeneic (C3H)
BMTmice:
Groups of 6 C57BL/6 mice received iv infusion of 300mg/Kg
cyclophosphamide (in 0.5 ml PBS). A control group received PBS only, as did
a control group of 6 C3H mice. 24 hrs later cyclophosphamide treated
C57BL/6 mice received iv injection of 20x1 06 T-depleted bone marrow cells
pooled from C57BL/6 mice (syngeneic transplant), or C3H mice (allogeneic
transplantation). All groups of animals received intraperitoneal injection (in
0.5
ml PBS) of 5x106 EL4 or 5x105 C1498 tumor cells (see Figure 1) 28 days
later. Animals were monitored daily post tumor inoculation.
Data in Figure 1 (one of 2 such studies) show clearly that while
C3H mice rejected both EL4 and C1498 (allogeneic) leukemia cell growth,
100% mortality was seen within 9-12 days in normal C57BL/6 mice, or in
syngeneic bone marrow reconstituted mice. Interestingly, despite the absence
of overt GVHD (as defined by weight loss and overall health), two-thirds of
C3H reconstituted C57BL/6 mice rejected EL4 tumor cells, reflecting the
existence of a graft versus leukemia effect (GVL) (panel a of Figure 1), and
there was a marked delay of death for mice inoculated with C1498 leukemia
cells (panel b of Figure). In separate studies similar findings were made
using
tumor inocula (for EL4/C1498 respectively) ranging from 2x106-10x106, or
1.5x105-10x105 (RMG-unpublished).
Immunization of normal C57BL16 mice for protection against EL4 tumor
ra owth:
Blazar and co-workers reported immunization for protection from
tumor growth in C57BL/6 mice using tumor cells transfected to over-express
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mouse CD80 (Blazar et al. 1997). Using CD80 and CD86 transfected EL4
cells obtained from this same group, or C1498 cells tranfected with
CD80/CD86 in our laboratory, we immunized groups of 6 C57BL/6 mice ip
with Complete Freund's adjuvant (CFA) alone, or with CFA mixed with 5x106
mitomycin-C treated tumor cells, or CD80/CD86 transfected tumor. Animals
received 2 injections at 14 day intervals. 10 days after the last immunization
all mice received 5x106 EL4 tumor cells, or 5x105 C1498 cells, and mortality
followed. Data are shown in Figure 2 (1 of 2 studies), for EL4 only.
In agreement with a number of other reports, mice pre-
immunized with CD80-transfected EL4 survive significantly longer after
challenge with viable EL4 tumor cells than non-immunized animals, or those
immunized with non-transfected cells or CD86 transfected cells (p<0.05)-see
also Figure 5. Similar data were obtained using CD80-transfected C1498 cells
(RMG-unpublished). In separate studies (not shown) mice immunized with
tumor cells in the absence of Freund's Adjuvant failed to show any protection
from tumor growth. However, equivalent protection (to that seen using
Freund's Adjuvant) was also seen using concomitant immunization with
poly(I:C) (100 g/mouse) as adjuvant (data not shown).
Role of CD4+ and/or CD8+ cells in modulation of tumor growth after BMT.
In order to investigate the effector cells responsible for leukemia
growth-inhibition in mice transplanted with allogeneic bone marrow (see
Figure 1), BMT recipients received weekly injections of 100 g/mouse anti-
CD4 (GK1.5) or anti-CD8 (2.43) mAb, followed at 28 days by leukemia cell
injection as described for Figure 1. Depletion of CD4 and CD8 cells in all
mice
with these treatments was >98% as defined by FACS analysis (not shown).
As shown in Figure 3 (data from one of 3 studies), and in
agreement with data reported elsewhere (27), in this BMT model tumor
growth inhibition for EL4 cells is predominantly a function of CD8 rather than
CD4 cells, while for C1498 leukemia cells growth inhibition was equally, but
not completely, inhibited by infusion of either anti-CD4 or anti-CD8 mAb (see
pane b of Figure).
Evidence that tumor rejection in BMT mice is regulated by CD200:
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Previous studies in rodent transplant models have implicated
expression of a novel molecule, CD200, in the regulation of an immune
rejection response. Specifically, blocking functional expression of CD200 by a
monoclonal antibody to murine CD200 prevented the increased graft survival
which followed donor-specific pretransplant immunization, while a soluble
form of CD200 linked to murine IgG Fc (CD200Fc) was a potent
immunosuppressant (3,5). In order to investigate whether expression of
CD200 was involved in regulation of tumor immunity, we studied first the
effect of infusion of CD200Fc on suppression of resistance to growth of EL4
or C1498 tumor in BMT mice as described in Figure 1, and second the effect
of CD200Fc infusion in mice immunized with CD80-transfected tumor cells as
described in Figure 2. Note this CD200FC lacks binding sites for mouse
complement and FcR (see Materials and Methods, and (5)). In all cases
control groups of mice received infusion of equivalent amounts of pooled
normal mouse IgG. Data for these studies is shown in Figures 4 and 5
respectively (data from one of 2 studies in each case).
It is clear that suppression of growth of either EL4 or C1498
tumor cells in BMT mice is inhibited by infusion of CD200Fc, but not by
pooled normal mouse IgG (Figure 4). CD200Fc also caused increased
mortality in EL4 or C1498 injected normal C3H mice. Data in Figure 5 show
that resistance to EL4 tumor growth in EL4-CD80 immunized mice (as
documented in Figure 2) is also inhibited by infusion of CD200Fc. In separate
studies (not shown) a similar inhibition of immunity induced by CD80
transfected C1498 was demonstrated using CD200Fc.
Effect of anti-CD200 mAb on resistance to tumor growth in mice immunized
with CD801CD86 transfected tumor cells:
As further evidence for a role for CD200 expression in tumor
immunity in EL4-CD80/EL4-CD86, or C1498-CD80/C1498-CD86 immunized
mice we examined the effect of infusion of an anti-CD200 mAb on EL4 or
C1498 tumor growth in this model. Infusion of anti-CD200 into mice
preimmunized with EL4-CD86, or C1498-CD86 uncovered evidence for
resistance to tumor growth. Separate studies (not shown) revealed that anti-
CD200 produced no significant perturbation of EL4 growth in the EL4 or EL4-
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CD80 immunized mice, or of C1498 growth in C1498 or C1498-CD86
immunized mice. We interpreted these data to suggest that immunization with
EL4-CD86 or C1498-CD86 elicited an antagonism of tumor immunity resulting
from increased expression of CD200. Thus blocking the functional increase of
CD200 expression with anti-CD200 reversed the inhibitory effect. Note that in
studies not shown we have reproduced these same effects of anti-CD200 (in
mice immunized with CD86-transfected tumor cells) with F(ab')2 anti-CD200
(RMG-unpublished).
To confirm that indeed immunization with CD86-transfected
tumor cells was associated with increased expression of CD200, we repeated
the study shown in Figure 6, and sacrificed 3 mice/group at 4 days following
EL4 tumor injection. RNA was isolated from the spleen of all mice, and
assayed by quantitative PCR for expression of CD200 (using GAPDH as
"housekeeping" mRNA control). Data for this study are shown in Figure 7, and
show convincingly that CD200 mRNA expression was >5-fold increased
following preimmunization with EL4-CD86, a condition associated with
increased tumor growth compared with EL4-immunized mice (Figures 2 and
6). In dual-staining FACS studies (not shown), with PE-anti-CD200 and FITC-
DEC205, the predominant CD200+ population seen in control and immunized
mice were DEC205+ (>80%)-see also (6). Similar results were obtained using
C1498 tumor cells (data not shown).
Given this increase in CD200 expression following
preimmunization with CD86-transfected cells, and the evidence that CD200 is
associated with delivery of an immunosuppressive, signal to antigen
encountered at the same time, we also examined the response of spleen cells
taken from these C57BL/6 mice to allostimulation (with mitomycin-C treated
BALB/c spleen cells), in the presence/absence of anti-CD200 mAb. Data from
one of 3 studies are shown in Table 1. Interestingly, mice preimmunized with
EL4-CD86 cells show a decreased ability to generate CTL on
alloimmunization with third-party antigen (BALB/c), and decreased type-1
cytokine production (IL-2, IFNy), with some trend to increased type-2
cytokines (IL-4 and IL-10). These effects were reversed by inclusion of anti-
CD200 in culture, consistent with the hypothesis that they result from
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increased delivery of an immunosuppressive signal via CD200 in spleen cells
obtained from these animals (3).
Evidence for an interaction between CD200 and CD200r+ cells in inhibition of
EL4 tumor growth:
Inhibition resulting from infusion of CD200Fc into mice follows
an interaction with immunosuppressive CD200r+ cells (13). At least one
identifiable functionally active population of suppressive CD200r+ cells was
described as a small, F4/80+ cell in a pool of splenic cells following LPS
stimulation (13)-F4/80 is a known cell surface marker for tissue macrophages.
In a further study we investigated whether signaling induced by
CD200:CD200r interaction (where CD200r+ cells were from lymphocyte-
depleted, LPS stimulated, spleen cells) was behind the suppression of tumor
immunity seen following CD200Fc injection. All groups of 6 recipient mice
received tumor cells ip. In addition to infusion of CD200Fc as
immunosuppressant (in Figure 8), one group of C3H reconstituted animals
received CD200r+ cells (A in Figure 8: >65% of these cells stained with an
anti-CD200r mAb), while a final group received a mixture of both CD200Fc
and CD200r+ cells (A). It is clear from the Figure that it is this final
group, in
which interaction between CD200 and CD200r is possible, which showed
maximum inhibition of tumor immunity compared with the C3H reconstituted
control mice (=).
Role of CD4+ and/or CD8+ cells in CD200 regulated modulation of tumor
growth after BMT.=
Data in Figure 3 above, and elsewhere (27) show that tumor
growth inhibition for EL4 cells is predominantly a function of CD8 rather than
CD4 cells, while for C1498 leukemia cells growth inhibition was equally, but
not completely, inhibited by infusion of either anti-CD4 or anti-CD8 mAb. To
investigate the role of CD200 in the protection mediated by different T cell
subclasses, the following additional studies were performed. In the first,
C57BL/6 recipients of C3H BMT received (at 28 days post BMT) inoculations
of EL4 or C1498 tumor cells along with anti-CD4, anti-CD8 or CD200Fc
alone, or combinations of (anti-CD4+CD200Fc) or (anti-CD8+CD200Fc).
Survival was followed as before (see Figure 9-one of 2 studies). In a second
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study (Figure 10-data from one of 2 such experiments) a similar treatment
regimen of mAbs or CD200Fc alone or in combination was used to modify
growth of EL4 tumor cells in mice preimmunized with EL4-CD80 cells as
described earlier in Figure 2.
Data in panel a of Figure 9 confirm the effects previously
documented in Figures 3 and 4, that CD200Fc and anti-CD8 each
significantly impaired the growth inhibition in BMT recipients of EL4 cells,
while anti-CD4 mAb was less effective. Combinations of CD200Fc and either
anti-T cell mAb led to even more pronounced inhibition of tumor immunity in
the BMT recipients, to levels seen with non-allogeneic transplanted mice.
Data with C1498 tumor cells (panel b) were somewhat analogous, though as
in Figure 3, anti-CD4 alone produced equivalent suppression of growth
inhibition to anti-CD8 with this tumor. As was the case for the EL4 tumor,
combinations of CD200Fc and either anti-T cell mAb caused essentially
complete suppression of C1498 tumor growth inhibition. Both sets of data,
from panels a and b, are consistent with the notion that CD200Fc blocks
(residual) growth-inhibitory functional activity in both CD4 and CD8 cells,
thus
further inhibiting tumor immunity remaining after depletion of T cell subsets.
Data in Figure 10, using EL4 immunized BL/6 mice, also
showed combinations of CD200Fc and anti-CD4 treatment produced optimal
suppression of tumor immunity to EL4 cells, consistent with an effect of
CD200Fc on CD8+ cells. Anti-CD8 alone abolished tumor immunity in these
studies, so any potential additional effects of CD200Fc on CD4+ cells could
not be evaluated.
DISCUSSION
In the studies described above, the inventors have studied
whether expression of the molecule CD200, previously reported to down-
regulate rejection of tissue/organ allografts in rodents (3), was implicated
in
immunity to tumor cells in syngeneic hosts. Two model systems were used.
The one, in which tumor cells are injected into mice which had received an
allogeneic bone marrow transplant following cyclophosphamide pre-
conditioning, has been favoured as a model for studying potential innovative
treatments of leukemia/lymphoma in man (27, 30-32). In the other EL4 or
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C1498 tumor cells were infused into BL/6 mice which had been preimmunized
with tumor cells transfected to overexpress the costimulatory molecules CD80
or CD86. These studies were stimulated by the growing interest in such
therapy for immunization of human tumor patients with autologous transfected
tumor cells (11, 23-26).
In both sets of models we found evidence for inhibition of tumor
growth (Figures 1 and 2) which could be further modified by treatment
designed to regulate expression of CD200. Infusion of CD200Fc suppressed
tumor immunity (led to increased tumor growth, and faster mortality) in both
models (Figures 4 and 5), while anti-CD200 improved tumor immunity in mice
immunized with CD86-transfected EL4 or C1498 tumor cells (Figure 6). The
inventors interpreted this latter finding as suggesting that the failure to
control
tumor growth following immunization with EL4-CD86 or C1498-CD86 was
associated with overexpression of endogenous CD200, a hypothesis which
was confirmed by quantitative PCR analysis of tissue taken from such mice
(Figure 7). CD200 was predominantly expressed on DEC205+ cells in the
spleen of these mice (see text), which was associated with a decreased ability
of these spleen cell populations to respond to allostimulation in vitro (see
Table 1). Non antigen-specific inhibition following CD200 expression formed
the basis of our previous reports that a soluble form of CD200 (CD200Fc) was
a potent immunosuppressant (3). Consistent with the hypothesis that
increased expression of CD200 in mice immunized with CD86-transfected
tumor cells was responsible for the inhibition of alloreactivity seen in Table
1,
suppression was abolished by addition of anti-CD200 mAb (see lower half of
Table 1). Earlier reports have already documented an immunosuppressive
effect of CD200Fc on alloimmune responses (5), production of antibody in
mice following immunization with sheep erythrocytes (5).
Maximum inhibition of tumor immunity was achieved by
concomitant infusion of CD200Fc and CD200r+ cells (F4/80+ macrophages-
see Figure 8). The inventors next investigated the cell type responsible for
tumor growth inhibition whose activity was regulated by CD200:CD200r
interactions. The data confirmed previous reports that EL4 growth inhibition
was predominantly associated with CD8 immune cells, while immunity to
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C1498 was a function of both CD4+ and CD8+ cells (27). For both tumors in
BMT models suppression of tumor growth inhibition was maximal following
combined treatment with CD200Fc and either anti-T cell mAb, consistent with
the idea that CD200 suppression acts on both CD4+ and CD8+ T cells.
A number of studies have examined immunity to EL4 or C1498
tumor cells in similar models to those described above (27, 33), concluding
that CD8+ cells are important in (syngeneic) immunity to each tumor, and
CD4+ T cells are also important in immunity to C1498 (27). Evidence to date
suggests that NK cell mediated killing is not relevant to tumor growth
inhibition
in BMT mice of the type used above (27). Other reports have addressed the
issue of the relative efficiency of induction of tumor immunity in a number of
models following transfection with CD80 or CD86, and also concluded that
CD80 may be superior in induction of anti-tumor immunity (27, 34), while
CD86 may lead to preferential induction of type-2 cytokines (8). This is of
interest given the cytokine production profile seen in EL4-CD86 immunized
mice (Table 1), which is similar to the profile seen following CD200Fc
treatment of allografted mice (3). EL4-CD86 immunized mice show increased
expression of CD200 (Figure 7), with no evidence for increased resistance to
tumor growth (Figure 2). Resistance is seen in these mice following treatment
with anti-CD200 (Figure 6A). Somewhat better protection from tumor growth
is seen using viable tumor cells for immunization, rather than mitomycin-C
treated cells as above (27). Whether this would improve the degree of
protection from tumor growth in our model, and/or significantly alter the role
of
CD200:CD200' interactions in its regulation, remains to be seen.
There are few studies exploring the manner in which
suppression mediated by CD200:CD200r interactions occurs. In a recent
study in CD200 KO mice Hoek et al observed a profound increase in the
presence of activated macrophages and/or macrophage-like cells (14), and
we and others had previously found that CD200r was expressed on
macrophages (13,35). The inventors also reported that CD200r was present
on a subpopulation of T cells, including the majority of activated y8TCR+
cells
(13). ySTCR+ cells may mediate their suppressive function via cytokine
production (36), while unpublished data (RMG-in preparation) suggests that
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the CD200' macrophage cell population may exert its activity via
mechanisms involving the indoleamine 2,3-dioxygenase (IDO) tryptophan
catabolism pathway (37). We suggest that the mechanism by which CD200Fc
leads to suppression of tumor growth inhibition in the models described is
likely to be a function both of the tumor effector cell population involved
(Figures 9, 10) as well as the CD200r cell population implicated in
suppression.
In a limited series of studies (not shown) we have used other
BMT combinations (B10 congenic mice repopulated with B10.D2, 1310.13R. or
B10.A bone marrow) to show a similar resistance to growth of EL4 or C1498
tumor cells, which is abolished by infusion of CD200Fc. Studies are in
progress to examine whether DBA/2 or BALB/c mice can be immunized to
resist growth of P815 syngeneic (H2d) tumor cells by P815 cells tranfected
with CD80/CD86, and whether this too can be abolished by CD200Fc. Taken
together, however, our data are consistent with the hypothesis that the
immunomodulation following CD200:CD200r interactions, described initially in
a murine allograft model system, is important also in rodent models of tumor
immunity. This has important clinical implications.
EXAMPLE 2
TRIM IN THE FSL SARCOMA LUNG METASTASIS MODEL:CUES FROM
PREGNANCY
Allogeneic leukocyte-induced transfusion-related
immunomodulation (TRIM) has been shown to enhance tumor growth (38,
39). OX-2 is expressed on a variety of cells in transfused blood (i.e. a
subpopulation of dendritic cells and possibly B cells) (40, 41), thus the
effect
of anti-OX-2 on the TRIM enhancement of FSL10 lung nodules was
examined.
MATERIALS and METHODS
Enhancement of lung nodules by TRIM
A dose response curve demonstrated a plateau in the TRIM
enhancement of lung metastases with 50, 100 or 200 gl of BALB/c
heparinized blood given 4 days after tail vein injection of the cultured tumor
cell by tail vein (see Figure 11). A dose of 200 l of BALB/c heparinized
blood
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(about 15-20% of blood volume) was given 4 days after tail vein injection of
the cultured tumor cells as a physiologically suitable model in which to
screen
for treatments that have a major abrogating effect on TRIM.
All animals were monitored for signs of illness daily, and 21
days after tumor inoculation, the mice were sacrificed, the lungs were
removed and fixed in Bouin's solution, and the number of surface nodules
was counted. To deal with variation in number of metastases between mice,
20-25 mice per group were used and medians were calculated (using log-
transformed data, where 0 nodules was set at 0.1 for that animal). It was then
possible to assess the significance of differences in log mean sem with
respect to our a priori hypotheses using Student's t test, and to construct 95
% confidence intervals for the medians. Differences in the proportion of mice
in different groups with no visible metastases was assessed by the %2
statistic, or by Fisher's Exact test where appropriate.
Figure 11A shows the median number of lung nodules in
C57BI/6J mice receiving the indicated dose of freshly-prepared allogeneic
BALB/c strain blood by tail vein. The effect is seen if the blood is given 7
days
prior to, or 4 days after a tail vein injection of 1 x 106 FSL sarcoma cells.
FSL10 is a methlycholanthrene-induced fibrosarcoma generated in C57BI/6
mice and maintained by standard tissue culture in vitro. Such cells are weakly
antigenic. Group size is 20-25 per group, and P values showing increased
numbers to lung nodules are on the figure. Figure 11 B shows proportion of
mice with no tumor nodules. P values were determined by Student's t test for
A, and by Chi-square or Fisher's Exact test for B.
The Role of Dendritic Cells
MAb to a myeloid DC/APC surface marker (5 pg anti-CD11c) or
lymphoid dendritic cells (DC) (5 g supernatant of DEC205 hybridoma, an
amount shown to be sufficient using in vitro assays of DC function (42) was
added to 200 l of BALB/c blood or to PBS. The TRIM enhancement of tumor
growth was analyzed using the same method as above.
RESULTS
Enhancement of lung nodules by TRIM
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Figure 12A represents the effect of adding anti-OX-2
monoclonal antibody (3B6, 1 ug per million leukocytes) to the blood (or PBS
control) before tumor cell transfer. A control is the same amount of 3B6 in
PBS. The total dose was 3.3 ug per mouse. Figure 12A shows this amount of
anti-CD200 in PBS had no effect, whereas when added to blood, the
stimulation of tumor nodule number was prevented, - indeed, it was reduced
below control levels. Figure 12B shows % with no lung nodules. In this and
subsequent studies, 2 x 10 6 FSL cells were used, and the blood was always
given 4 days after this.
As illustrated in Figure 12, the enhancement of lung nodules in
mice given 2x105 sarcoma cells by 200 gl of BALB/c blood compared to
phosphate buffered saline control (PBS) given 4 days after tumor injection,
was completely blocked by adding 3.3 gg of anti-OX-2 (3B6 monoclonal
antibody (mAb)) to the blood before the transfusion. The mAb in PBS had no
effect. Interestingly, the proportion of mice with lung metastases was boosted
by allogeneic blood compared to PBS but was reduced by blood to which
anti-OX-2 had been added. The median number of nodules was greater in
this study in part because we had doubled the tumor cell inoculum, but we do
see experiment-to-experiment variation in the number of nodules in the
control group which has been important in executing large experiments, as
will be discussed.
The Role of Dendritic Cells
Figure 13 is a repetition of the experiment in Figure 12A which
confirms the effect of anti-OX-2, but with addition of antibodies to dentritic
cells. Anti-CD11c was used for myeloid dendritic cells, and DEC205 for
lymphoid dendritic cells. The latter are usually CD8-positive. It can be
readily
appreciated that monoclonal antibody to lymphoid dendritic cells had no effect
on the stimulation of lung metastases, whereas anti-CD11c blocked the effect.
The reason the number of nodules is not below control is thought to be due to
the existence of OX-2-positive and OX-2-negative CD11c-type dendritic cells.
The latter stimulate immunity, and this is seen when anti-OX-2 is used to
block one of the subsets. Anti-CD11c leads to loss of both subsets.
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The result shows that anit-CD11c, but not DEC205, abrogated
the TRIM effect (* indicates significant increase over control, ** indicates
significant abrogation of TRIM, *** indicates significant decrease below
control, P < 0.05). There was no effect of anti-OX-2, DEC205 or anti-CD 11 c
in
PBS injected as a control (data not shown). Due to the large number of
treatment groups in this experiment, it could. not be done in a single day.
Therefore, 5 mice in each group were treated in 4 experiments and the data
was examined and pooled. The result therefore compensates for any effect of
day-to-day variation in tumor cells, mice or blood used for transfusion.
While the present invention has been described with reference
to what are presently considered to be the preferred examples, it is to be
understood that the invention is not limited to the disclosed examples.
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TABLE I
Preimmunization of mice with EL4-CD86 causes increased CD200 expression
which leads to generalized suppression to newly encountered alloantigen
Tumor used for Immunizations % Lysisb Cytokines in Supernatantc
IL-2 IL-4 IFNy IL-10
NONE (control) 43+5.5 980+125 50+10 455+65 35+10
EL4 41+6.2 890+135 60+15 515 70 30+10
EL4-CD80 46+6.3 955+140 45+15 525+55 30+10
EL4-CD86 16 4.2* 420+75* 125+20* 240 40* 120+20*
NONE (control) + 46+5.8 950+105 55+15 490+60 40+10
EL4 + 44 4.9 940+115 50+20 530 60 35+10
EL4-CD80 + 44+6.0 905+120 60+15 555 75 35+10
EL4-CD86 + 39+4.29 870+125 75+20 540+65 40+15
Footnotes:
a. Spleen cells were pooled from 3 C57BL/6 mice/group, pretreated as described
in the
text, by immunization with 5x106 mitomycin-C treated EL4 tumor cells, or
CD80/CD86-transfected tumor cells, in Complete Freund's Adjuvant 4 days
earlier.
5x106 spleen cells were incubated in triplicate with equal numbers of
mitomycin-C
treated BALB/c spleen stimulator cells. + indicates anti-CD200 (anti-OX2)
added to
cultures (5 g/ml)
b. % specific lysis in 4-hr 51Cr release assays with 72-hr cultured BALB/c
spleen Con A
blast cells (effector:target ratio shown is 100:1).
c. Cytokines in culture supernatants assayed in triplicate by ELISA at 40 hrs
(see
Materials and Methods). Data represent pg/ml except for IL-10 (ng/ml). *p<0.05
compared with all groups.
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- 43 --
tctgcgactg cccgtccagc ccctgccatc acctggaagg gtactgggac aggaattgag 540
aatagtaccg agagtcactt ccattcaaat gggactacat ctgtcaccag catcctccgg 600
gtcaaagacc ccaaaactca agttgggaag gaagtgattt gccaggtttt atacctgggg 660
aatgtgattg actacaagca gagtctggac aaaggatttt ggttttcagt tccactgttg 720
ctaagcattg tttctctggt aattct:tctg atcttgatct ccatcttact atactggaaa 780
cgtcaccgaa atcaggagcg gggtgaatca tcacagggga tgcaaagaat gaaataa 837
<210> 4
<211> 278
<212> PRT
<213> Mus musculus
<400> 4
Met Gly Ser Leu Val Phe Arg Arg Pro Phe Cys His Leu Ser Thr Tyr
1 5 10 15
Ser Leu Ile Trp Gly Met Ala Ala Val Ala Leu Ser Thr Ala Gln Val
20 25 30
Glu Val Val Thr Gln Asp Glu Arg Lys Ala Leu His Thr Thr Ala Ser
35 40 45
Leu Arg Cys Ser Leu Lys Thr Ser Gln Glu Pro Leu Ile Val Thr Trp
50 55 60
Gln Lys Lys Lys Ala Val Ser Pro Glu Asn Met Val Thr Tyr Her Lys
65 70 75 80
Thr His Gly Val Val Ile Gln Pro Ala Tyr Lys Asp Arg Ile Asn Val
85 90 95
Thr Glu Leu Gly Leu Trp Asn Ser Ser Ile Thr Phe Trp Asn Thr Thr
100 105 110
Leu Glu Asp Glu Gly Cys Tyr Met Cys Leu Phe Asn Thr Phe Gly Ser
115 120 125
Gln Lys Val Ser Gly Thr Ala Cys Leu Thr Leu Tyr Val Gln Pro Ile
130 135 140
CA 02417874 2003-07-04
- 44 -
Val His Leu His 'Tyr Asn Tyr Phe Glu Asp His Leu Asn Ile Thr Cys
145 150 155 160
Ser Ala Thr Ala Arg Pro Ala Pro Ala Ile Ser Trp Lys Gly Thr Gly
165 170 175
Thr Gly Ile Glu Asn Ser Thr Glu Ser His Phe His Ser Asn Gly Thr
180 185 190
Thr Ser Val Thr Ser Ile Leu Arg Val Lys Asp Pro Lys Thr Gln Val
195 2C0 205
Gly Lys Glu Val Ile Cys Gln Val Leu Tyr Leu Gly Asn Val Ile Asp
210 215 220
Tyr Lys Gln Ser Leu Asp Lys Gly Phe Trp Phe Ser Val Pro Leu Leu
225 230 235 240
Leu Ser Ile Val Her Leu Val Ile Leu Leu Val Leu Ile Ser Ile Leu
245 250 255
Leu Tyr Trp Lys Arg His Arg Asn Gln Glu Arg Giy Glu Ser Her Gln
260 265 270
Gly Met Gln Arg Met Lys
275
<210> 5
<211> 825
<212> DNA
<213> Homo sapiens
<400> 5
gtgatcagga tgcccttctc tcatctctcc tcctacagcc tggtttgggt catggcagca 60
gtggtgctgt gcacagcaca agtgcaagtg gtgacccagg atgaaagaga gcagctgtac 120
acacctgctt ccttaaaatg ctctc::gcaa aatgcccagg aagccctcat tgtgacatgg 180
cagaaaaaga aagctgtaag cccagaaaac atggtcacct tcagcgagaa ccatggggtg 240
gtgatccagc ctgcctataa ggacaagata aacattaccc agctgggact ccaaaactca 300
accatcacct tctggaatat caccctggag gatgaagggt gttacatgtg tctcttcaat 360
acctttggtt ttgggaagat ctcagg_3aacg gcctgcctcca ccgtctatgt acagcccata 420
CA 02417874 2003-07-04
- 45 -
gtatcccttc actacaaatt ctctgaagac cacctaaata tcacttgctc tgccactgcc 480
cgcccagccc ccatggtctt ctggaaggtc cctcggtcag ggattgaaaa tagtacagtg 540
actctgtctc acccaaatgg gacca,:::gtct gttaccagca tcctccatat caaagaccct 600
aagaatcagg tggggaagga ggtgat.ctgc caggtgctgc acctggggac tgtgaccgac 660
tttaagcaaa ccgtcaacaa aggatattgg ttttcagttc cgctattgct aagcattgtt 720
tccctggtaa ttcttctcat cctaa::.ctca atcttactgt actggaaacg tcaccggaat 780
caggaccgag gtgaattgtc acagggagtt caaaaaatga cataa 825
<210> 6
<211> 274
<212> PRT
<213> Homo sapiens
<400> 6
Val Ile Arg Met Pro Phe Ser His Leu Ser Thr Tyr Ser Leu Val Trp
1 5 10 15
Val Met Ala Ala Val Val Leu Cys Thr Ala Gln Val Gln Val Val Thr
20 25 30
Gln Asp Glu Arg G:.u Gln Leu Tyr Thr Thr Ala Ser Leu Lys Cys Ser
35 40 45
Leu Gln Asn Ala Gln Glu Ala Leu Ile Val Thr Trp Gln Lys Lys Lys
50 55 60
Ala Val Ser Pro G].u Asn Met Val. Thr Phe Ser Glu Asn His Gly Val
65 70 75 80
Val Ile Gln Pro A].a Tyr Lys Asp Lys Ile Asn Ile Thr Gln Leu Gly
8_ ; 90 95
Leu Gln Asn Ser Thr Ile Thr Phe Trp Asn Ile Thr Leu Glu Asp Glu
100 105 110
Gly Cys Tyr Met Cys Leu Phe Asn Thr Phe Gly Phe Gly Lys Ile Ser
115 120 125
Gly Thr Ala Cys Leu Thr Val Tyr Val Gln Pro Ile Val Ser Leu His
130 135 140
CA 02417874 2003-07-04
- 46 -
Tyr Lys Phe Ser Glu Asp His Leu Asn Ile Thr Cys Ser Ala Thr Ala
145 150 155 160
Arg Pro Ala Pro Met Val Phe Trp Lys Val Pro Arg Ser Gly Ile Glu
165 170 175
Asn Ser Thr Val Thr Leu Ser His Pro Asn Gly Thr Thr Ser Val Thr
180 185 190
Ser Ile Leu His Ile Lys Asp Pro Lys Asn Gln Val Gly Lys Gl.u Val
195 200 205
Ile Cys Gln Val Leu His Leu Gly Thr Val Thr Asp Phe Lys Gin Thr
210 215 220
Val Asn Lys Gly Tyr Trp Phe Ser Val Pro Leu Leu Leu Ser Ile Val
225 230 235 240
Ser Leu Val Ile Leu Leu Val Leu Ile Ser Ile Leu Leu Tyr Trp Lys
245 250 255
Arg His Arg Asn Gln Asp Arg Gly Glu Leu Ser Gln Gly Val Gln Lys
260 265 270
Met Thr
