U.S. patent application number 15/156207 was filed with the patent office on 2016-11-10 for use of mtor inhibitors to enhance t cell immune responses.
The applicant listed for this patent is Emory University. Invention is credited to Rafi R. Ahmed, Koichi Araki, Christian P. Larsen.
Application Number | 20160324834 15/156207 |
Document ID | / |
Family ID | 41664188 |
Filed Date | 2016-11-10 |
United States Patent
Application |
20160324834 |
Kind Code |
A1 |
Ahmed; Rafi R. ; et
al. |
November 10, 2016 |
Use of mTOR Inhibitors to Enhance T Cell Immune Responses
Abstract
It is disclosed herein that treatment of a subject with an mTOR
inhibitor enhances antigen-specific T cell immune responses. Thus,
provided herein is a method of enhancing an antigen-specific T cell
response in a subject by administering to the subject a
therapeutically effective amount of an mTOR inhibitor. The antigen
can be any antigen, such as an antigen from a pathogen or a
vaccine, or a tumor antigen. In some embodiments, the method
further comprises administering to the subject a vaccine, such as a
virus vaccine or a cancer vaccine. The mTOR inhibitor can be
administered either before or after vaccination to enhance the
quantity and quality of the T cell immune response and
immunological memory. In some examples, the mTOR inhibitor is
rapamycin or a rapamycin analog.
Inventors: |
Ahmed; Rafi R.; (Atlanta,
GA) ; Larsen; Christian P.; (Atlanta, GA) ;
Araki; Koichi; (Atlanta, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Emory University |
Atlanta |
GA |
US |
|
|
Family ID: |
41664188 |
Appl. No.: |
15/156207 |
Filed: |
May 16, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14468087 |
Aug 25, 2014 |
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15156207 |
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13057057 |
Feb 1, 2011 |
8840899 |
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PCT/US2009/052886 |
Aug 5, 2009 |
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14468087 |
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61086350 |
Aug 5, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2760/10034
20130101; A61K 2039/55516 20130101; A61K 45/06 20130101; C12N
2730/10134 20130101; A61P 33/00 20180101; C12N 2740/13043 20130101;
A61P 31/12 20180101; A61K 2039/53 20130101; A61K 2039/5258
20130101; A61P 31/04 20180101; A61K 2039/55561 20130101; C12N
2710/24122 20130101; A61K 39/12 20130101; A61K 31/4745 20130101;
C12N 2740/13023 20130101; A61P 35/00 20180101; A61K 2039/5256
20130101; C12N 2770/10022 20130101; A61K 39/0011 20130101; A61P
31/10 20180101; A61K 31/436 20130101; A61K 39/12 20130101; C12N
2710/24143 20130101; C07K 14/005 20130101; C12N 2710/10343
20130101; A61K 39/39 20130101; A61K 31/4745 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/436 20060101
A61K031/436; A61K 39/00 20060101 A61K039/00 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
numbers 5R37AI030048 and 5P01AI044644 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A method of treating cancer comprising administering an
effective amount of a composition comprising an mTOR inhibitor in
combination with a purified antigen or a vaccine.
2. The method of claim 1, wherein the purified antigen is a tumor
antigen.
3. The method of claim 2, wherein the tumor is a hematologic cancer
or a solid tumor.
4. The method of claim 3, wherein the hematologic cancer is a
leukemia or a lymphoma.
5. The method of claim 3, wherein the solid tumor is a carcinoma,
melanoma, sarcoma or central nervous system tumor.
6. The method of claim 1, wherein the tumor antigen is selected
from fibroblast growth factor 5, tyrosinase, epithelial tumor
antigen (ETA), carcinoembryonic antigen (CEA), beta-human chorionic
gonadotropin, alphafetoprotein (AFP), human telomerase reverse
transcriptase, thyroglobulin, intestinal carboxyl esterase,
macrophage colony stimulating factor, prostase, prostate-specific
antigen (PSA), human epidermal growth factor receptor 2, survivin,
telomerase, prostate-carcinoma tumor antigen-I, neutrophil
elastase, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor,
and mesothelin,
7. The method of claim 1, wherein the mTOR inhibitor is rapamycin
or analogs.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
14/468,087 filed Aug. 25, 2014, which is a division of U.S.
application Ser. No. 13/057,057 filed Feb. 1, 2011 that granted as
U.S. Pat. No. 8,840,899 on Sep. 23, 2014, which is the National
Stage of International Application No. PCT/US2009/052886 filed Aug.
5, 2009, which claims the benefit of priority to U.S. Provisional
Application No. 61/086,350 filed Aug. 5, 2008. The entirety of each
of these applications is hereby incorporated by reference for all
purposes.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA
THE OFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)
[0003] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is 08100USDIV2_ST25.txt.
The text file is 78 KB, was created on May 16, 2016, and is being
submitted electronically via EFS-Web.
FIELD
[0004] This disclosure concerns the use of mammalian target of
rapamycin (mTOR) inhibitors, such as rapamycin or analogs thereof,
to enhance T cell immune responses.
BACKGROUND
[0005] Mammalian target of rapamycin (mTOR) is a serine/threonine
protein kinase known to play a role in regulating cell growth, cell
proliferation, cell motility, cell survival, protein synthesis and
transcription. Dysregulation of the mTOR pathway is implicated as a
contributing factor to various human diseases, particularly various
types of cancer. Rapamycin is a natural product produced by the
bacterium Streptomyces hygroscopicus that can inhibit mTOR through
association with its intracellular receptor FK-506 binding protein
12 (FKBP12). The FKBP12-rapamycin complex binds directly to the
FKBP12-rapamycin binding domain of mTOR.
[0006] It has been demonstrated that mTOR functions as a catalytic
subunit for two distinct molecular complexes, mTOR complex 1
(mTORC1) and mTOR complex 2 (mTORC2). In addition to mTOR, mTORC1
is composed of regulatory associated protein of mTOR (Raptor) and
mammalian LST8/G-protein .beta.-subunit like protein
(mLST8/G.beta.L). This complex functions as a nutrient/energy/redox
sensor and plays a role in regulating protein synthesis. The
activity of mTORC1 is stimulated by insulin, growth factors, serum,
phosphatidic acid, amino acids (particularly leucine) and oxidative
stress (Hay and Sonenberg, Genes Dev. 18(16):1926-1945, 2004;
Wullschleger et al., Cell 124(3):471-484). In contrast, mTORC1 is
known to be inhibited by low nutrient levels, growth factor
deprivation, reductive stress, caffeine, rapamycin,
farnesylthiosalicylic acid and curcumin (Beevers et al., Int. J.
Cancer 119(4):757-764, 2006; McMahon et al., Mol. Endocrinol.
19(1):175-183). The components of mTORC2 are rapamycin-insensitive
companion of mTOR (Rictor), G.beta.L, mammalian stress-activated
protein kinase interacting protein 1 and mTOR. mTORC2 has been
shown to function as an important regulator of the cytoskeleton
through its stimulation of F-actin stress fibers, paxillin, RhoA,
Rac1, Cdc42 and protein kinase C alpha (Sarbassov et al., Curr.
Biol. 14(14): 1296-302, 2004; Sarbassov et al., Science 307(5712):
1098-101, 2005). Unlike mTORC1, mTORC2 is not sensitive to
rapamycin.
[0007] A number of mTOR inhibitors are currently being used, or are
currently being investigated in clinical trials, to treat a variety
of conditions. Inhibitors of mTOR, such as rapamycin, are known to
exhibit immunosuppressive and anti-proliferative properties.
Accordingly, mTOR inhibitors are routinely administered to
transplant recipients to prevent organ or bone marrow
rejection.
[0008] Vaccines are widely used to treat or prevent disease,
including infectious disease and cancer. In order for a vaccine to
be effective, sufficient immunological memory against the target
pathogen or cancer must be elicited, which often requires more than
one dose of vaccine. The ability to induce adequate immunological
memory in a subject by administration of a single vaccine dose is
desirable to achieve rapid vaccination, as well as to reduce cost
and improve compliance. Thus, a need remains for methods of
enhancing immune responses against candidate vaccines.
SUMMARY
[0009] As disclosed herein, mTOR inhibitors have surprisingly been
demonstrated to enhance antigen-specific T cell immune responses,
which are critical for establishing immunity. To enhance
antigen-specific T cell immune responses in a subject exposed to an
antigen, an mTOR inhibitor is administered during the contraction
phase of a T cell response, or the inhibitor is administered at any
time prior to or subsequent to antigen challenge when administered
at a low dose.
[0010] Provided herein is a method of enhancing an antigen-specific
T cell response in a subject by administering to the subject a
therapeutically effective amount of an antigen and a
therapeutically effective amount of an mTOR inhibitor, thereby
enhancing an antigen-specific T cell immune response in the
subject. In some embodiments, enhancing an antigen-specific T cell
response in a subject includes increasing the number or quality of
antigen-specific T cells in the subject. In some embodiments, the
antigen is part of a vaccine. The antigen can be any antigen,
including, but not limited to, an antigen from a pathogen, such as
a virus, bacteria, fungus or parasite, or a tumor antigen.
[0011] Also provided is a method of increasing the proportion of
antigen-specific CD127.sup.HighKLRG-1.sup.Low CD8.sup.+ T cells in
a subject by administering to the subject a therapeutically
effective amount of an antigen and an mTOR inhibitor. Further
provided is a method of increasing expression of CD127, CD62L,
Bcl-2 and CD27, and decreasing expression of KLRG-1, in CD8+ T
cells of a subject by administering to the subject an antigen and
an mTOR inhibitor.
[0012] The foregoing and other features and advantages will become
more apparent from the following detailed description of several
embodiments, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 is a schematic model of virus-specific CD8.sup.+ T
cell responses during an acute viral infection. Virus-specific
CD8.sup.+ T cells expand upon viral infection and become effector T
cells. The expansion phase is followed by a contraction phase in
which 90-95% of the effector cells die. The remaining effector
cells differentiate into memory T cells during the contraction
phase, and these memory cells further differentiate into high
quality memory T cells during the maintenance phase.
[0014] FIG. 2A is a schematic of the experimental design for
testing the effect of rapamycin treatment on virus-specific
CD8.sup.+ T cells during the expansion phase. Mice were infected
with lymphocytic choriomeningitis virus (LCMV) on Day 0 and treated
with rapamycin every day starting one day prior to infection (Day
-1). Peripheral blood mononuclear cells (PBMCs) from untreated (No
Rapa) and rapamycin-treated (Rapa Tx) mice were analyzed on Day 8.
FIG. 2B shows fluorescence activated cell sorting (FACS) plots of
PBMCs obtained from untreated and rapamycin-treated mice. PBMCs
were stained with LCMV GP33 epitope-specific tetramer DbGP33,
anti-CD8 and anti-CD44. The percentage of virus-specific CD8.sup.+
T cells is indicated. FIG. 2C is a graph showing LCMV titers in
rapamycin-treated and untreated mice.
[0015] FIG. 3A is a schematic of the experimental design for
testing the effect of rapamycin treatment on virus-specific
CD8.sup.+ T cells during the maintenance phase. Carboxyfluorescein
succinimidyl ester (CFSE)-labeled GP33-epitope specific memory
CD8.sup.+ T cells were adoptively transferred into mice on Day 0.
Mice were treated with rapamycin every day starting one day prior
to adoptive transfer (Day -1). Splenocytes from untreated and
rapamycin-treated mice were analyzed on Day 42. FIG. 3B is a graph
showing the number of virus-specific CD8.sup.+ T cells in the
spleen of untreated and rapamycin-treated mice on Day 42. FIG. 3C
shows FACS plots of CFSE-labeled virus-specific CD8.sup.+ T cells
obtained from untreated and rapamycin-treated mice on Day 42
post-transfer. The percentage of adoptively transferred cells that
divided more than twice is indicated.
[0016] FIG. 4A is a schematic of the experimental design for
testing the effect of rapamycin treatment on virus-specific
CD8.sup.+ T cells during the contraction phase. Mice were infected
with LCMV on Day 0 and treated with rapamycin starting on Day 8
after infection. Splenocytes from untreated and rapamycin-treated
mice were analyzed on Day 35. FIG. 4B is a graph showing the number
of virus-specific CD8.sup.+ T cells obtained from the spleen of
untreated and rapamycin-treated mice. Tetramer staining was used to
detect GP33, GP276 and NP396 epitope-specific CD8.sup.+ T cells,
while CD8.sup.+ T cells specific for epitopes NP205 and GP118 were
detected by IFN-.gamma. staining following peptide stimulation.
[0017] FIG. 5A shows a series of FACS plots evaluating expression
of T cell markers (CD127, CD62L, KLRG-1, CD27 and Bcl-2) on
splenocytes obtained from untreated and rapamycin-treated mice
according to the procedure shown in FIG. 4A. FIG. 5B is a series of
graphs that summarizes the phenotype of DbGP33, DbGP276 and DbNP396
tetramer-positive CD8.sup.+ T cells.
[0018] FIG. 6 is a series of graphs showing that rapamycin
treatment induces high quality memory T cells during the
contraction phase in PBMCs, liver and lymph nodes (LN).
[0019] FIG. 7A is a schematic of the experimental design for
demonstrating that rapamycin treatment enhances CD62L re-expression
during the T cell contraction phase. LCMV-specific transgenic (P14)
effector CD8.sup.+ T cells (Thy-1.1.sup.+) were isolated on Day 8
post-infection. CD62L.sup.high cells were depleted from the
isolated effector P14 cells and the remaining CD62L.sup.low
effector CD8.sup.+ T cells were transferred into Thy-1.2.sup.+
naive mice. Mice were then treated with rapamycin daily. FIG. 7B is
a graph showing conversion of CD62L expression from low to high in
PBMC from Day 0 to Day 26 post-transfer. FIG. 7C is a graph showing
the number of CD62L.sup.high P14 cells in the spleen of untreated
and rapamycin-treated mice on Day 26 post-transfer. FIG. 7D shows
FACS plots of CD62L expression of Thy-1.1.sup.+ P14 cells on Day 26
post-transfer. The percentage of cells that are CFSE.sup.high
(cells that have not divided) and CD62L.sup.high is indicated.
[0020] FIG. 8A is a schematic of the experimental design for
evaluating protective immunity by rapamycin-induced memory
CD8.sup.+ T cells. CD62L.sup.Low LCMV-specific Day 8 effector P14
cells were transferred into naive mice on Day 0 and mice were
treated with rapamycin for 25 days. Mice were challenged with
vaccinia virus (VV) GP33 on Day 28. FIG. 8B shows FACS plots of
splenocytes obtained from untreated and rapamycin-treated mice on
Day 5 post-challenge. The percentage of DbGP33 tetramer-positive
P14 cells is indicated. FIG. 8C is a graph showing the number of
DbGP33 tetramer-positive P14 cells in the spleen of untreated and
rapamycin-treated mice on Day 5 post-challenge. FIG. 8D is a graph
showing viral titer in the ovaries (PFU/gram) of naive, untreated
and rapamycin-treated mice on Day 5 post-challenge.
[0021] FIG. 9A is a schematic of the experimental design for
evaluating homeostatic proliferation of rapamycin-induced memory
CD8.sup.+ T cells. CFSE-labeled P14 memory cells derived from
rapamycin-treated or untreated mice were adoptively transferred
into naive mice and analyzed up to 30 days post-transfer. FIG. 9B
is a graph showing the percentage of divided P14 memory cells in
PBMC over time. FIG. 9C shows FACS plots of CFSE-labeled CD8.sup.+
T cells obtained from the spleen of untreated and rapamycin-treated
mice on Day 30 post-transfer. The percentage of P14 cells that
divided more than twice is indicated.
[0022] FIG. 10A is a schematic of the experimental design to
demonstrate that rapamycin treatment enhances high quality memory T
cells during the contraction phase of recall responses.
LCMV-specific memory P14 cells were transferred into naive mice on
Day -1. The next day, mice were infected with VVgp33 and either
untreated or treated with rapamycin daily from Day 8
post-infection. FIG. 10B is a graph showing the number of P14 cells
in the spleen of untreated and rapamycin-treated mice on Day 31.
FIG. 10C is a series of FACS plots showing the phenotypic
differences of P14 cells on Day 31 post-infection in the spleen, as
measured by expression of high quality T cell markers CD127, CD62L,
KLRG-1, CD27 and Bcl-2.
[0023] FIG. 11A is a schematic of the experimental design to
demonstrate that low dose rapamycin treatment enhances the number
of virus-specific CD8.sup.+ T cells. Rapamycin treatment was
initiated one day prior to infection with LCMV. FIG. 11B is a graph
showing the number of DbGP33 tetramer-positive LCMV-specific
CD8.sup.+ T responses in PBMC obtained from untreated and
rapamycin-treated mice up to 30 days post-infection. FIG. 11C is a
graph showing the number of virus-specific CD8.sup.+ T cells in the
spleen of untreated and rapamycin-treated mice at Day 35. Tetramer
staining was used to detect GP33, GP276 and NP396 epitope-specific
CD8.sup.+ T cells, while CD8.sup.+ T cells specific for epitopes
NP205 and GP118 were detected by IFN-.gamma. staining following
peptide stimulation.
[0024] FIG. 12A is a series of FACS plots showing phenotypic
changes (expression of CD127, CD62L, KLRG-1 and Bcl-2) of
virus-specific CD8.sup.+ T cells obtained from the spleen of
untreated and rapamycin-treated mice on Day 35 post-infection. FIG.
12B is a series of FACS plots showing the percentage of
virus-specific CD8.sup.+ T cells co-expressing CD127 and either
CD62L or KLRG-1. FIG. 12C is a series of graphs showing the
kinetics of phenotypic changes of DbGP33 tetramer-positive
LCMV-specific CD8.sup.+ T cells in PBMC obtained from untreated and
rapamycin-treated mice at intervals from Day 0 to Day 30.
[0025] FIG. 13A is a schematic of the experimental design to
demonstrate that low dose rapamycin treatment induces high quality
memory T cells during recall responses. Thy-1.1.sup.+ P14 memory T
cells were adoptively transferred into Thy-1.2.sup.+ recipient mice
and low dose rapamycin treatment was initiated on the same day (Day
-1). The following day (Day 0), recipient mice were infected with
LCMV. FIG. 13B is a series of FACS plots showing LCMV-specific
CD8.sup.+ T cell responses after infection. Shown is the percentage
of Thy-1.1.sup.+ P14 cells in PBMCs obtained from untreated and
rapamycin-treated mice on Days 8, 14, 22 and 35 post-infection.
FIG. 13C is a series of FACS plots showing the percentage of
LCMV-specific CD8.sup.+ T cells expressing CD62L and CD127 on Days
8, 14, 22 and 35 post-infection.
[0026] FIG. 14A is a schematic of the experimental design to
demonstrate that low dose rapamycin treatment induces high quality
memory T cells upon immunization with virus-like particles (VLPs).
Rapamycin treatment was initiated one day prior to immunization
with VLPs. FIG. 14B is a graph showing DbGP33 tetramer-positive
CD8.sup.+ T cell responses in PBMCs obtained from untreated and
rapamycin-treated mice. FIG. 14C is a graph showing the number of
DbGP33 tetramer-positive CD8.sup.+ T cells obtained from the spleen
of untreated and rapamycin-treated mice on Day 34 post-infection.
FIGS. 14D and 14E are FACS plots showing the phenotypic analysis of
DbGP33 tetramer-positive CD8.sup.+ T cells in the spleen on Day 34
post-immunization.
[0027] FIG. 15 is a schematic depiction of the mammalian target of
rapamycin (mTOR) pathway. mTOR is part of two distinct complexes,
mTOR complex 1 (mTORC1) and complex 2 (mTORC2). mTORC1 is sensitive
to rapamycin.
[0028] FIG. 16A is a schematic of the experimental design for
raptor knockdown in virus-specific CD8.sup.+ T cells. Retrovirus
encoding a control or raptor-specific short hairpin RNA (shRNA)
(RNAi) was constructed and used to transduce LCMV-specific P14
cells. Transduced P14 cells were adoptively transferred into naive
mice, and the mice were then infected with LCMV. FIG. 16B is a
series of graphs showing the phenotypic changes of adoptively
transferred P14 cells obtained from the spleen on Day 35 after LCMV
infection. GFP-positive cells are retrovirus-transduced cells.
[0029] FIG. 17A is a schematic of the experimental design to
demonstrate that LCMV-specific CD8.sup.+ T cells become rapamycin
sensitive after FKBP12 knockdown. Control or FKBP12 RNAi retrovirus
transduced LCMV-specific P14 cells were adoptively transferred into
naive mice and the mice were infected with LCMV. Rapamycin
treatment was initiated on the day prior to LCMV infection (Day
-1). FIG. 17B is a series of graphs showing the phenotypic changes
of adoptively transferred P14 cells on Day 16 post-LCMV infection
in PBMC. Green fluorescent protein (GFP)-positive cells are
retrovirus-transduced cells.
[0030] FIG. 18 shows FACS plots demonstrating that low dose
rapamycin treatment enhances the number of virus-specific memory
CD4.sup.+ T cells. Rapamycin treatment was initiated one day prior
to infection with LCMV. Spleen cells were stimulated with LCMV GP61
peptide specific for CD4.sup.+ T cells and intracellular cytokine
staining was performed. The percentage of CD4.sup.+ T cells
expressing IL-2 and/or IFN-.gamma. in the presence and absence of
peptide stimulation is indicated.
[0031] FIG. 19A is a schematic of the experimental design to
demonstrate that low dose rapamycin treatment improves the quantity
and quality of memory T cells induced by recombinant adenovirus
serotype 5 (rAd5) that expresses LCMV glycoprotein (rAd5-LCMV-GP).
Rapamycin treatment was initiated one day prior to vaccination with
rAd5-LCMV-GP. FIG. 19B is a graph showing the number of DbGP33
tetramer-positive CD8.sup.+ T cells in the spleen on Day 35
post-vaccination. FIG. 19C is a series of FACS plots showing
phenotypic analysis of DbGP33 tetramer-positive CD8.sup.+ T cells
in the spleen on Day 35 post-vaccination.
[0032] FIG. 20A is a graph showing the kinetics of endogenous GP33
epitope-specific CD8.sup.+ T cells in PBMCs of LCMV-infected B6
mice treated with rapamycin from Day -1 to Day 30 post-infection
(shaded area) (No Rapa, n=3 mice; Rapa Tx, n=6). FIG. 20B is a
series of FACS plots showing phenotypic analysis of endogenous
DbGP33 tetramer-positive cells in the spleen at Day 36 post
infection. FIG. 20C is schematic diagram and pair of FACS plots
showing GP33 epitope-specific P14 transgenic memory CD8.sup.+ T
cells (Day 34 post-infection) were generated in the presence or
absence of rapamycin, labeled with CFSE and then adoptively
transferred into naive mice to monitor their homeostatic
proliferation. CFSE dilution of P14 cells at 30 days post transfer
is shown in the FACS plots and the number represents percentage of
memory cells that divided more than two times. FIG. 20D is a
schematic diagram and a pair of graphs showing that memory P14
cells derived from rapamycin-treated or untreated mice were
adoptively transferred and mice were challenged with vaccinia virus
expressing the GP33 epitope (VVGP33). Kinetics of P14 cells in
PBMCs after challenge (left graph) and the total P14 cell numbers
in spleen on Day 30 post-infection (right graph) are shown (No
Rapa, n=4; Rapa Tx, n=6). Error bars indicate standard error of the
mean (SEM).
[0033] FIG. 21A is a graph showing kinetics of endogenous GP33
epitope-specific CD8.sup.+ T cells in PBMCs of LCMV-infected B6
mice treated with rapamycin from Day -1 to Day 8 post-infection
(shaded area) (n=3-6 for each time point). FIG. 21B is a graph
showing the average number of DbGP33 tetramer-positive cells on Day
36 post-infection in spleens of LCMV-infected mice treated with
rapamycin (No Rapa, n=9; Rapa Tx Day -1 to Day 8, n=3). FIG. 21C is
a series of FACS plots showing CD127, KLRG-1, and Bcl-2 expression
on endogenous DbGP33 tetramer-positive cells in PBMCs at 8 days
post-LCMV infection in B6 mice. Rapamycin was administered from Day
-1 to Day 8 post-infection. FIG. 21D is a series of FACS plots
showing phenotypic analysis of DbGP33 tetramer-positive cells in
spleens of LCMV-infected mice (rapamycin treatment from Day -1 to
Day 8 post-infection). Error bars indicate SEM.
[0034] FIG. 22A is a graph showing kinetics of endogenous GP33
epitope-specific CD8.sup.+ T cells in PBMCs of LCMV-infected B6
mice treated with rapamycin from Day 8 to Day 36 post-infection
(shaded area) (No Rapa, n=9 mice; Rapa Tx, n=9). FIG. 22B is a
series of graphs showing phenotypic changes in endogenous DbGP33
tetramer-positive CD8.sup.+ T cells in the spleen on Day 36
post-LCMV infection (n=12 for each group). B6 Mice were treated
with rapamycin during the effector to memory T cell transition
period (Days 8-35 post-infection). FIG. 22C is a pair of FACS plots
and a schematic diagram showing CD62L-negative Day 8 P14 transgenic
effector CD8.sup.+ T cells were purified, labeled with CFSE, and
then adoptively transferred into naive mice. Half of these mice
were treated with rapamycin after transfer and CD62L conversion in
the antigen-specific CD8.sup.+ T cells was analyzed longitudinally
in the blood (FIG. 22D). FIG. 22E is a series of FACS plots showing
CFSE profile and CD62L expression on antigen-specific memory
CD8.sup.+ T cells in the spleen at Day 27 after transfer of
CD62L-negative effector T cells. FIG. 22F is a schematic diagram
showing CD62L-negative Day 8 P14 transgenic effector CD8.sup.+ T
cells were adoptively transferred into naive mice. These mice were
treated with rapamycin for 25 days, and were challenged with VVGP33
on Day 28 post-transfer. At 5 days after challenge, P14 expansion
in spleen (FIG. 22G) and viral titers in ovary (FIG. 22H) were
analyzed (n=4-6 for each group). Flow data were gated on CD8.sup.+
T cells. Error bars indicate SEM.
[0035] FIGS. 23A-23C are graphs showing knockdown of specific genes
using a retrovirus based RNAi system. Retrovirus-transduced
LCMV-specific P14 transgenic CD8.sup.+ T cells (marked by GFP
expression) were adoptively transferred into naive mice, followed
by LCMV infection. Phenotypic analysis of retrovirus transduced
cells (GFP.sup.+) and non-transduced (GFP.sup.-) P14 cells in PBMCs
was performed on Days 14-16 post infection. FIGS. 23A and 23B are a
series of graphs showing changes in expression of phenotypic
markers following mTOR (A) or raptor (B) RNAi treatment. Each line
shows expression of the indicated phenotypic markers on transduced
and non-transduced antigen-specific CD8.sup.+ T cells in individual
animals. The same control data are shown in A and B. FIG. 23C is a
series of graphs showing changes in expression of phenotypic
markers following FKBP12 RNAi treatment. FKBP12 RNAi expressing
retrovirus- or control retrovirus-transduced P14 transgenic
CD8.sup.+ T cells (marked by GFP expression) were adoptively
transferred into naive mice, followed by LCMV infection. Half of
the mice were treated with rapamycin throughout infection.
Phenotypic analysis of retrovirus-transduced cells (GFP.sup.+) and
non-transduced (GFP.sup.-) P14 cells in the PBMCs was performed on
Days 14-16 post-infection.
[0036] FIG. 24A is a schematic diagram showing LCMV-specific P14
transgenic memory CD8.sup.+ T cells (Thy-1.1) were adoptively
transferred into Thy-1.2 naive mice and these mice were infected
with LCMV in the presence or absence of rapamycin (Day -1 to Day 32
post-infection). FIG. 24B is a graph showing kinetics of P14 recall
responses upon infection. Flow data are gated on lymphocytes in
PBMCs. FIGS. 24C and 24D are a pair of graphs showing CD127
expression and CD62L expression, respectively, on P14 cells during
recall responses. Error bars and shaded area indicate SEM and
rapamycin treatment, respectively.
[0037] FIGS. 25A-25C demonstrate that mTOR regulates memory
CD8.sup.+ T cell responses in non-human primates. Rhesus macaques
were vaccinated with MVA (booster immunization) in the presence or
absence of rapamycin (n=3 for each group). PBMCs from vaccinated
macaques were stimulated with vaccinia virus and analyzed for
IFN-.gamma. production. FIG. 25A is a series of FACS plots
analyzing IFN-.gamma. production from representative macaques
(gated on CD3.sup.+CD8.sup.+ cells). FIG. 25B is a graph showing
kinetics of IFN-.gamma. producing vaccinia virus-specific CD8.sup.+
T cells in individual animals. The shaded area shows rapamycin
treatment. FIG. 25C is a graph showing IFN-.gamma. producing
vaccinia virus-specific CD8.sup.+ T cell contraction rate over
time. The number of vaccinia virus-specific CD8.sup.+ T cells at
the peak between days 7-21 post-vaccination was taken as 100% for
individual animals, and contraction rate was calculated as a
percentage of this peak response. Lines and shaded area show
nonlinear regression (one phase exponential decay) and rapamycin
treatment, respectively.
SEQUENCE LISTING
[0038] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. For double-stranded DNA
sequences, only one strand of each nucleic acid sequence is shown,
but the complementary strand is understood as included by any
reference to the displayed strand. In the accompanying sequence
listing:
[0039] SEQ ID NO: 1 is the nucleotide sequence of a shRNA specific
for raptor.
[0040] SEQ ID NO: 2 is the nucleotide sequence of a shRNA specific
for FKBP12.
[0041] SEQ ID NOs: 3 and 4 are the nucleotide and amino acid
sequences, respectively, of human mTOR deposited under GENBANK.TM.
Accession No. NM_004958 on Apr. 4, 2002.
[0042] SEQ ID NOs: 5 and 6 are the nucleotide and amino acid
sequences, respectively, of human mTOR deposited under GENBANK.TM.
Accession No. BC117166 on Jun. 26, 2006.
[0043] SEQ ID NOs: 7 and 8 are the nucleotide and amino acid
sequences, respectively, of LCMV glycoprotein deposited under
GENBANK.TM. Accession No. M20869 on Aug. 2, 1993.
[0044] SEQ ID NO: 9 is the amino acid sequence of the LCMV gp33-41
epitope.
[0045] SEQ ID NOs: 10 and 11 are the nucleotide sequences of the
sense and antisense strands, respectively, of an mTOR-specific
shRNA.
[0046] SEQ ID NOs: 12 and 13 are the nucleotide sequences of the
sense and antisense strands, respectively, of a raptor-specific
shRNA.
[0047] SEQ ID NOs: 14 and 15 are the nucleotide sequences of the
sense and antisense strands, respectively, of a FKBP12-specific
shRNA.
[0048] SEQ ID NOs: 16 and 17 are the nucleotide sequences of the
sense and antisense strands, respectively, of an S6K1-specific
shRNA.
[0049] SEQ ID NOs: 18 and 19 are the nucleotide sequences of the
sense and antisense strands, respectively, of an eIF4E-specific
shRNA.
DETAILED DESCRIPTION
I. Abbreviations
[0050] Ad Adenovirus
[0051] AFP Alphafetoprotein
[0052] CD Cluster of differentiation
[0053] CEA Carcinoembryonic antigen
[0054] CFSE Carboxyfluorescein succinimidyl ester
[0055] DC Dendritic cell
[0056] FACS Fluorescence activated cell sorting
[0057] FBS Fetal bovine serum
[0058] FKBP12 FK506-binding protein 12
[0059] G.beta.L G-protein .beta.-subunit
[0060] GFP Green fluorescent protein
[0061] GP Glycoprotein
[0062] HBV Hepatitis B virus
[0063] HBcAg Hepatitis B core antigen
[0064] HCV Hepatitis C virus
[0065] HIV Human immunodeficiency virus
[0066] HPV Human papillomavirus
[0067] IFN Interferon
[0068] IGF Insulin growth factor
[0069] IP Intraperitoneally
[0070] KLRG Killer cell lectin-like receptor G
[0071] LCMV Lymphocytic choriomeningitis virus
[0072] LN Lymph node
[0073] MHC Major histocompatibility complex
[0074] miRNA Micro RNA
[0075] mTOR Mammalian target of rapamycin
[0076] mTORC1 mTOR complex 1
[0077] mTORC2 mTOR complex 2
[0078] MVA Modified Vaccinia Ankara
[0079] NP Nucleoprotein
[0080] ODN Oligodeoxynucleotide
[0081] PBMC Peripheral blood mononuclear cell
[0082] PFU Plaque forming unit
[0083] PRAME Preferentially expressed antigen of melanoma
[0084] PrCP Peridinin chlorophyll protein
[0085] PSA Prostate specific antigen
[0086] Raptor Regulatory associated protein of mTOR
[0087] Rictor Rapamycin-insensitive companion of mTOR
[0088] RNA Ribonucleic acid
[0089] RNAi RNA interference
[0090] SEM Standard error of the mean
[0091] shRNA Short hairpin RNA
[0092] VLP Virus-like particle
[0093] VV Vaccinia virus
[0094] WT Wilms tumor
II. Terms and Methods
[0095] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0096] In order to facilitate review of the various embodiments of
the disclosure, the following explanations of specific terms are
provided:
[0097] Acute infection: An infection (such as a viral infection)
having a relatively short time course.
[0098] Adjuvant: A substance or vehicle that non-specifically
enhances the immune response to an antigen. Adjuvants can include a
suspension of minerals (alum, aluminum hydroxide, or phosphate) on
which antigen is adsorbed; or water-in-oil emulsion in which
antigen solution is emulsified in mineral oil (for example,
Freund's incomplete adjuvant), sometimes with the inclusion of
killed mycobacteria (Freund's complete adjuvant) to further enhance
antigenicity. Immunostimulatory oligonucleotides (such as those
including a CpG motif) can also be used as adjuvants (for example,
see U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371;
6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants also
include biological molecules, such as costimulatory molecules.
Exemplary biological adjuvants include IL-2, RANTES, GM-CSF,
TNF-.alpha., IFN-.gamma., G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L
and 41 BBL.
[0099] Administration: The introduction of a composition into a
subject by a chosen route. For example, if the chosen route is
intravenous, the composition (for example, rapamycin) is
administered by introducing the composition into a vein of the
subject.
[0100] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals. Similarly, the
term "subject" includes both human and veterinary subjects.
[0101] Antibody: A polypeptide ligand comprising at least a light
chain or heavy chain immunoglobulin variable region which
specifically recognizes and binds an epitope of an antigen.
Antibodies are composed of a heavy and a light chain, each of which
has a variable region, termed the variable heavy (V.sub.H) region
and the variable light (V.sub.L) region. Together, the V.sub.H
region and the V.sub.L region are responsible for binding the
antigen recognized by the antibody.
[0102] Antibodies include intact immunoglobulins and the variants
and portions of antibodies well known in the art, such as Fab
fragments, Fab' fragments, F(ab)'.sub.2 fragments, single chain Fv
proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A
scFv protein is a fusion protein in which a light chain variable
region of an immunoglobulin and a heavy chain variable region of an
immunoglobulin are bound by a linker, while in dsFvs, the chains
have been mutated to introduce a disulfide bond to stabilize the
association of the chains. The term also includes genetically
engineered forms such as chimeric antibodies (for example,
humanized murine antibodies), heteroconjugate antibodies (such as,
bispecific antibodies). See also, Pierce Catalog and Handbook,
1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,
Immunology, 3.sup.rd Ed., W. H. Freeman & Co., New York,
1997.
[0103] Typically, a naturally occurring immunoglobulin has heavy
(H) chains and light (L) chains interconnected by disulfide bonds.
There are two types of light chain, lambda (.lamda.) and kappa (k).
There are five main heavy chain classes (or isotypes) which
determine the functional activity of an antibody molecule: IgM,
IgD, IgG, IgA and IgE.
[0104] Each heavy and light chain contains a constant region and a
variable region, (the regions are also known as "domains"). In
combination, the heavy and the light chain variable regions
specifically bind the antigen. Light and heavy chain variable
regions contain a "framework" region interrupted by three
hypervariable regions, also called "complementarity-determining
regions" or "CDRs." The extent of the framework region and CDRs
have been defined (see, Kabat et al., Sequences of Proteins of
Immunological Interest, U.S. Department of Health and Human
Services, 1991). The Kabat database is now maintained online. The
sequences of the framework regions of different light or heavy
chains are relatively conserved within a species, such as humans.
The framework region of an antibody, that is the combined framework
regions of the constituent light and heavy chains, serves to
position and align the CDRs in three-dimensional space.
[0105] The CDRs are primarily responsible for binding to an epitope
of an antigen. The CDRs of each chain are typically referred to as
CDR1, CDR2, and CDR3, numbered sequentially starting from the
N-terminus, and are also typically identified by the chain in which
the particular CDR is located. Thus, a V.sub.H CDR3 is located in
the variable domain of the heavy chain of the antibody in which it
is found, whereas a V.sub.L CDR1 is the CDR1 from the variable
domain of the light chain of the antibody in which it is found.
Antibodies with different specificities (i.e. different combining
sites for different antigens) have different CDRs. Although it is
the CDRs that vary from antibody to antibody, only a limited number
of amino acid positions within the CDRs are directly involved in
antigen binding. These positions within the CDRs are called
specificity determining residues (SDRs).
[0106] References to "V.sub.H" or "VH" refer to the variable region
of an immunoglobulin heavy chain, including that of an Fv, scFv,
dsFv or Fab. References to "V.sub.L" or "VL" refer to the variable
region of an immunoglobulin light chain, including that of an Fv,
scFv, dsFv or Fab.
[0107] Antigen: A compound, composition, or substance that can
stimulate the production of antibodies or a T-cell response in an
animal, including compositions that are injected or absorbed into
an animal. An antigen reacts with the products of specific humoral
or cellular immunity, including those induced by heterologous
immunogens.
[0108] Antigen-specific T cell response: Refers to a T cell immune
response that is directed against a particular antigen.
Antigen-specific T cells are T cells that are capable of
specifically recognizing (via T cell receptors) and responding to
an antigen. As used herein, "enhancing" an antigen-specific T cell
response includes, but is not limited to, increasing the number,
quality and/or activity of T cells, such as CD4.sup.+ and/or
CD8.sup.+ memory T cells. There are three phases of an
antigen-specific CD8.sup.+ T cell response after exposure to
antigen (such as during a viral infection). First, during the
expansion phase naive antigen-specific CD8.sup.+ T cells
exponentially expand and become effector T cells. These effector T
cells stop proliferating approximately 1 to 2 weeks after exposure
and enter the contraction phase. During the contraction phase,
effector CD8.sup.+ T cells gradually acquire memory T cell
phenotype and function. The contraction phase is also referred to
as the "cell death phase" as a significant number of activated T
cells (often about 90%) die during this phase. The maintenance
phase, which is also referred to as the "memory phase," follows the
contraction phase and is characterized by long-term survival of
antigen-specific memory cells.
[0109] Antisense oligonucleotide: As used herein, an "antisense
oligonucleotide" is a single-stranded antisense compound that is a
nucleic acid-based oligomer. An antisense oligonucleotide can
include one or more chemical modifications to the sugar, base,
and/or internucleoside linkages. Generally, antisense
oligonucleotides are "DNA-like" such that when the antisense
oligonucleotide hybridizes to a target mRNA, the duplex is
recognized by RNase H (an enzyme that recognizes DNA:RNA duplexes),
resulting in cleavage of the mRNA.
[0110] Binding affinity: Affinity of an antibody for an antigen. In
one embodiment, affinity is calculated by a modification of the
Scatchard method described by Frankel et al. (Mol. Immunol.,
16:101-106, 1979). In some cases, binding affinity is measured by
an antigen/antibody dissociation rate. In another cases, a high
binding affinity is measured by a competition radioimmunoassay or
ELISA.
[0111] CD8.sup.+ effector T cells/CD4.sup.+ effector T cells:
Activated T cells that express CD8 or CD4, respectively. During an
immune response, effector T cells divide rapidly and secrete
cytokines to modulate the immune response. T effector cells are
also known as T helper cells.
[0112] CD8.sup.+ memory T cells/CD4.sup.+ memory T cells:
Antigen-specific CD8.sup.+ or CD4.sup.+ T cells that persist
long-term after an immune response. Upon re-exposure to the
antigen, memory T cells expand and become T effector cells.
[0113] Chronic infection: An infection (such as a viral infection)
that persists for a relatively long period of time. Chronic
infections typically result in little to no change in symptoms over
time and/or progress very slowly.
[0114] High quality T cells: As used herein, a "high quality T
cell" is an antigen-specific T cell that exhibits superior
properties relative to standard T cells, such as increased
proliferation in response to antigen or increased viral clearance.
High quality T cells can be identified by detecting the expression
level of specific cell-surface markers. In some embodiments, a high
quality T cell is a T cell expressing one or more of
CD127.sup.high, CD62L.sup.high, KLRG-1.sup.low, CD27.sup.high and
Bcl-2.sup.high. In some embodiments, high quality T cells are
CD127.sup.high, CD27.sup.high and Bcl-2.sup.high. In some
embodiments, high quality T cells are KLRG-1.sup.low, CD27.sup.high
and Bcl-2.sup.high. In some embodiments, high quality T cells are
CD127.sup.high, CD62L.sup.high, KLRG-1.sup.low and Bcl-2.sup.high.
In some embodiments, high quality T cells are CD127.sup.high,
CD62L.sup.high and KLRG-1.sup.low. Similarly, "enhancing the
quality of a T cell" refers to increasing functional activity of a
T cell, such as increased proliferation in response to antigen or
increased viral clearance. In some embodiments, enhancing the
quality of a T cell includes increasing expression of one or more
of CD127, CD62L, CD27 and Bcl-2, and/or decreasing expression of
KLRG-1. In some embodiments, the increase or decrease in expression
is about 1.5-fold, about 2-fold, about 3-fold, about 5-fold or
about 10-fold. High quality T cells can be identified according to
standard methods known in the art, such as by FACS.
[0115] Immune response: A response of a cell of the immune system,
such as a B cell or T cell, to a stimulus. In some embodiments, the
response is specific for a particular antigen (an "antigen-specific
response"). In some embodiments, an immune response is a T cell
response, such as a CD8.sup.+ response. In another embodiment, the
response is a B cell response, and results in the production of
antigen-specific antibodies. As used herein, "stimulating an immune
response" refers to promoting or enhancing the response of the
cells of the immune system to a stimulus, such as an antigen.
[0116] Immunogen: A compound, composition, or substance which is
capable, under appropriate conditions, of stimulating an immune
response, such as the production of antibodies or a T-cell response
in an animal, including compositions that are injected or absorbed
into an animal.
[0117] Increasing the proportion of CD127.sup.HighKLRG-1.sup.Low
CD8.sup.+ T cells: As used herein, "increasing the proportion of
CD127.sup.HighKLRG-1.sup.Low CD8.sup.+ T cells" refers to
increasing the ratio of CD127.sup.HighKLRG-1.sup.Low to
CD127.sup.LowKLRG-1.sup.High CD8.sup.+ T cells in the subject
exposed to an antigen (such as an infectious agent, tumor or
vaccine). As described herein, day 8 (the end of the T cell
expansion phase) effector CD8.sup.+ T cell populations are
characterized by two subsets: (1) terminal effector T cells
(CD127.sup.LowKLRG-1.sup.High) a large percentage of which die over
the following 2-4 weeks; and (2) memory precursor cells
(CD127.sup.HighKLRG-1.sup.Low) which survive and differentiate to
produce long-lived memory T cells. As disclosed herein, treatment
of a subject exposed to an antigen with an mTOR inhibitor increases
the proportion of antigen-specific CD127.sup.HighKLRG-1.sup.Low
CD8.sup.+ T cells in the subject. T cells expressing CD127 and
KLRG-1, and their relative expression levels, can be identified
according to standard methods known in the art, such as by
FACS.
[0118] Inhibit expression or activity: As used herein, a compound
that inhibits expression or activity of mTOR is a compound that
reduces the level of mTOR mRNA or protein in a cell or tissue, or
reduces (including eliminates) one or more activities of mTOR. For
example, an antisense compound targeting mTOR inhibits expression
of mTOR by promoting the degradation of mTOR mRNA, thereby reducing
the level of mTOR protein. In some embodiments, mTOR expression is
inhibited at least 10%, at least 25%, at least 50%, at least 75%,
at least 90%, or at least 95% relative to a control, such as
untreated control cells. As another example, an antibody or small
molecule that specifically binds or targets mTOR may inhibit
activity of mTOR by directly inhibiting its kinase activity or by
preventing mTOR protein from interacting with another protein. In
some embodiments, mTOR activity is inhibited at least 10%, at least
25%, at least 50%, at least 75%, at least 90%, or at least 95%
relative to an untreated control.
[0119] Isolated: An "isolated" biological component, such as a
nucleic acid, protein (including antibodies) or organelle that has
been substantially separated or purified away from other biological
components in the environment (such as a cell) in which the
component naturally occurs, i.e., other chromosomal and
extra-chromosomal DNA and RNA, proteins and organelles. Nucleic
acids and proteins that have been "isolated" include nucleic acids
and proteins purified by standard purification methods. The term
also embraces nucleic acids and proteins prepared by recombinant
expression in a host cell as well as chemically synthesized nucleic
acids.
[0120] Low dose rapamycin: Refers to a dose of rapamycin that does
not suppress antigen-specific T cell immune responses when
administered during the expansion, contraction or maintenance
phases of a T cell response. Generally, a low dose of rapamycin is
about 0.01 to about 0.15 mg/kg, such as about 0.05 to about 0.1, or
a dose that results in a blood concentration of approximately 5 to
20 ng/ml. In some examples a low dose of rapamycin is about 0.01,
about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about
0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12,
about 0.13, about 0.14 or about 0.15 mg/kg. In this context,
"about" refers to a value within 0.005 mg/kg.
[0121] MicroRNA (miRNA): Single-stranded RNA molecules that
regulate gene expression. miRNAs are generally 21-23 nucleotides in
length. miRNAs are processed from primary transcripts known as
pri-miRNA to short stem-loop structures called pre-miRNA and
finally to functional miRNA. Mature miRNA molecules are partially
complementary to one or more messenger RNA molecules, and their
primary function is to down-regulate gene expression. MicroRNAs
regulate gene expression through the RNAi pathway.
[0122] mTOR: A serine/threonine kinase that regulates the
expression of proteins involved in cell growth and proliferation
via phosphorylation of specific substrates. As such, mTOR plays an
integral role in the response to numerous hormones and growth
factors. Synonyms for mTOR include FRAP1, FKBP12-rapamycin
complex-associated protein, FK506-binding protein 12-rapamycin
complex-associated protein 1, rapamycin target protein and RAPT1.
Nucleotide and amino acid sequences of mTOR are known in the art
(for example, GENBANK.TM. Accession No. NM_004958, deposited on
Apr. 4, 2002 (SEQ ID NOs: 3 and 4), and GENBANK.TM. Accession No.
BC117166, deposited on Jun. 26, 2006 (SEQ ID NOs: 5 and 6)).
[0123] mTOR inhibitor: A molecule that inhibits expression or
activity of mTOR. mTOR inhibitors include, but are not limited to
small molecule, antibody, peptide and nucleic acid inhibitors. For
example, an mTOR inhibitor can be a molecule that inhibits the
kinase activity of mTOR or inhibits binding of mTOR to a ligand.
Inhibitors of mTOR also include molecules that down-regulate
expression of mTOR, such as an antisense compound. A number of mTOR
inhibitors are known in the art and are discussed below. In some
embodiments, the mTOR inhibitor is rapamycin or a rapamycin
analog.
[0124] Pathogen: A biological agent that causes disease or illness
to its host. Pathogens include, for example, bacteria, viruses,
fungi, protozoa and parasites. Pathogens are also referred to as
infectious agents or infectious microorganisms.
[0125] Examples of pathogenic viruses include, but are not limited
to those in the following virus families: Retroviridae (for
example, human immunodeficiency virus (HIV), human T-cell leukemia
viruses; Picornaviridae (for example, polio virus, hepatitis A
virus, hepatitis C virus, enteroviruses, human coxsackie viruses,
rhinoviruses, echoviruses, foot-and-mouth disease virus);
Caliciviridae (such as strains that cause gastroenteritis,
including Norwalk virus); Togaviridae (for example, alphaviruses
(including chikungunya virus, equine encephalitis viruses, Simliki
Forest virus, Sindbis virus, Ross River virus), rubella viruses);
Flaviridae (for example, dengue viruses, yellow fever viruses, West
Nile virus, St. Louis encephalitis virus, Japanese encephalitis
virus, Powassan virus and other encephalitis viruses);
Coronaviridae (for example, coronaviruses, severe acute respiratory
syndrome (SARS) virus; Rhabdoviridae (for example, vesicular
stomatitis viruses, rabies viruses); Filoviridae (for example,
Ebola virus, Marburg virus); Paramyxoviridae (for example,
parainfluenza viruses, mumps virus, measles virus, respiratory
syncytial virus); Orthomyxoviridae (for example, influenza viruses,
including avian flu and swine flu); Bunyaviridae (for example,
Hantaan viruses, Sin Nombre virus, Rift Valley fever virus, bunya
viruses, phleboviruses and Nairo viruses); Arenaviridae (such as
Lassa fever virus and other hemorrhagic fever viruses, Machupo
virus, Junin virus); Reoviridae (e.g., reoviruses, orbiviurses,
rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus);
Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses,
polyoma viruses, BK-virus); Adenoviridae (adenoviruses);
Herpesviridae (herpes simplex virus (HSV)-1 and HSV-2;
cytomegalovirus; Epstein-Barr virus; varicella zoster virus; and
other herpes viruses, including HSV-6); Poxviridae (variola
viruses, vaccinia viruses, pox viruses); and Iridoviridae (such as
African swine fever virus); Astroviridae; and unclassified viruses
(for example, the etiological agents of spongiform
encephalopathies, the agent of delta hepatitis (thought to be a
defective satellite of hepatitis B virus).
[0126] Examples of bacterial pathogens include, but are not limited
to: Helicobacter pylori, Escherichia coli, Vibrio cholerae, Borelia
burgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M.
tuberculosis, M. avium, M. intracellulare, M. kansaii, M.
gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria
meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group
A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans group), Streptococcus faecalis,
Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus
pneumoniae, pathogenic Campylobacter sp., Enterococcus sp.,
Haemophilus influenzae, Bacillus anthracis, corynebacterium
diphtherias, corynebacterium sp., Erysipelothrix rhusiopathiae,
Clostridium perfringens, Clostridium tetani, Enterobacter
aerogenes, Klebsiella pneumoniae, Pasteurella multocida,
Bacteroides sp., Fusobacterium nucleatum, Streptobacillus
moniliformis, Treponema pallidium, Treponema pertenue, Leptospira,
Bordetella pertussis, Shigella flexnerii, Shigella dysenteriae and
Actinomyces israelli.
[0127] Examples of fungal pathogens include, but are not limited
to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides
immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida
albicans.
[0128] Other pathogens (such as parasitic pathogens) include, but
are not limited to: Plasmodium falciparum, Plasmodium vivax,
Trypanosoma cruzi and Toxoplasma gondii.
[0129] Pharmaceutical agent: A chemical compound or composition
capable of inducing a desired therapeutic or prophylactic effect
when properly administered to a subject or a cell.
[0130] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers of use are conventional. Remington's
Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co.,
Easton, Pa., 15th Edition, 1975, describes compositions and
formulations suitable for pharmaceutical delivery of the
compositions disclosed herein.
[0131] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(such as powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0132] Polypeptide: A polymer in which the monomers are amino acid
residues that are joined together through amide bonds. When the
amino acids are alpha-amino acids, either the L-optical isomer or
the D-optical isomer can be used, the L-isomers being preferred.
The term polypeptide or protein as used herein encompasses any
amino acid sequence and includes modified sequences such as
glycoproteins. The term polypeptide is specifically intended to
cover naturally occurring proteins, as well as those that are
recombinantly or synthetically produced.
[0133] The term polypeptide fragment refers to a portion of a
polypeptide that exhibits at least one useful epitope. The phrase
"functional fragment(s) of a polypeptide" refers to all fragments
of a polypeptide that retain an activity, or a measurable portion
of an activity, of the polypeptide from which the fragment is
derived. Fragments, for example, can vary in size from a
polypeptide fragment as small as an epitope capable of binding an
antibody molecule to a large polypeptide capable of participating
in the characteristic induction or programming of phenotypic
changes within a cell. An epitope is a region of a polypeptide
capable of binding an immunoglobulin generated in response to
contact with an antigen. Thus, smaller peptides containing the
biological activity of insulin, or conservative variants of the
insulin, are thus included as being of use.
[0134] Conservative amino acid substitution tables providing
functionally similar amino acids are well known to one of ordinary
skill in the art. The following six groups are examples of amino
acids that are considered to be conservative substitutions for one
another:
[0135] 1) Alanine (A), Serine (S), Threonine (T);
[0136] 2) Aspartic acid (D), Glutamic acid (E);
[0137] 3) Asparagine (N), Glutamine (Q);
[0138] 4) Arginine (R), Lysine (K);
[0139] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0140] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0141] In some circumstances, variations in the cDNA sequence that
result in amino acid changes, whether conservative or not, are
minimized in order to preserve the functional and immunologic
identity of the encoded protein. The immunologic identity of the
protein may be assessed by determining whether it is recognized by
an antibody; a variant that is recognized by such an antibody is
immunologically conserved. Any cDNA sequence variant will
preferably introduce no more than twenty, and preferably fewer than
ten amino acid substitutions into the encoded polypeptide. Variant
amino acid sequences may, for example, be 80%, 90%, or even 95% or
98% identical to the native amino acid sequence. Programs and
algorithms for determining percentage identity can be found at the
National Center for Biotechnology Information web site.
[0142] Preventing, treating or ameliorating a disease: "Preventing"
a disease refers to inhibiting the full development of a disease.
"Treating" refers to a therapeutic intervention that ameliorates a
sign or symptom of a disease or pathological condition after it has
begun to develop. "Ameliorating" refers to the reduction in the
number or severity of signs or symptoms of a disease.
[0143] Purified: The term purified does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified antigen is one in which the specified antigen
is more enriched than it is in its generative environment, for
instance within a cell extract. Preferably, a preparation of a
specified antigen is purified such that the antigen represents at
least 75% of the total content of the preparation. In some
embodiments, a purified preparation contains at least 80%, at least
85%, at least 90%, at least 95%, at least 99% or more of the
specified antigen. As used herein, "purified" antigens include
recombinantly produced antigens.
[0144] Rapamycin: A small molecule with known immunosuppressive and
anti-proliferative properties. Rapamycin, also known as sirolimus,
is a macrolide that was first discovered as a product of the
bacterium Streptomyces hygroscopicus. Rapamycin binds and inhibits
the activity of mTOR. The chemical formula of rapamycin is
C.sub.51H.sub.79NO.sub.13 and the International Union of Pure and
Applied Chemistry (IUPAC) name is
(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,-
21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[-
(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6-
,
8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]-oxaazacyclohe-
ntriacontine-1,5,11,28,29(4H,6H,31H)-pentone.
[0145] Ribozyme: A catalytic RNA molecule. In some cases, ribozymes
can bind to specific sites on other RNA molecules and catalyze the
hydrolysis of phosphodiester bonds in the RNA molecules.
[0146] RNA interference (RNAi): Refers to a cellular process that
inhibits expression of genes, including cellular and viral genes.
RNAi is a form of antisense-mediated gene silencing involving the
introduction of double stranded RNA-like oligonucleotides leading
to the sequence-specific reduction of RNA transcripts.
Double-stranded RNA molecules that inhibit gene expression through
the RNAi pathway include siRNAs, miRNAs, and shRNAs.
[0147] Sample: As used herein, a "sample" obtained from a subject
refers to a cell, fluid or tissue sample. Bodily fluids include,
but are not limited to, blood, serum, urine, saliva and spinal
fluid. Cell samples include, for example, PBMCs, white blood cells,
lymphocytes, or other cells of the immune system.
[0148] Short hairpin RNA (shRNA): A sequence of RNA that makes a
tight hairpin turn and can be used to silence gene expression via
the RNAi pathway. The shRNA hairpin structure is cleaved by the
cellular machinery into siRNA.
[0149] Small interfering RNA (siRNA): A double-stranded nucleic
acid molecule that modulates gene expression through the RNAi
pathway. siRNA molecules are generally 20-25 nucleotides in length
with 2-nucleotide overhangs on each 3' end. However, siRNAs can
also be blunt ended. Generally, one strand of a siRNA molecule is
at least partially complementary to a target nucleic acid, such as
a target mRNA. siRNAs are also referred to as "small inhibitory
RNAs."
[0150] Small molecule inhibitor: A molecule, typically with a
molecular weight less than about 1000 Daltons, or in some
embodiments, less than about 500 Daltons, wherein the molecule is
capable of inhibiting, to some measurable extent, an activity of a
target molecule.
[0151] Subject: Living multi-cellular vertebrate organisms, a
category that includes both human and veterinary subjects,
including human and non-human mammals.
[0152] Therapeutically effective amount: A quantity of a specific
substance sufficient to achieve a desired effect in a subject being
treated. For instance, this can be the amount of an mTOR inhibitor
necessary to enhance a T cell response.
[0153] Tumor antigen: A tumor antigen is an antigen produced by
tumor cells that can stimulate tumor-specific T-cell immune
responses. Exemplary tumor antigens include, but are not limited
to, RAGE-1, tyrosinase, MAGE-1, MAGE-2, NY-ESO-1, Melan-A/MART-1,
glycoprotein (gp) 75, gp100, beta-catenin, preferentially expressed
antigen of melanoma (PRAME), MUM-1, Wilms tumor (WT)-1,
carcinoembryonic antigen (CEA), and PR-1. Additional tumor antigens
are known in the art (for example see Novellino et al., Cancer
Immunol. Immunother. 54(3):187-207, 2005) and are described below.
Tumor antigens are also referred to as "cancer antigens."
[0154] Tumor, cancer, neoplasia or malignancy: The result of
abnormal and uncontrolled growth of cells. Neoplasia, malignancy,
cancer and tumor are often used interchangeably and refer to
abnormal growth of a tissue or cells that results from excessive
cell division. Hematological cancers are cancers of the blood or
bone marrow. Examples of hematological tumors include leukemias,
including acute leukemias (such as acute lymphocytic leukemia,
acute myelocytic leukemia, acute myelogenous leukemia and
myeloblastic, promyelocytic, myelomonocytic, monocytic and
erythroleukemia), chronic leukemias (such as chronic myelocytic
(granulocytic) leukemia, chronic myelogenous leukemia, and chronic
lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's
disease, non-Hodgkin's lymphoma (indolent and high grade forms),
multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain
disease, myelodysplastic syndrome, hairy cell leukemia and
myelodysplasia.
[0155] Solid tumors are abnormal masses of tissue that usually do
not contain cysts or liquid areas and are named for the type of
cells that form them. Examples of solid tumors, such as sarcomas
and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, lymphoid malignancy, pancreatic cancer, breast
cancer, lung cancers, ovarian cancer, prostate cancer,
hepatocellular carcinoma, squamous cell carcinoma, basal cell
carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid
carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous
gland carcinoma, papillary carcinoma, papillary adenocarcinomas,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,
cervical cancer, testicular tumor, seminoma, bladder carcinoma,
melanoma, and CNS tumors (such as a glioma, astrocytoma,
medulloblastoma, craniopharyogioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,
neuroblastoma and retinoblastoma).
[0156] Vaccine: A preparation of immunogenic material capable of
stimulating an immune response, administered for the prevention,
amelioration, or treatment of infectious or other types of disease,
such as cancer. The immunogenic material may include
live-attenuated or killed microorganisms (such as bacteria or
viruses), or antigenic proteins, peptides or DNA derived from them.
In some cases, the vaccine is a subunit vaccine, which is an
immunizing agent that has been treated to remove traces of nucleic
acid (such as viral nucleic acid) so that only protein subunits
remain. The subunits have less risk of causing adverse reactions.
The vaccine can also be a live vaccine, which is a vaccine prepared
from living attenuated organisms or from viruses that have been
attenuated but can still replicate in the cells of the host
organism.
[0157] The immunogenic material for a cancer vaccine may include,
for example, a protein or peptide expressed by a tumor or cancer
cell. Vaccines may elicit both prophylactic (preventative) and
therapeutic responses. Methods of administration vary according to
the vaccine, but may include inoculation, ingestion, inhalation or
other forms of administration.
[0158] Unless otherwise explained, 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 disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. Hence "comprising A or B" means including A,
or B, or A and B. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present disclosure, suitable
methods and materials are described below. All publications, patent
applications, patents, GENBANK.TM. Accession numbers and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
explanations of terms, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
III. Overview of Several Embodiments
[0159] There are three phases of a virus-specific CD8.sup.+ T cell
response after virus infection (FIG. 1). First, naive
virus-specific CD8.sup.+ T cells exponentially expand and become
effector T cells. These effector T cells stop proliferating
approximately 1 to 2 weeks after infection and enter the
contraction phase. During the contraction phase, effector CD8.sup.+
T cells gradually acquire memory T cell phenotype and function.
[0160] Disclosed herein is the surprising finding that treatment
with an mTOR inhibitor enhances T cell immune responses. To enhance
antigen-specific T cell immune responses in a subject, an mTOR
inhibitor is administered during the contraction phase of a T cell
response, or is administered at any time prior to or subsequent to
antigen challenge when administered at a low dose. Thus, provided
herein is a method of enhancing an antigen-specific T cell response
in a subject exposed to an antigen by administering to the subject
a therapeutically effective amount of an mTOR inhibitor, thereby
enhancing a T cell immune response in a subject.
[0161] The subject in need of treatment can be any subject exposed
to an antigen, such as antigen from a pathogen during a viral,
bacterial, fungal or parasitic infection. In some cases, the
subject in need of treatment is a subject with a tumor who is
exposed to a tumor antigen expressed by the tumor or cancer cells.
The subject in need of treatment can also be exposed to an antigen
that is a component of a vaccine, such as for prophylactic
treatment of a disease (including, for example, an infectious
disease or cancer).
[0162] In particular embodiments, provided herein is a method of
enhancing an antigen-specific T cell response in a subject in need
of treatment by administering to the subject a therapeutically
effective amount of an an antigen and a therapeutically effective
amount of an mTOR inhibitor, thereby enhancing a T cell immune
response in a subject.
[0163] In some embodiments, the T cells are CD8.sup.+ T cells or
CD4.sup.+ T cells, or both. The CD8.sup.+ or CD4.sup.+ T cells can
be effector T cells or memory T cells.
[0164] In some embodiments, enhancing an antigen-specific T cell
response in a subject exposed to an antigen includes increasing the
number of CD8.sup.+ T cells, enhancing the quality of CD8.sup.+ T
cells, or both.
[0165] In some embodiments, the CD8.sup.+ T cells are CD8.sup.+
effector T cells. In some embodiments, enhancing the quality of
CD8.sup.+ effector T cells is characterized by an increase in the
proportion of antigen-specific CD127.sup.HighKLRG-1.sup.Low
CD8.sup.+ T cells in a subject relative to a control, such as the
proportion of antigen-specific CD127.sup.HighKLRG-1.sup.Low
CD8.sup.+ T cells prior to or in the absence of treatment with an
mTOR inhibitor. The control can also be a historical or reference
value.
[0166] In some embodiments, the CD8.sup.+ T cells are CD8.sup.+
memory T cells. In some embodiments, enhancing the quality of
CD8.sup.+ memory T cells is characterized by an increase in
expression of CD127, an increase in expression of CD62L, an
increase in expression of Bcl-2, an increase in expression of CD27,
a decrease in expression of KLRG, or a combination thereof. In some
examples, a decrease in expression of a T cell marker is a decrease
of at least about 10%, at least about 20%, at least about 30%, at
least about 40%, or at least about 50%. In some examples, an
increase in expression of a T cell marker is an increase of at
least about 10%, at least about 20%, at least about 30%, at least
about 40%, or at least about 50%. The increase or decrease in
expression of the T cell marker is relative to a control, such as
expression level prior to or in the absence of treatment with an
mTOR inhibitor. The control can also be a historical or reference
value.
[0167] In some embodiments, enhancing an antigen-specific T cell
response comprises increasing the number of CD4.sup.+ T cells,
enhancing the quality of CD4.sup.+ T cells, or both. In some
embodiments, the CD4.sup.+ T cells are CD4.sup.+ memory T
cells.
[0168] In some embodiments, the antigen administered to the subject
is a component of a vaccine.
[0169] The antigen can be any type of antigen against which an
immune response is desired, such as an antigen from a pathogen, or
a tumor antigen or antigen that is part of a vaccine. Accordingly,
a subject can be exposed to an antigen, such as occurs during an
infection with a pathogen or with development of cancer, or a
subject can be administered the antigen, such as by prophylactic or
therapeutic immunization with a vaccine. In some embodiments, the
antigen is from a pathogen, such as, but not limited to a virus,
bacterium, fungus or parasite. The antigen from the pathogen is any
protein or other molecule capable of eliciting an immune response
in a subject exposed to the antigen. In some examples, the antigen
is a virus, such as human immunodeficiency virus (HIV) or hepatitis
B virus (HBV). In some embodiments, the subject has an acute
infection. For example, influenza viruses and rhinoviruses
typically cause acute infections. In other embodiments, the subject
has a chronic infection. Examples of chronic infections include,
but are not limited to, hepatitis C virus infection (HCV) and HIV
infection.
[0170] In some embodiments, the antigen is a tumor antigen. In one
embodiment, the tumor is a hematologic cancer. In some examples,
the hematologic cancer is leukemia or lymphoma, such as lymphocytic
leukemia, myelogenous leukemia, myelocytic leukemia, Hodgkin's
disease, non-Hodgkin's lymphoma and multiple myeloma. In another
embodiment, the tumor is a solid tumor. In some examples, the solid
tumor is a carcinoma, melanoma, sarcoma or central nervous system
tumor. Examples of solid tumors include, but are not limited to
hepatocellular carcinoma, malignant melanoma, colon cancer,
pancreatic cancer, breast cancer, lung cancers, ovarian cancer,
prostate cancer and retinoblastoma.
[0171] In some embodiments of the methods of enhancing a T cell
response in a subject, the subject is naturally exposed to the
antigen such as via an infection with a pathogen, or due to the
development of a tumor. In these cases, the mTOR inhibitor is
generally administered following exposure to the antigen to augment
T cell immune responses in the exposed subject.
[0172] In some embodiments, the method of an enhancing a T cell
response in a subject further comprises administering a vaccine to
the subject. In one embodiment, the vaccine is a vaccine against a
pathogen, such as a virus. In another embodiment, the vaccine is a
cancer vaccine.
[0173] In some embodiments, the mTOR inhibitor is administered
prior to administration of the antigen (such as by administration
of a vaccine). In one embodiment, the mTOR inhibitor is
administered up to three days prior to administration of the
antigen. In another embodiment, the mTOR inhibitor is administered
up to one day prior to administration of the antigen.
[0174] In some embodiments, the mTOR inhibitor is administered
after administration of the antigen. In one embodiment, the mTOR
inhibitor is administered up to 20 days following administration of
the antigen. In some examples, the mTOR inhibitor is administered 7
to 20 days following administration of the antigen. In other
examples, the mTOR inhibitor is administered 10 to 15 days
following administration of the antigen. In some embodiments, the
mTOR inhibitor is administered on the same day as the antigen,
including, but not limited to, within 5 minutes, within 10 minutes,
or within 15 minutes of administration of the antigen. In some
examples, the mTOR inhibitor is administered simultaneously, such
as within 0 to 5 minutes of administration of the vaccine. In other
examples, the mTOR inhibitor is administered within 5 to 15 minutes
of administration of the antigen.
[0175] In some embodiment, the mTOR inhibitor is administered in a
single dose. In other embodiments, the mTOR inhibitor is
administered in multiple doses. In some examples, the mTOR
inhibitor is administered in 1 to 40 doses, such as 5 to 30 doses,
10 to 25 doses, or 15 to 20 doses. When administered in multiple
doses, the mTOR inhibitor can be administered prior to, on the same
day as, or following administration of the antigen, or a
combination thereof. For example, a subject can be administered the
mTOR inhibitor daily for three days prior to administration of the
antigen and daily for one week following immunization. As another
example, a subject can be administered the mTOR inhibitor on the
same day as the antigen and then administered the mTOR inhibitor
daily for up to one week. In some embodiments, the mTOR inhibitor
is administered daily. In some examples, the mTOR inhibitor is
administered daily for one week. In other embodiments, the mTOR
inhibitor is administered weekly.
[0176] In some embodiments, the mTOR inhibitor is administered
continuously, such as part of a patch or other transdermal delivery
means.
[0177] In some embodiments, the mTOR inhibitor is rapamycin or a
rapamycin analog. In one embodiment, the dose of rapamycin is about
0.2 to about 1.0 mg/kg, such as about 0.4 to about 0.8 mg/kg. In
some examples, the dose of rapamycin is about 0.2, about 0.3, about
0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.0 or about
1.0 mg/kg. In another embodiment, the dose of rapamycin is a low
dose of rapamycin. In one embodiment, the low dose of rapamycin is
about 0.01 to about 0.15 mg/kg, such as about 0.05 to about 0.1
mg/kg. In some examples, a low dose of rapamycin is about 0.01,
about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about
0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12,
about 0.13, about 0.14 or about 0.15 mg/kg. In this context,
"about" refers to a value within 0.005 mg/kg.
[0178] When a low dose of rapamycin (or another mTOR inhibitor) is
used, administration can occur at any time relative to exposure to
or administration of the antigen. When a higher dose of inhibitor
is used, it is typically effective when delivered to a subject
after exposure to the antigen, such as up to about 10, up to about
15 days or up to about 20 days following exposure, which correlates
with the T cell contraction phase of an immune response in humans
(Miller et al., Immunity 28(5):710-722, 2008). In some examples,
the higher dose of rapamycin is administered 7 to 20 days following
exposure to or administration of antigen.
[0179] In some embodiments, the method further comprises measuring
the number of antigen-specific T cells in a sample obtained from
the subject. In other embodiments, the method further comprises
measuring the expression of one or more of CD127, CD62L, Bcl-2,
CD27 and KLRG-1 in T cells from a sample obtained from the
subject.
[0180] Also provided herein is a method of increasing the
proportion of antigen-specific CD127.sup.HighKLRG-1.sup.Low
CD8.sup.+ T cells in a subject, comprising administering to the
subject a therapeutically effective amount of an antigen and an
mTOR inhibitor, thereby increasing the proportion of
antigen-specific CD127.sup.HighKLRG-1.sup.Low CD8.sup.+ T cells in
the subject. In some embodiments, an increase in the proportion of
antigen-specific CD127.sup.HighKLRG-1.sup.Low CD8.sup.+ T cells is
relative to the proportion of CD127.sup.HighKLRG-1.sup.Low
CD8.sup.+ T cells in the absence of treatment. In some embodiments,
the subject has an acute or chronic infection, or has a tumor.
[0181] In some examples, the method of increasing the proportion of
antigen-specific CD127.sup.HighKLRG-1.sup.Low CD8.sup.+ T cells in
a subject exposed to an antigen, comprises (i) selecting a subject
in need of treatment; (ii) administering to the subject an mTOR
inhibitor; and (iii) measuring the proportion of antigen-specific
CD127.sup.HighKLRG-1.sup.Low CD8.sup.+ T cells in the subject
relative to the proportion of CD127.sup.HighKLRG-1.sup.Low
CD8.sup.+ T cells in the absence of treatment.
[0182] Further provided is a method of increasing expression of
CD127, CD62L, Bcl-2 and CD27, and decreasing expression of KLRG-1,
in CD8.sup.+ T cells of a subject, comprising administering to the
subject a therapeutically effective amount of an antigen and an
mTOR inhibitor, thereby increasing expression of CD127, CD62L,
Bcl-2 and CD27, and decreasing expression of KLRG-1, in CD8.sup.+ T
cells of the subject. In some embodiments, the increase or decrease
in expression of the T cell markers is relative to expression in
the absence of treatment. In some embodiments, the subject has an
acute or chronic infection, or has a tumor.
[0183] In some examples, the method of increasing expression of
CD127, CD62L, Bcl-2 and CD27, and decreasing expression of KLRG-1,
in CD8.sup.+ T cells of a subject exposed to an antigen comprises
(i) selecting a subject in need of treatment; (ii) administering to
the subject an mTOR inhibitor; and (iii) measuring expression of
CD127, CD62L, Bcl-2, CD27 and KLRG-1 in CD8.sup.+ T cells of a
subject relative to expression in the absence of treatment.
[0184] Also provided is the use of an mTOR inhibitor and a vaccine
comprising an antigen in the manufacture of a medicament for
enhancing an antigen-specific T cell response in a subject, wherein
enhancing an antigen-specific T cell response in a subject
comprises increasing the number of antigen-specific T cells or
enhancing the quality of antigen-specific T cells in the
subject.
[0185] Also provided herein are compositions comprising an mTOR
inhibitor and an antigen and/or a vaccine. In some embodiments, the
vaccine is a live vaccine. In some embodiments, the vaccine is a
subunit vaccine. In some embodiments, the compositions comprise an
mTOR inhibitor, purified antigen and an adjuvant. The antigen can
be any antigen, such as an antigen from a pathogen, a tumor antigen
or a vaccine antigen. Suitable antigens are described herein and
are well known in the art. In some embodiments, the compositions
provided herein further comprise a pharmaceutically acceptable
carrier. Further provided is the use of such compositions in the
manufacture of a medicament for enhancing an antigen-specific T
cell response in a subject.
IV. Mammalian Target of Rapamycin (mTOR) Inhibitors
[0186] Inhibitors of mTOR for use with the methods claimed herein
can be any type of molecule that inhibits expression or activity of
mTOR. For example, mTOR inhibitors, include, but are not limited to
small molecules, synthetic compounds, antibodies, peptides and
nucleic acids (including, for example, antisense oligonucleotides,
small interfering RNA (siRNA), short hairpin RNA, microRNA,
ribozymes and the like).
[0187] A. Small Molecule Inhibitors
[0188] A number of small molecule mTOR inhibitors are known, some
of which are currently being used to treat a variety of diseases.
In addition, a number of mTOR inhibitors are under investigation in
clinical trials for treating of diseases such as cancer. The best
characterized mTOR inhibitor is rapamycin, a naturally occurring
small molecule with known immunosuppressive and anti-proliferative
properties. Rapamycin, also known as sirolimus, is a macrolide that
was first discovered as a product of the bacterium Streptomyces
hygroscopicus. Rapamycin binds and inhibits the activity of mTOR.
Rapamycin is also marketed under the name RAPAMUNE.TM.. Provided in
Table 1 below is a list of some of the mTOR inhibitors currently
being tested in clinical trials.
TABLE-US-00001 TABLE 1 mTOR inhibitors under evaluation in clinical
trials Compound Company Description RAD001 Novartis Orally
available derivative of rapamycin OSI-027 OSI Pharmaceuticals
Inhibits the kinase activity associated with both the mTORC1 and
mTORC2 complexes AP23573 Ariad Pharmaceuticals Rapamycin analog
AP23675 Ariad Pharmaceuticals Rapamycin analog AP23841 Ariad
Pharmaceuticals Rapamycin analog ABI-009 Abraxis Bioscience Inc.
mTOR inhibitor MK8669 Merck & Co. mTOR inhibitor TOP216
Topotarget A/S mTOR inhibitor TAFA93 Isotechnika Inc. Prodrug of
rapamycin TORISEL .TM. Wyeth Pharmaceuticals mTOR inhibitor
CERTICAN .TM. Novartis AG mTOR inhibitor
[0189] Additional mTOR inhibitors, including rapamycin derivatives
and analogs have been described, such as, for example, those
disclosed in PCT Publication Nos. WO 2007/135411, WO 98/02441, WO
01/14387 and WO 03/64383; and European Patent No. EP1880723.
[0190] B. Antisense Compounds
[0191] In addition to small molecule inhibitors, antisense
compounds that specifically target and down-regulate expression of
mTOR can be used with the methods provided herein. Generally, the
principle behind antisense technology is that an antisense compound
hybridizes to a target nucleic acid and effects the modulation of
gene expression activity, or function, such as transcription,
translation or splicing. The modulation of gene expression can be
achieved by, for example, target RNA degradation or occupancy-based
inhibition. An example of modulation of target RNA function by
degradation is RNase H-based degradation of the target RNA upon
hybridization with a DNA-like antisense compound, such as an
antisense oligonucleotide. Antisense oligonucleotides can also be
used to modulate gene expression, such as splicing, by
occupancy-based inhibition, such as by blocking access to splice
sites.
[0192] Another example of modulation of gene expression by target
degradation is RNA interference (RNAi) using small interfering RNAs
(siRNAs). RNAi is a form of antisense-mediated gene silencing
involving the introduction of double stranded RNA-like
oligonucleotides leading to the sequence-specific reduction of
targeted endogenous mRNA levels. Another type of antisense compound
that utilizes the RNAi pathway is a microRNA. MicroRNAs are
naturally occurring RNAs involved in the regulation of gene
expression. However, these compounds can be synthesized to regulate
gene expression via the RNAi pathway. Similarly, short hairpin RNAs
(shRNAs) are RNA molecules that form a tight hairpin turn and can
be used to silence gene expression via the RNAi pathway. The shRNA
hairpin structure is cleaved by the cellular machinery into
siRNA.
[0193] Other compounds that are often classified as antisense
compounds are ribozymes. Ribozymes are catalytic RNA molecules that
can bind to specific sites on other RNA molecules and catalyze the
hydrolysis of phosphodiester bonds in the RNA molecules. Ribozymes
modulate gene expression by direct cleavage of a target nucleic
acid, such as a messenger RNA.
[0194] Each of the above-described antisense compounds provides
sequence-specific target gene regulation. This sequence-specificity
makes antisense compounds effective tools for the selective
modulation of a target nucleic acid of interest, such as mTOR.
[0195] Any type of antisense compound that specifically targets and
regulates expression of mTOR is contemplated for use with the
disclosed methods. Such antisense compounds include single-stranded
compounds, such as antisense oligonucleotides, and double-stranded
compounds, including compounds with at least partial
double-stranded structure, including siRNAs, miRNAs, shRNAs and
ribozymes. Methods of designing, preparing and using antisense
compounds that specifically target mTOR are within the abilities of
one of skill in the art.
[0196] Furthermore, sequences for mTOR are publicly available.
Exemplary human mTOR nucleotide sequences are provided herein as
SEQ ID NO: 3 (GENBANK.TM. Accession No. NM_004958, deposited on
Apr. 4, 2002) and SEQ ID NO: 5 (GENBANK.TM. Accession No. BC117166,
deposited on Jun. 26, 2006). Antisense compounds specifically
targeting mTOR can be prepared by designing compounds that are
complementary to an mTOR nucleotide sequence, particularly the mTOR
mRNA sequence. Antisense compounds targeting mTOR need not be 100%
complementary to mTOR to specifically hybridize and regulate
expression the target gene. For example, the antisense compound, or
antisense strand of the compound if a double-stranded compound, can
be at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 99% or 100% complementary to the selected mTOR
nucleic acid sequence. Methods of screening antisense compounds for
specificity are well known in the art (see, for example, U.S.
Patent Application Publication No. 2003-0228689). Exemplary mTOR
shRNA sequences are provided herein as SEQ ID NOs: 10 and 11.
[0197] C. Antibodies Specific for mTOR
[0198] An mTOR polypeptide or a fragment or conservative variant
thereof can be used to produce antibodies which are immunoreactive
or specifically bind to an epitope of an mTOR. Polyclonal
antibodies, antibodies which consist essentially of pooled
monoclonal antibodies with different epitopic specificities, as
well as distinct monoclonal antibody preparations are included.
[0199] The preparation of polyclonal antibodies is well known to
those skilled in the art. See, for example, Green et al.,
"Production of Polyclonal Antisera," in: Immunochemical Protocols,
pages 1-5, Manson, ed., Humana Press, 1992; Coligan et al.,
"Production of Polyclonal Antisera in Rabbits, Rats, Mice and
Hamsters," in: Current Protocols in Immunology, section 2.4.1,
1992.
[0200] The preparation of monoclonal antibodies likewise is
conventional (see, for example, Kohler & Milstein, Nature
256:495, 1975; Coligan et al., sections 2.5.1-2.6.7; and Harlow et
al. in: Antibodies: a Laboratory Manual, page 726, Cold Spring
Harbor Pub., 1988). Briefly, monoclonal antibodies can be obtained
by injecting mice with a composition comprising an antigen,
verifying the presence of antibody production by removing a serum
sample, removing the spleen to obtain B lymphocytes, fusing the B
lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones that produce antibodies to
the antigen, and isolating the antibodies from the hybridoma
cultures. Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography (see, e.g., Coligan et al., sections
2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al., Purification
of Immunoglobulin G (IgG), in: Methods in Molecular Biology, Vol.
10, pages 79-104, Humana Press, 1992).
[0201] Methods of in vitro and in vivo multiplication of monoclonal
antibodies are well known to those skilled in the art.
Multiplication in vitro may be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium,
optionally supplemented by a mammalian serum such as fetal calf
serum or trace elements and growth-sustaining supplements such as
normal mouse peritoneal exudate cells, spleen cells, thymocytes or
bone marrow macrophages. Production in vitro provides relatively
pure antibody preparations and allows scale-up to yield large
amounts of the desired antibodies. Large-scale hybridoma
cultivation can be carried out by homogenous suspension culture in
an airlift reactor, in a continuous stirrer reactor, or in
immobilized or entrapped cell culture. Multiplication in vivo may
be carried out by injecting cell clones into mammals
histocompatible with the parent cells, such as syngeneic mice, to
cause growth of antibody-producing tumors. Optionally, the animals
are primed with a hydrocarbon, especially oils such as pristane
(tetramethylpentadecane) prior to injection. After one to three
weeks, the desired monoclonal antibody is recovered from the body
fluid of the animal.
[0202] Antibodies can also be derived from a subhuman primate
antibody. General techniques for raising therapeutically useful
antibodies in baboons can be found, for example, in PCT Publication
No. WO 91/11465, 1991; and Losman et al., Int. J. Cancer 46:310,
1990.
[0203] Alternatively, an antibody that specifically binds an mTOR
polypeptide can be derived from a humanized monoclonal antibody.
Humanized monoclonal antibodies are produced by transferring
complementarity determining regions from another species such as a
mouse from heavy and light variable chains of the immunoglobulin
into a human variable domain, and then substituting human residues
in the framework regions of the murine counterparts. The use of
antibody components derived from humanized monoclonal antibodies
obviates potential problems associated with the immunogenicity of
murine constant regions. General techniques for cloning murine
immunoglobulin variable domains are described, for example, by
Orlandi et al., Proc. Natl. Acad. Sci. U.S.A. 86:3833, 1989.
Techniques for producing humanized monoclonal antibodies are
described, for example, by Jones et al., Nature 321:522, 1986;
Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science
239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci. U.S.A.
89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer
et al., J. Immunol. 150:2844, 1993.
[0204] Antibodies can be derived from human antibody fragments
isolated from a combinatorial immunoglobulin library. See, for
example, Barbas et al., in: Methods: a Companion to Methods in
Enzymology, Vol. 2, page 119, 1991; Winter et al., Ann. Rev.
Immunol. 12:433, 1994. Cloning and expression vectors that are
useful for producing a human immunoglobulin phage library can be
obtained, for example, from STRATAGENE Cloning Systems (La Jolla,
Calif.).
[0205] In addition, antibodies can be derived from a human
monoclonal antibody. Such antibodies can be obtained from
transgenic mice that have been "engineered" to produce specific
human antibodies in response to antigenic challenge. In this
technique, elements of the human heavy and light chain loci are
introduced into strains of mice derived from embryonic stem cell
lines that contain targeted disruptions of the endogenous heavy and
light chain loci. The transgenic mice can synthesize human
antibodies specific for human antigens, and the mice can be used to
produce human antibody-secreting hybridomas. Methods for obtaining
human antibodies from transgenic mice are described by Green et
al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856,
1994; and Taylor et al., Int. Immunol. 6:579, 1994.
[0206] Antibodies include intact molecules as well as fragments
thereof, such as Fab, F(ab')2, and Fv which are capable of binding
the epitopic determinant. These antibody fragments retain some
ability to selectively bind with their antigen or receptor and are
defined as follows:
[0207] (1) Fab, the fragment which contains a monovalent
antigen-binding fragment of an antibody molecule, can be produced
by digestion of whole antibody with the enzyme papain to yield an
intact light chain and a portion of one heavy chain;
[0208] (2) Fab', the fragment of an antibody molecule can be
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain; two Fab' fragments are obtained per antibody
molecule;
[0209] (3) (Fab').sub.2, the fragment of the antibody that can be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held
together by two disulfide bonds;
[0210] (4) Fv, defined as a genetically engineered fragment
containing the variable region of the light chain and the variable
region of the heavy chain expressed as two chains; and
[0211] (5) Single chain antibody (SCA), defined as a genetically
engineered molecule containing the variable region of the light
chain, the variable region of the heavy chain, linked by a suitable
polypeptide linker as a genetically fused single chain
molecule.
[0212] Methods of making these fragments are known in the art (see
for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988). An epitope is any
antigenic determinant on an antigen to which the paratope of an
antibody binds. Epitopic determinants usually consist of chemically
active surface groupings of molecules such as amino acids or sugar
side chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics.
[0213] Antibody fragments can be prepared by proteolytic hydrolysis
of the antibody or by expression in E. coli of DNA encoding the
fragment. Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
This fragment can be further cleaved using a thiol reducing agent,
and optionally a blocking group for the sulfhydryl groups resulting
from cleavage of disulfide linkages, to produce 3.5S Fab'
monovalent fragments. Alternatively, an enzymatic cleavage using
pepsin produces two monovalent Fab' fragments and an Fc fragment
directly (see U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647,
and references contained therein; Nisonhoff et al., Arch. Biochem.
Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et
al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967;
and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).
[0214] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0215] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association may be noncovalent (Inbar et
al., Proc. Natl. Acad. Sci. U.S.A. 69:2659, 1972). Alternatively,
the variable chains can be linked by an intermolecular disulfide
bond or cross-linked by chemicals such as glutaraldehyde (see, for
example, Sandhu, Crit. Rev. Biotech. 12:437, 1992). Preferably, the
Fv fragments comprise V.sub.H and V.sub.L chains connected by a
peptide linker. These single-chain antigen binding proteins (sFv)
are prepared by constructing a structural gene comprising DNA
sequences encoding the V.sub.H and V.sub.L domains connected by an
oligonucleotide. The structural gene is inserted into an expression
vector, which is subsequently introduced into a host cell such as
E. coli. The recombinant host cells synthesize a single polypeptide
chain with a linker peptide bridging the two V domains. Methods for
producing sFvs are known in the art (see Whitlow et al., Methods: a
Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et
al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack et al.,
Bio/Technology 11:1271, 1993; and Sandhu, supra).
[0216] Antibodies can be prepared using an intact polypeptide or
fragments containing small peptides of interest as the immunizing
antigen. The polypeptide or a peptide used to immunize an animal
can be derived from substantially purified polypeptide produced in
host cells, in vitro translated cDNA, or chemical synthesis which
can be conjugated to a carrier protein, if desired. Such commonly
used carriers which are chemically coupled to the peptide include
keyhole limpet hemocyanin, thyroglobulin, bovine serum albumin, and
tetanus toxoid. The coupled peptide is then used to immunize the
animal (e.g., a mouse, a rat, or a rabbit).
[0217] Polyclonal or monoclonal antibodies can be further purified,
for example, by binding to and elution from a matrix to which the
polypeptide or a peptide to which the antibodies were raised is
bound. Those of skill in the art will know of various techniques
common in the immunology arts for purification and/or concentration
of polyclonal antibodies, as well as monoclonal antibodies (see,
for example, Coligan et al., Unit 9, Current Protocols in
Immunology, Wiley Interscience, 1991).
V. Antigens and Vaccines
[0218] As described herein, administration of an mTOR inhibitor in
conjunction with exposure to an antigen or administration of a
vaccine, enhances antigen-specific T cell immune responses. The
antigen can be any type of antigen against which an immune response
is desired in a subject, or any antigen to which a subject is
exposed. In some cases, a subject is exposed to the antigen during
an infection, such as a viral, bacterial, fungal or parasitic
infection. In other cases, the subject has a tumor and is exposed
to a tumor-specific antigen. Alternatively, the antigen can be
administered to a subject, such as in the form of a vaccine. In
some embodiments, the vaccine is a vaccine against a pathogen, or a
cancer vaccine.
[0219] A. Antigens
[0220] In some embodiments, the antigen is an antigen from a
pathogen, such as a virus, bacterium, fungus or parasite. Viral
pathogens include, but are not limited to retroviruses, such as
human immunodeficiency virus (HIV) and human T-cell leukemia
viruses; picornaviruses, such as polio virus, hepatitis A virus;
hepatitis C virus, enteroviruses, human coxsackie viruses,
rhinoviruses, echoviruses, and foot-and-mouth disease virus;
caliciviruses, such as strains that cause gastroenteritis (e.g.,
Norwalk virus); togaviruses, such as alphaviruses (including
chikungunya virus, equine encephalitis viruses, Sindbis virus,
Semliki Forest virus, and Ross River virus) and rubella virus;
flaviviruses, such as dengue viruses, yellow fever viruses, West
Nile virus, St. Louis encephalitis virus, Japanese encephalitis
virus, Powassan virus and other encephalitis viruses;
coronaviruses, including severe acute respiratory syndrome (SARS)
virus; rhabdoviruses, such as vesicular stomatitis virus and rabies
virus; filoviruses, such as Ebola virus and Marburg virus);
paramyxoviruses, such as parainfluenza virus, mumps virus, measles
virus, and respiratory syncytial virus; orthomyxoviruses, such as
influenza viruses (including avian influenza viruses and swine
influenza viruses); bunyaviruses, such as Hantaan virus; Sin Nombre
virus, and Rift Valley fever virus, phleboviruses and Nairo
viruses; arenaviruses, such as Lassa fever virus and other
hemorrhagic fever viruses, Machupo virus and Junin virus;
reoviruses, such as mammalian reoviruses, orbiviurses and
rotaviruses; birnaviruses; hepadnaviruses, such as hepatitis B
virus; parvoviruses; papovaviruses, such as papilloma viruses,
polyoma viruses and BK-virus; adenoviruses; herpesviruses, such as
herpes simplex virus (HSV)-1 and HSV-2, cytomegalovirus,
Epstein-Barr virus, varicella zoster virus, and other herpes
viruses, including HSV-6); pox viruses, such as variola viruses and
vaccinia viruses; irodoviruses, such as African swine fever virus;
astroviruses; and unclassified viruses (for example, the
etiological agents of spongiform encephalopathies, the agent of
delta hepatitis (thought to be a defective satellite of hepatitis B
virus).
[0221] Bacterial pathogens include, but are not limited to
Helicobacter pylori, Escherichia coli, Vibrio cholerae, Borelia
burgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M.
tuberculosis, M. avium, M. intracellulare, M. kansai and, M.
gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria
meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group
A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans group), Streptococcus faecalis,
Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus
pneumoniae, pathogenic Campylobacter sp., Enterococcus sp.,
Haemophilus influenzae, Bacillus anthracis, Corynebacterium
diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,
Clostridium perfringers, Clostridium tetani, Enterobacter
aerogenes, Klebsiella pneumoniae, Pasteurella multocida,
Bacteroides sp., Fusobacterium nucleatum, Streptobacillus
moniliformis, Treponema pallidium, Treponema pertenue, Leptospira,
Bordetella pertussis, Shigella flexnerii, Shigella dysenteriae and
Actinomyces israelli.
[0222] Fungal pathogens include, but are not limited to
Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides
immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida
albicans. Parasitic pathogens include, but are not limited to
Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi and
Toxoplasma gondii.
[0223] In some cases, the antigen is a tumor-associated antigen.
Tumor antigens are proteins that are produced by tumor cells that
elicit an immune response, particularly T-cell mediated immune
responses. The tumor antigen can be any tumor-associated antigen,
which are well known in the art and include, for example,
carcinoembryonic antigen (CEA), .beta.-human chorionic
gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP,
thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse
transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut
hsp70-2, macrophage colony stimulating factor, prostase,
prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53,
prostein, PSMA, Her2/neu, survivin and telomerase,
prostate-carcinoma tumor antigen-1, MAGE, ELF2M, neutrophil
elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II,
IGF-I receptor and mesothelin. A list of selected tumor antigens
and their associated tumors are shown below in Table 2.
TABLE-US-00002 TABLE 2 Exemplary tumors and their tumor antigens
Tumor Tumor Associated Target Antigens Acute myelogenous leukemia
Wilms tumor 1 (WT1), PRAME, PR1, proteinase 3, elastase, cathepsin
G Chronic myelogenous leukemia WT1, PRAME, PR1, proteinase 3,
elastase, cathepsin G Myelodysplastic syndrome WT1, PRAME, PR1,
proteinase 3, elastase, cathepsin G Acute lymphoblastic leukemia
PRAME Chronic lymphocytic leukemia Survivin Non-Hodgkin's lymphoma
Survivin Multiple myeloma NY-ESO-1 Malignant melanoma MAGE, MART,
Tyrosinase, PRAME GP100 Breast cancer WT1, herceptin, epithelial
tumor antigen (ETA) Lung cancer WT1 Ovarian cancer CA-125 Prostate
cancer PSA Pancreatic cancer CA19-9, RCAS1 Colon cancer CEA Renal
cell carcinoma (RCC) Fibroblast growth factor 5 Germ cell tumors
AFP
[0224] In some embodiments, a subject is administered an mTOR
inhibitor following diagnosis of the subject (e.g. a diagnosis of
the presence of an infection or cancer). The mTOR inhibitor can be
administered in a single dose or in multiple doses over time. In
some examples, a subject having an infection or cancer is
administered an mTOR inhibitor daily for at least one week, at
least one month or at least three months.
[0225] B. Vaccines
[0226] In some embodiments, the antigen is delivered as part of a
vaccine. A number of vaccines against infectious diseases are
currently approved for use in the United States, examples of which
are listed below in Table 3.
TABLE-US-00003 TABLE 3 Approved Vaccines for Immunization and
Distribution in the U.S. Product Name Trade Name Anthrax Vaccine
Adsorbed BIOTHRAX BCG Vaccine TICE BCG BCG Vaccine MYCOBAX
Diphtheria & Tetanus Toxoids Adsorbed None Diphtheria &
Tetanus Toxoids Adsorbed None Diphtheria & Tetanus Toxoids
& Acellular Pertussis TRIPEDIA Vaccine Adsorbed Diphtheria
& Tetanus Toxoids & Acellular Pertussis INFANRIX Vaccine
Adsorbed Diphtheria & Tetanus Toxoids & Acellular Pertussis
DAPTACEL Vaccine Adsorbed Diphtheria & Tetanus Toxoids &
Acellular Pertussis PEDIARIX Vaccine Adsorbed, Hepatitis B
(recombinant) and Inactivated Poliovirus Vaccine Combined
Diphtheria and Tetanus Toxoids and Acellular KINRIX Pertussis
Adsorbed and Inactivated Poliovirus Vaccine Diphtheria and Tetanus
Toxoids and Acellular PENTACEL Pertussis Adsorbed, Inactivated
Poliovirus and Haemophilus b Conjugate (Tetanus Toxoid Conjugate)
Vaccine Haemophilus b Conjugate Vaccine (Diphtheria HIBTITER CRM197
Protein Conjugate) Haemophilus b Conjugate Vaccine (Meningococcal
PEDVAXHIB Protein Conjugate) Haemophilus b Conjugate Vaccine
(Tetanus Toxoid ACTHIB Conjugate) Haemophilus b Conjugate Vaccine
(Meningococcal COMVAX Protein Conjugate) & Hepatitis B Vaccine
(Recombinant) Hepatitis A Vaccine, Inactivated HAVRIX Hepatitis A
Vaccine, Inactivated VAQTA Hepatitis A Inactivated and Hepatitis B
TWINRIX (Recombinant) Vaccine Hepatitis B Vaccine (Recombinant)
RECOMBIVAX HB Hepatitis B Vaccine (Recombinant) ENGERIX-B Human
Papillomavirus (Types 6, 11, 16, 18) GARDASIL Recombinant Vaccine
Influenza Virus Vaccine AFLURIA Influenza Virus Vaccine, H5N1 None
Influenza Virus Vaccine, Trivalent, Types A and B FLULAVAL
Influenza Virus Vaccine, Live, Intranasal FLUMIST Influenza Virus
Vaccine, Trivalent, Types A and B FLUARIX Influenza Virus Vaccine,
Trivalent, Types A and B FLUVIRIN Influenza Virus Vaccine,
Trivalent, Types A and B FLUZONE Japanese Encephalitis Virus
Vaccine Inactivated JE-VAX Measles Virus Vaccine, Live ATTENUVAX
Measles and Mumps Virus Vaccine, Live M-M-Vax Measles, Mumps, and
Rubella Virus Vaccine, Live M-M-R II Measles, Mumps, Rubella and
Varicella Virus PROQUAD Vaccine, Live Meningococcal Polysaccharide
(Serogroups A, C, Y MENACTRA and W-135) Diphtheria Toxoid Conjugate
Vaccine Meningococcal Polysaccharide Vaccine, Groups A, MENOMUNE-
C, Y and W-135 Combined A/C/Y/W-135 Mumps Virus Vaccine Live
MUMPSVAX Plague Vaccine None Pneumococcal Vaccine, Polyvalent
PNEUMOVAX 23 Pneumococcal 7-valent Conjugate Vaccine PREVNAR
(Diphtheria CRM197 Protein) Poliovirus Vaccine Inactivated (Human
Diploid POLIOVAX Cell) Poliovirus Vaccine Inactivated (Monkey
Kidney IPOL Cell) Rabies Vaccine IMOVAX Rabies Vaccine RABAVERT
Rabies Vaccine Adsorbed No Trade Name Rotavirus Vaccine, Live, Oral
ROTARIX Rotavirus Vaccine, Live, Oral, Pentavalent ROTATEQ Rubella
Virus Vaccine Live MERUVAX II Smallpox (Vaccinia) Vaccine, Live
ACAM2000 Smallpox Vaccine, Dried, Calf Lymph Type DRYVAX Tetanus
& Diphtheria Toxoids Adsorbed for Adult None Use Tetanus &
Diphtheria Toxoids Adsorbed for Adult DECAVAC Use Tetanus &
Diphtheria Toxoids Adsorbed for Adult TENIVAC Use Tetanus Toxoid
None Tetanus Toxoid Adsorbed None Tetanus Toxoid Adsorbed None
Tetanus Toxoid, Reduced Diphtheria Toxoid and ADACEL Acellular
Pertussis Vaccine, Adsorbed Tetanus Toxoid, Reduced Diphtheria
Toxoid and BOOSTRIX Acellular Pertussis Vaccine, Adsorbed Typhoid
Vaccine Live Oral Ty21a VIVOTIF Typhoid Vi Polysaccharide Vaccine
TYPHIM VI Varicella Virus Vaccine Live VARIVAX Yellow Fever Vaccine
YF-VAX Zoster Vaccine, Live ZOSTAVAX
[0227] With the exception of the HPV and HBV vaccines that prevent
cervical cancer and liver cancer, respectively, as a result of
inhibiting virus infection, there are currently no cancer vaccines
approved for clinical use. However, a number of vaccine candidates
are being evaluated for a wide variety of different types of
cancer. For example, candidate tumor vaccines include, but are not
limited to, antigen/adjuvant vaccines (cancer-specific antigenic
protein fragments in combination with an adjuvant); whole-cell
tumor vaccines (tumor cells taken from a subject's own tumor or the
tumor of another patient); dendritic cell (DC) vaccines (DCs are
isolated from a patient, stimulated ex vivo with the patient's
cancer antigens and re-injected in the patient); DNA vaccines
(nucleic acids encoding the sequence of a tumor antigen); and
idiotype vaccines (antibodies specifically produced by a cancer
cell). Accordingly, mTOR inhibitors can be used in conjunction with
any such vaccine developed for eliciting immune responses against
cancer.
[0228] A subject to be vaccinated can be administered an mTOR
inhibitor prior to vaccination, at the same time as vaccination,
following vaccination, or a combination thereof. An mTOR inhibitor
can be administered in a single dose or multiple doses.
Administration of mTOR inhibitors is discussed in detail below.
VI. Administration of mTOR Inhibitors
[0229] Administration of an mTOR inhibitor in accordance with the
methods described herein can occur prior to, at the same time as,
or following exposure to or delivery of an antigen or vaccine. The
timing of administration depends in part on the dose of mTOR
inhibitor administered. Low doses of mTOR inhibitor, such as
rapamycin or a rapamycin analog, can be administered at any time
relative to exposure of an antigen. Higher doses of mTOR inhibitor
are more effective when delivered following exposure to an antigen
or delivery of a vaccine, such as up to about 10 days, up to about
15 days, or up to about 20 days following exposure/delivery, which
correlates with the T cell contraction phase of an immune response
(Miller et al., Immunity 28(5):710-722, 2008).
[0230] In some embodiments, the mTOR inhibitor is administered up
to three days prior to administration of a vaccine or exposure to
an antigen. In some embodiments, the mTOR inhibitor is administered
up to one day prior to administration of a vaccine or exposure to
an antigen.
[0231] In some embodiments, the mTOR inhibitor is administered on
the same day as the vaccine. As used herein, "on the same day"
refers to administration that occurs within 24 hours (either before
or after) administration of the vaccine. In some examples, the mTOR
inhibitor is administered at the same time as the vaccine, such as
within 0 to 5 minutes, within 5 to 10 minutes or within 10 to 15
minutes of administration of the vaccine. In some examples, the
mTOR inhibitor is administered within about 15 minutes to 1 hour of
administration of the vaccine. In other examples, the mTOR
inhibitor is administered within about 30 minutes to about 2 hours
of administration of the vaccine.
[0232] In some embodiments, the mTOR inhibitor is administered
following vaccination or exposure to an antigen. In some
embodiments, the mTOR inhibitor is administered up to 10 days, up
to 15 days or up to 20 days after vaccination or exposure to an
antigen. In some examples, the mTOR inhibitor is administered 7 to
20 days, or 10 to 15 days, following vaccination or exposure to
antigen.
[0233] As described above, administration of an mTOR inhibitor can
be accomplished by single or multiple doses. The dose administered
to a subject should be sufficient to induce a beneficial
therapeutic response (i.e. to establish sufficient immunological
memory) in a subject over time, such as preventing or inhibiting
infection by a pathogen, or inhibiting development or spread of a
tumor. A therapeutically effective dose can also be determined by
measuring the immune response, such as by detecting the number and
quality of antigen-specific T cells, such as CD8.sup.+ or CD4.sup.+
memory T cells. As used herein, "multiple doses" means two or more
doses, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35 or more doses. In some examples, the mTOR inhibitor is
administered in 1 to 40 doses, such as about 5 to 30 doses, about
10 to 25 doses or about 15 to 20 doses.
[0234] The dose required will vary from subject to subject
depending on the species, age, weight and general condition of the
subject, the severity of the disease or disorder being treated, the
particular composition being used and its mode of administration.
In some embodiments, the mTOR inhibitor is rapamycin and the dose
is about 0.01 to about 0.15 mg/kg, such as about 0.05 to about 0.1
mg/kg when rapamycin is administered prior to or concomitant with
the antigen or vaccine. In some examples, the dose of rapamycin is
about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10,
0.11, 0.12, 0.13, 0.14 or 0.15 mg/kg. In this context, "about"
refers to a value within 0.005 mg/kg. A dose of about 0.01 to about
0.15 mg/kg typically results in a blood concentration of
approximately 5 to 20 ng/ml. When the mTOR inhibitor is
administered prior to or concomitant with the antigen at this
relatively low dose, the inhibitor can also be administered for any
period of time after administration of the antigen or vaccine. In
other embodiments, the mTOR inhibitor is rapamycin and the dose is
about 0.2 to about 1.0 mg/kg, such as about 0.4 to about 0.8 mg/kg,
when rapamycin is administered after administration of the antigen
or vaccine, such as during the T cell contraction phase of an
immune response. In some examples, the dose of rapamycin is about
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg/kg. In this
context, "about" refers to a value within 0.05 mg/kg. A dose of
about 0.2 to about 1.0 mg/kg typically results in a blood
concentration of approximately 40 to 100 ng/ml. An appropriate dose
can be determined by one of ordinary skill in the art using only
routine experimentation.
[0235] When administered in multiple doses, the dosing schedule of
the mTOR inhibitor can vary. In some embodiments, the mTOR
inhibitor is administered twice a day, daily, weekly or monthly. In
some embodiments, the mTOR inhibitor is administered daily for
about one week. In other embodiments, the mTOR inhibitor is
administered daily for about one month.
[0236] In some embodiments, the mTOR inhibitor is administered
continuously, such as part of a patch or other transdermal
application.
[0237] The mTOR inhibitors can be administered by any suitable
route. The route of administration will be determined by a variety
of factors, including the type of inhibitor used, the composition
of inhibitor (e.g., liquid or solid form), and the immune response
desired. Methods of administration include, but are not limited to,
intradermal, topical, intramuscular, transdermal, intraperitoneal,
parenteral, intravenous, subcutaneous, vaginal, rectal, intranasal,
inhalation, oral or mist-spray delivery to the lungs. Parenteral
administration, such as subcutaneous, intravenous or intramuscular
administration, is generally achieved by injection. Injectables can
be prepared in conventional forms, either as liquid solutions or
suspensions, solid forms suitable for solution or suspension in
liquid prior to injection, or as emulsions. Injection solutions and
suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described. Administration can be
systemic or local. Sterile injectable solutions are prepared by
incorporating the active compounds in the required amount in the
appropriate solvent with any other ingredients as required,
followed by filtered sterilization.
[0238] In some embodiments, the mTOR inhibitor is administered
topically or transdermally, for example in a patch, pad, bandage,
cream, gel, lotion, spray, foam or paste. When administered as a
patch, pad, bandage or the like, the patch, pad or bandage can be
replaced at regular intervals to maintain a constant dose of mTOR
inhibitor. Alternatively, the patch, pad or bandage can be applied
for a given time period, such as one day, two days, three days,
four days, five days, six days or seven days, or until the mTOR
inhibitor is depleted from the patch, pad or bandage. Patches
suitable for transdermal delivery of therapeutic agents are known
in the art (see, for example, U.S. Patent Application Publication
Nos. 2005/0142176; 2008/0274166; 2009/0028929; and
2009/0048567).
[0239] The mTOR inhibitors are administered in any suitable manner,
such as with pharmaceutically acceptable carriers. Pharmaceutically
acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method
used to administer the composition. Accordingly, there is a wide
variety of suitable formulations of pharmaceutical compositions of
the present disclosure.
[0240] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like. Pharmaceutically acceptable carriers include, but are
not limited to, saline, buffered saline, dextrose, water, glycerol,
ethanol, and combinations thereof. The carrier and composition can
be sterile, and the formulation suits the mode of administration.
The composition can also contain minor amounts of wetting or
emulsifying agents, or pH buffering agents. The composition can be
a liquid solution, suspension, emulsion, tablet, pill, capsule,
sustained release formulation, or powder. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulations can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, and
magnesium carbonate. Any of the common pharmaceutical carriers,
such as sterile saline solution or sesame oil, can be used. The
medium can also contain conventional pharmaceutical adjunct
materials such as, for example, pharmaceutically acceptable salts
to adjust the osmotic pressure, buffers, preservatives and the
like. Other media that can be used with the compositions and
methods provided herein are normal saline and sesame oil.
[0241] The mTOR inhibitors can be formulated in a neutral or salt
form. Pharmaceutically-acceptable salts include the acid addition
salts (formed with the free amino groups of the protein) which are
formed with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or organic acids such as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective.
[0242] Generally, dispersions are prepared by incorporating the
various sterilized active ingredients into a sterile vehicle which
contains the basic dispersion medium and the required components.
In the case of sterile powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum-drying and freeze-drying techniques which yield a powder of
the active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof. The compositions of
the present invention may also be administered into the epidermis
using the Powderject System (Chiron, Emeryville, Calif.). The
Powderject delivery technique works by the acceleration of fine
particles to supersonic speed within a helium gas jet and delivers
pharmaceutical agents and vaccines to skin and mucosal injection
sites, without the pain or the use of needles.
[0243] In some embodiments, the mTOR inhibitors are administered in
combination with other therapeutic agents. For example, the mTOR
inhibitors (or vaccine administered in conjunction with the mTOR
inhibitor) can be administered with an adjuvant, such as Freund
incomplete adjuvant or Freund's complete adjuvant.
[0244] Optionally, one or more cytokines, such as IL-2, IL-6,
IL-12, RANTES, GM-CSF, TNF-.alpha., or IFN-.gamma., one or more
growth factors, such as GM-CSF or G-CSF; one or more molecules such
as OX-40L or 41 BBL, or combinations of these molecules, can be
used as biological adjuvants (see, for example, Salgaller et al.,
1998, J. Surg. Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J.
Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl
1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90).
These molecules can be administered systemically (or locally) to
the host.
[0245] A number of means for inducing cellular responses, both in
vitro and in vivo, are known. Lipids have been identified as agents
capable of assisting in priming CTL in vivo against various
antigens. For example, as described in U.S. Pat. No. 5,662,907,
palmitic acid residues can be attached to the alpha and epsilon
amino groups of a lysine residue and then linked (for example, via
one or more linking residues, such as glycine, glycine-glycine,
serine, serine-serine, or the like) to an immunogenic peptide. The
lipidated peptide can then be injected directly in a micellar form,
incorporated in a liposome, or emulsified in an adjuvant. As
another example, E. coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine can be used to prime
tumor specific CTL when covalently attached to an appropriate
peptide (see, Deres et al., Nature 342:561, 1989). Further, as the
induction of neutralizing antibodies can also be primed with the
same molecule conjugated to a peptide which displays an appropriate
epitope, two compositions can be combined to elicit both humoral
and cell-mediated responses where that is deemed desirable.
[0246] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the disclosure to the particular features or
embodiments described.
EXAMPLES
Example 1
Rapamycin Treatment During the Contraction Phase of a CD8.sup.+ T
Cell Response Induces High Quality Memory T Cells
[0247] This example describes the finding that rapamycin treatment
(i) induces high quality memory T cells during the contraction
phase of a T cell response; (ii) enhances differentiation of memory
T cells during the T cell contraction phase; and (iii) enhances
high quality memory T cells during the contraction phase of recall
T cell responses. This example further demonstrates that memory
CD8.sup.+ T cells induced in rapamycin treated animals are high
quality memory T cells.
[0248] There are three phases of a virus-specific CD8.sup.+ T cell
response after virus infection (FIG. 1). First, naive
virus-specific CD8.sup.+ T cells exponentially expand and become
effector T cells. These effector T cells stop proliferating
approximately 1 to 2 weeks after infection and enter the
contraction phase. During the contraction phase, effector CD8.sup.+
T cells gradually acquire memory T cell phenotype and function.
[0249] Rapamycin is a macrolide with immunosuppressive and
anti-proliferative properties. The effect of rapamycin treatment on
T cell responses induced by vaccination was evaluated in mice
infected with lymphocytic choriomeningitis virus (LCMV). To test
the effect of rapamycin treatment on the expansion phase of a T
cell response, mice were injected intraperitoneally with
2.times.10.sup.5 plaque forming units (PFU) of LCMV on Day 0 and
treated with rapamycin once a day for 9 days, starting one day
prior to vaccination (Day -1). Rapamycin was administered
intraperitoneally at a dose of 0.6 mg/kg (resulting in blood
concentration of approximately 40-100 ng/ml). On Day 8, peripheral
blood mononuclear cells (PBMCs) were isolated and evaluated by FACS
analysis to determine the percentage of virus-specific CD8.sup.+ T
cells in rapamycin-treated and untreated mice (FIG. 2A). PBMCs were
incubated with antibodies specific for CD8 and CD44 (a memory T
cell marker), and a LCMV GP33 epitope-specific MHC class I tetramer
(DbGP33; Murali-Krishna et al., Immunity 8(2):177-87, 1998). The
anti-CD8 antibody was conjugated with the fluorochrome peridinin
chlorophyll protein (PerCP) (BD Biosciences) and the anti-CD44
antibody was conjugated with the fluorophore Pacific Blue.RTM.
(eBioscience, San Diego, Calif.). As shown in FIG. 2B, mice treated
with rapamycin during the T cell expansion phase had a smaller
percentage of virus-specific CD8.sup.+ T cells (2.49%) relative to
untreated mice (6.98%), suggesting that rapamycin inhibited
antigen-driven T cell proliferation. In addition, LCMV titer in
rapamycin-treated mice was significantly increased relative to
untreated mice (FIG. 2C), demonstrating the immunosuppressive
effect of rapamycin when administered during the T cell expansion
phase.
[0250] To determine the effect of rapamycin treatment during the T
cell maintenance phase, mice were adoptively transferred with
carboxyfluorescein succinimidyl ester (CFSE)-labeled GP33
epitope-specific memory CD8.sup.+ T cells (TCR-transgenic P14
cells; Pircher et al., Nature 342:559-561, 1989). CFSE-labeled
cells were transferred intravenously via tail vein injection. Mice
were either untreated or treated with rapamycin daily for 43 days
at a dose of 0.6 mg/kg, beginning one day prior to adoptive
transfer (Day -1) (FIG. 3A). On Day 42, the number of transferred
virus-specific CD8.sup.+ T cells in the spleen of treated and
untreated mice was determined. To isolate spleen lymphocytes,
spleens were homogenized and red blood cells were lysed with 0.83%
ammonium chloride. Isolated splenocytes were then evaluated by
FACS. As shown in FIG. 3B, rapamycin treatment decreased the number
of virus-specific CD8.sup.+ T cells in the spleen. Proliferation of
virus-specific CD8.sup.+ T cells was also evaluated on Day 42 by
detecting fluorescence intensity of CFSE-labeled T cells by FACS.
As shown in FIG. 3C, rapamycin treatment reduced the percentage of
virus-specific CD8.sup.+ T cells that divided more than twice
(17.8%), relative to untreated mice (42.5%).
[0251] To determine the effect of rapamycin treatment during the T
cell contraction phase, mice were injected intraperitoneally with
2.times.10.sup.5 PFU of LCMV on Day 0 and administered 0.6 mg/kg of
rapamycin daily beginning on Day 8 (FIG. 4A). On Day 35
post-infection, the number of virus-specific CD8.sup.+ T cells in
the spleen of rapamycin-treated and untreated mice was determined
by FACS using anti-CD8 antibody, anti-CD44 antibody and tetramer
staining. Tetramer staining was used to detect GP33, GP276 and
NP396 epitope-specific CD8.sup.+ T cells. To detect NP205 and GP118
epitope-specific CD8.sup.+ T cells, interferon (IFN)-.gamma.
positive cells were measured by intracellular staining after
peptide stimulation. As shown in FIG. 4B, rapamycin treatment
during the T cell contraction phase did not reduce the number of
virus-specific CD8.sup.+ T cells.
[0252] Thus, in accordance with the known immunosuppressive
properties of rapamycin, treatment with rapamycin exhibited an
immunosuppressive effect during the T cell expansion (FIG. 2) and
maintenance (FIG. 3) phases. However, rapamycin treatment did not
alter the number of virus-specific CD8.sup.+ T cells during the
contraction phase (FIG. 4).
[0253] To examine whether rapamycin treatment effects the quality
of virus-specific CD8.sup.+ T cells during the contraction phase,
phenotypic analysis of virus-specific CD8.sup.+ T cells in spleen,
PBMCs and liver was performed by evaluating markers for high
quality memory T cells (including CD127.sup.High, CD62L.sup.High,
KLRG-1.sup.Low, CD27.sup.High and Bcl-2.sup.High) by FACS. As shown
in FIG. 5, a number of significant differences were identified
between control and rapamycin-treated animals. In rapamycin-treated
animals, GP33, GP276 and NP396 epitope-specific CD8.sup.+ T cells
phenotypically showed high quality memory T cells (CD127.sup.High,
CD62L.sup.High, KLRG-1.sup.Low, CD27.sup.High, Bcl-2.sup.High)
compared to untreated animals. In addition to spleen, a similar
phenotypic trend of virus-specific CD8.sup.+ T cells was identified
in PBMCs and liver (FIG. 6). In lymph node, no significant change
in cell surface markers was observed between untreated and
rapamycin-treated animals (FIG. 6). These results were expected
because virus-specific CD8.sup.+ T cells in lymph node typically
exhibit a high quality phenotype compared to other tissues.
However, in rapamycin-treated animals, Bcl-2 expression was higher
than control even in lymph node. Taken together, rapamycin
treatment during contraction phase enhanced generation of high
quality memory T cells.
[0254] The results described above indicate that rapamycin
treatment induces high quality memory T cells during the T cell
contraction phase. To investigate how rapamycin accumulates high
quality memory T cells, CD62L-negative virus-specific effector T
cells were transferred into naive mice as described below and
illustrated in FIG. 7A. LCMV-specific transgenic (P14) effector
CD8.sup.+ T cells (Thy-1.1.sup.+) were isolated from transgenic P14
mice (fully backcrossed with C57BL/6 mice) on Day 8 after LCMV
infection. CD62L.sup.high cells were depleted from the isolated
effector P14 cells using microbeads (Miltenyi Biotec, Auburn,
Calif.) (Wherry et al., Nat. Immunol. 4(3):225-234, 2003). The
remaining CD62L.sup.low effector CD8.sup.+ T cells were transferred
into Thy-1.2.sup.+ naive mice. Mice were then left untreated or
treated with 0.6 mg/kg rapamycin daily for 26 days. In some
experiments, Day 8 effector P14 cells were labeled with CFSE.
Virus-specific CD8.sup.+ T cells in rapamycin-treated mice quickly
re-expressed CD62L compared with control mice (FIG. 7B), and the
absolute number of CD62L.sup.High virus-specific CD8.sup.+ T cells
was greater in rapamycin-treated mice (FIG. 7C). In addition, CD62L
re-expression occurred with no or minimal cell division. Therefore,
when CFSE-labeled CD62L negative effector T cells were transferred,
most cells still retained CFSE 26 days post transfer (FIG. 7D).
These results demonstrate that rapamycin treatment enhances CD62L
re-expression without cell division during the contraction phase.
Furthermore, these data suggest that rapamycin treatment improves
differentiation of memory T cells during the contraction phase.
[0255] Next, studies were undertaken to determine whether
rapamycin-induced memory T cells are effective for rapidly
controlling virus infection. To address this issue, the following
experiments were designed. CD62L-negative LCMV-specific effector
CD8.sup.+ T cells (Day 8 effector P14 cells) were transferred into
naive mice. These mice were either left untreated or treated with
rapamycin for 25 days, then challenged with vaccinia virus (VV)
that expresses the GP33 epitope (VVgp33; Wherry et al., Nat.
Immunol. 4(3):225-234, 2003) on Day 28 (FIG. 8A). VVgp33 was
administered intraperitoneally at a dose of 5.times.10.sup.6 PFU.
To evaluate the T cell recall response, the percentage of
virus-specific CD8.sup.+ T cells in the spleen was determined by
FACS on Day 5 after VV challenge. As shown in FIG. 8B, a greater
percentage of virus-specific CD8.sup.+ T cells (DbGP33
tetramer-positive P14 cells) was detected in rapamycin treated mice
(4.93%) relative to untreated mice (1.69%). In addition, the
absolute number of DbGP33 tetramer-positive P14 cells was greater
in rapamycin-treated mice than in untreated mice (FIG. 8C),
suggesting that rapamycin-induced memory T cells expanded rapidly
compared with control.
[0256] To evaluate the effect of rapamycin treatment on virus
infection, viral titers were determined in the ovaries of naive
mice, untreated mice and rapamycin-treated mice on Day 5
post-challenge. As shown in FIG. 8D, rapamycin treatment led to a
reduction in viral titer relative to untreated and naive mice.
These results suggest that rapamycin-induced memory CD8.sup.+ T
cells are high quality memory T cells capable of effectively
inhibiting virus infection.
[0257] In addition to better viral control, homeostatic
proliferation is another characteristic of high quality memory T
cells. To investigate the ability of rapamycin induced memory T
cells to undergo homeostatic proliferation, CFSE-labeled memory T
cells derived from rapamycin-treated or untreated mice were
adoptively transferred into naive mice (FIG. 9A). As shown in FIG.
9B, the percentage of divided memory T cells in PBMC was increased
with rapamycin treatment. Cell division of P14 memory cells was
also evaluated in the spleen 30 days post-transfer. As shown in
FIG. 9C, rapamycin treatment increased the percentage of cells that
divided more than twice (46.1%), relative to the control (36.8%).
These data demonstrate that rapamycin-treated memory T cells
exhibit better homeostatic proliferation in both PBMC and spleen,
suggesting that rapamycin induces effective memory T cells. Taken
together, rapamycin-induced memory CD8.sup.+ T cells are bona fide
high quality memory T cells.
[0258] Next, experiments were performed to determine whether
rapamycin has any effect on virus-specific CD8.sup.+ T cells after
a recall response. To investigate this, mice were treated with 0.6
mg/kg rapamycin during the contraction phase of a recall response.
LCMV-specific memory P14 cells were transferred into naive mice on
Day -1. On Day 0, mice were infected with 5.times.10.sup.6 PFU
VVgp33. Mice were either untreated or treated with rapamycin each
day starting on Day 8 after VV infection (FIG. 10A). The number of
P14 cells in the spleen of untreated and rapamycin-treated mice was
determined on Day 31 post-infection by FACS. As shown in FIG. 10B
and FIG. 10C, the number of virus-specific CD8.sup.+ T cells was
similar between control and rapamycin-treated animals and there was
no significant difference in CD127 and CD62L expression. However,
expression of KLRG-1 was lower and expression of CD27 and Bcl-2 was
higher on virus-specific CD8.sup.+ T cells from rapamycin-treated
mice, relative to control mice (FIG. 10C). These results suggest
that rapamycin enhances high quality memory T cells during the
contraction phase of T cell recall responses.
Example 2
Low Dose Rapamycin Treatment Enhances the Number of
Antigen-Specific CD8.sup.+ T Cells and Induces High Quality Memory
T Cells
[0259] As described above, rapamycin treatment during the T cell
expansion phase inhibits antigen-driven T cell proliferation. This
example describes the effect of a lower dose of rapamycin on T cell
responses. In particular, this example demonstrates that low dose
rapamycin treatment (i) enhances the number of virus-specific
CD8.sup.+ T cells; (ii) induces high quality memory T cells during
a primary T cell response; (iii) induces high quality memory T
cells during a recall response; and (iv) induces high quality
memory T cells upon immunization with a non-infectious
immunogen.
[0260] Mice were injected intraperitoneally with 2.times.10.sup.6
PFU LCMV on Day 0 and treated daily with a low dose of rapamycin
(0.075 mg/kg, which results in a blood concentration of
approximately 5-20 ng/ml) beginning one day prior to infection (Day
-1) (FIG. 11A). To evaluate the number of virus-specific CD8.sup.+
T cells in untreated and rapamycin-treated mice, PBMCs were
isolated and subjected to FACS analysis. As shown in FIG. 11B, a
similar number of GP33 epitope-specific CD8.sup.+ T cells was
detected in PBMCs from treated and untreated mice isolated on Day 8
post-infection. However, mice treated with low dose rapamycin
maintained a higher number of GP33 epitope-specific CD8.sup.+ T
cells compared to untreated animals from Day 8 until the conclusion
of the 30-day evaluation period. Also examined was the number of
virus-specific CD8.sup.+ T cells in the spleen of untreated and
rapamycin-treated mice 35 days post-infection. Tetramer staining
was used to detect GP33, GP276 and NP396 epitope-specific CD8.sup.+
T cells. For NP205 and GP118 epitope-specific CD8.sup.+ T cells,
IFN-.gamma. positive cells were measured by intracellular staining
after peptide stimulation. As shown in FIG. 11C, rapamycin
treatment enhanced the number of all epitope-specific CD8.sup.+ T
cells examined.
[0261] To investigate the quality of virus-specific CD8.sup.+ T
cells in low dose rapamycin-treated mice, phenotypic analysis of T
cells was performed by FACS as described above. As shown in FIG.
12, there were significant differences between control and
rapamycin-treated animals. In treated animals, virus-specific
CD8.sup.+ T cells exhibited a CD127.sup.High CD62L.sup.High
KLRG-1.sup.Low Bcl-2.sup.High phenotype compared to untreated mice
(FIG. 12A and FIG. 12B). These differences were detected by Day 8
post-infection (FIG. 12C). These data suggest that rapamycin
regulates phenotypic changes during T cell differentiation. In
addition, these results suggest that low dose rapamycin treatment
induces high quality memory T cells.
[0262] Next, the effect of low dose rapamycin treatment on
virus-specific CD8.sup.+ T cells after a recall response was
evaluated. To investigate this, mice were treated with a low dose
of rapamycin (0.075 mg/kg) during recall responses. Thy-1.1.sup.+
P14 memory cells were adoptively transferred into Thy-1.2.sup.+
recipient mice and rapamycin treatment was initiated on the same
day (Day -1). The next day (Day 0), recipient mice were injected
intraperitoneally with 2.times.10.sup.6 PFU LCMV (FIG. 13A). The
percentage of LCMV-specific CD8.sup.+ T cells in PBMCs isolated
from treated and untreated mice on Days 8, 14, 22 and 35 was
evaluated by FACS. As shown in FIG. 13B, the percentage of
virus-specific CD8.sup.+ T cells was greater in rapamycin treated
animals than in control mice at each day tested. Moreover,
rapamycin treatment enhanced expression of CD127 and CD62L (FIG.
13C). These results indicate that low dose rapamycin induces high
quality memory T cells during recall responses as well as primary
responses.
[0263] To determine whether low dose rapamycin treatment has an
effect on T cell responses against noninfectious immunogens,
virus-like particles (VLPs) that present GP33 epitope were used to
immunize mice (Storni et al., J. Immunol. 168(6):2880-2886, 2002;
Storni et al., J. Immunol. 171(2):795-801, 2003). Mice were
immunized with 50 .mu.g of VLPs by subcutaneous injection, and mice
were either untreated or treated with rapamycin beginning one day
prior to immunization (Day -1) (FIG. 14A). After VLP immunization,
GP33 epitope-specific CD8.sup.+ T cells expanded similarly in the
rapamycin-treated and control groups (FIG. 14B). However,
rapamycin-treated mice maintained a higher number of
antigen-specific CD8.sup.+ T cells compared to control in PBMC
(FIG. 14B) and spleen (FIG. 14C). In addition, the phenotype of
antigen-specific CD8.sup.+ T cells isolated from the spleen of
rapamycin-treated mice 34 days post-infection exhibited markers of
high quality memory T cells (CD127.sup.High, CD62L.sup.High
KLRG-1.sup.Low, Bcl-2.sup.High) (FIG. 14D and FIG. 14E). These
results suggest that low dose rapamycin induces high quality memory
T cells not only upon infection, but also upon immunization with
non-infectious immunogen.
Example 3
Rapamycin Intrinsically Affects Virus-Specific CD8.sup.+ T Cells
for Generation of High Quality Memory T Cells and Enhances the
Number of Virus-Specific Memory CD4.sup.+ T Cells
[0264] It is known in the art that rapamycin inhibits mTOR, which
is ubiquitously expressed and plays a role in a number of cellular
processes, including translation, cell survival, autophagy and
actin cytoskeleton dynamics. Therefore, it is possible that
rapamycin not only affects T cells, but also non-T cells in vivo.
How rapamycin induces high quality memory T cells in vivo was
previously unknown. To test whether generation of high quality
memory T cells by rapamycin is a CD8-intrinsic effect, knockdown of
rapamycin-related molecules was performed using a retrovirus-based
RNA interference (RNAi) system. mTOR is part of two distinct
complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2)
(FIG. 15). Recent studies suggest that mTORC1 is sensitive to
rapamycin. Therefore, to inhibit the mTORC1 pathway, experiments
were performed to inhibit expression of raptor, which is part of
mTORC1 (FIG. 16A).
[0265] Retrovirus encoding a control or raptor shRNA
(GCCCGAGTCTGTGAATGTAAT; SEQ ID NO: 1) was constructed by cloning
the shRNA into the pMKO.1-GFP retrovirus vector (Addgene,
Cambridge, Mass.). Retrovirus was generated by co-transfection of
pMKO.1-GFP-raptor (or pMKO.1-GFP-control) and pCL-Eco (packaging
vector) plasmid into HEK-293T cells. For transduction, P14
transgenic mice were infected with 1.times.10.sup.6 PFU LCMV
intravenously. One day later, activated splenocytes were
spin-transduced with freshly made retroviral supernatants from
HEK-293T cells (90 minutes at 37.degree. C., 3000 rpm). After
transduction, transduced splenocytes were adoptively transferred
intravenously to naive recipient mice that were subsequently
infected intraperitoneally with LCMV at a dose of 2.times.10.sup.5
PFU. Virus-specific memory T cell phenotype in mouse spleen was
evaluated in control and raptor RNAi-treated animals 35 days
post-infection by FACS. Raptor knockdown resulted in a high quality
memory T cell phenotype (CD127.sup.High, CD62L.sup.high
KLRG-1.sup.Low) in LCMV-infected mice (FIG. 16B).
[0266] A FKBP12 knockdown-retrovirus vector was also constructed by
cloning the FKBP12 shRNA (GCCAAACTGATAATCTCCTCA; SEQ ID NO: 2) into
the pMKO.1-GFP retrovirus vector (Addgene). FKBP12 forms a complex
with rapamycin, and this complex inhibits mTORC1 (FIG. 15). Thus,
virus-specific CD8.sup.+ T cells with FKBP12 knockdown should be
rapamycin insensitive. To test this hypothesis,
retrovirus-transduced LCMV-specific P14 cells were adoptively
transferred into naive mice and mice were then infected with LCMV
at a dose of 2.times.10.sup.5 PFU. Rapamycin treatment (0.075
mg/kg) was initiated one day prior to infection (FIG. 17A). The
phenotypic changes of adoptively transferred P14 cells were
evaluated by FACS on Day 16 post-infection in PBMC. As shown in
FIG. 17B, the effect of rapamycin treatment was diminished by
FKBP12 knockdown. Taken together, these results demonstrate that
rapamycin intrinsically affects virus-specific CD8.sup.+ T cells
for generation of high quality memory T cells.
[0267] In addition to CD8.sup.+ T cells, low dose rapamycin
treatment enhanced the number of virus-specific memory CD4.sup.+ T
cells (FIG. 18). Mice were infected with 2.times.10.sup.5 PFU LCMV
on Day 0 and were either untreated or treated with rapamycin
beginning one day prior to infection (Day -1). Spleen cells were
isolated on Day 35 and stimulated with LCMV GP61 peptide specific
for CD4.sup.+ T cells. Intracellular cytokine (IL-2 and
IFN-.gamma.) staining was performed and the cells were subjected to
FACS. As shown in FIG. 18, rapamycin treatment resulted in a higher
number of IFN-.gamma..sup.+ cells upon peptide stimulation relative
to cells from untreated mice.
Example 4
Low Dose Rapamycin Treatment Improves Quantity and Quality of
Memory T Cells
[0268] This example describes the finding that low dose rapamycin
treatment improves the quantity and quality of memory T cells
generated by recombinant adenovirus serotype 5 (rAd5) that
expresses LCMV glycoprotein (rAd5-LCMV-GP). The nucleotide and
amino acid sequences of LCMV GP, deposited under GENBANK.TM.
Accession No. M20869 on Aug. 2, 1993, are set forth herein as SEQ
ID NOs: 7 and 8, respectively.
[0269] Mice were vaccinated with rAd5-LCMV-GP (1.times.10.sup.10
viral particles) by intramuscular injection on Day 0 and either
left untreated or treated with rapamycin at a dose of 0.075 mg/kg
from Day -1 to Day 34 (FIG. 19A). The number of DbGP33
tetramer-positive CD8.sup.+ T cells in the spleen of treated and
untreated mice was determined by FACS on Day 35 post-vaccination.
As shown in FIG. 19B, rapamycin treatment significantly increased
the number of virus-specific CD8.sup.+ T cells. The phenotype of
virus-specific CD8.sup.+ T cells was also evaluated by FACS (FIG.
19C). In accordance with data described above, CD8.sup.+ T cells
from rapamycin-treated mice exhibited markers of high quality
memory T cells (CD127.sup.High, CD62L.sup.High, KLRG-1.sup.Low,
Bcl-2.sup.High).
Example 5
mTOR Regulates Memory CD8.sup.+ T Cell Differentiation
[0270] Rapamycin is a commonly used immunosuppressive drug in
transplant recipients and specifically inhibits the intracellular
kinase mTOR (Wullschleger et al., Cell 124:471-484, 2006). Several
recent studies have shown that rapamycin has various effects on the
immune system such as inhibiting type I interferon production by
plasmacytoid dendritic cells (Cao et al., Nat Immunol
9(10):1157-1164, 2008), modulating T cell trafficking (Sinclair et
al., Nat Immunol 9(5):513-521, 2008), and regulating Foxp3
expression in regulatory T cells (Sauer et al., Proc Natl Acad Sci
USA 105:7797-7802, 2008; Haxhinasto et al., J Exp Med 205:565-574,
2008). However, the role of the mTOR pathway in regulating
CD8.sup.+ T cell responses is not known. To address this issue, the
following experiments were performed.
Materials and Methods
[0271] Mice, viral infection, VLP, and virus titrations. Twelve- to
sixteen-week old C57BL/6j mice were purchased from The Jackson
Laboratory (Bar Harbor, Me.). Thy-1.1 P14 transgenic mice bearing
the DbGP33-specific TCR were fully backcrossed to C57BL/6 mice in
the animal colony. LCMV Armstrong (2.times.10.sup.5 PFU, IP) and
recombinant vaccinia virus GP33 (VVGP33, 5.times.10.sup.6 PFU, IP),
which expresses the LCMV GP33 epitope, were used for infection.
VVGP33 titers were determined in the ovary by plaque assay as
described previously (Wherry et al., Nat Immunol 4, 225-234, 2003).
For VLP immunization, mice were subcutaneously administered 50
.mu.g of VLP, which was derived from the hepatitis B core antigen
(HBcAg) genetically fused to the LCMV gp33-41 epitope (KAVYNFA.TM.;
SEQ ID NO: 9) and packaged with CpG-ODN.
[0272] Administration of rapamycin in mice. Rapamycin (Wyeth,
Madison, N.J.) was administered to mice IP daily during the
treatment period. Three different treatment periods were used: 1)
throughout LCMV infection (Day -1 to the memory phase, Day 35
post-infection); 2) the T cell expansion phase (Day -1 to Day 8
post-infection); or 3) T cell contraction phase (Day 8 to the
memory phase, Day 35 post-infection). The daily dose of rapamycin
was 75 .mu.g/kg (blood levels approximately 5-20 ng/ml) for
treatments 1) and 2) and 600 .mu.g/kg (blood levels approximately
40-100 ng/ml) for treatment 3) (the contraction phase treatment)
because the higher dose inhibits T cell responses during the
expansion phase of the CD8.sup.+ T cell response (as evidenced by a
decrease in antigen-specific CD8.sup.+ T cells). Control mice
received sham treatment during the same time periods described
above (daily injection of the buffer without rapamycin).
[0273] Rhesus macaques and vaccination. Six colony-bred, SPF Rhesus
macaques (Macaca mulatta) were inoculated with DRYVAX.TM. (Wyeth,
Madison, N.J.) by scarification. Briefly, a bifurcated needle was
immersed in the vaccine suspension and used to poke the skin 15
consecutive times. At 105 days post DRYVAX.TM. vaccination, animals
were vaccinated with 10.sup.8 PFU Modified Vaccinia Ankara (MVA)
vaccine intramuscularly.
[0274] Administration of rapamycin in rhesus macaques. Daily
administration of rapamycin (approximately 10-50 .mu.g/kg/day) was
given intramuscularly to three of the six DRYVAX.TM. immunized
rhesus macaques approximately 5 days before MVA vaccination. Blood
levels of rapamycin were maintained within a range of about 5-15
ng/ml. The other three macaques were left untreated as
controls.
[0275] Generation and isolation of effector and memory T cell
subsets. To generate LCMV-specific P14 effector T cells, mice
adoptively transferred with 1.times.10.sup.5 P14 naive T cells were
infected with LCMV. On Day 8 post-infection, effector P14 cells
were isolated from the spleen, and CD62L-negative CD8.sup.+ T cells
were purified using anti-CD62L magnetic beads and a CD8.sup.+ T
cell isolation kit (Miltenyi Biotec, Auburn, Calif.). These cells
were then used for a CD62L conversion experiment and a protective
immune response experiment. For the CD62L conversion experiment,
CFSE-labeled CD62L-negative P14 effector cells (about
7-10.times.10.sup.6 cells) were adoptively transferred into naive
mice. For the protective immune response experiment,
3.times.10.sup.5 CD62L-negative P14 effector cells were adoptively
transferred into naive mice, and rapamycin was administered for 25
days. On Day 28 post-transfer, mice were challenged with VVGP33 to
examine protective immune responses.
[0276] To obtain memory P14 cells generated in rapamycin treated
mice, B6 mice adoptively transferred with 1.times.10.sup.5 P14
naive T cells were infected with LCMV, and these mice were then
treated with rapamycin from Day -1 to Day 33 post-infection. On Day
34 post-infection, memory P14 T cells generated in the presence of
rapamycin were isolated from the spleen. Control memory P14 cells
were obtained using the same method without the rapamycin
treatment. For the homeostatic experiment, CFSE-labeled memory P14
cells (1.times.10.sup.6 cells) obtained from either
rapamycin-treated or untreated mice were adoptively transferred
into separate naive recipients. For the recall response experiment,
1.times.10.sup.4 P14 memory T cells derived from either
rapamycin-treated or untreated mice were adoptively transferred
into separate naive mice, and the day after transfer these mice
were infected with VVGP33. To investigate effects of rapamycin
during secondary T cell responses, 2.5.times.10.sup.4 P14 memory T
cells (>60 days post infection) were adoptively transferred into
naive mice, and rapamycin treatment was started. The day after
transfer, these mice were infected LCMV.
[0277] Flow cytometry. MHC class I tetramers were made as described
previously (Murali-Krishna et al., Immunity 8, 177-187, 1998). All
antibodies for flow cytometry were purchased from BD Biosciences
except for CD127, KLRG-1, and CD27. Antibodies to CD127 and CD27
were purchased from eBiosciences (San Diego, Calif.) and
anti-KLRG-1 was purchased from Southern Biotech (Birmingham, Ala.).
Single cell suspensions of spleen cells, lymph nodes, livers, or
PBMCs from mice were prepared and direct ex-vivo staining was
carried out as described previously (Wherry et al., Nat Immunol 4,
225-234, 2003). For in vivo BrdU incorporation, LCMV-infected mice
were fed 0.8 mg/ml BrdU in their drinking water every day. BrdU in
virus-specific CD8.sup.+ T cells was measured using the BrdU flow
kit (BD Biosciences), according to the manufacturer's instructions.
To detect vaccinia virus-specific CD8.sup.+ T cells generated in
rhesus macaques, 1.5.times.10.sup.6 PBMCs isolated by density
gradient centrifugation were incubated at 37.degree. C. for 15
hours with vaccinia virus at a multiplicity of infection of 1 in a
volume of 300 .mu.l RPMI containing 10% heat inactivated FBS.
Brefeldin A (5 .mu.g/mL) was added for the final 5 hours of
incubation. IFN-.gamma. producing vaccinia virus-specific CD8.sup.+
T cells were detected by intracellular cytokine staining.
[0278] Retrovirus based RNAi. The pMKO.1 GFP retroviral vector
(Addgene plasmid 10676, Cambridge, Mass.) was used for these
experiments. Double stranded oligonucleotides for short hairpin RNA
(shRNA) against mTOR, raptor, and FKBP12 were cloned into pMKO.1
GFP between the AgeI and EcoRI restriction sites. The sequences for
mTOR, raptor, FKBP12, S6K1 and eIF4E shRNAs are shown in Table
4.
TABLE-US-00004 TABLE 4 shRNAs specific for mTOR, raptor, FKBP12,
S6K1 and eIF4E Sense/ SEQ ID Target Antisense Sequence NO: mTOR
Sense CCGGGCCAGAATCCATCCATTCATT 10 CTCGAGAATGAATGGATGGATTCTG
GCTTTTTG mTOR Antisense AATTCAAAAAGCCAGAATCCATCCA 11
TTCATTCTCGAGAATGAATGGATGG ATTCTGGC Raptor Sense
CCGGGCCCGAGTCTGTGAATGTAAT 12 CTCGAGATTACATTCACAGACTCGG GCTTTTTG
Raptor Antisense AATTCAAAAAGCCCGAGTCTGTGAA 13
TGTAATCTCGAGATTACATTCACAG ACTCGGGC FKBP12 Sense
CCGGGCCAAACTGATAATCTCCTCA 14 CTCGAGTGAGGAGATTATCAGTTTG GCTTTTTG
FKBP12 Antisense AATTCAAAAAGCCAAACTGATAATC 15
TCCTCACTCGAGTGAGGAGATTATC AGTTTGGC S6K1 Sense
CCGGGCATGGAACATTGTGAGAAAT 16 CTCGAGATTTCTCACAATGTTCCATG CTTTTTG
S6K1 Antisense AATTCAAAAAGCATGGAACATTGTG 17
AGAAATCTCGAGATTTCTCACAATG TTCCATGC eIF4E Sense
CCGGCCGAAGATAGTGATTGGTTAT 18 CTCGAGATAACCAATCACTATCTTC GGTTTTTG
eIF4E Antisense AATTCAAAAACCGAAGATAGTGATT 19
GGTTATCTCGAGATAACCAATCACT ATCTTCGG
[0279] Recombinant retrovirus was made by co-transfection with
pMKO.1 GFP and pCL-Eco (Imgenex, San Diego, Calif.) in 293T cells
using TransIT-293 (Mirus, Madison, Wis.). Forty-eight hours after
transfection, culture supernatants were collected. To transduce P14
cells with the recombinant retrovirus, P14 transgenic mice were
infected with 1.times.10.sup.6 PFU of LCMV intravenously. After 24
hours, P14 transgenic spleen cells were isolated and then
spin-transduced at 37.degree. C. for 90 minutes with freshly
collected retrovirus containing 8 .mu.g/ml of polybrene (Sigma, St.
Louis, Mo.). Retroviral transduced P14 spleen cells
(5.times.10.sup.5) were adoptively transferred into naive mice,
followed by LCMV infection (2.times.10.sup.5 PFU, IP). The
GFP.sup.+ P14 CD8.sup.+ T cells were purified by FACS on Day 7 or 8
post-infection, and protein expression levels were analyzed by
western blotting. Expression of mTOR, raptor, FKBP12, S6K1 and
eIF4E was significantly reduced in cells transduced with retrovirus
containing the respective shRNAs.
Results
[0280] The role of the mTOR pathway in regulating CD8.sup.+ T cell
responses is not well understood. To address this issue, B6 mice
were treated with rapamycin during the course of an acute LCMV
infection and the virus-specific CD8.sup.+ T cell response was
monitored (FIG. 20A). It was observed that rapamycin enhanced the
LCMV-specific CD8.sup.+ T cell response. Increased numbers of
antigen-specific CD8.sup.+ T cells were seen in both lymphoid and
non-lymphoid tissues, including in PBMCs (FIG. 20A), spleen, lymph
nodes and liver. The striking thing about this result was the
decreased contraction of the T cell response in the rapamycin
treated group. Similar frequencies of virus-specific effector
CD8.sup.+ T cells were observed in both groups of mice at the peak
of the T cell response on Day 8 post-infection, but there was
minimal contraction of the T cells in the rapamycin-treated group
(FIG. 20A). To determine whether the decreased T cell contraction
seen between about Days 8-30 post-infection in the
rapamycin-treated group was due to increased cell proliferation
and/or reduced cell death, mice were infected with LCMV in the
presence or absence of rapamycin and then given BrdU during the T
cell contraction phase from approximately Days 10-22. It was found
that there was minimal incorporation of BrdU by antigen-specific
CD8.sup.+ T cells in either group of mice, indicating that the
decreased contraction of T cells in the presence of rapamycin was
not due to increased cell proliferation. Thus, it appears that the
major effect of rapamycin is to enhance the survival of
antigen-specific CD8.sup.+ T cells.
[0281] Next, the phenotype of the memory CD8.sup.+ T cells present
in the two groups of mice at Day 36 post-infection was examined
(FIG. 20B). To investigate this, phenotypic analysis of
virus-specific memory CD8.sup.+ T cells was performed using four
markers that are useful in defining memory CD8.sup.+ T cells: CD127
(IL-7 receptor .alpha. and essential for memory T cell maintenance;
Kaech et al., Nat Immunol 4:1191-1198, 2003; Huster et al., Proc
Natl Acad Sci USA 101:5610-5615, 2004; Schluns et al., Nat Immunol
1:426-432, 2000; Tan et al., J Exp Med 195:1523-32, 2002); CD62L
(lymph node homing receptor and associated with high proliferative
capacity; Wherry et al., Nat Immunol 4, 225-234, 2003); KLRG-1
(inversely-correlated with long lived memory cells; Sarkar et al.,
J Exp Med 205(3):625-40, 2008; Joshi et al., Immunity 27: 281-295,
2007); and Bcl-2 (anti-apoptotic and expressed at high levels in
memory T cells; Schluns et al., Nat Immunol 1:426-432, 2000).
Memory CD8.sup.+ T cells generated in the presence of rapamycin
expressed higher levels of CD127, CD62L, and Bcl-2, and had a
higher frequency of KLRG-1.sup.Low cells compared to control mice
(FIG. 20B). These data strongly suggest that inhibition of the mTOR
pathway using rapamycin not only increases the magnitude of the
virus-specific CD8.sup.+ T cell response (FIG. 20A), but also
improves the functional qualities of the memory CD8.sup.+ T cells
since memory cells with the CD127.sup.High
CD62L.sup.HighBcl-2.sup.High and KLRG-1.sup.Low phenotype are
associated with long-lived protective immunity (Wherry et al., Nat
Immunol 4, 225-234, 2003; Sarkar et al., J Exp Med 205(3):625-40,
2008; Joshi et al., Immunity 27: 281-295, 2007). To directly test
this, the ability of these memory CD8.sup.+ T cells to undergo
homeostatic proliferation, a property essential for long-term
memory maintenance, and to make recall responses upon re-exposure
to antigen, was examined. As shown in FIG. 20C and FIG. 20D,
virus-specific memory CD8.sup.+ T cells generated in mice treated
with rapamycin were superior to memory cells generated in untreated
mice in both of these hallmark memory properties.
[0282] In the experiment shown in FIG. 20, mice were continuously
treated with rapamycin during the entire course of the T cell
response (Day -1 to 35 post-infection). It was next examined how
rapamycin would affect the CD8.sup.+ T cell response if it was only
given during the T cell expansion phase (Days -1 to 8
post-infection). These results (FIGS. 21A and 21B) were strikingly
similar to what was observed earlier (FIG. 20A); even if the
rapamycin treatment was discontinued during the contraction phase
(about Days 8-30) there was only minimal death of the effector
CD8.sup.+ T cells generated in the presence of the drug. Previous
studies have shown that the Day 8 effector CD8.sup.+ T cell
population consists of two subsets, the terminal effector T cells
(CD127.sup.Low, KLRG-1.sup.High) that mostly die over the ensuing
2-4 weeks, and the memory precursor cells (CD127.sup.High,
KLRG-1.sup.Low) that mostly survive and further differentiate to
give rise to the pool of long-lived memory cells (Kaech et al., Nat
Immunol 4:1191-1198, 2003; Sarkar et al., J Exp Med 205(3):625-40,
2008; Joshi et al., Immunity 27: 281-295, 2007). These results
suggested that rapamycin enhances the formation of these memory
precursor cells. This was indeed the case and Day 8 virus-specific
effector CD8.sup.+ T cells generated in rapamycin-treated mice
contained a higher proportion of CD127.sup.High KLRG-1.sup.Low
cells and these cells also expressed higher levels of Bcl-2 (FIG.
21C). However, it was observed that the phenotype of memory
CD8.sup.+ T cells at Day 36 post-infection was similar in the
drug-treated and control mice (FIG. 21D). This was different from
the results obtained upon continuous rapamycin treatment (compare
FIG. 20B versus FIG. 21D). Taken together, these results clearly
show that rapamycin enhances the formation of memory precursors
during the naive to effector T cell differentiation phase, but that
rapamycin may also regulate the effector to memory transition
phase.
[0283] To test this hypothesis, mice were treated with rapamycin
only during the T cell contraction phase (approximately Days 8-35)
following acute LCMV infection (FIG. 22). It was found that the
number of memory cells generated were not affected by the drug
(FIG. 22A), but the phenotype of these memory CD8.sup.+ T cells was
strikingly different (FIG. 22B). Thus, rapamycin treatment during
the effector to memory transition phase enhanced the memory
differentiation program resulting in a significantly higher number
of virus-specific CD8.sup.+ T cells with the phenotype
characteristic of highly functional memory cells (p value;
<0.0001-0.0022) (FIG. 22B).
[0284] It was important to determine if this represented cell
proliferation and outgrowth of a subset of effector CD8.sup.+ T
cells already expressing these memory markers or if rapamycin truly
increased the expression of these markers in the surviving effector
T cells during this effector to memory differentiation phase. To
address this issue, highly purified (>99.7%) and CFSE-labeled
population of Day 8 CD62L.sup.Low antigen-specific effector
CD8.sup.+ T cells were transferred into naive mice and both cell
division and memory differentiation of these transferred effector
cells was monitored in the presence or absence of rapamycin (FIG.
22C). It was found that there was no cell division during this
effector to memory transition phase (approximately Days 1-25
post-transfer), but that the memory T cells that differentiated in
the presence of rapamycin re-expressed CD62L much faster (FIG. 22D
and FIG. 22E). More importantly, these memory CD8.sup.+ T cells
were functionally superior and exhibited better recall responses
and protective immunity (viral control) following challenge with
vaccinia virus expressing the LCMV GP33 epitope (FIGS. 22F-22H).
Thus, inhibiting mTOR during the effector to memory transition
phase improves the functional qualities of memory T cells.
[0285] The results described above demonstrate that rapamycin can
enhance both the magnitude and quality of the CD8.sup.+ T cell
response following a primary viral infection. It was next examined
whether similar effects would be seen during a secondary response.
As shown in FIG. 24, rapamycin also enhanced recall responses when
drug treatment was only done during secondary LCMV infection. Thus,
rapamycin regulates both primary and secondary T cell responses,
which has important implications in designing strategies for
improving memory T cell qualities during prime-boost vaccine
regimens.
[0286] To determine if these findings from the mouse model of LCMV
infection could be generalized to other systems, the effect of
rapamycin treatment following immunization of mice with a
non-replicating vaccine was examined. In these experiments, mice
were vaccinated with VLPs (virus-like particles) derived from
hepatitis B core antigen genetically fused to the LCMV GP33 epitope
(Storni et al., J Immunol 172:1777-1785, 2004). Rapamycin again
enhanced both the magnitude and the quality of the VLP-induced
memory CD8.sup.+ T cells. It should be noted that the effects of
rapamycin treatment were very long-lasting; memory T cell numbers
remained 10-fold higher even 165 days after stopping the drug
treatment.
[0287] The applicability of this approach was also tested in a
non-human primate model. Rhesus macaques previously immunized with
vaccinia virus were boosted with MVA in the presence or absence of
rapamycin and antigen-specific CD8.sup.+ T cell responses were
analyzed by intracellular IFN-.gamma. staining. Clear differences
were found in frequencies of antigen-specific CD8.sup.+ T cells
between rapamycin-treated and untreated monkeys. In the presence of
rapamycin, maintenance of a higher number of memory CD8.sup.+ T
cells was observed (FIG. 25A and FIG. 25B) and slower T cell
contraction was evident compared to control animals (FIG. 25C).
These results demonstrate that rapamycin enhances T cell immunity
in both mice and non-human primates following vaccination with
either live or inactivated vaccines.
[0288] These results clearly establish that mTOR is a major
regulator of memory CD8.sup.+ T cell differentiation. However, one
unanswered question is whether mTOR is acting intrinsically in
antigen-specific CD8.sup.+ T cells to regulate memory
differentiation or if the observed effects of rapamycin on memory
formation are mediated by some other cells of the immune system. It
is important to resolve this issue since mTOR is ubiquitously
expressed by many cells and several recent studies have shown that
rapamycin can modulate the functional properties of several other
cells of the immune system (Cao et al., Nat Immunol
9(10):1157-1164, 2008; Sauer et al., Proc Natl Acad Sci USA
105:7797-7802, 2008; Haxhinasto et al., J Exp Med 205:565-574,
2008; Ohtani et al., Blood 112:635-643, 2008; Weichhart et al.,
Immunity 29(4):565-577, 2008).
[0289] To address this question, a retrovirus-based RNA
interference (RNAi) system was used to specifically knock-down
various genes of the mTOR pathway (mTOR, raptor, S6K1, eIF4E, and
FKBP12) in antigen-specific CD8.sup.+ T cells. Retroviruses marked
by GFP and expressing RNAi for a particular gene or a control
retrovirus were used to infect LCMV-specific transgenic CD8.sup.+ T
cells (P14 cells) and these transduced cells were then adoptively
transferred into naive mice, followed by LCMV infection. This
system allowed for comparison of the phenotypic changes that occur
during memory T cell differentiation in GFP-positive
retrovirus-transduced versus GFP-negative non-transduced
antigen-specific CD8.sup.+ T cells in the same environment (i.e.,
the same mouse). Thus, any differences in memory differentiation
that are seen between these two cell populations can be ascribed to
the intrinsic effects of that particular gene in antigen-specific T
cells.
[0290] First, mTOR itself was knocked down in antigen-specific
CD8.sup.+ T cells. It was found that mTOR RNAi
retrovirus-transduced GFP.sup.+ P14 cells showed significantly
higher expression of the canonical memory T cell markers (CD127,
CD62L, Bcl-2, CD27) and lower expression of KLRG-1 compared to
non-transduced or control vector-transduced P14 cells (FIG. 23A).
These data show that mTOR acts intrinsically in antigen-specific
CD8.sup.+ T cells to regulate memory differentiation. However,
since mTOR forms two distinct complexes, the rapamycin-sensitive
mTOR complex 1 (mTORC1) and the rapamycin-insensitive mTORC2 (see
FIG. 15 and Wullschleger et al., Cell 124:471-484, 2006), mTOR
knockdown does not completely mimic rapamycin treatment. To
distinguish between these two pathways, the raptor gene, which is
an essential component of the mTORC1 complex (Hara et al., Cell
110:177-189, 2002; Kim et al., Cell 110:163-175, 2002) was knocked
down. As shown in FIG. 23B, inhibition of raptor in
antigen-specific T cells gave results identical to what was
observed upon knockdown of mTOR identifying the mTORC1 complex as
the regulator of memory differentiation.
[0291] To gain more insight into mechanisms by which mTOR regulates
memory formation, the roles of S6K1 and eIF4E were examined. It was
found that knockdown of these mTORC1 downstream effectors
significantly enhanced memory CD8.sup.+ T cell differentiation.
Thus, these results show that mTOR is exerting its effect through
these two downstream molecules.
[0292] To further explore the role of mTOR in T cell intrinsic
versus external effects on memory differentiation,
rapamycin-insensitive antigen-specific CD8.sup.+ T cells were
generated by knockdown of the FKBP12 protein. This intracellular
protein binds rapamycin and it is this FKBP12--rapamycin complex
that inhibits the mTORC1 pathway. Thus, by knocking down FKBP12 in
P14 CD8 cells, these cells were made insensitive to any intrinsic
effects of rapamycin, but the drug could still act effectively on
all the other cells in the mouse. This system allows one to examine
if inhibition of mTOR in other cells can effect memory CD8.sup.+ T
cell differentiation. As shown in FIG. 23C, inhibiting mTOR in
other cells when the antigen-specific cells themselves were
rapamycin-insensitive did not affect memory differentiation. The
effect of rapamycin on memory differentiation almost disappeared
upon knockdown of FKBP12 from the P14 cells and these cells did not
show increased expression of the characteristic memory markers
(e.g., CD127, CD62L, Bcl2) (see last column of the figures in FIG.
23C). Thus, taken together the results shown in FIGS. 23A-23C
establish that mTOR not only acts intrinsically in antigen-specific
CD8.sup.+ T cells, but that inhibiting mTOR in other cells has
minimal to no effect on memory T cell differentiation.
[0293] During the past few years considerable progress has been
made in understanding the lineage relationships between naive,
effector and memory T cells and in defining the phenotypic and
functional changes that underlie memory CD8.sup.+ T cell
differentiation (Williams et al., Annu Rev Immunol 25:171-192,
2007); Kaech et al., Immunity 27, 393-405, 2007). However, much
less is known about the intracellular molecules and pathways that
regulate the generation of memory T cells. In this example, a
molecular pathway has been identified that regulates memory T cell
differentiation. In addition, these findings provide a strategy for
modulating the formation of memory cells. In particular, the
ability to increase the functional qualities of memory T cells
provides a new approach for enhancing the efficacy of vaccines
against infectious diseases and cancer.
Example 6
Rapamycin Treatment with Hepatitis B Virus (HBV) Vaccination
[0294] This example describes the use of rapamycin in conjunction
with the HBV vaccine to enhance immunological memory specific for
HBV, thereby minimizing the need for booster immunizations.
Currently, it is recommended that the HBV vaccine be administered
in three doses. For infants, the first dose is typically
administered at birth, followed by booster doses at 1-2 months and
at 6-18 months. For adults, booster doses of the HBV vaccine are
recommended 1-2 months and 4-6 months following primary
immunization.
[0295] An adult subject with no prior exposure to HBV is
administered a primary dose of HBV vaccine RECOMBIVAX HB.TM..
Beginning on the day of immunization, the subject is orally
administered rapamycin (in either tablet or liquid form) daily for
7 days. HBV immune responses can be evaluated in the subject
following primary immunization and administration of rapamycin to
determine whether a booster dose of HBV vaccine is required to
establish sufficient immunological memory to prevent HBV
infection.
Example 7
Rapamycin Treatment of a Subject with Chronic Hepatitis C Virus
Infection
[0296] This example describes the use of rapamycin in the treatment
of a subject diagnosed with chronic hepatitis C virus (HCV)
infection. Patients with chronic HCV infection are at risk of
developing liver inflammation, fibrosis, cirrhosis or liver cancer.
Thus, it is desirable to treat HCV patients to reduce or eliminate
HCV titers, replication and spread.
[0297] The subject diagnosed with chronic HCV is treated with a low
dose of rapamycin (approximately 0.075 mg/kg) daily for 30 days.
Rapamycin is administered orally (in either tablet or liquid form).
HCV-specific immune responses, or the titer of HCV in the subject,
can be evaluated after 30 days to determine if additional doses of
rapamycin are required. The subject is optionally treated with
anti-viral medication, such as interferon alpha or ribavirin.
Example 8
Rapamycin Treatment of a Subject Infected with Influenza Virus
[0298] This example describes the use of rapamycin in the treatment
of a subject with an acute influenza virus infection. To enhance
the immune responses against influenza virus, a subject with an
acute infection is administered rapamycin within 15 days of
exposure to the virus. The subject is administered rapamycin daily
at a dose of approximately 0.6 mg/kg. Rapamycin is administered
orally (in either tablet or liquid form). Rapamycin is administered
daily for approximately 14 days, or until the symptoms of infection
have cleared. The subject is optionally treated with anti-flu virus
medication, such as oseltamivir, zanamavir, amantadine or
rimantadine.
[0299] This disclosure provides a method of enhancing
antigen-specific T cell immune responses in a subject. It will be
apparent that the precise details of the methods described may be
varied or modified without departing from the spirit of the
described disclosure. We claim all such modifications and
variations that fall within the scope and spirit of the claims
below.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 19 <210> SEQ ID NO 1 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 1 gcccgagtct gtgaatgtaa t 21 <210> SEQ
ID NO 2 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
2 gccaaactga taatctcctc a 21 <210> SEQ ID NO 3 <211>
LENGTH: 8733 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <300> PUBLICATION INFORMATION: <308> DATABASE
ACCESSION NUMBER: Genbank/004958 <309> DATABASE ENTRY DATE:
2002-04-04 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(8733)
<400> SEQUENCE: 3 gctcccggct tagaggacag cggggaaggc gggcggtggg
gcagggggcc tgaagcggcg 60 gtaccggtgc tggcggcggc agctgaggcc
ttggccgaag ccgcgcgaac ctcagggcaa 120 gatgcttgga accggacctg
ccgccgccac caccgctgcc accacatcta gcaatgtgag 180 cgtcctgcag
cagtttgcca gtggcctaaa gagccggaat gaggaaacca gggccaaagc 240
cgccaaggag ctccagcact atgtcaccat ggaactccga gagatgagtc aagaggagtc
300 tactcgcttc tatgaccaac tgaaccatca catttttgaa ttggtttcca
gctcagatgc 360 caatgagagg aaaggtggca tcttggccat agctagcctc
ataggagtgg aaggtgggaa 420 tgccacccga attggcagat ttgccaacta
tcttcggaac ctcctcccct ccaatgaccc 480 agttgtcatg gaaatggcat
ccaaggccat tggccgtctt gccatggcag gggacacttt 540 taccgctgag
tacgtggaat ttgaggtgaa gcgagccctg gaatggctgg gtgctgaccg 600
caatgagggc cggagacatg cagctgtcct ggttctccgt gagctggcca tcagcgtccc
660 taccttcttc ttccagcaag tgcaaccctt ctttgacaac atttttgtgg
ccgtgtggga 720 ccccaaacag gccatccgtg agggagctgt agccgccctt
cgtgcctgtc tgattctcac 780 aacccagcgt gagccgaagg agatgcagaa
gcctcagtgg tacaggcaca catttgaaga 840 agcagagaag ggatttgatg
agaccttggc caaagagaag ggcatgaatc gggatgatcg 900 gatccatgga
gccttgttga tccttaacga gctggtccga atcagcagca tggagggaga 960
gcgtctgaga gaagaaatgg aagaaatcac acagcagcag ctggtacacg acaagtactg
1020 caaagatctc atgggcttcg gaacaaaacc tcgtcacatt acccccttca
ccagtttcca 1080 ggctgtacag ccccagcagt caaatgcctt ggtggggctg
ctggggtaca gctctcacca 1140 aggcctcatg ggatttggga cctcccccag
tccagctaag tccaccctgg tggagagccg 1200 gtgttgcaga gacttgatgg
aggagaaatt tgatcaggtg tgccagtggg tgctgaaatg 1260 caggaatagc
aagaactcgc tgatccaaat gacaatcctt aatttgttgc cccgcttggc 1320
tgcattccga ccttctgcct tcacagatac ccagtatctc caagatacca tgaaccatgt
1380 cctaagctgt gtcaagaagg agaaggaacg tacagcggcc ttccaagccc
tggggctact 1440 ttctgtggct gtgaggtctg agtttaaggt ctatttgcct
cgcgtgctgg acatcatccg 1500 agcggccctg cccccaaagg acttcgccca
taagaggcag aaggcaatgc aggtggatgc 1560 cacagtcttc acttgcatca
gcatgctggc tcgagcaatg gggccaggca tccagcagga 1620 tatcaaggag
ctgctggagc ccatgctggc agtgggacta agccctgccc tcactgcagt 1680
gctctacgac ctgagccgtc agattccaca gctaaagaag gacattcaag atgggctact
1740 gaaaatgctg tccctggtcc ttatgcacaa accccttcgc cacccaggca
tgcccaaggg 1800 cctggcccat cagctggcct ctcctggcct cacgaccctc
cctgaggcca gcgatgtggg 1860 cagcatcact cttgccctcc gaacgcttgg
cagctttgaa tttgaaggcc actctctgac 1920 ccaatttgtt cgccactgtg
cggatcattt cctgaacagt gagcacaagg agatccgcat 1980 ggaggctgcc
cgcacctgct cccgcctgct cacaccctcc atccacctca tcagtggcca 2040
tgctcatgtg gttagccaga ccgcagtgca agtggtggca gatgtgctta gcaaactgct
2100 cgtagttggg ataacagatc ctgaccctga cattcgctac tgtgtcttgg
cgtccctgga 2160 cgagcgcttt gatgcacacc tggcccaggc ggagaacttg
caggccttgt ttgtggctct 2220 gaatgaccag gtgtttgaga tccgggagct
ggccatctgc actgtgggcc gactcagtag 2280 catgaaccct gcctttgtca
tgcctttcct gcgcaagatg ctcatccaga ttttgacaga 2340 gttggagcac
agtgggattg gaagaatcaa agagcagagt gcccgcatgc tggggcacct 2400
ggtctccaat gccccccgac tcatccgccc ctacatggag cctattctga aggcattaat
2460 tttgaaactg aaagatccag accctgatcc aaacccaggt gtgatcaata
atgtcctggc 2520 aacaatagga gaattggcac aggttagtgg cctggaaatg
aggaaatggg ttgatgaact 2580 ttttattatc atcatggaca tgctccagga
ttcctctttg ttggccaaaa ggcaggtggc 2640 tctgtggacc ctgggacagt
tggtggccag cactggctat gtagtagagc cctacaggaa 2700 gtaccctact
ttgcttgagg tgctactgaa ttttctgaag actgagcaga accagggtac 2760
acgcagagag gccatccgtg tgttagggct tttaggggct ttggatcctt acaagcacaa
2820 agtgaacatt ggcatgatag accagtcccg ggatgcctct gctgtcagcc
tgtcagaatc 2880 caagtcaagt caggattcct ctgactatag cactagtgaa
atgctggtca acatgggaaa 2940 cttgcctctg gatgagttct acccagctgt
gtccatggtg gccctgatgc ggatcttccg 3000 agaccagtca ctctctcatc
atcacaccat ggttgtccag gccatcacct tcatcttcaa 3060 gtccctggga
ctcaaatgtg tgcagttcct gccccaggtc atgcccacgt tccttaacgt 3120
cattcgagtc tgtgatgggg ccatccggga atttttgttc cagcagctgg gaatgttggt
3180 gtcctttgtg aagagccaca tcagacctta tatggatgaa atagtcaccc
tcatgagaga 3240 attctgggtc atgaacacct caattcagag cacgatcatt
cttctcattg agcaaattgt 3300 ggtagctctt gggggtgaat ttaagctcta
cctgccccag ctgatcccac acatgctgcg 3360 tgtcttcatg catgacaaca
gcccaggccg cattgtctct atcaagttac tggctgcaat 3420 ccagctgttt
ggcgccaacc tggatgacta cctgcattta ctgctgcctc ctattgttaa 3480
gttgtttgat gcccctgaag ctccactgcc atctcgaaag gcagcgctag agactgtgga
3540 ccgcctgacg gagtccctgg atttcactga ctatgcctcc cggatcattc
accctattgt 3600 tcgaacactg gaccagagcc cagaactgcg ctccacagcc
atggacacgc tgtcttcact 3660 tgtttttcag ctggggaaga agtaccaaat
tttcattcca atggtgaata aagttctggt 3720 gcgacaccga atcaatcatc
agcgctatga tgtgctcatc tgcagaattg tcaagggata 3780 cacacttgct
gatgaagagg aggatccttt gatttaccag catcggatgc ttaggagtgg 3840
ccaaggggat gcattggcta gtggaccagt ggaaacagga cccatgaaga aactgcacgt
3900 cagcaccatc aacctccaaa aggcctgggg cgctgccagg agggtctcca
aagatgactg 3960 gctggaatgg ctgagacggc tgagcctgga gctgctgaag
gactcatcat cgccctccct 4020 gcgctcctgc tgggccctgg cacaggccta
caacccgatg gccagggatc tcttcaatgc 4080 tgcatttgtg tcctgctggt
ctgaactgaa tgaagatcaa caggatgagc tcatcagaag 4140 catcgagttg
gccctcacct cacaagacat cgctgaagtc acacagaccc tcttaaactt 4200
ggctgaattc atggaacaca gtgacaaggg ccccctgcca ctgagagatg acaatggcat
4260 tgttctgctg ggtgagagag ctgccaagtg ccgagcatat gccaaagcac
tacactacaa 4320 agaactggag ttccagaaag gccccacccc tgccattcta
gaatctctca tcagcattaa 4380 taataagcta cagcagccgg aggcagcggc
cggagtgtta gaatatgcca tgaaacactt 4440 tggagagctg gagatccagg
ctacctggta tgagaaactg cacgagtggg aggatgccct 4500 tgtggcctat
gacaagaaaa tggacaccaa caaggacgac ccagagctga tgctgggccg 4560
catgcgctgc ctcgaggcct tgggggaatg gggtcaactc caccagcagt gctgtgaaaa
4620 gtggaccctg gttaatgatg agacccaagc caagatggcc cggatggctg
ctgcagctgc 4680 atggggttta ggtcagtggg acagcatgga agaatacacc
tgtatgatcc ctcgggacac 4740 ccatgatggg gcattttata gagctgtgct
ggcactgcat caggacctct tctccttggc 4800 acaacagtgc attgacaagg
ccagggacct gctggatgct gaattaactg cgatggcagg 4860 agagagttac
agtcgggcat atggggccat ggtttcttgc cacatgctgt ccgagctgga 4920
ggaggttatc cagtacaaac ttgtccccga gcgacgagag atcatccgcc agatctggtg
4980 ggagagactg cagggctgcc agcgtatcgt agaggactgg cagaaaatcc
ttatggtgcg 5040 gtcccttgtg gtcagccctc atgaagacat gagaacctgg
ctcaagtatg caagcctgtg 5100 cggcaagagt ggcaggctgg ctcttgctca
taaaacttta gtgttgctcc tgggagttga 5160 tccgtctcgg caacttgacc
atcctctgcc aacagttcac cctcaggtga cctatgccta 5220 catgaaaaac
atgtggaaga gtgcccgcaa gatcgatgcc ttccagcaca tgcagcattt 5280
tgtccagacc atgcagcaac aggcccagca tgccatcgct actgaggacc agcagcataa
5340 gcaggaactg cacaagctca tggcccgatg cttcctgaaa cttggagagt
ggcagctgaa 5400 tctacagggc atcaatgaga gcacaatccc caaagtgctg
cagtactaca gcgccgccac 5460 agagcacgac cgcagctggt acaaggcctg
gcatgcgtgg gcagtgatga acttcgaagc 5520 tgtgctacac tacaaacatc
agaaccaagc ccgcgatgag aagaagaaac tgcgtcatgc 5580 cagcggggcc
aacatcacca acgccaccac tgccgccacc acggccgcca ctgccaccac 5640
cactgccagc accgagggca gcaacagtga gagcgaggcc gagagcaccg agaacagccc
5700 caccccatcg ccgctgcaga agaaggtcac tgaggatctg tccaaaaccc
tcctgatgta 5760 cacggtgcct gccgtccagg gcttcttccg ttccatctcc
ttgtcacgag gcaacaacct 5820 ccaggataca ctcagagttc tcaccttatg
gtttgattat ggtcactggc cagatgtcaa 5880 tgaggcctta gtggaggggg
tgaaagccat ccagattgat acctggctac aggttatacc 5940 tcagctcatt
gcaagaattg atacgcccag acccttggtg ggacgtctca ttcaccagct 6000
tctcacagac attggtcggt accaccccca ggccctcatc tacccactga cagtggcttc
6060 taagtctacc acgacagccc ggcacaatgc agccaacaag attctgaaga
acatgtgtga 6120 gcacagcaac accctggtcc agcaggccat gatggtgagc
gaggagctga tccgagtggc 6180 catcctctgg catgagatgt ggcatgaagg
cctggaagag gcatctcgtt tgtactttgg 6240 ggaaaggaac gtgaaaggca
tgtttgaggt gctggagccc ttgcatgcta tgatggaacg 6300 gggcccccag
actctgaagg aaacatcctt taatcaggcc tatggtcgag atttaatgga 6360
ggcccaagag tggtgcagga agtacatgaa atcagggaat gtcaaggacc tcacccaagc
6420 ctgggacctc tattatcatg tgttccgacg aatctcaaag cagctgcctc
agctcacatc 6480 cttagagctg caatatgttt ccccaaaact tctgatgtgc
cgggaccttg aattggctgt 6540 gccaggaaca tatgacccca accagccaat
cattcgcatt cagtccatag caccgtcttt 6600 gcaagtcatc acatccaagc
agaggccccg gaaattgaca cttatgggca gcaacggaca 6660 tgagtttgtt
ttccttctaa aaggccatga agatctgcgc caggatgagc gtgtgatgca 6720
gctcttcggc ctggttaaca cccttctggc caatgaccca acatctcttc ggaaaaacct
6780 cagcatccag agatacgctg tcatcccttt atcgaccaac tcgggcctca
ttggctgggt 6840 tccccactgt gacacactgc acgccctcat ccgggactac
agggagaaga agaagatcct 6900 tctcaacatc gagcatcgca tcatgttgcg
gatggctccg gactatgacc acttgactct 6960 gatgcagaag gtggaggtgt
ttgagcatgc cgtcaataat acagctgggg acgacctggc 7020 caagctgctg
tggctgaaaa gccccagctc cgaggtgtgg tttgaccgaa gaaccaatta 7080
tacccgttct ttagcggtca tgtcaatggt tgggtatatt ttaggcctgg gagatagaca
7140 cccatccaac ctgatgctgg accgtctgag tgggaagatc ctgcacattg
actttgggga 7200 ctgctttgag gttgctatga cccgagagaa gtttccagag
aagattccat ttagactaac 7260 aagaatgttg accaatgcta tggaggttac
aggcctggat ggcaactaca gaatcacatg 7320 ccacacagtg atggaggtgc
tgcgagagca caaggacagt gtcatggccg tgctggaagc 7380 ctttgtctat
gaccccttgc tgaactggag gctgatggac acaaatacca aaggcaacaa 7440
gcgatcccga acgaggacgg attcctactc tgctggccag tcagtcgaaa ttttggacgg
7500 tgtggaactt ggagagccag cccataagaa aacggggacc acagtgccag
aatctattca 7560 ttctttcatt ggagacggtt tggtgaaacc agaggcccta
aataagaaag ctatccagat 7620 tattaacagg gttcgagata agctcactgg
tcgggacttc tctcatgatg acactttgga 7680 tgttccaacg caagttgagc
tgctcatcaa acaagcgaca tcccatgaaa acctctgcca 7740 gtgctatatt
ggctggtgcc ctttctggta actggaggcc cagatgtgcc catcacgttt 7800
tttctgaggc ttttgtactt tagtaaatgc ttccactaaa ctgaaaccat ggtgagaaag
7860 tttgactttg ttaaatattt tgaaatgtaa atgaaaagaa ctactgtata
ttaaaagttg 7920 gtttgaacca actttctagc tgctgttgaa gaatatattg
tcagaaacac aaggcttgat 7980 ttggttccca ggacagtgaa acatagtaat
accacgtaaa tcaagccatt cattttgggg 8040 aacagaagat ccataacttt
agaaatacgg gttttgactt aactcacaag agaactcatc 8100 ataagtactt
gctgatggaa gaatgaccta gttgctcctc tcaacatggg tacagcaaac 8160
tcagcacagc caagaagcct caggtcgtgg agaacatgga ttaggatcct agactgtaaa
8220 gacacagaag atgctgacct cacccctgcc acctatccca agacctcact
ggtctgtgga 8280 cagcagcaga aatgtttgca agataggcca aaatgagtac
aaaaggtctg tcttccatca 8340 gacccagtga tgctgcgact cacacgcttc
aattcaagac ctgaccgcta gtagggaggt 8400 ttattcagat cgctggcagc
ctcggctgag cagatgcaca gaggggatca ctgtgcagtg 8460 ggaccaccct
cactggcctt ctgcagcagg gttctgggat gttttcagtg gtcaaaatac 8520
tctgtttaga gcaagggctc agaaaacaga aatactgtca tggaggtgct gaacacaggg
8580 aaggtctggt acatattgga aattatgagc agaacaaata ctcaactaaa
tgcacaaagt 8640 ataaagtgta gccatgtcta gacaccatgt tgtatcagaa
taatttttgt gccaataaat 8700 gacatcagaa ttttaaacat atgtaaaaaa aaa
8733 <210> SEQ ID NO 4 <211> LENGTH: 2549 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <300>
PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:
Genbank/004958 <309> DATABASE ENTRY DATE: 2002-04-04
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(2459) <400>
SEQUENCE: 4 Met Leu Gly Thr Gly Pro Ala Ala Ala Thr Thr Ala Ala Thr
Thr Ser 1 5 10 15 Ser Asn Val Ser Val Leu Gln Gln Phe Ala Ser Gly
Leu Lys Ser Arg 20 25 30 Asn Glu Glu Thr Arg Ala Lys Ala Ala Lys
Glu Leu Gln His Tyr Val 35 40 45 Thr Met Glu Leu Arg Glu Met Ser
Gln Glu Glu Ser Thr Arg Phe Tyr 50 55 60 Asp Gln Leu Asn His His
Ile Phe Glu Leu Val Ser Ser Ser Asp Ala 65 70 75 80 Asn Glu Arg Lys
Gly Gly Ile Leu Ala Ile Ala Ser Leu Ile Gly Val 85 90 95 Glu Gly
Gly Asn Ala Thr Arg Ile Gly Arg Phe Ala Asn Tyr Leu Arg 100 105 110
Asn Leu Leu Pro Ser Asn Asp Pro Val Val Met Glu Met Ala Ser Lys 115
120 125 Ala Ile Gly Arg Leu Ala Met Ala Gly Asp Thr Phe Thr Ala Glu
Tyr 130 135 140 Val Glu Phe Glu Val Lys Arg Ala Leu Glu Trp Leu Gly
Ala Asp Arg 145 150 155 160 Asn Glu Gly Arg Arg His Ala Ala Val Leu
Val Leu Arg Glu Leu Ala 165 170 175 Ile Ser Val Pro Thr Phe Phe Phe
Gln Gln Val Gln Pro Phe Phe Asp 180 185 190 Asn Ile Phe Val Ala Val
Trp Asp Pro Lys Gln Ala Ile Arg Glu Gly 195 200 205 Ala Val Ala Ala
Leu Arg Ala Cys Leu Ile Leu Thr Thr Gln Arg Glu 210 215 220 Pro Lys
Glu Met Gln Lys Pro Gln Trp Tyr Arg His Thr Phe Glu Glu 225 230 235
240 Ala Glu Lys Gly Phe Asp Glu Thr Leu Ala Lys Glu Lys Gly Met Asn
245 250 255 Arg Asp Asp Arg Ile His Gly Ala Leu Leu Ile Leu Asn Glu
Leu Val 260 265 270 Arg Ile Ser Ser Met Glu Gly Glu Arg Leu Arg Glu
Glu Met Glu Glu 275 280 285 Ile Thr Gln Gln Gln Leu Val His Asp Lys
Tyr Cys Lys Asp Leu Met 290 295 300 Gly Phe Gly Thr Lys Pro Arg His
Ile Thr Pro Phe Thr Ser Phe Gln 305 310 315 320 Ala Val Gln Pro Gln
Gln Ser Asn Ala Leu Val Gly Leu Leu Gly Tyr 325 330 335 Ser Ser His
Gln Gly Leu Met Gly Phe Gly Thr Ser Pro Ser Pro Ala 340 345 350 Lys
Ser Thr Leu Val Glu Ser Arg Cys Cys Arg Asp Leu Met Glu Glu 355 360
365 Lys Phe Asp Gln Val Cys Gln Trp Val Leu Lys Cys Arg Asn Ser Lys
370 375 380 Asn Ser Leu Ile Gln Met Thr Ile Leu Asn Leu Leu Pro Arg
Leu Ala 385 390 395 400 Ala Phe Arg Pro Ser Ala Phe Thr Asp Thr Gln
Tyr Leu Gln Asp Thr 405 410 415 Met Asn His Val Leu Ser Cys Val Lys
Lys Glu Lys Glu Arg Thr Ala 420 425 430 Ala Phe Gln Ala Leu Gly Leu
Leu Ser Val Ala Val Arg Ser Glu Phe 435 440 445 Lys Val Tyr Leu Pro
Arg Val Leu Asp Ile Ile Arg Ala Ala Leu Pro 450 455 460 Pro Lys Asp
Phe Ala His Lys Arg Gln Lys Ala Met Gln Val Asp Ala 465 470 475 480
Thr Val Phe Thr Cys Ile Ser Met Leu Ala Arg Ala Met Gly Pro Gly 485
490 495 Ile Gln Gln Asp Ile Lys Glu Leu Leu Glu Pro Met Leu Ala Val
Gly 500 505 510 Leu Ser Pro Ala Leu Thr Ala Val Leu Tyr Asp Leu Ser
Arg Gln Ile 515 520 525 Pro Gln Leu Lys Lys Asp Ile Gln Asp Gly Leu
Leu Lys Met Leu Ser 530 535 540 Leu Val Leu Met His Lys Pro Leu Arg
His Pro Gly Met Pro Lys Gly 545 550 555 560 Leu Ala His Gln Leu Ala
Ser Pro Gly Leu Thr Thr Leu Pro Glu Ala 565 570 575 Ser Asp Val Gly
Ser Ile Thr Leu Ala Leu Arg Thr Leu Gly Ser Phe 580 585 590 Glu Phe
Glu Gly His Ser Leu Thr Gln Phe Val Arg His Cys Ala Asp 595 600 605
His Phe Leu Asn Ser Glu His Lys Glu Ile Arg Met Glu Ala Ala Arg 610
615 620 Thr Cys Ser Arg Leu Leu Thr Pro Ser Ile His Leu Ile Ser Gly
His 625 630 635 640 Ala His Val Val Ser Gln Thr Ala Val Gln Val Val
Ala Asp Val Leu 645 650 655 Ser Lys Leu Leu Val Val Gly Ile Thr Asp
Pro Asp Pro Asp Ile Arg 660 665 670 Tyr Cys Val Leu Ala Ser Leu Asp
Glu Arg Phe Asp Ala His Leu Ala 675 680 685 Gln Ala Glu Asn Leu Gln
Ala Leu Phe Val Ala Leu Asn Asp Gln Val 690 695 700 Phe Glu Ile Arg
Glu Leu Ala Ile Cys Thr Val Gly Arg Leu Ser Ser 705 710 715 720 Met
Asn Pro Ala Phe Val Met Pro Phe Leu Arg Lys Met Leu Ile Gln 725 730
735 Ile Leu Thr Glu Leu Glu His Ser Gly Ile Gly Arg Ile Lys Glu Gln
740 745 750 Ser Ala Arg Met Leu Gly His Leu Val Ser Asn Ala Pro Arg
Leu Ile 755 760 765 Arg Pro Tyr Met Glu Pro Ile Leu Lys Ala Leu Ile
Leu Lys Leu Lys 770 775 780 Asp Pro Asp Pro Asp Pro Asn Pro Gly Val
Ile Asn Asn Val Leu Ala 785 790 795 800 Thr Ile Gly Glu Leu Ala Gln
Val Ser Gly Leu Glu Met Arg Lys Trp 805 810 815 Val Asp Glu Leu Phe
Ile Ile Ile Met Asp Met Leu Gln Asp Ser Ser 820 825 830 Leu Leu Ala
Lys Arg Gln Val Ala Leu Trp Thr Leu Gly Gln Leu Val 835 840 845 Ala
Ser Thr Gly Tyr Val Val Glu Pro Tyr Arg Lys Tyr Pro Thr Leu 850 855
860 Leu Glu Val Leu Leu Asn Phe Leu Lys Thr Glu Gln Asn Gln Gly Thr
865 870 875 880 Arg Arg Glu Ala Ile Arg Val Leu Gly Leu Leu Gly Ala
Leu Asp Pro 885 890 895 Tyr Lys His Lys Val Asn Ile Gly Met Ile Asp
Gln Ser Arg Asp Ala 900 905 910 Ser Ala Val Ser Leu Ser Glu Ser Lys
Ser Ser Gln Asp Ser Ser Asp 915 920 925 Tyr Ser Thr Ser Glu Met Leu
Val Asn Met Gly Asn Leu Pro Leu Asp 930 935 940 Glu Phe Tyr Pro Ala
Val Ser Met Val Ala Leu Met Arg Ile Phe Arg 945 950 955 960 Asp Gln
Ser Leu Ser His His His Thr Met Val Val Gln Ala Ile Thr 965 970 975
Phe Ile Phe Lys Ser Leu Gly Leu Lys Cys Val Gln Phe Leu Pro Gln 980
985 990 Val Met Pro Thr Phe Leu Asn Val Ile Arg Val Cys Asp Gly Ala
Ile 995 1000 1005 Arg Glu Phe Leu Phe Gln Gln Leu Gly Met Leu Val
Ser Phe Val 1010 1015 1020 Lys Ser His Ile Arg Pro Tyr Met Asp Glu
Ile Val Thr Leu Met 1025 1030 1035 Arg Glu Phe Trp Val Met Asn Thr
Ser Ile Gln Ser Thr Ile Ile 1040 1045 1050 Leu Leu Ile Glu Gln Ile
Val Val Ala Leu Gly Gly Glu Phe Lys 1055 1060 1065 Leu Tyr Leu Pro
Gln Leu Ile Pro His Met Leu Arg Val Phe Met 1070 1075 1080 His Asp
Asn Ser Pro Gly Arg Ile Val Ser Ile Lys Leu Leu Ala 1085 1090 1095
Ala Ile Gln Leu Phe Gly Ala Asn Leu Asp Asp Tyr Leu His Leu 1100
1105 1110 Leu Leu Pro Pro Ile Val Lys Leu Phe Asp Ala Pro Glu Ala
Pro 1115 1120 1125 Leu Pro Ser Arg Lys Ala Ala Leu Glu Thr Val Asp
Arg Leu Thr 1130 1135 1140 Glu Ser Leu Asp Phe Thr Asp Tyr Ala Ser
Arg Ile Ile His Pro 1145 1150 1155 Ile Val Arg Thr Leu Asp Gln Ser
Pro Glu Leu Arg Ser Thr Ala 1160 1165 1170 Met Asp Thr Leu Ser Ser
Leu Val Phe Gln Leu Gly Lys Lys Tyr 1175 1180 1185 Gln Ile Phe Ile
Pro Met Val Asn Lys Val Leu Val Arg His Arg 1190 1195 1200 Ile Asn
His Gln Arg Tyr Asp Val Leu Ile Cys Arg Ile Val Lys 1205 1210 1215
Gly Tyr Thr Leu Ala Asp Glu Glu Glu Asp Pro Leu Ile Tyr Gln 1220
1225 1230 His Arg Met Leu Arg Ser Gly Gln Gly Asp Ala Leu Ala Ser
Gly 1235 1240 1245 Pro Val Glu Thr Gly Pro Met Lys Lys Leu His Val
Ser Thr Ile 1250 1255 1260 Asn Leu Gln Lys Ala Trp Gly Ala Ala Arg
Arg Val Ser Lys Asp 1265 1270 1275 Asp Trp Leu Glu Trp Leu Arg Arg
Leu Ser Leu Glu Leu Leu Lys 1280 1285 1290 Asp Ser Ser Ser Pro Ser
Leu Arg Ser Cys Trp Ala Leu Ala Gln 1295 1300 1305 Ala Tyr Asn Pro
Met Ala Arg Asp Leu Phe Asn Ala Ala Phe Val 1310 1315 1320 Ser Cys
Trp Ser Glu Leu Asn Glu Asp Gln Gln Asp Glu Leu Ile 1325 1330 1335
Arg Ser Ile Glu Leu Ala Leu Thr Ser Gln Asp Ile Ala Glu Val 1340
1345 1350 Thr Gln Thr Leu Leu Asn Leu Ala Glu Phe Met Glu His Ser
Asp 1355 1360 1365 Lys Gly Pro Leu Pro Leu Arg Asp Asp Asn Gly Ile
Val Leu Leu 1370 1375 1380 Gly Glu Arg Ala Ala Lys Cys Arg Ala Tyr
Ala Lys Ala Leu His 1385 1390 1395 Tyr Lys Glu Leu Glu Phe Gln Lys
Gly Pro Thr Pro Ala Ile Leu 1400 1405 1410 Glu Ser Leu Ile Ser Ile
Asn Asn Lys Leu Gln Gln Pro Glu Ala 1415 1420 1425 Ala Ala Gly Val
Leu Glu Tyr Ala Met Lys His Phe Gly Glu Leu 1430 1435 1440 Glu Ile
Gln Ala Thr Trp Tyr Glu Lys Leu His Glu Trp Glu Asp 1445 1450 1455
Ala Leu Val Ala Tyr Asp Lys Lys Met Asp Thr Asn Lys Asp Asp 1460
1465 1470 Pro Glu Leu Met Leu Gly Arg Met Arg Cys Leu Glu Ala Leu
Gly 1475 1480 1485 Glu Trp Gly Gln Leu His Gln Gln Cys Cys Glu Lys
Trp Thr Leu 1490 1495 1500 Val Asn Asp Glu Thr Gln Ala Lys Met Ala
Arg Met Ala Ala Ala 1505 1510 1515 Ala Ala Trp Gly Leu Gly Gln Trp
Asp Ser Met Glu Glu Tyr Thr 1520 1525 1530 Cys Met Ile Pro Arg Asp
Thr His Asp Gly Ala Phe Tyr Arg Ala 1535 1540 1545 Val Leu Ala Leu
His Gln Asp Leu Phe Ser Leu Ala Gln Gln Cys 1550 1555 1560 Ile Asp
Lys Ala Arg Asp Leu Leu Asp Ala Glu Leu Thr Ala Met 1565 1570 1575
Ala Gly Glu Ser Tyr Ser Arg Ala Tyr Gly Ala Met Val Ser Cys 1580
1585 1590 His Met Leu Ser Glu Leu Glu Glu Val Ile Gln Tyr Lys Leu
Val 1595 1600 1605 Pro Glu Arg Arg Glu Ile Ile Arg Gln Ile Trp Trp
Glu Arg Leu 1610 1615 1620 Gln Gly Cys Gln Arg Ile Val Glu Asp Trp
Gln Lys Ile Leu Met 1625 1630 1635 Val Arg Ser Leu Val Val Ser Pro
His Glu Asp Met Arg Thr Trp 1640 1645 1650 Leu Lys Tyr Ala Ser Leu
Cys Gly Lys Ser Gly Arg Leu Ala Leu 1655 1660 1665 Ala His Lys Thr
Leu Val Leu Leu Leu Gly Val Asp Pro Ser Arg 1670 1675 1680 Gln Leu
Asp His Pro Leu Pro Thr Val His Pro Gln Val Thr Tyr 1685 1690 1695
Ala Tyr Met Lys Asn Met Trp Lys Ser Ala Arg Lys Ile Asp Ala 1700
1705 1710 Phe Gln His Met Gln His Phe Val Gln Thr Met Gln Gln Gln
Ala 1715 1720 1725 Gln His Ala Ile Ala Thr Glu Asp Gln Gln His Lys
Gln Glu Leu 1730 1735 1740 His Lys Leu Met Ala Arg Cys Phe Leu Lys
Leu Gly Glu Trp Gln 1745 1750 1755 Leu Asn Leu Gln Gly Ile Asn Glu
Ser Thr Ile Pro Lys Val Leu 1760 1765 1770 Gln Tyr Tyr Ser Ala Ala
Thr Glu His Asp Arg Ser Trp Tyr Lys 1775 1780 1785 Ala Trp His Ala
Trp Ala Val Met Asn Phe Glu Ala Val Leu His 1790 1795 1800 Tyr Lys
His Gln Asn Gln Ala Arg Asp Glu Lys Lys Lys Leu Arg 1805 1810 1815
His Ala Ser Gly Ala Asn Ile Thr Asn Ala Thr Thr Ala Ala Thr 1820
1825 1830 Thr Ala Ala Thr Ala Thr Thr Thr Ala Ser Thr Glu Gly Ser
Asn 1835 1840 1845 Ser Glu Ser Glu Ala Glu Ser Thr Glu Asn Ser Pro
Thr Pro Ser 1850 1855 1860 Pro Leu Gln Lys Lys Val Thr Glu Asp Leu
Ser Lys Thr Leu Leu 1865 1870 1875 Met Tyr Thr Val Pro Ala Val Gln
Gly Phe Phe Arg Ser Ile Ser 1880 1885 1890 Leu Ser Arg Gly Asn Asn
Leu Gln Asp Thr Leu Arg Val Leu Thr 1895 1900 1905 Leu Trp Phe Asp
Tyr Gly His Trp Pro Asp Val Asn Glu Ala Leu 1910 1915 1920 Val Glu
Gly Val Lys Ala Ile Gln Ile Asp Thr Trp Leu Gln Val 1925 1930 1935
Ile Pro Gln Leu Ile Ala Arg Ile Asp Thr Pro Arg Pro Leu Val 1940
1945 1950 Gly Arg Leu Ile His Gln Leu Leu Thr Asp Ile Gly Arg Tyr
His 1955 1960 1965 Pro Gln Ala Leu Ile Tyr Pro Leu Thr Val Ala Ser
Lys Ser Thr 1970 1975 1980 Thr Thr Ala Arg His Asn Ala Ala Asn Lys
Ile Leu Lys Asn Met 1985 1990 1995 Cys Glu His Ser Asn Thr Leu Val
Gln Gln Ala Met Met Val Ser 2000 2005 2010 Glu Glu Leu Ile Arg Val
Ala Ile Leu Trp His Glu Met Trp His 2015 2020 2025 Glu Gly Leu Glu
Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn 2030 2035 2040 Val Lys
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met 2045 2050 2055
Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala 2060
2065 2070 Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp Cys Arg Lys
Tyr 2075 2080 2085 Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala
Trp Asp Leu 2090 2095 2100 Tyr Tyr His Val Phe Arg Arg Ile Ser Lys
Gln Leu Pro Gln Leu 2105 2110 2115 Thr Ser Leu Glu Leu Gln Tyr Val
Ser Pro Lys Leu Leu Met Cys 2120 2125 2130 Arg Asp Leu Glu Leu Ala
Val Pro Gly Thr Tyr Asp Pro Asn Gln 2135 2140 2145 Pro Ile Ile Arg
Ile Gln Ser Ile Ala Pro Ser Leu Gln Val Ile 2150 2155 2160 Thr Ser
Lys Gln Arg Pro Arg Lys Leu Thr Leu Met Gly Ser Asn 2165 2170 2175
Gly His Glu Phe Val Phe Leu Leu Lys Gly His Glu Asp Leu Arg 2180
2185 2190 Gln Asp Glu Arg Val Met Gln Leu Phe Gly Leu Val Asn Thr
Leu 2195 2200 2205 Leu Ala Asn Asp Pro Thr Ser Leu Arg Lys Asn Leu
Ser Ile Gln 2210 2215 2220 Arg Tyr Ala Val Ile Pro Leu Ser Thr Asn
Ser Gly Leu Ile Gly 2225 2230 2235 Trp Val Pro His Cys Asp Thr Leu
His Ala Leu Ile Arg Asp Tyr 2240 2245 2250 Arg Glu Lys Lys Lys Ile
Leu Leu Asn Ile Glu His Arg Ile Met 2255 2260 2265 Leu Arg Met Ala
Pro Asp Tyr Asp His Leu Thr Leu Met Gln Lys 2270 2275 2280 Val Glu
Val Phe Glu His Ala Val Asn Asn Thr Ala Gly Asp Asp 2285 2290 2295
Leu Ala Lys Leu Leu Trp Leu Lys Ser Pro Ser Ser Glu Val Trp 2300
2305 2310 Phe Asp Arg Arg Thr Asn Tyr Thr Arg Ser Leu Ala Val Met
Ser 2315 2320 2325 Met Val Gly Tyr Ile Leu Gly Leu Gly Asp Arg His
Pro Ser Asn 2330 2335 2340 Leu Met Leu Asp Arg Leu Ser Gly Lys Ile
Leu His Ile Asp Phe 2345 2350 2355 Gly Asp Cys Phe Glu Val Ala Met
Thr Arg Glu Lys Phe Pro Glu 2360 2365 2370 Lys Ile Pro Phe Arg Leu
Thr Arg Met Leu Thr Asn Ala Met Glu 2375 2380 2385 Val Thr Gly Leu
Asp Gly Asn Tyr Arg Ile Thr Cys His Thr Val 2390 2395 2400 Met Glu
Val Leu Arg Glu His Lys Asp Ser Val Met Ala Val Leu 2405 2410 2415
Glu Ala Phe Val Tyr Asp Pro Leu Leu Asn Trp Arg Leu Met Asp 2420
2425 2430 Thr Asn Thr Lys Gly Asn Lys Arg Ser Arg Thr Arg Thr Asp
Ser 2435 2440 2445 Tyr Ser Ala Gly Gln Ser Val Glu Ile Leu Asp Gly
Val Glu Leu 2450 2455 2460 Gly Glu Pro Ala His Lys Lys Thr Gly Thr
Thr Val Pro Glu Ser 2465 2470 2475 Ile His Ser Phe Ile Gly Asp Gly
Leu Val Lys Pro Glu Ala Leu 2480 2485 2490 Asn Lys Lys Ala Ile Gln
Ile Ile Asn Arg Val Arg Asp Lys Leu 2495 2500 2505 Thr Gly Arg Asp
Phe Ser His Asp Asp Thr Leu Asp Val Pro Thr 2510 2515 2520 Gln Val
Glu Leu Leu Ile Lys Gln Ala Thr Ser His Glu Asn Leu 2525 2530 2535
Cys Gln Cys Tyr Ile Gly Trp Cys Pro Phe Trp 2540 2545 <210>
SEQ ID NO 5 <211> LENGTH: 8155 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <300> PUBLICATION
INFORMATION: <308> DATABASE ACCESSION NUMBER: Genbank
BC117166 <309> DATABASE ENTRY DATE: 2006-06-26 <313>
RELEVANT RESIDUES IN SEQ ID NO: (1)..(8155) <400> SEQUENCE: 5
cgcgaacctc agggcaagat gcttggaacc ggacctgccg ccgccaccac cgctgccacc
60 acatctagca atgtgagcgt cctgcagcag tttgccagtg gcctaaagag
ccggaatgag 120 gaaaccaggg ccaaagccgc caaggagctc cagcactatg
tcaccatgga actccgagag 180 atgagtcaag aggagtctac tcgcttctat
gaccaactga accatcacat ttttgaattg 240 gtttccagct cagatgccaa
tgagaggaaa ggtggcatct tggccatagc tagcctcata 300 ggagtggaag
gtgggaatgc cacccgaatt ggcagatttg ccaactatct tcggaacctc 360
ctcccctcca atgacccagt tgtcatggaa atggcatcca aggccattgg ccgtcttgcc
420 atggcagggg acacttttac cgctgagtac gtggaatttg aggtgaagcg
agccctggaa 480 tggctgggtg ctgaccgcaa tgagggccgg agacatgcag
ctgtcctggt tctccgtgag 540 ctggccatca gcgtccctac cttcttcttc
cagcaagtgc aacccttctt tgacaacatt 600 tttgtggccg tgtgggaccc
caaacaggcc atccgtgagg gagctgtagc cgcccttcgt 660 gcctgtctga
ttctcacaac ccagcgtgag ccgaaggaga tgcagaagcc tcagtggtac 720
aggcacacat ttgaagaagc agagaaggga tttgatgaga ccttggccaa agagaagggc
780 atgaatcggg atgatcggat ccatggagcc ttgttgatcc ttaacgagct
ggtccgaatc 840 agcagcatgg agggagagcg tctgagagaa gaaatggaag
aaatcacaca gcagcagctg 900 gtacacgaca agtactgcaa agatctcatg
ggcttcggaa caaaacctcg tcacattacc 960 cccttcacca gtttccaggc
tgtacagccc cagcagtcaa atgccttggt ggggctgctg 1020 gggtacagct
ctcaccaagg cctcatggga tttgggacct cccccagtcc agctaagtcc 1080
accctggtgg agagccggtg ttgcagagac ttgatggagg agaaatttga tcaggtgtgc
1140 cagtgggtgc tgaaatgcag gaatagcaag aactcgctga tccaaatgac
aatccttaat 1200 ttgttgcccc gcttggctgc attccgacct tctgccttca
cagataccca gtatctccaa 1260 gataccatga accatgtcct aagctgtgtc
aagaaggaga aggaacgtac agcggccttc 1320 caagccctgg ggctactttc
tgtggctgtg aggtctgagt ttaaggtcta tttgcctcgc 1380 gtgctggaca
tcatccgagc ggccctgccc ccaaaggact tcgcccataa gaggcagaag 1440
gcaatgcagg tggacgccac agtcttcact tgcatcagca tgctggctcg agcaatgggg
1500 ccaggcatcc agcaggatat caaggagctg ctggagccca tgctggcagt
gggactaagc 1560 cctgccctca ctgcagtgct ctacgacctg agccgtcaga
ttccacagct aaagaaggac 1620 attcaagatg ggctactgaa aatgctgtcc
ctggtcctta tgcacaaacc ccttcgccac 1680 ccaggcatgc ccaagggcct
ggcccatcag ctggcctctc ctggcctcac gaccctccct 1740 gaggccagcg
atgtgggcag catcactctt gccctccgaa cgcttggcag ctttgaattt 1800
gaaggccact ctctgaccca atttgttcgc cactgtgcgg atcattttct gaacagtgag
1860 cacaaggaga tccgcatgga ggctgcccgc acctgctccc gcctgctcac
accctccatc 1920 cacctcatca gtggccatgc tcatgtggtt agccagaccg
cagtgcaagt ggtggcagat 1980 gtgcttagca aactgctcgt agttgggata
acagatcctg accctgacat tcgctactgt 2040 gtcttggcgt ccctggacga
gcgctttgat gcacacctgg cccaggcgga gaacttgcag 2100 gccttgtttg
tggctctgaa tgaccaggtg tttgagatcc gggagctggc catctgcact 2160
gtgggccgac tcagtagcat gaaccctgcc tttgtcatgc ctttcctgcg caagatgctc
2220 atccagattt tgacagagtt ggagcacagt gggattggaa gaatcaaaga
gcagagtgcc 2280 cgcatgctgg ggcacctggt ctccaatgcc ccccgactca
tccgccccta catggagcct 2340 attctgaagg cattaatttt gaaactgaaa
gatccagacc ctgatccaaa cccaggtgtg 2400 atcaataatg tcctggcaac
aataggagaa ttggcacagg ttagtggcct ggaaatgagg 2460 aaatgggttg
atgaactttt tattatcatc atggacatgc tccaggattc ctctttgttg 2520
gccaaaaggc aggtggctct gtggaccctg ggacagttgg tggccagcac tggctatgta
2580 gtagagccct acaggaagta ccctactttg cttgaggtgc tactgaattt
tctgaagact 2640 gagcagaacc agggtacacg cagagaggcc atccgtgtgt
tagggctttt aggggctttg 2700 gatccttaca agcacaaagt gaacattggc
atgatagacc agtcccggga tgcctctgct 2760 gtcagcctgt cagaatccaa
gtcaagtcag gattcctctg actatagcac tagtgaaatg 2820 ctggtcaaca
tgggaaactt gcctctggat gagttctacc cagctgtgtc catggtggcc 2880
ctgatgcgga tcttccgaga ccagtcactc tctcatcatc acaccatggt tgtccaggcc
2940 atcaccttca tcttcaagtc cctgggactc aaatgtgtgc agttcctgcc
ccaggtcatg 3000 cccacgttcc ttaatgtcat tcgagtctgt gatggggcca
tccgggaatt tttgttccag 3060 cagctgggaa tgttggtgtc ctttgtgaag
agccacatca gaccttatat ggatgaaata 3120 gtcaccctca tgagagaatt
ctgggtcatg aacacctcaa ttcagagcac gatcattctt 3180 ctcattgagc
aaattgtggt agctcttggg ggtgaattta agctctacct gccccagctg 3240
atcccacaca tgctgcgtgt cttcatgcat gacaacagcc caggccgcat tgtctctatc
3300 aagttactgg ctgcaatcca gctgtttggc gccaacctgg atgactacct
gcatttactg 3360 ctgcctccta ttgttaagtt gtttgatgcc cctgaagctc
cactgccatc tcgaaaggca 3420 gcgctagaga ctgtggaccg cctgacggag
tccctggatt tcactgacta tgcctcccgg 3480 atcattcacc ctattgttcg
aacactggac cagagcccag aactgcgctc cacagccatg 3540 gacacgctgt
cttcacttgt ttttcagctg gggaagaagt accaaatttt cattccaatg 3600
gtgaataaag ttctggtgcg acaccgaatc aatcatcagc gctatgatgt gctcatctgc
3660 agaattgtca agggatacac acttgctgat gaagaggagg atcctttgat
ttaccagcat 3720 cggatgctta ggagtggcca aggggatgca ttggctagtg
gaccagtgga aacaggaccc 3780 atgaagaaac tgcacgtcag caccatcaac
ctccaaaagg cctggggcgc tgccaggagg 3840 gtctccaaag atgactggct
ggaatggctg agacggctga gcctggagct gctgaaggac 3900 tcatcatcgc
cctccctgcg ctcctgctgg gccctggcac aggcctacaa cccgatggcc 3960
agggatctct tcaatgctgc atttgtgtcc tgctggtctg aactgaatga agatcaacag
4020 gatgagctca tcagaagcat cgagttggcc ctcacctcac aagacatcgc
tgaagtcaca 4080 cagaccctct taaacttggc tgaattcatg gaacacagtg
acaagggccc cctgccactg 4140 agagatgaca atggcattgt tctgctgggt
gagagagctg ccaagtgccg agcatatgcc 4200 aaagcactac actacaaaga
actggagttc cagaaaggcc ccacccctgc cattctagaa 4260 tctctcatca
gcattaataa taagctacag cagccggagg cagcggccgg agtgttagaa 4320
tatgccatga aacactttgg agagctggag atccaggcta cctggtatga gaaactgcac
4380 gagtgggagg atgcccttgt ggcctatgac aagaaaatgg acaccaacaa
ggacgaccca 4440 gagctgatgc tgggccgcat gcgctgcctc gaggccttgg
gggaatgggg tcaactccac 4500 cagcagtgct gtgaaaagtg gaccctggtt
aatgatgaga cccaagccaa gatggcccgg 4560 atggctgctg cagctgcatg
gggtttaggt cagtgggaca gcatggaaga atacacctgt 4620 atgatccctc
gggacaccca tgatggggca ttttatagag ctgtgctggc actgcatcag 4680
gacctcttct ccttggcaca acagtgcatt gacaaggcca gggacctgct ggatgctgaa
4740 ttaactgcga tggcaggaga gagttacagt cgggcatatg gggccatggt
ttcttgccac 4800 atgctgtccg agctggagga ggttatccag tacaaacttg
tccccgagcg acgagagatc 4860 atccgccaga tctggtggga gagactgcag
ggctgccagc gtatcgtaga ggactggcag 4920 aaaatcctta tggtgcggtc
ccttgtggtc agccctcatg aagacatgag aacctggctc 4980 aagtatgcaa
gcctgtgcgg caagagtggc aggctggctc ttgctcataa aactttagtg 5040
ttgctcctgg gagttgatcc gtctcggcaa cttgaccatc ctctgccaac agttcaccct
5100 caggtgacct atgcctacat gaaaaacatg tggaagagtg cccgcaagat
cgatgccttc 5160 cagcacatgc agcattttgt ccagaccatg cagcaacagg
cccagcatgc catcgctact 5220 gaggaccagc agcataagca ggaactgcac
aagctcatgg cccgatgctt cctgaaactt 5280 ggagagtggc agctgaatct
acagggcatc aatgagagca caatccccaa agtgctgcag 5340 tactacagcg
ccgccacaga gcacgaccgc agctggtaca aggcctggca tgcgtgggca 5400
gtgatgaact tcgaagctgt gctacactac aaacatcaga accaagcccg cgatgagaag
5460 aagaaactgc gtcatgccag cggggccaac atcaccaacg ccaccactgc
cgccaccacg 5520 gccgccactg ccaccaccac tgccagcacc gagggcagca
acagtgagag tgaggccgag 5580 agcaccgaga acagccccac cccatcgccg
ctgcagaaga aggtcactga ggatctgtcc 5640 aaaaccctcc tgatgtacac
ggtgcctgcc gtccagggct tcttccgttc catctccttg 5700 tcacgaggca
acaacctcca ggatacactc agagttctca ccttatggtt tgattatggt 5760
cactggccag atgtcaatga ggccttagtg gagggggtga aagccatcca gattgatacc
5820 tggctacagg ttatacctca gctcattgca agaattgata cgcccagacc
cttggtggga 5880 cgtctcattc accagcttct cacagacatt ggtcggtacc
acccccaggc cctcatctac 5940 ccactgacag tggcttctaa gtctaccacg
acagcccggc acaatgcagc caacaagatt 6000 ctgaagaaca tgtgtgagca
cagcaacacc ctggtccagc aggccatgat ggtgagcgag 6060 gagctgatcc
gagtggccat cctctggcat gagatgtggc atgaaggcct ggaagaggca 6120
tctcgtttgt actttgggga aaggaacgtg aaaggcatgt ttgaggtgct ggagcccttg
6180 catgctatga tggaacgggg cccccagact ctgaaggaaa catcctttaa
tcaggcctat 6240 ggtcgagatt taatggaggc ccaagagtgg tgcaggaagt
acatgaaatc agggaatgtc 6300 aaggacctca cccaagcctg ggacctctat
tatcatgtgt tccgacgaat ctcaaagcag 6360 ctgcctcagc tcacatcctt
agagctgcaa tatgtttccc caaaacttct gatgtgccgg 6420 gaccttgaat
tggctgtgcc aggaacatat gaccccaacc agccaatcat tcgcattcag 6480
tccatagcac cgtctttgca agtcatcaca tccaagcaga ggccccggaa attgacactt
6540 atgggcagca acggacatga gtttgttttc cttctaaaag gccatgaaga
tctgcgccag 6600 gatgagcgtg tgatgcagct cttcggcctg gttaacaccc
tcctggccaa tgacccaaca 6660 tctcttcgga aaaacctcag catccagaga
tacgctgtca tccctttatc gaccaactcg 6720 ggcctcattg gctgggttcc
ccactgtgac acactgcacg ccctcatccg ggactacagg 6780 gagaagaaga
agatccttct caacatcgag catcgcatca tgttgcggat ggctccggac 6840
tatgaccact tgactctgat gcagaaggtg gaggtgtttg agcatgccgt caataataca
6900 gctggggacg acctggccaa gctgctatgg ctgaaaagcc ccagctccga
ggtgtggttt 6960 gaccgaagaa ccaattatac ccgttcttta gcggtcatgt
caatggttgg gtatatttta 7020 ggcctgggag atagacaccc atccaacctg
atgctggacc gtctgagtgg gaagatcctg 7080 cacattgact ttggggactg
ctttgaggtt gctatgaccc gagagaagtt tccagagaag 7140 attccattta
gactaacaag aatgttgacc aatgctatgg aggttacagg cctggatggc 7200
aactacagaa tcacatgcca cacagtgatg gaggtgctgc gagagcacaa ggacagtgtc
7260 atggccgtgc tggaagcctt tgtctatgac cccttgctga actggaggct
gatggacaca 7320 aataccaaag gcaacaagcg atcccgaacg aggacggatt
cctactctgc tggccagtca 7380 gtcgaaattt tggacggtgt ggaacttgga
gagccagccc ataagaaaac ggggaccaca 7440 gtgccagaat ctattcattc
tttcattgga gacggtttgg tgaaaccaga ggccctaaat 7500 aagaaagcta
tccagattat taacagggtt cgagataagc tcactggtcg ggacttctct 7560
catgatgaca ctttggatgt tccaacgcaa gttgagctgc tcatcaaaca agcgacatcc
7620 catgaaaacc tctgccagtg ctatattggc tggtgccctt tctggtaact
ggaggcccag 7680 atgtgcccat cacgtttttt ctgaggcttt tgtactttag
taaatgcttc cactaaactg 7740 aaaccatggt gagaaagttt gactttgtta
aatattttga aatgtaaatg aaaagaacta 7800 ctgtatatta aaagttggtt
tgaaccaact ttctagctgc tgttgaagaa tatattgtca 7860 gaaacacaag
gcttgatttg gttcccagga cagtgaaaca tagtaatacc acgtaaatca 7920
agccattcat tttggggaac agaagatcca taactttaga aatacgggtt ttgacttaac
7980 tcacaagaga actcatcata agtacttgct gatggaagaa tgacctagtt
gctcctctca 8040 acatgggtac agcaaactca gcacagccaa gaagcctcag
gtcgtggaga acatggatta 8100 ggatcctaga ctgtaaagac acagaagatg
ctgacctcac ccctgccacc tatcc 8155 <210> SEQ ID NO 6
<211> LENGTH: 2549 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION:
<308> DATABASE ACCESSION NUMBER: Genbank BC117166 <309>
DATABASE ENTRY DATE: 2006-06-26 <313> RELEVANT RESIDUES IN
SEQ ID NO: (1)..(2549) <400> SEQUENCE: 6 Met Leu Gly Thr Gly
Pro Ala Ala Ala Thr Thr Ala Ala Thr Thr Ser 1 5 10 15 Ser Asn Val
Ser Val Leu Gln Gln Phe Ala Ser Gly Leu Lys Ser Arg 20 25 30 Asn
Glu Glu Thr Arg Ala Lys Ala Ala Lys Glu Leu Gln His Tyr Val 35 40
45 Thr Met Glu Leu Arg Glu Met Ser Gln Glu Glu Ser Thr Arg Phe Tyr
50 55 60 Asp Gln Leu Asn His His Ile Phe Glu Leu Val Ser Ser Ser
Asp Ala 65 70 75 80 Asn Glu Arg Lys Gly Gly Ile Leu Ala Ile Ala Ser
Leu Ile Gly Val 85 90 95 Glu Gly Gly Asn Ala Thr Arg Ile Gly Arg
Phe Ala Asn Tyr Leu Arg 100 105 110 Asn Leu Leu Pro Ser Asn Asp Pro
Val Val Met Glu Met Ala Ser Lys 115 120 125 Ala Ile Gly Arg Leu Ala
Met Ala Gly Asp Thr Phe Thr Ala Glu Tyr 130 135 140 Val Glu Phe Glu
Val Lys Arg Ala Leu Glu Trp Leu Gly Ala Asp Arg 145 150 155 160 Asn
Glu Gly Arg Arg His Ala Ala Val Leu Val Leu Arg Glu Leu Ala 165 170
175 Ile Ser Val Pro Thr Phe Phe Phe Gln Gln Val Gln Pro Phe Phe Asp
180 185 190 Asn Ile Phe Val Ala Val Trp Asp Pro Lys Gln Ala Ile Arg
Glu Gly 195 200 205 Ala Val Ala Ala Leu Arg Ala Cys Leu Ile Leu Thr
Thr Gln Arg Glu 210 215 220 Pro Lys Glu Met Gln Lys Pro Gln Trp Tyr
Arg His Thr Phe Glu Glu 225 230 235 240 Ala Glu Lys Gly Phe Asp Glu
Thr Leu Ala Lys Glu Lys Gly Met Asn 245 250 255 Arg Asp Asp Arg Ile
His Gly Ala Leu Leu Ile Leu Asn Glu Leu Val 260 265 270 Arg Ile Ser
Ser Met Glu Gly Glu Arg Leu Arg Glu Glu Met Glu Glu 275 280 285 Ile
Thr Gln Gln Gln Leu Val His Asp Lys Tyr Cys Lys Asp Leu Met 290 295
300 Gly Phe Gly Thr Lys Pro Arg His Ile Thr Pro Phe Thr Ser Phe Gln
305 310 315 320 Ala Val Gln Pro Gln Gln Ser Asn Ala Leu Val Gly Leu
Leu Gly Tyr 325 330 335 Ser Ser His Gln Gly Leu Met Gly Phe Gly Thr
Ser Pro Ser Pro Ala 340 345 350 Lys Ser Thr Leu Val Glu Ser Arg Cys
Cys Arg Asp Leu Met Glu Glu 355 360 365 Lys Phe Asp Gln Val Cys Gln
Trp Val Leu Lys Cys Arg Asn Ser Lys 370 375 380 Asn Ser Leu Ile Gln
Met Thr Ile Leu Asn Leu Leu Pro Arg Leu Ala 385 390 395 400 Ala Phe
Arg Pro Ser Ala Phe Thr Asp Thr Gln Tyr Leu Gln Asp Thr 405 410 415
Met Asn His Val Leu Ser Cys Val Lys Lys Glu Lys Glu Arg Thr Ala 420
425 430 Ala Phe Gln Ala Leu Gly Leu Leu Ser Val Ala Val Arg Ser Glu
Phe 435 440 445 Lys Val Tyr Leu Pro Arg Val Leu Asp Ile Ile Arg Ala
Ala Leu Pro 450 455 460 Pro Lys Asp Phe Ala His Lys Arg Gln Lys Ala
Met Gln Val Asp Ala 465 470 475 480 Thr Val Phe Thr Cys Ile Ser Met
Leu Ala Arg Ala Met Gly Pro Gly 485 490 495 Ile Gln Gln Asp Ile Lys
Glu Leu Leu Glu Pro Met Leu Ala Val Gly 500 505 510 Leu Ser Pro Ala
Leu Thr Ala Val Leu Tyr Asp Leu Ser Arg Gln Ile 515 520 525 Pro Gln
Leu Lys Lys Asp Ile Gln Asp Gly Leu Leu Lys Met Leu Ser 530 535 540
Leu Val Leu Met His Lys Pro Leu Arg His Pro Gly Met Pro Lys Gly 545
550 555 560 Leu Ala His Gln Leu Ala Ser Pro Gly Leu Thr Thr Leu Pro
Glu Ala 565 570 575 Ser Asp Val Gly Ser Ile Thr Leu Ala Leu Arg Thr
Leu Gly Ser Phe 580 585 590 Glu Phe Glu Gly His Ser Leu Thr Gln Phe
Val Arg His Cys Ala Asp 595 600 605 His Phe Leu Asn Ser Glu His Lys
Glu Ile Arg Met Glu Ala Ala Arg 610 615 620 Thr Cys Ser Arg Leu Leu
Thr Pro Ser Ile His Leu Ile Ser Gly His 625 630 635 640 Ala His Val
Val Ser Gln Thr Ala Val Gln Val Val Ala Asp Val Leu 645 650 655 Ser
Lys Leu Leu Val Val Gly Ile Thr Asp Pro Asp Pro Asp Ile Arg 660 665
670 Tyr Cys Val Leu Ala Ser Leu Asp Glu Arg Phe Asp Ala His Leu Ala
675 680 685 Gln Ala Glu Asn Leu Gln Ala Leu Phe Val Ala Leu Asn Asp
Gln Val 690 695 700 Phe Glu Ile Arg Glu Leu Ala Ile Cys Thr Val Gly
Arg Leu Ser Ser 705 710 715 720 Met Asn Pro Ala Phe Val Met Pro Phe
Leu Arg Lys Met Leu Ile Gln 725 730 735 Ile Leu Thr Glu Leu Glu His
Ser Gly Ile Gly Arg Ile Lys Glu Gln 740 745 750 Ser Ala Arg Met Leu
Gly His Leu Val Ser Asn Ala Pro Arg Leu Ile 755 760 765 Arg Pro Tyr
Met Glu Pro Ile Leu Lys Ala Leu Ile Leu Lys Leu Lys 770 775 780 Asp
Pro Asp Pro Asp Pro Asn Pro Gly Val Ile Asn Asn Val Leu Ala 785 790
795 800 Thr Ile Gly Glu Leu Ala Gln Val Ser Gly Leu Glu Met Arg Lys
Trp 805 810 815 Val Asp Glu Leu Phe Ile Ile Ile Met Asp Met Leu Gln
Asp Ser Ser 820 825 830 Leu Leu Ala Lys Arg Gln Val Ala Leu Trp Thr
Leu Gly Gln Leu Val 835 840 845 Ala Ser Thr Gly Tyr Val Val Glu Pro
Tyr Arg Lys Tyr Pro Thr Leu 850 855 860 Leu Glu Val Leu Leu Asn Phe
Leu Lys Thr Glu Gln Asn Gln Gly Thr 865 870 875 880 Arg Arg Glu Ala
Ile Arg Val Leu Gly Leu Leu Gly Ala Leu Asp Pro 885 890 895 Tyr Lys
His Lys Val Asn Ile Gly Met Ile Asp Gln Ser Arg Asp Ala 900 905 910
Ser Ala Val Ser Leu Ser Glu Ser Lys Ser Ser Gln Asp Ser Ser Asp 915
920 925 Tyr Ser Thr Ser Glu Met Leu Val Asn Met Gly Asn Leu Pro Leu
Asp 930 935 940 Glu Phe Tyr Pro Ala Val Ser Met Val Ala Leu Met Arg
Ile Phe Arg 945 950 955 960 Asp Gln Ser Leu Ser His His His Thr Met
Val Val Gln Ala Ile Thr 965 970 975 Phe Ile Phe Lys Ser Leu Gly Leu
Lys Cys Val Gln Phe Leu Pro Gln 980 985 990 Val Met Pro Thr Phe Leu
Asn Val Ile Arg Val Cys Asp Gly Ala Ile 995 1000 1005 Arg Glu Phe
Leu Phe Gln Gln Leu Gly Met Leu Val Ser Phe Val 1010 1015 1020 Lys
Ser His Ile Arg Pro Tyr Met Asp Glu Ile Val Thr Leu Met 1025 1030
1035 Arg Glu Phe Trp Val Met Asn Thr Ser Ile Gln Ser Thr Ile Ile
1040 1045 1050 Leu Leu Ile Glu Gln Ile Val Val Ala Leu Gly Gly Glu
Phe Lys 1055 1060 1065 Leu Tyr Leu Pro Gln Leu Ile Pro His Met Leu
Arg Val Phe Met 1070 1075 1080 His Asp Asn Ser Pro Gly Arg Ile Val
Ser Ile Lys Leu Leu Ala 1085 1090 1095 Ala Ile Gln Leu Phe Gly Ala
Asn Leu Asp Asp Tyr Leu His Leu 1100 1105 1110 Leu Leu Pro Pro Ile
Val Lys Leu Phe Asp Ala Pro Glu Ala Pro 1115 1120 1125 Leu Pro Ser
Arg Lys Ala Ala Leu Glu Thr Val Asp Arg Leu Thr 1130 1135 1140 Glu
Ser Leu Asp Phe Thr Asp Tyr Ala Ser Arg Ile Ile His Pro 1145 1150
1155 Ile Val Arg Thr Leu Asp Gln Ser Pro Glu Leu Arg Ser Thr Ala
1160 1165 1170 Met Asp Thr Leu Ser Ser Leu Val Phe Gln Leu Gly Lys
Lys Tyr 1175 1180 1185 Gln Ile Phe Ile Pro Met Val Asn Lys Val Leu
Val Arg His Arg 1190 1195 1200 Ile Asn His Gln Arg Tyr Asp Val Leu
Ile Cys Arg Ile Val Lys 1205 1210 1215 Gly Tyr Thr Leu Ala Asp Glu
Glu Glu Asp Pro Leu Ile Tyr Gln 1220 1225 1230 His Arg Met Leu Arg
Ser Gly Gln Gly Asp Ala Leu Ala Ser Gly 1235 1240 1245 Pro Val Glu
Thr Gly Pro Met Lys Lys Leu His Val Ser Thr Ile 1250 1255 1260 Asn
Leu Gln Lys Ala Trp Gly Ala Ala Arg Arg Val Ser Lys Asp 1265 1270
1275 Asp Trp Leu Glu Trp Leu Arg Arg Leu Ser Leu Glu Leu Leu Lys
1280 1285 1290 Asp Ser Ser Ser Pro Ser Leu Arg Ser Cys Trp Ala Leu
Ala Gln 1295 1300 1305 Ala Tyr Asn Pro Met Ala Arg Asp Leu Phe Asn
Ala Ala Phe Val 1310 1315 1320 Ser Cys Trp Ser Glu Leu Asn Glu Asp
Gln Gln Asp Glu Leu Ile 1325 1330 1335 Arg Ser Ile Glu Leu Ala Leu
Thr Ser Gln Asp Ile Ala Glu Val 1340 1345 1350 Thr Gln Thr Leu Leu
Asn Leu Ala Glu Phe Met Glu His Ser Asp 1355 1360 1365 Lys Gly Pro
Leu Pro Leu Arg Asp Asp Asn Gly Ile Val Leu Leu 1370 1375 1380 Gly
Glu Arg Ala Ala Lys Cys Arg Ala Tyr Ala Lys Ala Leu His 1385 1390
1395 Tyr Lys Glu Leu Glu Phe Gln Lys Gly Pro Thr Pro Ala Ile Leu
1400 1405 1410 Glu Ser Leu Ile Ser Ile Asn Asn Lys Leu Gln Gln Pro
Glu Ala 1415 1420 1425 Ala Ala Gly Val Leu Glu Tyr Ala Met Lys His
Phe Gly Glu Leu 1430 1435 1440 Glu Ile Gln Ala Thr Trp Tyr Glu Lys
Leu His Glu Trp Glu Asp 1445 1450 1455 Ala Leu Val Ala Tyr Asp Lys
Lys Met Asp Thr Asn Lys Asp Asp 1460 1465 1470 Pro Glu Leu Met Leu
Gly Arg Met Arg Cys Leu Glu Ala Leu Gly 1475 1480 1485 Glu Trp Gly
Gln Leu His Gln Gln Cys Cys Glu Lys Trp Thr Leu 1490 1495 1500 Val
Asn Asp Glu Thr Gln Ala Lys Met Ala Arg Met Ala Ala Ala 1505 1510
1515 Ala Ala Trp Gly Leu Gly Gln Trp Asp Ser Met Glu Glu Tyr Thr
1520 1525 1530 Cys Met Ile Pro Arg Asp Thr His Asp Gly Ala Phe Tyr
Arg Ala 1535 1540 1545 Val Leu Ala Leu His Gln Asp Leu Phe Ser Leu
Ala Gln Gln Cys 1550 1555 1560 Ile Asp Lys Ala Arg Asp Leu Leu Asp
Ala Glu Leu Thr Ala Met 1565 1570 1575 Ala Gly Glu Ser Tyr Ser Arg
Ala Tyr Gly Ala Met Val Ser Cys 1580 1585 1590 His Met Leu Ser Glu
Leu Glu Glu Val Ile Gln Tyr Lys Leu Val 1595 1600 1605 Pro Glu Arg
Arg Glu Ile Ile Arg Gln Ile Trp Trp Glu Arg Leu 1610 1615 1620 Gln
Gly Cys Gln Arg Ile Val Glu Asp Trp Gln Lys Ile Leu Met 1625 1630
1635 Val Arg Ser Leu Val Val Ser Pro His Glu Asp Met Arg Thr Trp
1640 1645 1650 Leu Lys Tyr Ala Ser Leu Cys Gly Lys Ser Gly Arg Leu
Ala Leu 1655 1660 1665 Ala His Lys Thr Leu Val Leu Leu Leu Gly Val
Asp Pro Ser Arg 1670 1675 1680 Gln Leu Asp His Pro Leu Pro Thr Val
His Pro Gln Val Thr Tyr 1685 1690 1695 Ala Tyr Met Lys Asn Met Trp
Lys Ser Ala Arg Lys Ile Asp Ala 1700 1705 1710 Phe Gln His Met Gln
His Phe Val Gln Thr Met Gln Gln Gln Ala 1715 1720 1725 Gln His Ala
Ile Ala Thr Glu Asp Gln Gln His Lys Gln Glu Leu 1730 1735 1740 His
Lys Leu Met Ala Arg Cys Phe Leu Lys Leu Gly Glu Trp Gln 1745 1750
1755 Leu Asn Leu Gln Gly Ile Asn Glu Ser Thr Ile Pro Lys Val Leu
1760 1765 1770 Gln Tyr Tyr Ser Ala Ala Thr Glu His Asp Arg Ser Trp
Tyr Lys 1775 1780 1785 Ala Trp His Ala Trp Ala Val Met Asn Phe Glu
Ala Val Leu His 1790 1795 1800 Tyr Lys His Gln Asn Gln Ala Arg Asp
Glu Lys Lys Lys Leu Arg 1805 1810 1815 His Ala Ser Gly Ala Asn Ile
Thr Asn Ala Thr Thr Ala Ala Thr 1820 1825 1830 Thr Ala Ala Thr Ala
Thr Thr Thr Ala Ser Thr Glu Gly Ser Asn 1835 1840 1845 Ser Glu Ser
Glu Ala Glu Ser Thr Glu Asn Ser Pro Thr Pro Ser 1850 1855 1860 Pro
Leu Gln Lys Lys Val Thr Glu Asp Leu Ser Lys Thr Leu Leu 1865 1870
1875 Met Tyr Thr Val Pro Ala Val Gln Gly Phe Phe Arg Ser Ile Ser
1880 1885 1890 Leu Ser Arg Gly Asn Asn Leu Gln Asp Thr Leu Arg Val
Leu Thr 1895 1900 1905 Leu Trp Phe Asp Tyr Gly His Trp Pro Asp Val
Asn Glu Ala Leu 1910 1915 1920 Val Glu Gly Val Lys Ala Ile Gln Ile
Asp Thr Trp Leu Gln Val 1925 1930 1935 Ile Pro Gln Leu Ile Ala Arg
Ile Asp Thr Pro Arg Pro Leu Val 1940 1945 1950 Gly Arg Leu Ile His
Gln Leu Leu Thr Asp Ile Gly Arg Tyr His 1955 1960 1965 Pro Gln Ala
Leu Ile Tyr Pro Leu Thr Val Ala Ser Lys Ser Thr 1970 1975 1980 Thr
Thr Ala Arg His Asn Ala Ala Asn Lys Ile Leu Lys Asn Met 1985 1990
1995 Cys Glu His Ser Asn Thr Leu Val Gln Gln Ala Met Met Val Ser
2000 2005 2010 Glu Glu Leu Ile Arg Val Ala Ile Leu Trp His Glu Met
Trp His 2015 2020 2025 Glu Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe
Gly Glu Arg Asn 2030 2035 2040 Val Lys Gly Met Phe Glu Val Leu Glu
Pro Leu His Ala Met Met 2045 2050 2055 Glu Arg Gly Pro Gln Thr Leu
Lys Glu Thr Ser Phe Asn Gln Ala 2060 2065 2070 Tyr Gly Arg Asp Leu
Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr 2075 2080 2085 Met Lys Ser
Gly Asn Val Lys Asp Leu Thr Gln Ala Trp Asp Leu 2090 2095 2100 Tyr
Tyr His Val Phe Arg Arg Ile Ser Lys Gln Leu Pro Gln Leu 2105 2110
2115 Thr Ser Leu Glu Leu Gln Tyr Val Ser Pro Lys Leu Leu Met Cys
2120 2125 2130 Arg Asp Leu Glu Leu Ala Val Pro Gly Thr Tyr Asp Pro
Asn Gln 2135 2140 2145 Pro Ile Ile Arg Ile Gln Ser Ile Ala Pro Ser
Leu Gln Val Ile 2150 2155 2160 Thr Ser Lys Gln Arg Pro Arg Lys Leu
Thr Leu Met Gly Ser Asn 2165 2170 2175 Gly His Glu Phe Val Phe Leu
Leu Lys Gly His Glu Asp Leu Arg 2180 2185 2190 Gln Asp Glu Arg Val
Met Gln Leu Phe Gly Leu Val Asn Thr Leu 2195 2200 2205 Leu Ala Asn
Asp Pro Thr Ser Leu Arg Lys Asn Leu Ser Ile Gln 2210 2215 2220 Arg
Tyr Ala Val Ile Pro Leu Ser Thr Asn Ser Gly Leu Ile Gly 2225 2230
2235 Trp Val Pro His Cys Asp Thr Leu His Ala Leu Ile Arg Asp Tyr
2240 2245 2250 Arg Glu Lys Lys Lys Ile Leu Leu Asn Ile Glu His Arg
Ile Met 2255 2260 2265 Leu Arg Met Ala Pro Asp Tyr Asp His Leu Thr
Leu Met Gln Lys 2270 2275 2280 Val Glu Val Phe Glu His Ala Val Asn
Asn Thr Ala Gly Asp Asp 2285 2290 2295 Leu Ala Lys Leu Leu Trp Leu
Lys Ser Pro Ser Ser Glu Val Trp 2300 2305 2310 Phe Asp Arg Arg Thr
Asn Tyr Thr Arg Ser Leu Ala Val Met Ser 2315 2320 2325 Met Val Gly
Tyr Ile Leu Gly Leu Gly Asp Arg His Pro Ser Asn 2330 2335 2340 Leu
Met Leu Asp Arg Leu Ser Gly Lys Ile Leu His Ile Asp Phe 2345 2350
2355 Gly Asp Cys Phe Glu Val Ala Met Thr Arg Glu Lys Phe Pro Glu
2360 2365 2370 Lys Ile Pro Phe Arg Leu Thr Arg Met Leu Thr Asn Ala
Met Glu 2375 2380 2385 Val Thr Gly Leu Asp Gly Asn Tyr Arg Ile Thr
Cys His Thr Val 2390 2395 2400 Met Glu Val Leu Arg Glu His Lys Asp
Ser Val Met Ala Val Leu 2405 2410 2415 Glu Ala Phe Val Tyr Asp Pro
Leu Leu Asn Trp Arg Leu Met Asp 2420 2425 2430 Thr Asn Thr Lys Gly
Asn Lys Arg Ser Arg Thr Arg Thr Asp Ser 2435 2440 2445 Tyr Ser Ala
Gly Gln Ser Val Glu Ile Leu Asp Gly Val Glu Leu 2450 2455 2460 Gly
Glu Pro Ala His Lys Lys Thr Gly Thr Thr Val Pro Glu Ser 2465 2470
2475 Ile His Ser Phe Ile Gly Asp Gly Leu Val Lys Pro Glu Ala Leu
2480 2485 2490 Asn Lys Lys Ala Ile Gln Ile Ile Asn Arg Val Arg Asp
Lys Leu 2495 2500 2505 Thr Gly Arg Asp Phe Ser His Asp Asp Thr Leu
Asp Val Pro Thr 2510 2515 2520 Gln Val Glu Leu Leu Ile Lys Gln Ala
Thr Ser His Glu Asn Leu 2525 2530 2535 Cys Gln Cys Tyr Ile Gly Trp
Cys Pro Phe Trp 2540 2545 <210> SEQ ID NO 7 <211>
LENGTH: 3376 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <300> PUBLICATION INFORMATION: <308> DATABASE
ACCESSION NUMBER: Genback M20869 <309> DATABASE ENTRY DATE:
1993-08-02 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(3376)
<400> SEQUENCE: 7 cgcaccgggg atcctaggct ttttggattg cgctttcctc
tagatcaact gggtgtcagg 60 ccctatccta cagaaggatg ggtcagattg
tgacaatgtt tgaggctctg cctcacatca 120 tcgatgaggt gatcaacatt
gtcattattg tgcttatcgt gatcacgggt atcaaggctg 180 tctacaattt
tgccacctgt gggatattcg cattgatcag tttcctactt ctggctggca 240
ggtcctgtgg catgtacggt cttaagggac ccgacattta caaaggagtt taccaattta
300 agtcagtgga gtttgatatg tcacatctga acctgaccat gcccaacgca
tgttcagcca 360 acaactccca ccattacatc agtatgggga cttctggact
agaattgacc ttcaccaatg 420 attccatcat cagtcacaac ttttgcaatc
tgacctctgc cttcaacaaa aagacctttg 480 accacacact catgagtata
gtttcgagcc tacacctcag tatcagaggg aactccaact 540 ataaggcagt
atcctgcgac ttcaacaatg gcataaccat ccaatacaac ttgacattct 600
cagatcgaca aagtgctcag agccagtgta gaaccttcag aggtagagtc ctagatatgt
660 ttagaactgc cttcgggggg aaatacatga ggagtggctg gggctggaca
ggctcagatg 720 gcaagaccac ctggtgtagc cagacgagtt accaatacct
gattatacaa aatagaacct 780 gggaaaacca ctgcacatat gcaggtcctt
ttgggatgtc caggattctc ctttcccaag 840 agaagactaa gttcttcact
aggagactag cgggcacatt cacctggact ttgtcagact 900 cttcaggggt
ggagaatcca ggtggttatt gcctgaccaa atggatgatt cttgctgcag 960
agcttaagtg tttcgggaac acagcagttg cgaaatgcaa tgtaaatcat gatgccgaat
1020 tctgtgacat gctgcgacta attgactaca acaaggctgc tttgagtaag
ttcaaagagg 1080 acgtagaatc tgccttgcac ttattcaaaa caacagtgaa
ttctttgatt tcagatcaac 1140 tactgatgag gaaccacttg agagatctga
tgggggtgcc atattgcaat tactcaaagt 1200 tttggtacct agaacatgca
aagaccggcg aaactagtgt ccccaagtgc tggcttgtca 1260 ccaatggttc
ttacttaaat gagacccact tcagtgatca aatcgaacag gaagccgata 1320
acatgattac agagatgttg aggaaggatt acataaagag gcaggggagt acccccctag
1380 cattgatgga ccttctgatg ttttccacat ctgcatatct agtcagcatc
ttcctgcacc 1440 ttgtcaaaat accaacacac aggcacataa aaggtggctc
atgtccaaag ccacaccgat 1500 taaccaacaa aggaatttgt agttgtggtg
catttaaggt gcctggtgta aaaaccgtct 1560 ggaaaagacg ctgaagaaca
gcgcctccct gactctccac ctcgaaagag gtggagagtc 1620 agggaggccc
agagggtctt agagtgtcac aacatttggg cctctaaaaa ttaggtcatg 1680
tggcagaatg ttgtgaacag ttttcagatc tgggagcctt gctttggagg cgctttcaaa
1740 aatgatgcag tccatgagtg cacagtgcgg ggtgatctct ttcttctttt
tgtcccttac 1800 tattccagta tgcatcttac acaaccagcc atatttgtcc
cacactttgt cttcatactc 1860 cctcgaagct tccctggtca tttcaacatc
gataagctta atgtccttcc tattctgtga 1920 gtccagaagc tttctgatgt
catcggagcc ttgacagctt agaaccatcc cctgcggaag 1980 agcacctata
actgacgagg tcaacccggg ttgcgcattg aagaggtcgg caagatccat 2040
gccgtgtgag tacttggaat cttgcttgaa ttgtttttga tcaacgggtt ccctgtaaaa
2100 gtgtatgaac tgcccgttct gtggttggaa aattgctatt tccactggat
cattaaatct 2160 accctcaatg tcaatccatg taggagcgtt ggggtcaatt
cctcccatga ggtcttttaa 2220 aagcattgtc tggctgtagc ttaagcccac
ctgaggtgga cctgctgctc caggcgctgg 2280 cctgggtgaa ttgactgcag
gtttctcgct tgtgagatca attgttgtgt tttcccatgc 2340 tctccccaca
atcgatgttc tacaagctat gtatggccat ccttcacctg aaaggcaaac 2400
tttatagagg atgttttcat aagggttcct gtccccaact tggtctgaaa caaacatgtt
2460 gagttttctc ttggccccga gaactgcctt caagaggtcc tcgctgttgc
ttggcttgat 2520 caaaattgac tctaacatgt tacccccatc caacagggct
gcccctgcct tcacggcagc 2580 accaagacta aagttatagc cagaaatgtt
gatgctggac tgctgttcag tgatgacccc 2640 cagaactggg tgcttgtctt
tcagcctttc aagatcatta agatttggat acttgactgt 2700 gtaaagcaag
ccaaggtctg tgagcgcttg tacaacgtca ttgagcggag tctgtgactg 2760
tttggccata caagccatag ttagacttgg cattgtgcca aattgattgt tcaaaagtga
2820 tgagtctttc acatcccaaa ctcttaccac accacttgca ccctgctgag
gctttctcat 2880 cccaactatc tgtaggatct gagatctttg gtctagttgc
tgtgttgtta agttccccat 2940 atatacccct gaagcctggg gcctttcaga
cctcatgatc ttggccttca gcttctcaag 3000 gtcagccgca agagacatca
gttcttctgc actgagcctc cccactttca aaacattctt 3060 ctttgatgtt
gactttaaat ccacaagaga atgtacagtc tggttgagac ttctgagtct 3120
ctgtaggtct ttgtcatctc tcttttcctt cctcatgatc ctctgaacat tgctgacctc
3180 agagaagtcc aacccattca gaaggttggt tgcatcctta atgacagcag
ccttcacatc 3240 tgatgtgaag ctctgcaatt ctcttctcaa tgcttgcgtc
cattggaagc tcttaacttc 3300 cttagacaag gacatcttgt tgctcaatgg
tttctcaaga caaatgcgca atcaaatgcc 3360 taggatccac tgtgcg 3376
<210> SEQ ID NO 8 <211> LENGTH: 498 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <300> PUBLICATION
INFORMATION: <308> DATABASE ACCESSION NUMBER: Genbank
<309> DATABASE ENTRY DATE: 1993-08-02 <313> RELEVANT
RESIDUES IN SEQ ID NO: (1)..(498) <400> SEQUENCE: 8 Met Gly
Gln Ile Val Thr Met Phe Glu Ala Leu Pro His Ile Ile Asp 1 5 10 15
Glu Val Ile Asn Ile Val Ile Ile Val Leu Ile Val Ile Thr Gly Ile 20
25 30 Lys Ala Val Tyr Asn Phe Ala Thr Cys Gly Ile Phe Ala Leu Ile
Ser 35 40 45 Phe Leu Leu Leu Ala Gly Arg Ser Cys Gly Met Tyr Gly
Leu Lys Gly 50 55 60 Pro Asp Ile Tyr Lys Gly Val Tyr Gln Phe Lys
Ser Val Glu Phe Asp 65 70 75 80 Met Ser His Leu Asn Leu Thr Met Pro
Asn Ala Cys Ser Ala Asn Asn 85 90 95 Ser His His Tyr Ile Ser Met
Gly Thr Ser Gly Leu Glu Leu Thr Phe 100 105 110 Thr Asn Asp Ser Ile
Ile Ser His Asn Phe Cys Asn Leu Thr Ser Ala 115 120 125 Phe Asn Lys
Lys Thr Phe Asp His Thr Leu Met Ser Ile Val Ser Ser 130 135 140 Leu
His Leu Ser Ile Arg Gly Asn Ser Asn Tyr Lys Ala Val Ser Cys 145 150
155 160 Asp Phe Asn Asn Gly Ile Thr Ile Gln Tyr Asn Leu Thr Phe Ser
Asp 165 170 175 Arg Gln Ser Ala Gln Ser Gln Cys Arg Thr Phe Arg Gly
Arg Val Leu 180 185 190 Asp Met Phe Arg Thr Ala Phe Gly Gly Lys Tyr
Met Arg Ser Gly Trp 195 200 205 Gly Trp Thr Gly Ser Asp Gly Lys Thr
Thr Trp Cys Ser Gln Thr Ser 210 215 220 Tyr Gln Tyr Leu Ile Ile Gln
Asn Arg Thr Trp Glu Asn His Cys Thr 225 230 235 240 Tyr Ala Gly Pro
Phe Gly Met Ser Arg Ile Leu Leu Ser Gln Glu Lys 245 250 255 Thr Lys
Phe Phe Thr Arg Arg Leu Ala Gly Thr Phe Thr Trp Thr Leu 260 265 270
Ser Asp Ser Ser Gly Val Glu Asn Pro Gly Gly Tyr Cys Leu Thr Lys 275
280 285 Trp Met Ile Leu Ala Ala Glu Leu Lys Cys Phe Gly Asn Thr Ala
Val 290 295 300 Ala Lys Cys Asn Val Asn His Asp Ala Glu Phe Cys Asp
Met Leu Arg 305 310 315 320 Leu Ile Asp Tyr Asn Lys Ala Ala Leu Ser
Lys Phe Lys Glu Asp Val 325 330 335 Glu Ser Ala Leu His Leu Phe Lys
Thr Thr Val Asn Ser Leu Ile Ser 340 345 350 Asp Gln Leu Leu Met Arg
Asn His Leu Arg Asp Leu Met Gly Val Pro 355 360 365 Tyr Cys Asn Tyr
Ser Lys Phe Trp Tyr Leu Glu His Ala Lys Thr Gly 370 375 380 Glu Thr
Ser Val Pro Lys Cys Trp Leu Val Thr Asn Gly Ser Tyr Leu 385 390 395
400 Asn Glu Thr His Phe Ser Asp Gln Ile Glu Gln Glu Ala Asp Asn Met
405 410 415 Ile Thr Glu Met Leu Arg Lys Asp Tyr Ile Lys Arg Gln Gly
Ser Thr 420 425 430 Pro Leu Ala Leu Met Asp Leu Leu Met Phe Ser Thr
Ser Ala Tyr Leu 435 440 445 Val Ser Ile Phe Leu His Leu Val Lys Ile
Pro Thr His Arg His Ile 450 455 460 Lys Gly Gly Ser Cys Pro Lys Pro
His Arg Leu Thr Asn Lys Gly Ile 465 470 475 480 Cys Ser Cys Gly Ala
Phe Lys Val Pro Gly Val Lys Thr Val Trp Lys 485 490 495 Arg Arg
<210> SEQ ID NO 9 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic peptide <400>
SEQUENCE: 9 Lys Ala Val Tyr Asn Phe Ala Thr Met 1 5 <210> SEQ
ID NO 10 <211> LENGTH: 58 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
10 ccgggccaga atccatccat tcattctcga gaatgaatgg atggattctg gctttttg
58 <210> SEQ ID NO 11 <211> LENGTH: 58 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 11 aattcaaaaa gccagaatcc atccattcat
tctcgagaat gaatggatgg attctggc 58 <210> SEQ ID NO 12
<211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 12
ccgggcccga gtctgtgaat gtaatctcga gattacattc acagactcgg gctttttg 58
<210> SEQ ID NO 13 <211> LENGTH: 58 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 13 aattcaaaaa gcccgagtct gtgaatgtaa
tctcgagatt acattcacag actcgggc 58 <210> SEQ ID NO 14
<211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 14
ccgggccaaa ctgataatct cctcactcga gtgaggagat tatcagtttg gctttttg 58
<210> SEQ ID NO 15 <211> LENGTH: 58 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 15 aattcaaaaa gccaaactga taatctcctc
actcgagtga ggagattatc agtttggc 58 <210> SEQ ID NO 16
<211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 16
ccgggcatgg aacattgtga gaaatctcga gatttctcac aatgttccat gctttttg 58
<210> SEQ ID NO 17 <211> LENGTH: 58 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 17 aattcaaaaa gcatggaaca ttgtgagaaa
tctcgagatt tctcacaatg ttccatgc 58 <210> SEQ ID NO 18
<211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 18
ccggccgaag atagtgattg gttatctcga gataaccaat cactatcttc ggtttttg 58
<210> SEQ ID NO 19 <211> LENGTH: 58 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 19 aattcaaaaa ccgaagatag tgattggtta
tctcgagata accaatcact atcttcgg 58
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 19 <210>
SEQ ID NO 1 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 1 gcccgagtct gtgaatgtaa t 21 <210> SEQ
ID NO 2 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
2 gccaaactga taatctcctc a 21 <210> SEQ ID NO 3 <211>
LENGTH: 8733 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <300> PUBLICATION INFORMATION: <308> DATABASE
ACCESSION NUMBER: Genbank/004958 <309> DATABASE ENTRY DATE:
2002-04-04 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(8733)
<400> SEQUENCE: 3 gctcccggct tagaggacag cggggaaggc gggcggtggg
gcagggggcc tgaagcggcg 60 gtaccggtgc tggcggcggc agctgaggcc
ttggccgaag ccgcgcgaac ctcagggcaa 120 gatgcttgga accggacctg
ccgccgccac caccgctgcc accacatcta gcaatgtgag 180 cgtcctgcag
cagtttgcca gtggcctaaa gagccggaat gaggaaacca gggccaaagc 240
cgccaaggag ctccagcact atgtcaccat ggaactccga gagatgagtc aagaggagtc
300 tactcgcttc tatgaccaac tgaaccatca catttttgaa ttggtttcca
gctcagatgc 360 caatgagagg aaaggtggca tcttggccat agctagcctc
ataggagtgg aaggtgggaa 420 tgccacccga attggcagat ttgccaacta
tcttcggaac ctcctcccct ccaatgaccc 480 agttgtcatg gaaatggcat
ccaaggccat tggccgtctt gccatggcag gggacacttt 540 taccgctgag
tacgtggaat ttgaggtgaa gcgagccctg gaatggctgg gtgctgaccg 600
caatgagggc cggagacatg cagctgtcct ggttctccgt gagctggcca tcagcgtccc
660 taccttcttc ttccagcaag tgcaaccctt ctttgacaac atttttgtgg
ccgtgtggga 720 ccccaaacag gccatccgtg agggagctgt agccgccctt
cgtgcctgtc tgattctcac 780 aacccagcgt gagccgaagg agatgcagaa
gcctcagtgg tacaggcaca catttgaaga 840 agcagagaag ggatttgatg
agaccttggc caaagagaag ggcatgaatc gggatgatcg 900 gatccatgga
gccttgttga tccttaacga gctggtccga atcagcagca tggagggaga 960
gcgtctgaga gaagaaatgg aagaaatcac acagcagcag ctggtacacg acaagtactg
1020 caaagatctc atgggcttcg gaacaaaacc tcgtcacatt acccccttca
ccagtttcca 1080 ggctgtacag ccccagcagt caaatgcctt ggtggggctg
ctggggtaca gctctcacca 1140 aggcctcatg ggatttggga cctcccccag
tccagctaag tccaccctgg tggagagccg 1200 gtgttgcaga gacttgatgg
aggagaaatt tgatcaggtg tgccagtggg tgctgaaatg 1260 caggaatagc
aagaactcgc tgatccaaat gacaatcctt aatttgttgc cccgcttggc 1320
tgcattccga ccttctgcct tcacagatac ccagtatctc caagatacca tgaaccatgt
1380 cctaagctgt gtcaagaagg agaaggaacg tacagcggcc ttccaagccc
tggggctact 1440 ttctgtggct gtgaggtctg agtttaaggt ctatttgcct
cgcgtgctgg acatcatccg 1500 agcggccctg cccccaaagg acttcgccca
taagaggcag aaggcaatgc aggtggatgc 1560 cacagtcttc acttgcatca
gcatgctggc tcgagcaatg gggccaggca tccagcagga 1620 tatcaaggag
ctgctggagc ccatgctggc agtgggacta agccctgccc tcactgcagt 1680
gctctacgac ctgagccgtc agattccaca gctaaagaag gacattcaag atgggctact
1740 gaaaatgctg tccctggtcc ttatgcacaa accccttcgc cacccaggca
tgcccaaggg 1800 cctggcccat cagctggcct ctcctggcct cacgaccctc
cctgaggcca gcgatgtggg 1860 cagcatcact cttgccctcc gaacgcttgg
cagctttgaa tttgaaggcc actctctgac 1920 ccaatttgtt cgccactgtg
cggatcattt cctgaacagt gagcacaagg agatccgcat 1980 ggaggctgcc
cgcacctgct cccgcctgct cacaccctcc atccacctca tcagtggcca 2040
tgctcatgtg gttagccaga ccgcagtgca agtggtggca gatgtgctta gcaaactgct
2100 cgtagttggg ataacagatc ctgaccctga cattcgctac tgtgtcttgg
cgtccctgga 2160 cgagcgcttt gatgcacacc tggcccaggc ggagaacttg
caggccttgt ttgtggctct 2220 gaatgaccag gtgtttgaga tccgggagct
ggccatctgc actgtgggcc gactcagtag 2280 catgaaccct gcctttgtca
tgcctttcct gcgcaagatg ctcatccaga ttttgacaga 2340 gttggagcac
agtgggattg gaagaatcaa agagcagagt gcccgcatgc tggggcacct 2400
ggtctccaat gccccccgac tcatccgccc ctacatggag cctattctga aggcattaat
2460 tttgaaactg aaagatccag accctgatcc aaacccaggt gtgatcaata
atgtcctggc 2520 aacaatagga gaattggcac aggttagtgg cctggaaatg
aggaaatggg ttgatgaact 2580 ttttattatc atcatggaca tgctccagga
ttcctctttg ttggccaaaa ggcaggtggc 2640 tctgtggacc ctgggacagt
tggtggccag cactggctat gtagtagagc cctacaggaa 2700 gtaccctact
ttgcttgagg tgctactgaa ttttctgaag actgagcaga accagggtac 2760
acgcagagag gccatccgtg tgttagggct tttaggggct ttggatcctt acaagcacaa
2820 agtgaacatt ggcatgatag accagtcccg ggatgcctct gctgtcagcc
tgtcagaatc 2880 caagtcaagt caggattcct ctgactatag cactagtgaa
atgctggtca acatgggaaa 2940 cttgcctctg gatgagttct acccagctgt
gtccatggtg gccctgatgc ggatcttccg 3000 agaccagtca ctctctcatc
atcacaccat ggttgtccag gccatcacct tcatcttcaa 3060 gtccctggga
ctcaaatgtg tgcagttcct gccccaggtc atgcccacgt tccttaacgt 3120
cattcgagtc tgtgatgggg ccatccggga atttttgttc cagcagctgg gaatgttggt
3180 gtcctttgtg aagagccaca tcagacctta tatggatgaa atagtcaccc
tcatgagaga 3240 attctgggtc atgaacacct caattcagag cacgatcatt
cttctcattg agcaaattgt 3300 ggtagctctt gggggtgaat ttaagctcta
cctgccccag ctgatcccac acatgctgcg 3360 tgtcttcatg catgacaaca
gcccaggccg cattgtctct atcaagttac tggctgcaat 3420 ccagctgttt
ggcgccaacc tggatgacta cctgcattta ctgctgcctc ctattgttaa 3480
gttgtttgat gcccctgaag ctccactgcc atctcgaaag gcagcgctag agactgtgga
3540 ccgcctgacg gagtccctgg atttcactga ctatgcctcc cggatcattc
accctattgt 3600 tcgaacactg gaccagagcc cagaactgcg ctccacagcc
atggacacgc tgtcttcact 3660 tgtttttcag ctggggaaga agtaccaaat
tttcattcca atggtgaata aagttctggt 3720 gcgacaccga atcaatcatc
agcgctatga tgtgctcatc tgcagaattg tcaagggata 3780 cacacttgct
gatgaagagg aggatccttt gatttaccag catcggatgc ttaggagtgg 3840
ccaaggggat gcattggcta gtggaccagt ggaaacagga cccatgaaga aactgcacgt
3900 cagcaccatc aacctccaaa aggcctgggg cgctgccagg agggtctcca
aagatgactg 3960 gctggaatgg ctgagacggc tgagcctgga gctgctgaag
gactcatcat cgccctccct 4020 gcgctcctgc tgggccctgg cacaggccta
caacccgatg gccagggatc tcttcaatgc 4080 tgcatttgtg tcctgctggt
ctgaactgaa tgaagatcaa caggatgagc tcatcagaag 4140 catcgagttg
gccctcacct cacaagacat cgctgaagtc acacagaccc tcttaaactt 4200
ggctgaattc atggaacaca gtgacaaggg ccccctgcca ctgagagatg acaatggcat
4260 tgttctgctg ggtgagagag ctgccaagtg ccgagcatat gccaaagcac
tacactacaa 4320 agaactggag ttccagaaag gccccacccc tgccattcta
gaatctctca tcagcattaa 4380 taataagcta cagcagccgg aggcagcggc
cggagtgtta gaatatgcca tgaaacactt 4440 tggagagctg gagatccagg
ctacctggta tgagaaactg cacgagtggg aggatgccct 4500 tgtggcctat
gacaagaaaa tggacaccaa caaggacgac ccagagctga tgctgggccg 4560
catgcgctgc ctcgaggcct tgggggaatg gggtcaactc caccagcagt gctgtgaaaa
4620 gtggaccctg gttaatgatg agacccaagc caagatggcc cggatggctg
ctgcagctgc 4680 atggggttta ggtcagtggg acagcatgga agaatacacc
tgtatgatcc ctcgggacac 4740 ccatgatggg gcattttata gagctgtgct
ggcactgcat caggacctct tctccttggc 4800 acaacagtgc attgacaagg
ccagggacct gctggatgct gaattaactg cgatggcagg 4860 agagagttac
agtcgggcat atggggccat ggtttcttgc cacatgctgt ccgagctgga 4920
ggaggttatc cagtacaaac ttgtccccga gcgacgagag atcatccgcc agatctggtg
4980 ggagagactg cagggctgcc agcgtatcgt agaggactgg cagaaaatcc
ttatggtgcg 5040 gtcccttgtg gtcagccctc atgaagacat gagaacctgg
ctcaagtatg caagcctgtg 5100 cggcaagagt ggcaggctgg ctcttgctca
taaaacttta gtgttgctcc tgggagttga 5160 tccgtctcgg caacttgacc
atcctctgcc aacagttcac cctcaggtga cctatgccta 5220 catgaaaaac
atgtggaaga gtgcccgcaa gatcgatgcc ttccagcaca tgcagcattt 5280
tgtccagacc atgcagcaac aggcccagca tgccatcgct actgaggacc agcagcataa
5340 gcaggaactg cacaagctca tggcccgatg cttcctgaaa cttggagagt
ggcagctgaa 5400 tctacagggc atcaatgaga gcacaatccc caaagtgctg
cagtactaca gcgccgccac 5460 agagcacgac cgcagctggt acaaggcctg
gcatgcgtgg gcagtgatga acttcgaagc 5520 tgtgctacac tacaaacatc
agaaccaagc ccgcgatgag aagaagaaac tgcgtcatgc 5580 cagcggggcc
aacatcacca acgccaccac tgccgccacc acggccgcca ctgccaccac 5640
cactgccagc accgagggca gcaacagtga gagcgaggcc gagagcaccg agaacagccc
5700 caccccatcg ccgctgcaga agaaggtcac tgaggatctg tccaaaaccc
tcctgatgta 5760 cacggtgcct gccgtccagg gcttcttccg ttccatctcc
ttgtcacgag gcaacaacct 5820 ccaggataca ctcagagttc tcaccttatg
gtttgattat ggtcactggc cagatgtcaa 5880 tgaggcctta gtggaggggg
tgaaagccat ccagattgat acctggctac aggttatacc 5940 tcagctcatt
gcaagaattg atacgcccag acccttggtg ggacgtctca ttcaccagct 6000
tctcacagac attggtcggt accaccccca ggccctcatc tacccactga cagtggcttc
6060 taagtctacc acgacagccc ggcacaatgc agccaacaag attctgaaga
acatgtgtga 6120 gcacagcaac accctggtcc agcaggccat gatggtgagc
gaggagctga tccgagtggc 6180 catcctctgg catgagatgt ggcatgaagg
cctggaagag gcatctcgtt tgtactttgg 6240 ggaaaggaac gtgaaaggca
tgtttgaggt gctggagccc ttgcatgcta tgatggaacg 6300
gggcccccag actctgaagg aaacatcctt taatcaggcc tatggtcgag atttaatgga
6360 ggcccaagag tggtgcagga agtacatgaa atcagggaat gtcaaggacc
tcacccaagc 6420 ctgggacctc tattatcatg tgttccgacg aatctcaaag
cagctgcctc agctcacatc 6480 cttagagctg caatatgttt ccccaaaact
tctgatgtgc cgggaccttg aattggctgt 6540 gccaggaaca tatgacccca
accagccaat cattcgcatt cagtccatag caccgtcttt 6600 gcaagtcatc
acatccaagc agaggccccg gaaattgaca cttatgggca gcaacggaca 6660
tgagtttgtt ttccttctaa aaggccatga agatctgcgc caggatgagc gtgtgatgca
6720 gctcttcggc ctggttaaca cccttctggc caatgaccca acatctcttc
ggaaaaacct 6780 cagcatccag agatacgctg tcatcccttt atcgaccaac
tcgggcctca ttggctgggt 6840 tccccactgt gacacactgc acgccctcat
ccgggactac agggagaaga agaagatcct 6900 tctcaacatc gagcatcgca
tcatgttgcg gatggctccg gactatgacc acttgactct 6960 gatgcagaag
gtggaggtgt ttgagcatgc cgtcaataat acagctgggg acgacctggc 7020
caagctgctg tggctgaaaa gccccagctc cgaggtgtgg tttgaccgaa gaaccaatta
7080 tacccgttct ttagcggtca tgtcaatggt tgggtatatt ttaggcctgg
gagatagaca 7140 cccatccaac ctgatgctgg accgtctgag tgggaagatc
ctgcacattg actttgggga 7200 ctgctttgag gttgctatga cccgagagaa
gtttccagag aagattccat ttagactaac 7260 aagaatgttg accaatgcta
tggaggttac aggcctggat ggcaactaca gaatcacatg 7320 ccacacagtg
atggaggtgc tgcgagagca caaggacagt gtcatggccg tgctggaagc 7380
ctttgtctat gaccccttgc tgaactggag gctgatggac acaaatacca aaggcaacaa
7440 gcgatcccga acgaggacgg attcctactc tgctggccag tcagtcgaaa
ttttggacgg 7500 tgtggaactt ggagagccag cccataagaa aacggggacc
acagtgccag aatctattca 7560 ttctttcatt ggagacggtt tggtgaaacc
agaggcccta aataagaaag ctatccagat 7620 tattaacagg gttcgagata
agctcactgg tcgggacttc tctcatgatg acactttgga 7680 tgttccaacg
caagttgagc tgctcatcaa acaagcgaca tcccatgaaa acctctgcca 7740
gtgctatatt ggctggtgcc ctttctggta actggaggcc cagatgtgcc catcacgttt
7800 tttctgaggc ttttgtactt tagtaaatgc ttccactaaa ctgaaaccat
ggtgagaaag 7860 tttgactttg ttaaatattt tgaaatgtaa atgaaaagaa
ctactgtata ttaaaagttg 7920 gtttgaacca actttctagc tgctgttgaa
gaatatattg tcagaaacac aaggcttgat 7980 ttggttccca ggacagtgaa
acatagtaat accacgtaaa tcaagccatt cattttgggg 8040 aacagaagat
ccataacttt agaaatacgg gttttgactt aactcacaag agaactcatc 8100
ataagtactt gctgatggaa gaatgaccta gttgctcctc tcaacatggg tacagcaaac
8160 tcagcacagc caagaagcct caggtcgtgg agaacatgga ttaggatcct
agactgtaaa 8220 gacacagaag atgctgacct cacccctgcc acctatccca
agacctcact ggtctgtgga 8280 cagcagcaga aatgtttgca agataggcca
aaatgagtac aaaaggtctg tcttccatca 8340 gacccagtga tgctgcgact
cacacgcttc aattcaagac ctgaccgcta gtagggaggt 8400 ttattcagat
cgctggcagc ctcggctgag cagatgcaca gaggggatca ctgtgcagtg 8460
ggaccaccct cactggcctt ctgcagcagg gttctgggat gttttcagtg gtcaaaatac
8520 tctgtttaga gcaagggctc agaaaacaga aatactgtca tggaggtgct
gaacacaggg 8580 aaggtctggt acatattgga aattatgagc agaacaaata
ctcaactaaa tgcacaaagt 8640 ataaagtgta gccatgtcta gacaccatgt
tgtatcagaa taatttttgt gccaataaat 8700 gacatcagaa ttttaaacat
atgtaaaaaa aaa 8733 <210> SEQ ID NO 4 <211> LENGTH:
2549 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION
NUMBER: Genbank/004958 <309> DATABASE ENTRY DATE: 2002-04-04
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(2459) <400>
SEQUENCE: 4 Met Leu Gly Thr Gly Pro Ala Ala Ala Thr Thr Ala Ala Thr
Thr Ser 1 5 10 15 Ser Asn Val Ser Val Leu Gln Gln Phe Ala Ser Gly
Leu Lys Ser Arg 20 25 30 Asn Glu Glu Thr Arg Ala Lys Ala Ala Lys
Glu Leu Gln His Tyr Val 35 40 45 Thr Met Glu Leu Arg Glu Met Ser
Gln Glu Glu Ser Thr Arg Phe Tyr 50 55 60 Asp Gln Leu Asn His His
Ile Phe Glu Leu Val Ser Ser Ser Asp Ala 65 70 75 80 Asn Glu Arg Lys
Gly Gly Ile Leu Ala Ile Ala Ser Leu Ile Gly Val 85 90 95 Glu Gly
Gly Asn Ala Thr Arg Ile Gly Arg Phe Ala Asn Tyr Leu Arg 100 105 110
Asn Leu Leu Pro Ser Asn Asp Pro Val Val Met Glu Met Ala Ser Lys 115
120 125 Ala Ile Gly Arg Leu Ala Met Ala Gly Asp Thr Phe Thr Ala Glu
Tyr 130 135 140 Val Glu Phe Glu Val Lys Arg Ala Leu Glu Trp Leu Gly
Ala Asp Arg 145 150 155 160 Asn Glu Gly Arg Arg His Ala Ala Val Leu
Val Leu Arg Glu Leu Ala 165 170 175 Ile Ser Val Pro Thr Phe Phe Phe
Gln Gln Val Gln Pro Phe Phe Asp 180 185 190 Asn Ile Phe Val Ala Val
Trp Asp Pro Lys Gln Ala Ile Arg Glu Gly 195 200 205 Ala Val Ala Ala
Leu Arg Ala Cys Leu Ile Leu Thr Thr Gln Arg Glu 210 215 220 Pro Lys
Glu Met Gln Lys Pro Gln Trp Tyr Arg His Thr Phe Glu Glu 225 230 235
240 Ala Glu Lys Gly Phe Asp Glu Thr Leu Ala Lys Glu Lys Gly Met Asn
245 250 255 Arg Asp Asp Arg Ile His Gly Ala Leu Leu Ile Leu Asn Glu
Leu Val 260 265 270 Arg Ile Ser Ser Met Glu Gly Glu Arg Leu Arg Glu
Glu Met Glu Glu 275 280 285 Ile Thr Gln Gln Gln Leu Val His Asp Lys
Tyr Cys Lys Asp Leu Met 290 295 300 Gly Phe Gly Thr Lys Pro Arg His
Ile Thr Pro Phe Thr Ser Phe Gln 305 310 315 320 Ala Val Gln Pro Gln
Gln Ser Asn Ala Leu Val Gly Leu Leu Gly Tyr 325 330 335 Ser Ser His
Gln Gly Leu Met Gly Phe Gly Thr Ser Pro Ser Pro Ala 340 345 350 Lys
Ser Thr Leu Val Glu Ser Arg Cys Cys Arg Asp Leu Met Glu Glu 355 360
365 Lys Phe Asp Gln Val Cys Gln Trp Val Leu Lys Cys Arg Asn Ser Lys
370 375 380 Asn Ser Leu Ile Gln Met Thr Ile Leu Asn Leu Leu Pro Arg
Leu Ala 385 390 395 400 Ala Phe Arg Pro Ser Ala Phe Thr Asp Thr Gln
Tyr Leu Gln Asp Thr 405 410 415 Met Asn His Val Leu Ser Cys Val Lys
Lys Glu Lys Glu Arg Thr Ala 420 425 430 Ala Phe Gln Ala Leu Gly Leu
Leu Ser Val Ala Val Arg Ser Glu Phe 435 440 445 Lys Val Tyr Leu Pro
Arg Val Leu Asp Ile Ile Arg Ala Ala Leu Pro 450 455 460 Pro Lys Asp
Phe Ala His Lys Arg Gln Lys Ala Met Gln Val Asp Ala 465 470 475 480
Thr Val Phe Thr Cys Ile Ser Met Leu Ala Arg Ala Met Gly Pro Gly 485
490 495 Ile Gln Gln Asp Ile Lys Glu Leu Leu Glu Pro Met Leu Ala Val
Gly 500 505 510 Leu Ser Pro Ala Leu Thr Ala Val Leu Tyr Asp Leu Ser
Arg Gln Ile 515 520 525 Pro Gln Leu Lys Lys Asp Ile Gln Asp Gly Leu
Leu Lys Met Leu Ser 530 535 540 Leu Val Leu Met His Lys Pro Leu Arg
His Pro Gly Met Pro Lys Gly 545 550 555 560 Leu Ala His Gln Leu Ala
Ser Pro Gly Leu Thr Thr Leu Pro Glu Ala 565 570 575 Ser Asp Val Gly
Ser Ile Thr Leu Ala Leu Arg Thr Leu Gly Ser Phe 580 585 590 Glu Phe
Glu Gly His Ser Leu Thr Gln Phe Val Arg His Cys Ala Asp 595 600 605
His Phe Leu Asn Ser Glu His Lys Glu Ile Arg Met Glu Ala Ala Arg 610
615 620 Thr Cys Ser Arg Leu Leu Thr Pro Ser Ile His Leu Ile Ser Gly
His 625 630 635 640 Ala His Val Val Ser Gln Thr Ala Val Gln Val Val
Ala Asp Val Leu 645 650 655 Ser Lys Leu Leu Val Val Gly Ile Thr Asp
Pro Asp Pro Asp Ile Arg 660 665 670 Tyr Cys Val Leu Ala Ser Leu Asp
Glu Arg Phe Asp Ala His Leu Ala 675 680 685 Gln Ala Glu Asn Leu Gln
Ala Leu Phe Val Ala Leu Asn Asp Gln Val 690 695 700 Phe Glu Ile Arg
Glu Leu Ala Ile Cys Thr Val Gly Arg Leu Ser Ser 705 710 715 720 Met
Asn Pro Ala Phe Val Met Pro Phe Leu Arg Lys Met Leu Ile Gln 725 730
735 Ile Leu Thr Glu Leu Glu His Ser Gly Ile Gly Arg Ile Lys Glu Gln
740 745 750 Ser Ala Arg Met Leu Gly His Leu Val Ser Asn Ala Pro Arg
Leu Ile 755 760 765 Arg Pro Tyr Met Glu Pro Ile Leu Lys Ala Leu Ile
Leu Lys Leu Lys 770 775 780 Asp Pro Asp Pro Asp Pro Asn Pro Gly Val
Ile Asn Asn Val Leu Ala 785 790 795 800 Thr Ile Gly Glu Leu Ala Gln
Val Ser Gly Leu Glu Met Arg Lys Trp 805 810 815 Val Asp Glu Leu Phe
Ile Ile Ile Met Asp Met Leu Gln Asp Ser Ser 820 825 830
Leu Leu Ala Lys Arg Gln Val Ala Leu Trp Thr Leu Gly Gln Leu Val 835
840 845 Ala Ser Thr Gly Tyr Val Val Glu Pro Tyr Arg Lys Tyr Pro Thr
Leu 850 855 860 Leu Glu Val Leu Leu Asn Phe Leu Lys Thr Glu Gln Asn
Gln Gly Thr 865 870 875 880 Arg Arg Glu Ala Ile Arg Val Leu Gly Leu
Leu Gly Ala Leu Asp Pro 885 890 895 Tyr Lys His Lys Val Asn Ile Gly
Met Ile Asp Gln Ser Arg Asp Ala 900 905 910 Ser Ala Val Ser Leu Ser
Glu Ser Lys Ser Ser Gln Asp Ser Ser Asp 915 920 925 Tyr Ser Thr Ser
Glu Met Leu Val Asn Met Gly Asn Leu Pro Leu Asp 930 935 940 Glu Phe
Tyr Pro Ala Val Ser Met Val Ala Leu Met Arg Ile Phe Arg 945 950 955
960 Asp Gln Ser Leu Ser His His His Thr Met Val Val Gln Ala Ile Thr
965 970 975 Phe Ile Phe Lys Ser Leu Gly Leu Lys Cys Val Gln Phe Leu
Pro Gln 980 985 990 Val Met Pro Thr Phe Leu Asn Val Ile Arg Val Cys
Asp Gly Ala Ile 995 1000 1005 Arg Glu Phe Leu Phe Gln Gln Leu Gly
Met Leu Val Ser Phe Val 1010 1015 1020 Lys Ser His Ile Arg Pro Tyr
Met Asp Glu Ile Val Thr Leu Met 1025 1030 1035 Arg Glu Phe Trp Val
Met Asn Thr Ser Ile Gln Ser Thr Ile Ile 1040 1045 1050 Leu Leu Ile
Glu Gln Ile Val Val Ala Leu Gly Gly Glu Phe Lys 1055 1060 1065 Leu
Tyr Leu Pro Gln Leu Ile Pro His Met Leu Arg Val Phe Met 1070 1075
1080 His Asp Asn Ser Pro Gly Arg Ile Val Ser Ile Lys Leu Leu Ala
1085 1090 1095 Ala Ile Gln Leu Phe Gly Ala Asn Leu Asp Asp Tyr Leu
His Leu 1100 1105 1110 Leu Leu Pro Pro Ile Val Lys Leu Phe Asp Ala
Pro Glu Ala Pro 1115 1120 1125 Leu Pro Ser Arg Lys Ala Ala Leu Glu
Thr Val Asp Arg Leu Thr 1130 1135 1140 Glu Ser Leu Asp Phe Thr Asp
Tyr Ala Ser Arg Ile Ile His Pro 1145 1150 1155 Ile Val Arg Thr Leu
Asp Gln Ser Pro Glu Leu Arg Ser Thr Ala 1160 1165 1170 Met Asp Thr
Leu Ser Ser Leu Val Phe Gln Leu Gly Lys Lys Tyr 1175 1180 1185 Gln
Ile Phe Ile Pro Met Val Asn Lys Val Leu Val Arg His Arg 1190 1195
1200 Ile Asn His Gln Arg Tyr Asp Val Leu Ile Cys Arg Ile Val Lys
1205 1210 1215 Gly Tyr Thr Leu Ala Asp Glu Glu Glu Asp Pro Leu Ile
Tyr Gln 1220 1225 1230 His Arg Met Leu Arg Ser Gly Gln Gly Asp Ala
Leu Ala Ser Gly 1235 1240 1245 Pro Val Glu Thr Gly Pro Met Lys Lys
Leu His Val Ser Thr Ile 1250 1255 1260 Asn Leu Gln Lys Ala Trp Gly
Ala Ala Arg Arg Val Ser Lys Asp 1265 1270 1275 Asp Trp Leu Glu Trp
Leu Arg Arg Leu Ser Leu Glu Leu Leu Lys 1280 1285 1290 Asp Ser Ser
Ser Pro Ser Leu Arg Ser Cys Trp Ala Leu Ala Gln 1295 1300 1305 Ala
Tyr Asn Pro Met Ala Arg Asp Leu Phe Asn Ala Ala Phe Val 1310 1315
1320 Ser Cys Trp Ser Glu Leu Asn Glu Asp Gln Gln Asp Glu Leu Ile
1325 1330 1335 Arg Ser Ile Glu Leu Ala Leu Thr Ser Gln Asp Ile Ala
Glu Val 1340 1345 1350 Thr Gln Thr Leu Leu Asn Leu Ala Glu Phe Met
Glu His Ser Asp 1355 1360 1365 Lys Gly Pro Leu Pro Leu Arg Asp Asp
Asn Gly Ile Val Leu Leu 1370 1375 1380 Gly Glu Arg Ala Ala Lys Cys
Arg Ala Tyr Ala Lys Ala Leu His 1385 1390 1395 Tyr Lys Glu Leu Glu
Phe Gln Lys Gly Pro Thr Pro Ala Ile Leu 1400 1405 1410 Glu Ser Leu
Ile Ser Ile Asn Asn Lys Leu Gln Gln Pro Glu Ala 1415 1420 1425 Ala
Ala Gly Val Leu Glu Tyr Ala Met Lys His Phe Gly Glu Leu 1430 1435
1440 Glu Ile Gln Ala Thr Trp Tyr Glu Lys Leu His Glu Trp Glu Asp
1445 1450 1455 Ala Leu Val Ala Tyr Asp Lys Lys Met Asp Thr Asn Lys
Asp Asp 1460 1465 1470 Pro Glu Leu Met Leu Gly Arg Met Arg Cys Leu
Glu Ala Leu Gly 1475 1480 1485 Glu Trp Gly Gln Leu His Gln Gln Cys
Cys Glu Lys Trp Thr Leu 1490 1495 1500 Val Asn Asp Glu Thr Gln Ala
Lys Met Ala Arg Met Ala Ala Ala 1505 1510 1515 Ala Ala Trp Gly Leu
Gly Gln Trp Asp Ser Met Glu Glu Tyr Thr 1520 1525 1530 Cys Met Ile
Pro Arg Asp Thr His Asp Gly Ala Phe Tyr Arg Ala 1535 1540 1545 Val
Leu Ala Leu His Gln Asp Leu Phe Ser Leu Ala Gln Gln Cys 1550 1555
1560 Ile Asp Lys Ala Arg Asp Leu Leu Asp Ala Glu Leu Thr Ala Met
1565 1570 1575 Ala Gly Glu Ser Tyr Ser Arg Ala Tyr Gly Ala Met Val
Ser Cys 1580 1585 1590 His Met Leu Ser Glu Leu Glu Glu Val Ile Gln
Tyr Lys Leu Val 1595 1600 1605 Pro Glu Arg Arg Glu Ile Ile Arg Gln
Ile Trp Trp Glu Arg Leu 1610 1615 1620 Gln Gly Cys Gln Arg Ile Val
Glu Asp Trp Gln Lys Ile Leu Met 1625 1630 1635 Val Arg Ser Leu Val
Val Ser Pro His Glu Asp Met Arg Thr Trp 1640 1645 1650 Leu Lys Tyr
Ala Ser Leu Cys Gly Lys Ser Gly Arg Leu Ala Leu 1655 1660 1665 Ala
His Lys Thr Leu Val Leu Leu Leu Gly Val Asp Pro Ser Arg 1670 1675
1680 Gln Leu Asp His Pro Leu Pro Thr Val His Pro Gln Val Thr Tyr
1685 1690 1695 Ala Tyr Met Lys Asn Met Trp Lys Ser Ala Arg Lys Ile
Asp Ala 1700 1705 1710 Phe Gln His Met Gln His Phe Val Gln Thr Met
Gln Gln Gln Ala 1715 1720 1725 Gln His Ala Ile Ala Thr Glu Asp Gln
Gln His Lys Gln Glu Leu 1730 1735 1740 His Lys Leu Met Ala Arg Cys
Phe Leu Lys Leu Gly Glu Trp Gln 1745 1750 1755 Leu Asn Leu Gln Gly
Ile Asn Glu Ser Thr Ile Pro Lys Val Leu 1760 1765 1770 Gln Tyr Tyr
Ser Ala Ala Thr Glu His Asp Arg Ser Trp Tyr Lys 1775 1780 1785 Ala
Trp His Ala Trp Ala Val Met Asn Phe Glu Ala Val Leu His 1790 1795
1800 Tyr Lys His Gln Asn Gln Ala Arg Asp Glu Lys Lys Lys Leu Arg
1805 1810 1815 His Ala Ser Gly Ala Asn Ile Thr Asn Ala Thr Thr Ala
Ala Thr 1820 1825 1830 Thr Ala Ala Thr Ala Thr Thr Thr Ala Ser Thr
Glu Gly Ser Asn 1835 1840 1845 Ser Glu Ser Glu Ala Glu Ser Thr Glu
Asn Ser Pro Thr Pro Ser 1850 1855 1860 Pro Leu Gln Lys Lys Val Thr
Glu Asp Leu Ser Lys Thr Leu Leu 1865 1870 1875 Met Tyr Thr Val Pro
Ala Val Gln Gly Phe Phe Arg Ser Ile Ser 1880 1885 1890 Leu Ser Arg
Gly Asn Asn Leu Gln Asp Thr Leu Arg Val Leu Thr 1895 1900 1905 Leu
Trp Phe Asp Tyr Gly His Trp Pro Asp Val Asn Glu Ala Leu 1910 1915
1920 Val Glu Gly Val Lys Ala Ile Gln Ile Asp Thr Trp Leu Gln Val
1925 1930 1935 Ile Pro Gln Leu Ile Ala Arg Ile Asp Thr Pro Arg Pro
Leu Val 1940 1945 1950 Gly Arg Leu Ile His Gln Leu Leu Thr Asp Ile
Gly Arg Tyr His 1955 1960 1965 Pro Gln Ala Leu Ile Tyr Pro Leu Thr
Val Ala Ser Lys Ser Thr 1970 1975 1980 Thr Thr Ala Arg His Asn Ala
Ala Asn Lys Ile Leu Lys Asn Met 1985 1990 1995 Cys Glu His Ser Asn
Thr Leu Val Gln Gln Ala Met Met Val Ser 2000 2005 2010 Glu Glu Leu
Ile Arg Val Ala Ile Leu Trp His Glu Met Trp His 2015 2020 2025 Glu
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn 2030 2035
2040 Val Lys Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met
2045 2050 2055 Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn
Gln Ala 2060 2065 2070 Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp
Cys Arg Lys Tyr 2075 2080 2085 Met Lys Ser Gly Asn Val Lys Asp Leu
Thr Gln Ala Trp Asp Leu 2090 2095 2100
Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Gln Leu Pro Gln Leu 2105
2110 2115 Thr Ser Leu Glu Leu Gln Tyr Val Ser Pro Lys Leu Leu Met
Cys 2120 2125 2130 Arg Asp Leu Glu Leu Ala Val Pro Gly Thr Tyr Asp
Pro Asn Gln 2135 2140 2145 Pro Ile Ile Arg Ile Gln Ser Ile Ala Pro
Ser Leu Gln Val Ile 2150 2155 2160 Thr Ser Lys Gln Arg Pro Arg Lys
Leu Thr Leu Met Gly Ser Asn 2165 2170 2175 Gly His Glu Phe Val Phe
Leu Leu Lys Gly His Glu Asp Leu Arg 2180 2185 2190 Gln Asp Glu Arg
Val Met Gln Leu Phe Gly Leu Val Asn Thr Leu 2195 2200 2205 Leu Ala
Asn Asp Pro Thr Ser Leu Arg Lys Asn Leu Ser Ile Gln 2210 2215 2220
Arg Tyr Ala Val Ile Pro Leu Ser Thr Asn Ser Gly Leu Ile Gly 2225
2230 2235 Trp Val Pro His Cys Asp Thr Leu His Ala Leu Ile Arg Asp
Tyr 2240 2245 2250 Arg Glu Lys Lys Lys Ile Leu Leu Asn Ile Glu His
Arg Ile Met 2255 2260 2265 Leu Arg Met Ala Pro Asp Tyr Asp His Leu
Thr Leu Met Gln Lys 2270 2275 2280 Val Glu Val Phe Glu His Ala Val
Asn Asn Thr Ala Gly Asp Asp 2285 2290 2295 Leu Ala Lys Leu Leu Trp
Leu Lys Ser Pro Ser Ser Glu Val Trp 2300 2305 2310 Phe Asp Arg Arg
Thr Asn Tyr Thr Arg Ser Leu Ala Val Met Ser 2315 2320 2325 Met Val
Gly Tyr Ile Leu Gly Leu Gly Asp Arg His Pro Ser Asn 2330 2335 2340
Leu Met Leu Asp Arg Leu Ser Gly Lys Ile Leu His Ile Asp Phe 2345
2350 2355 Gly Asp Cys Phe Glu Val Ala Met Thr Arg Glu Lys Phe Pro
Glu 2360 2365 2370 Lys Ile Pro Phe Arg Leu Thr Arg Met Leu Thr Asn
Ala Met Glu 2375 2380 2385 Val Thr Gly Leu Asp Gly Asn Tyr Arg Ile
Thr Cys His Thr Val 2390 2395 2400 Met Glu Val Leu Arg Glu His Lys
Asp Ser Val Met Ala Val Leu 2405 2410 2415 Glu Ala Phe Val Tyr Asp
Pro Leu Leu Asn Trp Arg Leu Met Asp 2420 2425 2430 Thr Asn Thr Lys
Gly Asn Lys Arg Ser Arg Thr Arg Thr Asp Ser 2435 2440 2445 Tyr Ser
Ala Gly Gln Ser Val Glu Ile Leu Asp Gly Val Glu Leu 2450 2455 2460
Gly Glu Pro Ala His Lys Lys Thr Gly Thr Thr Val Pro Glu Ser 2465
2470 2475 Ile His Ser Phe Ile Gly Asp Gly Leu Val Lys Pro Glu Ala
Leu 2480 2485 2490 Asn Lys Lys Ala Ile Gln Ile Ile Asn Arg Val Arg
Asp Lys Leu 2495 2500 2505 Thr Gly Arg Asp Phe Ser His Asp Asp Thr
Leu Asp Val Pro Thr 2510 2515 2520 Gln Val Glu Leu Leu Ile Lys Gln
Ala Thr Ser His Glu Asn Leu 2525 2530 2535 Cys Gln Cys Tyr Ile Gly
Trp Cys Pro Phe Trp 2540 2545 <210> SEQ ID NO 5 <211>
LENGTH: 8155 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <300> PUBLICATION INFORMATION: <308> DATABASE
ACCESSION NUMBER: Genbank BC117166 <309> DATABASE ENTRY DATE:
2006-06-26 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(8155)
<400> SEQUENCE: 5 cgcgaacctc agggcaagat gcttggaacc ggacctgccg
ccgccaccac cgctgccacc 60 acatctagca atgtgagcgt cctgcagcag
tttgccagtg gcctaaagag ccggaatgag 120 gaaaccaggg ccaaagccgc
caaggagctc cagcactatg tcaccatgga actccgagag 180 atgagtcaag
aggagtctac tcgcttctat gaccaactga accatcacat ttttgaattg 240
gtttccagct cagatgccaa tgagaggaaa ggtggcatct tggccatagc tagcctcata
300 ggagtggaag gtgggaatgc cacccgaatt ggcagatttg ccaactatct
tcggaacctc 360 ctcccctcca atgacccagt tgtcatggaa atggcatcca
aggccattgg ccgtcttgcc 420 atggcagggg acacttttac cgctgagtac
gtggaatttg aggtgaagcg agccctggaa 480 tggctgggtg ctgaccgcaa
tgagggccgg agacatgcag ctgtcctggt tctccgtgag 540 ctggccatca
gcgtccctac cttcttcttc cagcaagtgc aacccttctt tgacaacatt 600
tttgtggccg tgtgggaccc caaacaggcc atccgtgagg gagctgtagc cgcccttcgt
660 gcctgtctga ttctcacaac ccagcgtgag ccgaaggaga tgcagaagcc
tcagtggtac 720 aggcacacat ttgaagaagc agagaaggga tttgatgaga
ccttggccaa agagaagggc 780 atgaatcggg atgatcggat ccatggagcc
ttgttgatcc ttaacgagct ggtccgaatc 840 agcagcatgg agggagagcg
tctgagagaa gaaatggaag aaatcacaca gcagcagctg 900 gtacacgaca
agtactgcaa agatctcatg ggcttcggaa caaaacctcg tcacattacc 960
cccttcacca gtttccaggc tgtacagccc cagcagtcaa atgccttggt ggggctgctg
1020 gggtacagct ctcaccaagg cctcatggga tttgggacct cccccagtcc
agctaagtcc 1080 accctggtgg agagccggtg ttgcagagac ttgatggagg
agaaatttga tcaggtgtgc 1140 cagtgggtgc tgaaatgcag gaatagcaag
aactcgctga tccaaatgac aatccttaat 1200 ttgttgcccc gcttggctgc
attccgacct tctgccttca cagataccca gtatctccaa 1260 gataccatga
accatgtcct aagctgtgtc aagaaggaga aggaacgtac agcggccttc 1320
caagccctgg ggctactttc tgtggctgtg aggtctgagt ttaaggtcta tttgcctcgc
1380 gtgctggaca tcatccgagc ggccctgccc ccaaaggact tcgcccataa
gaggcagaag 1440 gcaatgcagg tggacgccac agtcttcact tgcatcagca
tgctggctcg agcaatgggg 1500 ccaggcatcc agcaggatat caaggagctg
ctggagccca tgctggcagt gggactaagc 1560 cctgccctca ctgcagtgct
ctacgacctg agccgtcaga ttccacagct aaagaaggac 1620 attcaagatg
ggctactgaa aatgctgtcc ctggtcctta tgcacaaacc ccttcgccac 1680
ccaggcatgc ccaagggcct ggcccatcag ctggcctctc ctggcctcac gaccctccct
1740 gaggccagcg atgtgggcag catcactctt gccctccgaa cgcttggcag
ctttgaattt 1800 gaaggccact ctctgaccca atttgttcgc cactgtgcgg
atcattttct gaacagtgag 1860 cacaaggaga tccgcatgga ggctgcccgc
acctgctccc gcctgctcac accctccatc 1920 cacctcatca gtggccatgc
tcatgtggtt agccagaccg cagtgcaagt ggtggcagat 1980 gtgcttagca
aactgctcgt agttgggata acagatcctg accctgacat tcgctactgt 2040
gtcttggcgt ccctggacga gcgctttgat gcacacctgg cccaggcgga gaacttgcag
2100 gccttgtttg tggctctgaa tgaccaggtg tttgagatcc gggagctggc
catctgcact 2160 gtgggccgac tcagtagcat gaaccctgcc tttgtcatgc
ctttcctgcg caagatgctc 2220 atccagattt tgacagagtt ggagcacagt
gggattggaa gaatcaaaga gcagagtgcc 2280 cgcatgctgg ggcacctggt
ctccaatgcc ccccgactca tccgccccta catggagcct 2340 attctgaagg
cattaatttt gaaactgaaa gatccagacc ctgatccaaa cccaggtgtg 2400
atcaataatg tcctggcaac aataggagaa ttggcacagg ttagtggcct ggaaatgagg
2460 aaatgggttg atgaactttt tattatcatc atggacatgc tccaggattc
ctctttgttg 2520 gccaaaaggc aggtggctct gtggaccctg ggacagttgg
tggccagcac tggctatgta 2580 gtagagccct acaggaagta ccctactttg
cttgaggtgc tactgaattt tctgaagact 2640 gagcagaacc agggtacacg
cagagaggcc atccgtgtgt tagggctttt aggggctttg 2700 gatccttaca
agcacaaagt gaacattggc atgatagacc agtcccggga tgcctctgct 2760
gtcagcctgt cagaatccaa gtcaagtcag gattcctctg actatagcac tagtgaaatg
2820 ctggtcaaca tgggaaactt gcctctggat gagttctacc cagctgtgtc
catggtggcc 2880 ctgatgcgga tcttccgaga ccagtcactc tctcatcatc
acaccatggt tgtccaggcc 2940 atcaccttca tcttcaagtc cctgggactc
aaatgtgtgc agttcctgcc ccaggtcatg 3000 cccacgttcc ttaatgtcat
tcgagtctgt gatggggcca tccgggaatt tttgttccag 3060 cagctgggaa
tgttggtgtc ctttgtgaag agccacatca gaccttatat ggatgaaata 3120
gtcaccctca tgagagaatt ctgggtcatg aacacctcaa ttcagagcac gatcattctt
3180 ctcattgagc aaattgtggt agctcttggg ggtgaattta agctctacct
gccccagctg 3240 atcccacaca tgctgcgtgt cttcatgcat gacaacagcc
caggccgcat tgtctctatc 3300 aagttactgg ctgcaatcca gctgtttggc
gccaacctgg atgactacct gcatttactg 3360 ctgcctccta ttgttaagtt
gtttgatgcc cctgaagctc cactgccatc tcgaaaggca 3420 gcgctagaga
ctgtggaccg cctgacggag tccctggatt tcactgacta tgcctcccgg 3480
atcattcacc ctattgttcg aacactggac cagagcccag aactgcgctc cacagccatg
3540 gacacgctgt cttcacttgt ttttcagctg gggaagaagt accaaatttt
cattccaatg 3600 gtgaataaag ttctggtgcg acaccgaatc aatcatcagc
gctatgatgt gctcatctgc 3660 agaattgtca agggatacac acttgctgat
gaagaggagg atcctttgat ttaccagcat 3720 cggatgctta ggagtggcca
aggggatgca ttggctagtg gaccagtgga aacaggaccc 3780 atgaagaaac
tgcacgtcag caccatcaac ctccaaaagg cctggggcgc tgccaggagg 3840
gtctccaaag atgactggct ggaatggctg agacggctga gcctggagct gctgaaggac
3900 tcatcatcgc cctccctgcg ctcctgctgg gccctggcac aggcctacaa
cccgatggcc 3960 agggatctct tcaatgctgc atttgtgtcc tgctggtctg
aactgaatga agatcaacag 4020 gatgagctca tcagaagcat cgagttggcc
ctcacctcac aagacatcgc tgaagtcaca 4080 cagaccctct taaacttggc
tgaattcatg gaacacagtg acaagggccc cctgccactg 4140 agagatgaca
atggcattgt tctgctgggt gagagagctg ccaagtgccg agcatatgcc 4200
aaagcactac actacaaaga actggagttc cagaaaggcc ccacccctgc cattctagaa
4260 tctctcatca gcattaataa taagctacag cagccggagg cagcggccgg
agtgttagaa 4320 tatgccatga aacactttgg agagctggag atccaggcta
cctggtatga gaaactgcac 4380 gagtgggagg atgcccttgt ggcctatgac
aagaaaatgg acaccaacaa ggacgaccca 4440
gagctgatgc tgggccgcat gcgctgcctc gaggccttgg gggaatgggg tcaactccac
4500 cagcagtgct gtgaaaagtg gaccctggtt aatgatgaga cccaagccaa
gatggcccgg 4560 atggctgctg cagctgcatg gggtttaggt cagtgggaca
gcatggaaga atacacctgt 4620 atgatccctc gggacaccca tgatggggca
ttttatagag ctgtgctggc actgcatcag 4680 gacctcttct ccttggcaca
acagtgcatt gacaaggcca gggacctgct ggatgctgaa 4740 ttaactgcga
tggcaggaga gagttacagt cgggcatatg gggccatggt ttcttgccac 4800
atgctgtccg agctggagga ggttatccag tacaaacttg tccccgagcg acgagagatc
4860 atccgccaga tctggtggga gagactgcag ggctgccagc gtatcgtaga
ggactggcag 4920 aaaatcctta tggtgcggtc ccttgtggtc agccctcatg
aagacatgag aacctggctc 4980 aagtatgcaa gcctgtgcgg caagagtggc
aggctggctc ttgctcataa aactttagtg 5040 ttgctcctgg gagttgatcc
gtctcggcaa cttgaccatc ctctgccaac agttcaccct 5100 caggtgacct
atgcctacat gaaaaacatg tggaagagtg cccgcaagat cgatgccttc 5160
cagcacatgc agcattttgt ccagaccatg cagcaacagg cccagcatgc catcgctact
5220 gaggaccagc agcataagca ggaactgcac aagctcatgg cccgatgctt
cctgaaactt 5280 ggagagtggc agctgaatct acagggcatc aatgagagca
caatccccaa agtgctgcag 5340 tactacagcg ccgccacaga gcacgaccgc
agctggtaca aggcctggca tgcgtgggca 5400 gtgatgaact tcgaagctgt
gctacactac aaacatcaga accaagcccg cgatgagaag 5460 aagaaactgc
gtcatgccag cggggccaac atcaccaacg ccaccactgc cgccaccacg 5520
gccgccactg ccaccaccac tgccagcacc gagggcagca acagtgagag tgaggccgag
5580 agcaccgaga acagccccac cccatcgccg ctgcagaaga aggtcactga
ggatctgtcc 5640 aaaaccctcc tgatgtacac ggtgcctgcc gtccagggct
tcttccgttc catctccttg 5700 tcacgaggca acaacctcca ggatacactc
agagttctca ccttatggtt tgattatggt 5760 cactggccag atgtcaatga
ggccttagtg gagggggtga aagccatcca gattgatacc 5820 tggctacagg
ttatacctca gctcattgca agaattgata cgcccagacc cttggtggga 5880
cgtctcattc accagcttct cacagacatt ggtcggtacc acccccaggc cctcatctac
5940 ccactgacag tggcttctaa gtctaccacg acagcccggc acaatgcagc
caacaagatt 6000 ctgaagaaca tgtgtgagca cagcaacacc ctggtccagc
aggccatgat ggtgagcgag 6060 gagctgatcc gagtggccat cctctggcat
gagatgtggc atgaaggcct ggaagaggca 6120 tctcgtttgt actttgggga
aaggaacgtg aaaggcatgt ttgaggtgct ggagcccttg 6180 catgctatga
tggaacgggg cccccagact ctgaaggaaa catcctttaa tcaggcctat 6240
ggtcgagatt taatggaggc ccaagagtgg tgcaggaagt acatgaaatc agggaatgtc
6300 aaggacctca cccaagcctg ggacctctat tatcatgtgt tccgacgaat
ctcaaagcag 6360 ctgcctcagc tcacatcctt agagctgcaa tatgtttccc
caaaacttct gatgtgccgg 6420 gaccttgaat tggctgtgcc aggaacatat
gaccccaacc agccaatcat tcgcattcag 6480 tccatagcac cgtctttgca
agtcatcaca tccaagcaga ggccccggaa attgacactt 6540 atgggcagca
acggacatga gtttgttttc cttctaaaag gccatgaaga tctgcgccag 6600
gatgagcgtg tgatgcagct cttcggcctg gttaacaccc tcctggccaa tgacccaaca
6660 tctcttcgga aaaacctcag catccagaga tacgctgtca tccctttatc
gaccaactcg 6720 ggcctcattg gctgggttcc ccactgtgac acactgcacg
ccctcatccg ggactacagg 6780 gagaagaaga agatccttct caacatcgag
catcgcatca tgttgcggat ggctccggac 6840 tatgaccact tgactctgat
gcagaaggtg gaggtgtttg agcatgccgt caataataca 6900 gctggggacg
acctggccaa gctgctatgg ctgaaaagcc ccagctccga ggtgtggttt 6960
gaccgaagaa ccaattatac ccgttcttta gcggtcatgt caatggttgg gtatatttta
7020 ggcctgggag atagacaccc atccaacctg atgctggacc gtctgagtgg
gaagatcctg 7080 cacattgact ttggggactg ctttgaggtt gctatgaccc
gagagaagtt tccagagaag 7140 attccattta gactaacaag aatgttgacc
aatgctatgg aggttacagg cctggatggc 7200 aactacagaa tcacatgcca
cacagtgatg gaggtgctgc gagagcacaa ggacagtgtc 7260 atggccgtgc
tggaagcctt tgtctatgac cccttgctga actggaggct gatggacaca 7320
aataccaaag gcaacaagcg atcccgaacg aggacggatt cctactctgc tggccagtca
7380 gtcgaaattt tggacggtgt ggaacttgga gagccagccc ataagaaaac
ggggaccaca 7440 gtgccagaat ctattcattc tttcattgga gacggtttgg
tgaaaccaga ggccctaaat 7500 aagaaagcta tccagattat taacagggtt
cgagataagc tcactggtcg ggacttctct 7560 catgatgaca ctttggatgt
tccaacgcaa gttgagctgc tcatcaaaca agcgacatcc 7620 catgaaaacc
tctgccagtg ctatattggc tggtgccctt tctggtaact ggaggcccag 7680
atgtgcccat cacgtttttt ctgaggcttt tgtactttag taaatgcttc cactaaactg
7740 aaaccatggt gagaaagttt gactttgtta aatattttga aatgtaaatg
aaaagaacta 7800 ctgtatatta aaagttggtt tgaaccaact ttctagctgc
tgttgaagaa tatattgtca 7860 gaaacacaag gcttgatttg gttcccagga
cagtgaaaca tagtaatacc acgtaaatca 7920 agccattcat tttggggaac
agaagatcca taactttaga aatacgggtt ttgacttaac 7980 tcacaagaga
actcatcata agtacttgct gatggaagaa tgacctagtt gctcctctca 8040
acatgggtac agcaaactca gcacagccaa gaagcctcag gtcgtggaga acatggatta
8100 ggatcctaga ctgtaaagac acagaagatg ctgacctcac ccctgccacc tatcc
8155 <210> SEQ ID NO 6 <211> LENGTH: 2549 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <300>
PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:
Genbank BC117166 <309> DATABASE ENTRY DATE: 2006-06-26
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(2549) <400>
SEQUENCE: 6 Met Leu Gly Thr Gly Pro Ala Ala Ala Thr Thr Ala Ala Thr
Thr Ser 1 5 10 15 Ser Asn Val Ser Val Leu Gln Gln Phe Ala Ser Gly
Leu Lys Ser Arg 20 25 30 Asn Glu Glu Thr Arg Ala Lys Ala Ala Lys
Glu Leu Gln His Tyr Val 35 40 45 Thr Met Glu Leu Arg Glu Met Ser
Gln Glu Glu Ser Thr Arg Phe Tyr 50 55 60 Asp Gln Leu Asn His His
Ile Phe Glu Leu Val Ser Ser Ser Asp Ala 65 70 75 80 Asn Glu Arg Lys
Gly Gly Ile Leu Ala Ile Ala Ser Leu Ile Gly Val 85 90 95 Glu Gly
Gly Asn Ala Thr Arg Ile Gly Arg Phe Ala Asn Tyr Leu Arg 100 105 110
Asn Leu Leu Pro Ser Asn Asp Pro Val Val Met Glu Met Ala Ser Lys 115
120 125 Ala Ile Gly Arg Leu Ala Met Ala Gly Asp Thr Phe Thr Ala Glu
Tyr 130 135 140 Val Glu Phe Glu Val Lys Arg Ala Leu Glu Trp Leu Gly
Ala Asp Arg 145 150 155 160 Asn Glu Gly Arg Arg His Ala Ala Val Leu
Val Leu Arg Glu Leu Ala 165 170 175 Ile Ser Val Pro Thr Phe Phe Phe
Gln Gln Val Gln Pro Phe Phe Asp 180 185 190 Asn Ile Phe Val Ala Val
Trp Asp Pro Lys Gln Ala Ile Arg Glu Gly 195 200 205 Ala Val Ala Ala
Leu Arg Ala Cys Leu Ile Leu Thr Thr Gln Arg Glu 210 215 220 Pro Lys
Glu Met Gln Lys Pro Gln Trp Tyr Arg His Thr Phe Glu Glu 225 230 235
240 Ala Glu Lys Gly Phe Asp Glu Thr Leu Ala Lys Glu Lys Gly Met Asn
245 250 255 Arg Asp Asp Arg Ile His Gly Ala Leu Leu Ile Leu Asn Glu
Leu Val 260 265 270 Arg Ile Ser Ser Met Glu Gly Glu Arg Leu Arg Glu
Glu Met Glu Glu 275 280 285 Ile Thr Gln Gln Gln Leu Val His Asp Lys
Tyr Cys Lys Asp Leu Met 290 295 300 Gly Phe Gly Thr Lys Pro Arg His
Ile Thr Pro Phe Thr Ser Phe Gln 305 310 315 320 Ala Val Gln Pro Gln
Gln Ser Asn Ala Leu Val Gly Leu Leu Gly Tyr 325 330 335 Ser Ser His
Gln Gly Leu Met Gly Phe Gly Thr Ser Pro Ser Pro Ala 340 345 350 Lys
Ser Thr Leu Val Glu Ser Arg Cys Cys Arg Asp Leu Met Glu Glu 355 360
365 Lys Phe Asp Gln Val Cys Gln Trp Val Leu Lys Cys Arg Asn Ser Lys
370 375 380 Asn Ser Leu Ile Gln Met Thr Ile Leu Asn Leu Leu Pro Arg
Leu Ala 385 390 395 400 Ala Phe Arg Pro Ser Ala Phe Thr Asp Thr Gln
Tyr Leu Gln Asp Thr 405 410 415 Met Asn His Val Leu Ser Cys Val Lys
Lys Glu Lys Glu Arg Thr Ala 420 425 430 Ala Phe Gln Ala Leu Gly Leu
Leu Ser Val Ala Val Arg Ser Glu Phe 435 440 445 Lys Val Tyr Leu Pro
Arg Val Leu Asp Ile Ile Arg Ala Ala Leu Pro 450 455 460 Pro Lys Asp
Phe Ala His Lys Arg Gln Lys Ala Met Gln Val Asp Ala 465 470 475 480
Thr Val Phe Thr Cys Ile Ser Met Leu Ala Arg Ala Met Gly Pro Gly 485
490 495 Ile Gln Gln Asp Ile Lys Glu Leu Leu Glu Pro Met Leu Ala Val
Gly 500 505 510 Leu Ser Pro Ala Leu Thr Ala Val Leu Tyr Asp Leu Ser
Arg Gln Ile 515 520 525 Pro Gln Leu Lys Lys Asp Ile Gln Asp Gly Leu
Leu Lys Met Leu Ser 530 535 540 Leu Val Leu Met His Lys Pro Leu Arg
His Pro Gly Met Pro Lys Gly 545 550 555 560 Leu Ala His Gln Leu Ala
Ser Pro Gly Leu Thr Thr Leu Pro Glu Ala 565 570 575 Ser Asp Val Gly
Ser Ile Thr Leu Ala Leu Arg Thr Leu Gly Ser Phe 580 585 590 Glu Phe
Glu Gly His Ser Leu Thr Gln Phe Val Arg His Cys Ala Asp 595 600 605
His Phe Leu Asn Ser Glu His Lys Glu Ile Arg Met Glu Ala Ala Arg
610 615 620 Thr Cys Ser Arg Leu Leu Thr Pro Ser Ile His Leu Ile Ser
Gly His 625 630 635 640 Ala His Val Val Ser Gln Thr Ala Val Gln Val
Val Ala Asp Val Leu 645 650 655 Ser Lys Leu Leu Val Val Gly Ile Thr
Asp Pro Asp Pro Asp Ile Arg 660 665 670 Tyr Cys Val Leu Ala Ser Leu
Asp Glu Arg Phe Asp Ala His Leu Ala 675 680 685 Gln Ala Glu Asn Leu
Gln Ala Leu Phe Val Ala Leu Asn Asp Gln Val 690 695 700 Phe Glu Ile
Arg Glu Leu Ala Ile Cys Thr Val Gly Arg Leu Ser Ser 705 710 715 720
Met Asn Pro Ala Phe Val Met Pro Phe Leu Arg Lys Met Leu Ile Gln 725
730 735 Ile Leu Thr Glu Leu Glu His Ser Gly Ile Gly Arg Ile Lys Glu
Gln 740 745 750 Ser Ala Arg Met Leu Gly His Leu Val Ser Asn Ala Pro
Arg Leu Ile 755 760 765 Arg Pro Tyr Met Glu Pro Ile Leu Lys Ala Leu
Ile Leu Lys Leu Lys 770 775 780 Asp Pro Asp Pro Asp Pro Asn Pro Gly
Val Ile Asn Asn Val Leu Ala 785 790 795 800 Thr Ile Gly Glu Leu Ala
Gln Val Ser Gly Leu Glu Met Arg Lys Trp 805 810 815 Val Asp Glu Leu
Phe Ile Ile Ile Met Asp Met Leu Gln Asp Ser Ser 820 825 830 Leu Leu
Ala Lys Arg Gln Val Ala Leu Trp Thr Leu Gly Gln Leu Val 835 840 845
Ala Ser Thr Gly Tyr Val Val Glu Pro Tyr Arg Lys Tyr Pro Thr Leu 850
855 860 Leu Glu Val Leu Leu Asn Phe Leu Lys Thr Glu Gln Asn Gln Gly
Thr 865 870 875 880 Arg Arg Glu Ala Ile Arg Val Leu Gly Leu Leu Gly
Ala Leu Asp Pro 885 890 895 Tyr Lys His Lys Val Asn Ile Gly Met Ile
Asp Gln Ser Arg Asp Ala 900 905 910 Ser Ala Val Ser Leu Ser Glu Ser
Lys Ser Ser Gln Asp Ser Ser Asp 915 920 925 Tyr Ser Thr Ser Glu Met
Leu Val Asn Met Gly Asn Leu Pro Leu Asp 930 935 940 Glu Phe Tyr Pro
Ala Val Ser Met Val Ala Leu Met Arg Ile Phe Arg 945 950 955 960 Asp
Gln Ser Leu Ser His His His Thr Met Val Val Gln Ala Ile Thr 965 970
975 Phe Ile Phe Lys Ser Leu Gly Leu Lys Cys Val Gln Phe Leu Pro Gln
980 985 990 Val Met Pro Thr Phe Leu Asn Val Ile Arg Val Cys Asp Gly
Ala Ile 995 1000 1005 Arg Glu Phe Leu Phe Gln Gln Leu Gly Met Leu
Val Ser Phe Val 1010 1015 1020 Lys Ser His Ile Arg Pro Tyr Met Asp
Glu Ile Val Thr Leu Met 1025 1030 1035 Arg Glu Phe Trp Val Met Asn
Thr Ser Ile Gln Ser Thr Ile Ile 1040 1045 1050 Leu Leu Ile Glu Gln
Ile Val Val Ala Leu Gly Gly Glu Phe Lys 1055 1060 1065 Leu Tyr Leu
Pro Gln Leu Ile Pro His Met Leu Arg Val Phe Met 1070 1075 1080 His
Asp Asn Ser Pro Gly Arg Ile Val Ser Ile Lys Leu Leu Ala 1085 1090
1095 Ala Ile Gln Leu Phe Gly Ala Asn Leu Asp Asp Tyr Leu His Leu
1100 1105 1110 Leu Leu Pro Pro Ile Val Lys Leu Phe Asp Ala Pro Glu
Ala Pro 1115 1120 1125 Leu Pro Ser Arg Lys Ala Ala Leu Glu Thr Val
Asp Arg Leu Thr 1130 1135 1140 Glu Ser Leu Asp Phe Thr Asp Tyr Ala
Ser Arg Ile Ile His Pro 1145 1150 1155 Ile Val Arg Thr Leu Asp Gln
Ser Pro Glu Leu Arg Ser Thr Ala 1160 1165 1170 Met Asp Thr Leu Ser
Ser Leu Val Phe Gln Leu Gly Lys Lys Tyr 1175 1180 1185 Gln Ile Phe
Ile Pro Met Val Asn Lys Val Leu Val Arg His Arg 1190 1195 1200 Ile
Asn His Gln Arg Tyr Asp Val Leu Ile Cys Arg Ile Val Lys 1205 1210
1215 Gly Tyr Thr Leu Ala Asp Glu Glu Glu Asp Pro Leu Ile Tyr Gln
1220 1225 1230 His Arg Met Leu Arg Ser Gly Gln Gly Asp Ala Leu Ala
Ser Gly 1235 1240 1245 Pro Val Glu Thr Gly Pro Met Lys Lys Leu His
Val Ser Thr Ile 1250 1255 1260 Asn Leu Gln Lys Ala Trp Gly Ala Ala
Arg Arg Val Ser Lys Asp 1265 1270 1275 Asp Trp Leu Glu Trp Leu Arg
Arg Leu Ser Leu Glu Leu Leu Lys 1280 1285 1290 Asp Ser Ser Ser Pro
Ser Leu Arg Ser Cys Trp Ala Leu Ala Gln 1295 1300 1305 Ala Tyr Asn
Pro Met Ala Arg Asp Leu Phe Asn Ala Ala Phe Val 1310 1315 1320 Ser
Cys Trp Ser Glu Leu Asn Glu Asp Gln Gln Asp Glu Leu Ile 1325 1330
1335 Arg Ser Ile Glu Leu Ala Leu Thr Ser Gln Asp Ile Ala Glu Val
1340 1345 1350 Thr Gln Thr Leu Leu Asn Leu Ala Glu Phe Met Glu His
Ser Asp 1355 1360 1365 Lys Gly Pro Leu Pro Leu Arg Asp Asp Asn Gly
Ile Val Leu Leu 1370 1375 1380 Gly Glu Arg Ala Ala Lys Cys Arg Ala
Tyr Ala Lys Ala Leu His 1385 1390 1395 Tyr Lys Glu Leu Glu Phe Gln
Lys Gly Pro Thr Pro Ala Ile Leu 1400 1405 1410 Glu Ser Leu Ile Ser
Ile Asn Asn Lys Leu Gln Gln Pro Glu Ala 1415 1420 1425 Ala Ala Gly
Val Leu Glu Tyr Ala Met Lys His Phe Gly Glu Leu 1430 1435 1440 Glu
Ile Gln Ala Thr Trp Tyr Glu Lys Leu His Glu Trp Glu Asp 1445 1450
1455 Ala Leu Val Ala Tyr Asp Lys Lys Met Asp Thr Asn Lys Asp Asp
1460 1465 1470 Pro Glu Leu Met Leu Gly Arg Met Arg Cys Leu Glu Ala
Leu Gly 1475 1480 1485 Glu Trp Gly Gln Leu His Gln Gln Cys Cys Glu
Lys Trp Thr Leu 1490 1495 1500 Val Asn Asp Glu Thr Gln Ala Lys Met
Ala Arg Met Ala Ala Ala 1505 1510 1515 Ala Ala Trp Gly Leu Gly Gln
Trp Asp Ser Met Glu Glu Tyr Thr 1520 1525 1530 Cys Met Ile Pro Arg
Asp Thr His Asp Gly Ala Phe Tyr Arg Ala 1535 1540 1545 Val Leu Ala
Leu His Gln Asp Leu Phe Ser Leu Ala Gln Gln Cys 1550 1555 1560 Ile
Asp Lys Ala Arg Asp Leu Leu Asp Ala Glu Leu Thr Ala Met 1565 1570
1575 Ala Gly Glu Ser Tyr Ser Arg Ala Tyr Gly Ala Met Val Ser Cys
1580 1585 1590 His Met Leu Ser Glu Leu Glu Glu Val Ile Gln Tyr Lys
Leu Val 1595 1600 1605 Pro Glu Arg Arg Glu Ile Ile Arg Gln Ile Trp
Trp Glu Arg Leu 1610 1615 1620 Gln Gly Cys Gln Arg Ile Val Glu Asp
Trp Gln Lys Ile Leu Met 1625 1630 1635 Val Arg Ser Leu Val Val Ser
Pro His Glu Asp Met Arg Thr Trp 1640 1645 1650 Leu Lys Tyr Ala Ser
Leu Cys Gly Lys Ser Gly Arg Leu Ala Leu 1655 1660 1665 Ala His Lys
Thr Leu Val Leu Leu Leu Gly Val Asp Pro Ser Arg 1670 1675 1680 Gln
Leu Asp His Pro Leu Pro Thr Val His Pro Gln Val Thr Tyr 1685 1690
1695 Ala Tyr Met Lys Asn Met Trp Lys Ser Ala Arg Lys Ile Asp Ala
1700 1705 1710 Phe Gln His Met Gln His Phe Val Gln Thr Met Gln Gln
Gln Ala 1715 1720 1725 Gln His Ala Ile Ala Thr Glu Asp Gln Gln His
Lys Gln Glu Leu 1730 1735 1740 His Lys Leu Met Ala Arg Cys Phe Leu
Lys Leu Gly Glu Trp Gln 1745 1750 1755 Leu Asn Leu Gln Gly Ile Asn
Glu Ser Thr Ile Pro Lys Val Leu 1760 1765 1770 Gln Tyr Tyr Ser Ala
Ala Thr Glu His Asp Arg Ser Trp Tyr Lys 1775 1780 1785 Ala Trp His
Ala Trp Ala Val Met Asn Phe Glu Ala Val Leu His 1790 1795 1800 Tyr
Lys His Gln Asn Gln Ala Arg Asp Glu Lys Lys Lys Leu Arg 1805 1810
1815 His Ala Ser Gly Ala Asn Ile Thr Asn Ala Thr Thr Ala Ala Thr
1820 1825 1830 Thr Ala Ala Thr Ala Thr Thr Thr Ala Ser Thr Glu Gly
Ser Asn 1835 1840 1845 Ser Glu Ser Glu Ala Glu Ser Thr Glu Asn Ser
Pro Thr Pro Ser 1850 1855 1860 Pro Leu Gln Lys Lys Val Thr Glu Asp
Leu Ser Lys Thr Leu Leu 1865 1870 1875 Met Tyr Thr Val Pro Ala Val
Gln Gly Phe Phe Arg Ser Ile Ser 1880 1885 1890
Leu Ser Arg Gly Asn Asn Leu Gln Asp Thr Leu Arg Val Leu Thr 1895
1900 1905 Leu Trp Phe Asp Tyr Gly His Trp Pro Asp Val Asn Glu Ala
Leu 1910 1915 1920 Val Glu Gly Val Lys Ala Ile Gln Ile Asp Thr Trp
Leu Gln Val 1925 1930 1935 Ile Pro Gln Leu Ile Ala Arg Ile Asp Thr
Pro Arg Pro Leu Val 1940 1945 1950 Gly Arg Leu Ile His Gln Leu Leu
Thr Asp Ile Gly Arg Tyr His 1955 1960 1965 Pro Gln Ala Leu Ile Tyr
Pro Leu Thr Val Ala Ser Lys Ser Thr 1970 1975 1980 Thr Thr Ala Arg
His Asn Ala Ala Asn Lys Ile Leu Lys Asn Met 1985 1990 1995 Cys Glu
His Ser Asn Thr Leu Val Gln Gln Ala Met Met Val Ser 2000 2005 2010
Glu Glu Leu Ile Arg Val Ala Ile Leu Trp His Glu Met Trp His 2015
2020 2025 Glu Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg
Asn 2030 2035 2040 Val Lys Gly Met Phe Glu Val Leu Glu Pro Leu His
Ala Met Met 2045 2050 2055 Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr
Ser Phe Asn Gln Ala 2060 2065 2070 Tyr Gly Arg Asp Leu Met Glu Ala
Gln Glu Trp Cys Arg Lys Tyr 2075 2080 2085 Met Lys Ser Gly Asn Val
Lys Asp Leu Thr Gln Ala Trp Asp Leu 2090 2095 2100 Tyr Tyr His Val
Phe Arg Arg Ile Ser Lys Gln Leu Pro Gln Leu 2105 2110 2115 Thr Ser
Leu Glu Leu Gln Tyr Val Ser Pro Lys Leu Leu Met Cys 2120 2125 2130
Arg Asp Leu Glu Leu Ala Val Pro Gly Thr Tyr Asp Pro Asn Gln 2135
2140 2145 Pro Ile Ile Arg Ile Gln Ser Ile Ala Pro Ser Leu Gln Val
Ile 2150 2155 2160 Thr Ser Lys Gln Arg Pro Arg Lys Leu Thr Leu Met
Gly Ser Asn 2165 2170 2175 Gly His Glu Phe Val Phe Leu Leu Lys Gly
His Glu Asp Leu Arg 2180 2185 2190 Gln Asp Glu Arg Val Met Gln Leu
Phe Gly Leu Val Asn Thr Leu 2195 2200 2205 Leu Ala Asn Asp Pro Thr
Ser Leu Arg Lys Asn Leu Ser Ile Gln 2210 2215 2220 Arg Tyr Ala Val
Ile Pro Leu Ser Thr Asn Ser Gly Leu Ile Gly 2225 2230 2235 Trp Val
Pro His Cys Asp Thr Leu His Ala Leu Ile Arg Asp Tyr 2240 2245 2250
Arg Glu Lys Lys Lys Ile Leu Leu Asn Ile Glu His Arg Ile Met 2255
2260 2265 Leu Arg Met Ala Pro Asp Tyr Asp His Leu Thr Leu Met Gln
Lys 2270 2275 2280 Val Glu Val Phe Glu His Ala Val Asn Asn Thr Ala
Gly Asp Asp 2285 2290 2295 Leu Ala Lys Leu Leu Trp Leu Lys Ser Pro
Ser Ser Glu Val Trp 2300 2305 2310 Phe Asp Arg Arg Thr Asn Tyr Thr
Arg Ser Leu Ala Val Met Ser 2315 2320 2325 Met Val Gly Tyr Ile Leu
Gly Leu Gly Asp Arg His Pro Ser Asn 2330 2335 2340 Leu Met Leu Asp
Arg Leu Ser Gly Lys Ile Leu His Ile Asp Phe 2345 2350 2355 Gly Asp
Cys Phe Glu Val Ala Met Thr Arg Glu Lys Phe Pro Glu 2360 2365 2370
Lys Ile Pro Phe Arg Leu Thr Arg Met Leu Thr Asn Ala Met Glu 2375
2380 2385 Val Thr Gly Leu Asp Gly Asn Tyr Arg Ile Thr Cys His Thr
Val 2390 2395 2400 Met Glu Val Leu Arg Glu His Lys Asp Ser Val Met
Ala Val Leu 2405 2410 2415 Glu Ala Phe Val Tyr Asp Pro Leu Leu Asn
Trp Arg Leu Met Asp 2420 2425 2430 Thr Asn Thr Lys Gly Asn Lys Arg
Ser Arg Thr Arg Thr Asp Ser 2435 2440 2445 Tyr Ser Ala Gly Gln Ser
Val Glu Ile Leu Asp Gly Val Glu Leu 2450 2455 2460 Gly Glu Pro Ala
His Lys Lys Thr Gly Thr Thr Val Pro Glu Ser 2465 2470 2475 Ile His
Ser Phe Ile Gly Asp Gly Leu Val Lys Pro Glu Ala Leu 2480 2485 2490
Asn Lys Lys Ala Ile Gln Ile Ile Asn Arg Val Arg Asp Lys Leu 2495
2500 2505 Thr Gly Arg Asp Phe Ser His Asp Asp Thr Leu Asp Val Pro
Thr 2510 2515 2520 Gln Val Glu Leu Leu Ile Lys Gln Ala Thr Ser His
Glu Asn Leu 2525 2530 2535 Cys Gln Cys Tyr Ile Gly Trp Cys Pro Phe
Trp 2540 2545 <210> SEQ ID NO 7 <211> LENGTH: 3376
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION
NUMBER: Genback M20869 <309> DATABASE ENTRY DATE: 1993-08-02
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(3376) <400>
SEQUENCE: 7 cgcaccgggg atcctaggct ttttggattg cgctttcctc tagatcaact
gggtgtcagg 60 ccctatccta cagaaggatg ggtcagattg tgacaatgtt
tgaggctctg cctcacatca 120 tcgatgaggt gatcaacatt gtcattattg
tgcttatcgt gatcacgggt atcaaggctg 180 tctacaattt tgccacctgt
gggatattcg cattgatcag tttcctactt ctggctggca 240 ggtcctgtgg
catgtacggt cttaagggac ccgacattta caaaggagtt taccaattta 300
agtcagtgga gtttgatatg tcacatctga acctgaccat gcccaacgca tgttcagcca
360 acaactccca ccattacatc agtatgggga cttctggact agaattgacc
ttcaccaatg 420 attccatcat cagtcacaac ttttgcaatc tgacctctgc
cttcaacaaa aagacctttg 480 accacacact catgagtata gtttcgagcc
tacacctcag tatcagaggg aactccaact 540 ataaggcagt atcctgcgac
ttcaacaatg gcataaccat ccaatacaac ttgacattct 600 cagatcgaca
aagtgctcag agccagtgta gaaccttcag aggtagagtc ctagatatgt 660
ttagaactgc cttcgggggg aaatacatga ggagtggctg gggctggaca ggctcagatg
720 gcaagaccac ctggtgtagc cagacgagtt accaatacct gattatacaa
aatagaacct 780 gggaaaacca ctgcacatat gcaggtcctt ttgggatgtc
caggattctc ctttcccaag 840 agaagactaa gttcttcact aggagactag
cgggcacatt cacctggact ttgtcagact 900 cttcaggggt ggagaatcca
ggtggttatt gcctgaccaa atggatgatt cttgctgcag 960 agcttaagtg
tttcgggaac acagcagttg cgaaatgcaa tgtaaatcat gatgccgaat 1020
tctgtgacat gctgcgacta attgactaca acaaggctgc tttgagtaag ttcaaagagg
1080 acgtagaatc tgccttgcac ttattcaaaa caacagtgaa ttctttgatt
tcagatcaac 1140 tactgatgag gaaccacttg agagatctga tgggggtgcc
atattgcaat tactcaaagt 1200 tttggtacct agaacatgca aagaccggcg
aaactagtgt ccccaagtgc tggcttgtca 1260 ccaatggttc ttacttaaat
gagacccact tcagtgatca aatcgaacag gaagccgata 1320 acatgattac
agagatgttg aggaaggatt acataaagag gcaggggagt acccccctag 1380
cattgatgga ccttctgatg ttttccacat ctgcatatct agtcagcatc ttcctgcacc
1440 ttgtcaaaat accaacacac aggcacataa aaggtggctc atgtccaaag
ccacaccgat 1500 taaccaacaa aggaatttgt agttgtggtg catttaaggt
gcctggtgta aaaaccgtct 1560 ggaaaagacg ctgaagaaca gcgcctccct
gactctccac ctcgaaagag gtggagagtc 1620 agggaggccc agagggtctt
agagtgtcac aacatttggg cctctaaaaa ttaggtcatg 1680 tggcagaatg
ttgtgaacag ttttcagatc tgggagcctt gctttggagg cgctttcaaa 1740
aatgatgcag tccatgagtg cacagtgcgg ggtgatctct ttcttctttt tgtcccttac
1800 tattccagta tgcatcttac acaaccagcc atatttgtcc cacactttgt
cttcatactc 1860 cctcgaagct tccctggtca tttcaacatc gataagctta
atgtccttcc tattctgtga 1920 gtccagaagc tttctgatgt catcggagcc
ttgacagctt agaaccatcc cctgcggaag 1980 agcacctata actgacgagg
tcaacccggg ttgcgcattg aagaggtcgg caagatccat 2040 gccgtgtgag
tacttggaat cttgcttgaa ttgtttttga tcaacgggtt ccctgtaaaa 2100
gtgtatgaac tgcccgttct gtggttggaa aattgctatt tccactggat cattaaatct
2160 accctcaatg tcaatccatg taggagcgtt ggggtcaatt cctcccatga
ggtcttttaa 2220 aagcattgtc tggctgtagc ttaagcccac ctgaggtgga
cctgctgctc caggcgctgg 2280 cctgggtgaa ttgactgcag gtttctcgct
tgtgagatca attgttgtgt tttcccatgc 2340 tctccccaca atcgatgttc
tacaagctat gtatggccat ccttcacctg aaaggcaaac 2400 tttatagagg
atgttttcat aagggttcct gtccccaact tggtctgaaa caaacatgtt 2460
gagttttctc ttggccccga gaactgcctt caagaggtcc tcgctgttgc ttggcttgat
2520 caaaattgac tctaacatgt tacccccatc caacagggct gcccctgcct
tcacggcagc 2580 accaagacta aagttatagc cagaaatgtt gatgctggac
tgctgttcag tgatgacccc 2640 cagaactggg tgcttgtctt tcagcctttc
aagatcatta agatttggat acttgactgt 2700 gtaaagcaag ccaaggtctg
tgagcgcttg tacaacgtca ttgagcggag tctgtgactg 2760 tttggccata
caagccatag ttagacttgg cattgtgcca aattgattgt tcaaaagtga 2820
tgagtctttc acatcccaaa ctcttaccac accacttgca ccctgctgag gctttctcat
2880 cccaactatc tgtaggatct gagatctttg gtctagttgc tgtgttgtta
agttccccat 2940 atatacccct gaagcctggg gcctttcaga cctcatgatc
ttggccttca gcttctcaag 3000 gtcagccgca agagacatca gttcttctgc
actgagcctc cccactttca aaacattctt 3060 ctttgatgtt gactttaaat
ccacaagaga atgtacagtc tggttgagac ttctgagtct 3120 ctgtaggtct
ttgtcatctc tcttttcctt cctcatgatc ctctgaacat tgctgacctc 3180
agagaagtcc aacccattca gaaggttggt tgcatcctta atgacagcag ccttcacatc
3240
tgatgtgaag ctctgcaatt ctcttctcaa tgcttgcgtc cattggaagc tcttaacttc
3300 cttagacaag gacatcttgt tgctcaatgg tttctcaaga caaatgcgca
atcaaatgcc 3360 taggatccac tgtgcg 3376 <210> SEQ ID NO 8
<211> LENGTH: 498 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <300> PUBLICATION INFORMATION: <308>
DATABASE ACCESSION NUMBER: Genbank <309> DATABASE ENTRY DATE:
1993-08-02 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(498)
<400> SEQUENCE: 8 Met Gly Gln Ile Val Thr Met Phe Glu Ala Leu
Pro His Ile Ile Asp 1 5 10 15 Glu Val Ile Asn Ile Val Ile Ile Val
Leu Ile Val Ile Thr Gly Ile 20 25 30 Lys Ala Val Tyr Asn Phe Ala
Thr Cys Gly Ile Phe Ala Leu Ile Ser 35 40 45 Phe Leu Leu Leu Ala
Gly Arg Ser Cys Gly Met Tyr Gly Leu Lys Gly 50 55 60 Pro Asp Ile
Tyr Lys Gly Val Tyr Gln Phe Lys Ser Val Glu Phe Asp 65 70 75 80 Met
Ser His Leu Asn Leu Thr Met Pro Asn Ala Cys Ser Ala Asn Asn 85 90
95 Ser His His Tyr Ile Ser Met Gly Thr Ser Gly Leu Glu Leu Thr Phe
100 105 110 Thr Asn Asp Ser Ile Ile Ser His Asn Phe Cys Asn Leu Thr
Ser Ala 115 120 125 Phe Asn Lys Lys Thr Phe Asp His Thr Leu Met Ser
Ile Val Ser Ser 130 135 140 Leu His Leu Ser Ile Arg Gly Asn Ser Asn
Tyr Lys Ala Val Ser Cys 145 150 155 160 Asp Phe Asn Asn Gly Ile Thr
Ile Gln Tyr Asn Leu Thr Phe Ser Asp 165 170 175 Arg Gln Ser Ala Gln
Ser Gln Cys Arg Thr Phe Arg Gly Arg Val Leu 180 185 190 Asp Met Phe
Arg Thr Ala Phe Gly Gly Lys Tyr Met Arg Ser Gly Trp 195 200 205 Gly
Trp Thr Gly Ser Asp Gly Lys Thr Thr Trp Cys Ser Gln Thr Ser 210 215
220 Tyr Gln Tyr Leu Ile Ile Gln Asn Arg Thr Trp Glu Asn His Cys Thr
225 230 235 240 Tyr Ala Gly Pro Phe Gly Met Ser Arg Ile Leu Leu Ser
Gln Glu Lys 245 250 255 Thr Lys Phe Phe Thr Arg Arg Leu Ala Gly Thr
Phe Thr Trp Thr Leu 260 265 270 Ser Asp Ser Ser Gly Val Glu Asn Pro
Gly Gly Tyr Cys Leu Thr Lys 275 280 285 Trp Met Ile Leu Ala Ala Glu
Leu Lys Cys Phe Gly Asn Thr Ala Val 290 295 300 Ala Lys Cys Asn Val
Asn His Asp Ala Glu Phe Cys Asp Met Leu Arg 305 310 315 320 Leu Ile
Asp Tyr Asn Lys Ala Ala Leu Ser Lys Phe Lys Glu Asp Val 325 330 335
Glu Ser Ala Leu His Leu Phe Lys Thr Thr Val Asn Ser Leu Ile Ser 340
345 350 Asp Gln Leu Leu Met Arg Asn His Leu Arg Asp Leu Met Gly Val
Pro 355 360 365 Tyr Cys Asn Tyr Ser Lys Phe Trp Tyr Leu Glu His Ala
Lys Thr Gly 370 375 380 Glu Thr Ser Val Pro Lys Cys Trp Leu Val Thr
Asn Gly Ser Tyr Leu 385 390 395 400 Asn Glu Thr His Phe Ser Asp Gln
Ile Glu Gln Glu Ala Asp Asn Met 405 410 415 Ile Thr Glu Met Leu Arg
Lys Asp Tyr Ile Lys Arg Gln Gly Ser Thr 420 425 430 Pro Leu Ala Leu
Met Asp Leu Leu Met Phe Ser Thr Ser Ala Tyr Leu 435 440 445 Val Ser
Ile Phe Leu His Leu Val Lys Ile Pro Thr His Arg His Ile 450 455 460
Lys Gly Gly Ser Cys Pro Lys Pro His Arg Leu Thr Asn Lys Gly Ile 465
470 475 480 Cys Ser Cys Gly Ala Phe Lys Val Pro Gly Val Lys Thr Val
Trp Lys 485 490 495 Arg Arg <210> SEQ ID NO 9 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide <400> SEQUENCE: 9 Lys Ala Val Tyr Asn Phe
Ala Thr Met 1 5 <210> SEQ ID NO 10 <211> LENGTH: 58
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 10 ccgggccaga atccatccat
tcattctcga gaatgaatgg atggattctg gctttttg 58 <210> SEQ ID NO
11 <211> LENGTH: 58 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
11 aattcaaaaa gccagaatcc atccattcat tctcgagaat gaatggatgg attctggc
58 <210> SEQ ID NO 12 <211> LENGTH: 58 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 12 ccgggcccga gtctgtgaat gtaatctcga
gattacattc acagactcgg gctttttg 58 <210> SEQ ID NO 13
<211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 13
aattcaaaaa gcccgagtct gtgaatgtaa tctcgagatt acattcacag actcgggc 58
<210> SEQ ID NO 14 <211> LENGTH: 58 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 14 ccgggccaaa ctgataatct cctcactcga
gtgaggagat tatcagtttg gctttttg 58 <210> SEQ ID NO 15
<211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 15
aattcaaaaa gccaaactga taatctcctc actcgagtga ggagattatc agtttggc 58
<210> SEQ ID NO 16 <211> LENGTH: 58 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 16 ccgggcatgg aacattgtga gaaatctcga
gatttctcac aatgttccat gctttttg 58 <210> SEQ ID NO 17
<211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 17
aattcaaaaa gcatggaaca ttgtgagaaa tctcgagatt tctcacaatg ttccatgc 58
<210> SEQ ID NO 18 <211> LENGTH: 58 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 18 ccggccgaag atagtgattg gttatctcga
gataaccaat cactatcttc ggtttttg 58 <210> SEQ ID NO 19
<211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 19
aattcaaaaa ccgaagatag tgattggtta tctcgagata accaatcact atcttcgg
58
* * * * *