U.S. patent application number 13/670096 was filed with the patent office on 2013-07-18 for tim-3 ligands and methods thereof.
This patent application is currently assigned to BETH ISRAEL DEACONESS MEDICAL CENTER, INC.. The applicant listed for this patent is Beth Israel Deaconess Medical Center, Inc., The Brigham and Women's Hospital, Inc.. Invention is credited to Eugene K. Cha, Sumone Chakravarti, Vijay K. Kuchroo, Catherine Sabatos, Alberto Sanchez-Fueyo, Terry Strom, Xin Xiao Zheng, Chen Zhu.
Application Number | 20130183688 13/670096 |
Document ID | / |
Family ID | 34421718 |
Filed Date | 2013-07-18 |
United States Patent
Application |
20130183688 |
Kind Code |
A1 |
Kuchroo; Vijay K. ; et
al. |
July 18, 2013 |
TIM-3 LIGANDS AND METHODS THEREOF
Abstract
The invention relates to isolated polypeptides and nucleic acids
encoding polypeptides which comprise a tim-3 IgV domain and a tim-3
intracellular domain, wherein the polypeptides do not comprise a
tim-3 mucin domain or a tim-3 transmembrane domain. In addition,
the invention relates to methods of modulating immune responses in
a subject, comprising administering to the subject a
therapeutically effective amount of an agent that modulates tim-3
activity. Immune responses include, but are not limited to, immune
tolerance, transplantation tolerance, Th1 responses and Th2
responses.
Inventors: |
Kuchroo; Vijay K.; (Newton,
MA) ; Strom; Terry; (Brookline, MA) ; Cha;
Eugene K.; (Muttontown, NY) ; Chakravarti;
Sumone; (Victoria, AU) ; Sabatos; Catherine;
(San Francisco, CA) ; Zhu; Chen; (Brookline,
MA) ; Zheng; Xin Xiao; (Wellesley, MA) ;
Sanchez-Fueyo; Alberto; (Barcelona, ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Brigham and Women's Hospital, Inc.;
Beth Israel Deaconess Medical Center, Inc.; |
Boston
Boston |
MA
MA |
US
US |
|
|
Assignee: |
BETH ISRAEL DEACONESS MEDICAL
CENTER, INC.
Boston
MA
THE BRIGHAM AND WOMEN'S HOSPITAL, INC.
Boston
MA
|
Family ID: |
34421718 |
Appl. No.: |
13/670096 |
Filed: |
November 6, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10958169 |
Oct 4, 2004 |
8329660 |
|
|
13670096 |
|
|
|
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60508319 |
Oct 3, 2003 |
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Current U.S.
Class: |
435/7.92 |
Current CPC
Class: |
A61P 7/00 20180101; A61P
29/00 20180101; A61P 21/04 20180101; A61P 37/02 20180101; C07K
2319/30 20130101; A61P 1/00 20180101; C07K 14/4713 20130101; C07K
14/70503 20130101; A61P 11/06 20180101; C07K 16/18 20130101; A61P
1/16 20180101; A61P 27/14 20180101; A61P 11/02 20180101; A61P 13/12
20180101; A61P 25/00 20180101; A61P 27/02 20180101; A61P 35/02
20180101; A61P 17/06 20180101; A61P 5/14 20180101; G01N 33/505
20130101; C07K 16/28 20130101; A61P 19/02 20180101; A61P 37/06
20180101; A61P 43/00 20180101; A61P 7/04 20180101; A61P 1/04
20180101; A61P 7/06 20180101; A61P 35/00 20180101; A61P 3/10
20180101; A61P 37/08 20180101 |
Class at
Publication: |
435/7.92 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Goverment Interests
GOVERNMENT FUNDING
[0002] Work described herein was funded by grant numbers
1R01NS045937-01, 2R01N535685-06, 2R37NS30843-11, 1R01A144880-03,
2P01A139671-07, 1P01N538037-04 and 1F31GM20927-01 from the National
Institutes of Health. Accordingly, the United States government has
certain rights in the invention.
Claims
1. A method of identifying an agent that modulates binding between
a tim-3 polypeptide and a galectin-9 polypeptide, the method
comprising: (a) contacting the tim-3 polypeptide and the galectin-9
polypeptide in the presence of a test agent; and (b) determining
the effect of the test agent on the binding of the tim-3
polypeptide and the galectin-9 polypeptide; thereby identifying an
agent that modulates the binding between a tim-3 polypeptide and a
galectin-9 polypeptide.
2. The method of claim 1, wherein step (b) comprises comparing
formation of a tim-3/galectin-9 complex in the presence of the test
agent with formation of a tim-3/galectin-9 complex in the absence
of the test agent.
3. The method of claim 1, wherein the test agent is a small
molecule compound, an antibody or a polypeptide.
4. The method of claim 1, wherein the test agent increases binding
between the tim-3 and galectin-9 polypeptides.
5. The method of claim 1, wherein the test agent decreases binding
between the tim-3 and galectin-9 polypeptides.
6. The method of claim 1, wherein the tim-3 polypeptide or the
galectin-9 polypeptide or both are expressed in a cell.
7. The method of claim 2, wherein comparing formation of the a
tim-3/galectin-9 complex comprises detecting expression of a
reporter gene, wherein the expression of the reporter gene is
dependent on the formation of the tim-3/galectin-9 complex.
8. The method of claim 1, wherein the galectin-9 polypeptide
comprises (i) amino acids 1-323 of SEQ ID NO: 10; or (ii) amino
acids 1-355 of SEQ ID NO: 19.
9. The method of claim 1, wherein the tim-3 polypeptide comprises
amino acids 30-128 of SEQ ID NO: 13.
10. A method of identifying an agent that modulates an immune
response, the method comprising (a) contacting the tim-3
polypeptide and the galectin-9 polypeptide in the presence of a
test agent; and (b) determining the effect of the test agent on the
binding of the tim-3 polypeptide and the galectin-9 polypeptide;
thereby identifying an agent that modulates an immune response.
11. The method of claim 10, wherein step (b) comprises comparing
formation of a tim-3/galectin-9 complex in the presence of the test
agent with formation of a tim-3/galectin-9 complex in the absence
of the test agent.
12. The method of claim 11, wherein comparing formation of the a
tim-3/galectin-9 complex comprises detecting expression of a
reporter gene, wherein the expression of the reporter gene is
dependent on the formation of the tim-3/galectin-9 complex.
13. The method of claim 10, wherein the immune response is a Th1
immune response.
14. The method of claim 10, wherein the immune response is a Th2
immune response.
15. An assay comprising: (a) combining a tim-3 polypeptide, a
galectin-9 polypeptide, and a test agent under conditions where the
tim-3 and galectin-9 polypeptides interact to form a
tim-3/galectin-9 complex in the absence of the test agent; and (b)
measuring a parameter of tim-3/galectin-9 complex formation in the
presence of the test agent; wherein a change in the parameter of
tim-3/galectin-9 complex formation in the presence of the test
agent relative to tim-3/galectin-9 complex formation in the absence
of the test agent is indicative that the test agent inhibits or
potentiates tim-3/galectin-9 complex formation.
16. The assay of claim 15, wherein the parameter of
tim-3/galectin-9 complex formation is assembly or stability of
tim-3/galectin-9 complex formation.
17. The assay of claim 15, wherein the test agent is a small
molecule compound, an antibody or a polypeptide.
18. The assay of claim 15, wherein the tim-3 polypeptide or the
galectin-9 polypeptide or both are expressed in a cell.
19. The assay of claim 15, wherein the galectin-9 polypeptide
comprises (iii) amino acids 1-323 of SEQ ID NO: 10; or (iv) amino
acids 1-355 of SEQ ID NO: 19.
20. The assay of claim 15, wherein the tim-3 polypeptide comprises
amino acids 30-128 of SEQ ID NO: 13.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application that claims
benefit under 35 USC .sctn.120 of U.S. application Ser. No.
10/958,169 filed on Oct. 4, 2004, which claims benefit under 35
U.S.C. .sctn.119(e) of U.S. Provisional Application No. 60/508,319,
filed Oct. 3, 2003, the contents of each of which are herein
incorporated by reference in their entireties.
BACKGROUND OF THE INVENTION
[0003] Upon stimulation by antigen, naive CD4.sup.+ T helper cells
develop into two main effector pathways, Th1 and Th2 cells, defined
by their cytokine profiles (Mosmann et al. J Immunol 136, 2348-2357
(1986), Mosmann et al. Immunol Today 19, 138-146 (1996), Abbas, et
al. Nature 383, 787-793 (1996)).
[0004] Th1 cells produce cytokines (interferon (IFN-y, interleukin
(IL)-2, tumor-necrosis factor TNF-.alpha. and lymphotoxin) that are
most frequently associated with cell-mediated immune responses
against intracellular pathogens. The pathological consequences of
an inappropriate Th1 response are delayed type hypersensitivity
(DTH) reactions (Sher, et al. Annu Rev Immunol. 10, 385-409
(1992)), induction of organ-specific autoimmune disease (Liblau, et
al. Immunol Today 16, 34-38 (1995)), rheumatoid arthritis,
inflammatory bowel disease (IBD), type I diabetes multiple
sclerosis, and allograft rejection.
[0005] Th2 cells produce cytokines (IL-4, IL-10 and IL-13)
necessary for the clearance of extracellular helminthic infections,
and inappropriate Th2 cell activation promotes the onset of atopic
and allergic diseases (Abbas, et al. Nature 383, 787-793 (1996),
Sher, et al. Annu Rev Immunol. 10, 385-409 (1992)), such as
allergic asthma.
[0006] In addition to their distinct roles in disease, the two T
helper subsets also cross-regulate each other's expansion and
functions. Thus, preferential induction of Th2 cells inhibits
autoimmune diseases (Nicholson, L., et al. Immunity 3, 397-405
(1995), Kuchroo, et al. Cell 80, 707-718 (1995)), while predominant
induction of Th1 cells can regulate asthma, atopy and allergies
(Lack, et al. J Immunol 152, 2546-2554 (1994); Hofstra, et al. J
Immunol 161, 5054-5060 (1998)).
[0007] Applicants have recently identified a novel cell surface
protein, Tim-3, which is expressed on Th1 but not Th2 cells. Tim-3
(T cell Immunoglobulin and Mucin domain containing molecule) is a
type I membrane protein of 281 amino acids whose extracellular
domain comprises of an IgV-like domain followed by a mucin-like
region. The human orthologue of Tim-3 shares 63% amino acid
identity with murine Tim-3. Tim-3 is polymorphic and, along with
other Tim family members, has been linked to murine asthma
(McIntire, J. et al. Nat Immunol 2, 1109-1116 (2001)). In addition,
the Tim gene family region is syntenic with a major asthma
susceptibility locus in humans (McIntire, J. et al. Nat Immunol 2,
1109-1116 (2001)). These studies underscore the importance of Tim-3
and the Tim gene family in regulation of immune-mediated
diseases.
[0008] In vivo during an ongoing immune response, administration of
anti-Tim-3-antibody increased macrophage activation and expansion
(Monney, L. et al. Nature 415, 536-541 (2002). Anti-Tim-3 antibody
treatment also exacerbated the autoimmune disease experimental
autoimmune encephalomyelitis (EAE), significantly increasing
mortality and causing enhanced demyelination and infiltration of
activated macrophages to the central nervous system (CNS).
[0009] Accordingly, a need remains to identify agents which
modulate tim-3 function and thus modulate immune responses. Some
aspects of the present invention provide such agents, methods to
identify such agents, and methods of modulating immune responses
using such agents.
SUMMARY OF THE INVENTION
[0010] The invention provides novel polypeptides, nucleic acids and
compositions, including those useful in modulating immune
responses. The invention further provides methods of modulating
immune responses in a subject in need thereof, and methods of
identifying agents which may be used to modulate immune
responses.
[0011] The invention provides an isolated polypeptide comprising a
tim-3 IgV domain and a tim-3 intracellular domain, wherein the
polypeptide does not comprise a tim-3 mucin domain or a tim-3
transmembrane domain. The invention also provides the nucleic acids
which encode such polypeptides.
[0012] The invention also provides an isolated nucleic acid which
hybridizes under high stringency conditions to a nucleic acid
encoding soluble tim-3 but which does not hydridize under high
stringency conditions to a nucleic acid encoding full-length tim-3.
Such nucleic acid is useful, among other things, to modulate immune
responses.
[0013] Furthermore, the invention provides a pharmaceutical package
comprising (i) a polypeptide which comprises the IgV domain of
tim-3; and (ii) instructions for administering the composition to a
subject for treating a hyperplastic condition.
[0014] The invention additionally provides an isolated antibody or
fragment thereof which binds to a polypeptide having an amino acid
sequence set forth in SEQ ID NO: 2 but which does not bind to a
polypeptide having the amino acid sequence set forth in SEQ ID NO:
13 Likewise, the invention provides an isolated antibody or
fragment thereof which binds to a polypeptide having an amino acid
sequence set forth in SEQ ID NO: 4 but which does not bind to a
polypeptide having the amino acid sequence set forth in SEQ ID NO:
14.
[0015] The invention provides methods of modulating immune
responses in a subject, such as immune tolerance in a subject,
comprising administering to the subject a therapeutically effective
amount of an agent that modulates tim-3 activity. The invention
also provides methods for modulating immune responses, such as Th1
and Th2 responses, in a subject.
[0016] The invention further provides methods of identifying agents
which modulate immune responses, and methods of identifying agents
which modulate the binding interaction between tim-3 and its
ligands, such as galectin-9.
[0017] The invention further provides agents for the manufacture of
medicaments to treat any of the disorders described herein. Any
methods disclosed herein for treating or preventing a disorder by
administering an agent to a subject may be applied to the use of
the agent in the manufacture of a medicament to treat that
disorder. For example, in one specific embodiment, a tim-3IgV-HSA
fusion protein may be used in the manufacture of a medicament for
the treatment of Th2-mediated disorder, whereas a pegylated
galectin-9 polypeptide may be used, in the manufacture of a
medicament for the treatment of a Th1-mediated disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A-1C show the identification of alternatively spliced
soluble form of Tim-3. FIG. 1A Full-length and alternatively
spliced forms of Tim-3 were amplified from cDNA generated from
concanavalin A-stimulated splenocytes. Primers were designed in the
5' and 3' UTR of the Tim-3 gene and subjected to RT-PCR. Products
were resolved on a 1.2% agarose gel and visualized using ethidium
bromide (lane 1). Amplicons of 800 bp and 1 kb were cloned into the
pEF6 vector (lanes 2 & 3). In order to confirm the cloning,
plasmid DNA was digested with the Smal restriction endonuclease
(New England Biolabs). Reactions were terminated with loading dye
and run on a 1.2% agarose gel and visualized with ethidium bromide
(lanes 2 and 3). Clones containing inserts of 1 kb or 800 bp were
sequenced. Lane 1, con-A activated splenocytes cDNA amplified by
PCR with Tim-3-F and Tim-3-R primer; Lane 2, 1 kb cloned product,
fl-Tim-3; Lane 3, 800 bp cloned product, s-Tim-3. FIG. 1B The
predicted amino acid translation of the nucleotide sequence from
the 1 kb amplicon conformed to the full-length form of Tim-3
(fl-Tim-3) consisting of signal peptide, IgV, mucin, transmembrane
and cytoplasmic domains. Alignment of the amino acid translation of
the nucleotide sequence from the 800 bp amplicon with fl-Tim-3
demonstrated a novel isoform (s-Tim-3) containing only the signal
peptide, IgV and cytoplasmic domains. FIG. 1C Schematic
representation of Tim-3 gene structure, and the alternatively
spliced transcripts corresponding to fl-Tim-3 and s-Tim-3. The
murine Tim-3 gene consists of 7 exons. The fl-Tim-3 transcript is
comprised of all 7 exons. In contrast, the s-Tim-3 transcript is
comprised of only exon 1, exon 2, exon 6 and exon 7. Coding region
signal peptide, IgV, mucin, transmembrane (tm) and cytoplasmic
domains of the transcript are shown.
[0019] FIGS. 2A-2B show that Tim-3-Ligand is expressed on CD4+ T
cells. FIG. 2A Whole spleen cells from C57BL/6 mice were CD4+
column purified, stained with and gated for CD4 expression, and
co-stained for various cell-surface markers (CD25, CD62L, CD44,
CD45RB and CD54, upper and lower graphs in order from left to
right). Cells were co-stained for Tim-3-Ig with biotinylated ex- or
s-Tim-3-Ig, followed by streptavidin-PE as a secondary reagent for
detection. Cells were gated on CD4+, and surface marker positive or
negative populations. Legend: (Thin dashed line) hIgG; (Solid thick
line), s-Tim-3-Ig; and (Thick dotted line), ex-Tim-3-Ig. FIG. 2B
Th1 (AE7) and Th2 (LR1F1) clones were stained with biotinylated
s-Tim-3-Ig or hIgG, followed by streptavidin-PE. Cells were stained
as resting cells (day 0) (upper and lower graphs: left), as
activated cells at 4 days post-activation (day 4) (upper and lower
graphs: center) and as resting cells at 10 days post-activation
(day 10) (upper and lower graphs: right). Legend (Thin dashed line)
hIgG; (Solid thick line), s-Tim-3-Ig;
[0020] FIGS. 3A-3B show that administration of Tim-3-Ig induced
hyperproliferation and spontaneous production of Th1 cytokines.
FIG. 3A SJL/J mice were immunized with PLP 139-151/CFA and injected
intraperitoneally every other day from day 0-8 with 100 p.g
ex-Tim-3-Ig or s-Tim-3-Ig, or hIgG (100 .mu.g) or PBS (100 .mu.l)
as controls. Mice were sacrificed on day 10, and spleens were
removed and cultured in vitro for 48 h without peptide
restimulation. Proliferation was measured in triplicate wells after
48 h by .sup.3H-thymidine incorporation (upper left graph).
Supernatants were taken at 48 h and used in cytokine ELISAs; IL-2
(upper right graph) and IFN-.gamma. (lower graph) production are
shown; no IL-4, IL-10 or TNF-a was detected. Data shown for
individual mice; representative of 10 experiments for proliferation
and five experiments for cytokines. FIG. 3B The spleen cells taken
from immunized, fusion-protein treated mice on day 10 were
stimulated in vitro with 0-100 .mu.g of PLP 139-151 peptide.
Proliferation was measured after 48 h by .sup.3H-thymidine
incorporation in triplicate wells (upper left graph). Supernatants
were taken at 48 h from in vitro cultures of whole spleen cells
restimulated with PLP 139-151 peptide, and cytokine ELISAs for
IL-2, IL-4, IL-10, TNF-.alpha. and IFN-.gamma. were performed. IL-2
(upper right graph) and IFN-.gamma. (lower graph) production are
shown; no IL-4, IL-10 or TNF-.alpha. was detected. Legend:
.tangle-solidup., PBS; .box-solid., hIgG; , ex-Tim-3-Ig; and
.smallcircle., s-Tim-3-Ig-treated. Data shown for individual mice;
representative of 10 experiments for proliferation and five
experiments for cytokines.
[0021] FIGS. 4A-4C show that spontaneous hyperproliferation and
cytokine release is mediated by T cells. FIG. 4A SJL/J mice were
immunized with PLP 139-151/CFA and treated with 100 .mu.g
intraperitoneally (every other day from days 0-8) of ex-Tim-3-Ig or
hIgG as control, and sacrificed on day 10. T, B and CD1 1b.sup.+
cells were purified from the spleen. Either whole splenocytes
(5.times.10.sup.5) or T cells (10.sup.5) were incubated in the
presence or absence of either 2.times.105 B cells, 2.times.10.sup.5
CD1 1b.sup.+ cells or both, and the proliferative response was
measured (3H-thymidine incorporation in triplicate wells). Results
are represented as average for four mice combined; representative
of three experiments. FIG. 4B Supernatants were taken from the in
vitro cultures described in FIG. 4A at 48 h, and cytokine ELISAs
for IL-2, IL-4, IL-10, TNF-.alpha. and IFN-.gamma. were performed.
IL-2 (left graph) and IFN-.gamma. (right graph) production are
shown; no IL-4, IL-10 or TNF-.alpha. was detected. Results are
represented as average for four mice combined. FIG. 4C SJL/J mice
were immunized with PLP 139-151/CFA and given 100 .mu.g
intraperitoneally every other day from day 0-8 of ex-Tim-3-Ig,
s-Tim-3-Ig or hIgG as control, and spleens were taken on day 10.
Whole spleen cells (5.times.10.sup.5 to 10.sup.6 cells) were
cultured in vitro with 10 .mu.M BrdU. After 48 h, cells were
stained with mAbs to CD3 (all dot plots), CD25 (center dot plots),
CD69 (lower dot plots) and BrdU (upper dot plots). Dot plots
(logarithmic scale) represent cell populations and BrdU
incorporation. Results are for individual mice; representative of
four experiments.
[0022] FIG. 5 shows that Tim-3-Ig treatment in vivo inhibited
induction of tolerance. SJL/J mice were immunized with PLP
139-151/CFA and concurrently given intraperitoneal injections of
500 mg soluble PLP 139-151 (or PBS as a control vehicle) to induce
tolerance. Mice were injected intraperitoneally every other day
from day 0-8 with 100 mg ex-Tim-3-Ig or hIgG as control. Mice were
sacrificed on day 10, and lymph nodes were taken and cells
(2.times.10.sup.5/well) cultured in vitro with increasing
concentrations of PLP 139-151 peptide (data shown for 100 mg/ml PLP
139-151). Proliferation was measured in triplicate wells after 48 h
by .sup.3H-thymidine incorporation. Supernatants were taken from
the in vitro cultures at 48 h, and cytokine ELISAs for IL-2, IL-4,
IL-10 and IFN-g were performed. P values were obtained by student's
t test. Legend: .dagger. P<0.01 when non-tolerized (PBS) hIgG
group compared with tolerized (PLP) hIgG group. P<0.01 when
tolerized (PLP) hIgG group compared with tolerized (PLP) Tim-3-Ig
group. .dagger-dbl. P<0.01 when non-tolerized (PBS) hIgG group
compared with tolerized (PLP) hIgG group. .diamond. P<0.05 when
tolerized (PLP) hIgG group compared with tolerized (PLP) Tim-3-Ig
group. .sctn. P<0.5 when non-tolerized (PBS) hIgG group compared
with tolerized (PLP) hIgG group. {hacek over ( )}P<0.1 when
tolerized (PLP) hIgG group compared with tolerized (PLP) Tim-3-Ig
group.
[0023] FIGS. 6A-6B show that administration of Tim-3-Ig prevented
tolerance induction. FIG. 6A SJL/J mice were immunized with PLP
139-151/CFA and concurrently given intraperitoneal injections of
500 .mu.g soluble PLP 139-151 (or PBS as a control vehicle) to
induce tolerance. Mice were injected intraperitoneally every other
day from day 0-8 with 100 .mu.g ex-Tim-3-Ig (right graph), or PBS
(100 .mu.l) (left and right graphs) or hIgG (100 .mu.g) (left
graph) as controls. Mice were sacrificed on day 10, and spleens
were taken and cells (5.times.10.sup.5/well) cultured in vitro with
increasing concentrations of PLP 139-151 peptide. Proliferation was
measured in triplicate wells after 48 h by .sup.3H-thymidine
incorporation. Mice given control PBS as tolerogen, .DELTA., PBS;
.quadrature. hIgG, and .smallcircle., ex-Tim-3-Ig treated; mice
given PLP as tolerogen, .tangle-solidup., PBS, .box-solid.hIgG, and
, ex-Tim-3-Ig treated. Average given for two mice/treatment group.
FIG. 6B Supernatants were taken from the in vitro cultures
described in FIG. 6A at 48 h, and cytokine ELISAs for IL-2 (upper
left graph), IL-4 (lower left graph), IL-10 (lower right graph),
TNF-.alpha. (center graph) and IFN-.gamma. (upper right graph) were
performed. Symbols as described in FIG. 6A. Average given for two
mice/treatment group.
[0024] FIGS. 7A-7B show the expression of TIM-3 and TIM-3L. FIG. 7A
Ex-Tim-3-Ig and, more significantly, the truncated s-TIM-3-Ig
fusion protein bind to resting CD4+ T cells and a portion of CD11c+
but not CD11b+, B220+, or CD8+ T cells. Isotype control staining is
shown in closed histograms; open histograms show binding with
ex-TIM-3-Fc (dotted line) and s-TIM-3-Ig (solid line). FIG. 7B
CD4+CD25-, but not regulatory CD4+C25+, T cells, downregulate
TIM-3L after in vitro stimulation. Both CD4+CD25- and CD4+CD25+ T
cells bind s-TIM-3-Ig in resting condition (upper panel). While no
changes in the expression of TIM-3L are observed after 24 hours of
in vitro stimulation with anti-CD3, anti-CD28 and rIL-2 (middle
panel), at 48 hours TIM-3L is only detected on CD4+CD25+ T cells.
Isotype control staining is shown in closed histograms; open
histograms show binding with s-TIM-3-Ig. Data shown in this figure
are representative of 3 independent experiments.
[0025] FIGS. 8A-8C show that TIM-3 contributes to the tolerizing
effects of DST plus anti-CD154 treatment in an MHC-mismatched islet
allograft model. FIG. 8A TIM-3 (upper graph) and IFN-.gamma. (lower
graph) gene transcripts are upregulated in rejecting but not in
long-term surviving islet allografts. Data are expressed as
relative fold difference between target samples and a calibrator
(isolated islets). Values plotted represent the mean.+-.SE obtained
from 4 independent experiments. FIG. 8B Concurrent administration
of ex-Tim-3-Ig together with DST plus anti-CD154 prevents the
induction of transplantation tolerance to islet allografts. FIG. 8C
Administration of ex-Tim-3-Ig significantly decreases the
tolerance-promoting capacity of CTLA4Ig.
[0026] FIGS. 9A-9D show that TIM-3 modulates the
alloantigen-specific effects of DST plus anti-CD154 treatment on
CD4+CD25+ regulatory T cells. FIG. 9A depletion of CD4+CD25+ T
cells before DST plus anti-CD154 administration results in rapid
islet allograft rejection. MST=median survival time (days). FIG. 9B
ex-Tim-3-Ig does not interfere with the capacity of CD4+CD25+
regulatory T cells to suppress CD4+CD25-T cells in an adoptive
transfer skin allograft model if administered at the time of
transplantation. FIG. 9C 1.times.10.sup.5 CD4+CD25- T cells from
naive or DST plus anti-CD154 treated mice rapidly induce skin
allograft rejection (left hand panel). Both CD4+CD25+ T cells
harvested from naive hosts and CD4+CD25+ T cells obtained from DST
plus anti-CD154 treated mice are capable of preventing skin
allograft rejection when adoptively transferred at high ratios of
regulatory to effector T cells (4.times.10.sup.5:1.times.10.sup.5;
middle panel). Only CD4+CD25+ T cells harvested from treated mice
exert significant graft-protecting effects when transferred with an
equal number of CD4+CD25- T cells (4.times.105:4.times.105; right
hand panel). In the middle and right hand panel CD4+CD25- T cells
are derived only from naive untreated mice. FIG. 9D The concurrent
administration of ex-Tim-3-Ig together with DST plus anti-CD154
abolishes the enhanced immunosuppressive effects conferred to
CD4+CD25+ T cells by DST plus anti-CD154 treatment (the group of
naive CD4+CD25+ T cells is used as baseline for statistical
comparisons). MST=median survival time (days).
[0027] FIGS. 10A-10B show the identification of galectin-9 as a
TIM-3 binding protein. FIG. 10A TK-1 cells express the highest
level of TIM-3 ligand(s). Three T cell lines, TK-1, S49.1G.3PHA
100/0, and BW1547.G.1.4, were stained by biotinylated sTIM-3-Ig
(0.4 mg/ml) at 1:20 (top row graphs) and 1:50 dilutions (center row
graphs), and detected by anti-human IgG-Fc-PE for the TIM-3 ligand
expression. All T cell lines are stained positively by TIM-3
fusions FACS analysis, among which up to 90% of TK-1 were
positively stained by both flTIM-3-Ig and sTIM-3-Ig (bottom row
graphs). FIG. 10B Demonstration of the specific interaction between
a 35 kD protein and TIM-3 by pull-down assays. Extracellular
membrane associated proteins on live TK-1 cells were biotinylated
by NHS-LC-Biotin in PBS pH7.9 buffer at room temperature. Reaction
was stopped by DMEM medium after 1 hour incubation. Cell lysate
from the biotinylated TK-1 cells was used in pull-down experiment
by incubating with 5 .mu.g flTIM-3-Ig, sTIM-3-Ig, TIM-2-Ig, and
hIgG respectively plus Protein G-agarose beads at 4.degree. C.
Protein samples eluted from beads were treated with or without
PNGase F to remove N-linked sugar chains, and applied to SDS-PAGE
and Western blot. Only signals from biotinylated membrane
associated proteins bound to avidin conjugated peroxidase and were
detected by chemiluminescent signal.
[0028] FIGS. 11A-11C show shows specific interaction between
galectin-9 and TIM-3. FIG. 11A Both regular and long isoforms for
galectin-9 (galectin-9 and galectin-9-L respectively) are able to
bind to TIM-3. Protein sequences between two galectin-9 isoforms
are identical except a 31 amino acid peptide insert in the hinge
region of galectin-9-L. Murine cDNAs for isoforms of galectin-9
from TK-1 cells were subcloned into a eukaryotic bicistronic
expression vector pIRES2-EGFP. The plasmids were transiently
transfected into CHO cells respectively. Cells were fixed for
intracellular staining with flTIM-3-Ig (top row graphs), sTIM-3-Ig
(center row graphs), and hIgG (bottom row graphs) and detected by
anti-human IgG-Fc-PE 48 hours after transfection. EGFP positive
signals in FACS analyses indicated transfected cells. The
anti-human IgG-Fc-PE was used to detect the binding from Ig fusions
or hIgG. FIG. 11B Murine cDNAs for galectin-9 (first column
graphs), -4 (second column graphs), -3 (third column graphs), and
-1 (fourth column graphs) were subcloned into pIRES2-EGFP. The
plasmids and the empty vector (fifth column graphs) that only
produces EGFP were transiently transfected into CHO for
intracellular staining by s-TIM-3-Ig respectively. The anti-human
IgG-Fc-PE was used to detect sTIM-3-Ig binding. FIG. 11C Galectin-9
cDNA was subcloned into prokaryotic expression vector pTrcHis 2B.
Recombinant galectin-9 (r-galectin-9) was expressed in BL21 E. coli
strain and purified through lactose agarose column. Two .mu.g of
purified galecin-9 was used to incubate with Ig fusion proteins for
pull-down experiment. Elutes from protein G beads were applied to
SDS-PAGE and stained by Coomassie blue. Lanes 1 to 3 represent
elutes from sTIM-3-Ig, TIM-2-Ig, and TIM-4-Ig and their captured
protein respectively. Lanes 4 to 6 stand for Ig fusion protein
only. The molecular weight of r-galectin-9 is 38 kD.
[0029] FIGS. 12A-12C show galectin-9 recognizes the
.beta.-galactoside bond in oligosaccharide chains of TIM-3. FIG.
12A Galectin-9 expression plasmid was transiently transfected into
CHO cells and fixed for intracellular staining by sTIM-3-Ig.
Incubation buffers with different concentrations from 0 to 100 mM
of .alpha.-lactose were used in the experiments. Bars in the
histograph represent percentage of double positive cell population
for EGFP and sTIM-3-Ig staining in presence of different
concentration of a-lactose. FIG. 12B Two .mu.g of r-galectin-9 was
mixed with 4 .mu.g sTIM-3-Ig in presence of different concentration
of a-lactose from 0 to 100 mM in a pull-down experiment. Elutes
from protein G beads were applied to SDS-PAGE and stained by
Coomassie blue. A band at 50 kD represents sTIM-3-Ig, and the one
at 38 kD stands for r-galectin-9. FIG. 12C CRD domains in
galectin-9 are required for interaction with TIM-3. Constructs with
mutation of R64A in N-terminal CRD domain (second column graphs),
R238A in C-terminal CRD domain (third column graphs), or both
(fourth column graphs) were generated and subcloned into expression
vector pIRES2-EGFP. CHO cells were transiently transfected by wt
(first column graphs) and mutant galection-9 expression plasmids
(second through fourth column graphs) for intracellular staining by
sTIM-3-Ig. EGFP and anti-human IgG-Fc-PE double positive standard
for interaction between galectin-9 and sTIM-3-Ig.
[0030] FIGS. 13A-13B show galectin-9 induces apoptosis in activated
murine Th1 cells and its apoptotic effect is functionally related
to its interaction to TIM-3. FIG. 13A Spleen CD4+CD62L+ T cells
from D011.10 transgenic mice were in vitro polarized into Th1 and
Th2 cells for at least 3 rounds. The Th1 (top row graphs) and Th2
cells (bottom row graphs) 3 day after stimulation by VOA peptide
and APCs were treated with PBS (first column graphs), galectin-3
(second column graphs), and different doses of galectin-9 (third
through fifth column graphs). After 8 hours, cells were stained
with PI and Annexin V-FITC for detecting apoptotic cells. PI
positive populations (both PI+Annexin V+ and PI+Annexin V-) are
dead cells or late stage apoptotic cells. PI-Annexin V+ population
is early stage apoptotic cells; and double negative population is
life cells. FIG. 13B Th1 (right graph) and Th2 (left graphs cells
were treated with PBS, 0.75 mM galectin-9, 0.75 mM galectin-9 for
0, 2, 4, 8, and 12 hours. Cells were harvested for nucleosome
enrichment assays. The fragmented nucleosomal structures released
into cell lysate supernatant are induced by apoptotic procedure,
and were detected by antibodies against histone and genomic DNA in
the ELISA assay and read at OD 405 nm. As in FIG. 13A, Th2 cells
were resistant to apoptotic effects from galectin-9.
[0031] FIGS. 14A-14B show administration of galectin-9 in mice
results in reduction of IFN-.gamma. and IL-2 production by
downregulating IFN-g and IL-2 producing cells. However it does not
affect the whole spleen cell proliferation. C57BL/6J mice were
immunized with 100 mg MOG35-55 peptide plus 200 ml CFA.
R-galectin-9 was i.p. injected to the mice everyday from day 3 to
day 9. Mice were sacrificed and spleen cells were harvested for
cell proliferation assay (FIG. 14A, left graph), ELISA (FIG. 14A,
right graph), and ELISPOT (FIG. 14B, all graphs).
DETAILED DESCRIPTION OF THE INVENTION
(i) Overview
[0032] The present invention relates to reagents, compositions and
methods for modulating the activation of Th1 cells, and for
modulating immune responses, including but not limited to immune
tolerance and transplantation tolerance.
[0033] The invention derives in part from the discovery of a novel
splice isoform of tim-3, in mice and humans, encoding a soluble
form of tim-3 comprising the IgV domain and the intracellular
domain of tim-3, but lacking the mucin and the transmembrane
domain. The invention provides isolated nucleic acids encoding the
tim-3 soluble isoform or fragments thereof. The invention
additionally provides fusion proteins which comprise soluble tim-3
and another polypeptide, such as the IgFc domain of
immunoglobulins. Furthermore, the invention provides reagents which
modulate the expression and/or function of the soluble tim-3
protein but not that of full-length tim-3.
[0034] The invention also derives in part from the discovery that
blocking the activation of tim-3 by a ligand, results in an
increase in Th1 cell activation, including but not limited to, Th1
cell proliferation and production of the cytokines IL-2 and
IFN-.gamma.. Furthermore, the invention derives from the discovery
that tim-3 activity, such as a tim-3 interaction with a ligand, is
required to establish immune tolerance and transplantation
tolerance in a mouse model. In addition, the invention also derives
from the discovery that galectin-9 is a tim-3 ligand. Galectin-9 is
a member of galectin family which is ubiquitously expressed on a
variety of cell types and which binds .beta.-galactoside. Two forms
of galectin-9 have been described in humans, a long and a short
form. The human short isoform lacks 31 amino acids that are located
between the N-terminal carbohydrate-binding domain and the link
peptide in the long isoform (residues 149-180 of the human long
isoform). The human and mouse short galectin-9 amino acid sequences
are listed in SEQ ID NO: 10 and 17 respectively, while their
corresponding DNA coding sequences are listed as SEQ ID NO:9 and
16. The mouse and human long galectin-9 amino acid sequences are
listed in SEQ ID NO: 18 and 19 respectively.
[0035] In the human short galectin-9 isoform, the N- and C-terminal
carbohydrate recognition domains (CRD) stretch from 16-146 and from
195-322, respectively, these are joined by a linker peptide
stretching from 149-174 (See Genbank Accession No.
NP.sub.--002299). The two CRDs in the human long isoform stretch
from residues 16-146 and 227-354, respectively.
[0036] The Galectin-9 amino acid and DNA coding sequences are also
described in U.S. Pat. Nos. 6,468,768 and 6,027,916, hereby
incorporated by reference. The invention thus provides methods for
modulating immune responses, such as increasing or decreasing Th1
and/or Th2 responses, and also provides methods for increasing or
decreasing immune tolerance and transplantation tolerance.
[0037] One aspect of the invention provides an isolated polypeptide
comprising a tim-3 IgV domain and a tim-3 intracellular domain,
wherein the polypeptide does not comprise a tim-3 mucin domain or a
tim-3 transmembrane domain. In specific embodiments, the
polypeptide is a mammalian polypeptide, such as a human or a mouse
polypeptide. In one embodiment, the tim-3 IgV domain comprises
amino acids 22-131 of SEQ ID NO:13 or 22-132 of SEQ ID NO:14. In
one embodiment, the tim-3 intracellular domain comprises amino
acids 226-301 of SEQ ID NO:13 or amino acids 217-281 of SEQ ID
NO:14. In some embodiments, the isolated polypeptide further
comprises the Fc domain of an immunoglobulin or other carrier
protein.
[0038] The invention further provides compositions comprising the
isolated polypeptide described herein. In one embodiment, the
compositions further comprise a pharmaceutically acceptable
carrier. The invention also provides nucleic acids encoding the
polypeptides described herein. The invention also provides an
isolated nucleic acid which hybridizes under high stringency
conditions to a nucleic acid encoding soluble tim-3 but which does
not hydridize under high stringency conditions to a nucleic acid
encoding full-length tim-3, such as a nucleic acid comprising the
sequence set forth in SEQ ID NO: 5.
[0039] Another aspect of the invention provides pharmaceutical
packages. A specific aspect provides a pharmaceutical package
comprising (i) a polypeptide which comprises the IgV domain of
tim-3; and (ii) instructions for administering the composition to a
subject for treating a hyperplastic condition or for treating a
Th2-mediated condition. In a specific embodiment, the hyperplastic
condition is renal cell cancer, Kaposi's sarcoma, chronic leukemia,
prostate cancer, breast cancer, sarcoma, pancreatic cancer,
leukemia, ovarian carcinoma, rectal cancer, throat cancer,
melanoma, colon cancer, bladder cancer, lymphoma, mastocytoma, lung
cancer, mammary adenocarcinoma, pharyngeal squamous cell carcinoma,
testicular cancer, Hodgkin's lymphoma, gastrointestinal cancer, or
stomach cancer.
[0040] In a specific embodiment, the pharmaceutical package
comprising (i) a polypeptide which comprises a galectin-9
polypeptide; and (ii) instructions for administering the
composition to a subject for treating an autoimmune disorder.
[0041] The invention further provides immunological reagents. In
specific aspect provides an isolated antibody or fragment thereof
which binds to a polypeptide having an amino acid sequence set
forth in SEQ ID NO: 2 but which does not bind to a polypeptide
having the amino acid sequence set forth in SEQ ID NO: 13, or that
binds to a polypeptide having an amino acid sequence set forth in
SEQ ID NO: 4 but which does not bind to a polypeptide having the
amino acid sequence set forth in SEQ ID NO: 14, such as an antibody
that binds to a polypeptide comprising an amino acid sequence set
forth in SEQ ID NO: 6 or 8. In preferred embodiments, the antibody
is a monoclonal antibody. The invention further provides hybridoma
cell lines which secretes said monoclonal antibodies.
[0042] On aspect of the invention provides methods of modulating
immune responses in a subject. One aspect of the invention provides
a method of modulating an immune response in a subject in need
thereof, the method comprising administering to the subject a
therapeutically effective amount of an agent that modulates tim-3
activity. In one preferred embodiment, modulating an immune
response comprises increasing a Th1 response or decreasing a Th2
response and wherein the agent decreases tim-3 activity.
[0043] In one embodiment of the methods described herein for
increasing a Th1 response or decreasing a Th2 response by
decreasing tim-3 activity, the subject is afflicted with a
hyperplastic condition or with a Th2-mediated disorder, such as
asthma, an allergy, allergic rhinitis, gastrointestinal allergy,
food allergy, eosinophilia, conjunctivitis or glomerulonephritis.
In another specific embodiment, the agent inhibits expression of
soluble tim-3. The agent that decreases tim-3 activity may be an
antibody or a fragment thereof, such as an antibody that binds to
tim-3, such as one that binds to the extracellular domain of tim-3,
such as to amino acids 30-128 of SEQ ID NO: 13.
[0044] In one embodiment of the methods described herein for
increasing a Th1 response or decreasing a Th2 response by
decreasing tim-3 activity, the agent reduces the binding of
galectin-9 to tim-3. In a specific embodiment, the agent comprises
a polypeptide comprising (i) amino acids 30-128 of SEQ ID NO: 13;
or (iii) an amino acid sequence that is at least 90% identical to
amino acids 30-128 of SEQ ID NO: 13. In some embodiments, the
polypeptide agents are modified to increase their in vivo
stability, such as by pegylation or by fusion to a plasma protein,
such as human serum albumin or an Fc domain of an immunoglobulin.
In a specific embodiment, the agent comprises a polypeptide
comprising the IgV domain of tim-3, the intracellular domain of
tim-3, and the Fc domain of an immunoglobulin, but does not contain
comprise the mucin domain of tim-3 or the transmembrane domain of
tim-3. In a specific embodiment, the agent decreases the expression
level of a tim-3 polypeptide or a galectin-9 polypeptide. In a
specific embodiment, the agent is an double stranded RNA antisense
oligonucleotide. In a specific embodiment, the agent inhibits
binding of full-length tim-3 to galectin-9, such as one that
inhibits binding of (i) a polypeptide comprising amino acids 30-128
of SEQ ID NO: 13; to (ii) galectin-9 (e.g. SED ID NO:10 or in SED
ID NO:19). In another embodiment, the agent that reduces
tim-3/galectin-9 binding comprises a carbohydrate, such as lactose,
.beta.-galactoside, a glycosylated polypeptide, pectin or modified
pectin.
[0045] In one preferred embodiment of the methods for modulating
immune responses in a subject, modulating an immune response
comprises decreasing a Th1 response or increasing a Th2 response,
and wherein the agent increases tim-3 activity. In a specific
embodiment, the subject is afflicted with an autoimmune disease,
with host versus graft disease (HVGD), or the subject is an organ
transplant recipient.
[0046] In one embodiment of the methods described herein for
decreasing a Th1 response or increasing a Th2 response by
increasing tim-3 activity, the agent is an antibody, an antibody
fragment, or a polypeptide. In a specific embodiment, the antibody
is a bispecific antibody specific for tim-3 and galectin-9. In
another specific embodiment, the binding of the agent to tim-3
increases the phosphorylation of the intracellular domain of tim-3.
In another specific embodiment, the agent is a tim-3 ligand, such
as a recombinant version of a naturally occurring ligand. In
another specific embodiment, the binding of the tim-3 ligand to
full-length tim-3 increases the phosphorylation of the
intracellular domain of tim-3. In another specific embodiment, the
tim-3 ligand comprises a galectin-9 polypeptide. In another
specific embodiment, the agent is a polypeptide comprising at least
one of the two carbohydrate recognition domains (CRD) of
galectin-9. In another specific embodiment, the polypeptide
comprises two CRD domains of galectin-9. In another specific
embodiment, the agent comprises a polypeptide comprising an amino
acid sequence which is at least 80%, 90% or 95% identical to the
amino acid sequence set forth in SEQ ID NO:10 or SEQ NO:18.
[0047] One aspect of the invention provides methods of identifying
agents that modulates the binding between a tim-3 polypeptide and a
galectin-9 polypeptide. One specific aspect provides a method of
identifying an agent that modulates the binding between a tim-3
polypeptide and a galectin-9 polypeptide, the method comprising:
(a) contacting the tim-3 polypeptide and the galectin-9 polypeptide
in the presence of a test agent; and (b) determining the effect of
the test agent on the binding of the tim-3 polypeptide and the
galectin-9 polypeptide; thereby identifying a agent that modulates
the binding between a tim-3 polypeptide and a galectin-9
polypeptide.
[0048] Another aspects provides a method of identifying an agent
that modulates an immune response, such as a method comprising (a)
contacting the tim-3 polypeptide and the galectin-9 polypeptide in
the presence of a test agent; and (b) determining the effect of the
test agent on the binding of the tim-3 polypeptide and the
galectin-9 polypeptide. In some embodiments, step (b) comprises
comparing formation of a tim-3/galectin-9 complex in the presence
of the test agent with an appropriate control. An appropriate
control may comprise the formation of a complex between the first
polypeptide and the second polypeptide in the absence of the test
agent. The agent may increases or decrease the binding between
tim-3 and galectin-9, and may comprise a small compound, an
antibody or a polypeptide. In some embodiments of the methods for
identifying an agent that modulates an immune response, the immune
response is a Th1 immune response or a Th2 immune response.
[0049] The methods for identifying an agent that modulates an
immune response or to identify agents that modulates the binding
between a tim-3 polypeptide and a galectin-9 polypeptide may be
carried out in vitro or in vivo. In a specific embodiment, the
tim-3 polypeptide or the galectin-9 polypeptide or both are
expressed in a cell. In a specific embodiment, detecting the
formation of the complex comprises detecting the expression of a
reporter gene, wherein the expression of the reporter gene is
dependent on the formation of the complex. In another specific
embodiment, the tim-3 polypeptide or the galectin-9 polypeptide or
both are labeled with a fluorescent molecule. In another specific
embodiment, the galectin-9 polypeptide in the foregoing methods
comprises (i) amino acids 1-323 of SEQ ID NO: 10; or (ii) amino
acids 1-355 of SEQ ID NO: 19; or (iii) an amino acid sequence that
is at least 90% identical to amino acids 1-323 of SEQ ID NO: 10;
(iii) an amino acid sequence that is at least 90% identical to
amino acids 1-355 of SEQ ID NO: 19. In another specific embodiment,
the tim-3 polypeptide of the foregoing methods comprises (i) amino
acids 30-128 of SEQ ID NO: 13; or (ii) an amino acid sequence that
is at least 90% identical to amino acids 30-128 of SEQ ID NO:
13.
[0050] The invention further provides a method of conducting a drug
discovery business comprising: (a) identifying compounds that
affect the binding between tim-3 and galectin-9; (b) conducting
therapeutic profiling of compounds identified in step (a), or
further analogs thereof, for efficacy and toxicity in animals; and
(c) formulating a pharmaceutical preparation including one or more
compounds identified in step (b) as having an acceptable
therapeutic profile. The invention further provides a method of
conducting a drug discovery business comprising: (a) identifying
compounds that affect the binding between tim-3 and galectin-9; (b)
optionally conducting therapeutic profiling of compounds identified
in step (a), or further analogs thereof, for efficacy and toxicity
in animals; and (c) licensing, to a third party, the rights for
further drug development and/or sales for compounds identified in
step (a), or analogs thereof. A specific embodiment further
comprises collecting royalties based on sales of said compounds
identified in step (a) or analogs thereof.
[0051] The invention further provides method of increasing tim-3
activity, comprising contacting a cell which expresses soluble
tim-3 with an amount of a double stranded RNA sufficient to
decrease the expression of soluble tim-3, wherein the double
stranded RNA does not inhibit the expression of full-length tim-3
in the cell, thereby increasing tim-3 activity. In a specific
embodiment, the double stranded RNA hybridizes under high
stringency conditions to a nucleic acid comprising SEQ ID NO: 1 but
does not hybridize under high stringency conditions to a nucleic
acid comprising SEQ ID NO: 11. In another specific embodiment, the
double stranded RNA comprises the nucleic acid sequence set forth
in SEQ ID NO: 15. In another specific embodiment, the double
stranded RNA is an siRNA or a hairpin RNA. In another specific
embodiment, the contacting is effected by administering the double
stranded RNA to a subject.
[0052] The invention further provides a method of detecting soluble
tim-3 gene expression comprising detecting the presence of a
nucleic acid encoding soluble tim-3, wherein the detection of a
nucleic acid encoding the soluble tim-3 indicates soluble tim-3
gene expression.
[0053] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are described in the literature. See, for
example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et
al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins eds. 1984); Transcription And Translation
(B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal
Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells
And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To
Molecular Cloning (1984); the treatise, Methods In Enzymology
(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian
Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). The
contents of these reference is hereby incorporated by reference in
their entirety.
(2) Definitions
[0054] For convenience, certain terms employed in the
specification, examples, and appended claims, are collected here.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0055] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0056] The term "including" is used herein to mean, and is used
interchangeably with, the phrase "including but not limited"
to.
[0057] The term "or" is used herein to mean, and is used
interchangeably with, the term "and/or," unless context clearly
indicates otherwise.
[0058] The term "such as" is used herein to mean, and is used
interchangeably, with the phrase "such as but not limited to".
[0059] The term "nucleic acid" refers to polynucleotides such as
deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic
acid (RNA). The term should also be understood to include, as
equivalents, analogs of either RNA or DNA made from nucleotide
analogs, and, as applicable to the embodiment being described,
single (sense or antisense) and double-stranded
polynucleotides.
[0060] The terms "polypeptide" and "protein" are used
interchangeably herein. The term "preventing" is art-recognized,
and when used in relation to a condition, such as a local
recurrence (e.g., pain), a disease such as cancer, a syndrome
complex such as heart failure or any other medical condition, is
well understood in the art, and includes administering, prior to
onset of the condition, a composition that reduces the frequency
of, reduces the severity of, or delays the onset of symptoms of a
medical condition in a subject relative to a subject which does not
receive the composition. Thus, prevention of cancer includes, for
example, reducing the number of detectable cancerous growths in a
population of patients receiving a prophylactic treatment relative
to an untreated control population, and/or delaying the appearance
of detectable cancerous growths in a treated population versus an
untreated control population, e.g., by a statistically and/or
clinically significant amount. Prevention of an infection includes,
for example, reducing the number of diagnoses of the infection in a
treated population versus an untreated control population, and/or
delaying the onset of symptoms of the infection in a treated
population versus an untreated control population. Prevention of
pain includes, for example, reducing the frequency of, reducing the
severity of, or alternatively delaying, pain sensations experienced
by subjects in a treated population versus an untreated control
population.
[0061] The term "effective amount" as used herein is defined as an
amount effective, at dosages and for periods of time necessary to
achieve the desired result. The effective amount of a compound of
the invention may vary according to factors such as the disease
state, age, sex, and weight of the animal. Dosage regimens 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.
[0062] A "subject" as used herein refers to any vertebrate animal,
preferably a mammal, and more preferably a human. Examples of
subjects include humans, non-human primates, rodents, guinea pigs,
rabbits, sheep, pigs, goats, cows, horses, dogs, cats, birds, and
fish.
[0063] A "variant" of a protein of interest, as used herein, refers
to an amino acid sequence that is altered by one or more amino
acids. The variant may have "conservative" changes, wherein a
substituted amino acid has similar structural or chemical
properties, (e.g., replacement of leucine with isoleucine). More
rarely, a variant may have "nonconservative" changes (e.g.,
replacement of a glycine with a tryptophan). Similar minor
variations may also include amino acid deletions or insertions, or
both. Guidance in determining which amino acid residues may be
substituted, inserted, or deleted without abolishing biological or
immunological activity may be found using computer programs well
known in the art, for example, DNASTAR software.
[0064] As used herein, a "Th1-associated disorder" is a disease or
condition associated with aberrant, e.g., increased TM cell
activity (e.g., increased Th1 cell responses) or number compared to
a reference, e.g., a normal control. Examples of Th1-associated
disorders include, e.g., autoimmune disorders (e.g., multiple
sclerosis, rheumatoid arthritis, type I diabetes and Crohn's
disease.
[0065] As used herein, a "Th2-associated disorder" is a disease or
condition associated with aberrant, e.g., increased Th2 cell
activity (e.g., increased Th2 cell responses) or number compared to
a reference, e.g., a normal control. Examples of Th2 disorders
include, e.g., asthma, allergy, and disorders associated with
antibody components (e.g., rheumatoid arthritis).
[0066] The term "analog" as used herein includes, but is not
limited, to amino acid sequences containing one or more amino acid
substitutions, insertions, and/or deletions from a reference
sequence. Amino acid substitutions may be of a conserved or
non-conserved nature. Conserved amino acid substitutions involve
replacing one or more amino acids of the proteins of the invention
with amino acids of similar charge, size, and/or hydrophobicity
characteristics. When only conserved substitutions are made the
resulting analog should be functionally equivalent. Non-conserved
substitutions involve replacing one or more amino acids of the
amino acid sequence with one or more amino acids which possess
dissimilar charge, size, and/or hydrophobicity characteristics.
Amino acid insertions may consist of single amino acid residues or
sequential amino acids ranging from 2 to 15 amino acids in length.
Deletions may consist of the removal of one or more amino acids, or
discrete portions from the amino acid sequence. The deleted amino
acids may or may not be contiguous.
[0067] The term "recombinant" is used herein to mean any nucleic
acid comprising sequences which are not adjacent in nature. A
recombinant nucleic acid may be generated in vitro, for example by
using the methods of molecular biology, or in vivo, for example by
insertion of a nucleic acid at a novel chromosomal location by
homologous or non-homologous recombination.
[0068] The term "agonist" refers to an agent that mimics or
up-regulates (e.g., potentiates or supplements) the bioactivity of
a protein, e.g., polypeptide X. An agonist may be a wild-type
protein or derivative thereof having at least one bioactivity of
the wild-type protein. An agonist may also be a compound that
upregulates expression of a gene or which increases at least one
bioactivity of a protein. An agonist may also be a compound which
increases the interaction of a polypeptide with another molecule,
e.g., a target peptide or nucleic acid.
[0069] The term "antagonist" refers to an agent that downregulates
(e.g., suppresses or inhibits) at least one bioactivity of a
protein. An antagonist may be a compound which inhibits or
decreases the interaction between a protein and another molecule,
e.g., a target peptide or enzyme substrate. An antagonist may also
be a compound that downregulates expression of a gene or which
reduces the amount of expressed protein present.
[0070] The term "therapeutic effect" refers to a local or systemic
effect in animals, particularly mammals, and more particularly
humans caused by a pharmacologically active substance. The term
thus means any substance intended for use in the diagnosis, cure,
mitigation, treatment or prevention of disease or in the
enhancement of desirable physical or mental development and
conditions in an animal or human. The phrase
"therapeutically-effective amount" means that amount of such a
substance that produces some desired local or systemic effect at a
reasonable benefit/risk ratio applicable to any treatment. In
certain embodiments, a therapeutically-effective amount of a
compound will depend on its therapeutic index, solubility, and the
like. For example, certain compounds discovered by the methods of
the present invention may be administered in a sufficient amount to
produce a reasonable benefit/risk ratio applicable to such
treatment.
[0071] The term "subject in need of treatment for a disorder" is a
subject diagnosed with that disorder or suspected of having that
disorder.
[0072] Other technical terms used herein have their ordinary
meaning in the art that they are used, as exemplified by a variety
of technical dictionaries, such as the McGraw-Hill Dictionary of
Chemical Terms and the Stedman's Medical Dictionary.
(3) Nucleic Acids
[0073] In certain aspects, the invention provides isolated and/or
recombinant nucleic acids encoding human soluble tim-3 polypeptides
or fragments thereof, such as, for example, SEQ ID NO: 1 and 5. One
aspect of the invention provides an isolated nucleic acid
comprising the nucleotide sequence set forth in SEQ ID NO:1. SEQ ID
NO:1 is the DNA coding sequence of the human soluble tim-3 isoform.
Human soluble tim-3 comprises the IgV domain (residues 22-131 of
SEQ ID NO:13) fused to the intracellular domain (residues 226-301
of SEQ ID NO:13) of the human full-length tim-3. In one embodiment,
the soluble human tim-3 nucleic acid provided by the present
invention additionally encodes the signal sequence of full length
tim-3 (amino acid residues 1-21 of SEQ ID NO:13) at the N-terminus
of the translated polypeptide. Another aspect of the invention
provides an isolated nucleic acid encoding a human soluble tim-3
polypeptide comprising the amino acid sequence set forth in SEQ ID
NO:2.
[0074] Similarly, another aspect of the invention provides isolated
and/or recombinant nucleic acids encoding mouse soluble tim-3
polypeptides or fragments thereof, such as, for example, SEQ ID NO:
3 and 7. One aspect of the invention provides an isolated nucleic
acid comprising the nucleotide sequence set forth in SEQ ID NO:3.
SEQ ID NO:3 is the DNA coding sequence of the mouse soluble tim-3
isoform. Mouse soluble tim-3 contains the IgV domain (residues
22-132 of SEQ ID NO:14) fused to the intracellular domain (residues
217-281 of SEQ ID NO:14) of the mouse full-length tim-3. In one
embodiment, the mouse soluble tim-3 nucleic acids provided by the
present invention additionally encode the signal sequence of full
length tim-3 (amino acid residues 1-21 of SEQ ID NO:14) at the
N-terminus of the translated polypeptide. Another aspect of the
invention provides an isolated nucleic acid encoding a mouse
soluble tim-3 polypeptide comprising the amino acid sequence set
forth in SEQ ID NO:4.
[0075] Nucleic acids of the invention are further understood to
include nucleic acids that comprise variants of SEQ ID NO: 1, 3, 5,
and 7. Variant nucleotide sequences include sequences that differ
by one or more nucleotides such as by substitutions, additions or
deletions, such as allelic variants; and will, therefore, include
coding sequences that differ from the nucleotide sequence of the
coding sequence designated in SEQ ID NO: 1, 3, 5, and 7, e.g. due
to the degeneracy of the genetic code. For example, nucleic acids
encoding soluble tim-3 polypeptides may be nucleic acids comprising
a sequence that is at least 90%, 95%, 99% or 100% identical to the
sequence of SEQ ID NO: 1 and 3, or a sequence that encodes the
polypeptide of SEQ ID NO: 2 and 4.
[0076] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991).
[0077] In one preferred embodiment, the percent identity between
two amino acid sequences is determined using the Needleman and
Wunsch (J Mol. Biol. (48):444-453 (1970)) algorithm which has been
incorporated into the GAP program in the GCG software package
(available at http://www.gcg.com), using either a Blossom 62 matrix
or a PAM250 matrix.
[0078] In yet another embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (Devereux, J., et al., Nucleic Acids Res.
12(1):387 (1984)) (available at http://www.gcg.com), using a
NWSgapdna-CMP matrix. In another embodiment, the percent identity
between two amino acid or nucleotide sequences is determined using
the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989))
which has been incorporated into the ALIGN program (version
2.0).
[0079] In other embodiments, variants will also include sequences
that hybridize under highly stringent conditions to a coding
sequence of a nucleic acid sequence designated in SEQ ID NO: 1 and
3. In one embodiment, the variant nucleotide sequences encode a
soluble tim-3 polypeptide which lacks a mucin and a transmembrane
domain. In another embodiment, the variant nucleotide sequences
encode a soluble tim-3 polypeptide capable of binding tim-3
ligands, such as but not limited to galectin-9.
[0080] One of ordinary skill in the art will understand readily
that appropriate stringency conditions which promote DNA
hybridization can be varied. For example, one could perform the
hybridization at 6.0.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by a wash of 2.0.times.SSC at
50.degree. C. For example, the salt concentration in the wash step
can be selected from a low stringency of about 2.0.times.SSC at
50.degree. C. to a high stringency of about 0.2.times.SSC at
50.degree. C. In addition, the temperature in the wash step can be
increased from low stringency conditions at room temperature, about
22.degree. C., to high stringency conditions at about 65.degree. C.
Both temperature and salt may be varied, or temperature or salt
concentration may be held constant while the other variable is
changed. In one embodiment, the invention provides nucleic acids
which hybridize under low stringency conditions of 6.times.SSC at
room temperature followed by a wash at 2.times.SSC at room
temperature.
[0081] Isolated nucleic acids which differ from SEQ ID NO: 1 and 3
due to degeneracy in the genetic code are also within the scope of
the invention. For example, a number of amino acids are designated
by more than one triplet. Codons that specify the same amino acid,
or synonyms (for example, CAU and CAC are synonyms for histidine)
may result in "silent" mutations which do not affect the amino acid
sequence of the protein. One skilled in the art will appreciate
that these variations in one or more nucleotides of the nucleic
acids encoding a particular protein may exist among individuals of
a given species due to natural allelic variation. Any and all such
nucleotide variations and resulting amino acid polymorphisms are
within the scope of this invention.
[0082] The invention also provides nucleic acids which hybridize
under high stringency conditions to nucleic acids encoding soluble
tim-3, and which do not encode the tim-3 transmembrane or the tim-3
mucin domain. In one embodiment, these nucleic acids encode at
least the IgV domain of tim-3. These nucleic acids may encode
soluble tim-3 proteins having mutations, deletions or insertions
within the IgV or intracellular domains. In one embodiment, these
nucleic acids encode polypeptides capable of binding to tim-3
ligands, such as galectin-9. In one embodiment, these nucleic acids
encode fusion proteins comprising soluble tim-3, with or without
mutations, and one or more domains from another protein(s), such as
serum albumin or the Fc domain of an immunoglobulin.
[0083] The invention also provides nucleic acids which hybridize
under high stringency or under physiological conditions to nucleic
acids encoding soluble tim-3 but which do not hybridize under high
stringency or under physiological conditions to nucleic acids
encoding full-length tim-3. In one embodiment, the invention
provides an isolated nucleic acid which hybridizes under high
stringency conditions to a nucleic acid set forth in SEQ ID NO:1
(encoding human soluble tim-3) but not to a nucleic acid set forth
in SEQ ID NO 11 (encoding full-length human tim-3). In another
embodiment, the invention provides an isolated nucleic acid which
hybridizes under high stringency conditions to a nucleic acid set
forth in SEQ ID NO:3 (encoding mouse soluble tim-3) but not to a
nucleic acid set forth in SEQ ID NO 12 (encoding full-length mouse
tim-3). In a preferred embodiment, such isolated nucleic acids span
the novel splice junction found in the soluble tim-3 cDNA that
joins the IgV and intracellular domain-coding sequences of human
and mouse tim-3. In another preferred embodiment, the nucleic acids
have the nucleotide sequence set forth in SEQ ID NO: 5 and 7. These
nucleic acids can be used in the methods described herein to detect
nucleic acids encoding soluble tim-3, in detecting the presence of
nucleic acids encoding tim-3 in a Th1 cell.
[0084] In certain aspects, nucleic acids encoding soluble tim-3
polypeptides and variants thereof may be used to increase soluble
tim-3 expression in an organism or cell by delivery of the nucleic
acid, such as by direct delivery. A nucleic acid therapy construct
of the present invention can be delivered, for example, as an
expression plasmid which, when transcribed in the cell, produces
RNA which encodes a soluble tim-3 polypeptide.
[0085] The invention also provides methods of detecting soluble
tim-3 gene expression comprising detecting the presence of a
nucleic acid encoding soluble tim-3, wherein the detection of a
nucleic acid encoding the soluble tim-3 indicates soluble tim-3
gene expression. In a preferred embodiment, the nucleic acid is an
mRNA. Detection of an mRNA encoding soluble tim-3 can be done using
methods commonly known in the art for the detection of mRNA
molecules, including northern blots, PCR-amplification of reverse
transcribed mRNA, or DNA microarrays. In another embodiment,
soluble tim-3 gene expression is detected using an antibody,
preferably on antibody that binds to soluble tim-3 but not to
full-length tim-3.
(4) RNA Interference, Ribozymes and Antisense
[0086] In certain aspects, the invention relates to RNAi, ribozyme,
antisense and other nucleic acid-related methods and compositions
for manipulating (typically decreasing the gene expression)
full-length tim-3, soluble tim-3 or tim-3 ligands. In one aspect of
the invention, the methods and compositions are specific to only
one form of tim-3, either the full-length or the soluble form. In
another aspect of the invention, the tim-3 ligand that is
manipulated is galectin-9.
[0087] Certain embodiments of the invention make use of materials
and methods for effecting knockdown of one form of tim-3 or of its
ligands, such as galectin-9, by means of RNA interference (RNAi).
RNAi is a process of sequence-specific post-transcriptional gene
repression which can occur in eukaryotic cells. In general, this
process involves degradation of an mRNA of a particular sequence
induced by double-stranded RNA (dsRNA) that is homologous to that
sequence. For example, the expression of a long dsRNA corresponding
to the sequence of a particular single-stranded mRNA (ss mRNA) will
labilize that message, thereby "interfering" with expression of the
corresponding gene. Accordingly, any selected gene may be repressed
by introducing a dsRNA which corresponds to all or a substantial
part of the mRNA for that gene. It appears that when a long dsRNA
is expressed, it is initially processed by a ribonuclease III into
shorter dsRNA oligonucleotides of in some instances as few as 21 to
22 base pairs in length. Furthermore, RNAi may be effected by
introduction or expression of relatively short homologous dsRNAs.
Indeed the use of relatively short homologous dsRNAs may have
certain advantages as discussed below.
[0088] Mammalian cells have at least two pathways that are affected
by double-stranded RNA (dsRNA). In the RNAi (sequence-specific)
pathway, the initiating dsRNA is first broken into short
interfering (si) RNAs, as described above. The siRNAs have sense
and antisense strands of about 21 nucleotides that form
approximately 19 nucleotide si RNAs with overhangs of two
nucleotides at each 3' end. Short interfering RNAs are thought to
provide the sequence information that allows a specific messenger
RNA to be targeted for degradation. In contrast, the nonspecific
pathway is triggered by dsRNA of any sequence, as long as it is at
least about 30 base pairs in length. The nonspecific effects occur
because dsRNA activates two enzymes: PKR, which in its active form
phosphorylates the translation initiation factor elF2 to shut down
all protein synthesis, and 2',5' oligoadenylate synthetase
(2',5'-AS), which synthesizes a molecule that activates RNAse L, a
nonspecific enzyme that targets all mRNAs. The nonspecific pathway
may represents a host response to stress or viral infection, and,
in general, the effects of the nonspecific pathway are preferably
minimized under preferred methods of the present invention.
Significantly, longer dsRNAs appear to be required to induce the
nonspecific pathway and, accordingly, dsRNAs shorter than about 30
bases pairs are preferred to effect gene repression by RNAi (see
Hunter et al. (1975) J Biol Chem 250: 409-17; Manche et al. (1992)
Mol Cell Biol 12: 5239-48; Minks et al. (1979) J Biol Chem 254:
10180-3; and Elbashir et al. (2001) Nature 411: 494-8).
[0089] RNAi has been shown to be effective in reducing or
eliminating the expression of a gene in a number of different
organisms including Caenorhabditis elegans (see e.g. Fire et al.
(1998) Nature 391: 806-11), mouse eggs and embryos (Wianny et al.
(2000) Nature Cell Biol 2: 70-5; Svoboda et al. (2000) Development
127: 4147-56), and cultured RAT-1 fibroblasts (Bahramina et al.
(1999) Mol Cell Biol 19: 274-83), and appears to be an anciently
evolved pathway available in eukaryotic plants and animals (Sharp
(2001) Genes Dev. 15: 485-90). RNAi has proven to be an effective
means of decreasing gene expression in a variety of cell types
including HeLa cells, NIF1/3T3 cells, COS cells, 293 cells and
BHK-21 cells, and typically decreases expression of a gene to lower
levels than that achieved using antisense techniques and, indeed,
frequently eliminates expression entirely (see Bass (2001) Nature
411: 428-9). In mammalian cells, siRNAs are effective at
concentrations that are several orders of magnitude below the
concentrations typically used in antisense experiments (Elbashir et
al. (2001) Nature 411: 494-8).
[0090] The double stranded oligonucleotides used to effect RNAi are
preferably less than 30 base pairs in length and, more preferably,
comprise about 25, 24, 23, 22, 21, 20, 19, 18 or 17 base pairs of
ribonucleic acid. Optionally the dsRNA oligonucleotides of the
invention may include 3' overhang ends. Exemplary 2-nucleotide 3'
overhangs may be composed of ribonucleotide residues of any type
and may even be composed of 2'-deoxythymidine resides, which lowers
the cost of RNA synthesis and may enhance nuclease resistance of
siRNAs in the cell culture medium and within transfected cells (see
Elbashi et al. (2001) Nature 411: 494-8). Longer dsRNAs of 50, 75,
100 or even 500 base pairs or more may also be utilized in certain
embodiments of the invention. Exemplary concentrations of dsRNAs
for effecting RNAi are about 0.05 nM, 0.1 nM, 0.5 nM, 1.0 nM, 1.5
nM, 25 nM or 100 nM, although other concentrations may be utilized
depending upon the nature of the cells treated, the gene target and
other factors readily discernable to the skilled artisan. Exemplary
dsRNAs may be synthesized chemically or produced in vitro or in
vivo using appropriate expression vectors. Exemplary synthetic RNAs
include 21 nucleotide RNAs chemically synthesized using methods
known in the art (e.g. Expedite RNA phosphoramidites and thymidine
phosphoramidite (Proligo, Germany). Synthetic oligonucleotides are
preferably deprotected and gel-purified using methods known in the
art (see e.g. Elbashir et al. (2001) Genes Dev. 15: 188-200).
Longer RNAs may be transcribed from promoters, such as T7 RNA
polymerase promoters, known in the art. A single RNA target, placed
in both possible orientations downstream of an in vitro promoter,
will transcribe both strands of the target to create a dsRNA
oligonucleotide of the desired target sequence.
[0091] If tim-3 or galectin-9 is the target of the double stranded
RNA, any of the above RNA species will be designed to include a
portion of nucleic acid sequence represented in a tim-3 nucleic
acid or a galectin-9 nucleic acid, such as, for example, a nucleic
acid that hybridizes, under stringent and/or physiological
conditions, to any of SEQ ID NO: 1, 3, 9, 10, 11 or 12 or
complements thereof. If the goal is to reduce the activity of
full-length tim-3, a double stranded RNA reagent that can
specifically downregulate the expression of full-length tim-3 but
not soluble tim-3 is preferred. Thus, the RNA reagent can decrease
the level of full-length tim-3, while soluble tim-3 can titrate
tim-3 ligands, such as galectin-9, to prevent their interaction
with full-length tim-3. In this embodiment, the RNA species can be
designed to include regions of the mRNA which encode the mucin or
transmembrane domains, which are present in full-length tim-3 but
absent in soluble tim-3.
[0092] A related aspect of the invention provides a method of
increasing tim-3 activity by inhibiting expression of soluble
tim-3, comprising contacting a cell which expresses soluble tim-3
with an amount of a double stranded RNA sufficient to inhibit the
expression of soluble tim-3, wherein the double stranded RNA does
not inhibit expression of full-length tim-3. In one embodiment, the
double stranded RNA hybridizes under high stringency conditions to
a nucleic acid comprising SEQ ID NO:1 but does not hybridize under
the same conditions to a nucleic acid comprising SEQ ID NO:11. In
another embodiment, the double stranded RNA hybridizes under
physiological conditions to a nucleic acid comprising SEQ ID NO:1
but does not hybridize under the same conditions to a nucleic acid
comprising SEQ ID NO:11. In a preferred embodiment, one strand of
the double stranded RNA comprises the nucleic acid sequence set
forth in SEQ ID NO:15. The methods of the present invention can be
performed in isolated cells expressing tim-3 or in a subject,
preferably a human.
[0093] When the target is galectin-9, the RNA species may include a
portion of nucleic acid sequence represented in a galectin-9
nucleic acid, such as, for example, a nucleic acid that hybridizes
under stringent and/or physiological conditions to SEQ ID NO: 9 or
to a complement thereof.
[0094] The specific sequence utilized in design of the
oligonucleotides may be any contiguous sequence of nucleotides
contained within the expressed gene message of the target. Programs
and algorithms, known in the art, may be used to select appropriate
target sequences. In addition, optimal sequences may be selected
utilizing programs designed to predict the secondary structure of a
specified single stranded nucleic acid sequence and allowing
selection of those sequences likely to occur in exposed single
stranded regions of a folded mRNA. Methods and compositions for
designing appropriate oligonucleotides may be found, for example,
in U.S. Pat. No. 6,251,588, the contents of which are incorporated
herein by reference. Messenger RNA (mRNA) is generally thought of
as a linear molecule which contains the information for directing
protein synthesis within the sequence of ribonucleotides, however
studies have revealed a number of secondary and tertiary structures
that exist in most mRNAs. Secondary structure elements in RNA are
formed largely by Watson-Crick type interactions between different
regions of the same RNA molecule. Important secondary structural
elements include intramolecular double stranded regions, hairpin
loops, bulges in duplex RNA and internal loops. Tertiary structural
elements are formed when secondary structural elements come in
contact with each other or with single stranded regions to produce
a more complex three dimensional structure. A number of researchers
have measured the binding energies of a large number of RNA duplex
structures and have derived a set of rules which can be used to
predict the secondary structure of RNA (see e.g. Jaeger et al.
(1989) Proc. Natl. Acad. Sci. USA 86:7706 (1989); and Turner et al.
(1988) Annu. Rev. Biophys. Biophys. Chem. 17:167). The rules are
useful in identification of RNA structural elements and, in
particular, for identifying single stranded RNA regions which may
represent preferred segments of the mRNA to target for silencing
RNAi, ribozyme or antisense technologies. Accordingly, preferred
segments of the mRNA target can be identified for design of the
RNAi mediating dsRNA oligonucleotides as well as for design of
appropriate ribozyme and hammerhead ribozyme compositions of the
invention.
[0095] The dsRNA oligonucleotides may be introduced into the cell
by transfection with an heterologous target gene using carrier
compositions such as liposomes, which are known in the art--e.g.
Lipofectamine 2000 (Life Technologies) as described by the
manufacturer for adherent cell lines. Transfection of dsRNA
oligonucleotides for targeting endogenous genes may be carried out
using Oligofectamine (Life Technologies). Transfection efficiency
may be checked using fluorescence microscopy for mammalian cell
lines after co-transfection of hGFP-encoding pAD3 (Kehlenback et
al. (1998) J Cell Biol 141: 863-74). The effectiveness of the RNAi
may be assessed by any of a number of assays following introduction
of the dsRNAs. These include Western blot analysis using antibodies
which recognize the tim-3 polypeptides, including the soluble
and/or full-length forms, or tim-3 ligands, such as galectin-9,
following sufficient time for turnover of the endogenous pool after
new protein synthesis is repressed, reverse transcriptase
polymerase chain reaction and Northern blot analysis to determine
the level of existing target mRNA, such as full-length tim-3,
soluble tim-3 or galectin-9 mRNA.
[0096] Further compositions, methods and applications of RNAi
technology are provided in U.S. Pat. Nos. 6,278,039, 5,723,750 and
5,244,805, which are incorporated herein by reference.
[0097] Ribozyme molecules designed to catalytically cleave tim-3 or
galectin-9 mRNA transcripts can also be used to prevent translation
of subject tim-3 or galectin mRNAs (see, e.g., PCT International
Publication WO90/11364, published Oct. 4, 1990; Sarver et al.
(1990) Science 247:1222-1225 and U.S. Pat. No. 5,093,246).
Ribozymes are enzymatic RNA molecules capable of catalyzing the
specific cleavage of RNA. (For a review, see Rossi (1994) Current
Biology 4: 469-471). The mechanism of ribozyme action involves
sequence specific hybridization of the ribozyme molecule to
complementary target RNA, followed by an endonucleolytic cleavage
event. The composition of ribozyme molecules preferably includes
one or more sequences complementary to a tim-3 or a galectin-9
mRNA, and the well known catalytic sequence responsible for mRNA
cleavage or a functionally equivalent sequence (see, e.g., U.S.
Pat. No. 5,093,246, which is incorporated herein by reference in
its entirety).
[0098] While ribozymes that cleave mRNA at site specific
recognition sequences can be used to destroy target mRNAs, the use
of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave
mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. Preferably, the
target mRNA has the following sequence of two bases: 5'-UG-3'. The
construction and production of hammerhead ribozymes is well known
in the art and is described more fully in Haseloff and Gerlach
(1988) Nature 334:585-591; and see PCT Appln. No. WO89/05852, the
contents of which are incorporated herein by reference). Hammerhead
ribozyme sequences can be embedded in a stable RNA such as a
transfer RNA (tRNA) to increase cleavage efficiency in vivo
(Perriman et al. (1995) Proc. Natl. Acad. Sci. USA, 92: 6175-79; de
Feyter, and Gaudron, Methods in Molecular Biology, Vol. 74, Chapter
43, "Expressing Ribozymes in Plants", Edited by Turner, P. C,
Humana Press Inc., Totowa, N.J.). In particular, RNA polymerase
III-mediated expression of tRNA fusion ribozymes are well known in
the art (see Kawasaki et al. (1998) Nature 393: 284-9; Kuwabara et
al. (1998) Nature Biotechnol. 16: 961-5; and Kuwabara et al. (1998)
Mol. Cell 2: 617-27; Koseki et al. (1999) J Virol 73: 1868-77;
Kuwabara et al. (1999) Proc Natl Acad Sci USA 96: 1886-91; Tanabe
et al. (2000) Nature 406: 473-4). There are typically a number of
potential hammerhead ribozyme cleavage sites within a given target
cDNA sequence. Preferably the ribozyme is engineered so that the
cleavage recognition site is located near the 5' end of the target
mRNA- to increase efficiency and minimize the intracellular
accumulation of non-functional mRNA transcripts. Furthermore, the
use of any cleavage recognition site located in the target sequence
encoding different portions of the C-terminal amino acid domains
of, for example, long and short forms of target would allow the
selective targeting of one or the other form of the target, and
thus, have a selective effect on one form of the target gene
product.
[0099] Gene targeting ribozymes necessarily contain a hybridizing
region complementary to two regions, each of at least 5 and
preferably each 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
or 20 contiguous nucleotides in length of a target mRNA. The tim-3
target mRNA include any of SEQ ID NO: 1, 3, 5 or 7. Galectin mRNA
sequences include SEQ ID NO: 9.
[0100] In addition, ribozymes possess highly specific
endoribonuclease activity, which autocatalytically cleaves the
target sense mRNA. The present invention extends to ribozymes which
hybridize to a sense mRNA encoding a tim-3 or galectin-9 protein
such as a therapeutic drug target candidate gene, thereby
hybridizing to the sense mRNA and cleaving it, such that it is no
longer capable of being translated to synthesize a functional
polypeptide product.
[0101] The ribozymes of the present invention also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as the
one which occurs naturally in Tetrahymena thermophila (known as the
IVS, or L-19 IVS RNA) and which has been extensively described by
Thomas Cech and collaborators (Zaug, et al. (1984) Science
224:574-578; Zaug, et al. (1986) Science 231:470-475; Zaug, et al.
(1986) Nature 324:429-433; published International patent
application No. WO88/04300 by University Patents Inc.; Been, et al.
(1986) Cell 47:207-216). The Cech-type ribozymes have an eight base
pair active site which hybridizes to a target RNA sequence
whereafter cleavage of the target RNA takes place. The invention
encompasses those Cech-type ribozymes which target eight base-pair
active site sequences that are present in a target gene or nucleic
acid sequence.
[0102] Ribozymes can be composed of modified oligonucleotides
(e.g., for improved stability, targeting, etc.) and should be
delivered to cells which express the target gene in vivo. A
preferred method of delivery involves using a DNA construct
"encoding" the ribozyme under the control of a strong constitutive
pol III or pol II promoter, so that transfected cells will produce
sufficient quantities of the ribozyme to destroy endogenous target
messages and inhibit translation. Because ribozymes, unlike
antisense molecules, are catalytic, a lower intracellular
concentration is required for efficiency.
[0103] In certain embodiments, a ribozyme may be designed by first
identifying a sequence portion sufficient to cause effective
knockdown by RNAi. The same sequence portion may then be
incorporated into a ribozyme. In this aspect of the invention, the
gene-targeting portions of the ribozyme or RNAi are substantially
the same sequence of at least 5 and preferably 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 or more contiguous nucleotides
of a tim-3 or galectin-9 nucleic acid.
[0104] In a long target RNA chain, significant numbers of target
sites are not accessible to the ribozyme because they are hidden
within secondary or tertiary structures (Birikh et al. (1997) Eur J
Biochem 245: 1-16). To overcome the problem of target RNA
accessibility, computer generated predictions of secondary
structure are typically used to identify targets that are most
likely to be single-stranded or have an "open" configuration (see
Jaeger et al. (1989) Methods Enzymol 183: 281-306). Other
approaches utilize a systematic approach to predicting secondary
structure which involves assessing a huge number of candidate
hybridizing oligonucleotides molecules (see Milner et al. (1997)
Nat Biotechnol 15: 537-41; and Patzel and Sczakiel (1998) Nat
Biotechnol 16: 64-8). Additionally, U.S. Pat. No. 6,251,588, the
contents of which are hereby incorporated herein, describes methods
for evaluating oligonucleotide probe sequences so as to predict the
potential for hybridization to a target nucleic acid sequence. The
method of the invention provides for the use of such methods to
select preferred segments of a target mRNA sequence that are
predicted to be single-stranded and, further, for the opportunistic
utilization of the same or substantially identical target mRNA
sequence, preferably comprising about 10-20 consecutive nucleotides
of the target mRNA, in the design of both the RNAi oligonucleotides
and ribozymes of the invention.
(5) Polypeptides
[0105] In certain aspects, the present disclosure makes available
isolated and/or purified forms of the soluble tim-3 polypeptides,
which are isolated from, or are otherwise substantially free of,
other proteins which might normally be associated with the protein
or a particular complex including the protein. In certain
embodiments, a soluble tim-3 polypeptide is a polypeptide that
comprises an amino acid sequence that is at least 90%, 95%, 97%,
99% or 100% identical to the amino acid sequence of SEQ ID NO: 2,
4, 6 or 8. The amino acid identity between two polypeptides can be
determined by first aligning the two polypeptide sequences using an
alignment algorithm, such as one based on the PAM250 matrix.
[0106] In certain embodiments, a soluble tim-3 polypeptide is a
polypeptide comprising a portion of an amino acid sequence that is
at least 90%, 95%, 97%, 99% or 100% identical to any of SEQ ID NO:
2, 4, 6 and 8, and preferably wherein said portion is a functional
portion, such as a portion that is sufficient to modulate
activation of Th1 cells or that is able to bind to a tim-3 ligand.
In one embodiment, the portion comprises the IgV domain of tim-3.
In some embodiments, the soluble tim-3 polypeptides contain
conservative amino acid substitutions. In certain embodiments a
soluble tim-3 polypeptide is a polypeptide obtained when a nucleic
acid comprising a nucleic acid sequence at least 90%, 95%, 97%, 99%
or 100% identical to a nucleic acid sequence of SEQ ID NO: 1, 3, 5
and 7 is expressed in cell. In certain embodiments a soluble tim-3
polypeptide is purified or partially purified.
[0107] In some embodiments, the IgV domain of soluble tim-3
comprises amino acids 22-131 of SEQ ID NO:13. In other embodiments,
it comprises amino acids 22-132 of SEQ ID NO:14. In one embodiment,
the intracellular domain of the soluble tim-3 polypeptides provided
by the invention comprises amino acids 226-301 of SEQ ID NO:13,
while in another embodiment it comprises amino acids 217-281 of SEQ
ID NO:14.
[0108] The invention further encompasses fusion proteins comprising
a soluble tim-3 polypeptide and a heterologous protein. In one
embodiment, the soluble tim-3 protein comprises the IgV domain but
lacks the mucin, transmembrane, and intracellular domains. In
certain embodiments, fusion proteins comprising a soluble tim-3
polypeptide and an immunoglobulin element are provided. In one
embodiment, the invention provides a polypeptide comprising the IgV
domain of tim-3, the intracellular domain of tim-3, and the Fc
domain of an immunoglobulin, wherein the protein does not contain
the mucin domain of tim-3 or the transmembrane domain of tim-3. The
tim-3 polypeptide can be mouse or human. An exemplary
immunoglobulin element is a constant region like the Fc domain of a
human IgG1 heavy chain (Browning et al., J. Immunol., 154, pp.
33-46 (1995)). Soluble receptor-IgG fusion proteins are common
immunological reagents and methods for their construction are known
in the art (see e.g., U.S. Pat. Nos. 5,225,538, 5,766,883 and
5,876,969), all of which are incorporated by reference. In some
embodiments, soluble peptides of the present invention are fused to
Fc variants.
[0109] In a related embodiment, the modified proteins of the
invention comprise tim-3 and galectin-9 fusion proteins with an Fc
region of an immunoglobulin. As is known, each immunoglobulin heavy
chain constant region comprises four or five domains. The domains
are named sequentially as follows: CH1-hinge-CH2-CH3(-CH4). The DNA
sequences of the heavy chain domains have cross-homology among the
immunoglobulin classes, e.g., the CH2 domain of IgG is homologous
to the CH2 domain of IgA and IgD, and to the CH3 domain of IgM and
IgE. As used herein, the term, "immunoglobulin Fc region" is
understood to mean the carboxyl-terminal portion of an
immunoglobulin chain constant region, preferably an immunoglobulin
heavy chain constant region, or a portion thereof. For example, an
immunoglobulin Fc region may comprise 1) a CH1 domain, a CH2
domain, and a CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a
CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or
5) a combination of two or more domains and an immunoglobulin hinge
region. In a preferred embodiment the immunoglobulin Fc region
comprises at least an immunoglobulin hinge region a CH2 domain and
a CH3 domain, and preferably lacks the CH1 domain.
[0110] In one embodiment, the class of immunoglobulin from which
the heavy chain constant region is derived is IgG (Ig.gamma.)
(.gamma. subclasses 1, 2, 3, or 4). Other classes of
immunoglobulin, IgA (Ig.alpha.), IgD (Ig.delta.), IgE (Ig.epsilon.)
and IgM (Ig.mu.), may be used. The choice of appropriate
immunoglobulin heavy chain constant regions is discussed in detail
in U.S. Pat. Nos. 5,541,087, and 5,726,044. The choice of
particular immunoglobulin heavy chain constant region sequences
from certain immunoglobulin classes and subclasses to achieve a
particular result is considered to be within the level of skill in
the art. The portion of the DNA construct encoding the
immunoglobulin Fc region preferably comprises at least a portion of
a hinge domain, and preferably at least a portion of a CH3 domain
of Fc .gamma. or the homologous domains in any of IgA, IgD, IgE, or
IgM.
[0111] Furthermore, it is contemplated that substitution or
deletion of amino acids within the immunoglobulin heavy chain
constant regions may be useful in the practice of the invention.
One example would be to introduce amino acid substitutions in the
upper CH2 region to create a Fc variant with reduced affinity for
Fc receptors (Cole et al. (1997) J. IMMUNOL. 159:3613). One of
ordinary skill in the art can prepare such constructs using well
known molecular biology techniques.
[0112] In a further embodiment, the fusion proteins comprise a
soluble tim-3 polypeptide and a second heterologous polypeptide to
increase the in vivo stability of the fusion protein, or to
modulate its biological activity or localization, or to facilitate
purification of the fusion protein. Other exemplary heterologous
proteins that can be used to generate tim-3 soluble fusion proteins
include, but not limited to, glutathione-S-transferase (GST), an
enzymatic activity such as alkaline phosphatase (AP), or an epitope
tag such as hemagluttin (HA).
[0113] Preferably, stable plasma proteins, which typically have a
half-life greater than 20 hours in the circulation, are used to
construct fusions proteins with tim-3 or galectin-9. Such plasma
proteins include but are not limited to: immunoglobulins, serum
albumin, lipoproteins, apolipoproteins and transferrin. Sequences
that can target the soluble tim-3 or galectin-9 molecules to a
particular cell or tissue type may also be attached to the soluble
tim-3 to create a specifically-localized soluble tim-3 fusion
protein.
[0114] In one preferred embodiment, the invention provides tim-3 or
galectin-9 fusions to albumin. As used herein, "albumin" refers
collectively to albumin protein or amino acid sequence, or an
albumin fragment or variant, having one or more functional
activities (e.g., biological activities) of albumin. In particular,
"albumin" refers to human albumin or fragments thereof (see EP 201
239, EP 322 094 WO 97/24445, WO95/23857) especially the mature form
of human albumin, or albumin from other vertebrates. In particular,
the albumin fusion proteins of the invention may include naturally
occurring polymorphic variants of human albumin and fragments of
human albumin (See WO95/23857), for example those fragments
disclosed in EP 322 094 (namely HA (Pn), where n is 369 to 419).
The albumin may be derived from any vertebrate, especially any
mammal, for example human, cow, sheep, or pig. Non-mammalian
albumins include, but are not limited to, hen and salmon. The
albumin portion of the albumin fusion protein may be from a
different animal than the tim-3 or galectin-9 protein.
[0115] In some embodiments, the albumin protein portion of an
albumin fusion protein corresponds to a fragment of serum albumin.
Fragments of serum albumin polypeptides include polypeptides having
one or more residues deleted from the amino terminus or from the
C-terminus. Generally speaking, an HA fragment or variant will be
at least 100 amino acids long, preferably at least 150 amino acids
long. The HA variant may consist of or alternatively comprise at
least one whole domain of HA. Domains, of human albumin are
described in U.S. Patent Publication No. 2004/0171123.
[0116] In certain embodiments, the invention includes nucleic acids
encoding soluble tim-3 polypeptides. In further embodiments, this
invention also pertains to a host cell comprising soluble tim-3
polypeptides and related derivatives. The host cell may be any
prokaryotic or eukaryotic cell. For example, a polypeptide of the
present invention may be expressed in bacterial cells such as E.
coli, insect cells (e.g., using a baculovirus expression system),
yeast, or mammalian cells. In one embodiment, the soluble tim-3
polypeptide is made and secreted by a mammalian cell, and the
soluble tim-3 polypeptide is purified from the culture medium.
Other suitable host cells are known to those skilled in the art.
Accordingly, some embodiments of the present invention further
pertain to methods of producing the soluble tim-3 polypeptides.
[0117] It is also possible to modify the structure of the subject
tim-3 polypeptides for such purposes as enhancing therapeutic or
prophylactic efficacy, or stability (e.g., ex vivo shelf life and
resistance to proteolytic degradation in vivo). Such modified
polypeptides, when designed to retain at least one activity of the
naturally-occurring form of the protein, are considered functional
equivalents of the tim-3polypeptides described in more detail
herein. Such modified polypeptides can be produced, for instance,
by amino acid substitution, deletion, or addition.
[0118] For instance, it is reasonable to expect, for example, that
an isolated replacement of a leucine with an isoleucine or valine,
an aspartate with a glutamate, a threonine with a serine, or a
similar replacement of an amino acid with a structurally related
amino acid (i.e. conservative mutations) will not have a major
effect on the biological activity of the resulting molecule.
Conservative replacements are those that take place within a family
of amino acids that are related in their side chains. Genetically
encoded amino acids are can be divided into four families: (1)
acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine;
(3) nonpolar=alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan; and (4) uncharged
polar=glycine, asparagine, glutamine, cysteine, serine, threonine,
tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes
classified jointly as aromatic amino acids. In similar fashion, the
amino acid repertoire can be grouped as (1) acidic=aspartate,
glutamate; (2) basic=lysine, arginine histidine, (3)
aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,
threonine, with serine and threonine optionally be grouped
separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine,
tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6)
sulfur-containing=cysteine and methionine. (see, for example,
Biochemistry, 2nd ed., Ed. by L. Stryer, W.H. Freeman and Co.,
1981). Whether a change in the amino acid sequence of a polypeptide
results in a functional homolog can be readily determined by
assessing the ability of the variant polypeptide to produce a
response in cells in a fashion similar to the wild-type protein.
For instance, such variant forms of a tim-3 polypeptide can be
assessed, e.g., for their ability to modulate the secretion of
cytokines by Th1 cells, or their ability to bind to a tim-3
ligands, such as galectin-9. Polypeptides in which more than one
replacement has taken place can readily be tested in the same
manner.
[0119] In certain aspects, functional variants or modified forms of
the subject soluble tim-3 and galectin-9 polypeptides include
fusion proteins having at least a portion of the soluble
polypeptide and one or more fusion domains. Well known examples of
such fusion domains include, but are not limited to, polyhistidine,
Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,
protein G, and an immunoglobulin heavy chain constant region (Fe),
maltose binding protein (MBP), which are particularly useful for
isolation of the fusion proteins by affinity chromatography. For
the purpose of affinity purification, relevant matrices for
affinity chromatography, such as glutathione-, amylase-, and
nickel- or cobalt-conjugated resins are used. Another fusion domain
well known in the art is green fluorescent protein (GFP). Fusion
domains also include "epitope tags," which are usually short
peptide sequences for which a specific antibody is available. Well
known epitope tags for which specific monoclonal antibodies are
readily available include FLAG, influenza virus haemagglutinin
(HA), and c-myc tags. In some cases, the fusion domains have a
protease cleavage site, such as for Factor Xa or Thrombin, which
allows the relevant protease to partially digest the fusion
proteins and thereby liberate the recombinant proteins therefrom.
The liberated proteins can then be isolated from the fusion domain
by subsequent chromatographic separation.
[0120] Some of the tim-3 or galectin-9 polypeptides provided by the
invention, or used in the methods of the present invention, may
further comprise post-translational modifications. Exemplary
post-translational protein modification include phosphorylation,
acetylation, methylation, ADP-ribosylation, ubiquitination,
glycosylation, carbonylation, sumoylation, biotinylation or
addition of a polypeptide side chain or of a hydrophobic group. As
a result, the modified soluble polypeptides may contain non-amino
acid elements, such as lipids, poly- or mono-saccharide, and
phosphates.
[0121] In one specific embodiment of the present invention,
modified forms of the subject tim-3 and galectin-9 soluble
polypeptides comprise linking the subject soluble polypeptides to
nonproteinaceous polymers. In one specific embodiment, the polymer
is polyethylene glycol ("PEG"), polypropylene glycol, or
polyoxyalkylenes, in the manner as set forth in U.S. Pat. No.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0122] PEG is a well-known, water soluble polymer that is
commercially available or can be prepared by ring-opening
polymerization of ethylene glycol according to methods well known
in the art (Sandler and Karo, Polymer Synthesis, Academic Press,
New York, Vol. 3, pages 138-161). The term "PEG" is used broadly to
encompass any polyethylene glycol molecule, without regard to size
or to modification at an end of the PEG, and can be represented by
the formula: X--O(CH.sub.2CH.sub.2O).sub.n-1CH.sub.2CH.sub.2OH (1),
where n is 20 to 2300 and X is H or a terminal modification, e.g.,
a C.sub.1-4 alkyl. In one embodiment, the PEG of the invention
terminates on one end with hydroxy or methoxy, i.e., X is H or CH3
("methoxy PEG"). A PEG can contain further chemical groups which
are necessary for binding reactions; which results from the
chemical synthesis of the molecule; or which is a spacer for
optimal distance of parts of the molecule. In addition, such a PEG
can consist of one or more PEG side-chains which are linked
together. PEGs with more than one PEG chain are called multiarmed
or branched PEGs. Branched PEGs can be prepared, for example, by
the addition of polyethylene oxide to various polyols, including
glycerol, pentaerythriol, and sorbitol. For example, a four-armed
branched PEG can be prepared from pentaerythriol and ethylene
oxide. Branched PEG are described in, for example, EP-A 0 473 084
and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEG
side-chains (PEG2) linked via the primary amino groups of a lysine
(Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69).
[0123] PEG conjugation to peptides or proteins generally involves
the activation of PEG and coupling of the activated
PEG-intermediates directly to target proteins/peptides or to a
linker, which is subsequently activated and coupled to target
proteins/peptides (see Abuchowski, A. et al, J. Biol. Chem., 252,
3571 (1977) and J. Biol. Chem., 252, 3582 (1977), Zalipsky, et al.,
and Harris et. al., in: Poly(ethylene glycol) Chemistry:
Biotechnical and Biomedical Applications; (J. M. Harris ed.) Plenum
Press: New York, 1992; Chap. 21 and 22).
[0124] One skilled in the art can select a suitable molecular mass
for PEG, e.g., based on how the pegylated tim-3 or galectin-9
polypeptide will be used therapeutically, the desired dosage,
circulation time, resistance to proteolysis, immunogenicity, and
other considerations. For a discussion of PEG and its use to
enhance the properties of proteins, see N. V. Katre, Advanced Drug
Delivery Reviews 10: 91-114 (1993).
[0125] In one embodiment of the invention, PEG molecules may be
activated to react with amino groups on tim-3 or galectin-9
polypeptides, such as with lysines (Bencham C. O. et al., Anal.
Biochem., 131, 25 (1983); Veronese, F. M. et al., Appl. Biochem.,
11, 141 (1985).; Zalipsky, S. et al., Polymeric Drugs and Drug
Delivery Systems, adrs 9-110 ACS Symposium Series 469 (1999);
Zalipsky, S. et al., Europ. Polym. J., 19, 1177-1183 (1983);
Delgado, C. et al., Biotechnology and Applied Biochemistry, 12,
119-128 (1990)). In another embodiment, PEG molecules may be
coupled to sulfhydryl groups on tim-3 or galectin-9 (Sartore, L.,
et al., Appl. Biochem. Biotechnol., 27, 45 (1991); Morpurgo et al.,
Biocon. Chem., 7, 363-368 (1996); Goodson et al., Bio/Technology
(1990) 8, 343; U.S. Pat. No. 5,766,897). U.S. Pat. Nos. 6,610,281
and 5,766,897 describes exemplary reactive PEG species that may be
coupled to sulfhydryl groups. In some embodiments, the pegylated
tim-3 or galectin-9 proteins comprise a PEG molecule covalently
attached to the alpha amino group of the N-terminal amino acid.
Site specific N-terminal reductive amination is described in
Pepinsky et al., (2001) JPET, 297,1059, and U.S. Pat. No.
5,824,784. The use of a PEG-aldehyde for the reductive amination of
a protein utilizing other available nucleophilic amino groups is
described in U.S. Pat. No. 4,002,531, in Wieder et al., (1979) J.
Biol. Chem. 254, 12579, and in Chamow et al., (1994) Bioconjugate
Chem. 5, 133.
(6) Immunological Reagents
[0126] The invention additionally provides immunological reagents
capable of binding to tim-3 polypeptides, including mouse and human
polypeptides, and in preferred embodiments, immunological reagents
that bind to soluble tim-3 but not to full-length tim-3.
[0127] One aspect of the invention provides an isolated antibody or
fragment thereof which binds to a polypeptide having an amino acid
sequence set forth in SEQ ID NO:2 but which does not bind to a
polypeptide having the amino acid sequence set forth in SEQ ID
NO:13. SEQ ID NO:2 is the human soluble tim-3 protein while SEQ ID
NO:13 is the human full-length tim-3 protein. Accordingly, such
antibodies would bind to soluble human tim-3 but not to full-length
human tim-3.
[0128] Another aspect of the invention provides an isolated
antibody or fragment thereof which binds to a polypeptide having an
amino acid sequence set forth in SEQ ID NO:4 but which does not
bind to a polypeptide having the amino acid sequence set forth in
SEQ ID NO:14. SEQ ID NO:4 is the mouse soluble tim-3 protein while
SEQ ID NO:14 is the mouse full-length tim-3 protein. Accordingly,
such antibodies would bind to soluble mouse tim-3 but not to
full-length mouse tim-3.
[0129] Generating specific antisera that binds to soluble tim-3 but
not to full-length tim-3 can be achieved, for example, by
generating immunogens to a peptide having an amino acid sequence
contained in the soluble tim-3 protein which is absent from the
full-length protein. Such a peptide may span the junction of the
IgV domain and intracellular domains of soluble tim-3. This
junction is not present in the full-length tim-3 sequence, where
the mucin and transmembrane domains are inserted between the IgV
and intracellular domains. In one embodiment, the 16 amino acid
human soluble tim-3 peptide according to SEQ ID NO:6 is used as an
immunogen to generate such antibodies. To generate the equivalent
antibodies to mouse tim-3, the peptide according to SEQ ID NO: 8
can be used. One skilled in the art can manipulate this peptide by
extending at either the C- or N-terminus or both with additional
residues, or by shortening as desired to increase immunogenicity or
solubility. In addition, cysteine groups or other residues can be
added to the peptide to conjugate to a carrier or confer additional
desirable properties.
[0130] Similarly, peptides that bind to full-length tim-3, but not
to soluble tim-3, can be generated using peptides whose sequences
are found in the full-length but not in the soluble form of tim-3.
For instance, such peptides can correspond to the sequences of the
tim-3 transmembrane domain, or more preferably, to sequences of the
tim-3 mucin domain. Alternatively, these peptides may contain
sequences that span the junction between the IgV domain and the
mucin domain of full-length tim-3, or between the transmembrane
domain and the intracellular domains also of full-length tim-3.
[0131] Chickens, mammals, such as a mouse, a hamster, a goat, a
guinea pig or a rabbit, can be immunized with an immunogenic form
of the any of the peptides variants described (e.g., an antigenic
fragment which is capable of eliciting an antibody response).
Techniques for conferring immunogenicity on a protein or peptide
include conjugation to carriers or other techniques well known in
the art. For instance, a peptidyl portion of one of the subject
proteins 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 immunoassays can
be used with the immunogen as antigen to assess the levels of
antibodies.
[0132] Following immunization, antisera can be obtained and, if
desired, polyclonal antibodies against the target protein can be
further isolated from the serum. To produce monoclonal antibodies,
antibody producing cells (lymphocytes) can be harvested from an
immunized animal and fused by standard somatic cell fusion
procedures with immortalizing cells such as myeloma cells to yield
hybridoma cells. Such techniques are well known in the art, and
include, for example, the hybridoma technique (originally developed
by Kohler and Milstein, Nature, 256: 495-497, 1975), as well as the
human B cell hybridoma technique (Kozbar et al., Immunology Today,
4: 72, 1983), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, Inc. pp. 77-96, 1985). Hybridoma
cells can be screened immunochemically for production of antibodies
specifically reactive to the tim-3 peptide and the monoclonal
antibodies isolated. Accordingly, another aspect of the invention
provides hybridoma cell lines which produce the antibodies
described herein.
[0133] The antibodies can then be tested for their binding
specificity to full-length tim-3 and soluble tim-3. For example,
antibodies generated against the peptide according to SEQ ID NO: 6,
which corresponds to the IgV-Intracellular domain junction of human
soluble tim-3, can be tested for their ability to bind to either
form of tim-3 by immobilizing the tim-3 polypeptide on a membrane
and performing a western blot with the antibodies. One skilled in
the art may employ any routine method to determine if antibodies
bind to a protein.
[0134] In other embodiments, monoclonal antibodies can be generated
against the entire tim-3 soluble protein, and then screened for
whether or not they bind to the full-length tim-3 protein, in order
to identify antibodies specific to only soluble tim-3.
[0135] The term antibody as used herein is intended to include
fragments which are also specifically reactive with a protein
described herein or a complex comprising such protein. Antibodies
can be fragmented using conventional techniques and the fragments
screened in the same manner as described above for whole
antibodies. For example, F(ab').sub.2 fragments can be generated by
treating antibody with pepsin. The resulting F(ab').sub.2 fragment
can be treated to reduce disulfide bridges to produce Fab'
fragments. The antibody of the present invention is further
intended to include bispecific and chimeric molecules, as well as
single chain (scFv) antibodies.
[0136] The subject antibodies include trimeric antibodies and
humanized antibodies, which can be prepared as described, e.g., in
U.S. Pat. No. 5,585,089. Also within the scope of the invention are
single chain antibodies. All of these modified forms of antibodies
as well as fragments of antibodies are intended to be included in
the term "antibody".
(7) Identification of Tim-3/Galectin-9 Immunomodulatory Agents
[0137] Another aspect of the invention provides methods for
identifying agents which modulate immunomodulatory responses, such
as, for example, identifying agents which modulate the interaction
between tim-3 and galectin-9. In one aspect, the invention provides
methods for identifying agents which block the binding of tim-3 to
tim-3 ligands, such as to galectin-9. Agents which block this
interaction would be predicted to prevent activation of tim-3,
leading to increased Th1 responses and/or reduced Th2 responses. In
another aspect, the invention provides methods for identifying
agents which promote the binding of tim-3 to tim-3 ligands, such as
to galectin-9, or which mimic the ligand binding to tim-3. Such
would be predicted to promote activation of tim-3, leading to
decreased Th1 responses and/or increased Th2 responses.
Accordingly, agents identified using the methods described herein
may be used to modulate T.sub.H1 and T.sub.H2 responses in a
subject in need thereof.
[0138] In one aspect, the identification of a tim-3/galectin-9
complex in the present invention facilitates rational design of
agonists and antagonists based on the structural features of the
tim-3 and galectin-9 proteins, which can be determined using X-ray
crystallography, neuron diffraction, nuclear magnetic resonance
spectrometry, and other techniques. Methods for rational drug
design are well known in the art (see Chemical and Structural
Approaches to Rational Drug Design, David B Weiner, William V.
Williams, CRC Press (1994); Rational Drug Design: Novel Methodology
and Practical Applications, Vol. 719, Abby L. Parrill (Editor),
American Chemical Society (1999); Structure-based Ligand Design,
Klaus Gubemator, Wiley, John & Sons, Incorporated (1998)). A
related aspect of the invention provides reconstituted protein
preparations comprising a tim-3 polypeptide and/or a galectin-9
polypeptide. In a specific embodiment, at least 1% of the protein
in the reconstituted compositions is a tim-3 polypeptide and a
galectin-9 polypeptide, or more preferably at least 2%, 3%, 4%, 5%,
10%, 20%, 30%, 40% or 50%. In a one embodiment, the proteins in the
reconstituted compositions are recombinant proteins.
[0139] Another aspect of the invention provides methods for
screening agents that promote or block the formation of a complex
between tim-3 and galectin-9. Such methods may be performed in
vitro or in a cell, and they may be performed using full-length
proteins or fragments thereof, such as soluble fragments (i.e.
those lacking a transmembrane domain), of one or both of the
proteins. In some embodiments of the methods described herein,
soluble protein comprising the IgV, and optionally the mucin
domain, of tim-3 are used. A variety of other reagents may be
included in the screening assay. These include reagents like salts,
neutral proteins, e.g. albumin, detergents, etc. that are used to
facilitate optimal protein-protein binding and/or reduce
non-specific or background interactions. Reagents that improve the
efficiency of the assay, such as protease inhibitors, nuclease
inhibitors, anti-microbial agents, etc. may also be used. The
mixture of components are added in any order that provides for the
requisite binding. Incubations are performed at any suitable
temperature, typically between 4.degree. C. and 40.degree. C.
Incubation periods are selected for optimum activity, but may also
be optimized to facilitate rapid high-throughput screening.
Typically between 0.1 and 1 hours will be sufficient for in vitro
assays.
[0140] The methods for the identification of agents of the present
invention are well suited for screening libraries of compounds in
multi-well plates (e.g., 96-well plates), with a different test
compound or group of test compounds in each well. In particular,
the methods may be employed with combinatorial libraries. These
methods may be "miniaturized" in an assay system through any
acceptable method of miniaturization, including but not limited to
multi-well plates, such as 24, 48, 96 or 384-wells per plate,
micro-chips or slides. The assay may be reduced in size to be
conducted on a micro-chip support, advantageously involving smaller
amounts of reagents and other materials. Any miniaturization of the
process which is conducive to high-throughput screening is within
the scope of the invention.
[0141] One specific aspect of the invention provides methods for
identifying therapeutic agents that modulate Th1 function.
Accordingly, one aspect of the invention provides a method for
assessing the ability of an agent to modulate Th1 activation,
comprising: 1) combining: a tim-3 polypeptide or fragment thereof,
a galectin-9 polypeptide or fragment thereof, and an agent, under
conditions wherein the tim-3 and galectin-9 polypeptides physically
interact in the absence of the agent, 2) determining if the agent
interferes with the interaction, and optionally, 3) for an agent
that interferes with the interaction, further assessing its ability
to promote the activation of Th1 cells.
[0142] Another specific aspect of the invention provides a method
of identifying an agent that modulates the binding between a tim-3
polypeptide and a galectin-9 polypeptide comprising: (a) contacting
the tim-3 polypeptide and the galectin-9 polypeptide in the
presence of a test agent; and (b) determining the effect of the
test agent on the binding of the tim-3 polypeptide and the
galectin-9 polypeptide; thereby identifying a agent that modulates
the binding between a tim-3 polypeptide and a galectin-9
polypeptide.
[0143] A related specific aspect of the invention provides a method
of identifying an agent that modulates an immune response, the
method comprising (a) contacting the tim-3 polypeptide and the
galectin-9 polypeptide in the presence of a test agent; and (b)
determining the effect of the test agent on the binding of the
tim-3 polypeptide and the galectin-9 polypeptide; thereby
identifying an agent that modulates an immune response.
[0144] In one embodiment of the methods described herein for
detecting the interaction between a tim-3 polypeptide and a
galectin-9 polypeptide, the galectin-9 polypeptide comprises (i)
amino acids 1-323 of SEQ ID NO: 10; (ii) amino acids 1-355 of SEQ
ID NO: 19; or (iii) an amino acid sequence that is at least 90%
identical to amino acids 1-323 of SEQ ID NO: 10; (iii) an amino
acid sequence that is at least 90% identical to amino acids 1-355
of SEQ ID NO: 19. In another embodiment, the tim-3 polypeptide
comprises (i) amino acids 30-128 of SEQ ID NO: 13; or (iii) an
amino acid sequence that is at least 90% identical to amino acids
30-128 of SEQ ID NO: 13.
[0145] In some aspect of the invention the agents are identified
through in vitro assays. A variety of assay formats will suffice
and, in light of the present disclosure, those not expressly
described herein will nevertheless be comprehended by one of
ordinary skill in the art. Assay formats which approximate such
conditions as formation of protein complexes, enzymatic activity,
may be generated in many different forms, and include assays based
on cell-free systems, e.g. purified proteins or cell lysates, as
well as cell-based assays which utilize intact cells. Simple
binding assays can also be used to detect agents which bind to
tim-3 or galectin-9. Such binding assays may also identify agents
that act by disrupting the interaction between a tim-3 polypeptide
and a galectin-9 polypeptide.
[0146] Agents to be tested can be produced, for example, by
bacteria, yeast or other organisms (e.g. natural products),
produced chemically (e.g. small molecules, including
peptidomimetics), or produced recombinantly. Because tim-3 and
galectin-9 are transmembrane proteins, preferred embodiments of the
assays and methods described to identify agents which modulate
complex formation between tim-3 and galectin-9 employ soluble forms
of these proteins rather than full-length protein. Soluble forms
include those lacking the transmembrane domain and/or those
comprising the IgV domain or fragments thereof which retain their
ability to bind their cognate binding partners.
[0147] In many drug screening programs which test libraries of
compounds and natural extracts, high throughput assays are
desirable in order to maximize the number of compounds surveyed in
a given period of time. Assays of the present invention which are
performed in cell-free systems, which may be developed with
purified or semi-purified proteins or with lysates, are often
preferred as "primary" screens in that they can be generated to
permit rapid development and relatively easy detection of an
alteration in a molecular target which is mediated by a test
compound. Moreover, the effects of cellular toxicity and/or
bioavailability of the test compound can be generally ignored in
the in vitro system, the assay instead being focused primarily on
the effect of the drug on the molecular target as may be manifest
in an alteration of binding affinity with other proteins or changes
in enzymatic properties of the molecular target.
[0148] In preferred in vitro embodiments of the present assay, a
reconstituted tim-3/galectin-9 complex comprises a reconstituted
mixture of at least semi-purified proteins. By semi-purified, it is
meant that the proteins utilized in the reconstituted mixture have
been previously separated from other cellular or viral proteins.
For instance, in contrast to cell lysates, the proteins involved in
tim-3/galectin-9 complex formation are present in the mixture to at
least 50% purity relative to all other proteins in the mixture, and
more preferably are present at 90-95% purity. In certain
embodiments of the subject method, the reconstituted protein
mixture is derived by mixing highly purified proteins such that the
reconstituted mixture substantially lacks other proteins (such as
of cellular or viral origin) which might interfere with or
otherwise alter the ability to measure tim-3/galectin-9 complex
assembly and/or disassembly.
[0149] Assaying tim-3/galectin-9 complexes, in the presence and
absence of a candidate agent, can be accomplished in any vessel
suitable for containing the reactants. Examples include microtitre
plates, test tubes, and micro-centrifuge tubes. In a screening
assay, the effect of a test agent may be assessed by, for example,
assessing the effect of the test agent on kinetics, steady-state
and/or endpoint of the reaction.
[0150] In one embodiment of the present invention, drug screening
assays can be generated which detect inhibitory agents on the basis
of their ability to interfere with assembly or stability of the
tim-3/galectin-9 complex. In an exemplary binding assay, the
compound of interest is contacted with a mixture comprising a
tim-3/galectin-9 complex. Detection and quantification of
tim-3/galectin-9 complexes provides a means for determining the
compound's efficacy at inhibiting (or potentiating) interaction
between the two polypeptides. The efficacy of the compound can be
assessed by generating dose response curves from data obtained
using various concentrations of the test compound. Moreover, a
control assay can also be performed to provide a baseline for
comparison. In the control assay, the formation of complexes is
quantitated in the absence of the test compound.
[0151] Complex formation may be detected by a variety of
techniques. For instance, modulation in the formation of complexes
can be quantitated using, for example, detectably labeled proteins
(e.g. radiolabeled, fluorescently labeled, or enzymatically
labeled), by immunoassay, or by chromatographic detection. Surface
plasmon resonance systems, such as those available from Biacore
.COPYRGT. International AB (Uppsala, Sweden), may also be used to
detect protein-protein interaction.
[0152] The proteins and peptides described herein may be
immobilized. Often, it will be desirable to immobilize the peptides
and polypeptides to facilitate separation of complexes from
uncomplexed forms of one of the proteins, as well as to accommodate
automation of the assay. The peptides and polypeptides can be
immobilized on any solid matrix, such as a plate, a bead or a
filter. The peptide or polypeptide can be immobilized on a matrix
which contains reactive groups that bind to the polypeptide.
Alternatively or in combination, reactive groups such as cysteines
in the protein can react and bind to the matrix. In another
embodiment, the polypeptide may be expressed as a fusion protein
with another polypeptide which has a high binding affinity to the
matrix, such as a fusion protein to streptavidin which binds biotin
with high affinity.
[0153] In an illustrative embodiment, a fusion protein can be
provided which adds a domain that permits the protein to be bound
to an insoluble matrix. For example, a GST-TIM-3-IgV-domain fusion
protein, which comprises the IgV domain of tim-3 fused to
glutathione transferase, can be adsorbed onto glutathione sepharose
beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized
microtitre plates, which are then combined with galectin-9 or a
fragment thereof, e.g. an .sup.35S-labeled polypeptide, and the
test compound and incubated under conditions conducive to complex
formation. Following incubation, the beads are washed to remove any
unbound interacting protein, and the matrix bead-bound radiolabel
determined directly (e.g. beads placed in scintillant), or in the
supernatant after the complexes are dissociated, e.g. when
microtitre plate is used. Alternatively, after washing away unbound
protein, the complexes can be dissociated from the matrix,
separated by SDS-PAGE gel, and the level of interacting polypeptide
found in the matrix-bound fraction quantitated from the gel using
standard electrophoretic techniques.
[0154] It will be understood that various modifications of the
above-described assay are included within the scope of the present
invention. For example, the roles of the proteins can be
switched--that is, the galectin-9 protein may be immobilized to the
solid support and a solution containing the tim-3 protein may be
contacted with the bound galectin-9 protein. Additionally, the
immobilized protein or the free protein may be exposed to a test
compound prior to the binding assay, and the effects of this
pre-exposure may be assessed relative to controls. Compounds
identified in this manner also inhibit the binding of the tim-3 to
galectin-9 or vice versa. Alternatively, the test compound may be
added subsequent to the mixing of tim-3 and galectin-9. A compound
effective to reduce the level of binding in such an assay displaces
tim-3 protein from the galectin-9 protein or vice versa.
[0155] In addition to Western blots, other, more rapid, detection
schemes, such as multi-well ELISA-type approaches, may be employed.
For example, a partially-purified (e.g., by the GST methods above)
tim-3 protein may be attached to the bottoms of wells in a
multi-well plate (e.g., 96-well plate) by introducing a solution
containing the protein into the plate and allowing the protein to
bind to the plastic. The excess protein-containing solution is then
washed out, and a blocking solution (containing, for example,
bovine serum albumin (BSA)) is introduced to block non-specific
binding sites. The plate is then washed several more times and a
solution containing an galectin-9 protein and, in the case of
experimental (vs. control) wells, a test compound added. Different
wells may contain different test compound, different concentrations
of the same test substance, different tim-3 proteins or galectin-9
protein, or different concentrations of tim-3 protein or galectin-9
protein. Further, it will be understood that various modifications
to this detection scheme may be made. For example, the wells of a
multi-well plate may be coated with a polypeptide containing the
galectin-9 protein, rather than the tim-3 protein, and binding
interactions assayed upon addition of a free tim-3 protein. The
wells may also be pre-coated with compound(s) that enhance
attachment of the protein to be immobilized and/or decrease the
level of non-specific binding. For example, the wells may be
derivatized to contain glutathione and may be pre-coated with BSA,
to promote attachment of the immobilized protein in a known
orientation with the binding site(s) exposed.
[0156] Detection methods useful in such assays include
antibody-based methods (i.e., an antibody directed against the
"free" protein), direct detection of a reporter moiety incorporated
into the "free" protein (such as a fluorescent label), and
proximity energy transfer methods (such as a radioactive "free"
protein resulting in fluorescence or scintillation of molecules
incorporated into the immobilized protein or the solid
support).
[0157] Yet another variation of the methods of the present
invention for identifying a compound capable of affecting binding
of a tim-3 protein to a galectin-9 protein is through the use of
affinity biosensor methods. Such methods may be based on the
piezoelectric effect, electrochemistry, or optical methods, such as
ellipsometry, optical wave guidance, and surface plasmon resonance
(SPR). SPR is particular advantageous for monitoring molecular
interactions in real-time, enabling a sensitive and comprehensive
analysis of the effects of test compounds on the binding
interactions between two proteins than the methods discussed above.
This advantage is somewhat offset, however, by the lower throughput
of the technique (as compared with multi-well plate-based
methods).
[0158] As hereinbefore mentioned, a test compound can be said to
have an effect on the binding between a tim-3 protein and a
galectin-9 protein if the compound has any effect on the binding of
tim-3 to the galectin-9 protein (i.e., if the compound increases or
decreases the binding), and the effect exceeds a threshold value
(which is set to a desired level by the practitioner of the
invention as described above; e.g., several-fold increase or
several :fold decrease in binding). Preferably the effect on
binding is a significant effect. The term "significant" as used
herein, specifically in terms of a "significant effect", refers to
a difference in a quantifiable parameter between two groups being
compared that is statistically-significant using standard
statistical tests. In some embodiments of the methods described
herein, step (b) comprises comparing formation of a
tim-3/galectin-9 complex in the presence of the test agent with an
appropriate control. In some embodiments, the appropriate control
comprises the formation of a complex between the first polypeptide
and the second polypeptide in the absence of the agent or compound
being tested.
[0159] Therefore, in an embodiment of the present invention, there
is provided a method of screening for compounds that affect the
binding between a tim-3 protein and a galectin-9 protein
comprising: (a) contacting the tim-3 protein and the galectin-9
protein in the presence of a test compound; (b) determining the
effect of the test compound on the binding of the tim-3 protein and
the galectin-9 protein; and (c) identifying the compound as
effective if its measured effect on the extent of binding is above
a threshold level.
[0160] The term "affect the binding between a tim-3 protein and a
galectin-9 protein" means the test compound produces a difference
in the binding between the tim-3 protein and the galectin-9 protein
in its presence as compared to the binding between the tim-3
protein and the galectin-9 protein in its absence (control).
Preferably this difference in binding is a significant difference.
In a specific embodiment, a significant difference comprises at
least a 10%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 200% or 500%
increase or decrease in binding. The compound may inhibit or
enhance the binding, or in terms of the affect on tim-3, act as an
antagonist, an agonist or act as a compound which enhances the
effects of other agonists or antagonists. The type of measurement
used to quantify the effect of a test compound on the binding
between a tim-3 protein and a galectin-9 protein will depend on the
type of assay and detection methods used and this can be readily
determined by a person having skill in the art. For example, when
using a biological screen that employs Western blotting as the
means for detection, the binding can be measured using
densitometry. The densitometry values may be normalized and a
threshold level may be set based on the amount of variation in the
signal between a series of control samples (i.e. without test
compound). The smaller the variation, the smaller the effect of a
test compound that can be reliably detected.
[0161] In still further embodiments of the present assays, the
tim-3/galectin-9 complex is generated in whole cells, taking
advantage of cell culture techniques to support the subject assay.
For example, as described below, the tim-3/galectin-9 complex can
be constituted in a eukaryotic cell culture system, such as a
mammalian cell or a yeast cell. Other cells know to one skilled in
the art may be used. Advantages to generating the subject assay in
a whole cell include the ability to detect inhibitors which are
functional in an environment more closely approximating that which
therapeutic use of the inhibitor would require, including the
ability of the agent to gain entry into the cell. Furthermore,
certain of the in vivo embodiments of the assay, such as examples
given below, are amenable to high through-put analysis of test
agents. The components of the tim-3/galectin-9 complex can be
endogenous to the cell selected to support the assay.
Alternatively, some or all of the components can be derived from
exogenous sources. For instance, fusion proteins can be introduced
into the cell by recombinant techniques (such as through the use of
an expression vector), as well as by microinjecting the fusion
protein itself or mRNA encoding the fusion protein.
[0162] In yet another embodiment, the tim-3 and galectin-9
polypeptides can be used to generate an interaction trap assay (see
also, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell
72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054;
Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al.
(1993) Oncogene 8:1693-1696), for subsequently detecting agents
which disrupt binding of the proteins to one and other.
[0163] The yeast two-hybrid protein interaction assay may also be
employed to identify compounds that affect the binding of a tim-3
protein to a galectin-9 protein. The assay is based on the finding
that most eukaryotic transcription activators are modular, i.e.,
that the activators typically contain activation domains that
activate transcription, and DNA binding domains that localize the
activator to the appropriate region of a DNA molecule.
[0164] In a two hybrid system, a first fusion protein contains one
of a pair of interacting proteins fused to a DNA binding domain,
and a second fusion protein contains the other of a pair of
interacting proteins fused to a transcription activation domain.
The two fusion proteins are independently expressed in the same
cell, and interaction between the "interacting protein" portions of
the fusions reconstitute the function of the transcription
activation factor, which is detected by activation of transcription
of a reporter gene. At least two different cell-based two hybrid
protein-protein interaction assay systems have been used to assess
binding interactions and/or to identify interacting proteins. Both
employ a pair of fusion hybrid proteins, where one of the pair
contains a first of two "interacting" proteins fused to a
transcription activation domain of a transcription activating
factor, and the other of the pair contains a second of two
"interacting" proteins fused to a DNA binding domain of a
transcription activating factor.
[0165] In another embodiment, one of the proteins is expressed on a
cell, such as on the cell surface, whereas the other protein is a
native or a recombinant protein that is purified or partially
purified and contacted with the cell, such as to allow formation of
a complex.
[0166] In some embodiments, the agents identified as modulating the
binding interaction between tim-3 and galectin-9 may be further
evaluated for functional effects, such as their effect on the
induction of a Th1/Th2 response by T cells in vitro or in vivo,
such as by using the assays described in the experimental section.
In some embodiments, animal models of disease conditions are
further used to characterize the agents identified using the
methods described herein, such as Experimental Autoimmune
Encephalomyelitis models, KKAy mice as model of type 2 diabetes
mellitus and Fabry disease models (alphaGAL knock-out). See also
Bedell et al., Genes Dev. 1997 Jan. 1; 11(1):11-43 for additional
mouse models of human disease which may be used.
[0167] The test agent or test compound can be any agent or compound
which one wishes to test including, but not limited to, proteins
(including antibodies), peptides, nucleic acids (including RNA,
DNA, antisense oligonucleotide, peptide nucleic acids),
carbohydrates, organic compounds, inorganic compounds, natural
products, library extracts, bodily fluids and other samples that
one wishes to test for affecting the binding between a tim-3 and
galectin-9 polypeptide. In particular the test compound may be a
peptide mimetic of a tim-3 protein or a fragment thereof. In some
embodiments the test agent is purified or partially purified agent,
whereas in other embodiments it is not purified.
[0168] Test agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 50 and less than
about 2,500 Daltons. Test agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The test agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Test agents are also found among biomolecules
including, but not limited to: peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0169] Test agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and oligopeptides.
Libraries of small organic/peptide may be generated using
combinatorial techniques such as those described in Blondelle et
al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. Pat. Nos.
5,359,115 and 5,362,899; the Ellman U.S. Pat. No. 5,288,514; the
Still et al. PCT publication WO 94/08051; Chen et al. (1994) JACS
116:2661; Kerr et al. (1993) JACS 115:252; PCT publications
WO92/10092, WO93/09668 and WO91/07087; and the Lerner et al. PCT
publication WO93/20242.
[0170] Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs.
[0171] In other embodiments, the test agents are peptidomimetics of
tim-3, galectin-9 or fragments thereof. Peptidomimetics are
compounds based on, or derived from, peptides and proteins.
Peptidomimetics that may be used in the present invention typically
can be obtained by structural modification of a known analog
peptide sequence using unnatural amino acids, conformational
restraints, isosteric replacement, and the like. The subject
peptidomimetics constitute the continuum of structural space
between peptides and non-peptide synthetic structures; analog
peptidomimetics may be useful, therefore, in delineating
pharmacophores and in helping to translate peptides into nonpeptide
compounds with the activity of the parent analog peptides.
[0172] Moreover, as is apparent from the present disclosure,
mimetopes of the subject tim-3 and galectin-9 sequences can be
provided. Such peptidomimetics can have such attributes as being
non-hydrolyzable (e.g., increased stability against proteases or
other physiological conditions which degrade the corresponding
peptide), increased specificity and/or potency, and increased cell
permeability for intracellular localization of the peptidomimetic.
For illustrative purposes, peptide analogs of the present invention
can be generated using, for example, benzodiazepines (e.g., see
Freidinger et al. in Peptides: Chemistry and Biology, G. R.
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),
substituted gamma lactam rings (Garvey et al. in Peptides:
Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988, p 123), C-7 mimics (Huffman et al. in Peptides:
Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988, p. 105), keto-methylene pseudopeptides (Ewenson
et al. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides:
Structure and Function (Proceedings of the 9th American Peptide
Symposium) Pierce Chemical Co. Rockland, Ill., 1985), a-turn
dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and
Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), a-aminoalcohols
(Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann
et al. (1986) Biochem Biophys Res Commun 134:71), diaminoketones
(Nataraj an et al. (1984) Biochem Biophys Res Commun 124:141), and
methyleneamino-modified (Roark et al. in Peptides: Chemistry and
Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,
1988, p 134). Also, see generally, Session III: Analytic and
synthetic methods, in Peptides: Chemistry and Biology, G. R.
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988).
[0173] In addition to a variety of sidechain replacements that can
be carried out to generate the subject analog peptidomimetics, the
present invention specifically contemplates the use of
conformationally restrained mimics of peptide secondary structure.
Numerous surrogates have been developed for the amide bond of
peptides. Frequently exploited surrogates for the amide bond
include the following groups (i) trans-olefins, (ii) fluoroalkene,
(iii) methyleneamino, (iv) phosphonamides, and (v)
sulfonamides.
[0174] In some embodiments, the test agents are preselected for
their ability to bind to a tim-3 or a galectin-9 protein prior to
determining if they can affect the binding between a tim-3 or a
galectin-9 polypeptide. In one embodiment, test agent may first be
selected for its ability to bind a tim-3 or a galectin-9
polypeptide. The test agent may be preselected by screening a
library of test agents, such as a peptide library or a phage
display library.
(8) Compositions, Formulations and Packaging
[0175] A further aspect of the invention provides compositions
comprising the nucleic acids, polypeptides or agents described
herein. In one embodiment, the compositions are pharmaceutical
compositions. Pharmaceutical compositions for use in accordance
with the present invention may be formulated in conventional manner
using one or more physiologically acceptable carriers or
excipients. Thus, the compounds and their physiologically
acceptable salts and solvates may be formulated for administration
by, for example, by aerosol, intravenous, oral or topical route.
The administration may comprise intralesional, intraperitoneal,
subcutaneous, intramuscular or intravenous injection; infusion;
liposome-mediated delivery; topical, intrathecal, gingival pocket,
per rectum, intrabronchial, nasal, transmucosal, intestinal, oral,
ocular or otic delivery.
[0176] An exemplary composition of the invention comprises an RNAi
mixed with a delivery system, such as a liposome system, and
optionally including an acceptable excipient. In a preferred
embodiment, the composition is formulated for injection.
[0177] Techniques and formulations generally may be found in
Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton,
Pa. For systemic administration, injection is preferred, including
intramuscular, intravenous, intraperitoneal, and subcutaneous. For
injection, the compounds of the invention can be formulated in
liquid solutions, preferably in physiologically compatible buffers
such as Hank's solution or Ringer's solution. In addition, the
compounds may be formulated in solid form and redissolved or
suspended immediately prior to use. Lyophilized forms are also
included.
[0178] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., ationd oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0179] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions may take the form of tablets
or lozenges formulated in conventional manner. For administration
by inhalation, the compounds for use according to the present
invention are conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebuliser, with the
use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of e.g., gelatin for use in
an inhaler or insufflator may be formulated containing a powder mix
of the compound and a suitable powder base such as lactose or
starch.
[0180] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0181] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0182] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0183] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration bile
salts and fusidic acid derivatives. In addition, detergents may be
used to facilitate permeation. Transmucosal administration may be
through nasal sprays or using suppositories. For topical
administration, the oligomers of the invention are formulated into
ointments, salves, gels, or creams as generally known in the art. A
wash solution can be used locally to treat an injury or
inflammation to accelerate healing.
[0184] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0185] For therapies involving the administration of nucleic acids,
the oligomers of the invention can be formulated for a variety of
modes of administration, including systemic and topical or
localized administration. Techniques and formulations generally may
be found in Remmington's Pharmaceutical Sciences, Meade Publishing
Co., Easton, Pa. For systemic administration, injection is
preferred, including intramuscular, intravenous, intraperitoneal,
intranodal, and subcutaneous for injection, the oligomers of the
invention can be formulated in liquid solutions, preferably in
physiologically compatible buffers such as Hank's solution or
Ringer's solution. In addition, the oligomers may be formulated in
solid form and redissolved or suspended immediately prior to use.
Lyophilized forms are also included.
[0186] Systemic administration can also be by transmucosal or
transdermal means, or the compounds can be administered orally. For
transmucosal or transdermal administration, penetrants appropriate
to the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art, and include, for
example, for transmucosal administration bile salts and fusidic
acid derivatives. In addition, detergents may be used to facilitate
permeation. Transmucosal administration may be through nasal sprays
or using suppositories. For oral administration, the oligomers are
formulated into conventional oral administration forms such as
capsules, tablets, and tonics. For topical administration, the
oligomers of the invention are formulated into ointments, salves,
gels, or creams as generally known in the art.
[0187] Toxicity and therapeutic efficacy of the agents and
compositions of the present invention can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD50 (the dose lethal to 50% of the
population) and the ED50 (the dose therapeutically effective in 50%
of the population). The dose ratio between toxic and therapeutic
effects is the therapeutic index and it can be expressed as the
ratio LD50/ED50. Compounds which exhibit large therapeutic induces
are preferred. While compounds that exhibit toxic side effects may
be used, care should be taken to design a delivery system that
targets such compounds to the site of affected tissue in order to
minimize potential damage to uninfected cells and, thereby, reduce
side effects.
[0188] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50 (i.e., the concentration of the test compound which
achieves a half-maximal inhibition of symptoms) as determined in
cell culture. Such information can be used to more accurately
determine useful doses in humans. Levels in plasma may be measured,
for example, by high performance liquid chromatography.
[0189] In one embodiment of the methods described herein, the
effective amount of the agent is between about 1 mg and about 50 mg
per kg body weight of the subject. In one embodiment, the effective
amount of the agent is between about 2 mg and about 40 mg per kg
body weight of the subject. In one embodiment, the effective amount
of the agent is between about 3 mg and about 30 mg per kg body
weight of the subject. In one embodiment, the effective amount of
the agent is between about 4 mg and about 20 mg per kg body weight
of the subject. In one embodiment, the effective amount of the
agent is between about 5 mg and about 10 mg per kg body weight of
the subject.
[0190] In one embodiment of the methods described herein, the agent
is administered at least once per day. In one embodiment, the agent
is administered daily. In one embodiment, the agent is administered
every other day. In one embodiment, the agent is administered every
6 to 8 days. In one embodiment, the agent is administered
weekly.
[0191] As for the amount of the compound and/or agent for
administration to the subject, one skilled in the art would know
how to determine the appropriate amount. As used herein, a dose or
amount would be one in sufficient quantities to either inhibit the
disorder, treat the disorder, treat the subject or prevent the
subject from becoming afflicted with the disorder. This amount may
be considered an effective amount. A person of ordinary skill in
the art can perform simple titration experiments to determine what
amount is required to treat the subject. The dose of the
composition of the invention will vary depending on the subject and
upon the particular route of administration used. In one
embodiment, the dosage can range from about 0.1 to about 100,000
ug/kg body weight of the subject. Based upon the composition, the
dose can be delivered continuously, such as by continuous pump, or
at periodic intervals. For example, on one or more separate
occasions. Desired time intervals of multiple doses of a particular
composition can be determined without undue experimentation by one
skilled in the art.
[0192] The effective amount may be based upon, among other things,
the size of the compound, the biodegradability of the compound, the
bioactivity of the compound and the bioavailability of the
compound. If the compound does not degrade quickly, is bioavailable
and highly active, a smaller amount will be required to be
effective. The effective amount will be known to one of skill in
the art; it will also be dependent upon the form of the compound,
the size of the compound and the bioactivity of the compound. One
of skill in the art could routinely perform empirical activity
tests for a compound to determine the bioactivity in bioassays and
thus determine the effective amount. In one embodiment of the above
methods, the effective amount of the compound comprises from about
1.0 ng/kg to about 100 mg/kg body weight of the subject. In another
embodiment of the above methods, the effective amount of the
compound comprises from about 100 ng/kg to about 50 mg/kg body
weight of the subject. In another embodiment of the above methods,
the effective amount of the compound comprises from about 1 ug/kg
to about 10 mg/kg body weight of the subject. In another embodiment
of the above methods, the effective amount of the compound
comprises from about 100 ug/kg to about 1 mg/kg body weight of the
subject.
[0193] As for when the compound, compositions and/or agent is to be
administered, one skilled in the art can determine when to
administer such compound and/or agent. The administration may be
constant for a certain period of time or periodic and at specific
intervals.
[0194] The compound may be delivered hourly, daily, weekly,
monthly, yearly (e.g. in a time release form) or as a one time
delivery. The delivery may be continuous delivery for a period of
time, e.g. intravenous delivery. In one embodiment of the methods
described herein, the agent is administered at least once per day.
In one embodiment of the methods described herein, the agent is
administered daily. In one embodiment of the methods described
herein, the agent is administered every other day. In one
embodiment of the methods described herein, the agent is
administered every 6 to 8 days. In one embodiment of the methods
described herein, the agent is administered weekly.
[0195] Another aspect of the invention provides a pharmaceutical
package comprising (i) a polypeptide which comprises the IgV domain
of tim-3; and (ii) instructions for administering the composition
to a subject afflicted with a hyperplastic condition selected from
the group comprising of renal cell cancer, Kaposi's sarcoma,
chronic leukemia, prostate cancer, breast cancer, sarcoma,
pancreatic cancer, leukemia, ovarian carcinoma, rectal cancer,
throat cancer, melanoma, colon cancer, bladder cancer, lymphoma,
mastocytoma, lung cancer, mammary adenocarcinoma, pharyngeal
squamous cell carcinoma, testicular cancer, gastrointestinal cancer
and stomach cancer.
(9) Methods of Regulating Immune Responses
[0196] One aspect of the invention provides methods of modulating
immune responses, such as but not limited to, modulating Th1 or Th2
responses, immune tolerance and transplantation tolerance. The term
modulating as used herein refers to increasing or decreasing. The
methods of regulating immune responses of the present application
are based, in part, on the discovery that titrating tim-3 ligands,
using a fusion protein comprising soluble tim-3, Th1 responses are
enhanced, the establishment of peripheral tolerance is abrogated,
and transplantation tolerance is decreased. By contrast, the
administration of galectin-9 to mammals, discovered by applicants
as a tim-3 ligand, results in reduced Th1 responses.
[0197] Another aspect of the invention provides a method of
reducing immune tolerance, increasing Th1-mediated immune
responses, and/or decreasing Th2-mediated immune responses in a
subject in need thereof, the method comprising administering to the
subject a therapeutically effective amount of an agent that
decreases the expression or activity of tim-3, galectin-9 or both
i.e. a tim-3 or galectin-9 agonist, or that increases the binding
of tim-3 to galectin-9.
[0198] Reducing immune tolerance and increasing Th1-mediated
responses, by decreasing tim-3 activity, is beneficial in cancer
immunotherapy. The immune system can develop tolerance against
tumor antigens, thus allowing tumors to evade immune surveillance.
In one aspect of the invention, an agent which decreases tim-3
activity is administered to a subject afflicted with a hyperplastic
condition.
[0199] The terms "cancer" and "tumor" are used interchangeably,
both terms referring to a hyperplastic condition. In one
embodiments, the cancer is selected from the group consisting of
Kaposi's sarcoma, chronic leukemia, prostate cancer, breast cancer,
sarcoma, pancreatic cancer, leukemia, ovarian carcinoma, rectal
cancer, throat cancer, melanoma, colon cancer, bladder cancer,
lymphoma, mastocytoma, lung cancer, mammary adenocarcinoma,
pharyngeal squamous cell carcinoma, and gastrointestinal or stomach
cancer. In another embodiment, the cancer is selected for the group
consisting of basal cell carcinoma, biliary tract cancer; bladder
cancer; bone cancer; brain and CNS cancer; breast cancer; cervical
cancer; choriocarcinoma; colon and rectum cancer; connective tissue
cancer; cancer of the digestive system; endometrial cancer;
esophageal cancer; eye cancer; cancer of the head and neck; gastric
cancer; intra-epithelial neoplasm; kidney cancer; larynx cancer;
leukemia; liver cancer; lung cancer (e.g., small cell and non-small
cell); lymphoma including Hodgkin's and non-Hodgkin's lymphoma;
melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip,
tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer;
prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer;
cancer of the respiratory system; sarcoma; skin cancer; stomach
cancer; testicular cancer; thyroid cancer; uterine cancer; cancer
of the urinary system, as well as other carcinomas and
sarcomas.
[0200] In another embodiment, the agents used to increase Th1
activation by inhibiting tim-3 activity are used to enhance the
immune response of a subject to a vaccine. In one particular,
embodiment, and agent which inhibits tim-3 activity, such as a
polypeptide comprising a tim-3 IgV domain or an antibody that
inhibits the formation of a complex between full-length tim-3 and a
tim-3 ligand, is administered with the vaccine to enhance the
immune response towards the vaccine. Vaccines include, but are not
limited to, those intended to prevent viral or bacterial
infections. In other embodiments, the agent is administered prior
to the vaccination, while in other embodiments the agent is
administered after the vaccine is administered to the subject.
[0201] In yet another aspect, the invention features a method of
decreasing, inhibiting, suppressing, ameliorating, or delaying a
Th2-associated response (e.g., an allergic or an asthmatic
response), in a subject in need thereof, the method comprising
administering to a subject an agent that decreases expression or
activity of tim-3, galectin-9 or both i.e. administering a tim-3 or
a galectin-9 antagonist, or an agent that decreases the binding of
galectin-9 to tim-3.
[0202] A "Th2-mediated disorder" as used herein refers to a disease
that is associated with the development of a Th2 immune response. A
"Th2 immune response" as used herein refers to the induction of at
least one Th2-cytokine or a Th2-antibody. In preferred embodiments
more than one Th2-cytokine or Th2-antibody is induced. Thus a
Th2-mediated disease is a disease associated with the induction of
a Th2 response and refers to the partial or complete induction of
at least one Th2-cytokine or Th2-antibody or an increase in the
levels of at least one Th2-cytokine or Th2-antibody. These
disorders are known in the art and include for instance, but are
not limited to, atopic conditions, such as asthma and allergy,
including allergic rhinitis, gastrointestinal allergies, including
food allergies, eosinophilia, conjunctivitis, glomerulonephritis,
certain pathogen susceptibilities such as helminthic (e.g.,
leishmaniasis) and certain viral infections, including human
immunodeficiency virus (HIV), and certain bacterial infections,
including tuberculosis and lepromatous leprosy. In a preferred
embodiment, the Th2-associated response is asthma or an
allergy.
[0203] Asthma, as defined herein, is reversible airflow limitation
in an individual over a period of time. Asthma is characterized by
the presence of cells such as eosinophils, mast cells, basophils,
and CD25.sup.+ T lymphocytes in the airway walls. There is a close
interaction between these cells, because of the activity of
cytokines which have a variety of communication and biological
effector properties. Chemokines attract cells to the site of
inflammation and cytokines activate them, resulting in inflammation
and damage to the mucosa. With chronicity of the process, secondary
changes occur, such as thickening of basement membranes and
fibrosis. The disease is characterized by increased airway
hyper-responsiveness to a variety of stimuli, and airway
inflammation. A patient diagnosed as asthmatic will generally have
multiple indications over time, including wheezing, asthmatic
attacks, and a positive response to methacholine challenge, i.e., a
PC20 on methacholine challenge of less than about 4 mg/ml.
Guidelines for diagnosis may be found, for example, in the National
Asthma Education Program Expert Panel Guidelines for Diagnosis and
Management of Asthma, National Institutes of Health, 1991, Pub. No.
91-3042.
[0204] As used herein, "allergy" shall refer to acquired
hypersensitivity to a substance (allergen). Allergic conditions
include eczema, allergic rhinitis or coryza, hay fever, bronchial
asthma, urticaria (hives) and food allergies, and other atopic
conditions. A "subject having an allergy" is a subject that has or
is at risk of developing an allergic reaction in response to an
allergen. An "allergen" refers to a substance that can induce an
allergic or asthmatic response in a susceptible subject. The list
of allergens is enormous and can include pollens, insect venoms,
animal dander, dust, fungal spores and drugs (e.g.,
penicillin).
[0205] Allergens of interest include antigens found in food, such
as strawberries, peanuts, milk proteins, egg whites, etc. Other
allergens of interest include various airborne antigens, such as
grass pollens, animal danders, house mite feces, etc. Molecularly
cloned allergens include Dermatophagoides pteryonys sinus (Der P1);
Lol pl-V from rye grass pollen; a number of insect venoms,
including venom from jumper ant Myrmecia pilosula; Apis mellifera
bee venom phospholipase A2 (PLA2 and antigen 5S; phospholipases
from the yellow jacket Vespula maculifrons and white faced hornet
Dolichovespula maculata; a large number of pollen proteins,
including birch pollen, ragweed pollen, Parol (the major allergen
of Parietaria officinalis) and the cross-reactive allergen Parjl
(from Parietaria judaica), and other atmospheric pollens including
Olea europaea, Artemisia sp., gramineae, etc. Other allergens of
interest are those responsible for allergic dermatitis caused by
blood sucking arthropods, e.g. Diptera, including mosquitos
(Anopheles sp., Aedes sp., Culiseta sp., Culex sp.); flies
(Phlebotomus sp., Culicoides sp.) particularly black flies, deer
flies and biting midges; ticks (Dermacenter sp., Ornithodoros sp.,
Otobius sp.); fleas, e.g. the order Siphonaptera, including the
genera Xenopsylla, Pulex and Ctenocephalides felis. The specific
allergen may be a polysaccharide, fatty acid moiety, protein,
etc.
[0206] One specific aspect of the invention provides a method of
preventing or reducing the likelihood of being afflicted with an
atopic disease in a subject, the method comprising administering to
the subject a therapeutically effective amount of a polypeptide,
said polypeptide comprising (i) SEQ ID NO: 10; (ii) SEQ ID NO: 18;
(iii) an amino acid sequence that is at least 90% identical or
similar to SEQ ID NO: 10; or (iv) an amino acid sequence that is at
least 90% identical or similar to SEQ ID NO: 18.
[0207] According to the present invention, agents which modulate
tim-3 or galectin-9 activity, or modulate complex formation between
tim-3 and galectin-9, may be used in combination with other
compositions and procedures for the modulation of an immune
responses or for treatment of a disorder or conditions. For
example, a tumor may be treated conventionally with surgery,
radiation or chemotherapy. Agents which decreases tim-3 activity,
such as a soluble tim-3-IgFc fusion protein, may be subsequently
administered to the patient to extend the dormancy of
micrometastases and to stabilize any residual primary tumor.
[0208] In some embodiments, the agents which decrease tim-3
activity preferentially inhibit expression of soluble tim-3
relative to expression of full-length tim-3. In some embodiments,
the agent which blocks tim-3 binding to galectin-9 is an antibody,
or an antibody fragment such as an antibody fragment which retains
high affinity binding to its antigen. Such an antibody may block
tim-3/galectin-9 binding by binding either to galectin-9 or to
tim-3, and in particular the extracellular domain of tim-3. In a
specific embodiment, the agent is an antibody or antibody fragment
that binds to a polypeptide comprising amino acids 30-128 of SEQ ID
NO: 13.
[0209] Monoclonal antibodies can be generated, by one skilled in
the art, which bind the tim-3 extracellular domain, and those
antibodies can be further tested for their ability to block binding
of a tim-3 ligand, such as galectin-9, to full-length tim-3 using
the methods provided by the instant invention. The preferred
antibodies would block the binding interactions between full-length
tim-3 and its ligands without themselves acting as an activator of
full-length tim-3 activity. Using the assays described in the
experimental procedures for example, one skilled in the art can
determine if a candidate antibody is an activator of tim-3
activity, such as by administering the antibody to an immunized
mouse and testing for in vitro proliferation and cytokine
production by Th1 cells isolated for the spleen of the mouse.
Preferred antibodies for decreasing immune tolerance would both
block binding of tim-3 ligands to tim-3 and not induce activation
of tim-3 i.e. not suppress Th1 cell proliferation and cytokine
release.
[0210] In one embodiment, the agent used to inhibit tim-3 function
comprises an antibody. In one embodiment, the antibody binds to the
IgV domain of tim-3 (amino acids 30-128 of SEQ ID NO:13) and blocks
binding of tim-3 ligands to full-length tim-3. In one embodiment,
the tim-3 ligand is galectin-9. Accordingly, in another embodiment,
the antibody binds to galectin-9. Upon binding, the antibody may
prevent the physical interaction between galectin-9 and full-length
tim-3.
[0211] In another embodiment, the agent which blocks binding of
full-length tim-3 to galectin-9 in the subject comprises a
recombinant tim-3 polypeptide that competes with the endogenous
tim-3 for binding to galectin-9. In a specific embodiment, the
agent comprises a polypeptide comprising (i) amino acids 30-128 of
SEQ ID NO: 13; or (iii) an amino acid sequence that is at least 90%
identical to amino acids 30-128 of SEQ ID NO: 13. In a specific
embodiment, the polypeptide agent is pegylated and/or is a fusion
protein with human serum albumin or with the Fc domain of an
immunoglobulin. In another embodiment, the agent used in the
methods to activate Th1 cells or to decrease immune tolerance in a
subject comprises a soluble tim-3 polypeptide with an amino acid
sequence according to SEQ ID NO:2. In a preferred embodiment, the
agent comprises the IgV domain of tim-3 (amino acids 30-128 of SEQ
ID NO:11). In yet another embodiment, the soluble tim-3 polypeptide
comprises the IgV domain of tim-3, optionally the intracellular
domain of tim-3, and the Fc domain of an immunoglobulin, but does
not contain the mucin domain of tim-3 or the transmembrane domain
of tim-3. In a preferred embodiment, these polypeptides bind
galectin-9. Variants of these polypeptides, such as those which
increase binding of tim-3 ligands, can also be used.
[0212] In some embodiments, the agent which decreases tim-3
activity inhibits binding of (i) a polypeptide comprising amino
acids 30-128 of SEQ ID NO: 13; to (ii) galectin-9, such as the long
or short forms of galectin-9.
[0213] Applicants have discovered that lactose inhibits binding of
galectin-9 to tim-3. Accordingly, in one embodiment, the agent
which inhibits binding of full-length tim-3 to galectin-9 comprises
a carbohydrate, such as lactose or pectin/modified pectin. Modified
pectins are described in U.S. Patent Pub. Nos. 2003/0004132 and
2002/0187959.
[0214] In another embodiment, the agent used in the methods
described herein to activate promote Th1 responses and/or to
inhibit Th2 responses decrease the expression level of full-length
tim-3 polypeptide or of a tim-3 ligand, such as galectin-9
polypeptide. In one embodiments, the agent is a double stranded RNA
species provided by the present invention, including those directed
at full-length tim-3 or to a tim-3 ligand, such as galectin-9.
[0215] In a preferred embodiment, the agent used in the methods
described herein to activate promote Th1 responses and/or to
inhibit Th2 responses, or the agent used to inhibit tim-3 function,
is a small organic molecule, e.g., other than a peptide or
oligonucleotide, having a molecular weight of less than about 2,000
Daltons, which blocks the binding of tim-3 to its ligands, such as
to galectin-9. Such agents can be identified, for example, using
the methods provided by the invention. In another embodiment, the
agent which inhibits tim-3 function is a peptide or peptide
derivative which structurally mimics the portion of galectin-9 that
binds tim-3. Such a peptide may act as an antagonistic competitor
by preventing functional interactions between tim-3 and its
ligand(s). In one embodiment, the ligand is galectin-9.
[0216] According to the present invention, agents which decrease
the activity of tim-3, such as by reducing its expression level or
reducing the formation of a tim-3/ligand complex, may be used in
combination with other compositions and procedures for the
treatment of hyperplastic conditions. For example, a tumor may be
treated conventionally with surgery, radiation or chemotherapy, and
double stranded RNA, antibodies or small molecule inhibitors
against tim-3 or against tim-3 ligands, such as galectin-9, may be
subsequently administered to the patient to extend the dormancy of
micrometastases and to stabilize any residual primary tumor.
[0217] Another aspect of the invention provides a method of
treating or preventing or reducing the likelihood of being
afflicted with a Th1-mediated disorder in a subject in need of such
treatment, the method comprising administering to the subject a
therapeutically effective amount of an agent that increases
expression or activity of tim-3, galectin-9 or both, or that
increases the binding of tim-3 to galectin-9.
[0218] The invention also features a method of decreasing,
inhibiting, suppressing, ameliorating, or delaying a Th1-mediated
immune response, in a subject in need thereof, comprising
administering to the subject a tim-3 or a galectin-9 agonist, e.g.,
a tim-3 or a galectin-9 agonist as described herein, in an amount
sufficient to decrease, inhibit, suppress, ameliorate, or delay
said Th1-mediated immune response in said subject.
[0219] In contrast to a Th2-mediated disorder, a "Th1-mediated
disorder" as used herein refers to a disease that is associated
with the development of a Th1 immune response. A "Th1 immune
response" as used herein refers to the induction of at least one
Th1-cytoldne or a Th1-antibody. In preferred embodiments more than
one Th1-cytokine or Th1-antibody is induced. Thus a Th1-mediated
disease is a disease associated with the induction of a Th1
response and refers to the partial or complete induction of at
least one Th1-cytokine or Th1-antibody or an increase in the levels
of at least one Th1-cytokine or Th1-antibody. These disorders are
known in the art and include for instance, but are not limited to,
autoimmune (especially organ-specific) disease, psoriasis, Th1
inflammatory disorders, infection with extracellular parasites
(e.g., response to helminths), solid organ allograft rejection
(e.g., acute kidney allograft rejection), symptoms associated with
hepatitis B (HBV) infection (e.g., HBV acute phase or recovery
phase), chronic hepatitis C (HCV) infection, insulin-dependent
diabetes mellitus (IDDM), multiple sclerosis (MS), subacute
lymphocytic thyroiditis ("silent thyroiditis"), Crohn's disease,
primary biliary cirrhosis, primary sclerosing cholangitis,
sarcoidosis, atherosclerosis, acute graft-versus-host disease
(GvHD), glomerulonephritis, anti-glomerular basement membrane
disease, Wegener's granulomatosis, inflammatory myopathies,
Sjogren's syndrome, Behget's syndrome, rheumatoid arthritis, Lyme
arthritis, and unexplained recurrent abortion.
[0220] In some embodiments the Th1-mediated disorder is selected
from the group consisting of atherosclerosis, infection with
extracellular parasites, symptoms associated with hepatitis B (HBV)
infection (e.g., HBV acute phase or recovery phase), chronic
hepatitis C (HCV) infection, silent thyroiditis, primary biliary
cirrhosis, primary sclerosing cholangitis, glomerulonephritis,
anti-glomerular basement membrane disease, Wegener's
granulomatosis, inflammatory myopathies, Sjogren's syndrome,
Behcet's syndrome, rheumatoid arthritis, and unexplained recurrent
abortion.
[0221] The methods described herein for decreasing Th1-mediated
immune responses may be particularly beneficial to treat autoimmune
diseases in a subject. In one embodiment, the methods of the
present invention for reducing a Th1 response in a subject are
directed at subjects afflicted with, or at high risk of developing
an autoimmune disease. "Autoimmune disease" is a class of diseases
in which a subject's own antibodies react with host tissue or in
which immune effector T cells are autoreactive to endogenous
self-peptides and cause destruction of tissue. Thus an immune
response is mounted against a subject's own antigens, referred to
as self-antigens.
[0222] A "self-antigen" as used herein refers to an antigen of a
normal host tissue. Normal host tissue does not include cancer
cells. Thus an immune response mounted against a self-antigen, in
the context of an autoimmune disease, is an undesirable immune
response and contributes to destruction and damage of normal
tissue, whereas an immune response mounted against a cancer antigen
is a desirable immune response and contributes to destruction of
the tumor or cancer.
[0223] Autoimmune diseases include but are not limited to
rheumatoid arthritis, Crohn's disease, multiple sclerosis, systemic
lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia
gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome,
pemphigus (e.g., pemphigus vulgaris), Grave's disease, autoimmune
hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderrna
with anti-collagen antibodies, mixed connective tissue disease,
polymyositis, pernicious anemia, idiopathic Addison's disease,
autoimmune-associated infertility, glomerulonephritis (e.g.,
crescentic glomerulonephritis, proliferative glomerulonephritis),
bullous pemphigoid, Sjogren's syndrome, insulin resistance, and
autoimmune diabetes mellitus (type 1 diabetes mellitus;
insulin-dependent diabetes mellitus). Recently autoimmune disease
has been recognized also to encompass atherosclerosis and
Alzheimer's disease. In one specific embodiment, the autoimmune
disease is selected from the group consisting of multiple
sclerosis, type-I diabetes, Hashinoto's thyroiditis, Crohn's
disease, rheumatoid arthritis, systemic lupus erythematosus,
gastritis, autoimmune hepatitis, hemolytic anemia, autoimmune
hemophilia, autoimmune lymphoproliferative syndrome (ALPS),
autoimmune uveoretinitis, glomerulonephritis, Guillain-Barre
syndrome, psoriasis and myasthenia gravis. In another embodiment,
the Th1-mediated disorder is host versus graft disease (HVGD). In a
related embodiment, the subject is an organ or tissue transplant
recipient.
[0224] Yet another aspect of the invention provides a method for
increasing transplantation tolerance in a subject, comprising
administering to the subject a therapeutically effective amount of
an agent that increases tim-3 or galectin-9 function, expression,
or binding of tim-3 to galectin-9. In one specific embodiment, the
subject is a recipient of an allogenic transplant. The transplant
can be any organ or tissue transplant, including but not limited to
heart, kidney, liver, skin, pancreas, bone marrow, skin or
cartilage. Transplantation tolerance, as used herein, refers to a
lack of rejection of the donor organ by the recipient's immune
system. Furthermore, the agents can be used for preventing or
reducing the likelihood of being afflicted with rejection of tissue
or cell transplants.
[0225] As used herein, the terms "agent" and "compound" include
both protein and non-protein moieties. An agent can be any chemical
(element, molecule, compound, drug), made synthetically, made by
recombinant techniques or isolated from a natural source. For
example, agents can be peptides, polypeptides, antibodies or
antibody fragments, peptoids, sugars, hormones, or nucleic acid
molecules (such as antisense or RNAi nucleic acid molecules). In
addition, agents can be small molecules or molecules of greater
complexity made by combinatorial chemistry, for example, and
compiled into libraries. These libraries can comprise, for example,
alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers
and other classes of organic compounds. Agents can also be natural
or genetically engineered products isolated from lysates or growth
media of cells--bacterial, animal or plant.
[0226] In one embodiment, the agent which increases tim-3 activity,
such as those used to promote a Th2 response and/or inhibit a Th1
response, promotes the binding of full-length tim-3 to a ligand. In
a preferred embodiment, the tim-3 ligand is galectin-9. The agent
may be a polypeptide, which may itself bind to tim-3 or may itself
bind to a tim-3 ligand, such as galectin-9, and increase the
binding interaction between tim-3 and the ligand.
[0227] In one embodiment of the methods described herein, the agent
which increases tim-3 activity is a tim-3 ligand. An example of
such a ligand is galectin-9. Accordingly, in some embodiments said
agent comprises a galectin-9 polypeptide. In another embodiment,
said agent comprises a polypeptide comprising at least one of the
two carbohydrate recognition domains (CRD) of galectin-9 i.e. at
least the N-terminal or the C-terminal, or both.
[0228] In another embodiment, the agent which increases tim-3
activity comprises a polypeptide comprising an amino acid sequence
which is at least 80% identical to the amino acid sequence set
forth in SEQ ID NO:10 or SEQ ID NO:18. In other embodiments, the
sequence is 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:18.
In a specific embodiment, the polypeptide comprises an amino acid
sequence which is at least 80%, 90% or 95% identical to the amino
acid sequence of at least one CRD of human galectin-9.
[0229] In other embodiments, the agent which increases tim-3
activity is a peptide mimetic or a small molecule which can
function as galectin-9 in activating the tim-3 receptor. The
peptide or small molecule may structurally resemble the surface of
galectin-9 that binds tim-3, such that the peptide or small
molecule can activate tim-3 upon binding it, leading to increased
tim-3 activity and activation of Th1 cells, which includes
increased cell proliferation and secretion of cytokines. In a
specific embodiment, the agent which increases tim-3 activity
promotes the tyrosine phosphorylation of the intracellular domain
of tim-3.
[0230] In another embodiment, the agent which increases tim-3
activity in the methods described herein in as an antibody or
fragment thereof. Antibodies can be generated which bind to tim-3
and mimic the binding of a ligand, resulting in intracellular
signaling. Upon generating an antibody that binds to the
extracellular domain of tim-3, a skilled artisan may test whether
the antibody activates tim-3, which would lead to a suppression of
Th1 cell proliferation, reduced cytokine release, and increased
tolerance. The methods described in the experimental procedure
could be used to determine if an antibody binding activates
full-length tim-3. In a specific embodiment, the antibody which
increases tim-3 activity is a bispecific antibody specific for
tim-3 and galectin-9.
[0231] In yet another embodiment, the agent which increases tim-3
activity reduces the expression or function of soluble tim-3, but
does not directly affect that of full-length tim-3. In one
embodiment, the agent is a double stranded RNA species which
specifically inhibits the expression of soluble tim-3, such as the
one comprising the nucleotide sequence according to SEQ ID NO: 15.
Such double stranded RNA may be administered as a double-stranded
hairpin RNA. In another embodiment, the agent which increases tim-3
activity is an antibody that specifically binds to soluble tim-3
but not to full-length tim-3, such as those provided by the
invention.
Description of Sequence Listings
[0232] SEQ ID NO: 1 is the coding sequence of human soluble tim-3
isoform
[0233] SEQ ID NO: 2 is human soluble tim-3 protein
[0234] SEQ ID NO: 3 is the coding sequence of mouse soluble tim-3
isoform
[0235] SEQ ID NO: 4 is mouse soluble tim-3 protein
[0236] SEQ ID NO: 5 is a 16 nucleotide DNA fragment identical to
the novel splice junction of human soluble tim-3 cDNA
[0237] SEQ ID NO: 6 is a 16 amino acid peptide identical to the
novel IgV domain/Intracellular domain junction of human soluble
tim-3 protein
[0238] SEQ ID NO: 7 is a 16 bp DNA fragment identical to the novel
splice junction of mouse soluble tim-3 cDNA
[0239] SEQ ID NO: 8 is a 16 amino acid peptide identical to the
novel IgV domain/intracellular domain junction of mouse soluble
tim-3 protein
[0240] SEQ ID NO: 9 is the coding sequence of human galectin-9,
short form.
[0241] SEQ ID NO: 10 is the amino acid sequence of human
galectin-9, short form.
[0242] SEQ ID NO: 11 is the coding sequence of full-length human
tim-3.
[0243] SEQ ID NO: 12 is the coding sequence of full-length mouse
tim-3.
[0244] SEQ ID NO: 13 is the amino acid sequence of full-length
human tim-3.
[0245] SEQ ID NO: 14 is the amino acid sequence of full-length
mouse tim-3.
[0246] SEQ ID NO: 15 is a 16 nucleotide RNA sequence corresponding
to the novel splice junction of human soluble tim-3.
[0247] SEQ ID NO: 16 is the coding sequence of mouse
galectin-9.
[0248] SEQ ID NO: 17 is the amino acid sequence of mouse
galectin-9.
[0249] SEQ ID NO: 18 is the amino acid sequence of mouse
galectin-9, long form. (see also Genbank Accession No.
NP.sub.--034838)
[0250] SEQ ID NO: 19 is the amino acid sequence of human
galectin-9, long form. (see also 10 Genbank Accession No.
NP.sub.--033665)
EXEMPLIFICATION
[0251] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention.
[0252] The examples are divided into two series, each series having
its own description of the methods used in the examples.
First Series of Experiments
A. Methodology
[0253] Cloning of s-Tim-3
[0254] Splenocytes were activated with 1 .mu.g/mL concanavalin A
(Sigma, St. Louis, Mo.). 48 hours post-activation, cells were
harvested and RNA extracted using TRIzol reagent (Invitrogen) as
per manufacturers' instructions. RNA was reverse transcribed using
Superscript II reverse transcription kit (Invitrogen, Los Angeles,
Calif.). Tim-3 primers, with SmaI restriction overhangs, were
designed in the 5' (TIM-3-F: 5' GCCCGGGAGGAGCTAAAGCTATCCCTACACA3')
and 3' (TIM3-R: 5' GCCCGGGCCAATGAGGTTGCCAAGTGACATA 3') UTR of the
Tim-3 gene, for the amplification of both fl-Tim-3 and s-Tim-3.
Reactions were carried out in a DNA Thermal cycler (Perkin-Elmer,
Chicago, Ill.) for 35 cycles with denaturation at 95.degree. C. for
1 min, annealing at 55.degree. C. for 30 sec and extension at
72.degree. C. for 90 sec per cycle. Reactions were completed with a
final 10 mM extension at 72.degree. C. All products were resolved
on a 1.2% agarose gel and visualized using ethidium bromide.
T Cell Sort and Tim-3-Ligand Staining
[0255] Whole spleen and lymph node cells were harvested from naive
C57BL/6 mice (Jackson Laboratories, Bar Harbor, Me.), and CD4+ T
cells were column purified (R&D Systems, Minneapolis, Minn.).
CD4.sup.+ T cells were stained with mAbs to CD25, CD62L, CD45RB,
CD44 and CD54 (Pharmingen, San Diego, Calif.). Cells were stained
with biotinylated ex-Tim-3-Ig, s-Tim-3-Ig or hIgG, and
streptavidin-PE was used as the secondary detection reagent for
detection in flow cytometry.
[0256] AE7, a pigeon cytochrome c specific, I-E.sup.k-restricted
Th1 clone.sup.34'.sup.35 and LR1F1, a PLP 139-151 altered peptide
Q144 (HSLGKQLGHPDKF) specific, I-A.sup.s restricted Th2
clone.sup.36 were maintained in DMEM supplemented with 0.1 mM
nonessential amino acids, sodium pyruvate (1 mM), L-glutamine (2
mM), MEM essential vitamin mixture (1.times.), penicillin (100
U/ml), streptomycin (100 U/ml), gentamicin (0.1 mg/ml), 10%
heat-inactivated fetal bovine serum (BioWhittaker, Incorporated,
Walkersville, Md.), asparagine (0.1 mM), folic acid (0.1 mg/ml) and
2-mercaptoethanol (5.times.10.sup.-5 M) (Sigma, St. Louis, Mo.),
0.6% T cell growth factor (T-Stim, Collaborative Biomedical
Research, Bedford, Mass.) and 0.06% recombinant IL-2. Cells were
maintained in a rest/stimulation protocol (stimulation media is
complete medium without T-Stim and r-IL-2) as described.sup.6,34,
and stained for s-Tim-3-Ig binding.
Proliferation Assays
[0257] Female SJL/J mice (6-12 weeks old) (Jackson Laboratory, Bar
Harbor, Me.) were injected subcutaneously (s.c.) in each flank with
50 .mu.g PLP 139-151 peptide (HSLGKWLGHPDKF) (Quality Controlled
Biochemicals, Boston, Mass.) emulsified in complete Freund's
adjuvant (CFA) (Difco, Kansas City, Mo.). Mice were injected
intraperitoneally every other day (beginning the same day as
immunization, day 0, and continuing through day 8) with either 100
.mu.g ex-Tim-3-Ig or s-Tim-3-Ig, or 100 .mu.g control hIgG or 100
.mu.l PBS. Mice were sacrificed on day 10, and spleens were
removed. Cells were plated at 5.times.10.sup.5 cells/well in round
bottom 96 well plates (Falcon, Becton Dickinson, Los Angeles,
Calif.) with PLP 139-151 added at 0-100 .mu.g/ml for 48 hrs, and
plates were pulsed with 1 .mu.Ci .sup.3[H]-thymidine/per well for
16-18 hrs. The incorporated radiolabeled thymidine was measured
utilizing a Beta Plate scintillation counter (Perkin Elmer Wallac
Inc, Atlanta, Ga.). The data are presented as mean cpm in
triplicate wells.
[0258] For cell separation experiments, CD11b.sup.+ and B220.sup.+
cells were purified through positive selection by MACS Sort
magnetic beads (Miltenyi Biotech, Auburn, Calif.), and CD3.sup.+ T
cells were purified by negative selection columns after depletion
of CD11b+ and B220.sup.+ cells (R&D Systems, Minneapolis,
Minn.). CD3.sup.+ T cells were plated at 10.sup.5 cells/well, and
CD11b.sup.+ and B220.sup.+ cells were plated at 2.times.10.sup.5
cells/well.
[0259] For tolerance induction experiments, SJL mice were immunized
s.c. with 50 .mu.g PLP 139-151/CFA and concurrently given an
intraperitoneal injection of 500 .mu.g soluble PLP 139-151 to
induce tolerance. Spleen cells were plated at 5.times.10.sup.5
cells/well and lymph node cells at 2.times.10.sup.5 cells/well.
Cytokine ELISAs
[0260] Cytokine production was measured for IL-2, IL-4, IL-10,
IFN-.gamma. and TNF-.alpha. by quantitative capture ELISA. Briefly,
purified rat mAb to mouse IL-2 (clone JES-1A12), IL-4 (clone
BVD4-1D11), IL-10 (clone JES5-2A5), IFN-.gamma. (clone R4-6A2) and
TNF-.alpha. (clone G281-2626) were obtained from Pharmingen (San
Diego, Calif.) and used to coat ELISA plates (Immulon 4, Dynatech
Laboratories, Chantilly, Va.). Recombinant mouse cytokines from
Pharmingen were used to construct standard curves, and biotinylated
rat mAbs to mouse IL-2 (clone JES6-5H4), IL-4 (clone BVD6-24G2),
IL-10 (clone SXC-1), EFN-.gamma. (clone XMG1.2) and TNF-.alpha.
(clone MP6-XT3) were used as the second antibody. Plates were
developed with TMB microwell peroxidase substrate (Kirkegaard and
Perry Laboratories, Gaithersburg, Md.) and read after the addition
of stop solution at 450 nm using a Benchmark microplate reader
(Bio-Rad Laboratories, Hercules, Calif.).
BrdU Incorporation
[0261] Spleens were taken from PLP 139-151/CFA immunized, Tim-3-Ig
or hIgG treated mice at day 10, and 5.times.10.sup.5 to
1.times.10.sup.6 whole spleen cells (96 well, round bottom plates)
were incubated for 48 hat 37.degree. C., 10% CO.sub.2 with 5-10
.mu.M 5-bromodeoxyuridine (Sigma, St. Louis, Mo.). After 48 h,
cells were stained with mAb to CD3-CyC, CD25-PE, and CD69-PE
(Pharmingen, San Diego, Calif.). Cells were subsequently fixed and
permeabilized (Cytoperm, Pharmingen, San Diego, Calif.), and
treated with DNAse I (Sigma, St. Louis, Mo.). Cells were then
stained with FITC-conjugated mAb to BrdU (or mouse IgG1 as isotype
control) and analyzed by FACS (Becton Dickinson, Los Angeles,
Calif.).
B. Examples of First Series of Experiments
Example 1
Identification of Soluble Tim-3 Containing the Extracellular
Immunoglobulin Domain but Lacking the Mucin Domain of Full-Length
Tim-3
[0262] Primers were designed in the 5' and 3' untranslated regions
(UTRs) of the murine Tim-3 gene and were used to amplify Tim-3 from
cDNA generated from concanavalin A (con-A) activated splenocytes by
PCR. In addition to the full-length form of Tim-3 of approximately
1 kb, another amplicon 800 bp in size was identified (FIG. 1A, lane
1). The predicted amino acid translation of the 1 kb amplicon
demonstrated an open reading frame (ORF) consistent with the
full-length, membrane-anchored form of Tim-3 (fl-Tim-3), containing
signal peptide, IgV, mucin, transmembrane and cytoplasmic domains
(FIG. 1B). Analysis of the ORF from the 800 bp product demonstrated
the presence of a novel isoform of Tim-3 which contained only the
signal peptide, IgV and cytoplasmic domains, and lacked the mucin
domain and transmembrane region (FIGS. 1B, 1C). The absence of the
mucin and transmembrane domains was consistent with the splicing of
exons 3, 4 and 5 from the murine Tim-3 gene (FIG. 1C). These data
suggest that the product encoded by the 800 bp amplicon is an
alternatively spliced, soluble form of Tim-3 (s-Tim-3), containing
the IgV portion of the extracellular domain of murine TIM-3 fused
to the cytoplasmic domain.
Example 2
Construction of Soluble Tim-3-Ig Fusion Proteins
[0263] To identify potential Tim-3-Ligand(s) and their functional
in vivo interactions with TIM-3, soluble fusion proteins were
designed for both the full-length and soluble forms of Tim-3(21).
The cDNA encoding the entire extracellular portion of mouse Tim-3
but without transmembrane and cytoplasmic tail, was fused to cDNA
encoding a human IgG1 Fc tail to form full-length Tim-3-Ig fusion
protein (ex-Tim-3-Ig). In light of the existence of a secreted form
of Tim-3 containing only the IgV portion of the extracellular
domain (FIGS. 1A, 1B), a second fusion protein was constructed
composed of cDNA encoding the IgV portion of mouse Tim-3 fused with
cDNA encoding the human IgG1 Fc tail to form soluble Tim-3-Ig
fusion protein (s-Tim-3-Ig). These constructs were stably
transfected into NS.1 B cells, and the proteins were purified from
the supernatants(21).
Example 3
CD4.sup.+ T Cells Express Tim-3-Ligand
[0264] To determine which cells express Tim-3-Ligand, whole spleen
cells from naive, unimmunized SJL/J, NOD, C57BL/6 and BALB/c mice
were stained with ex-Tim-3-Ig or s-Tim-3-Ig, and co-stained for
various cell surface markers. When analyzed by flow cytometry,
CD4.sup.+ T cells obtained from each strain were positive for
Tim-3-Ligand expression when stained with either s-Tim-3-Ig or
ex-Tim-3-Ig fusion proteins. When purified CD4.sup.+ T cells were
co-stained for a panel of cell surface activation markers, it was
found that both naive (CD25.sup.-, CD62L.sup.+, CD44.sup.lo,
CD45RB.sup.hi, CD54.sup.-) and activated, memory/regulatory
(CD25.sup.+, CD62L.sup.-, CD44.sup.hi, CD45RB.sup.lo, CD54.sup.+)
populations expressed Tim-3-Ligand as assessed by both ex-Tim-3-Ig
and s-Tim-3-Ig staining (FIG. 2A). When CD4.sup.+ T cells were
purified and sorted for CD25 expression, both CD4.sup.+25.sup.+ and
CD4.sup.+25.sup.- populations expressed Tim-3-Ligand. However, when
sorted cells were activated for 48 h in vitro with increasing
concentrations of anti-CD3/anti-CD28 and IL-2, CD4.sup.+CD25.sup.+
T cells retained expression of Tim-3-Ligand whereas
CD4.sup.+CD25.sup.- cells downregulated. A small fraction of
CD11c.sup.+ dendritic cells and CD11b.sup.+ macrophages from whole
spleen were also stained by the Tim-3-Ig fusion proteins, but B
cells (B220.sup.+, CD19+) from whole spleen did not stain for
Tim-3-Ligand with either fusion protein.
[0265] Since CD4.sup.+ T cells stained positively with the Tim-3-Ig
fusion proteins, a panel of long term Th1 or Th2 clones was tested
for the expression of Tim-3-Ligand to determine whether there was
selective expression of Tim-3-Ligand on Th1 or Th2 cells.
Interestingly, both Th1 and Th2 cells were positive for staining
with s-Tim-3-Ig in the resting state (day 0, FIG. 2B), while
activation of these cell lines downregulated Tim-3-Ligand
expression (day 4, FIG. 2B). Between 7-10 days after activation,
the Tim-3-Ligand expression was upregulated on the now quiescent T
cell clones (day 10, FIG. 2B). Thus, it appears that Tim-3-Ligand
is expressed on both resting Th1 and Th2 cells and that expression
is downregulated upon activation. These data are consistent with
the data from the second series of experiments which demonstrate
that CD4/CD25- T cells (which contain effector T cells)
downregulate Tim-3-Ligand expression upon activation.
Example 4
In Vivo Administration of Tim-3-Ig Induces Hyperproliferation
[0266] To determine the in vivo role of Tim-3/Tim-3-Ligand
interaction during the course of a Th1 immune response, SJL/J mice
were immunized with myelin proteolipid protein PLP 139-151 in
complete Freund's adjuvant (CFA) and treated with either
ex-Tim-3-Ig or s-Tim-3-Ig, and human IgG (hIgG) or PBS as controls.
The treated mice were sacrificed on day 10, and spleen cells were
restimulated in vitro to determine proliferation and cytokine
production. Whole spleen cells from control hIgG- or PBS-treated
mice showed a low background (basal) response in the absence of
antigenic restimulation (FIG. 3A) and demonstrated a dose-dependent
increase in proliferation with the addition of PLP 139-151 peptide
(FIG. 3B). Conversely, spleen cells from mice treated with
ex-Tim-3-Ig or s-Tim-3-Ig had a very high basal proliferation in
the absence of antigenic restimulation, which in some experiments
was as high as 120,000 cpm (FIG. 3A). However, no major enhancement
in the proliferative response was observed when specific antigen
(PLP 139-151) was titrated into cell cultures from ex-Tim-3-Ig- or
s-Tim-3-Ig-treated mice (FIG. 3B). Taken together, these data
suggest that spleen cells from immunized mice treated with either
fusion protein rapidly proliferate in vivo such that they continue
to proliferate in vitro without further antigenic stimulation
(FIGS. 3A, 3B).
Example 5
In Vivo Administration of Tim-3-Ig Induces Amplified Th1 Cytokine
Production
[0267] To determine whether the proliferating cells also produce
cytokines, supernatants were harvested from in vitro cultures at 48
hours and analyzed by cytokine ELISAs for IL-2, IL-4, IL-10,
TNF-.alpha. and IFN-.gamma.. Analysis revealed that spleen cells
from immunized SJL/J mice treated with ex-Tim-3-Ig or s-Tim-3-Ig
produced large quantities of the Th1 cytokines IL-2 and
IFN-.gamma., without antigenic restimulation (FIG. 3A). In
contrast, spleen cells from mice treated with control PBS or hIgG
did not produce IL-2 or IFN-.gamma. in the absence of antigenic
restimulation (FIG. 3A). Spleen cells from mice treated with
ex-Tim-3-Ig or s-Tim-3-Ig also secreted high amounts of and
IFN-.gamma. in vitro following PLP 139-151 peptide restimulation
(FIG. 3B). Spleen cells from mice treated with control PBS or hIgG
produced little or no IFN-.gamma., and only produced IL-2 upon PLP
139-151 peptide restimulation (FIG. 3B). Taken together, these data
suggest that in vivo treatment with either ex-Tim-3-Ig or
s-Tim-3-Ig induced hyperproliferation of Th1 cells and release of
Th1 cytokines ex vivo.
Example 6
Hyperproliferation and Th1 Cytokine Production is Mediated by T
Cells in Tim-3-Ig Treated Mice
[0268] To determine the phenotype of the cell types that
proliferate in immunized, Tim-3-Ig-treated mice, individual cell
populations of CD3.sup.+ T cells, CD11b.sup.+ macrophages and
B220.sup.+ B cells were purified from the spleens of immunized
mice. After purification, cell populations were cultured separately
or re-combined in vitro and assessed for their ability to
proliferate as detected by .sup.3[H]-thymidine incorporation. Of
all the cell types, purified CD3.sup.+ T cells from mice treated
with ex-Tim-3-Ig proliferated vigorously without peptide
restimulation, whereas CD3.sup.+ T cells from control hIgG-treated
mice demonstrated only low background levels of proliferation (FIG.
4A). Individual populations of B220.sup.+ B cells or CD11b.sup.+
cells from control hIgG-treated mice showed low proliferation
(approximately 10,000 cpm; FIG. 4A). While the B cells and
CD11b.sup.+ cells from ex-Tim-3-Ig-treated mice showed twice as
much proliferation (approximately 20,000 cpm; FIG. 4A) as those
from the hIgG-treated control mice, this proliferation was minimal
in comparison to that of the CD3.sup.+ T cells from the
ex-Tim-3-Ig-treated mice. This suggested that T cells, and not
B220.sup.+ B cells or CD11b.sup.+ macrophages, harvested from the
ex-Tim-3-Ig-treated mice were hyperproliferating. This is in
contrast to the observation in anti-TIM-3 antibody treated mice,
where the majority of activation and expansion was seen in the
CD11b.sup.+/F4-80.sup.+ macrophage population, and not in the T or
B cell compartments (20). The dependence on lymphocytes for the
hyperproliferation phenotype in the Tim-3-Ig-treated mice is
further supported by data obtained from administration of Tim-3-Ig
to immunized RAG-2.sup.-/- mice. When SJL-RAG-2.sup.-/- mice were
immunized with PLP 139-151 in CFA and treated with ex-Tim-3-Ig or
s-Tim-3-Ig, there was no proliferative background or response to
antigenic restimulation observed, suggesting that the background
response seen in spleen cells taken from immunized, fusion
protein-treated mice was dependent on the presence of T and/or B
cells. Administration of ex-Tim-3-Ig or s-Tim-3-Ig to unimmunized
mice also increased the background proliferative response, but this
was not due to a polyclonal response to the human Fc tail present
in the fusion proteins since the T cell response could not be
recalled with either human Ig or either of the Tim-3-Ig fusion
proteins. Furthermore, administration of human Ig in unimmunized
mice did not induce a basal proliferative response (FIG. 3A). This
suggests that a subtle ongoing immune response in the unimmunized
mice can also be uncovered by the administration of Tim-3-Ig.
[0269] When CD3.sup.+ T cells from hIgG-treated mice were mixed
with B220.sup.+ B cells or CD11b.sup.+ cells from hIgG-treated
mice, the proliferation remained low (approximately 20,000 cpm) and
was only slightly increased by the addition of B220.sup.+ B or
CD11b.sup.+ cells from ex-Tim-3-Ig-treated mice (approximately
30-40,000 cpm) (FIG. 4A). The greatest proliferation was discerned
when CD3.sup.+ T cells from ex-Tim-3-Ig-treated mice were cultured
with B220.sup.+ B cells from either the hIgG- or
ex-Tim-3-Ig-treated mice (450,000 cpm) (FIG. 4A). Interestingly,
when CD3.sup.+ T cells from ex-Tim-3-Ig-treated mice are mixed with
CD11b.sup.+ cells from either hIgG- or ex-Tim-3-Ig-treated mice,
the spontaneous proliferation seen in CD3.sup.+ T cells from
Tim-3-Ig-treated mice was markedly decreased (down to 30-38,000 cpm
from 90,000 cpm for purified CD3.sup.+ T cells alone) (FIG. 4A).
Taken together, these data suggest that administration of
ex-Tim-3-Ig during an ongoing immune response induced
hyperproliferation of CD3.sup.+ T cells, with a smaller effect on
the proliferation of APCs (B cells and macrophages). B220.sup.+ B
cells, regardless of their source, enhanced the proliferation of
the T cells from ex-Tim-3-Ig-treated mice, whereas CD11b.sup.+
macrophages inhibited the spontaneous hyperproliferation of
CD3.sup.+ T cells from ex-Tim-3-Ig-treated mice.
[0270] To determine the cytokine secretion patterns of these
individual cell populations, supernatants were taken at 48 h and
analyzed for IL-2, IL-4, IL-10, TNF-.alpha. and IFN-.gamma.
production by ELISA. While no individual population of cells from
ex-Tim-3-Ig-treated mice was found to produce IL-2, CD3.sup.+ T
cells from mice treated with ex-Tim-3-Ig were responsible for the
majority of the IFN-.gamma. production (FIG. 4B). Addition of
B220.sup.+ B cells or CD11b.sup.+ macrophages (from either
ex-Tim-3-Ig- or hIgG-treated mice) resulted in the secretion of
IL-2 by CD3.sup.+ T cells from ex-Tim-3-Ig-treated mice.
Conversely, addition of B220.sup.+ B cells or CD11b.sup.+
macrophages to the CD3.sup.+ T cells from ex-Tim-3-Ig-treated mice
resulted in a decrease in IFN-.gamma. production from these
CD3.sup.+ T cells (FIG. 4B). In short, these data demonstrate that
hyperproliferating CD3.sup.+ T cells from ex-Tim-3-Ig-treated mice
produced large amounts of IFN-.gamma., but required APCs to produce
IL-2.
[0271] To further characterize the cells that hyperproliferate in
response to in vivo treatment with ex-Tim-3-Ig, the phenotype of
cells incorporating 5-bromodeoxyuridine (BrdU), a thymidine analog
that is incorporated by cycling cells, was determined. The
percentage of activated CD3.sup.+CD25.sup.+ cells was increased 2-3
fold in the Tim-3-Ig-treated groups over the hIgG-treated control
group. From this data (FIG. 4C), it was also clear that there was
an increased percentage of CD3.sup.+ T cells from the
Tim-3-Ig-treated mice that incorporated BrdU when compared to
hIgG-treated controls. This data was consistent with an increase in
the percentage of CD3.sup.+CD25.sup.+ and CD3.sup.+CD69.sup.+ cells
observed in whole spleen cells from Tim-3-Ig-treated mice as
compared to cells from the control hIgG-treated mice. This
observation supported the in vitro proliferation and cytokine data
(FIGS. 4A, 4B), and confirmed that T cells from Tim-3-Ig-treated,
immunized mice are highly activated, rapidly proliferating cells
(FIG. 4C).
Example 7
Administration of Tim-3-Ig to Mice Abrogates Induction of
Tolerance
[0272] Since administration of Tim-3-Ig in vivo resulted in
hyperactivation of Th1 cells with amplified production of IL-2,
Applicants hypothesized that administration of Tim-3-Ig may prevent
or abrogate induction of peripheral tolerance. SJL/J mice were
tolerized with high dose soluble PLP 139-151 peptide and
concurrently immunized with PLP 139-151 emulsified in CFA. This
tolerization protocol renders PLP 139-151-specific T cells tolerant
to subsequent activation such that cells hypoproliferate and do not
produce IL-2(24). To test the effect of ex-Tim-3-Ig on the
induction of tolerance, tolerized mice were treated every other day
for 8 days with Tim-3-Ig or control PBS or hIgG, and lymph nodes
and spleens were taken at day 10 and examined for their in vitro
recall response to PLP 139-151 through proliferation and cytokine
production.
[0273] The induction of tolerance in the draining lymph nodes was
examined, in addition to the spleens, since it is in the draining
lymph nodes that T cells are induced and first activated. As seen
in FIG. 5, lymph node cells from immunized mice tolerized with
soluble PLP showed a significant decrease in proliferation to
restimulation with PLP 139-151 compared to the control
(PBS-tolerized hIgG-treated group). When Tim-3-Ig was
co-administered with the tolerogenic dose of PLP 139-151, a
significant proliferative response was observed which was
comparable to or even greater than that of the control,
non-tolerized mice, demonstrating that Tim-3-Ig abrogated or
interfered with tolerance induction (FIG. 5). While tolerization
with PLP 139-151 resulted in the complete loss of IL-2 and
IFN-.gamma. production in the control hIgG-treated group, mice
treated with Tim-3-Ig continued to produce Th1 cytokines when
restimulated in vitro with cognate antigen (FIG. 5).
[0274] The effects of tolerance induction in the spleens of the
mice were determined. As shown in FIG. 5A, concurrent in vivo
administration of soluble PLP 139-151 in PLP-immunized mice
resulted in a dramatic decrease in the proliferative response to
PLP 139-151 peptide, confirming the ability of soluble PLP 139-151
to induce T cell tolerance in vivo. Administration of hIgG or PBS
together with the soluble PLP 139-151 did not alter tolerance
induction (FIG. 6A). However, when ex-Tim-3-Ig was administered
together with a tolerogenic dose of PLP 139-151, the proliferation
of splenic cells from ex-Tim-3-Ig-treated mice was equivalent to
that obtained in splenic cells from non-tolerized control animals
(FIG. 6A). Hence, administration of Tim-3-Ig overcomes the
induction of or abrogates tolerance, and results in T cell
expansion.
[0275] Since high dose soluble antigen is known to predominantly
induce tolerance of Th1 cells and particularly inhibit IL-2
production, Applicants next investigated production of cytokines in
the spleens of the tolerized mice. Supernatants were harvested from
in vitro cultures at 48 h, and cytokine ELISAs for IL-2, IL-4,
IL-10, IFN-.gamma. and TNF-.alpha. were performed to determine if
spleen cells from tolerized and Tim-3-Ig-treated mice were
producing cytokines. As expected, administration of soluble PLP to
PLP-immunized mice inhibited IL-2 production (FIG. 6B).
Co-administration of ex-Tim-3-Ig resulted in a dramatic increase in
IL-2 production, such that IL-2 was produced at lower antigenic
concentrations in the ex-Tim-3-Ig treated mice when compared to
non-tolerized, PLP-immunized mice (FIG. 6B). Whereas PLP-immunized
mice did not show any significant production of IFN-.gamma. or
TNF-.alpha., Tim-3-Ig treatment of the tolerized mice resulted in a
dramatic production of both IFN-y and TNF-a from the splenic
cultures upon restimulation in vitro (FIG. 6B). Low levels of IL-4
and IL-10 were observed in supernatants from the tolerized,
ex-Tim-3-Ig-treated mice only at the highest doses of in vitro
restimulation with specific antigen (FIG. 6B).
C. Discussion of First Series of Experiments
[0276] The discovery of Tim-3 as a Th1-specific transmembrane
protein provided a novel means by which to phenotypically
distinguish Th1 from Th2 cells. The functional role of this cell
surface protein expressed on fully committed Th1 cells is beginning
to be elucidated. Initial studies demonstrated that administration
of anti-Tim-3 antibody during the course of an immune response
exacerbated the autoimmune disease EAE and resulted in the
activation and expansion of macrophages(20). The data presented
herein begins to explore the functional in vivo effects of
Tim-3/Tim-3-Ligand interaction by use of soluble Tim-3 fusion
proteins. Administration of ex-tim-3-Ig or s-Tim-3-Ig fusion
proteins in vivo during an ongoing immune response resulted in
hyperproliferation of T cells with spontaneous production of Th1
cytokines (IFN-.gamma. and IL-2), even in the absence of antigenic
re-stimulation. Furthermore, Tim-3-Ig abrogated or interfered with
the induction of tolerance mediated by the administration of high
dose soluble antigen.
[0277] Flow cytometric analysis of lymphoid cells harvested from
nave mice and stained with Tim-3-Ig revealed that a ligand for
Tim-3 is expressed on CD4+ T cells. Furthermore, T cell clones,
whether of Th1 or Th2 phenotype, bound Tim-3-Ig. Although a small
number of CD11c.sup.+ dendritic cells and CD11b.sup.+ macrophages
also appeared to stain with Tim-3-Ig, CD4.sup.+ T cells were the
predominant cell type expressing Tim-3-Ligand. This data was
superficially surprising since our initial observations regarding
Tim-3 showed that treatment of immunized SJL/J mice with anti-Tim-3
antibody increased basal proliferation of whole spleen cells via a
cell-to-cell interaction between macrophages and Th1 cells(20).
This suggested that the ligand for Tim-3 was potentially expressed
on macrophages and that anti-Tim-3 antibody either co-capped Tim-3
on Th1 cells to activate macrophages via its ligand, or blocked a
negative signal that resulted in macrophage activation. One
possible explanation is that a small but potentially critical
population of macrophages that may bear a ligand for Tim-3 were
activated in this manner by the antibody administration. An
alternate possibility is that the macrophage activation seen upon
anti-Tim-3 antibody treatment in vivo may be a secondary
consequence of disturbing the interaction of Tim-3 with the
Tim-3-Ligand expressed on other T cells(25).
[0278] Knowing that Tim-3 is expressed on terminally differentiated
Th1 cells and that a potential Tim-3-Ligand is expressed on
CD4.sup.+ T cells raises the question of how these partners
interact in vivo. There are at least two potential mechanisms
whereby the Tim-3 fusion proteins might interact with the
Tim-3-Ligand in vivo. First, Tim-3-Ig may bind to CD4.sup.+ T cells
in vivo during the course of immunization and signal through this
ligand. This signal may preferentially differentiate naive
CD4.sup.+ T cells into Th1 cells or increase the activation state
of these CD4.sup.+ T cells. Given the Th1 environment created by
immunization with PLP 139-151 in CFA, these activated cells may
preferentially polarize to the Th1 phenotype, thus accounting for
the significantly increased T cell proliferation and production of
the Th1 cytokines IFN-.gamma. and IL-2. However, direct
cross-linking of Tim-3-Ligand on T cells, with or without
CD3-crosslinking by plate-bound antibody, does not induce
hyperproliferation of Th1 cells or production of Th1 cytokines.
This suggests that activation may not in fact occur through
Tim-3-Ig ligation of Tim-3-Ligand, or may uniquely occur in vivo
under Th1-polarizing conditions. A second possibility is that
Tim-3-Ig may bind the Tim-3-Ligand on ligand-bearing cells (T
cells, macrophages and/or dendritic cells), thus blocking its
ability to interact with Tim-3 on polarized Th1 cells. If the
normal physiological function of the interaction between Tim-3 and
Tim-3-Ligand is to downregulate the Th1 response, blocking the
ligand by administration of Tim-3-Ig could account for the
increased Th1 cell proliferation and Th1 cytokine production
observed.
[0279] The data presented herein and that of the second
experimental series favors the hypothesis that the interaction of
Tim-3 with its ligand may be an inhibitory one and that Tim-3-Ig
administration blocks this negative interaction, resulting in the
expansion of Th1 cells. This possible mechanism is further
supported by the data that administration of Tim-3-Ig abrogated
induction of peripheral tolerance, suggesting that the
physiological interaction between Tim-3 and Tim-3-Ligand may serve
to limit expansion of Th1 cells and contribute to induction of
tolerance in effector Th1 cells. There are at least two possible
mechanisms whereby Tim-3-Ig may abrogate tolerance induction.
Tim-3-Ig may hyperactivate effector Th1 cells, and IL-2 thus
produced may prevent induction of anergy by high dose soluble
antigen. However, another possibility is that Tim-3-Ig may induce
proliferation and effector functions in non-tolerized T cells that
normally may not proliferate (due to low affinity TCR/MHC-peptide
interaction) such that the net result is T cell expansion rather
than nonresponsiveness. It is known that high dose tolerance often
tolerizes Th1 cells while sparing or expanding Th2 cells(26). As
administration of Tim-3-Ig abrogates this tolerance induction in
Th1 cells, Th2 cells may remain unaffected, leading to the
production of Th2 cytokines IL-4 and IL-10 observed upon
restimulation of spleen cells in vitro (FIG. 5B).
[0280] Our results suggest that the Tim-3/Tim-3-Ligand pathway may
be important for tolerance induction. Interestingly, it has also
been shown that CTLA-4 is required for tolerance induction (27).
Given the importance of Tim-3 and Tim-3-Ligand in tolerance
induction, it is possible that the Tim-3/Tim-3-Ligand pathway may
be functioning as a negative regulator of immune responses in a
manner similar to CTLA-4, but specifically for effector Th1
cell.
[0281] The discovery of a soluble, Ig domain-only form of Tim-3
raised the issue of the role of soluble Tim-3 in the regulation of
Th1 responses. This soluble splice variant of Tim-3 is similar to
other immunoregulatory/inhibitory receptors that are made as
soluble molecules by T cells (e.g. CTLA-4), and these soluble
alternatively spliced forms of the receptors have been shown to
play an important role in susceptibility and resistance to
autoimmune disease (23). It is not, however, clear at this stage
when s-Tim-3 is made during Th1 cell differentiation and what its
function might be. If, as the present data suggests, the
Tim-3/Tim-3-Ligand interaction is an inhibitory one, production of
s-Tim-3 may act to block this inhibitory effect, and promote
expansion and differentiation of Th1 cells. This could occur if
binding of s-Tim-3 to Tim-3-Ligand competes with the inhibitory
binding of membrane-bound ex-tim-3 to Tim-3-Ligand. This
competition could diminish or prevent the ligation of ex-Tim-3 to
Tim-3-Ligand, and subsequently decrease or abrogate downregulation
of the Th1 response. Potential regulation of the production of
ex-Tim-3 versus s-Tim-3 at the mRNA and protein levels could offer
a dynamic means of regulating the immune function of effector Th1
cells.
[0282] Since s-Tim-3 contains only the IgV domain but lacks the
mucin domain, this data further suggests that the majority of
Tim-3's immunoregulatory activity may be mediated by its Ig domain.
Preliminary data suggests that s-Tim-3-Ig shows high
affinity/avidity binding to CD4.sup.+ T cells and also mediates
stronger immunoregulatory effects in vivo. It has previously been
shown that members of the picornavirus family (such as poliovirus
and rhinoviruses) and human immunodeficiency virus (HIV)
preferentially bind to the Ig domain of receptors rather than
subsequent functional domains(28-31). Furthermore, havcr-1, the
receptor for picornavirus family member hepatitis A virus (HAV),
has been shown to bind HAV through the N-terminal cysteine
(Cys)-rich region of havcr-1 (which is part of the Ig domain of the
receptor)(28,29). Given that havcr-1 is the human homolog of murine
Tim-1(19), a member of the Tim gene family, it stands to reason
that Tim-3 may interact with its ligand in a similar manner,
through its Ig domain.
[0283] The potential functional role(s) of the mucin domain that is
part of the full-length membrane-anchored form of Tim-3 are
uncertain. It has been postulated that the Threonine/Serine/Proline
(TSP)-rich mucin-like region of havcr-1 may serve to extend the
Cys-rich region above the cell surface to facilitate binding to
HAV(28,32). The mucin domain of Tim-3 may function in a similar
structural rather than functional binding manner. Another
possibility is that the mucin domain of Tim-3 may present
carbohydrate moieties that can interact with selectins, and thus
facilitate the trafficking of effector Th1 cells. A role such as
this in facilitating lymphocyte homing has been postulated for the
mucin region of the structurally similar multi-domain receptor
MAdCAM-1(33).
[0284] Taken together, these data suggest that Tim-3 interaction
with its ligand may represent an inhibitory pathway which regulates
expansion and function of Th1 cells during normal T cell immune
responses. This pathway may also play a crucial role in regulating
peripheral tolerance of effector Th1 cells.
D. References
[0285] 1. Mosmann, T., Cherwinski, H., Bond, M., Giedlin, M. &
Coffman, R. Two types of murine helper T cell clone. I. Definition
according to profiles of lymphokine activities and secreted
proteins. J Immunol 136, 2348-2357 (1986). [0286] 2. Mosmann, T.
& Sad, S. The expanding universe of T-cell subsets: Th1, Th2
and more. Immunol Today 19, 138-146 (1996). [0287] 3. Abbas, A.,
Murphy, K. & Sher, A. Functional diversity of helper T
lymphocytes. Nature 383, 787-793 (1996). [0288] 4. Sher, A. &
Coffman, R. Regulation of immunity to parasites by T cells and T
cell-derived cytokines. Annu Rev Immunol. 10, 385-409 (1992).
[0289] 5. Liblau, R., Singer, S. & McDevitt, H. Th1 and Th2
CD4+ T cells in the pathogenesis of organ-specific autoimmune
diseases. Immunol Today 16, 34-38 (1995). [0290] 6. Nicholson, L.,
Greer, J., Sobel, R., Lees, M. & Kuchroo, V. An altered peptide
ligand mediates immune deviation and prevents autoimmune
encephalomyelitis. Immunity 3, 397-405 (1995). [0291] 7. Kuchroo,
V. et al. B7-1 and B7-2 costimulatory molecules activate
differentially the Th1/Th2 developmental pathways: application to
autoimmune disease therapy. Cell 80, 707-718 (1995). [0292] 8.
Lack, G. et al. Nebulized but not parenteral IFN-gamma decreases
IgE 5 production and normalizes airways function in a murine model
of allergen sensitization. J Immunol 152, 2546-2554 (1994). [0293]
9. Hofstra, C. et al. Prevention of Th2-like cell responses by
coadministration of IL-12 and IL-18 is associated with inhibition
of antigen-induced airway hyperresponsiveness, eosinophilia, and
serum IgE levels. J Immunol 161, 5054-5060 10 (1998). [0294] 10.
Syrbe, U., Siveke, J. & Hamann, A. Th1/Th2 subsets: distinct
differences in homing and chemokine receptor expression? Springer
Semin Immunopathol 21, 263-285 (1999). [0295] 11. Loetscher, P. et
al. CCR5 is characteristic of Th1 lymphocytes. Nature 391, 344-345
(1998). [0296] 12. Bonecchi, R. et al. Differential expression of
chemokine receptors and chemotactic responsiveness of type 1 T
helper cells (This) and Th2s. J Exp Med 187, 129-134 (1998). [0297]
13. Sallusto, F., Lenig, D., Mackay, C. & Lanzavecchia, A.
Flexible programs of chemokine receptor expression on human
polarized T helper 1 and 2 lymphocytes. J Exp Med 187, 875-883
(1998). [0298] 14. Venkataraman, C., Schaefer, G. & Schindler,
U. Cutting edge: Chandra, a novel four-transmembrane domain protein
differentially expressed in helper type 1 lymphocytes. J Immunol
165, 632-636 (2000). [0299] 15. Jourdan, P. et al. IL-4 induces
functional cell-surface expression of CXCR4 on human T cells. J
Immunol 160, 4153-4157 (1998). [0300] 16. Lohning, M. et al. Tl/ST2
is preferentially expressed on murine Th2 cells, independent of
interleukin 4, interleukin 5, and interleukin 10, and important for
Th2 effector function. Proc Natl Acad Sci USA 95, 6930-6935 (1998).
[0301] 17. McAdam, A. et al. Mouse inducible costimulatory molecule
(ICOS) expression is enhanced by CD28 costimulation and regulates
differentiation of CD4+ T cells. J Immunol 165, 5035-5040 (2000).
[0302] 18. Zingoni, A. et al. The chemokine receptor CCR8 is
preferentially expressed in Th2 but not Th1 cells. J Immunol 161,
547-551 (1998). [0303] 19. McIntire, J. et al. Identification of
Tapr (an airway hyperreactivity regulatory locus) and the linked
Tim gene family. Nat Immunol 2, 1109-1116 (2001). [0304] 20.
Monney, L. et al. Th1-specific cell surface protein Tim-3 regulates
macrophage activation and severity of an autoimmune disease. Nature
415, 536-541 (2002). [0305] 21. Zheng, X. et al. Administration of
noncytolytic IL-10/Fc in murine models of
lipopolysaccharide-induced septic shock and allogeneic islet
transplantation. J Immunol 154, 5590-5600 (1995). [0306] 22.
Sanchez-Fueyo, A. et al. The Ig superfamily member Tim-3 inhibits
Th1-15 mediated auto- and allo-immune response and promostes
immunological tolerance. (submitted). [0307] 23. Ueda, H. et al.
Association of the T-cell regulatory gene CTLA4 with susceptibility
to autoimmune disease. Nature 423, 506-511 (2003). [0308] 24.
Perez, V. et al. Induction of peripheral T cell tolerance in vivo
requires CTLA-4 engagement. Immunity 6, 411-417 (1997). [0309] 25.
Kuchroo, V., Umetsu, D., DeKruyff, R. & Freeman, G. The TIM
gene family: emerging roles in immunity and disease. Nat Rev
Immunol 3, 454-462 (2003). [0310] 26. Burstein, H. & Abbas, A.
In vivo role of interleukin 4 in T cell tolerance induced by
aqueous protein antigen. J Exp Med 177, 457-463 (1993). [0311] 27.
Greenwald, R., Boussiotis, V., Lorsbach, R., Abbas, A. &
Sharpe, A. CTLA-4 regulates induction of anergy in vivo. Immunity
14, 145-155 (2001). [0312] 28. Thompson, P., Lu, J. & Kaplan,
G. The Cys-rich region of hepatitis A virus cellular receptor 1 is
required for binding of hepatitis A virus and protective monoclonal
antibody 190/4. J Virol 72, 3751-3761 (1998). [0313] 29.
Silberstein, E., Dveksler, G. & Kaplan, G. Neutralization of
hepatitis A virus (HAV) by an immunoadhesin containing the
cysteine-rich region of HAV cellular receptor-1. J Virol 75,
717-725 (2001). [0314] 30. Xing, L. et al. Distinct cellular
receptor interactions in poliovirus and rhinoviruses. EMBO J 19,
1207-1216 (2000). [0315] 31. Kaplan, G. et al. Identification of a
surface glycoprotein on African green monkey kidney cells as a
receptor for hepatitis A virus. EMBO J 15, 4282-4296 (1996). [0316]
32. Jentoft, N. Why are proteins O-glycosylated? Trends Biochem Sci
15, 291-294 (1990). [0317] 33. Briskin, M., McEvoy, L. &
Butcher, E. MAdCAM-1 has homology to immunoglobulin and mucin-like
adhesion receptors and to IgA1. Nature 363, 461-464 (1993). [0318]
34. Kovac, Z. & Schwartz, R. The molecular basis of the
requirement for antigen processing of pigeon cytochrome c prior to
T cell activation. J Immunol 134, 3233-3240 (1985). [0319] 35.
Matis, L. et al. Clonal analysis of the major histocompatibility
complex restriction and the fine specificity of antigen recognition
in the T cell proliferative response to cytochrome C. J Immunol
130, 1527-1535 (1983). [0320] 36. Nicholson, L., Murtaza, A.,
Hafler, B., Sette, A. & Kuchroo, V. A T cell receptor
antagonist peptide induces T cells that mediate bystander
suppression and prevent autoimmune encephalomyelitis induced with
multiple myelin antigens. Proc Natl Acad Sci USA 94, 9279-9284
(1997).
Second Series of Experiments
A. Introduction
[0321] Differentiation and clonal expansion of T helper (Th)
precursor cells into T effector populations plays an important role
in the adaptive immune response and provides protection against
intracellular viruses and pathogenic bacteria. However,
unrestrained activation of Th effector cells has also been shown to
underlie a number of inflammatory disorders. In this context, Th1
effector cells are implicated in the pathogenesis of rheumatoid
arthritis, inflammatory bowel disease (IBD), and other autoimmune
disorders including type I diabetes and multiple sclerosis(1,2), as
well as allograft rejection(3,4). In contrast, Th2 cell activation
plays a critical role in the pathogenesis of allergic asthma(5) and
has been linked to the acquisition of transplant tolerance(3,4).
The extent of T cell activation and mode of differentiation is
largely determined by the duration and strength of T cell receptor
(TCR) mediated stimulation(6). In addition, a number of
costimulatory and accessory molecules, including TNF receptor(7)
and immunoglobulin (Ig) superfamily members(8), as well as
cytokines such as IL-2, regulate the extent of clonal expansion,
deletion, and/or anergy induction(9). However, while Applicants
appreciate many of the cellular and molecular mechanisms that
regulate the activation of naive T cells, the molecules that
determine the fate of effector T cell subpopulations remain to be
elucidated.
[0322] The Ig superfamily member TIM-3 (T cell Immunoglobulin
domain, Mucin domain) was initially described by Monney et al.(10)
as a transmembrane protein preferentially expressed on
differentiated Th1 cells. In a model of experimental allergic
encephalomyelitis (EAE), a Th1-mediated autoimmune disease, in vivo
administration of anti-TIM-3 monoclonal antibody (mAb) led to more
severe inflammatory events within the brain and more severe
clinical disease. Based on these observations, TIM-3 was proposed
as a negative regulator of tissue destructive immune responses in
EAE(10). However, it remained uncertain whether these data
reflected inhibition of a negative signal provided by TIM-3 or,
conversely, whether TIM-3 crosslinking in vivo with the mAb exerted
a positive signal to induce T cell activation and disease
exacerbation. Based on results reported herein, Applicants conclude
that TIM-3 engagement by its putative ligand provides an inhibitory
signal to dampen inflammatory responses in vivo. TIM-3 pathway
blockade via TIM-3-Ig fusion protein treatment accelerates diabetes
onset in the NOD (non obese diabetic) model, and abolishes the
capacity of both CTLA4 and combined donor specific transfusion
(DST) plus anti-CD154 (CD40L) treatment, potent, costimulatory
blockade-based, tolerance-promoting protocols in
transplantation(11-13), to induce tolerance to MHC-mismatched
allografts. While the precise mechanisms involved remain to be
fully elucidated, Applicants propose that TIM-3 regulates the
outcome of auto- and allo-immune responses at least in part by
modulating the capacity of regulatory T cells to dampen
inflammatory responses.
B. Methodology
Mice
[0323] All mice were obtained from The Jackson Laboratories (Bar
Harbor, Me.) and maintained under specific pathogen free conditions
in conventional animal facilities at Millennium Pharmaceuticals,
Inc. (Cambridge, Mass., USA), Beth Israel Deaconess Medical Center
(Boston, Mass., USA), or DRFZ (Berlin, Germany) according to the
relevant institutional and state guidelines.
Identification and Cloning of Murine TIM-3
[0324] A Th1-specific library was generated using the Clontech
PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, Calif.). RNA
from activated Th1 clones were used as "tester" and RNA from
activated Th2 clones used as "driver." Plasmid DNAs from individual
clones were spotted onto nylon filters and hybridization performed
with single stranded probes from Th1 and Th2 RNA. One of the clones
in the Th1 library consisted of an 857-bp cDNA and was used to
obtain the full-length clone using a cDNA library from murine Th1
cells. The human homologue was subsequently identified and
sequenced from a human spleen cDNA library. Murine antigen specific
Th1 (AE7 and Donis) and Th2 (D10.G4, DAX, CDC25) clones were
stimulated every 10-14 days with peptide, mitomycin C treated APCs,
and IL-2 (100 U/ml). Cells were activated with anti-CD3 mAb (2C11,
Pharmingen) and RNA isolated. Differential expression of TIM-3 cDNA
was subsequently confirmed by Northern Blot from resting and
anti-CD3 activated clones.
Generation of TIM-3 mAb and TIM-3 Fusion Proteins
[0325] A DNA sequence containing the extracellular domain of TIM-3
was PCR-amplified and cloned into a vector containing the CD5
signal sequence and the human 30 IgG1 constant region. COS cells
were transfected and the recombinant protein purified over a
protein A column. Wky rats were immunized with purified murine
TIM-3 fusion protein (100 .mu.g) in CFA and boosted i.p. and
subcutaneously. Splenocytes were fused with SP/2 myeloma cells and
the resulting clones screened for binding on TIM-3-transfected CHO
cells. One of these clones, 8H7, was selected based on specific
binding to TIM-3, but not ICOS transfectants. Anti-TIM-3 8H7 mAb
was isotyped as a rat IgG1 using specific antibodies (BD
Pharmingen, San Diego, Calif.). ex-Tim-3-Ig and s-TIM-3-Ig were
constructed as human IgG1 Fc tail fusion proteins and expressed in
NS.1 cells as described in the first experimental series.
Biotinylated TIM-3 related fusion proteins or human IgG1 together
with fluorochrome-conjugated streptavidin (BD PharMingen) were used
for TIM-3L staining experiments.
Islet Transplantation
[0326] Islet transplantation was performed as previously
described(51). Approximately 700 DBA/2 (H-2.sup.d) islets were
transplanted under the renal capsule of streptozotocin-induced
diabetic C57BL/6 (H-2.sup.b). Allograft function was monitored by
serial blood glucose measurements. The tolerizing protocol employed
consisted in the i.v. administration of 10.sup.7 DBA/2 splenocytes
28 days before transplantation (day -28), and 250 .mu.g of a
hamster mAb anti-mouse CD154 (MR1, IgG2a, ATCC HB11048, American
Type Culture Collection, Rockville, Md.) i.p. on days -28, -26,
-24. ex-Tim-3-Ig control hIgG1 were administered i.p at a dose of
250 .mu.g on days -28, -26, -24. In selected recipients, 200 .mu.g
of rat anti-mouse CD25 mAb (PC61, 5.3, IgG1, ATCC TB222) was also
administered i.p. on days -40, -38 and -36. Applicants have
previously determined that anti-CD25 mAb administered at such doses
eliminates more than 80% of CD4+CD25+ T cells in both secondary
lymphoid organs and peripheral blood. In some experiments tolerance
was induced by administering 0.1 .mu.g CTLA4Ig on days 0, 2, 4, 6,
8 after transplantation.
Real-Time PCR Experiments
[0327] Total RNA was extracted from islet grafts with trizol and
reverse transcription performed using Multiscribed Reverse
Transcriptase Enzyme (PE Applied Biosystems, Foster City, Calif.).
Real-time PCR was performed with the ABI 7700 sequence detector
system (PE Applied Biosystems). To quantify the levels of mRNA, the
expression of the target genes was normalized to the housekeeping
gene GAPDH, and data were expressed as relative fold difference
between cDNA of the study samples and a calibrated sample. TIM-3
primer/probe sequences are as follows: probe,
5'-ACAGCTGCCTGCCCAGTGCCC-3'; forward primer,
5'-GCCGGTGGACCTCAGTTTC-3'; reverse primer,
5'-TGGGAGCCAGCACAGATCA-3'. GAPDH, IFN-.gamma., IL-4 and IL-10
primer/probe sets were purchased from Applied Biosystems.
Adoptive Cell Transfer into Skin Allograft Recipients
[0328] Single cell suspensions of lymph node cells were obtained
from naive C3H/He mice (H-2.sup.k), or from C3H/He mice treated
with an i.v. injection of 10.sup.7 DBA/2 splenocytes (28 days
before; -28), and either anti-CD154 (250 .mu.g on days -28, -26 and
-24) or anti-CD154 plus ex-Tim3-Ig (250 .mu.g each on days -28, -26
and -24). Cells were stained with fluorochrome-conjugated anti-CD25
and anti-CD4 (all from BD PharMingen) and sorted on a MoFlo.RTM.
High-Performance Cell Sorter (Cytomation; Fort Collins, Colo.).
Purity of CD4+CD25- and CD4+CD25+ preparations was consistently
>90%. Varying numbers of CD4+CD25+ T regs and CD4+CD25-effector
T cells were injected into the tail vein of C3H/He Scid mice hosts
undergoing allogeneic DBA/2 skin transplantation 24 hours later. In
some experiments, 3 doses of 250 .mu.g ex-Tim-3-Ig were
administered on days 0, 2 and 4 after skin transplant.
Full-thickness DBAJ2 tail skin allografts were performed as
previously described(52), and graft survival was monitored daily.
Rejection was defined as a complete necrosis of the skin grafts.
Similar experiments were performed using wild type C57BL/6 mice as
donors of T cell populations, and C57BL/6Rag.sup.-/- as recipients
of DBA/2 skin allografts.
In Vitro Proliferation Assays
[0329] To assess the proliferation of CD4+ T cell subpopulations
CD4+CD25+ and CD4+CD25- T cells were sorted as previously described
and cultured in round-bottom 30 96-well microtiter plates at a cell
density of 5.times.10.sup.5/mL together with 2 .mu.g/mL anti-CD3
mAb, 5 .mu.g/mL anti-CD28 mAb and 100 U/mL of rLL-2 (all from
PharMingen). Cells were harvested and stained with biotinylated
s-TIM-3-Ig or control human IgG1 followed by streptavidin-CyChrome
at 24 and 48 hours and analyzed by flow cytometry.
C. Examples of Second Series of Experiments
Example 11
A TIM-3 Ligand is Expressed on CD4+ T Cells
[0330] The structure of TIM-3 is somewhat reminiscent of mucosal
addressin cell adhesion molecule-1 (MAdCam-1), which contains two
Ig domains and a mucin rich region (10). In order to assess the
distribution of putative TIM-3 ligand(s) (TIM-3L), human
IgG1-derived Fc fusion proteins incorporating the extracellular
domain of murine TIM-3 (full-length or ex-Tim-3-Ig), or a
truncated, soluble, Ig domain only (s-TIM-3-Ig)(16) were generated.
Both ex-Tim-3-Ig and s-TIM-3-Ig bound to resting CD4+ T cells, but
not CD8+, B220+ or CD11b+ cells (FIG. 8C), with s-TIM-3-Ig binding
more intensely. Some binding was also noted on splenic CD11c+
dendritic cells (FIG. 7A), but the pattern of staining was far less
consistent than that observed for CD4+ T cells. Based on the
amplified staining pattern observed with s-TIM-3-Ig, as compared to
ex-Tim-3-Ig, the Ig domain of TIM-3 appears to be responsible for
interactions with TIM-3L.
Example 12
CD4+CD25- T Cells, but not CD4+CD25+Regulatory T Cells,
Down-Regulate TIM-3L Expression after Activation
[0331] In resting conditions, TIM-3 related fusion proteins bind
similarly to both effector CD4+CD25- and regulatory CD4+CD25+ T
cell subpopulations (FIG. 7B, upper panel). In order to identify
potential mechanisms through which TIM-3 might selectively target
the function of either regulatory or effector CD4+ T cells, the
kinetics of TIM-3L expression, assessed by the binding of
s-TIM-3-Ig, on CD4+CD25- and CD4+CD25+ T cells after activation in
vitro was studied. Stimulation with anti-CD3 and anti-CD28 mAbs did
not alter TIM-3L expression throughout the first 24 hours of
culture (FIG. 7B, middle panel). In contrast, TIM-3L expression was
downregulated upon activated CD4+CD25- T cells at 48 hours of
culture, while TIM-3L expression persisted upon CD4+CD25+
regulatory T cells (FIG. 7B lower panel). Downregulation of TIM-3L
expression on CD4+CD25- T cells was also observed upon in vitro
activation with Concanavalin A or LPS. The preferential expression
of the putative TIM-3L on activated regulatory T cells is mirrored
by the expression of TIM-3 on activated Th1 cells. Hence,
Applicants have investigated the hypothesis that TIM-3/TIM-3L
interactions are crucial to the acquisition and/or maintenance of
immunoregulation and tolerance in Th1-mediated immune
responses.
Example 16
TIM-3 Pathway Blockade Prevents Acquisition of Tolerance in 10
Allograft Models
[0332] While transplantation tolerance can be achieved in both Th1
and Th2 polarized conditions(4,25,26), Th1 to Th2 immunodeviation
facilitates the occurrence of tolerance(4,27). Indeed, the effects
of many tolerizing immunosuppressive regimens are associated with
immunodeviation into Th2-type allograft response(4). To address the
role of Th1-specific TIM-3 cell surface proteins in the acquisition
of allograft tolerance, an islet allograft model was utilized in
which recipients are treated with the combination of donor specific
transfusion (DST) and anti-CD154 (CD40L) mAb to achieve CD4O-CD154
costimulatory blockade(11,12,28). In this model, a single dose of
DST and anti-CD154 administered 1 month before transplantation
ensures the indefinite survival of MHC-mismatched islet allografts.
Moreover, treated long-surviving transplant recipients readily
accept second grafts from the same donor while rapidly reject
third-party strain grafts, indicating that treatment induces
donor-specific allograft tolerance(12,28). In contrast, in
untreated control recipients an islet-invasive lymphocytic
infiltrate can be observed 7 days after transplantation, and most
allografts are completely destroyed by day 20 post-transplant
(12,28).
[0333] To determine whether TIM-3 expression is relevant in this
model, real-time PCR experiments were performed to compare
intragraft gene expression in treated and control recipients. While
7 days after transplantation high intragraft yIFN and TIM-3 gene
expression is observed in both treated and control hosts (FIG. 8A),
the allograft response in long-surviving tolerant recipients (120
days post-transplant) is characterized by blunted TIM-3 and yIFN
intragraft gene expression (FIG. 8A). Intragraft expression of IL-2
is closely associated with that of TIM-3 and .gamma.IFN in both
treated and untreated hosts at day 7. In contrast, a 2-3 fold
increase in the intragraft expression of IL-4 and IL-10 is observed
in DST plus anti-CD154 treated hosts at both day 7 and 120 as
compared with rejecting grafts. Thus, treatment is associated with
upregulation of Th2-type gene expression, while downregulation of
Th1 responses occurs during the maintenance phase (day 120), but
not the induction phase (day 7), of transplantation tolerance.
[0334] Whether the integrity of the TIM-3 pathway is required for
the achievement of transplantation tolerance was next assessed. The
administration of ex-Tim-3-Ig together with DST plus anti-CD154
completely prevents the acquisition of islet allograft tolerance
and rapidly precipitates rejection (FIG. 8B). In addition,
ex-Tim-3-Ig administration also abrogates induction of tolerance
mediated by monotherapy with a high dose of anti-CD154.
[0335] In order to determine whether the TIM-3 pathway is important
only in the mechanisms by which tolerance is achieved with
anti-CD154 +/- DST treatment, the effects of TIM-3 blockade in a
transplant model in which tolerance is achieved via CTLA4Ig
administration were tested. The mechanisms of action of CTLA4Ig in
vivo involve both the inhibition of antigen-reactive T cell
proliferation and the induction of immunoregulation and peripheral
tolerance (30-32). While CTLA4Ig treatment produced indefinite
engraftment of islet allografts in 7 out of 9 treated recipients,
co-administration of ex-Tim-3-Ig resulted in the rejection of the
majority of the allografts (FIG. 8C). Taken together with the
results of the first experimental series, these data clearly
indicate that TIM-3 is a critical regulator of immunological
tolerance.
Example 16
TIM-3 Blockade does not Eliminate the Capacity of Naive CD4+CD25+
Regulatory T Cells to Suppress the Ability of CD4+CD25- T Cells to
Reject Allografts
[0336] Applicants have previously determined that CD4+CD25+
regulatory T cells are critically involved in the maintenance of
the tolerant state induced with DST plus anti-CD154 (12).
Applicants have now tested whether this regulatory T cell subset is
also required for the induction phase of transplant tolerance.
Interestingly, the consequences of CD4+CD25+ T cell subset
depletion were similar to the effects induced by ex-Tim-3-Ig
administration, and rapid rejection of the islet allografts was
observed in anti-CD25 mAb treated hosts (FIG. 9A).
[0337] Based on these results, it was hypothesized that engagement
of TIM-3 expressed on Th1 effector cells by TIM-3L bearing
CD4+CD25+ regulatory T cells present in nave hosts might be a
pre-requisite for their immunosuppressive function. To determine
whether regulatory T cells harvested from naive mice can exert
their immunosuppressive function in the absence of TIM-3
engagement, adoptive transfer experiments were conducted assessing
the capacity of naive CD4+CD25+ T cells to prevent allograft
rejection mediated by CD4+CD25- T cells in the presence of TIM-3
blockade. In this model, the transfer of as few as 1.times.10.sup.5
CD4+CD25- or CD8+ T cells into immunodeficient MHC-mismatched skin
allograft recipients results in rapid skin allograft rejection,
while transferred naive CD4+CD25+ T cells do not induce rejection
and prevent CD4+CD25- effector T cell populations from destroying
the grafts(33,34). Administration of ex-Tim-3-Ig to C3H/He Scid
recipients of DBA/2 skin allografts did not inhibit the capacity of
transferred naive CD4+CD25+ T cells to prevent co-transferred
CD4+CD25- T cells from mounting allograft rejection (FIG. 9B).
Similar results were observed in vitro when naive CD4+CD25+ T cells
were co-cultured with either naive CD4+CD25- T cells or Th1
polarized CD4+ T cells, and stimulated with soluble anti-CD3 in the
presence of ex-Tim-3-Ig. In addition, the transfer of
1.times.10.sup.5 CD4+CD25- T cells alone resulted in equally rapid
skin graft rejection regardless of whether ex-Tim-3-Ig or a control
hIgG were administered.
[0338] Taken together these results indicate that CD4+CD25+
regulatory T cells harvested from neve alloantigen-inexperienced
mice can suppress Th1 dependent 25 cytopathic responses in the
absence of TIM-3 engagement. Hence, TIM-3 blockade does not abolish
the immunoregulatory effector function of alloantigen-inexperienced
CD4+CD25+ T cells, nor does it markedly enhance the capacity of
CD4+CD25- T cells to reject skin allografts.
Example 17
TIM-3 Regulates the Strengthening of the Potency of
Alloantigen-Specific CD4+CD25+Regulatory T Cell Populations
[0339] Some therapeutic regimens that cause transplant tolerance
are known to bolster the potency of alloantigen-specific CD4+CD25+
regulatory T cells(33-35). To determine the impact of DST plus
anti-CD154 treatment on the generation of alloantigen-specific
CD4+CD25+ T cells, additional adoptive transfer experiments were
performed to compare the immunosuppressive capacity of CD4+CD25+ T
cells harvested from naive hosts or from mice treated with DST plus
anti-CD154. CD4+CD25- or CD8+ T cells harvested from treated or
untreated naive mice did not differ in their capacity to promote
acute skin allograft rejection (data for CD4+CD25-T cells are shown
in FIG. 9C), indicating that in this model DST plus anti-CD154
treatment does not have a major impact on effector T cell
populations. In contrast, CD4+CD25+ T cells sorted from DST plus
anti-CD154 treated hosts exerted a much greater immunosuppressive
effect upon naive CD4+CD25- T cells than CD4+CD25+ T cells
harvested from naive mice. Thus, while naive CD4+CD25+ T cells
could prevent CD4+CD25- T cells from rejecting skin allografts when
transferred at a high ratio (4:1) of CD4+CD25+ to CD4+CD25- (FIG.
9C, middle panel), only CD4+CD25+ T cells harvested from DST plus
anti-CD154 treated hosts were capable of mediating effective
immunosuppression at 1:1 ratios of regulatory to effector T cells
(FIG. 9C, right hand panel). Remarkably, this enhanced
immunosuppressive phenotype noted after DST plus anti-CD154
treatment was strictly donor-specific, since CD4+CD25+ T cells
harvested from hosts tolerized to a different donor strain
(C57BL/6) with DST plus anti-CD154 did not mediate greater
graft-protective effects than naive CD4+CD25+ T cells in response
to donor DBA/2 skin allografts (median skin allograft survival time
14 and 12 days, n=5 and 7, respectively). Furthermore, both DST and
anti-CD154 were required to achieve enhanced immunoregulatory
effects, since the provision of alloantigen without CD4O-CD154
costimulation blockade had no detectable effects upon the
immunosuppressive function of CD4+CD25+ T cells in vivo.
[0340] These observations suggest that DST plus anti-CD154 exerts
its tolerizing effect, in large measure, by enhancing the
immunosuppressive function of donor-pecific CD4+CD25+ regulatory T
cells. Applicants then addressed whether TIM-3 pathway was crucial
to this therapeutic effect. CD4+CD25+ T cells were harvested from
DST plus anti-CD154 treated mice, and their immunosuppressive
function compared in vivo with that of regulatory T cells obtained
from hosts treated with DST, anti-CD154 and ex-Tim-3-Ig. The
enhanced alloantigen-specific suppressive phenotype conferred by
DST plus anti-CD154 treatment was abolished by concurrent
administration of ex-Tim-3-Ig (FIG. 9D), since CD4+CD25+ T cells
harvested from treated hosts receiving ex-Tim-3-Ig did not exert
greater suppressive effects than nave regulatory cells (FIG. 9D).
In these experiments ex-Tim-3-Ig was only administered to CD4+CD25+
T cell donors, and no treatment whatsoever was given to skin
transplant recipients, thereby minimizing the likelihood that
ex-Tim-3-Ig treatment interfered with interactions between
CD4+CD25+ and CD4+CD25- T cells. In fact, in our adoptive transfer
model Applicants have observed that the administration of
ex-Tim-3-Ig after transplantation does not abrogate the protective
effects of previously generated donor-specific CD4+CD25+ T cells.
Taken together, our data suggest that: i) the TIM-3 pathway
facilitates the acquisition of donor-specific immunoregulatory
properties by CD4+CD25+ T cells during tolerance induction, and ii)
TEM-3/TIM-3L interactions are critical for the generation, but not
the immunosuppressive effector function, of donor-specific
CD4+CD25+ immunoregulatory T cells.
Example 18
Identification of Galectin-9 as a TIM-3 Ligand
[0341] To analyze the distribution of expression of the putative
TIM-3 ligand(s), cell surface staining by TIM-3-Ig fusion proteins
were used to screen TIM-3 ligand expressing cell types. It has been
shown that both flTIM-3-Ig and sTIM-3-Ig bind to resting CD4+ T
cells and to a small population of splenic CD11c+ dendritic cells
and CD11b+ macrophages, but not CD8+ or B220+ cells (Sabatos et al.
Nat Immunol 4:1102-1110, Sanchez-Fueyo et al. Nat Immunol
4:1093-1101) indicating that TIM-3 ligand(s) is predominantly
expressed on resting CD4+ T cells. Thus, a number of T cell lines/T
cell lymphomas were screened. FACS results demonstrated that three
T cell lines tested so far bound sTim-3-Ig, but not human IgG1
control, among which TK-1 (a T cell lymphoma) had the highest
binding to sTim-3-Ig (FIG. 10A). Staining by flTEM-3-Ig showed
similar results (FIG. 10A).
[0342] In order to identify specific extracellular membrane
associated TIM-3 binding protein, live TK-1 cells were biotin
labeled and the whole cell lysates were subjected to the pull-down
assay by incubating with TIM-3-Ig fusion proteins. Results from
SDS-PAGE-Western blot demonstrated that both flTim-3-Ig and
sTim-3-Ig were able to precipitate a protein or protein complex
with the molecular weight ranged from 40 to 60 kD (FIG. 1b, lane 1,
2). Compared with the control groups from TIM-2-Ig or hIgG pulled
down proteins (FIG. 10b, lane 3, 4), the 40 to 60 kD band is
specific for flTEM-3-Ig and sTIM-3-Ig. When N-linked
oligosaccharide chains were removed by PNGase F, the molecular
weight of the 40 to 60 kD band was reduced to 35 kD. Same
experiment using cell lysates from .sup.35S-Met metabolic labeled
TK-1 cells further excluded the possible contamination of this 35
kD band. Mass spectrometry analyses of the 35 kD band extracted
from silver stained SDS-PAGE gel identified the protein as
galectin-9, a member of the galectin family that is expressed on
lymphocytes and other cell types.
Example 19
Specific Interaction Between Galectin-9 and TIM-3
[0343] Galectins are a group of .beta.-galactoside binding lectins
containing the Carbohydrate Recognition Domain (CRD) to bind sugar
moieties on the cell surface; they are major regulators of immune
cell homeostasis (Rabinovich et al. Trends Immunol 23:313-320;
Rabinovich et al. Biochim Biophys Acta 1572:274-284.). Galectins do
not have signal peptides for ER-Golgi pathway secretion. Like IL-1,
galectins secrete extra-cellular via alternative pathways. The
total RNA from TK-1 cells was prepare for RT-PCR to clone
galectin-9 cDNA. Two galectin-9 coding sequences were cloned and
were later found as regular galectin-9 cDNA and its long isoform
that has 31 amino acids insertion in the hinge region of the two
CRD domains of galectin-9 has been reported to be predominantly
expressed in intestine epithelia.
[0344] To confirm the interaction between galectin-9 and TIM-3,
both galectin-9 isoform cDNAs were subcloned into an expression
vector that bicistronically expresses EGFP protein. When galectin-9
expression plasmids were transiently transfected into CHO cells,
cells that express EGFP will simultaneously express galectin-9. In
transient transfection, expression of galectin-9 was not detected
on the cell surface. It is possible that expression of galectin-9
in transiently transfected CHO cells might be separated from the
alternative transporting machinery. Therefore the protein product
may accumulate in the cytoplasm. It is indeed that intracellular
staining revealed the same positive staining pattern by sTIM-3-Ig
and flTIM-3-Ig from transfected CHO cells that are EGFP positive,
suggesting no differences between regular galectin-9 and its long
isoform when interacting with TIM-3 fusions. However neither of
them was able to bind to hIgG control (FIG. 11A).
[0345] To demonstrate the binding specificity between galectin-9
and TIM-3, galectin-9 transiently transfected CHO cells were
subjected to intracellular staining. F1TIM-3-Ig and sTIM-3-Ig are
the only fusion proteins that have all positive staining in
galectin-9 expressing cells. By contrast, TIM-2-Ig, TIM-4-Ig, and
hIgG cannot bind to the same transfected CHO cells, suggesting
TIM-3 has the highest affinity to galectin-9 comparing with other
TIM family members. As an alternative approach, purified
recombinant galectin-9 can be specifically pulled down by
sTIM-3-Ig, but not TIM-2 and TIM-4 Ig fusions (FIG. 11C). On the
other hand, both flTIM-3-Ig and sTIM-3-Ig only showed positive
binding to galectin-9 expressing cells, whereas they did not bind
to galectin-1, 3, and 4 unless the cells expressed very high level
of these proteins, suggesting certain level of nonspecific
interaction between TIM-3 and these galectins (FIG. 11C).
[0346] In conclusion, galectin-9 is a protein that specifically
binds to the TIM-3 extracellular portion. As both flTIM-3-Ig and
sTIM-3-Ig can bind to galectin-9, the Ig V domain on TIM-3 may be
responsible for interaction with galectin-9. When lactose was added
in the incubation buffer, interaction between galectin-9 and TIM-3
was attenuated in both intracellular staining and pull-down assay
in a dose-dependent manner (FIG. 12A). Furthermore, mutant
galectin-9 constructs that disrupt either the N-Terminal CRD or the
C-terminal CRD partially lose interaction to TIM-3, whereas double
mutations in both CRD domains completely abrogated binding to TIM-3
(FIG. 12B). Therefore, the interaction between galectin-9 and TIM-3
is dependent on the CRD domains in of galectin-9.
Example 20
Roles of TIM-3-Galectin-9 in Regulation of Effector Th1 Cells
[0347] Applicants hypothesized functional roles of a
galectin-9-TIM-3 pathway in the regulation of Th1 cell expansion
and homeostasis by an apoptotic mechanism. To test the apoptotic
effects of galectin-9 in effector T cells, neve spleen CD4+ T cells
from D011.10 transgenic mice were purified by a CD4 T cell negative
selection column and sorted for CD4+CD62L+ cells. Cells were in
vitro stimulated with VOA peptide and irradiated APC, and were
polarized in vitro for at least 3 rounds into Th1 and Th2 cells.
When active Th1 cells were treated with recombinant galectin-9,
cell death was induced in the majority of cells. By contrast,
activated Th2 cells were resistant to galectin-9-induced cell death
(FIG. 13). Further experiments performed on AE7 (Th1 cell line) and
D1 0G4 (Th2 cell line) demonstrated the similar effects of cell
death induced by recombinant galectin-9 in vitro.
Example 21
Recombinant Galectin-9 Attenuates Th1 Cell Activation
[0348] To determine the in vivo effects of the interaction of
galectin-9 and TIM-3 during a Th1 immune response, C57BL/6J mice
were immunized with MOG 35-55 in complete Freund's adjuvant (CFA).
Recombinant galectin-9 (100 .mu.g) was i.p. injected to immunized
mice daily from day 3 to 9. Spleen cells were harvested for cell
proliferation and cytokine production assays. Comparing with a PBS
treated control group, galectin-9 administration did not alter
spleen cell proliferation by .sup.3H thymidine incorporation,
however, ELISA results indicated more than 50 percent reduction in
IFN-.gamma. production. ELISPOT further demonstrated that fewer
cells from galectin-9 treated spleen produced IFN.gamma. and IL-2.
Interestingly, IL-4 and IL-5 production was not changed compared
with PBS treated mice (FIG. 14).
Example 22
Therapeutic Effect of Galectin-9-TIM-3 in Treatment of EAE
[0349] TIM-3 has been demonstrated to have an important function in
regulating Th1 cell responses and peripheral tolerance. Its
expression is correlated to pathology of both EAE and human
multiple sclerosis (MS). Interestingly, expression of galectin-9 in
astrocytes is induced by IL-1.beta., indicating that this protein
may be involved in limiting inflammation in the CNS (Yoshidaet et
al. Neuroreport 12:3755-3758). As TIM3-TIM-3 ligand(s) interaction
may regulate Th1 responses and promote immunological tolerance, it
is possible that both these functions are mediated by the
interaction of TIM-3-galectin-9. To address this possibility,
recombinant galectin-9 will be administered to MOG peptide
35-55/CFA immunized C57BL/6J mice every other day from the day of
immunization. The disease will be monitored and brains and spinal
cords will be examined histopathologically. As a functional ligand
for TIM-3, galectin-9 administration is expected to reduce the
severity of EAE, and/or induce tolerance against EAE. As a control,
Applicants will also test the Tim-3-/- mice in the C57BL/6J
background. It is expected that Tim-3 deficient mice will not be
affected by administration of galectin-9.
D. Discussion of Second Series of Experiments
[0350] The expansion and differentiation of Th precursors into the
Th1 or Th2 pathways regulate the outcome of immune responses to
bacterial, viral, auto- and allo-antigens. The extent of these T
cell responses is influenced by cytokines and a group of accessory
molecules that includes TNF receptor(7) and Ig superfamily
members(8). TIM-3 is a novel Th1-specific Ig superfamily member
recently identified by Monney et al.(10), as a negative regulator
of tissue destructive immune responses in EAE. In this model,
anti-TIM-3 mAb treatment increased the number and activation level
of macrophages, and the severity of tissue injury within the brain.
Based on these results, it was proposed that anti-TIM-3 might
activate macrophages by enhancing the migration of Th1 cells into
the brain or by blocking the interaction between TIM-3 and a
putative inhibitory TIM-3L.
[0351] Applicants have identified TIM-3 by comparing the gene
expression profiles of activated Th1 and Th2 clones, and Applicants
now report that TIM-3 plays an important role restraining
Th1-mediated responses, both in auto- and allo-immune models, and
that these effects appear to be mediated, at least in part, by the
modulation of the immunosuppressive function of CD4+CD25+
regulatory T cell populations.
[0352] CD4+CD25+ regulatory T cells play a central role in the
maintenance of self-tolerance(36-38), as well as in the long-term
acceptance of allogeneic transplants(12,34,35). Although their
exact mechanism of action has not been defined as yet, both
cell-to-cell contact interactions and soluble factors are
implicated in their immunosuppressive function (39,40). Our results
indicate that TIM-3 is not the molecular pathway through which
these CD4+CD25+ T cells deliver their immunosuppressive effects,
since CD4+CD25+ T cells from naive or tolerant hosts can strongly
suppress CD4+CD25- T cells lacking effective expression of TIM-3
both in vivo and in vitro (FIG. 9B). However, a TIM-3/TIM-3L
sensitive pathway is responsible for the functional generation of
donor-specific CD4+CD25+ regulatory T cells that emerge after the
administration of tolerizing treatments such as DST plus anti-CD154
treatment (FIG. 9D). Hence, both TIM-3 dependent and independent
pathways are involved in CD4+CD25+ T cell dependent
immunoregulation.
[0353] Although the precise cellular and molecular interactions
involving TIM-3 and its ligand(s) remain to be fully elucidated,
the expression patterns seen for TIM-3 and TIM-3L (FIGS. 8A-D)
suggest that a direct interaction between TIM-3 positive Th1
effector cells and TIM-3L positive regulatory T cells might
constitute a mechanism through which CD4+CD25+ T cells acquire
enhanced immunosuppressive function. Alternatively, given that
TIM-3L expression is also observed on some dendritic cells, TIM-3
effects on regulatory T cells could be indirectly exerted through
the intervention of APCs. This last interpretation would be
consistent with observations concerning the role of dendritic cell
phenotype in the modulation of CD4+CD25+ T cell mediated
immunoregulation(41,42) Finally, the fact that TIM-3 is capable of
recruiting src kinases upon ligation, and therefore capable of
influencing downstream signaling events, indicates that TIM-3
ligation could also exert a direct inhibitory signal upon Th1 cells
themselves. Nevertheless, the absence of a significant increase in
the frequency of IL-2- and .gamma.IFN-producing effector T cells in
the spleens of ex-Tim-3-Ig treated transplant recipients, suggests
that in transplantation TIM-3 has a more potent effect upon
regulatory than upon effector T cell populations.
[0354] Our findings offer a novel insight into the mechanisms
through which DST plus anti-CD154 promote tolerance induction in
transplantation. The current understanding states that the
provision of alloantigen to host T cells in the context of an
anti-CD154 induced immunosuppressive environment directly
inactivates alloreactive CD4+ and CD8+ T lymphocytes by anergy
and/or apoptosis, and subsequently promotes the expansion of
regulatory T cells capable of self-perpetuating the tolerant
state(11-13,28,43). Applicants now provide evidence indicating that
in polyclonal systems, DST plus anti-CD154 acts primarily by
increasing the immunosuppressive function of CD4+CD25+ T cells in
an alloantigen-specific manner, while the effects of treatment on
the capacity of effector T cell populations to reject allografts
are much less potent.
[0355] The mechanisms through which prior encounter with
alloantigen in the absence of CD40-CD154 costimulation induce these
TIM-3-sensitive immunoregulatory networks are speculative.
Regulatory T cell clones capable of recognizing allogeneic peptides
might undergo selective expansion(44,45) and/or acquire a more
efficient immunosuppressive function, thereby strengthening
donor-specific immunoregulatory circuits. Indeed, the lack of
effect of DST monotherapy in our model suggests that CD154
blockade, known to promote effector T cell apoptosis and
anergy(13,43), spares or even enhances the function and survival of
regulatory T cells. Alternatively, CD40-CD154 costimulation
blockade might modulate the phenotype of resident APCs, resulting
in the recruitment into the immunoregulatory compartment of naive
donor-reactive T cells undergoing activation. Interestingly, lack
of CD40 expression on APCs has been reported to promote
antigen-specific tolerance(46-49), in some cases through the
generation of CD4+ regulatory T cells(49). Hence, based on our
findings, Applicants speculate that CD40 and TIM-3 have opposing
effects and that their balance serves as a checkpoint in the
decision between tolerance and immunity. The importance of
TIM-3/TIM-3L pathway facilitating immunological tolerance is not
exclusively restricted to the mechanisms through which DST plus
anti-CD154 achieve transplantation tolerance. Indeed, based on own
observations that ex-Tim-3-Ig abrogates tolerance induction after
CTLA4Ig treatment (FIG. 9D), Applicants can conclude that TIM-3
plays a fundamental role in regulating Th1-mediated immune
responses and facilitating the generation of immunological
tolerance.
[0356] In short, Applicants have described that the Ig superfamily
member TIM-3 functions to inhibit aggressive Th1 mediated auto- and
allo-immune responses. These effects appear to be mediated, at
least in part, by the regulation of the immunosuppressive potency
of CD4+CD25+ regulatory T cells. Hence, expression of TIM-3 upon
Th1 cells provides a key check point that serves to dampen
pro-inflammatory Th1-dependent T cell responses and limit the
associated tissue injury.
E. References
[0357] 1. Romagnani, S. Lymphokine production by human T cells in
disease states. Annu Rev Immunol 12, 227-57 (1994). [0358] 2.
Kamradt, T. & Mitchison, N. A. Tolerance and autoimmunity. N
Engl J Med 344, 655-64 (2001). [0359] 3. Strom, T. B. et al. The
Th1/Th2 paradigm and the allograft response. Curr Opin Immunol 8,
688-93 (1996). [0360] 4. Li, X. C., Zand, M. S., Li, Y., Zheng, X.
X. & Strom, T. B. On histocompatibility barriers, Th1 to Th2
immune deviation, and the nature of the allograft responses. J
Immunol 161, 2241-7 (1998). [0361] 5. Anderson, G. P. & Coyle,
A. J. TH2 and `TH2-like` cells in allergy and asthma:
pharmacological perspectives. Trends Pharmacol Sci 15, 324-32.
(1994). [0362] 6. Kundig, T. M. et al. Duration of TCR stimulation
determines costimulatory requirement of T cells. Immunity 5, 41-52.
(1996). [0363] 7. Locksley, R. M., Killeen, N. & Lenardo, M. J.
The TNF and TNF receptor superfamilies: integrating mammalian
biology. Cell 104, 487-501. (2001). [0364] 8. Salomon, B. &
Bluestone, J. A. Complexities of CD28/B7: CTLA-4 costimulatory
pathways in autoimmunity and transplantation. Annu Rev Immunol 19,
225-52 (2001). [0365] 9. Refaeli, Y., Van Parijs, L., London, C.
A., Tschopp, J. & Abbas, A. K. Biochemical mechanisms of
IL-2-regulated Fas-mediated T cell apoptosis. Immunity 8, 615-23.
(1998). [0366] 10. Monney, L. et al. Th1-specific cell surface
protein Tim-3 regulates macrophage activation and severity of an
autoimmune disease. Nature 415, 536-41. (2002). [0367] 11. Parker,
D. C. et al. Survival of mouse pancreatic islet allografts in
recipients treated with allogeneic small lymphocytes and antibody
to CD40 ligand. Proc Natl Acad Sci USA 92, 9560-4. [0368] 12.
Sanchez-Fueyo, A., Weber, M., Domenig, C., Strom, T. B. &
Zheng, X. X. Tracking the immunoregulatory mechanisms active during
allograft tolerance. J Immunol 168, 2274-81. (2002). [0369] 13.
Quezada, S. A. et al. Mechanisms of donor specific transfusion
tolerance: pre-emptive induction of clonal T cell exhaustion via
indirect presentation. Blood (2003). [0370] 14. Coyle, A. J. et al.
The CD28-related molecule ICOS is required for effective T
cell-dependent immune responses. Immunity 13, 95-105 (2000). [0371]
15. Wells, A. D., Gudmundsdottir, H. & Turka, L. A. Following
the fate of individual T cells throughout activation and clonal
expansion. Signals from T cell receptor and CD28 differentially
regulate the induction and duration of a proliferative response. J
Clin Invest 100, 3173-83. (1997). [0372] 16. Sabatos, C. A. et al.
Tim-3/Tim-3-Ligand interaction regulates Th1 responses and
induction of peripheral tolerance. Submitted. [0373] 17. Delovitch,
T. L. & Singh, B. The nonobese diabetic mouse as a model of
autoimmune diabetes: immune dysregulation gets the NOD. Immunity 7,
727-38. (1997). [0374] 18. Christianson, S. W., Shultz, L. D. &
Leiter, E. H. Adoptive transfer of diabetes into immunodeficient
NOD-scid/scid mice. Relative contributions of CD4+ and CD8+ T-cells
from diabetic versus prediabetic NOD.NON-Thy-la donors. Diabetes
42, 44-55. (1993). [0375] 19. Rudd, C. E. et al. Two-step TCR
zeta/CD3-CD4 and CD28 signaling in T cells: SH2/SH3 domains,
protein-tyrosine and lipid kinases. Immunol Today 15, 225-34
(1994). [0376] 20. Prasad, K. V. et al. T-cell antigen CD28
interacts with the lipid kinase phosphatidylinositol 3-kinase by a
cytoplasmic Tyr(P)-Met-Xaa-Met motif. Proc Natl Acad Sci USA 91,
2834-8 (1994). [0377] 21. Schneider, H., Cai, Y. C., Prasad, K. V.,
Shoelson, S. E. & Rudd, C. E. T cell antigen CD28 binds to the
GRB-2/SOS complex, regulators of p21ras. Eur J Immunol 25, 1044-50
(1995). [0378] 22. Rudd, C. E. Upstream-downstream: CD28
cosignaling pathways and T cell function. Immunity 4, 527-34
(1996). [0379] 23. McIntire, J. J. et al. Identification of Tapr
(an airway hyperreactivity regulatory locus) and the linked Tim
gene family. Nat Immunol 2, 1109-16 (2001). [0380] 24. Kuchroo, V.
K., Umetsu, D. T., DeKruyff, R. H. & Freeman, G. J. The TIM
gene family: emerging roles in immunity and disease. Nat Rev
Immunol 3, 454-62 (2003). [0381] 25. Li, X. C. et al. IL-2 and IL-4
double knockout mice reject islet allografts: a role for novel T
cell growth factors in allograft rejection. J Immunol 161, 890-6
(1998). [0382] 26. Kishimoto, K. et al. The role of CD154-CD40
versus CD28-B7 costimulatory pathways in regulating allogeneic Th1
and Th2 responses in vivo. J Clin Invest 106, 63-72. (2000). [0383]
27. Sho, M. et al. Physiological Mechanisms of Regulating
Alloimmunity: Cytokines, CTLA-4, CD25(+) Cells, and the
Alloreactive T Cell Clone Size. J Immunol 169, 3744-51. (2002).
[0384] 28. Zheng, X. X. et al. CTLA4 signals are required to
optimally induce allograft tolerance with combined donor-specific
transfusion and anti-CD154 monoclonal antibody treatment. J Immunol
162, 4983-90. (1999). [0385] 29. DeKruyff, R. H., Fang, Y. &
Umetsu, D. T. IL-4 synthesis by in vivo primed keyhole limpet
hemocyanin specific CD4+ T cells. I. Influence of antigen
concentration and antigen presenting cell type. J Immunol 149,
3468-3476 (1992). [0386] 30. Judge, T. A. et al. The in vivo
mechanism of action of CTLA4Ig. J Immunol 156, 2294-9 (1996).
[0387] 31. Waaga, A. M. et al. Regulatory functions of
self-restricted MHC class II allopeptide-specific Th2 clones in
vivo. J Clin Invest 107, 909-16 (2001). [0388] 32. Lee, R. S. et
al. CTLA4Ig-induced linked regulation of allogeneic T cell
responses. J Immunol 166, 1572-82 (2001). [0389] 33. Maurik Av, A.,
Herber, M., Wood, K. J. & Jones, N. D. Cutting Edge:
CD4(+)CD25(+) Alloantigen-Specific Immunoregulatory Cells That Can
Prevent CD8(+) T Cell-Mediated Graft Rejection: Implications for
Anti-CD154 Immunotherapy. J Immunol 169, 5401-4. (2002). [0390] 34.
Kingsley, C. I., Karim, M., Bushell, A. R. & Wood, K. J.
CD25+CD4+ regulatory T cells prevent graft rejection: CTLA-4- and
IL-10-dependent immunoregulation of alloresponses. J Immunol 168,
1080-6. (2002). [0391] 35. Graca, L. et al. Both CD4(+)CD25(+) and
CD4(+)CD25(-) regulatory cells mediate dominant transplantation
tolerance. J Immunol 168, 5558-65. (2002). [0392] 36. Sakaguchi, S.
& Sakaguchi, N. Thymus and autoimmunity: capacity of the normal
thymus to produce pathogenic self-reactive T cells and conditions
required for their induction of autoimmune disease. J Exp Med 172,
537-45. (1990). [0393] 37. Sakaguchi, S., Sakaguchi, N., Asano, M.,
Itoh, M. & Toda, M. Immunologic self-tolerance maintained by
activated T cells expressing IL-2 receptor alpha-chains (CD25).
Breakdown of a single mechanism of self-tolerance causes various
autoimmune diseases. J Immunol 155, 1151-64. (1995). [0394] 38.
Salomon, B. et al. B7/CD28 costimulation is essential for the
homeostasis of the CD4+CD25+ immunoregulatory T cells that control
autoimmune diabetes. Immunity 12, 431-40. (2000). [0395] 39. Wood,
K. J. & Sakaguchi, S. Regulatory Lymphocytes: Regulatory T
cells in transplantation tolerance. Nat Rev Immunol 3, 199-210
(2003). [0396] 40. Shevach, E. M. CD4+CD25+ suppressor T cells:
more questions than answers. Nat Rev Immunol 2, 389-400 (2002).
[0397] 41. Caramalho, I. et al. Regulatory T Cells Selectively
Express Toll-like Receptors and Are Activated by
Lipopolysaccharide. J Exp Med 197, 403-11 (2003). [0398] 42.
Pasare, C. & Medzhitov, R. Toll Pathway-Dependent Blockade of
CD4+CD25+ T Cell-Mediated Suppression by Dendritic Cells. Science
299, 1033-6 (2003). [0399] 43. Iwakoshi, N. N. et al. Treatment of
allograft recipients with donor-specific transfusion and anti-CD154
antibody leads to deletion of alloreactive CD8+ T cells and
prolonged graft survival in a CTLA4-dependent manner. J Immunol
164, 512-21. (2000). [0400] 44. Yamazaki, S. et al. Direct
Expansion of Functional CD25+CD4+Regulatory T Cells by
Antigen-processing Dendritic Cells. J Exp Med 198, 235-47 (2003).
[0401] 45. Walker, L. S., Chodos, A., Eggena, M., Dooms, H. &
Abbas, A. K. Antigen-dependent Proliferation of CD4+ CD25+
Regulatory T Cells In Vivo. J Exp Med 198, 249-58 (2003). [0402]
46. Buhlmann, J. E. et al. In the absence of a CD40 signal, B cells
are tolerogenic. Immunity 2, 645-53 (1995). [0403] 47. Foy, T. M.,
Aruffo, A., Bajorath, J., Buhlmann, J. E. & Noelle, R. J.
Immune regulation by CD40 and its ligand GP39. Annu Rev Immunol 14,
591-617 (1996). [0404] 48. Hollander, G. A. et al. Induction of
alloantigen-specific tolerance by B cells from CD40-deficient mice.
Proc Natl Acad Sci USA 93, 4994-8 (1996). [0405] 49. Martin, E.,
O'Sullivan, B., Low, P. & Thomas, R. Antigen-specific
suppression of a primed immune response by dendritic cells mediated
by regulatory T cells secreting interleukin-10. Immunity 18, 155-67
(2003). [0406] 50. Yoon, J. W. et al. Control of autoimmune
diabetes in NOD mice by GAD expression or suppression in beta
cells. Science 284, 1183-7 (1999). [0407] 51. Steiger, J.,
Nickerson, P. W., Steurer, W., Moscovitch-Lopatin, M. & Strom,
T. B. IL-2 knockout recipient mice reject islet cell allografis. J
Immunol 155, 489-98 (1995). [0408] 52. Li, Y. et al. Blocking both
signal 1 and signal 2 of T-cell activation prevents apoptosis of
alloreactive T cells and induction of peripheral allograft
tolerance. Nat Med 5, 1298-302 (1999).
EQUIVALENTS
[0409] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
claims. Various publications are cited throughout this application.
The contents of these publications are hereby incorporated by
reference into this application
TABLE-US-00001 Sequence listings SEQ ID NO: 1
ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTAC
TTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCC
TATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTC
TGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTC
AGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAA
TGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTC
TAGCAGACAGTGGGATCTACTGCTGCCGGATCCAAATCCCAGGCATAATG
AATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGGGTATTCTCAT
AGCAAAGAGAAGATACAGAATTTAAGCCTCATCTCTTTGGCCAACCTCCCT
CCCTCAGGATTGGCAAATGCAGTAGCAGAGGGAATTCGCTCAGAAGAAAA CATCTATAC
CATTGAAGAGAACGTATATGAAGTGGAGGAGC CCAATGAGT
ATTATTGCTATGTCAGCAGCAGGCAGCAACCCTCACAACCTTTGGGTTGTC
GCTTTGCAATGCCATAG SEQ ID NO: 2
MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCW
GKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLAD
SGIYCCRIQIPGIMNDEKFNLKLVIKPGYSHSKEKIQNLSLISLANLPPSGLANA
VAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP SEQ ID NO: 3
ATGTTTTCAGGTCTTACCCTCAACTGTGTCCTGCTGCTGCTGCAACTACTAC
TTGCAAGGTCATTGGAAGATGGTTATAAGGTTGAGGTTGGTAAAAATGCC
TATCTGCCCTGCAGTTACACTCTACCTACATCTGGGACACTTGTGCCTATGT
GCTGGGGCAAGGGATTCTGTCCTTGGTCACAGTGTACCAATGAGTTGCTCA
GAACTGATGAAAGAAATGTGACATATCAGAAATCCAGCAGATACCAGCTA
AAGGGCGATCTCAACAAAGGAGATGTGTCTCTGATCATAAAGAATGTGAC
TCTGGATGACCATGGGACCTACTGCTGCAGGATACAGTTCCCTGGTCTTAT
GAATGATAAAAAATTAGAACTGAAATTAGACATCAAAGCAGGGTATTCCT
GTAAGAAAAAGAAGTTATCGAGTTTGAGCCTTATTACACTGGCCAACTTGC
CTCCAGGAGGGTTGGCAAATGCAGGAGCAGTCAGGATTCGCTCTGAGGAA
AATATCTACACCATCGAGGAGAACGTATATGAAGTGGAGAATTCAAATGA
GTACTACTGCTACGTCAACAGCCAGCAGCCATCCTGA SEQ ID NO: 4
MFSGLTLNCVLLLLQLLLARSLEDGYKVEVGKNAYLPCSYTLPTSGTLVPMC
WGKGFCPWSQCTNELLRTDERNVTYQKSSRYQLKGDLNKGDVSLIIKNVTLD
DHGTYCCRIQFPGLMNDKKLELKLDIKAGYSCICKKKLSSLSLITLANLPPGGL
ANAGAVRIRSEENIYTIEENVYEVENSNEYYCYVNSQQPS SEQ ID NO: 5 CAAACCAG
GGTATTCT SEQ ID NO: 6 NLKLVIKP GYSHSKEK SEQ ID NO: 7 CAAAGCAG
GGTATTCC SEQ ID NO: 8 ELKLDIKA GYSCKKKK SEQ ID NO: 9
ATGGCCTTCAGCGGTTCCCAGGCTCCCTACCTGAGTCCAGCTGTCCCCTTTT
CTGGGACTATTCAAGGAGGTCTCCAGGACGGACTTCAGATCACTGTCAAT
GGGACCGTTCTCAGCTCCAGTGGAACCAGGTTTGCTGTGAACTTTCAGACT
GGCTTCAGTGGAAATGACATTGCCTTCCACTTCAACCCTCGGTTTGAAGAT
GGAGGGTACGTGGTGTGCAACACGAGGCAGAACGGAAGCTGGGGGCCCG
AGGAGAGGAGGACACACATGCCTTTCCAGAAGGGGATGCCCTTTGACCTC
TGCTTCCTGGTGCAGAGCTCAGATTTCAAGGTGATGGTGAACGGGATCCTC
TTCGTGCAGTACTTCCACCGCGTGCCCTTCCACCGTGTGGACACCATCTTC
GTCAATGGCTCTGTGCAGCTGTCCTACATCAGCTTCCAGCCTCCCGGCGTG
TGGCCTGCCAACCCGGCTCCCATTACCCAGACAGTCATCCACACAGTGCAG
AGCGCCCCTGGACAGATGTTCTCTACTCCCGCCATCCCACCTATGATGTAC
CCCCACCCCGCCTATCCGATGCCTTTCATCACCACCATTCTGGGAGGGCTG
TACCCATCCAAGTCCATCCTCCTGTCAGGCACTGTCCTGCCCAGTGCTCAG
AGGTTCCACATCAACCTGTGCTCTGGGAACCACATCGCCTTCCACCTGAAC
CTCCGTTTTGATGAGAATGCTGTGGTCCGCAACACCCAGATCGACAACTCC TGGGGGTC
TGAGGAGCGAAGTCTGCCCCGAAAAATGCCCTTCGTCCGTGG
CCAGAGCTTCTCAGTGTGGATCTTGTGTGGAGCTCACTGCCTCAAGGTGGC
CGTGGATGGTCAGCACCTGTTTGAATACTACCATCGCCTGAGGAACCTGCC
CACCATCAACAGACTGGAAGTGGGGGGCGACATCCAGCTGACCCATGTGC AGACATAG SEQ ID
NO: 10 MAFSGSQAPYLSPAVPFSGTIQGGLQDGLQITVNGTVLSSSGTRFAVNFQTGFS
GNDIAFHFNPRFED GGYVVCNTRQNGSWGPEERRTHMPFQKGMPFDLCFLVQ
SSDFKVMVNGILFVQYFHRVPFHRVDTIFVNGSVQLSYISFQPPGVWPANPAPI
TQTVIHTVQSAPGQMFSTPAIPPMMYPHPAYPMPFITTILGGLYPSKSILLSGTV LP
SAQRFHINLCS GNHIAFHLNLRFDENAVVRNTQIDNS WG SEERS LPRKMPF
VRGQSFSVWILCGAHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTH VQT SEQ ID NO:
11 ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTAC
TTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCC
TATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTC
TGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTC
AGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAA
TGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTC
TAGCAGACAGTGGGATCTACTGCTGCCGGATCCAAATCCCAGGCATAATG
AATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCAC
CCCTGCACCGACTCTGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCT
TACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCC
CTGATATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACT
CTAGATTGGCCAATGACTTACGGGACTCTGGAGCAACCATCAGAATAGGC
ATCTACATCGGAGCAGGGATCTGTGCTGGGCTGGCTCTGGCTCTTATCTTC
GGCGCTTTAATTTTCAAATGGTATTCTCATAGCAAAGAGAAGATACAGAAT
TTAAGCCTCATCTCTTTGGCCAACCTCCCTCCCTCAGGATTGGCAAATGCA
GTAGCAGAGGGAATTCGCTCAGAAGAAAACATCTATACCATTGAAGAGAA
CGTATATGAAGTGGAGGAGCCCAATGAGTATTATTGCTATGTCAGCAGCA
GGCAGCAACCCTCACAACCTTTGGGTTGTCGCTTTGCAATGCCATAG SEQ ID NO: 12
ATGTTTTCAGGTCTTACCCTCAACTGTGTCCTGCTGCTGCTGCAACTACTAC
TTGCAAGGTCATTGGAAGATGGTTATAAGGTTGAGGTTGGTAAAAATGCC
TATCTGCCCTGCAGTTACACTCTACCTACATCTGGGACACTTGTGCCTATGT
GCTGGGGCAAGGGATTCTGTCC TTGGTCACAGTGTACCAATGAGTTGCTCA
GAACTGATGAAAGAAATGTGACATATCAGAAATCCAGCAGATACCAGCTA
AAGGGCGATCTCAACAAAGGAGATGTGTCTCTGATCATAAAGAATGTGAC
TCTGGATGACCATGGGACCTACTGCTGCAGGATACAGTTCCCTGGTCTTAT
GAATGATAAAAAATTAGAACTGAAATTAGACATCAAAGCAGCCAAGGTCA
CTCCAGCTCAGACTGCCCATGGGGACTCTACTACAGCTTCTCCAAGAACCC
TAACCACGGAGAGAAATGGTTCAGAGACACAGACACTGGTGACCCTCCAT
AATAACAATGGAACAAAAATTTCCACATGGGCTGATGAAATTAAGGACTC TGGAGAAAC
GATCAGAACTGCT ATCCACATTGGAGTGGGAGTCTC TGCTG
GGTTGACCCTGGCACTTATCATTGGTGTCTTAATCCTTAAATGGTATTCCTG
TAAGAAAAAGAAGTTATCGAGTTTGAGCCTTATTACACTGGCCAACTTGCC TCCAGGAG
GGTTGGCAAATGCAGGAGCAGTCAGGATTCGCTCTGAGGAAA ATATCTACAC
CATCGAGGAGAACGTATATGAAGTGGAGAATTCAAATGAG
TACTACTGCTACGTCAACAGCCAGCAGCCATCCTGA SEQ ID NO: 13
MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCW
GKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLAD
SGIYCCRIQPGIMNDEKFNLKLVEKPAKVTPAPTLQRDFTAAFPRMLTTRGHG
PAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGAT1RIGIYIGAGICAGLA
LALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEEN
VYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP SEQ ID NO: 14
MFSGLTLNCVLLLLQLLLARSLEDGYKVEVGKNAYLPCSYTLPTSGTLVPMC
WGKGFCPWSQCTNELLRTDERNVTYQKSSRYQLKGDLNKGDVSLIIKNVTLD
DHGTYCCRIQFPGLMNDKKLELKLDIKAAKVTPAQTAHGDSTTASPRTLTTER
NGSETQTLVTLHNNNGTKIS TWADEIKDSGETIRTAIHIGVGVSAGLTLALIIGV
LILKWYSCKKKKLSSLSLITLANLPPGGLANAGAVRIRSEENIYTIEENVYEVE
NSNEYYCYVNSQQPS SEQ ID NO: 15 CAAACCAGGGUAUUCU SEQ ID NO: 16
ATGGCTCTCTTCAGTGCCCAGTCTCCATACATTAACCCGATCATCCCCTTTA
CTGGACCAATCCAAGGAGGGCTGCAGGAGGGACTTCAGGTGACCCTCCAG
GGGACTACCAAGAGTTTTGCACAAAGGTTTGTGGTGAACTTTCAGAACAG
CTTCAATGGAAATGACATTGCCTTCCACTTCAACCCCCGGTTTGAGGAAGG
AGGGTATGTGGTTTGCAACACGAAGCAGAACGGACAGTGGGGGCCTGAGG
AGAGAAAGATGCAGATGCCCTTCCAGAAGGGGATGCCCTTTGAGCTTTGC
TTCCTGGTGCAGAGGTCAGAGTTCAAGGTGATGGTGAACAAGAAATTCTTT
GTGCAGTACCAACACC GCGTAC C CTAC CAC CTCGTGGACAC CATC GCTGTC
TCCGGCTGCTTGAAGCTGTCCTTTATCACCTTCCAGACTCAGGACTTTCGTC
CTGCCCACCAGGCAC C CATGGCTCAAACTAC CATC CATATGGTTCACAGCA
CCCCTGGACAGATGTTCTCTACTCCTGGAATCCCTCCTGTGGTGTACCCCA CCCCAGC CTATAC
CATACCTTTCTACAC CCC CATTC CAAATGGGCTTTACC
CGTCCAAGTCCATCATGATATCAGGCAATGTCTTGCCAGATGCTACGAGGT
TCCATATCAACCTTCGCTGTGGAGGTGACATTGCTTTCCACCTGAACCCCC
GTTTCAATGAGAATGCTGTTGTCCGAAACACTCAGATCAACAACTCCTGGG
GGCAGGAAGAGCGAAGTCTGCTTGGGAGGATGCCCTTCAGTCGAGGCCAG
AGCTTCTCGGTGTGGATCATATGCGAAGGTCACTGCTTCAAGGTGGCTGTG
AATGGTCAACACATGTGTGAATATTACCACCGCCTGAAGAACTTGCAGGA
TATCAACACTCTAGAAGTGGCGGGT GATATCCAGCTGACCCAC GTGCAGA CATAG SEQ ID
NO: 17 MALFSAQSPYINPIIPFTGPIQGGLQEGLQVTLQGTTKSFAQRFVVNFQNSFNG
NDIAFHFNPRFEEGGYVVCNTKQNGQWGPEERKMQMPFQKGMPFELCFLVQ
RSEFKVMVNKKFFVQYQHRVPYHLVDTIAVSGCLKLSFITFQTQDFRPAHQAP
MAQTTIHMVHSTPGQMFSTPGIPPVVYPTPAYTIPFYTPIPNGLYP SKSIMISGN
VLPDATRFHINLRCGGDIAFHLNPRFNENAVVRNTQINNSWGQEERSLLGRMP
FSRGQSFSVWIICEGHCFKVAVNGQHMCEYYHRLKNLQDINTLEVAGDIQLT HVQT SEQ ID
NO: 18 MALFSAQSPYINPIIPFTGPIQGGLQEGLQVTLQGTTKSFAQRFVVNFQNSFNG
NDIAFHFNPRFEEGGYVVCNTKQNGQWGPEERKMQMPFQKGMPFELCFLVQ RSEFKVMVNKKFFV
Q Y QHRVP YHLVD T IA V S G C LKLS F ITF QN SAAPVQHVFS
TLQFSQPVQFPRTPKGRKQKTQNFRPAHQAPMAQTTIHMVHSTPGQMF S TPGI PP
VVYPTPAYTIPFYTP IPNGLYP SKS IMI S GNVLPDATRFHINLRC GGD IAFHLN
PRFNENAVVRNTQINNSWGQEERSLLGRMPFSRGQ SF SVWIICEGHC FKVAVN
GQHMCEYYHRLKNLQDINTLEVAGDIQLTHVQT SEQ ID NO: 19
MAFSGSQAPYLSPAVPFSGTIQGGLQDGLQITVNGTVLSSSGTRFAVNFQTGFS
GNDIAFHFNPRFEDGGYVVCNTRQNGSWGPEERKTHMPFQKGMPFDLCFLV
QSSDFKVMVNGILFVQYFHRVPFHRVDTISVNGSVQLSYISFQNPRTVPVQPAF
STVPFSQPVCFPPRPRGRRQKPPGVWPANPAPITQTVIHTVQSAPGQMFSTPAIP P MMYPHP
AYPMPFITT ILGGLYP SKS ILLS GTV LP SAQRFHINLCSGNHIAFHLN
PRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCEAHCLKVAV
DGQHLFEYYHRLRNLPTIIVIRLEVGGDIQLTHVQT
Sequence CWU 1
1
191624DNAHomo sapiens 1atgttttcac atcttccctt tgactgtgtc ctgctgctgc
tgctgctact acttacaagg 60tcctcagaag tggaatacag agcggaggtc ggtcagaatg
cctatctgcc ctgcttctac 120accccagccg ccccagggaa cctcgtgccc
gtctgctggg gcaaaggagc ctgtcctgtg 180tttgaatgtg gcaacgtggt
gctcaggact gatgaaaggg atgtgaatta ttggacatcc 240agatactggc
taaatgggga tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg
300actctagcag acagtgggat ctactgctgc cggatccaaa tcccaggcat
aatgaatgat 360gaaaaattta acctgaagtt ggtcatcaaa ccagggtatt
ctcatagcaa agagaagata 420cagaatttaa gcctcatctc tttggccaac
ctccctccct caggattggc aaatgcagta 480gcagagggaa ttcgctcaga
agaaaacatc tataccattg aagagaacgt atatgaagtg 540gaggagccca
atgagtatta ttgctatgtc agcagcaggc agcaaccctc acaacctttg
600ggttgtcgct ttgcaatgcc atag 6242207PRTHomo sapiens 2Met Phe Ser
His Leu Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu
Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln 20 25 30Asn
Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu 35 40
45Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly
50 55 60Asn Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr
Ser65 70 75 80Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys Gly Asp Val
Ser Leu Thr 85 90 95Ile Glu Asn Val Thr Leu Ala Asp Ser Gly Ile Tyr
Cys Cys Arg Ile 100 105 110Gln Ile Pro Gly Ile Met Asn Asp Glu Lys
Phe Asn Leu Lys Leu Val 115 120 125Ile Lys Pro Gly Tyr Ser His Ser
Lys Glu Lys Ile Gln Asn Leu Ser 130 135 140Leu Ile Ser Leu Ala Asn
Leu Pro Pro Ser Gly Leu Ala Asn Ala Val145 150 155 160Ala Glu Gly
Ile Arg Ser Glu Glu Asn Ile Tyr Thr Ile Glu Glu Asn 165 170 175Val
Tyr Glu Val Glu Glu Pro Asn Glu Tyr Tyr Cys Tyr Val Ser Ser 180 185
190Arg Gln Gln Pro Ser Gln Pro Leu Gly Cys Arg Phe Ala Met Pro 195
200 2053594DNAMouse 3atgttttcag gtcttaccct caactgtgtc ctgctgctgc
tgcaactact acttgcaagg 60tcattggaag atggttataa ggttgaggtt ggtaaaaatg
cctatctgcc ctgcagttac 120actctaccta catctgggac acttgtgcct
atgtgctggg gcaagggatt ctgtccttgg 180tcacagtgta ccaatgagtt
gctcagaact gatgaaagaa atgtgacata tcagaaatcc 240agcagatacc
agctaaaggg cgatctcaac aaaggagatg tgtctctgat cataaagaat
300gtgactctgg atgaccatgg gacctactgc tgcaggatac agttccctgg
tcttatgaat 360gataaaaaat tagaactgaa attagacatc aaagcagggt
attcctgtaa gaaaaagaag 420ttatcgagtt tgagccttat tacactggcc
aacttgcctc caggagggtt ggcaaatgca 480ggagcagtca ggattcgctc
tgaggaaaat atctacacca tcgaggagaa cgtatatgaa 540gtggagaatt
caaatgagta ctactgctac gtcaacagcc agcagccatc ctga 5944197PRTMouse
4Met Phe Ser Gly Leu Thr Leu Asn Cys Val Leu Leu Leu Leu Gln Leu1 5
10 15Leu Leu Ala Arg Ser Leu Glu Asp Gly Tyr Lys Val Glu Val Gly
Lys 20 25 30Asn Ala Tyr Leu Pro Cys Ser Tyr Thr Leu Pro Thr Ser Gly
Thr Leu 35 40 45Val Pro Met Cys Trp Gly Lys Gly Phe Cys Pro Trp Ser
Gln Cys Thr 50 55 60Asn Glu Leu Leu Arg Thr Asp Glu Arg Asn Val Thr
Tyr Gln Lys Ser65 70 75 80Ser Arg Tyr Gln Leu Lys Gly Asp Leu Asn
Lys Gly Asp Val Ser Leu 85 90 95Ile Ile Lys Asn Val Thr Leu Asp Asp
His Gly Thr Tyr Cys Cys Arg 100 105 110Ile Gln Phe Pro Gly Leu Met
Asn Asp Lys Lys Leu Glu Leu Lys Leu 115 120 125Asp Ile Lys Ala Gly
Tyr Ser Cys Lys Lys Lys Lys Leu Ser Ser Leu 130 135 140Ser Leu Ile
Thr Leu Ala Asn Leu Pro Pro Gly Gly Leu Ala Asn Ala145 150 155
160Gly Ala Val Arg Ile Arg Ser Glu Glu Asn Ile Tyr Thr Ile Glu Glu
165 170 175Asn Val Tyr Glu Val Glu Asn Ser Asn Glu Tyr Tyr Cys Tyr
Val Asn 180 185 190Ser Gln Gln Pro Ser 195516DNAHomo sapiens
5caaaccaggg tattct 16616PRTHomo sapiens 6Asn Leu Lys Leu Val Ile
Lys Pro Gly Tyr Ser His Ser Lys Glu Lys1 5 10 15716DNAMouse
7caaagcaggg tattcc 16816PRTMouse 8Glu Leu Lys Leu Asp Ile Lys Ala
Gly Tyr Ser Cys Lys Lys Lys Lys1 5 10 159972DNAHomo sapiens
9atggccttca gcggttccca ggctccctac ctgagtccag ctgtcccctt ttctgggact
60attcaaggag gtctccagga cggacttcag atcactgtca atgggaccgt tctcagctcc
120agtggaacca ggtttgctgt gaactttcag actggcttca gtggaaatga
cattgccttc 180cacttcaacc ctcggtttga agatggaggg tacgtggtgt
gcaacacgag gcagaacgga 240agctgggggc ccgaggagag gaggacacac
atgcctttcc agaaggggat gccctttgac 300ctctgcttcc tggtgcagag
ctcagatttc aaggtgatgg tgaacgggat cctcttcgtg 360cagtacttcc
accgcgtgcc cttccaccgt gtggacacca tcttcgtcaa tggctctgtg
420cagctgtcct acatcagctt ccagcctccc ggcgtgtggc ctgccaaccc
ggctcccatt 480acccagacag tcatccacac agtgcagagc gcccctggac
agatgttctc tactcccgcc 540atcccaccta tgatgtaccc ccaccccgcc
tatccgatgc ctttcatcac caccattctg 600ggagggctgt acccatccaa
gtccatcctc ctgtcaggca ctgtcctgcc cagtgctcag 660aggttccaca
tcaacctgtg ctctgggaac cacatcgcct tccacctgaa cctccgtttt
720gatgagaatg ctgtggtccg caacacccag atcgacaact cctgggggtc
tgaggagcga 780agtctgcccc gaaaaatgcc cttcgtccgt ggccagagct
tctcagtgtg gatcttgtgt 840ggagctcact gcctcaaggt ggccgtggat
ggtcagcacc tgtttgaata ctaccatcgc 900ctgaggaacc tgcccaccat
caacagactg gaagtggggg gcgacatcca gctgacccat 960gtgcagacat ag
97210323PRTHomo sapiens 10Met Ala Phe Ser Gly Ser Gln Ala Pro Tyr
Leu Ser Pro Ala Val Pro1 5 10 15Phe Ser Gly Thr Ile Gln Gly Gly Leu
Gln Asp Gly Leu Gln Ile Thr 20 25 30Val Asn Gly Thr Val Leu Ser Ser
Ser Gly Thr Arg Phe Ala Val Asn 35 40 45Phe Gln Thr Gly Phe Ser Gly
Asn Asp Ile Ala Phe His Phe Asn Pro 50 55 60Arg Phe Glu Asp Gly Gly
Tyr Val Val Cys Asn Thr Arg Gln Asn Gly65 70 75 80Ser Trp Gly Pro
Glu Glu Arg Arg Thr His Met Pro Phe Gln Lys Gly 85 90 95Met Pro Phe
Asp Leu Cys Phe Leu Val Gln Ser Ser Asp Phe Lys Val 100 105 110Met
Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His Arg Val Pro Phe 115 120
125His Arg Val Asp Thr Ile Phe Val Asn Gly Ser Val Gln Leu Ser Tyr
130 135 140Ile Ser Phe Gln Pro Pro Gly Val Trp Pro Ala Asn Pro Ala
Pro Ile145 150 155 160Thr Gln Thr Val Ile His Thr Val Gln Ser Ala
Pro Gly Gln Met Phe 165 170 175Ser Thr Pro Ala Ile Pro Pro Met Met
Tyr Pro His Pro Ala Tyr Pro 180 185 190Met Pro Phe Ile Thr Thr Ile
Leu Gly Gly Leu Tyr Pro Ser Lys Ser 195 200 205Ile Leu Leu Ser Gly
Thr Val Leu Pro Ser Ala Gln Arg Phe His Ile 210 215 220Asn Leu Cys
Ser Gly Asn His Ile Ala Phe His Leu Asn Leu Arg Phe225 230 235
240Asp Glu Asn Ala Val Val Arg Asn Thr Gln Ile Asp Asn Ser Trp Gly
245 250 255Ser Glu Glu Arg Ser Leu Pro Arg Lys Met Pro Phe Val Arg
Gly Gln 260 265 270Ser Phe Ser Val Trp Ile Leu Cys Gly Ala His Cys
Leu Lys Val Ala 275 280 285Val Asp Gly Gln His Leu Phe Glu Tyr Tyr
His Arg Leu Arg Asn Leu 290 295 300Pro Thr Ile Asn Arg Leu Glu Val
Gly Gly Asp Ile Gln Leu Thr His305 310 315 320Val Gln
Thr11906DNAHomo sapiens 11atgttttcac atcttccctt tgactgtgtc
ctgctgctgc tgctgctact acttacaagg 60tcctcagaag tggaatacag agcggaggtc
ggtcagaatg cctatctgcc ctgcttctac 120accccagccg ccccagggaa
cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180tttgaatgtg
gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc
240agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat
agagaatgtg 300actctagcag acagtgggat ctactgctgc cggatccaaa
tcccaggcat aatgaatgat 360gaaaaattta acctgaagtt ggtcatcaaa
ccagccaagg tcacccctgc accgactctg 420cagagagact tcactgcagc
ctttccaagg atgcttacca ccaggggaca tggcccagca 480gagacacaga
cactggggag cctccctgat ataaatctaa cacaaatatc cacattggcc
540aatgagttac gggactctag attggccaat gacttacggg actctggagc
aaccatcaga 600ataggcatct acatcggagc agggatctgt gctgggctgg
ctctggctct tatcttcggc 660gctttaattt tcaaatggta ttctcatagc
aaagagaaga tacagaattt aagcctcatc 720tctttggcca acctccctcc
ctcaggattg gcaaatgcag tagcagaggg aattcgctca 780gaagaaaaca
tctataccat tgaagagaac gtatatgaag tggaggagcc caatgagtat
840tattgctatg tcagcagcag gcagcaaccc tcacaacctt tgggttgtcg
ctttgcaatg 900ccatag 90612846DNAMouse 12atgttttcag gtcttaccct
caactgtgtc ctgctgctgc tgcaactact acttgcaagg 60tcattggaag atggttataa
ggttgaggtt ggtaaaaatg cctatctgcc ctgcagttac 120actctaccta
catctgggac acttgtgcct atgtgctggg gcaagggatt ctgtccttgg
180tcacagtgta ccaatgagtt gctcagaact gatgaaagaa atgtgacata
tcagaaatcc 240agcagatacc agctaaaggg cgatctcaac aaaggagatg
tgtctctgat cataaagaat 300gtgactctgg atgaccatgg gacctactgc
tgcaggatac agttccctgg tcttatgaat 360gataaaaaat tagaactgaa
attagacatc aaagcagcca aggtcactcc agctcagact 420gcccatgggg
actctactac agcttctcca agaaccctaa ccacggagag aaatggttca
480gagacacaga cactggtgac cctccataat aacaatggaa caaaaatttc
cacatgggct 540gatgaaatta aggactctgg agaaacgatc agaactgcta
tccacattgg agtgggagtc 600tctgctgggt tgaccctggc acttatcatt
ggtgtcttaa tccttaaatg gtattcctgt 660aagaaaaaga agttatcgag
tttgagcctt attacactgg ccaacttgcc tccaggaggg 720ttggcaaatg
caggagcagt caggattcgc tctgaggaaa atatctacac catcgaggag
780aacgtatatg aagtggagaa ttcaaatgag tactactgct acgtcaacag
ccagcagcca 840tcctga 84613301PRTHomo sapiens 13Met Phe Ser His Leu
Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu Thr Arg
Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln 20 25 30Asn Ala Tyr
Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu 35 40 45Val Pro
Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly 50 55 60Asn
Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser65 70 75
80Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr
85 90 95Ile Glu Asn Val Thr Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg
Ile 100 105 110Gln Ile Pro Gly Ile Met Asn Asp Glu Lys Phe Asn Leu
Lys Leu Val 115 120 125Ile Lys Pro Ala Lys Val Thr Pro Ala Pro Thr
Leu Gln Arg Asp Phe 130 135 140Thr Ala Ala Phe Pro Arg Met Leu Thr
Thr Arg Gly His Gly Pro Ala145 150 155 160Glu Thr Gln Thr Leu Gly
Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile 165 170 175Ser Thr Leu Ala
Asn Glu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu 180 185 190Arg Asp
Ser Gly Ala Thr Ile Arg Ile Gly Ile Tyr Ile Gly Ala Gly 195 200
205Ile Cys Ala Gly Leu Ala Leu Ala Leu Ile Phe Gly Ala Leu Ile Phe
210 215 220Lys Trp Tyr Ser His Ser Lys Glu Lys Ile Gln Asn Leu Ser
Leu Ile225 230 235 240Ser Leu Ala Asn Leu Pro Pro Ser Gly Leu Ala
Asn Ala Val Ala Glu 245 250 255Gly Ile Arg Ser Glu Glu Asn Ile Tyr
Thr Ile Glu Glu Asn Val Tyr 260 265 270Glu Val Glu Glu Pro Asn Glu
Tyr Tyr Cys Tyr Val Ser Ser Arg Gln 275 280 285Gln Pro Ser Gln Pro
Leu Gly Cys Arg Phe Ala Met Pro 290 295 30014281PRTMouse 14Met Phe
Ser Gly Leu Thr Leu Asn Cys Val Leu Leu Leu Leu Gln Leu1 5 10 15Leu
Leu Ala Arg Ser Leu Glu Asp Gly Tyr Lys Val Glu Val Gly Lys 20 25
30Asn Ala Tyr Leu Pro Cys Ser Tyr Thr Leu Pro Thr Ser Gly Thr Leu
35 40 45Val Pro Met Cys Trp Gly Lys Gly Phe Cys Pro Trp Ser Gln Cys
Thr 50 55 60Asn Glu Leu Leu Arg Thr Asp Glu Arg Asn Val Thr Tyr Gln
Lys Ser65 70 75 80Ser Arg Tyr Gln Leu Lys Gly Asp Leu Asn Lys Gly
Asp Val Ser Leu 85 90 95Ile Ile Lys Asn Val Thr Leu Asp Asp His Gly
Thr Tyr Cys Cys Arg 100 105 110Ile Gln Phe Pro Gly Leu Met Asn Asp
Lys Lys Leu Glu Leu Lys Leu 115 120 125Asp Ile Lys Ala Ala Lys Val
Thr Pro Ala Gln Thr Ala His Gly Asp 130 135 140Ser Thr Thr Ala Ser
Pro Arg Thr Leu Thr Thr Glu Arg Asn Gly Ser145 150 155 160Glu Thr
Gln Thr Leu Val Thr Leu His Asn Asn Asn Gly Thr Lys Ile 165 170
175Ser Thr Trp Ala Asp Glu Ile Lys Asp Ser Gly Glu Thr Ile Arg Thr
180 185 190Ala Ile His Ile Gly Val Gly Val Ser Ala Gly Leu Thr Leu
Ala Leu 195 200 205Ile Ile Gly Val Leu Ile Leu Lys Trp Tyr Ser Cys
Lys Lys Lys Lys 210 215 220Leu Ser Ser Leu Ser Leu Ile Thr Leu Ala
Asn Leu Pro Pro Gly Gly225 230 235 240Leu Ala Asn Ala Gly Ala Val
Arg Ile Arg Ser Glu Glu Asn Ile Tyr 245 250 255Thr Ile Glu Glu Asn
Val Tyr Glu Val Glu Asn Ser Asn Glu Tyr Tyr 260 265 270Cys Tyr Val
Asn Ser Gln Gln Pro Ser 275 2801516RNAArtificial
SequenceOligonucleotide corresponding to splice junction of human
soluble tim-3 15caaaccaggg uauucu 1616969DNAMouse 16atggctctct
tcagtgccca gtctccatac attaacccga tcatcccctt tactggacca 60atccaaggag
ggctgcagga gggacttcag gtgaccctcc aggggactac caagagtttt
120gcacaaaggt ttgtggtgaa ctttcagaac agcttcaatg gaaatgacat
tgccttccac 180ttcaaccccc ggtttgagga aggagggtat gtggtttgca
acacgaagca gaacggacag 240tgggggcctg aggagagaaa gatgcagatg
cccttccaga aggggatgcc ctttgagctt 300tgcttcctgg tgcagaggtc
agagttcaag gtgatggtga acaagaaatt ctttgtgcag 360taccaacacc
gcgtacccta ccacctcgtg gacaccatcg ctgtctccgg ctgcttgaag
420ctgtccttta tcaccttcca gactcaggac tttcgtcctg cccaccaggc
acccatggct 480caaactacca tccatatggt tcacagcacc cctggacaga
tgttctctac tcctggaatc 540cctcctgtgg tgtaccccac cccagcctat
accatacctt tctacacccc cattccaaat 600gggctttacc cgtccaagtc
catcatgata tcaggcaatg tcttgccaga tgctacgagg 660ttccatatca
accttcgctg tggaggtgac attgctttcc acctgaaccc ccgtttcaat
720gagaatgctg ttgtccgaaa cactcagatc aacaactcct gggggcagga
agagcgaagt 780ctgcttggga ggatgccctt cagtcgaggc cagagcttct
cggtgtggat catatgcgaa 840ggtcactgct tcaaggtggc tgtgaatggt
caacacatgt gtgaatatta ccaccgcctg 900aagaacttgc aggatatcaa
cactctagaa gtggcgggtg atatccagct gacccacgtg 960cagacatag
96917322PRTMouse 17Met Ala Leu Phe Ser Ala Gln Ser Pro Tyr Ile Asn
Pro Ile Ile Pro1 5 10 15Phe Thr Gly Pro Ile Gln Gly Gly Leu Gln Glu
Gly Leu Gln Val Thr 20 25 30Leu Gln Gly Thr Thr Lys Ser Phe Ala Gln
Arg Phe Val Val Asn Phe 35 40 45Gln Asn Ser Phe Asn Gly Asn Asp Ile
Ala Phe His Phe Asn Pro Arg 50 55 60Phe Glu Glu Gly Gly Tyr Val Val
Cys Asn Thr Lys Gln Asn Gly Gln65 70 75 80Trp Gly Pro Glu Glu Arg
Lys Met Gln Met Pro Phe Gln Lys Gly Met 85 90 95Pro Phe Glu Leu Cys
Phe Leu Val Gln Arg Ser Glu Phe Lys Val Met 100 105 110Val Asn Lys
Lys Phe Phe Val Gln Tyr Gln His Arg Val Pro Tyr His 115 120 125Leu
Val Asp Thr Ile Ala Val Ser Gly Cys Leu Lys Leu Ser Phe Ile 130 135
140Thr Phe Gln Thr Gln Asp Phe Arg Pro Ala His Gln Ala Pro Met
Ala145 150 155 160Gln Thr Thr Ile His Met Val His Ser Thr Pro Gly
Gln Met Phe Ser 165 170 175Thr Pro Gly Ile Pro Pro Val Val Tyr Pro
Thr Pro Ala Tyr Thr Ile 180 185 190Pro Phe Tyr Thr Pro Ile Pro Asn
Gly Leu Tyr Pro Ser Lys Ser Ile 195
200 205Met Ile Ser Gly Asn Val Leu Pro Asp Ala Thr Arg Phe His Ile
Asn 210 215 220Leu Arg Cys Gly Gly Asp Ile Ala Phe His Leu Asn Pro
Arg Phe Asn225 230 235 240Glu Asn Ala Val Val Arg Asn Thr Gln Ile
Asn Asn Ser Trp Gly Gln 245 250 255Glu Glu Arg Ser Leu Leu Gly Arg
Met Pro Phe Ser Arg Gly Gln Ser 260 265 270Phe Ser Val Trp Ile Ile
Cys Glu Gly His Cys Phe Lys Val Ala Val 275 280 285Asn Gly Gln His
Met Cys Glu Tyr Tyr His Arg Leu Lys Asn Leu Gln 290 295 300Asp Ile
Asn Thr Leu Glu Val Ala Gly Asp Ile Gln Leu Thr His Val305 310 315
320Gln Thr18353PRTMouse 18Met Ala Leu Phe Ser Ala Gln Ser Pro Tyr
Ile Asn Pro Ile Ile Pro1 5 10 15Phe Thr Gly Pro Ile Gln Gly Gly Leu
Gln Glu Gly Leu Gln Val Thr 20 25 30Leu Gln Gly Thr Thr Lys Ser Phe
Ala Gln Arg Phe Val Val Asn Phe 35 40 45Gln Asn Ser Phe Asn Gly Asn
Asp Ile Ala Phe His Phe Asn Pro Arg 50 55 60Phe Glu Glu Gly Gly Tyr
Val Val Cys Asn Thr Lys Gln Asn Gly Gln65 70 75 80Trp Gly Pro Glu
Glu Arg Lys Met Gln Met Pro Phe Gln Lys Gly Met 85 90 95Pro Phe Glu
Leu Cys Phe Leu Val Gln Arg Ser Glu Phe Lys Val Met 100 105 110Val
Asn Lys Lys Phe Phe Val Gln Tyr Gln His Arg Val Pro Tyr His 115 120
125Leu Val Asp Thr Ile Ala Val Ser Gly Cys Leu Lys Leu Ser Phe Ile
130 135 140Thr Phe Gln Asn Ser Ala Ala Pro Val Gln His Val Phe Ser
Thr Leu145 150 155 160Gln Phe Ser Gln Pro Val Gln Phe Pro Arg Thr
Pro Lys Gly Arg Lys 165 170 175Gln Lys Thr Gln Asn Phe Arg Pro Ala
His Gln Ala Pro Met Ala Gln 180 185 190Thr Thr Ile His Met Val His
Ser Thr Pro Gly Gln Met Phe Ser Thr 195 200 205Pro Gly Ile Pro Pro
Val Val Tyr Pro Thr Pro Ala Tyr Thr Ile Pro 210 215 220Phe Tyr Thr
Pro Ile Pro Asn Gly Leu Tyr Pro Ser Lys Ser Ile Met225 230 235
240Ile Ser Gly Asn Val Leu Pro Asp Ala Thr Arg Phe His Ile Asn Leu
245 250 255Arg Cys Gly Gly Asp Ile Ala Phe His Leu Asn Pro Arg Phe
Asn Glu 260 265 270Asn Ala Val Val Arg Asn Thr Gln Ile Asn Asn Ser
Trp Gly Gln Glu 275 280 285Glu Arg Ser Leu Leu Gly Arg Met Pro Phe
Ser Arg Gly Gln Ser Phe 290 295 300Ser Val Trp Ile Ile Cys Glu Gly
His Cys Phe Lys Val Ala Val Asn305 310 315 320Gly Gln His Met Cys
Glu Tyr Tyr His Arg Leu Lys Asn Leu Gln Asp 325 330 335Ile Asn Thr
Leu Glu Val Ala Gly Asp Ile Gln Leu Thr His Val Gln 340 345
350Thr19355PRTHomo sapiens 19Met Ala Phe Ser Gly Ser Gln Ala Pro
Tyr Leu Ser Pro Ala Val Pro1 5 10 15Phe Ser Gly Thr Ile Gln Gly Gly
Leu Gln Asp Gly Leu Gln Ile Thr 20 25 30Val Asn Gly Thr Val Leu Ser
Ser Ser Gly Thr Arg Phe Ala Val Asn 35 40 45Phe Gln Thr Gly Phe Ser
Gly Asn Asp Ile Ala Phe His Phe Asn Pro 50 55 60Arg Phe Glu Asp Gly
Gly Tyr Val Val Cys Asn Thr Arg Gln Asn Gly65 70 75 80Ser Trp Gly
Pro Glu Glu Arg Lys Thr His Met Pro Phe Gln Lys Gly 85 90 95Met Pro
Phe Asp Leu Cys Phe Leu Val Gln Ser Ser Asp Phe Lys Val 100 105
110Met Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His Arg Val Pro Phe
115 120 125His Arg Val Asp Thr Ile Ser Val Asn Gly Ser Val Gln Leu
Ser Tyr 130 135 140Ile Ser Phe Gln Asn Pro Arg Thr Val Pro Val Gln
Pro Ala Phe Ser145 150 155 160Thr Val Pro Phe Ser Gln Pro Val Cys
Phe Pro Pro Arg Pro Arg Gly 165 170 175Arg Arg Gln Lys Pro Pro Gly
Val Trp Pro Ala Asn Pro Ala Pro Ile 180 185 190Thr Gln Thr Val Ile
His Thr Val Gln Ser Ala Pro Gly Gln Met Phe 195 200 205Ser Thr Pro
Ala Ile Pro Pro Met Met Tyr Pro His Pro Ala Tyr Pro 210 215 220Met
Pro Phe Ile Thr Thr Ile Leu Gly Gly Leu Tyr Pro Ser Lys Ser225 230
235 240Ile Leu Leu Ser Gly Thr Val Leu Pro Ser Ala Gln Arg Phe His
Ile 245 250 255Asn Leu Cys Ser Gly Asn His Ile Ala Phe His Leu Asn
Pro Arg Phe 260 265 270Asp Glu Asn Ala Val Val Arg Asn Thr Gln Ile
Asp Asn Ser Trp Gly 275 280 285Ser Glu Glu Arg Ser Leu Pro Arg Lys
Met Pro Phe Val Arg Gly Gln 290 295 300Ser Phe Ser Val Trp Ile Leu
Cys Glu Ala His Cys Leu Lys Val Ala305 310 315 320Val Asp Gly Gln
His Leu Phe Glu Tyr Tyr His Arg Leu Arg Asn Leu 325 330 335Pro Thr
Ile Asn Arg Leu Glu Val Gly Gly Asp Ile Gln Leu Thr His 340 345
350Val Gln Thr 355
* * * * *
References